Ultrafast laser diode thermal desorption method for analysis of representative pharmaceuticals in soil leachate samples.

We developed and evaluated a novel analytical method combining ambient ionization technique - laser diode thermal desorption with chemical ionization (LDTD-APCI) and tandem mass spectrometry detection. The LDTD/APCI-MS/MS method was developed for determination of representative pharmaceuticals from different classes (carbamazepine, sulfamethoxazole, irbesartan, fexofenadine) in leachate samples from soil sorption experimentation. We then optimized laser pattern, laser energy and spiked sample volume, which are crucial parameters for this LDTD/APCI-MS/MS method. We further identified utility of a chelating agent (Na2-EDTA) to obtain the highest achievable and reproducible signal of target analytes. Achieved method performance parameters (LODs, LOQs, trueness and precision) were comparable with those obtained from LC-MS/MS. However, application of this novel LDTD/APCI-MS/MS method reduced analysis time by two orders of magnitude (to 12 s), compared to more conventional LC-MS/MS approaches, without use of organic solvents. We expect this novel method will reduce costs and increase throughput for future analyses of pharmaceuticals in the environment while advancing a timely principle of green chemistry.

Select antibiotics in leachate from closed and active landfills exceed thresholds for antibiotic resistance development.

Though antibiotic resistance (ABR) represents a major global health threat, contributions of landfill leachate to the life cycle of antibiotics and ABR development are poorly understood in rapidly urbanizing regions of developing countries. We selected one of the largest active landfills in Asia and two landfills that have been closed for 20 years to examine antibiotic occurrences in leachates and associated hazards during wet and dry season sampling events. We focused on some of the most commonly used human antibiotics in Hong Kong, one of the most populous Asian cities and the fourth most densely populated cities in the world. Seven antibiotics (cephalexin [CLX], chloramphenicol [CAP], ciprofloxacin [CIP], erythromycin [ERY], roxithromycin [ROX], trimethoprim [TMP], sulfamethoxazole [SMX]) were quantitated using HPLC-MS/MS generally following previously reported methods. Whereas CLX, CAP, ROX and SMX in leachates did not exceed ABR predicted no effect concentrations (PNECs), exceedances were observed for CIP, ERY and TMP in some study locations and on some dates. In fact, an ABR PNEC for CIP was exceeded in leachates during both sampling periods from all study locations, including leachates that are directly discharged to coastal systems. These findings highlight the importance of developing an advanced understanding of pharmaceutical access, usage and disposal practices, effectiveness of intervention strategies (e.g., leachate treatment technologies, drug take-back schemes), and contributions of landfill leachates to the life cycle of antibiotics and ABR development, particularly in rapidly urbanizing coastal regions with less advanced waste management systems than Hong Kong.

Effects of sertraline on behavioral indices of crayfish Orconectes virilis.

Sertraline, a selective serotonin re-uptake inhibitor, is a widely prescribed antidepressant in North America. Though sertraline is continuously released from wastewater treatment plant discharge to surface water, effects of aqueous exposure of sertraline on behavioral responses of aquatic animals are largely unknown. Our study explored the effects of aqueous exposures of sertraline on antagonistic bouts and predator response behavior of virile crayfish (Orconectes virilis). Crayfish were either exposed or not exposed to waterborne sertraline and then size-matched for paired antagonistic bouts to determine if sertraline affects the aggression of each crayfish. We investigated the effect of sertraline on responses to visual predator cues and determined whether sertraline acts as an olfactory cue. Our results demonstrate that crayfish exposed to sertraline are more aggressive when paired with control crayfish but, when sertraline crayfish are paired, there is no change in aggression. Attraction response to sertraline in behavioral mazes was also observed, which may represent a maladaptive behavior, and in an ecological context may result in crayfish moving to areas with elevated levels of sertraline. However, aqueous exposure to sertraline had no effect on predator responses of crayfish. Future research is warranted to determine whether such medicine released in wastewater treatment plant effluents produces long-term ecologically important consequences for aquatic animals residing in urbanized aquatic ecosystems.

Inactivation of infectious bursal disease and Newcastle disease viruses at temperatures below 0 C using chemical disinfectants.

This study evaluated the effectiveness of bleach, Surface Decontamination Foam (SDF), and Virkon in inactivating infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) at temperatures below 0 C. To simulate the influence of organic load on the effectiveness of disinfectants, as would be encountered in disinfecting farm vehicles and equipment, the viruses were suspended in preparations containing light or heavy levels of organic matter. A small volume of the viral suspension was applied to the upper surface of stainless steel carrier disks and these were then air dried. The dried virus inoculum was covered with disinfectant to which propylene glycol had been added to prevent freezing. The disks were incubated at various temperatures for periods up to 24 hr. With NDV, at -10 C all three disinfectants in both organic preparations achieved a 5 log 10 reduction within 5 min. Results with SDF were similar at -25 and -10 C. To achieve comparable reduction with Virkon and bleach at -25 C, contact periods up to 2 or 24 hr, respectively, were required. With IBDV, to achieve a 5 log 10 to reduction by treatment with Virkon or SDF at -20 C, contact periods of 2 or 24 hr, respectively, were required in both organic preparations. It was concluded that at temperatures as low as -20 to -25 C, SDF was the most effective disinfectant for killing NDV and Virkon was most effective for killing IBDV. The finding that a contact period of up to 2 hr was required to kill IBDV, whereas NDV was killed within 5 min, attests to the greater stability of the former virus.

Evaluation of an isotope dilution liquid chromatography tandem mass spectrometry method for pharmaceuticals in fish.

An isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was successfully developed and applied for analysis of 15 pharmaceuticals and 2 pharmaceutically active metabolites in fish tissues. This method relied on electrospray ionization (ESI), for which the influence of sample matrix on analyte ionization efficiencies remains a persistent challenge to environmental analysis. Statistically derived method detection limits (MDLs) for most analytes ranged from 1 to 10 ng/g, independent of sample matrix, and were as low as 0.04 ng/g for the most sensitive compounds in fillet tissue. MDLs for fish fillets were determined for both 10 µL and 100 µL injection volumes; however, results showed that detection limits did not scale linearly with injection volume. Direct comparison of spike recoveries from fish liver demonstrated that isotope dilution was superior to matrix-matched calibration in compensating for matrix interference. Spike recoveries for the isotope dilution approach generally ranged from 91 to 112%, independent of tissue (i.e., fillet or liver). The developed method was applied to examine target analytes in brown trout (Salmo trutta), collected upstream and downstream from a municipal effluent discharge to East Canyon Creek, Park City, UT, USA. Though no pharmaceuticals were detected in fish samples from the upstream location, 3 and 10 compounds (out of 17 target analytes) were detected in fish fillet and liver samples, respectively, from the downstream sampling site. Pharmaceuticals in fish fillets were observed at concentrations ranging from 0.14 to 12 ng/g, while levels were markedly higher in liver tissues (range: 0.27-600 ng/g).

Suggesting a testing strategy for possible endocrine effects of drug metabolites.

Most pharmaceuticals are extensively metabolized by organisms, which results in internal exposure to mixtures of parent compounds and various metabolites. Many of these metabolites are considered non-toxic, but some metabolites retain toxic properties of the parent compound or elicit other undesirable outcomes. Unfortunately, the effects of metabolites are often not considered when endocrine activities of chemicals are evaluated in vitro. In this study two approaches, an "effect-based" and a "compound-by-compound" testing design, were used to determine the effects of metabolites of the antidepressant sertraline on aromatase enzyme activity. In the "effect-based" approach, a mixture of sertraline metabolites, produced by liver microsomes, inhibited aromatase, but was less potent than sertraline. In the "compound-by-compound" testing design, three specific metabolites were evaluated individually and in mixtures. Though two N-desmethylated metabolites were more potent aromatase inhibitors than sertraline, hydroxyl ketone sertraline did not inhibit the enzyme and mixtures of these metabolites and sertraline were less potent than predicted from a concentration addition model. Our findings highlight the importance of considering aromatase inhibition, and potentially other biological activities, of pharmaceutical metabolites produced by liver microsome preparations and then comparing such observations to studies of specific metabolites available for testing in pure form. Subsequently, a five step integrated strategy for screening of the potential endocrine effects of drugs and their metabolites are proposed.

Infectious bursal disease virus as a surrogate for studies on survival of various poultry viruses in compost.

Infectious bursal disease virus (IBDV) is resistant to many environmental stresses and often persists on farms for months. This study investigated survival of a vaccine strain of IBDV in the bursa of Fabricius and splenic tissue from experimentally infected chickens and in splenic tissue and manure that had been inoculated with the virus. The specimens buried in compost were contained within nylon mesh bags, and the tissues were enclosed within the abdominal cavity of chicken carcasses. Extracts of composted specimens were inoculated into Vero cell cultures, and real-time reverse transcriptase PCR was used to quantify the virus in the cultures. By day 7 in compost, the temperature had been slightly above 55 C for 2.6 days and IBDV had been inactivated in specimens that had been inoculated with virus but had survived in tissues that had been taken from infected chickens. By day 14, the temperature had been above 55 C for 8.8 days and the virus was inactivated in all specimens. The results suggest that composting of poultry carcasses and manure would help to break the cycle of infection with IBDV and that the virus could be valuable as a surrogate for predicting the inactivation of less resistant viruses during composting.

Degradation of foot-and-mouth disease virus during composting of infected pig carcasses.

The objective of this study was to investigate the inactivation and degradation of foot-and-mouth disease (FMD) virus during composting of infected pig carcasses as measured by virus isolation in tissue culture and by real-time reverse transcriptase polymerase chain reaction (RRT-PCR). Three FMD-infected pig carcasses were composted in a mixture of chicken manure and wood shavings in a biocontainment level 3 facility. Compost temperatures had reached 50 degrees C and 70 degrees C by days 10 and 19, respectively. Under these conditions, FMD virus was inactivated in specimens in compost by day 10 and the viral RNA was degraded in skin and internal organ tissues by day 21. In comparison, at ambient temperatures close to 20 degrees C, FMD virus survived to day 10 in the skin tissue specimen from the pig that had the highest initial level of viral RNA in its tissues and the viral RNA persisted to day 21. Similarly, beta-actin mRNA, tested as a PCR control, persisted to day 21 in specimens held at ambient temperatures, but it was degraded in the remnants of tissues recovered from compost on day 21. Results from this study provide evidence that composting could be used for safe disposal of pig carcasses infected with FMD virus.

Survival of avian influenza and Newcastle disease viruses in compost and at ambient temperatures based on virus isolation and real-time reverse transcriptase PCR.

In four composting experiments, survival of avian influenza (AI) and Newcastle disease (ND) viruses was assessed by virus isolation in embryonated chicken eggs (ECEs) and by real-time reverse transcriptase-polymerase chain reaction. Specimens contained in nylon mesh bags consisted of 20-g samples of chicken manure, used litter, or feed that had been inoculated with allantoic fluid containing an AI virus (H6N2, Expt. 1) or an ND vaccine virus (Expt. 2). Other specimens consisted of 20-g samples of infected ECEs that had been homogenized and mixed with corn silage. As a control, allantoic fluid diluted in phosphate-buffered saline was contained in sealed vials. Except for the feed, in which the AI virus was inactivated soon after the specimen was inoculated, on day 0 the specimens buried in compost or placed outside at ambient temperatures contained at least 5.0 log10 of virus and 7.7 log10 of viral RNA. By day 7, temperatures in compost ranged from 50 C to 65 C, and viruses had been killed in all specimens in bags. In comparison, viruses in sealed vials remained viable to day 10. Viral RNA in mesh-bag specimens had been degraded to nondetectable levels by day 10, but it was still detected in sealed vials on day 21. In specimens that were held at ambient temperatures (13 C-28 C), the viruses in mesh-bag specimens were inactivated by day 21, but their RNA was still detected. In comparison, the viruses in sealed vials survived to day 21. In Expts. 3 and 4, viruses were inactivated in carcass specimens and in whole ECEs during composting. In an in vitro experiment, the time required for a 1-log10 reduction of viruses was significantly shorter (P < 0.05) in water extracts from compost than in phosphate buffers at temperatures of 25 C to 45 C. This study provided evidence that microbial activity during composting contributed to the rapid killing of AI and ND viruses and to the degradation of their viral RNA.

Development of methods for detection and quantification of avian influenza and Newcastle disease viruses in compost by real-time reverse transcription polymerase chain reaction and virus isolation.

Composting has been used for disposal of poultry carcasses and manure following outbreaks caused by avian influenza virus (AIV) and Newcastle disease virus (NDV), but methods are needed to test for survival of the viruses in compost to ensure biosecurity. Methods developed in the present study include extracting viruses from compost and purifying viral RNA. The extracted viruses were detected by virus isolation using embryonated chicken eggs, and the purified RNA was detected by real-time reverse transcription PCR (RRT-PCR). The virus isolation and the RRT-PCR methods were evaluated with 3 compost preparations that were produced from chicken manure mixed with corn silage, wood shavings, or wheat straw. The detection limits of both methods were 1,700 and 1,000 embryo lethal doses of AIV and NDV per gram of compost, respectively. The copy number of viral RNA quantified by RRT-PCR was highly correlated with the amount of virus in compost. The results suggested that the RRT-PCR method may be used as an alternative to the virus isolation method for rapid detection and accurate quantification of AIV and NDV in compost.

Structural characterization and serological specificities of lipopolysaccharides from Salmonella enterica serovar Gallinarum biovar Pullorum standard, intermediate and variant antigenic type strains.

The structure and serological specificities of the lipopolysaccharides (LPSs) from Salmonella enterica serovar Gallinarum biovar Pullorum were studied to provide an improved basis for the distinction between antigenic types and the development of improved diagnostic tests. The structure of the LPS O-polysaccharide (O-PS) from S. Pullorum standard, intermediate and variant antigenic type strains was determined by mass spectrometry, nuclear magnetic resonance spectroscopy and chemical analysis. The LPS of the three strains shared a common structural repeating oligosaccharide unit containing d-mannose, l-rhamnose, d-galactose and d-tyvelose (1:1:1:1). The O-PS of the variant type LPS contained an additional d-glucose residue linked to the O-4 position of the d-galactose residue. The O-PS of the intermediate type LPS was partially the same as that of the variant LPS, however, the molar ratio of the d-glucose component was lower with respect to the other glycose components. Serological specificities of the three antigenic type LPSs were examined with anti-S. Pullorum LPS monoclonal antibodies (Mabs). On immunoblots, Mabs to the standard type O-PS reacted with high molecular mass (HMM) and low molecular mass (LMM) LPS from the standard strain, and with LMM but not HMM LPS from the variant strain. Monoclonal antibodies to the variant type O-PS reacted with HMM but not LMM LPS from the variant strain, and did not react with HMM or LMM LPS from the standard strain. On ELISA, the standard, intermediate and variant antigenic type strains were differentiated by the relative reactivity with the anti-LPS O-PS Mabs. Several of the anti-LPS O-PS Mabs were specific for S. Pullorum and other serogroup D1 Salmonella, and are potentially useful for the development of improved diagnostic tests for these organisms.

In vitro study of Salmonella enteritidis and Salmonella typhimurium definitive type 104: survival in egg albumen and penetration through the vitelline membrane.

Salmonella enteritidis and Salmonella typhimurium definitive type 104 (DT104) have been detected in the chicken oviduct, and their survival in egg albumen at the chicken body temperature of 42 degrees C may be important in oviductal and transovarian contamination of intact shell eggs. Eight S. enteritidis and 24 S. typhimurium DT104 strains were tested for their in vitro survival in egg albumen. The concentration of the organisms declined more rapidly when incubated at 42 degrees C than at 37 degrees C and dropped to nondetectable levels within 96 h at the higher, but not at the lower, temperature. In another experiment, 3 S. enteritidis and 3 S. typhimurium DT104 strains were randomly selected, and dosages of 20 and 200 cells of each strain were inoculated onto the vitelline membranes of egg yolks, which were then submerged in the original albumen and incubated for 24 h at 42 degrees C. Under these conditions, the organisms survived in albumen but did not penetrate the vitelline membrane. However, in a similar experiment, penetration did occur when the specimens were incubated at 30 degrees C for 72 h. The results suggest that low numbers of S. enteritidis and S. typhimurium DT104 can be expected to survive in egg albumen during the 24-h period of egg formation in the oviduct but would be unlikely to invade the yolk.before oviposition. However, depending on storage conditions following oviposition, S. enteritidis, as well as S. typhimurium DT104, could survive longer and may eventually invade the egg yolks.

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples.

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples.

Catastrophic phase inversion in region II of an ionomeric polymer-water system.

Previous work has identified distinct regions, on a phase inversion map, for dispersions of polyurethane ionomer (PUI) and water. In this study, events that occur, before, during, and after catastrophic phase inversion (provoked by adding water to polyurethane ionomer (PUI) in the RII regions of the phase inversion map) have been studied in order to characterise the inversion mechanism. Before phase inversion, initial water addition leads to the hydration of ionic groups and eventually water drops start to form in the hydrophobic portions of the polymer matrix. At the phase inversion point, the PUI-water interface restructures and the ionomer disintegrates into a dispersion of spherical particles enclosed by a continuous aqueous phase. It is suggested that pseudo-drop structures are formed simultaneously during the production of the small polymer-in-water drops. After phase inversion, water addition dilutes the emulsion and destroys the apparent ionic-centre-rich environment surrounding any isolated ionic groups on a particle surface. The larger water-in-polymer drops are likely to have participated in the phase inversion and the smaller water drops form the primary water drops in the multiple emulsions. The resultant emulsions are stable over a period of a few months but very few multiple drops remain after 1(1/4) years.

Identification and serological specificity of a polysaccharide component from Mycoplasma bovis.

Whole-cell lysate and proteinase K digest preparations of the Mycoplasma bovis type strain (American Type Culture Collection 25523) were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Coomassie blue staining for protein revealed approximately 50 bands for the lysate but only a single band for the digest. Silver staining for polysaccharide revealed at least 19 bands for the digest. Fourteen monoclonal antibodies (MAbs) were produced using a screening procedure with an M. bovis digest. On immunoblots of digests of four M. bovis strains, an almost identical profile was seen with each strain for all 14 MAbs but differences were evident between strains. One MAb, M1557, was used to analyse 17 M. bovis strains on immunoblots. Ten to 20 bands were observed with 16 of the 17 strains, and differences were apparent among all 16 strains. In an enzyme-linked immunosorbent assay, M1557 reacted with 16 of the 17 M. bovis strains, but did not react with any of 41 non-M. bovis organisms tested. Strong reactions were observed with the MAbs and M. bovis colonies in immunofluorescence. The M. boris polysaccharide and MAbs to this component may be useful for the development of diagnostic assays for this organism.

Different dispersion regions during the phase inversion of an ionomeric polymer-water system.

Catastrophic phase inversion is induced by changing the phase ratio in a liquid-liquid dispersion and is widely used during the dispersion stage in the production of aqueous polyurethane ionomer (PUI) colloids. In the work reported here, water was added to polyurethane ionomer prepolymer (PUIp) until the water became the continuous phase. Three different dispersion regions have been discovered by changing the ionic group content. Stable emulsions containing small polymer drops were produced in Region I. Stable coarse emulsions containing a mixture of drop structures were produced in Region II, but only temporary dispersions could be produced in Region III. Conductivity measurements could not always be used to detect the phase inversion points effectively because the PUIp was swollen by water. Therefore, torque change measurements have been used in conjunction with the conductivity measurements to detect the phase inversion points for all three dispersion regions. Scanning electron microscopy (SEM) and optical microscopy were used to obtain images of these dispersions in the different regions. A catastrophic phase inversion map is used to represent the changes that occur in the PUIp-W dispersions. This map is plotted using the ionic group content as the ordinate and water content (at the phase inversion points) as the abscissa.

Toxicity of select beta adrenergic receptor-blocking pharmaceuticals (B-blockers) on aquatic organisms.

One class of pharmaceutical compounds identified in U.S. and European waters are the B-adrenergic receptor blocking compounds (B-blockers). However, little information is available on the potential aquatic toxicity of these compounds. Therefore, Hyalella azteca, Daphnia magna, Ceriodaphnia dubia, and Oryias latipes (Japanese medaka) were exposed to metoprolol, nadolol, and propranolol to determine potential toxicity. Average 48-h LC(50) for propranolol to H. azteca was 29.8 mg/L. The no-observed-effects concentration (NOEC) and lowest-observed-effects concentration (LOEC) for propranolol affecting reproduction of H. azteca were 0.001 and 0.1 mg/L, respectively. The average propranolol and metoprolol 48-h LC(50)s for D. magna were 1.6 and 63.9 mg/L, respectively. C. dubia 48-h LC(50)s were 0.85 and 8.8 mg/L for propranolol and metoprolol, respectively. The NOEC and LOEC of propranolol affecting reproduction in C. dubia were 0.125 and 0.25 mg/L, respectively. In O. latipes, the propranolol 48-h LC(50) was 24.3 mg/L. Medaka growth was decreased at 0.5 mg/L propranolol. A 2-week medaka reproductive study indicated significant changes in plasma steroid levels; however, no changes in the average number of eggs produced or number of viable eggs which hatched was observed. In a 4-week follow-up propranolol exposure, the total number of eggs produced by medaka and the number of viable eggs that hatched were decreased at concentrations as low as 0.5 microg/L. Based on this study and the expected aqueous environmental exposure levels, adverse effects of propranolol to invertebrate populations is unlikely; however, further reproductive studies are need to elucidate the risk to teleosts.

Monoclonal antibodies specific for Campylobacter fetus lipopolysaccharides.

Four monoclonal antibodies (mAbs) (M1357, M1360, M1823 and M1825) which reacted with Campylobacter fetus lipopolysaccharide (LPS) core region epitopes were produced and characterized. Reactivity of these mAbs with C. fetus core LPS epitopes was determined by enzyme-linked immunosorbent assay (ELISA) with whole cell proteinase K digests and phenol-water extracted LPS, and by immunoblotting with proteinase K digests. The specificities of the four mAbs were evaluated using an indirect ELISA. One of the mAbs reacted with 42 and three of the mAbs reacted with 41 of the 42 C. fetus strains examined. No reaction was observed between the four mAbs and 32 non-C. fetus bacteria tested, with the exception of one mAb with one organism. The four mAbs reacted with serotype A and B strains indicating the presence of shared epitopes in C. fetus LPS core oligosaccharides. The specificities of three mAbs previously produced to C. fetus LPS O-antigens (M1177, M1183 and M1194) were also evaluated and no reaction was observed with these mAbs and the 32 non-C. fetus bacteria tested. Strong immunofluorescence reactions were observed with the anti-O chain mAbs and selected C. fetus strains of the homologous serotype. These anti-LPS core oligosaccharide and anti-LPS O chain mAbs are highly specific for C. fetus and are potentially useful as immunodiagnostic reagents for detection, identification and characterization of C. fetus.