Contaminants and levels of occurrence in washed and shredded poly(ethylene terephthalate) from curbside collection. II: Validation of extraction procedures, particle size sampling and crystallinity.

Part I of this study showed that washed and dried, shredded poly(ethylene terephthalate) (PET) (flake) obtained from curbside collection when Soxhlet extracted contained 26 semivolatile contaminants below the US FDA threshold of 215 ppb and six above this level. This paper reports the validation of the Soxhlet extraction technique by comparison with total dissolution with trifluoroacetic acid (TFA). The work was carried out for two of the three particle size ranges obtained by grinding the PET flake (300-425 and 425-700 microm) and for the unground flake. Further validation was undertaken by comparison of contaminant levels determined by total dissolution with TFA and sonication with dichloromethane (DCM) using flake ground to the 0-300 microm size range. The levels of contaminants increased with decreasing particle size range, but X-ray diffraction measurements of degrees of crystallinity were similar for each PET particle size range, thus showing that the differences in contaminant levels were not due to variable percentages of the amorphous material from the tops and bottoms of shredded bottles, relative to the amounts of crystalline PET from the mid-sections of the bottles. Hence, it was postulated that the variations in contaminant levels were due to selective grinding of the more highly contaminated surfaces, whilst the larger particles incorporated the less contaminated interior material. The grinding was also strongly selective with respect to the amorphous flake. Analysis of the segregated amorphous and crystalline flake phases indicated that many contaminants were similarly absorbed into both phases, whilst some were preferred by the amorphous PET and others were preferred by the crystalline PET.

Contaminants and levels of occurrence in washed and shredded poly(ethylene terephthalate) from curbside collection. Part 1: Extraction conditions.

The aim was to determine which contaminants were present in washed and dried shredded poly(ethylene terephthalate) (PET, flake) obtained from curbside collection and whether the concentrations were above the US FDA threshold of 215 ppb. Thirty-two semi-volatile contaminants were extracted from the treated flake by Soxhlet extraction using dichloromethane as a PET swelling solvent and gas chromatography-mass spectrometry (GC-MS) was used for identification and quantification. Soxhlet extraction of flake ground to 0-300 pm was effectively completed in 24 h, whereas sonication reduced the extraction time to 3 h. In contrast, Soxhlet extractions of flake ground to a larger particle size range (300-425 pm) were completed in 4 h, possibly due to less aggregation in the extraction thimble. The levels of 26 contaminants were below 215 ppb, but six were not. Dodecanoic acid was present at about 1200 ppb, 2-butoxyethanol was approximately 1000 ppb, limonene, benzophenone and methyl salicylate were above 800 ppb, and 2-methyl-naphthalene near 215 ppb.

Effects of dietary fat and conjugated linoleic acid on plasma metabolite concentrations and metabolic responses to homeostatic signals in pigs.

Sixteen female cross-bred (Large White x Landrace) pigs (initial weight 65 kg) with venous catheters were randomly allocated to four treatment groups in a factorial design. The respective factors were dietary fat (25 or 100 g/kg) and dietary conjugated linoleic acid (CLA; 0 or 10 g CLA-55/kg). Pigs were fed every 3 h (close to ad libitum digestible energy intake) for 8 d and were bled frequently. Plasma glucose and non-esterified fatty acid (NEFA) responses to insulin and adrenaline challenges were determined on day 8. Plasma concentrations of NEFA were significantly increased (10.5 and 5.4 % for low- and high-fat diets respectively, P=0.015) throughout the experiment, suggesting that there was a possible increase in fat mobilisation. The increase in lipolysis, an indicator of ss-adrenergic stimulated lipolysis, was also evident in the NEFA response to adrenaline. However, the increase in plasma triacylglycerol (11.0 and 7.1 % for low- and high-fat diets respectively, P=0.008) indicated that CLA could have reduced fat accretion via decreased adipose tissue triacylglycerol synthesis from preformed fatty acids, possibly through reduced lipoprotein lipase activity. Plasma glucose, the primary substrate for de novo lipid synthesis, and plasma insulin levels were unaffected by dietary CLA suggesting that de novo lipid synthesis was largely unaffected (P=0.24 and P=0.30 respectively). In addition, the dietary CLA had no effect upon the ability of insulin to stimulate glucose removal.

Salt effects in capillary zone electrophoresis. V. Adsorption and retention of electrically neutral analytes.

Two electrically neutral analytes previously observed to be separated from the neutral marker in capillary zone electrophoresis (CZE) experiments [sulphanilamide (SAA) and sulphaguanidine (SGW)] have been examined to determine the basis for separation. The degree of separation increases markedly with buffer concentration and improves with increasing field strength. On the basis of the apparent electrophoretic mobilities in conventional CZE, migration times in a zero EOF environment were calculated for SAA, SGW and six other sulphonamides that were known to be ionized. These six markers were used to test the legitimacy of our predictions and to correct for small discrepancies between the predicted and observed migration times. It was concluded that SAA and SGW have negligible electrophoretic mobilities and that they are retained in the electrical double layer close to the capillary wall. A mechanism for adsorption is proposed and the generality of the phenomenon is discussed.

Comparison of silver-ion high-performance liquid chromatographic quantification of free and methylated conjugated linoleic acids.

Silver-ion high-performance liquid chromatography was used to fractionate a mixture of conjugated linoleic acid (CLA) isomers (as the free fatty acids, CLAFFA) in commercial CLA mixtures and biological samples. Due to the unchanged retention mechanism, it was assumed that the elution order of the isomers remained the same as that of methyl esters separated on the same column. The most abundant isomers, cis/trans 10, 12-18:2 and cis/trans 9,11-18:2, were separated better as free acids on a single column than in the methyl ester form. Quantification of the CLA standard was used as the reference profile to evaluate different methylation methods commonly used to prepare CLA methyl esters for quantitation. Acid-and base-catalyzed derivatization methods resulted in CLA intraisomerization and losses in total conjugated dienes content. Acid (HCl and BF3) methylations significantly elevated the level of trans,trans isomers and significantly reduced the cis/trans isomers. Base methylation, tetramethylguanidine/methanol, resulted in loss of trans,trans isomers, and a substantial loss of total underivatized conjugated dienes. Other catalysts such as the trimethylsilyldiazomethane produced additional peaks of unidentified artifacts. The analysis of CLAFFA appears to provide more accurate quantification of CLA isomers in commercial and biological samples.

Dietary conjugated linoleic acids increase lean tissue and decrease fat deposition in growing pigs.

Conjugated linoleic acids (CLA) decrease the body fat content of rodents; the aim of this study was to determine whether dietary CLA altered carcass composition of pigs. Female Large White x Landrace pigs (n = 66) were used in this study. To obtain initial body composition, six pigs were slaughtered at 57 kg live weight, whereas the remaining pigs were allocated to one of six dietary treatments (0, 1.25, 2.5, 5.0, 7.5 and 10.0 g/kg CLA, containing 55% of CLA isomers). The diets, containing 14.3 MJ digestible energy (DE) and 9. 3 g available lysine per kg, were fed ad libitum for 8 wk. Dietary CLA had no significant effect on average daily gain (861 vs. 911 g/d for pigs fed diets with and without CLA, P = 0.15) or feed intake (2. 83 vs. 2.80 kg/d, P = 0.74). The gain to feed ratio was increased by dietary CLA by 6.3% (0.328 vs. 0.348, P = 0.009). Fat deposition decreased linearly (-8.2 +/- 2.09 g/d for each gram per kilogram increase in CLA concentration; P < 0.001) with increasing inclusion of CLA. At the highest level of CLA inclusion, fat deposition was decreased by 88 g/d (-31%). Similarly, the ratio of fat to lean tissue deposition decreased linearly (-0.093 +/- 0.0216 for each gram per kilogram increase in CLA concentration; P < 0.001) with increasing dietary CLA. The carcass lean tissue deposition response to dietary CLA was quadratic in nature and was maximized (+25%) at 5. 0 g/kg dietary CLA. Overall, dietary CLA increased the gain to feed ratio and lean tissue deposition and decreased fat deposition in finisher pigs.

Salt effects in capillary zone electrophoresis. IV. Resolution versus time and the effect of potassium phosphate and its concentration in the high ionic strength separation of sulphonamides.

The effects of potassium phosphate buffer and its concentration upon the capillary zone electrophoretic separation of 23 sulphonamides and a neutral marker were examined at pH 7. The resolution between the pairs was improved with the increased concentration of the buffer from 65 mM to 174 mM. Nineteen sulphonamides, a hydrolysis product and several unidentified minor components were baseline resolved in both 101 and 138 mM phosphate buffers. In 174 mM buffer all 21 ionised sulphonamides and the other compounds were separated. A simple relationship between the resolution of analyte pairs (Rs) and the square root of the mean analysis time for the pair (square root of tapp) was derived, but few of the pairs displayed this behaviour. For the majority of pairs of compounds, Joule heating appeared to cause a maximum in the Rs versus square root of tapp relationship, while non-ideality and shifts in ionisation with increasing salt concentration appeared dominant in other cases.

Chromatographic separation of fluorescent thiol adducts of 4-chloro-7-sulphobenzofurazan. Use as substrates for enzymes of the mercapturic acid xenobiotic pathway.

Fluorescent adducts of 4-chloro-7-sulphobenzofurazan with cysteine, cysteinylglycine, reduced glutathione and N-acetylcysteine were prepared. Adducts were separated by HPLC on a 3-mm Nova-Pak C18 reversed-phase column using isocratic elution with a solvent of acetonitrile-0.15 M phosphoric acid (5:95) buffered at pH 2.5. The adducts were detected using a fluorescence detector set at an excitation wavelength of 365 nm and an emission wavelength of 510 nm and an ultraviolet detector at 254 nm. The adduct of reduced glutathione was also formed by the action of the enzyme glutathione-S-transferase. This adduct acted as a substrate for the enzyme gamma-glutamyltranspeptidase and the product of this reaction, the 4-chloro-7-sulphobenzofurazanyl derivative of cysteinylglycine, acted as a substrate for either dipeptidase or aminopeptidase M. The sequential enzymic effects could be detected by changes in the relative fluorescence intensity of the solutions to which the respective enzymes had been added but were more appropriately followed by changes in the HPLC elution profiles after enzymic treatment of solutions.

Formation and verification of the structure of the 1-fluorenylmethyl chloroformate derivative of sulfamethazine.

Sulfamethazine (SMZ) is derivatized with 1-fluorenylmethyl chloroformate (FMOC) to form the fluorescent adduct SMZ-FMOC. Conditions for formation are optimized with respect to pH, reagent concentration, and reagent ratio. Reagent and product profiles (including the hydrolysis byproduct FMOC-OH) versus time are followed by reversed phase HPLC with UV absorbance detection. FMOC-SMZ has been crystallized, its composition confirmed by microanalysis, and its structure corroborated by IR and NMR spectroscopy. From 10 down to 1 ppm, there is clear gentle curvature in the fluorescence intensity of SMZ-FMOC. The linear response range extends from above 100 ppb down to about 100 ppt, and an increase in sensitivity for the fluorescent detection of FMOC-SMZ (over the usual UV absorbance detection of SMZ) is calculated to be better than 3 orders of magnitude.

The supercritical fluid extraction of polar drugs (sulphonamides) from inert matrices and meat animal products.

The objective of this study was to examine the extension of supercritical fluid extraction (SFE) to the extraction of polar drugs. The ultimate aim was to extract veterinary residues from food animal products and thereby demonstrate the versatility of SFE. This technique is shown to have many facets that require careful thought and understanding if it is to be successfully used. Our initial studies indicate that polar drugs may be readily solubilized from relatively inert matrices such as sand, with high recoveries and very little discrimination between related compounds while using only moderate extraction conditions and times. SFE of the same drugs from spiked chicken liver and swine muscle is significantly more difficult and requires more drastic conditions. Close to complete recoveries are achieved for some drugs, while considerably less is found in the worst case. For sulphamerazine, sulphamethizole, sulphamethazine, sulphamethoxypyridazine, sulphamethoxazole, and the major metabolite N4-acetyl-sulphamethoxazole, the recoveries are 97, 66, 94, 79, 53, and 65%, respectively, from spiked liver and 95, 27, 86, 91, 96 and 70%, respectively, from spiked swine muscle. Incurred sulphamethazine is recovered from swine muscle in good general agreement with the reference values provided.

Serial propagation of porcine group C rotavirus (pararotavirus) in a continuous cell line and characterization of the passaged virus.

The Cowden strain of porcine group C rotavirus (pararotavirus) was adapted to serial passage in a continuous monkey kidney cell line (MA104). Key factors in its successful adaptation included use of virus passaged in primary porcine kidney cells as the initial inoculum, use of roller tubes, and addition of pancreatin to the maintenance medium. A cell culture immunofluorescence test was used to quantitate the virus at each passage level, since a possible cytopathic effect was obscured by the effects of pancreatin. The virus titers dropped after initial passage into MA104 cells but increased thereafter, with peak titers evident after 16 passages (10(7) immunofluorescence U/ml). Immune electron microscopy and genome electropherotyping were used to identify group C rotavirus particles and confirm group C rotavirus double-stranded RNA gel migration patterns, respectively, from infected cell culture supernatants. The electropherotype of the cell culture-propagated group C rotavirus was identical to that of the gut virulent virus from which it was derived. The cell culture-passaged group C rotavirus also retained its infectivity for gnotobiotic pigs. No group A rotavirus was detected in the intestinal contents of the pigs or in cell culture fluids from group C rotavirus-inoculated monolayers with the two former techniques or the cell culture immunofluorescence test. This is the first verified report of serial propagation of a non-group A rotavirus in a continuous cell line.

Effect of age, weaning and postweaning diet on small intestinal growth and jejunal morphology in young swine.

The effect of age, weaning and postweaning diet on small intestinal growth and morphology were investigated in young swine. Small intestine weight and villus height, measured at the midpoint of the small intestine (i.e., jejunum), were determined in suckling and weaned pigs. Scanning electron microscopy was performed on jejunal specimens from suckling pigs killed at 2, 10, 21, 28 and 35 d of age and in 21-d and 35-d weaned pigs at various ages postweaning. A 2 X 2 factorial arrangement of postweaning diets also was used to investigate jejunal morphological measurements in a 21-d-old weanling pig group. These dietary treatments evaluated the effects of 0 or 25% added dried whey and 0 or 6% added corn oil. The morphology results demonstrated that jejunal villus height declined during the suckling period, with a marked reduction at 3 and 7 d postweaning for both 21-d-old and 35-d-old weaned pigs. Transmission electron microscopy also demonstrated long, uniform microvilli on the jejunal villi in suckling pigs at 2 and 21 d, with markedly reduced lengths upon weaning. Jejunal villi were shorter in weaned compared with suckling pigs at the same approximate chronological age. Scanning electron microscopy in suckling pigs at 2 and 10 d of age demonstrated long, thin, fingerlike villi with subsequently reduced heights and larger diameters by 35 d of age. At weaning, villi were in close apposition, resulting in an overall smoother villus luminal surface. Villus height subsequently increased by 14 d postweaning, coinciding with the appearance of morphologically tongue-shaped villi. Starter diet composition initially did not influence the villus height reduction response postweaning. Dietary corn oil addition was subsequently associated with shortened villus length (P less than .05) during the starter phase.

Etiology of ammoniated hay toxicosis.

Some animals consuming hay treated with anhydrous ammonia have developed neurological signs including hyperexcitability, circling and convulsions. A series of experiments was conducted to identify tentatively the toxin and determine its mode of action. Three out of four sheep fed ammoniated orchardgrass hay (approximately 4% ammonia on a dry basis) developed convulsions. Two of the three sheep died within 18 h of the onset of signs. The concentrations of blood lactate and pyruvate were elevated in the symptomatic sheep (P less than .05). A proposed toxin, 4-methyl imidazole, did not induce the syndrome when 750 mg/d (approximately 10 times the dietary amount) were administered orally. Four out of five calves that received milk from cows fed ammoniated oat hay (approximately 5% ammonia on dry basis) displayed hyperexcitability and circling. Concentrations of blood lactate and pyruvate were also elevated in the calves. The crude alkaloid fraction of the toxic milk produced neurological signs similar to those of the calves when injected into mice. A fluorescent compound was found in the alkaloid fraction of toxic milk and ammoniated hay, but not in control milk or untreated hay. The fluorescent compound was quite labile; hence, characterization has been unsuccessful thus far.

Porcine pararotavirus: detection, differentiation from rotavirus, and pathogenesis in gnotobiotic pigs.

Some characteristics of a newly recognized porcine enteric virus are described. Tentatively, the virus was referred to as porcine pararotavirus (PaRV) because it resembled rotaviruses in respect to size, morphology, and tropism for villous enterocytes of the small intestine. However, it was antigenically distinct from porcine, human, and bovine rotaviruses and reoviruses 1, 2, and 3, and the electrophoretic migration pattern of PaRV double-stranded RNA was distinct from the electrophoretic migration patterns of the rotaviral and reoviral genomes. By passage in gnotobiotic pigs, PaRV was isolated from two suckling diarrheic pigs originating from two herds. After oral exposure of gnotobiotic pigs, villous enterocytes of the small intestines became infected as judged by immunofluorescence, resulting in villous atrophy and diarrhea. Mortality was high when gnotobiotic pigs less than 5 days old were infected. The C strain of this virus was serially passed 10 times in gnotobiotic pigs, and electron microscopy, immunofluorescence, and serological tests indicated no extraneous agents. The virus was serially passed five times in cell cultures which contained pancreatin in the medium, but replication was negligible or absent, as the number of immunofluorescent cells decreased with each passage. Since rotaviral infections are frequently diagnosed by direct electron microscopy of fecal specimens, the presence of other morphologically similar viruses, such as PaRV, should be considered. The use of immune electron microscopy is suggested as a means of helping recognize this situation.

Oral administration of zinc sulfate for control of ovine foot rot.

Zinc sulfate (ZnSO4) was added to the diet of adult ewes and lambs with contagious foot rot for periods of 4 weeks to 6 months and to the diet of lambs for 5 weeks before experimental exposure to foot rot. Intake averaged between 0.5 and 0.75 g/animal/day. A beneficial effect was demonstrated when lambs with foot rot were fed ZnSO4 and were maintained under dry conditions, but when conditions were wet, ZnSO4 did not reduce the number of infections or prevent new infections. When ZnSO4 was fed before exposure, the infection rate of medicated lambs in a wet environment was almost twice that of unmedicated lambs in the same pen.

Zinc sulfate foot bath for control of ovine foot rot.

A 10% zinc sulfate (ZnSO4) solution was used as a foot bath to control foot rot in pastured lambs. When the foot bath was placed where infected lambs would walk through it each day, clinical evidence of foot rot was greatly reduced within 15 days, and the total number of cases detected after thorough foot trimming was greatly reduced within 30 days. When the foot bath was placed under the salt feeding box, it was not effective. Feeding ZnSO4 at the rate of 0.5 to 0.75 g/head/day was not effective in controlling foot rot. Temperature and moisture conditions were favorable for foot rot transmission throughout the trials.

Rotavirus-like, calicivirus-like, and 23-nm virus-like particles associated with diarrhea in young pigs.

Virus particles morphologically similar to caliciviruses and rotaviruses were detected by electron microscopy (EM) in the intestinal contents of a 27-day-old diarrheic nursing pig. A third small spherical 23-nm virus-like particle was also observed. Calicivirus-like particles averaged 33 nm in diameter. Similar to rotaviruses, rotavirus-like particles were present as single-capsid 55-nm forms or double-capsid 70-nm particles. Most gnotobiotic pigs orally exposed to samples containing these three viruses developed diarrhea and villous atrophy of the small intestine, and all shed the three viruses in their intestinal contents. Attempts to propagate these viruses in cell culture were unsuccessful. The antigenic relationship of the rotavirus-like particles to known rotaviruses was explored by immune EM and immunofluorescent staining. By these techniques, the rotavirus-like particles did not cross-react with antisera to porcine, bovine, or human rotaviruses or to reovirus type 3. Antisera from gnotobiotic pigs exposed to all three viruses had enzyme-linked immunosorbent assay and virus neutralization titers of <4 against porcine rotavirus. Previous infection of gnotobiotic pigs with the mixture containing rotavirus-like particles failed to protect them against a subsequent challenge with porcine rotavirus. The antigenic relationship of the calicivirus-like particles to known caliciviruses was investigated by immune EM and virus neutralization. By these tests, the calicivirus-like particles did not react with antisera against feline calicivirus strain 255 or M-8. In a study conducted at Plum Island Animal Disease Center, antiserum against the three combined agents did not specifically neutralize any serotype of swine vesicular exanthema virus.