Assessment of toxicity and potential risk of the anticoagulant rodenticide diphacinone using Eastern screech-owls (Megascops asio).

In the United States, new regulatory restrictions have been placed on the use of some second-generation anticoagulant rodenticides. This action may be offset by expanded use of first-generation compounds (e.g., diphacinone; DPN). Single-day acute oral exposure of adult Eastern screech-owls (Megascops asio) to DPN evoked overt signs of intoxication, coagulopathy, histopathological lesions (e.g., hemorrhage, hepatocellular vacuolation), and/or lethality at doses as low as 130 mg/kg body weight, although there was no dose-response relation. However, this single-day exposure protocol does not mimic the multiple-day field exposures required to cause mortality in rodent pest species and non-target birds and mammals. In 7-day feeding trials, similar toxic effects were observed in owls fed diets containing 2.15, 9.55 or 22.6 ppm DPN, but at a small fraction (<5%) of the acute oral dose. In the dietary trial, the average lowest-observed-adverse-effect-level for prolonged clotting time was 1.68 mg DPN/kg owl/week (0.24 mg/kg owl/day; 0.049 mg/owl/day) and the lowest lethal dose was 5.75 mg DPN/kg owl/week (0.82 mg/kg owl/day). In this feeding trial, DPN concentration in liver ranged from 0.473 to 2.21 µg/g wet weight, and was directly related to the daily and cumulative dose consumed by each owl. A probabilistic risk assessment indicated that daily exposure to as little as 3-5 g of liver from DPN-poisoned rodents for 7 days could result in prolonged clotting time in the endangered Hawaiian short-eared owl (Asio flammeus sandwichensis) and Hawaiian hawk (Buteo solitarius), and daily exposure to greater quantities (9-13 g of liver) could result in low-level mortality. These findings can assist natural resource managers in weighing the costs and benefits of anticoagulant rodenticide use in pest control and eradication programs.

Plasma and Skin Per- and Polyfluoroalkyl Substance (PFAS) Levels in Dairy Cattle with Lifetime Exposures to PFAS-Contaminated Drinking Water and Feed.

Plasma and ear notch samples were removed from 164 Holstein cows and heifers, which had lifetime exposures to per- and polyfluoroalkyl substances (PFAS) through consumption of contaminated feed and water sources. A suite of nine PFAS including five perfluoroalkyl carboxylic acids (PFCA) and four perfluoroalkyl sulfonic acids (PFSA) was quantified in plasma and ear notch samples by liquid chromatography-mass spectrometry. Bioaccumulation of four- to nine-carbon PFCAs did not occur in plasma or skin, but PFSAs longer than four carbons accumulated in both plasma and skin. Exposure periods of at least 1 year were necessary for PFSAs to reach steady-state concentrations in plasma. Neither parity (P = 0.76) nor lactation status (P = 0.30) affected total PFSA concentrations in mature cow plasma. In contrast, lactation status greatly affected (P < 0.0001) total PFSA concentrations in ear notch samples. Skin samples could be used for biomonitoring purposes in instances when on-farm blood collection and plasma preparation are not practical.

Acute toxicity of diphacinone in Northern bobwhite: effects on survival and blood clotting.

The anticoagulant rodenticide diphacinone was slightly toxic (acute oral LD50 2014 mg/kg) to Northern bobwhite (Colinus virginianus) in a 14-day acute toxicity trial. Precise and sensitive assays of blood clotting (prothrombin time, Russell's Viper venom time, and thrombin clotting time) were adapted for use in quail, and this combination of assays is recommended to measure the effects of anticoagulant rodenticides. A single oral sublethal dose of diphacinone (434 mg/kg body weight) prolonged clotting time at 48 h post-dose compared to controls. At 783 mg/kg (approximate LD02), clotting time was prolonged at both 24 and 48 h post-dose. Prolongation of in vitro clotting time reflects impaired coagulation complex activity, and was detected before overt signs of toxicity were apparent at the greatest dosages (2868 and 3666 mg/kg) in the acute toxicity trial. These clotting time assays and toxicity data will assist in the development of a pharmacodynamic model to predict toxicity, and also facilitate rodenticide hazard and risk assessments in avian species.

Complete Genome Sequence of Multidrug-Resistant Salmonella enterica Serovar I 4,[5],12:i:- 2015 U.S. Pork Outbreak Isolate USDA15WA-1.

The genome of a multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar I 4,[5],12:i:- isolate from the 2015 U.S. pork outbreak was sequenced. The complete nucleotide sequence of USDA15WA-1 is 5,031,277 bp, including Salmonella genomic island 4 encoding tolerance to multiple metals and an MDR module inserted in the fljB region.

An assay for determining the susceptibility of Salmonella isolates to commercial and household biocides.

Poultry and meat products contaminated with Salmonella enterica are a major cause of foodborne illness in the United States. The food industries use a wide variety of antimicrobial interventions to reduce bacterial contamination. However, little is known about Salmonella susceptibility to these compounds and some studies have shown a concerning link between biocide resistance and antibiotic resistance. To investigate this, a 96 well panel of 17 common household and commercially used biocides was designed to determine the minimum inhibitory concentrations (MIC) of these compounds for Salmonella. The panel contained two-fold serial dilutions of chemicals including Dodecyltrimethylammonium chloride (DC), Benzalkonium chloride (BKC), Cetylpyridinium chloride (CPC), Hexadecyltrimethylammonium bromide (HB), Hexadecyltrimethylammonium chloride (HC), Acetic acid (AA), Lactic acid (LA), Citric acid (CA), Peroxyacetic acid (PXA), Acidified sodium chlorite (ASC), Sodium hypochlorite (SHB), 1,3 dibromo, 5,5 dimethylhydantoin (DBH), Chlorhexidine (CHX), Sodium metasilicate (SM), Trisodium phosphate (TSP), Arsenite (ARI), and Arsenate (ARA). The assay was used to test the susceptibility of 88 multidrug resistant (MDR) Salmonella isolates from animal sources. Bacteria are defined as multidrug resistant (MDR) if it exhibited non-susceptibility to at least one agent in three or more antimicrobial categories. The concentration of biocide at which ≥50% of the isolates could not grow was designated as the minimum inhibitory concentration or MIC50 and was used as the breakpoint in this study. The MIC50 (µg ml-1) for the tested MDR Salmonella was 256 for DC, 40 for BKC, 80 for CPC. HB and HC, 1,640 for AA, 5664 for LA, 3,156 for CA, 880 for PXA, 320 for ASC, 3.0 for CHX, 1,248 for DBH, 3,152 (6%) for SHB, 60,320 for SM, 37,712 for TSP, 56 for ARI and 832 for ARA. A few isolates were not susceptible at the MIC50 breakpoint to some chemicals indicating possible resistance. Isolates with MICs of two 2-fold dilutions above the MIC50 were considered resistant. Biocides for which resistant isolates were detected included CPC (n = 1 isolate), HB (1), CA (18), ASC (7), CHX (22), ARA (16), and ARI (4). There was no correlation detected between the biocide susceptibility of Salmonella isolates and antibiotic resistance. This assay can determine the MICs of bacteria to 17 biocides in a single test and will be useful in evaluating the efficacy of biocides and to detect the development of resistance to them.

Neutralization of Bactericidal Activity Related to Antimicrobial Carryover in Broiler Carcass Rinse Samples.

Studies were conducted to examine the ability of three chemicals to neutralize residual antibacterial activity of commercial antimicrobial chemicals used in poultry processing. Chemical antimicrobial interventions used in poultry processing may have potential for carryover into whole poultry carcass buffered peptone water (BPW) rinses collected for monitoring Salmonella contamination. Such carryover may lead to false-negative results due to continuing bactericidal action of the antimicrobial chemicals in the rinse. To simulate testing procedures used to detect Salmonella contamination, studies were conducted by separately adding test neutralizers (highly refined soy lecithin, sodium thiosulfate, or sodium bicarbonate) to BPW and using these solutions as carcass rinses. Control samples consisted of BPW containing no additional neutralizing agents. One of four antimicrobial solutions (cetylpyridinium chloride, peroxyacetic acid, acidified sodium chlorite, and a pH 1 hydrochloric:citric acid mix) was then added to the rinses. The four antimicrobial solutions were prepared at maximum allowable concentrations and diluted with modified BPW rinses to volumes simulating maximum carryover. These solutions were then inoculated with a mixed culture of five nalidixic acid-resistant Salmonella serovars at 106 CFU/mL. The inoculated rinse was stored at 4°C for 24 h, and Salmonella was enumerated by direct plating on brilliant green sulfa agar supplemented with nalidixic acid. Results indicate that incorporation of optimal concentrations of three neutralizing agents into BPW neutralized the demonstrated carryover effects of each of the four antimicrobial solutions tested, allowing recovery of viable Salmonella at 106 CFU/mL (P > 0.05), equivalent to recovery from carcass rinses with no antimicrobial carryover. Incorporation of these neutralizers in BPW for Salmonella monitoring may reduce false-negative results and aid regulatory agencies in accurate reporting of Salmonella contamination of poultry.

Determination of diphacinone residues in Hawaiian invertebrates.

A reversed-phase ion-pair liquid chromatographic analysis combined with a solid-phase extraction clean-up method is used to assess the quantity of diphacinone residue found in invertebrates. Three invertebrate species are exposed to commercially available diphacinone-fortified bait used for rat control. The invertebrate samples are collected, frozen, and shipped to the laboratory. The samples are homogenized after cryogenic freezing. A portion of the homogenized samples are extracted with acidified chloroform-acetone, followed by cleanup with a silica solid-phase extraction column. Diphacinone is detected by UV absorption at 325 nm after separation by the chromatographic system. The method limit of detection (MLOD) for snail and slug samples averaged 0.055 and 0.066 mg/kg, respectively. Diphacinone residues in snail tissue ranges from 0.83 to 2.5 mg/kg for Oxychilus spp. The mean recoveries from snails at 0.20 and 2.0 are 97 +/- 21% and 84 +/- 6%. Diphacinone residues in slug tissue ranges from 1.3 to 4.0 mg/kg for Deroceras laeve and < MLOD to 1.8 mg/kg for Limax maximus, respectively. The mean recoveries from slugs at 0.20 and 2.0 mg/kg are 91% +/- 15% and 86% +/- 5%.

Effect of Simulated Sanitizer Carryover on Recovery of Salmonella from Broiler Carcass Rinsates.

Numerous antimicrobial chemicals are currently utilized as processing aids with the aim of reducing pathogenic bacteria on processed poultry carcasses. Carryover of active sanitizer to a carcass rinse solution intended for recovery of viable pathogenic bacteria by regulatory agencies may cause false-negative results. This study was conducted to document the potential carryover effect of five sanitizing chemicals commonly used as poultry processing aids for broilers in a postchill dip. The effect of postdip drip time on the volume of sanitizer solution carryover was first determined by regression of data obtained from 10 carcasses. The five sanitizer solutions were diluted with buffered peptone water at 0-, 1-, and 5-min drip time equivalent volumes as determined by the regression analysis. These solutions were then spiked to 10(5) CFU/ml with a mixture of five nalidixic acid-resistant Salmonella enterica serovars, stored at 4°C for 24 h, and finally enumerated by plate count on brilliant green sulfa agar containing nalidixic acid. At the 0- and 1-min drip time equivalents, no Salmonella recovery was observed in three of the five sanitizers studied. At the 5-min drip time equivalent, one of these sanitizers still exhibited significant (P ≤ 0.05) bactericidal activity. These findings potentially indicate that the currently utilized protocol for the recovery of Salmonella bacteria from postchill sanitizer interventions may lead to false-negative results due to sanitizer carryover into the carcass rinsate.

Evaluation of cocoa- and coffee-derived methylxanthines as toxicants for the control of pest coyotes.

Methylxanthines were quantified in coffee, tea, and chocolate products. Tarajuilie tea from India, cocoa powder, and cocoa nibs contained the highest levels of methylxanthines. Theobromine, caffeine, and theophylline combined in the ratios observed in tea and chocolate were ingested by coyotes. Although both mixtures induced acute toxicity, the symptoms accompanying the chocolate methylxanthine mimic were preferable. Manipulation of the ratios of methylxanthines in the chocolate mimic led to the identification of a 5:1 theobromine/caffeine mixture as a promising coyote toxicant. This mixture was then administered to coyotes using the coyote lure operative device (CLOD). Mortality occurred in every coyote that ingested any portion of the CLOD contents. These results indicate that mixtures of theobromine and caffeine have the potential to be developed into a selective, effective, and socially acceptable toxicant for the control of pest coyotes.

Probabilistic risk assessment for snails, slugs, and endangered honeycreepers in diphacinone rodenticide baited areas on Hawaii, USA.

Three probabilistic models were developed for characterizing the risk of mortality and subacute coagulopathy to Poouli, an endangered nontarget avian species, in broadcast diphacinone-baited areas on Hawaii, USA. For single-day exposure, the risk of Poouli mortality approaches 0. For 5-d exposure, the mean probability of mortality increased to 3% for adult and 8% for juvenile Poouli populations. For Poouli that consume snails containing diphacinone residues for 14 d, the model predicted increased levels of coagulopathy for 0.42 and 11% of adult and juvenile Poouli populations, respectively. Worst-case deterministic risk characterizations predicted acceptable levels of risk for nonthreatened or endangered species such as northern bobwhite quail and mallards. Also, no acute toxicity was noted for snails and slugs that feed on diphacinone baits.

Stability of the tranquilizer drug propionylpromazine hydrochloride in formulated products.

An analytical method to evaluate propionylpromazine hydrochloride (PPZHCl) in tranquilizer formulations was developed using high-performance liquid chromatography (HPLC). During analysis of aged quality-control samples, a previously unreported chromatographic response was observed at a shorter retention time than PPZHCl. Further investigation of formulations stored in trap tap devices at temperatures ranging from 5 to 40 degrees C during field trials at four different locations confirmed the degradation of the active ingredient. Further investigation using HPLC/tandem mass spectrometry revealed two to five degradates, with the major degradates being oxidation products of the active ingredient, PPZHCl. As PPZHCl formulations must be stable when stored at 5 to 40 degrees C for 6 to 12 months, reformulation with the anti-oxidant ascorbic acid was utilized to achieve the required PPZHCl stability.

Probabilistic model for estimating field mortality of target and non-target bird populations when simultaneously exposed to avicide bait.

A probabilistic model was developed to estimate target and non-target avian mortality associated with the application of the avicide CPTH (3-chloro-p-toluidine hydrochloride) to minimize sprouting rice damage in the southern USA. CPTH exposures for individual birds were predicted by random sampling from species-specific non-parametric distributions of bait seed consumption and CPTH residues detected on individual bait seeds. Mortality was predicted from the species-specific exposure versus mortality relationship. Individual variations in this response were captured in the model by Monte Carlo sampling from species-specific distributions of slopes and median toxicity values (LD50) for each bird. The model was used to simultaneously predict mortality (percentage of exposed population and number of birds killed/weight of consumed bait) for a target (blackbird) and non-target (mourning dove) species feeding on bait sites for up to five consecutive days.

Characterizing 3-chloro-p-toluidine hydrochloride on rough-hulled rice and ethyl-cellulose-coated rice baits using high-performance liquid chromatography.

Methods are developed to extract and quantitate the avicide 3-chloro-p-toluidine hydrochloride (CPT HCl) from rough-hulled rice and ethyl-cellulose-coated rice baits using high-performance liquid chromatography. The mobile phase used in the ethyl-cellulose-coated rice matrix method is an acetonitrile(ACN)-phosphate buffer (60:40) at pH 8, and the rough-hulled rice matrix method uses an CAN-phosphate (70:30) buffer at pH 2. Increased retention time is observed for CPT HCl at the higher pH. The two methods have been useful in characterizing different bait formulations in an ongoing pesticide formulation improvement program.

Response factor considerations for the quantitative analysis of western redcedar (Thuja plicata) foliar monoterpenes.

A method is described for quantitative analysis of monoterpenes in western redcedar (Thuja plicata) foliage by gas chromatography with flame ionization detection. Response factors for monoterpenes identified in redcedar are evaluated to determine similarities among monoterpene responses. Evaluation demonstrates that redcedar monoterpenes yield detector responses that fall into two groups. One monoterpene from each group is used as a standard for quantitative analysis. Redcedar monoterpenes are quantitated by comparing analyte response with the response factor of one of the standards in single-point calibrations. Homogenized foliage samples are extracted with ethyl acetate and the extracts passed through a solid phase extraction column of graphitized carbon to remove plant pigments. Method bias and repeatability are evaluated by fortifying foliage samples with (1S)-(+)-carvone and (1S)-(+)-2-carene and subjecting the samples to the extraction and analysis procedures. Detection limits are also assessed from fortified samples. Excellent recovery (> 95.0%) and precision (< 5%) are obtained from the analysis of 2-carene from fortified samples. Carvone recovery is approximately 80% with excellent precision (< 4%). The method limits of detection obtained from 2-carene and carvone fortified samples are 4.7 and 13.5 microg/g, respectively.

Analysis of Nitrosamines in Cooked Bacon by QuEChERS Sample Preparation and Gas Chromatography-Tandem Mass Spectrometry with Backflushing.

Nitrites are added as a preservative to a variety of cured meats, including bacon, to kill bacteria, extend shelf life, and improve quality. During cooking, nitrites in the meat can be converted to carcinogenic nitrosamines (NAs), the formation of which is mitigated by the addition of antioxidants. In the past, the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) monitored NAs in pan-fried bacon, but FSIS terminated monitoring of NAs in the 1990s due to the very low levels found. FSIS recently chose to conduct a risk assessment of NAs in cooked bacon to determine if current levels warrant routine monitoring of NAs again. To meet FSIS needs, we developed, validated, and implemented a new method of sample preparation and analysis to test cooked bacon for five NAs of most concern, which consist of N-nitroso-dimethylamine, -diethylamine, -dibutylamine, -piperidine, and -pyrrolidine. Sample preparation was based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach and analysis by gas chromatography-tandem mass spectrometry. Ruggedness was improved markedly in the analysis of the complex fatty extracts by backflushing the guard column, injection liner, and half of the analytical column after every injection. Validation results were acceptable with recoveries of 70-120% and <20% RSDs for the five NAs, with a reporting limit of 0.1 ng/g. NA concentrations in 48 samples were all <15 ng/g, with most <1 ng/g and many <0.1 ng/g. Also, microwave cooking of bacon gave slightly lower concentrations overall compared to pan-frying.

Perfluorooctane Sulfonate Plasma Half-Life Determination and Long-Term Tissue Distribution in Beef Cattle (Bos taurus).

Perfluorooctane sulfonate (PFOS) is used in consumer products as a surfactant and is found in industrial and consumer waste, which ends up in wastewater treatment plants (WWTPs). PFOS does not breakdown during WWTP processes and accumulates in the biosolids. Common practices include application of biosolids to pastures and croplands used for feed, and as a result, animals such as beef cattle are exposed to PFOS. To determine plasma and tissue depletion kinetics in cattle, 2 steers and 4 heifers were dosed with PFOS at 0.098 mg/kg body weight and 9.1 mg/kg, respectively. Plasma depletion half-lives for steers and heifers were 120 ± 4.1 and 106 ± 23.1 days, respectively. Specific tissue depletion half-lives ranged from 36 to 385 days for intraperitoneal fat, back fat, muscle, liver, bone, and kidney. These data indicate that PFOS in beef cattle has a sufficiently long depletion half-life to permit accumulation in edible tissues.

Poly(methyl methacrylate) synthetic grit formulations sustain the delivery of nicarbazin, a contraceptive agent, in pest waterfowl.

Sixty-three mallards were fed one of ten poly(methyl methacrylate) based synthetic grit formulations containing varying concentrations of a proposed wildlife contraceptive (nicarbazin), plasticizer (acetyl tributylcitrate) and/or cross-linking agent (1,4-butanediol diacrylate). Release characteristics of the contraceptive agent were monitored for the purpose of developing a contraceptive formulation for control of pest waterfowl in urban settings. The addition of plasticizer increased the erosion rate (t(1/2)=0.97-2.85 days), cross-linking the polymer matrix slightly decreased the erosion rate (t(1/2)=4.45-5.05 days) and increasing the concentration of the contraceptive agent increased the erosion rate (t(1/2)=3.3 and 9.9 days at 60% and 7.5% active ingredient, respectively). The larger and smaller grit pieces had longer half lives at 11.0 and 11.6 days, respectively while the mid sized grit had a half life of 4.95 days. Control grit had a half life of 12.7 days based on weight loss. Analysis of blood and feces for monitoring release from the grit and approximate indirect plasma levels of the active ingredient proved feasible.

High-performance liquid chromatography-based determination of nicarbazin excretion in waterfowl.

A method for the high-performance liquid chromatography (HPLC) determination of nicarbazin uptake and excretion in ducks is presented. The method uses few clean-up steps and provides a rapid assessment of nicarbazin excretion by measuring the analyte 4,4'-dinitrocarbanalide (DNC). During method development the effect of extraction volume, number of extractions, mobile phase composition, column temperature, and injection volume were varied to optimize sensitivity and achieve as short a run time as possible. For our purposes, a 2 x 5.0 ml 1:1 dimethyl formamide (DMF):acetonitrile (ACN) extraction injected (40 ml) into an HPLC system equipped with a Keystone octadecylsilyl (ODS) C18 column and a UV variable wavelength detector (lambda=347 nm) with a mobile phase of 60:40 (v/v) ACN-H2O, at a flow-rate of 1.0 ml/min at a column temperature of 35 degrees C provided adequate resolution and an acceptable total run time. Studies conducted during method development for inter-day recovery efficiencies for 0.46, 1.8 and 88.5 microg fortified samples (n=3) had mean recoveries of 91, 94 and 97% and intra-day (n=3) recoveries at the same fortification levels of 103, 94, and 92%. The method has been used successfully in excretion studies of nicarbazin in ducks.

Development of a high performance liquid chromatography/mass spectroscopy method for the determination of strychnine concentrations in insects used to assess potential risks to insectivores.

A high performance liquid chromatography/mass spectrometry (HPLC/MS) method was developed in support of a study to assess potential tertiary risks posed to insectivores by strychnine baited pocket gophers (Thomomys sp.). Necropholous insects are primary consumers of pocket gopher carcasses. A field study was conducted to collect insects from strychnine-baited and control pocket gopher carcasses. The majority of the insects collected were from the orders Diptera (flies, assayed separately as adults and larvae), Coleoptera (beetles), and Hymenoptera (ants and wasps, assayed separately). Samples (0.5 g) were extracted in acetic acid (2%) and analyzed with the mass spectrometer configured for tandem mass spectrometry. For most of the samples the strychnine concentrations were less than the method limit of detection. However, strychnine concentrations as high as 0.338, 0.341, 0.698, and 0.034 microg/g were detected in ants, fly adults, fly larvae, and beetles, respectively. This information collected with the HPLC/MS method is critical for assessing potential non-target hazards for insectivores.

Determination of 4,4'-dinitrocarbanilide (DNC), a component of Nicarbazin, in Canada goose (Branta canadensis) eggshells using high-performance liquid chromatography.

A method was developed using high-performance liquid chromatography to assay 4,4'-dinitrocarbanilide (DNC), the active ingredient in Nicarbazin, in eggshells collected from Canada geese fed a formulated feed fortified with Nicarbazin at doses of 0, 125, 250, and 500 microg/g. The method was developed using chicken eggshells fortified with DNC. The method was used to quantify DNC in both the shell-associated membranes and the calcified shell extracellular matrix. These values were compared to those obtained for a composite sample consisting of both the membranes and the calcified shell extracellular matrix. The validated method was used to quantify DNC in eggshells from geese fed fortified feed to ascertain the effect of Nicarbazin feed concentration on shell DNC concentration. DNC levels in the eggshells were highly correlated with feed dose.