Shellfish Toxin Uptake and Depuration in Multiple Atlantic Canadian Molluscan Species: Application to Selection of Sentinel Species in Monitoring Programs.

Shellfish toxin monitoring programs often use mussels as the sentinel species to represent risk in other bivalve shellfish species. Studies have examined accumulation and depuration rates in various species, but little information is available to compare multiple species from the same harvest area. A 2-year research project was performed to validate the use of mussels as the sentinel species to represent other relevant eastern Canadian shellfish species (clams, scallops, and oysters). Samples were collected simultaneously from Deadmans Harbour, NB, and were tested for paralytic shellfish toxins (PSTs) and amnesic shellfish toxin (AST). Phytoplankton was also monitored at this site. Scallops accumulated PSTs and AST sooner, at higher concentrations, and retained toxins longer than mussels. Data from monitoring program samples in Mahone Bay, NS, are presented as a real-world validation of findings. Simultaneous sampling of mussels and scallops showed significant differences between shellfish toxin results in these species. These data suggest more consideration should be given to situations where multiple species are present, especially scallops.

Development and Validation of a Hybrid Screening and Quantitative Method for the Analysis of Eight Classes of Therapeutants in Aquaculture Products by Liquid Chromatography-Tandem Mass Spectrometry.

A method using reverse-phase ultra-high-performance liquid chromatography coupled with tandem mass spectrometry is described for eight classes of therapeutants that are used in marine aquaculture products. Validation studies to evaluate recovery, precision, method detection limits, and measurement uncertainty were performed at three levels, using three representative matrices [salmon (fatty fish), tilapia (lean fish), and shrimp (crustaceans)] to assess the method performance for use as a screening or determinative (quantitative and confirmatory) method. A total of 16 sulfonamides (plus 2 potentiators), 2 tetracyclines, 11 (fluoro)quinolones, 7 nitroimidazoles, 3 amphenicols, 5 steroids, and 3 stilbenes met the quantitative criteria for method validation. An additional 5 triphenylmethane dyes, 2 sulfonamides, 2 tetracyclines, and 1 amphenicol met the required performance for use as a screening method. Limits of detection (LODs) for the compounds that met the quantitative criteria ranged from 0.1 to 5 µg/kg, while LODs for compounds from the screening group ranged from 0.1 to 30 µg/kg. This method provides a comprehensive approach to the determination of different classes of compounds in aquaculture products.

The Development and Single-Laboratory Validation of a Method for the Determination of Steroid Residues in Fish and Fish Products.

Due to potential use in aquacultured fish products, the Canadian Food Inspection Agency has identified residue testing for steroids as a priority. These compounds are used in aquaculture primarily to direct sexual differentiation with both androgens and estrogens applied depending on the desired outcome. Published research is lacking with respect to steroid residue testing in fish; however, recent studies in other matrixes provided transferable cleanup techniques. A simple, rapid, and sensitive method was developed and validated for use in monitoring aquacultured fish products for the presence of methyltestosterone, nandrolone, epi-nandrolone, boldenone, and epi-boldenone residues. The developed method consists of solvent extraction followed by cleanup using hexane and dual cartridge SPE with analysis by ultra-HPLC-MS/MS. The method is capable of detecting and confirming steroid residue levels ranging from 0.05 to 25 ng/g in salmon and tilapia, depending on the analyte. Recoveries ranged from 88 to 130% for the analytes. Instrument repeatability was less than 13% for all compounds, while intermediate precision ranged from 5 to 25% RSD. HorRat values were within acceptable ranges.

Animal-free paralytic shellfish toxin testing--the Canadian perspective to improved health protection.

The performance characteristics of AOAC Official Method 2011.02 (the PCOX method) as a replacement for the AOAC mouse bioassay procedure have been well defined by validation studies, but these data do not communicate the complete story. The context provided by analyzing 9000 regulatory monitoring samples over 3 years demonstrates not only the reduction in animal use but also the increase in food safety that has been realized using a chemistry-based method. Detection of lower toxin levels provided early warning to enable directed sampling as toxin levels increased. The toxin profile information generated by a chemistry-based method was used to detect potential interferences qualitatively and can be used to assess the impact of changes recommended to monitoring programs. Such changes might include which toxins should be included in an action limit or the toxic equivalence factors used for these toxins.

Development and method validation for the determination of nitroimidazole residues in salmon, tilapia and shrimp muscle.

The use of nitroimidazoles in aquacultured fish has been banned in many countries due to the suspected mutagenic and carcinogenic effects of these compounds. In response to the need to conduct residue testing of these compounds in fish, a simple, rapid, and sensitive method was developed and validated that is suitable for regulatory monitoring of nitroimidazole residues and their hydroxy metabolites in fish muscle tissue. Following solvent extraction of homogenized tissue and clean-up using a C18 SPE cartridge, analyses were conducted by ultra-performance UPLC-MS/MS. A precursor ion and two product ions were monitored for each of the parent compounds and metabolites included in the method. The validated method has an analytical range from 1 to 50 ng/g in the representative species (tilapia, salmon, and shrimp), with an LOD and LOQ ranging from 0.07 to 1.0 nglg and 0.21 to 3.0 nglg, respectively, depending on the analyte. Recoveries ranged from 81 to 124% and repeatability was between 4 and 17%. HorRat values were within typical limits of acceptability for a single laboratory. Working standards were stable for 12 months, extracts were stable for 5 days, and tissues for 2 months under appropriate storage conditions. This method was determined to be suitable for routine use for screening, quantification, and confirmation of nitroimidazole residues in a residue monitoring program for fish with regulatory oversight.

Acute toxicities of saxitoxin, neosaxitoxin, decarbamoyl saxitoxin and gonyautoxins 1&4 and 2&3 to mice by various routes of administration.

Saxitoxin and its derivatives, the paralytic shellfish toxins (PSTs), are known to be toxic to humans, and maximum permitted levels in seafood have been established by regulatory authorities in many countries. Until recently, the mouse bioassay was the reference method for determining the levels of these toxins in seafood, but this has now been superseded by chemical methods of analysis. The latter methods are able to determine the levels of many PSTs in shellfish, but for risk assessment an estimate of the relative toxicities of the individual components of the PST mixture is required. The relative toxicities are expressed as "Toxicity Equivalence Factors" (TEFs). At present, TEFs are based on relative specific activities in the mouse bioassay, rather than on acute toxicity determinations, as measured by median lethal doses (LD50s). In the present study, the median lethal doses of saxitoxin, neosaxitoxin, decarbamoyl saxitoxin and equilibrium mixtures of gonyautoxins 1&4 and gonyautoxins 2&3 have been determined by intraperitoneal injection, gavage and feeding. The results indicate that specific activities in the MBA do not consistently correlate with acute toxicities by any of the routes of administration, and TEFs, particularly for neosaxitoxin, require revision.

Best practices for single-laboratory validation of chemical methods for trace elements in foods. Part I--background and general considerations.

The metals subgroup of AOAC INTERNATIONAL's Community on Chemical Contaminants and Residues in Food has been engaged for the past several years in discussions concerning the requirements for the single-laboratory validation (SLV) of methods for the determination of trace elements in foods. This paper reviews the general guidance currently available related to validation of chemical analytical methods and current typical validation practices found in publications on the analysis of elements in food and other matrixes, such as environmental and clinical samples. Based on the available guidance on SLV requirements and a review of current practices in elemental analysis, a general approach based on best practices is proposed for SLV of a method for elements in food to demonstrate the method as "fit-for-purpose."

Zoo and aquarium webcams: an informed view.

Providing webcams for public viewing is a relatively new but growing phenomenon among zoos and aquariums. Reasons for incorporating this programmatic feature are varied, and no guidelines exist to aid institutions considering webcam installations. Decision makers need to know how much effort the cameras require as well as how successful other zoos have found them to be. We evaluated existing webcams and provide an overview of their characteristics, including reliability. Quantitative evaluations provided by zoo/aquarium staff and by zoo members indicate generally positive perceptions of webcams, whereas staff acknowledge a notable level of effort required. Here, we strive to offer guidelines that will help institutions considering this venture.

Investigations into matrix components affecting the performance of the official bioassay reference method for quantitation of paralytic shellfish poisoning toxins in oysters.

Significant differences previously observed in the determination of paralytic shellfish poisoning toxins (PSTs) in oysters using official method AOAC 2005.06 and 959.08 were investigated in detail with regard to possible matrix effects. Method AOAC 2005.06 gave results 2-3 times higher than the mouse bioassay method, 959.08, differences thought to be due to underestimation of PSTs by the mouse bioassay. In order to prove the cause of these large differences, work was conducted here to examine the presence and effects of matrix components on the performance of each of the two assays. A range of oyster, cockle and mussel samples were extracted using the AOAC 959.08 hydrochloric acid (HCl) extraction method and analysed for PSP by both MBA and LC-FLD. In addition, extracts were analysed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for metals as well as being subjected to a range of nutritional testing methods. Whilst there was no evidence for effect of nutritional components on either assay, ICP-MS analysis revealed a relationship between samples exhibiting the largest differences in relative method performance, specifically those with the largest LC-FLD/MBA toxicity ratio, and samples containing the highest concentrations of zinc and manganese. In order to prove the potential effect of the metals on either the LC-FLD and/or MBA assays, HCl extracts of a range of shellfish were subjected to a number of matrix modifications. Firstly, a number of PSP-positive oyster samples were processed to reduce the concentrations of metals within the extracts, without significantly reducing the concentrations of PSTs. Secondly, a range of mussel and cockle extracts, plus a standard solution of saxitoxin di-hydrochloride were spiked at variable concentrations of zinc. All treated and non-treated extracts, plus a number of controls were subjected to ICP-MS, LC-FLD and MBA testing. Results proved the absence of any effect of metals on the performance of the LC-FLD, whilst showing a large suppressive effect of the metals on the MBA. As such, the results show the performance of the official MBA is potentially unsafe for application to the routine monitoring of PSP toxicity in oysters or in any other shellfish found to contain high concentrations of metal ions.

Rapid postcolumn methodology for determination of paralytic shellfish toxins in shellfish tissue.

A rapid liquid chromatographic (LC) method with postcolumn oxidation and fluorescence detection (excitation 330 nm, emission 390 nm) for the determination of paralytic shellfish toxins (PSTs) in shellfish tissue has been developed. Extracts prepared for mouse bioassay (MBA) were treated with trichloroacetic acid to precipitate protein, centrifuged, and pH-adjusted for LC analysis. Saxitoxin (STX), neoSTX (NEO), decarbamoylSTX (dcSTX), and the gonyautoxins, GTX1, GTX2, GTX3, GTX4, GTX5, dcGTX2, and dcGTX3, were separated on a polar-linked alkyl reversed-phase column using a step gradient elution; the N-sulfocarbamoyl GTXs, C1, C2, C3, and C4, were determined on a C-8 reversed-phase column in the isocratic mode. Relative toxicities were used to determine STX-dihydrochloride salt (diHCl) equivalents (STXeq). Calibration graphs were linear for all toxins studied with STX showing a correlation coefficient of 0.999 and linearity between 0.18 and 5.9 ng STX-diHCI injected (equivalent to 3.9-128 microg STXeq/100 g in tissue). Detection limits for individual toxins ranged from 0.07 microg STXeq/100 g for C1 and C3 to 4.1 microg STXeq/100 g for GTX1. Spike recoveries ranged from 76 to 112% in mussel tissue. The relative standard deviation (RSD) of repeated injections of GTX and STX working standard solutions was < 4%. Uncertainty of measurement at a level of 195 microg STXeq/100 g was 9%, and within-laboratory reproducibility expressed as RSD was 4.6% using the same material. Repeatability of a 65 microg STXeq/100 g sample was 3.0% RSD. Seventy-three samples were analyzed by the new postcolumn method and both AOAC Official Methods for PST determination: the MBA (y = 1.22x + 13.99, r2 = 0.86) and the precolumn LC oxidation method of Lawrence (y = 2.06x + 12.21, r2 = 0.82).

Determination of malachite green and leucomalachite green in a variety of aquacultured products by liquid chromatography with tandem mass spectrometry detection.

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for determining the residues of malachite green (MG) and leucomalachite green (LMG) in a number of aquatic species. MG and its metabolite were extracted from homogenized tissues with a perchloric acid-acetonitrile solution; the extract was centrifuged; and an aliquot was taken, concentrated, and passed through a chemically bonded octadecyl C18 solid-phase extraction column. The compounds of interest were eluted with acetonitrile, and the eluate was evaporated to dryness. The residue was dissolved in acetonitrile and diluted with water in preparation for analysis by LC/MS/MS. MG and its metabolite were determined by reversed-phase LC using a Luna C18 column with an ammonium hydroxide-formic acid buffer in acetonitrile gradient and MS/MS detection using multiple reaction monitoring. Calibration curves were linear for all analyses between 5 and 500 pg injected for both analytes, with recoveries ranging from 81% for LMG to 98% for MG in salmon spiked at the 1 ng/g level. Detection limits of 0.1 ng/g for both MG and LMG were easily obtainable using the recommended method. The operational errors, interferences, and recoveries for spiked samples compared favorably with those obtained by established methodology. The recommended method is simple, rapid, and specific for monitoring residues of MG and LMG in a number of aquatic species.

Outpatient chemotherapy administration: decreasing wait times for patients and families.

Increasingly, there is a trend to deliver chemotherapy, where possible, in the outpatient ambulatory setting. In the few studies that have explored the setting of cancer care, long wait times are frequently linked to dissatisfaction. Several factors contribute to lengthy waiting times for patients and their families: long registration processes, lag times associated with obtaining laboratory results, time required for patient assessments and preparation of chemotherapeutic agents, adequacy of nursing resources, and physical space constraints in relation to patient volumes. With the goal of improving care delivery in the outpatient clinic, a fast-tracking system was established. Program planning included establishing patient eligibility criteria, protocol and treatment appropriateness, interdepartmental collaboration, development of a communication plan for families and staff, negotiation of physical space, and allocation of human resources. This was instituted by re-allocating existing resources and establishing an autonomous nurse-managed chemotherapy clinic. This fast-tracking program has enabled us to use our existing resources with greater efficiency and improve patient care from safety and quality-of-life perspectives for those included in the program.