Richard J. Fussell

Uptake and depuration of pharmaceuticals in aquatic invertebrates.

The uptake and depuration of a range of pharmaceuticals in the freshwater shrimp (Gammarus pulex) and the water boatman (Notonecta glauca) was studied. For one compound, studies were also done using the freshwater snail Planobarius corneus. In G. pulex, bioconcentration factors (BCFs) ranged from 4.6 to 185,900 and increased in the order moclobemide < 5-fluoruracil < carbamazepine < diazepam < carvedilol < fluoxetine. In N. glauca BCFs ranged from 0.1 to 1.6 and increased in the order 5-fluorouracil < carbamazepine < moclobemide < diazepam < fluoxetine < carvedilol. For P. corneus, the BCF for carvedilol was 57.3. The differences in degree of uptake across the three organisms may be due to differences in mode of respiration, behaviour and the pH of the test system. BCFs of the pharmaceuticals for each organism were correlated to the pH-corrected liposome-water partition coefficient of the pharmaceuticals.

Identification in residue analysis based on liquid chromatography with tandem mass spectrometry: Experimental evidence to update performance criteria

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is one of the most widely used techniques for identification (and quantification) of residues and contaminants across a number of different chemical domains. Although the same analytical technique is used, the parameters and criteria for identification vary depending on where in the world the analysis is performed and for what purpose (e.g. determination of pesticides, veterinary drugs, forensic toxicology, sports doping). The rationale for these differences is not clear and in most cases the criteria are essentially based on expert opinions rather than underpinned by experimental data. In the current study, the variability of the two key identification parameters, retention time and ion ratio, was assessed and compared against requirements set out in different legal and guidance documents. The study involved the analysis of 120 pesticides, representing various chemical classes, polarities, molecular weights, and detector response factors, in 21 different fruit and vegetable matrices of varying degrees of complexity. The samples were analysed non-fortified, and fortified at 10, 50 and 200 μg kg(-1), in five laboratories using different LC-MS/MS instruments and conditions. In total, over 135,000 extracted-ion chromatograms were manually verified to provide an extensive data set for the assessment. The experimental data do not support relative tolerances for retention time, or different tolerances for ion ratios depending on relative abundance of the two product ions measured. Retention times in todays chromatographic systems are sufficiently stable to justify an absolute tolerance of ±0.1 min. Ion ratios are stable as long as sufficient response is obtained for both product ions. Ion ratio deviations are typically within ±20% (relative), and within ±45% (relative) in case the response of product ions are close to the limit of detection. Ion ratio tolerances up to 50% did not result in false positives and reduced the false negative rate for pesticides with product ions in the low S/N range to <5%. Without ion ratio criterion, two false positives were obtained in 105 non-fortified samples. Although the study has been conducted for pesticides residues in fruits and vegetables, the impact of these findings is believed to extend towards other application areas and possibly support adjustment or consolidation of criteria across other analytical domains.

Evaluation and validation of an accurate mass screening method for the analysis of pesticides in fruits and vegetables using liquid chromatography–quadrupole-time of flight–mass spectrometry with automated detection

This study reports the development and validation of a screening method for the detection of pesticides in 11 different fruit and vegetable commodities. The method was based on ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-QTOF-MS). The objective was to validate the method in accordance with the SANCO guidance document (12571/2013) on analytical quality control and validation procedures for pesticide residues analysis in food and feed. Samples were spiked with 199 pesticides, each at two different concentrations (0.01 and 0.05 mg kg(-1)) and extracted using the QuEChERS approach. Extracts were analysed by UPLC-QTOF-MS using generic acquisition parameters. Automated detection and data filtering were performed using the UNIFI™ software and the peaks detected evaluated against a proprietary scientific library containing information for 504 pesticides. The results obtained using different data processing parameters were evaluated for 4378 pesticide/commodities combinations at 0.01 and 0.05 mg kg(-1). Using mass accuracy (± 5 ppm) with retention time (± 0.2 min) and a low response threshold (100 counts) the validated Screening Detection Limits (SDLs) were 0.01 mg kg(-1) and 0.05 mg kg(-1) for 57% and 79% of the compounds tested, respectively, with an average of 10 false detects per sample analysis. Excluding the most complex matrices (onion and leek) the detection rates increased to 69% and 87%, respectively. The use of additional parameters such as isotopic pattern and fragmentation information further reduced the number of false detects but compromised the detection rates, particularly at lower residue concentrations. The challenges associated with the validation and subsequent implementation of a pesticide multi-residue screening method are also discussed.

Can the unauthorised use of ceftiofur be detected in poultry

Ceftiofur is a third-generation cephalosporin antibiotic used to treat cattle and swine for bacterial infection of the respiratory tract. It is not authorised for use in poultry within the European Union. Due to the complexity of the chemistry and metabolism of ceftiofur, maximum residue limits (MRLs) are based on desfuroylceftiofur (DFC) equivalents after chemical conversion of all compounds that have an intact ß-lactam ring. In practice the DFC is usually stabilised as the acetamide (desfuroylceftiofur acetamide – DFCA) for analysis. Because of recent evidence of off-label use in the European Union, a policy need emerged to develop a cost-effective method for the detection of ceftiofur residues in poultry tissues. One-day-old chicks were each dosed subcutaneously with ceftiofur and samples taken from day 1 to day 44 post-dosing. Residues of ceftiofur parent compound were detected in whole chicks, wing feathers and faeces. On the basis of this finding it was decided to evaluate ceftiofur parent, as the marker, instead of proceeding with the time-consuming conversion to DFCA. Expected metabolites, DFC and desfuroylceftiofur cysteine disulfide (DCCD), were not detected in whole chicks, muscle or liver, but DFC was found in wing feathers. These results indicate that determination of ceftiofur parent compound in either whole chicks or possibly wing feathers and faeces may allow the detection of the misuse of ceftiofur.

Investigation into the validity of extrapolation in setting maximum residue levels for pesticides in crops of similar morphology

Field trials were initiated to investigate if extrapolation procedures, which were adopted to limit costs of pesticide registration for minor crops, are valid. Three pairs of crops of similar morphology; carrots/swedes, cauliflower/calabrese (broccoli) and French beans/edible-podded peas; were grown in parallel at four different geographical locations within the UK. The crops were treated with both systemic and non-systemic pesticides under maximum registered use conditions, i.e. the maximum permitted application rates and the minimum harvest intervals. Once mature, the crops were harvested and analysed for residues of the applied pesticides. The limits of quantification were in the range 0.005–0.02 mg kg−1. Analysis of variance and bootstrap estimates showed that in general, the mean residue concentrations for the individual pesticides were significantly different between crop pairs grown on each site. Similarly, the mean residue concentrations of most of the pesticides in each crop across sites were significantly different. These findings demonstrate that the extrapolations of residue levels for most of the selected pesticide/crop combinations investigated; chlorfenvinphos and iprodione from carrots to swedes; carbendazim, chlorpyrifos, diflubenzuron and dimethoate from cauliflower to calabrese; and malathion, metalaxyl and pirimicarb from French beans to edible-podded peas; appear invalid.

Testing a cumulative and aggregate exposure model using biomonitoring studies and dietary records for Italian vineyard spray operators

The need for improved tools to estimate the cumulative and aggregate exposure to compounds such as plant protection products (PPPs) is recognised in the EU Regulation 1107/2009. A new model has been developed to estimate the exposure within a population to single compounds or compounds within a Cumulative Action Group, considering dietary and non-dietary sources and multiple exposure routes. To test the model a field study was carried out in Italy with operators applying tebuconazole fungicides, with measurements of dermal exposure collected. Whole urine samples were collected and analysed to provide values for the absorbed dose of tebuconazole, with duplicate diet samples collected and analysed as a measure of dietary exposures. The model provided predicted values of exposure for combined dietary and non-dietary routes of exposures which were compared to the measured absorbed dose values based on urinary analysis. The model outputs provided mean daily exposure values of 1.77 (± 1.96) µg a.s./kg BW which are comparable to measured mean values from the biomonitoring field study of 1.73 (± 1.31) µg a.s./kg BW. To supplement the limited measurement data available, comparisons against other models were also made and found to be comparable.

Variability of residue concentrations of ciprofloxacin in honey from treated hives

ABSTRACT Honey bees (Apis mellifera L.) were treated with a model veterinary drug compound (ciprofloxacin) in a 3-year study (2012–14) to investigate the variability of residue concentration in honey. Sucrose solution containing ciprofloxacin was administered to 45 hives (1 g of ciprofloxacin per hive) at the beginning of the honey flow in late May/mid-June 2012, 2013 and 2014. Buckfast honey bees (A. mellifera – hybrid) were used in years 2012 and 2013. Carniolan honey bees (A. mellifera carnica) were used instead of the Buckfast honey bees as a replacement due to unforeseen circumstances in the final year of the study (2014). Honey was collected over nine scheduled time points from May/June till late October each year. Up to five hives were removed and their honey analysed per time point. Honey samples were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine ciprofloxacin concentration. Statistical assessment of the data shows that the inter-hive variation of ciprofloxacin concentrations in 2012/13 is very different compared with that of 2014 with relative standard deviations (RSDs) of 138% and 61%, respectively. The average ciprofloxacin concentration for 2014 at the last time point was more than 10 times the concentration compared with samples from 2012/13 at the same time point. The difference between the 2012/13 data compared with the 2014 data is likely due to the different type of honey bees used in this study (2012/13 Buckfast versus 2014 Carniolan). Uncertainty estimates for honey with high ciprofloxacin concentration (upper 95th percentile) across all hives for 55-day withdrawal samples gave residual standard errors (RSEs) of 22%, 20% and 11% for 2012, 2013 and 2014, respectively. If the number of hives were to be reduced for future studies, RSEs were estimated to be 52% (2012), 54% (2013) and 26% (2014) for one hive per time point (nine total hives).

Investigation into the experimental protocols required to determine maximum residue limits (MRLs) in honey.

There is current debate within the EU, and internationally, on how withdrawal periods and maximum residue limits (MRLs) may be set for honey production. Whilst comprehensive EU guidelines exist for calculating the withdrawal times of veterinary medicines in most food-producing species, the analytical variables to be studied for bees/honey are not well defined. The objective of this study was therefore to investigate and understand the factors, for example sampling variability, that is important in the development of a harmonized protocol that can be used to generate the robust scientific data necessary to assist risk assessors in proposing MRLs for honey. Ten bee colonies were treated in the spring with a model compound (ciprofloxacin). One hive was used to study intra-hive variation in residue concentrations and the other nine were used in an inter-hive study over a 41-week sampling period. All samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The highest mean concentration from nine hives used in the inter-hive study was 4627 µg/kg eight days (D8) after treatment. The concentration of ciprofloxacin declined to an average concentration of 1756 µg/kg at D30 and 733 µg/kg at D283 (over-winter sample). A generalized additive model was used to fit a smooth curve for trend estimation. For some individual hives the concentration of ciprofloxacin increased slightly at the later sampling time-points. Consequently it was not possible to interpolate, with confidence, a finite withdrawal period for ciprofloxacin at theoretical MRLs between 25 and 500 µg/kg. The observed variation in concentration of ciprofloxacin between hives indicates that the validity of the EU guideline for bees/honey, which requires five samples from five hives to calculate a withdrawal period, may require revision.