Multiresidue Method Research Articles

Development and applicability of a multi-residue method for dyes, including new residue markers, to detect drug misuse in aquaculture

ABSTRACT A qualitative and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the sensitive and exhaustive analysis of residues from triarylmethane dyes, triarylmethane-derivative dyes, phenothiazines, phenoxazines and xanthenes in aquaculture samples. For a wider and more robust detection of dye misuse on farms, other residue markers were also included the leuco forms of brilliant green, crystal violet and malachite green; one direct metabolite of Victoria pure blue BO and methylene blue and three bile acids, which are endogenous markers of the effects of dye contamination in fish. We optimised the extraction method by comparing several extraction solvents and sample solvents reported in the literature to have the best extraction efficiency. The residues were determined using a positive electrospray ionisation source. We assessed the parameters of this LC-MS/MS method by evaluating the matrix effects, identification and quantitative parameters according to the criteria stipulated in the European Commission Decision No. 2002/657/EC. A study on the applicability of the method was conducted on various aquaculture species and on a positive catfish.

Monitoring of Organochlorine Pesticide and Polychlorinated Biphenyl Residues in Common Swifts (Apus apus) in the Region of Hannover, Lower Saxony, Germany.

The use of pesticides is associated with the decline of several avian species. In this study, we monitored the organochlorine contaminants in common swifts (Apus apus) in the years 2016 to 2018. These long-distance migrants breed in Europe and winter in Africa. Their only feeding source is aerial plankton. Pooled organ samples of 42 adult and 40 juvenile swifts were tested with the multi-residue method by gas chromatography-mass spectrometry (GC-TOF/MS). Predominantly, 4,4′-DDE, dieldrin, hexachlorobenzene (HCB), lindane and polychlorinated biphenyls (PCBs) were found in most of these common swifts. Only 4,4′-DDE (adult: 83 ± 70 μg/kg, juvenile: 17 ± 39 μg/kg) and dieldrin (adult: 2 ± 3 μg/kg, juvenile: 0.3 ± 1 μg/kg) concentrations were significantly different between adult and juvenile birds. All detected concentrations in our study were far lower than the previously recorded pesticide concentrations of common swifts in Italy and those which are known to cause toxicity and death in birds.

Optimization and evaluation of four multi-residue methods for the determination of pesticide residues in orange oil using LC-MS/MS and GC-MS/MS: a comparative study

ABSTRACT Orange oil is considered the largest produced essential oils worldwide due to its unique properties. Pesticide residues in orange oil are expected to be much higher than the original fruit due to orange peel’s cold-pressing during orange oil production. These residuesmaycause various health problems if consumed. The purpose of our study was to optimize and compare four multi-residues extraction methods (dilution, QuEChERS, ethyl acetate, and mini-Luke) for analysis of 387 pesticides in orange oil using LC-MS/MS and GC-MS/MS. To our knowledge, this is the first report on the use of ethyl acetate and the mini-Luke method for the analysis of orange oil. The comparison was based on recoveries, matrix effect, and the amount of co-extract matrix. The optimum mean recoveries were obtained by the ethyl acetate method, which successfully analyzes 371 out of 387 pesticides with acceptable recovery (70–120%). It also showed a narrow recovery distribution in the range of 90–110% for 69% of all studied pesticides. Regarding the matrix effect, the QuEChERS method gave the highest number of pesticides with an in significant matrix effect (80–120%) for both LC and GC amenable pesticides. The least amount of co-extract matrix components according to GC-MS/MS scan and gravimetric analysis has been achieved by the QuEChERS method. In conclusion, the Ethyl acetate method gives acceptable recovery for a wide range of pesticides with a narrow recovery distribution and a moderate amount of co-extract matrix. While the QuEChERS method provides better selectivity and cleaner extract but with a narrow scope and less precision.

Determination and dietary risk assessment of 284 pesticide residues in local fruit cultivars in Shanghai, China

The presence of pesticide residues has become one of the main risk factors affecting the safety and quality of agro-food. In this study, a multi-residue method for the analysis of 284 pesticides in five local fruit cultivars in Shanghai was developed based on ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). The limits of determination and the limits of quantitation of pesticides were 0.6–10 and 2–30 μg/kg, respectively. A total of 44, 10, 10, 18, and 7 pesticides were detected in strawberries, watermelons, melons, peaches, and grapes, respectively. The pesticide levels in 95.0% of the samples were below the maximum residual limits (MRLs) prescribed by China, and in 66.2% of the samples below the EU MRLs. The dietary risk assessment study showed big differences in the chronic and acute exposure risk values among different Chinese consumer groups. Through fruit consumption, children/females showed higher exposure risks than adults/males. But both the risk values were less than 100%, indicating that potential dietary risk induced by the pesticides was not significant for Chinese consumers. Nevertheless, certain measures are needed for both growers and the government in order to decrease the MRL-exceeding rate of pesticide residues and ensure the quality and safety of fruits for consumers.

Development of multi-residue extraction procedures using QuEChERS and liquid chromatography tandem mass spectrometry for the determination of different types of organic pollutants in mussel.

This study aimed to develop multi-residue methods for the extraction of organic pollutants in mussels (Mytilus galloprovincialis), including 11 pharmaceuticals, 5 pesticides, 5 perfluoroalkyl substances (PFASs) and 2 illicit drugs. The combination of 4 different QuEChERS methods and 12 clean-ups (a total of 44 combinations) was tested. QuEChERS included acidified (AQ), non-acidified (SQ) and their miniaturized versions. The clean-ups included 6 different conventional dispersive solid phase extraction (dSPE) plus 2 enhanced matrix removal (EMR-Lipid) and 4 SPE procedures (including sorbents focused on phospholipid removal and polymer-based). After sample analysis via HPLC-MS/MS, the three methods that provided the best results were validated in terms of linearity, accuracy, precision, sensitivity and matrix effect. The methods selected were the combination of (i) SQ and EMR-Lipid, (ii) AQ and Z-sep+ bulk-based dSPE and (iii) AQ and graphitized carbon black (GCB)-based dSPE. Recoveries at two concentration levels (50 and 500ng/g) ranged 54-124%, 59-124% and 60-127%, respectively, and limits of quantification (LOQs) were < 30ng/g for most analytes using any of the methods. The three methods were tested in non-spiked mussel samples purchased in local markets, but organic pollutants were not detected in any sample. However, the methods probed to successfully extract a wide range of organic pollutants families in mussel samples from the market and from bioaccumulation trials.

Multi-residue analytical methods for pesticides in teas: a review

Tea is one of the most popular drinks worldwide. In the process of tea planting, for controlling pests, a variety of pesticides are usually applied. Therefore, pesticide residues and their metabolites in tea leaves, as well as their adverse effects on tea drinkers' health, are becoming increasingly concerning. The tea-leaf matrix is very complex because it is rich in organic acids, polyphenols, natural pigments, catechins, flavonols, metallic/non-metallic elements, and other unconfirmed substances. Hence, it is a great challenge to conduct multi-residue analysis of pesticides in tea samples. Researchers worldwide have been devoted to the development of multi-residue analytical methods for pesticide detection in tea and have made great progress in recent years. In this review, the recent advances in the multi-residue analysis of pesticides in tea matrices, especially those regarding matrix effects and their elimination, sample preparation and detection techniques, were reviewed in depth, and future research directions were suggested as well.

Method validation for the determination of 314 pesticide residues using tandem MS systems (GC–MS/MS and LC-MS/MS) in raisins: Focus on risk exposure assessment and respective processing factors in real samples (a pilot survey)

A multi-residue method for the simultaneous analysis of a wide range of pesticides in raisins using liquid and gas chromatography-tandem mass spectrometry (LC-MS/MS and GC–MS/MS) has been validated. Pesticides are extracted from raisins with ethyl acetate, followed by centrifugation. The validation study was in accordance with DG SANTE guidelines. Validation experiments have been performed in both analytical instruments. A total number of 314 pesticides were spiked in raisins of organic farming at two spiking levels for GC–MS/MS (0.025 and 0.1 mg/kg), and at three spiking levels for LC-MS/MS (0.005, 0.05, and 0.1 mg/kg) with 6 replicates at each concentration. The scope of validation included linearity, limits of quantification (LOQ), accuracy, precision, and matrix effects (%) for each pesticide. The validated method was then applied for the analysis of 37 commercial raisin samples purchased from the market. For the evaluation of the results, processing factors (PFs) have been applied to derive the amount of residue in raisins, from the maximum residue levels (MRLs) of grapes, and which in this paper will be referred as to the MRL expressed in raisins. In all conventional samples, pesticides were detected at concentrations above the LOQ. In total, 55 different pesticides were detected. All conventional samples contained multiple pesticides ranging from 2 to 24. On the other hand, samples from organic farming were found to be free of the analysed pesticides. The 13.5% of the examined samples were considered as violations. The exposure assessment for the acute risk of the violating samples indicated that no potential risk derives from the detected and approved in the EU pesticides, while the detection of not approved pesticides in the EU, and the lack of toxicological reference values for certain pesticides raise concerns for the human health, especially for children. The results of the survey study indicate the need to include processed samples, and in particular dry fruits with a high consumption rate such as raisins, in the official controls of pesticide residues in food.

Pesticide Residues on Tomatoes Grown and Consumed in Mwea Irrigation Scheme, Kirinyaga County, Kenya

The effects of pesticides on human health are of great concern worldwide despite their usefulness in agriculture. The aim of the study was to evaluate pesticide residues on tomatoes grown and consumed in Kirinyaga County in order to determine whether the levels fall within recommended MRLs. Reported increased use of unspecified pesticides and undocumented residue levels in tomatoes produced in the country justified the need to evaluate pesticide residues in tomatoes grown in the area and consumed locally. The study, which was conducted in Mwea Irrigation Scheme, Kirinyaga County, Kenya between July 2017 and July 2018 used analytical study design. Tomato samples of Rambo variety mainly grown in open fields and greenhouses in the Irrigation Scheme were purposively sampled from thirty-five sampling sites in open fields, greenhouses, markets and consumers. The samples were taken to Kenya Plant Health Inspectorate Services (KEPHIS) laboratory in Nairobi for analysis. Analysis was done using Quick Easy Cheap Effective Rugged and Safe (QuEChERS) multi-residue analytical method for Low-Fat products. Four pesticide residues detected on tomatoes above the recommended EU and Cordex MRLs were: malathion (0.0315±0.0032 mg/kg) in open fields, carbendazim (1.2341±0.1667 mg/kg) and thiamethoxam (0.3736±0.0358 mg/kg) from greenhouses and acephate (0.0321±0.0032 mg/kg) from the market. Pesticide residue levels of tomatoes from consumers were all significantly (p&lt;0.001, p&lt;0.01) below the EU and Cordex permitted MRLs. Occurrence of pesticide residues on tomatoes from production to consumption levels is of great concern to consumers because of the perceived long term negative health effects. Implementation, strengthening and enforcement of the food policy in the country will enhance frequent monitoring of pesticide residue levels in fresh produce consumed locally in Kenya.

High resolution mass spectrometry workflow for the analysis of food contaminants: Application to plant toxins, mycotoxins and phytoestrogens in plant-based ingredients

ABSTRACT An analytical workflow including mass spectral library, generic sample preparation, chromatographic separation, and analysis by high-resolution mass spectrometry (HRMS) was developed to gain insight into the occurrence of plant toxins, mycotoxins and phytoestrogens in plant-based food. This workflow was applied to 156 compounds including 90 plant toxins (pyrrolizidine alkaloids, tropane alkaloids, glycoalkaloids, isoquinoline alkaloids and aristolochic acids), 54 mycotoxins (including ergot alkaloids and Alternaria toxins) and 12 phytoestrogens (including isoflavones, lignans and coumestan) in plant-based protein ingredients, cereal and pseudo-cereal products. A mass spectral library was built based on fragmentation spectra collected at 10 different collision energies in both positive and negative ionisation modes for each toxin. Emphasis was put on a generic QuEChERS-like sample preparation followed by ultra-high-pressure liquid chromatography using alkaline mobile phase allowing the separation of more than 50 toxic pyrrolizidine alkaloids. HRMS acquisition comprised a full-scan event for toxins detection followed by data-dependent MS2 for toxin identification against mass spectrum. Method performance was evaluated using fortified samples in terms of sensitivity, repeatability, reproducibility and recovery. All toxins were positively identified at levels ranging from 1 µg kg−1 to 100 µg kg−1. Quantitative results obtained by a standard addition approach met SANTE/12682/2019 criteria for 132 out of 156 toxins. Such a workflow using generic, sensitive and selective multi-residue method allows a better insight into the occurrence of regulated and non-regulated toxins in plant-based foods and to conduct safety evaluation and risk assessments when needed.

Miniaturized multiresidue method for the analysis of pesticides and persistent organic pollutants in non-target wildlife animal liver tissues using GC-MS/MS

In order to gain a better insight into pesticide and pollutant exposure of small (non-target) wildlife animals, a QuEChERS sample preparation method was first developed for 5 g liver tissues (e.g. hedgehog samples) and then downscaled for the analysis of 100 mg liver tissues (e.g. bat samples). The optimized (micro) QuEChERS methods used 1% acetic acid in acetonitrile as organic solvent for liquid-liquid extraction (LLE) and salting out was performed with anhydrous magnesium sulfate and sodium acetate (4:1). After a freezing-out step, sample clean-up was carried out with anhydrous magnesium sulfate, PSA, C18, and GCB (150:25:20:5). Overall, 209 pesticides and persistent organic pollutants (POPs) can be analysed within each sample with gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Both methods were validated with representative analytes according to the European Commission guideline SANTE/12682/2019. Limits of quantification were between 1 and 20 μg kg−1, and the methods proved to be linear up to 400 μg kg−1. Additionally, the analytes delivered satisfactory results regarding recovery and precision. As proof of concept, samples of six hedgehog livers were analysed with both methods to prove the accuracy of the micro QuEChERS method. Additionally, six livers of different bat species were analysed with the downscaled method. The newly developed micro QuEChERS method for multiresidue analysis requires only minute amounts of biomaterial and represents a sophisticated novel technique for determining the exposure of small wildlife animals to different contaminants.

Incidence of ionophore and non-ionophore anticoccidials residues in poultry meat and eggs and their risk characterization.

A multi-residue method was applied to investigate the incidence and the concentration of ionophores and non-ionophore anticoccidials residues in poultry meat and hen eggs for the three-year period 2017-2019 in Italy. The risk related to the ingestion of such molecules was also characterized for the entire population. The average incidences of positive samples ranged from 1.35 to 9.45% while the maximum average concentration was of 4.28 μg/kg for nonionophore molecules. No uncompliant sample was recorded. The overall risk characterization related to the intake of anticoccidials trought chicken meat and eggs reveal a minor concern for consumers of all age. However, the monitoring of coccidiostates residues through official control activity in poultry meat and egg is crucial and it should be continuously conducted to ensure safety of such products and safeguard consumers̛ health.

Determination of 72 Chemical Pesticides and Estimation of Measurement of Uncertainty in Rice Using LC-MS/MS and GC-MS/MS

A multi-residue method was developed and validated for the simultaneous determination of 39 and 33 chemical pesticides in rice using LC-MS/MS and GC-MS/MS, respectively. The coefficient of determination was 0.994 to 0.999 over the linear concentration range of 0.01 to 0.1 μg mL-1. The LOD and LOQ were in the range of 0.31 to 3.35 and 1.04 to 10.06 μgkg-1, repsectively. The mean recoveries at 0.01, 0.05, and 0.1 mgkg-1 were found within the range 70 to 120% with satisfactory precision (RSD < 15%). Measurement uncertainty (Uexp) values estimated at 0.05 mgkg-1 spiking level were found lower than 20 μgkg-1 for all tested pesticides. A rice and wheat powder check samples under the proficiency test were analyzed using a standardized method, and the Z score recorded within the acceptable range of −2 to +2. The proposed method was applied to analyze the pesticides in real samples collected from the market and quantified the residues. The developed method could be useful for monitoring 72 chemical pesticides in rice matrices and can be adopted for the pesticide monitoring program.

Multi-residue Methodologies for the Analysis of Non-polar Pesticides in Water and Sediment Matrices by GC–MS/MS

A multi-residue method for the analysis of non-polar pesticides by GC–MS/MS in water and sediment matrices has been successfully developed, including 33 and 27 compounds, respectively. Water analysis method is based on a classic liquid–liquid extraction with recoveries ranging between 39 and 102%, with RSDs lower than 13%, LODs of 0.42–15.2 ng L−1 and LOQs of 0.72–50.8 ng L−1. Sediment analysis method is based on a pressurized liquid extraction with recoveries ranging between 37 and 133%, RSDs lower than 18%, LODs of 0.01–0.16 ng g−1 dry weigth (dw) and LOQs of 0.02–0.54 ng g−1 dw. Reported LODs were lower than the maximum acceptable detection limits set by the EU Watch Lists for selected pesticides. Applicability of both methodologies has been evaluated in real water and sediment samples collected in Catalonian river basins reporting oxadiazon for the first time in sediments from Catalonian river basins with a range of n.d. to 382 ng g−1 dw and a mean concentration of 44.0 ng g−1 dw. The importance of the simultaneous evaluation of both water and sediment has been emphasised since ten out of the 15 detected pesticides in the sediments can pose a high risk to aquatic organisms according to the Risk Quotient (RQ) method. Further detailed work needs to be done to better understand and assess the environmental impact of pesticide-contaminated sediments on aquatic organisms.

Dilution of QuEChERS Extracts Without Cleanup Improves Results in the UHPLC-MS/MS Multiresidue Analysis of Pesticides in Tomato

AbstractTomato is well-known to be one of the most cultivated and consumed vegetables worldwide and frequently contain pesticide residues. Therefore, a simple multiresidue method was established and validated to determine 129 pesticides and metabolites in tomato samples using a modified acetate QuEChERS without cleanup for sample preparation and determination by ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Dilution of the raw extract in different proportions of mobile phase was evaluated and a dilution of 10 times presented adequate results improving analysis performance while minimizing the matrix effect. Validation performed according to SANTE guideline presented satisfactory results. Practical method limit of quantification was 0.01 mg kg−1 for most compounds. Recoveries between 70 and 120% with precision ≤ 20% were found for most compounds and spike levels evaluated. Matrix effect results were not significant for most part of compounds. Method proved to be simple, robust, and effective to be applied in routine analysis. Method applicability was performed by analysis of samples commercialized in Brazil and positive results were found demonstrating the importance of the proposed method.

Multiresidue method for the simultaneous analysis of antibiotics and mycotoxins in feeds by ultra-high performance liquid chromatography coupled to tandem mass spectrometry

AbstractResidues in animal feeds and foods of animal origin have been important safety issue concerning both human and animal health. A multiresidue method for determination of eight mycotoxins and ten antibiotics was developed and validated in animal feeds by using QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction followed by UHPLC-MS/MS. Optimisation of UHPLC-MS/MS parameters was performed to achieve good separation and resolution. The method was validated according to the European Commission Decision 2002/657/EC. Matrix matched calibration curves showed good r2 (≥0.995) values, and limit of quantification (LOQ) values varied between 1.2 and 5.2 μg kg−1. Average recoveries ranged from 60 to 102% with relative standard deviations of 2.2 and 15.6% for all type of feed samples except for tetracyclines, lincomycin, tylosin, ochratoxin A, and fumonisin (B1 and B2).

A new, simple, efficient and robust multi-residue method based on pressurised-liquid extraction of agricultural soils to analyze pesticides by liquid chromatography coupled with a high resolution quadrupole time-of-flight mass spectrometer

ABSTRACT A new method for the analysis of pesticides in agricultural soils by using liquid chromatography coupled with a high-resolution quadrupole time-of-flight mass spectrometer (LC-QTOF-MS) has been developed. Twenty-four pesticides including herbicides, insecticides or fungicides have been studied. The linearity of the external calibration of the pesticides obtained from levels of concentration ranging from 0.010 to 400 µg/Lgives satisfactory results with the correlation coefficients higher than 0.99. The quantification limits of the equipment range from 0.010 to 1.250 µg/L and correspond to the lowest standard in the calibration range for which the residual deviation is less than 60%. An experimental design has been used to optimise the parameters of pressurised-liquid extraction (PLE) of pesticides in soils. The optimised conditions have been found by using methanol as extraction solvent at a temperature of 80°C, a pressure of 150 Bars and 2 extraction cycles of 5 minutes. The mean recoveries of pesticides (Mean) are higher than 72% with a relative standard deviation(RSD) less than 21%. These results demonstrate the good efficiency of PLE for the extraction of pesticides in agricultural soils of different nature. The LC-QTOF-MS is a sensitive, linear and robust instrument for the quantification of pesticides in unpurified soil extracts by external calibration. This analytical tool is not subjected to matrix effects of soil extracts.

Wide-scope multi-residue analysis of pesticides in beef by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

Increasing pesticide contamination in foods of animal origin has made the wide-scope multi-residue analysis of pesticides an international concern. In this study, a sensitive and reliable multi-residue pesticide analysis method for beef was developed with the use of 129 pesticides to demonstrate the full scope of the method. The pesticides were extracted and purified using a modified QuEChERS technique and analysed using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. An orthogonal array design was employed for optimisation of the method, significantly scientising and simplifying the process. Method validation revealed satisfactory sensitivity with limits of quantification values ranging from 0.003 to 11.37 µg∙kg−1. Matrix effects ranged from 83.85% to 120.66%. Recovery at three spiking levels (20, 50, and 100 µg∙kg−1) were 70.51–128.12% and the intra-day and inter-day precisions were less than 20%, indicating good accuracy and precision.

Dynamics of the Degradation of Acetyl-CoA Carboxylase Herbicides in Vegetables.

(1) Background: Aryloxyphenoxy-propionates and cyclohexanediones are herbicides most widely used in dicot crops worldwide. The main objective of the study was to determine the dynamics of herbicide residues in carrot, lettuce, cauliflower, and onion in order to suggest a low level of residues in harvested vegetables. (2) Methods: Small plot field trials were carried out in four vegetables in the Czech Republic. The samples of vegetables were collected continuously during the growing season. Multiresidue methods for the determination of herbicide residues by LC-MS/MS were used. Non-linear models of degradation of individual herbicides in vegetables were calculated using the exponential decay formula. Action GAP pre-harvest intervals for the 25% and 50% maximum residue limit (MRL) and 10 µg kg−1 limit (baby food) were established for all tested herbicides. (3) Results: The degradation dynamics of fluazifop in carrot, onion, and cauliflower was significantly slower compared to quizalofop and haloxyfop. The highest amount (2796 µg kg−1) of fluazifop residues was detected in cauliflower 11 days after application. No residue of propaquizafop and cycloxydim was detected in any vegetable samples. (4) Conclusions: Aryloxyphenoxy-propionate herbicide (except propaquizafop) could contaminate vegetables easily, especially vegetables with a short growing season. Vegetables treated with fluazifop are not suitable for baby food. Lettuce and cauliflower treated by quizalofop are not suitable for baby food, but in onion and carrot, quizalofop could be used. Propaquizafop and cycloxydim are prospective herbicides for non-residual (baby food) vegetable production.

Monitoring of pesticide and antibacterial drug residues in animal products from two states in India by modified multi-residue analytical methods using GC–ECD and HPLC–DAD

Monitoring of pesticide and antibacterial drug residues in animal products from two states in India by modified multi-residue analytical methods using GC–ECD and HPLC–DAD

Analysis of multiple pesticide residues in rice by LC–MS/MS

A multiresidue method without cleanup step was validated for simultaneous determination of multiclass pesticide residues in rice. The extracted incurred residues of pesticides were separated on a reversed-phase column and quantified by tandem mass spectrometry (MS/MS). The method accuracy was in the range of 71–108%, and the precision was in the range of 4–20% for 25 out of 28 selected pesticides. The method quantification limits for 25 selected pesticides were below Codex and EU maximum residue limits (MRL). The selected 25 pesticides met the EU-SANTE and FAO/WHO Codex Alimentarius Commission method validation guidelines. The interday repeatability of the optimized method was between 4 and 18% (n = 6). The expanded uncertainty calculated for the optimized method ranged from 22 to 48%. The results obtained demonstrated the method's suitability for routine analysis. In total, 35 samples were analyzed of which, 22 samples (62%) were found to be contaminated with pesticides concentrations ranging from 0.010 to 0.297 mgkg−1.