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Doi:10.1016/j.tifs.2006.02.002


Trends in Food Science & Technology 17 (2006) 482–489 Methods for rapid substances are illegally added to act as growth promoters,improving feed conversion efficiency and increasing the lean to fat ratio. The gain in protein deposition is thusbased on an improved feed conversion rate. However,these substances may remain in all animal-treated derived Growth promoters exert some effects on meat quality, usually towards poorer quality. An increase in connective residues in animal tissue production and collagen cross-linking because areduction in protein degradation allows more time for collagen molecules to cross-link and thus, increase thetoughness of the meat (). In addition, muscle proteases areinhibited by the presence of some of these substances.
Fidel Toldra´* and Milagro Reig For instance, calpains are inhibited by b-agonists but protein synthesis is increased. The myofibrillar protein Department of Food Science, Instituto de fragmentation is also decreased in agonists-treated Agroquı´mica y Tecnologı´a de Alimentos (CSIC), P.O. Box 73, 46100 Burjassot, Valencia, Spain The result is a substantial reduction in tenderness (Tel.: C34 96 3900022; fax: C34 96 3636301; On the other hand, lipolysis rate is increased by activation of the hormone-sensitive lipaseand then breakdown of triacylglycerols ( Rapid methods and automation for the detection and Thus, the amount of fat is substantially characterisation of chemical and veterinary drug residues reduced with the subsequent loss in juiciness and poorer in foods of animal origin constitutes a dynamic area in food processing and is experiencing important developments Some substances, like thiouracyls, produce a notice- mainly from the standpoint of food safety. Residues from able retention of water that is suddenly lost when these substances may be present in edible tissues, milk and cooking the meat. The result is tougher meat with poor eggs for human consumption and may exert different levels juiciness. But what is more important, most of these of toxicity on consumers when consuming them. Thus, easy, substances present in residual amounts in animal foods rapid and sensitive tests are really needed for an effective at- may have some important toxic effects. Some of them line use. This manuscript is presenting latest developments may exert genotoxic, inmunotoxic, carcinogenic or for rapid detection of chemical and veterinary drugs residues endocrine effects on consumers, constituting an important in foods of animal origin.
health risk that must be controlled. Thus, the presence ofthese residues must be monitored in foods of animalorigin ().
Antibiotics act as growth promoters but can contribute Veterinary and chemical drugs having anabolic effect to an increased human exposure to antibiotics, develop- are used for therapeutic and prophylactic purposes as well as for improved breeding efficiency, although most increased allergies due to its presence in foods. In fact, of them are banned in the European Union and can only the presence of residual antibiotics in animal foods be administered in specific circumstances (therapeutic constitute an important health risk because the increased purposes) but under strict control. In general, these microbial resistance detected in latest years (. In addition, the * Corresponding author.
presence of residual amounts of antibiotics produces 0924-2244/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tifs.2006.02.002 F. Toldra´, M. Reig / Trends in Food Science & Technology 17 (2006) 482–489 important difficulties to food processors for the extent 96/23/EC. This Decision, which is in force since 1 and control of food fermentation.
September 2002, provides rules for the analytical It is evident that there are important benefits for the methods to be used in testing of official samples and farmer when using these illegal substances, mainly specific common criteria for the interpretation of consisting in an increased feed conversion yield and an analytical results of official control laboratories for such increased lean meat with less fat. But, it is also evident samples. This Decision includes concepts like the that there are important prejudices for the processing decision limit (CCa) and the detection capability (CCb) industry, like lower quality of products and problems in for assessing non-compliant samples. The guidelines fermentation and very important prejudices to the given in the Directive imply the use of sophisticated consumers, not only for the worse quality or the higher analytical instrumentation like GC–MS or LC–MS. These water content but because of the presence of residues and controls are based on screening and those suspected non- its associated harmful health effects on humans. For all compliant samples are then confirmed through methods these reasons, there is an evident interest of both official based on the use of gas or liquid chromatography organisms and food industry to control the presence of coupled to mass spectrometry, or other sophisticated these substances in farms and foods of animal origin.
methodologies and analytical instrumentation, for accu- Recently, the EC Quality of Life Programme supported rate characterisation and confirmation.
European collaborative projects in the area of antimicro-bials and hormone residues analysis ().
Screening methodologies These projects consisted in the development and The full procedure and the methodologies for validation of screening and confirmatory analytical confirmatory analysis are costly in time, equipments methods and sensors for a cost-effective and time and chemicals. In addition, they require trained personnel efficient control of synthetic glucocorticoids, nitrofurans, with high expertise. Control laboratories must face a coccidiostatics, b-lactam residues and androgen residues large number of samples, with a variety of analytes, to in live and post-mortem animals.
be analysed in relatively short periods of time. Thus, The use of substances having hormonal or thireostatic there is a need for screening methods that allow the action as well as b-agonists is banned in the European analysis of such a large number of samples in short Union. Main veterinary drugs and substances with periods of time. This means that high through-put anabolic effect are listed in . Only a few methods with low cost must be available. These methods substances are authorised for therapeutic purposes and must be able to detect an analyte or class of analytes at under the control of a responsible veterinarian ( the level of interest The presence of these Some false positives (false compliant) substances in foods are controlled by official inspection are acceptable, as they will be further submitted for and analytical services following EC Directive 96/23/EC confirmatory analysis but the method must avoid or on measures to monitor certain substances and residues reduce to a minimum the number of false negative results in live animals and animal products. Analytical method- (non-compliant) because they will not be further ology, including criteria for identification and confir- analysed. The main requirements, that are generally mation, for the monitoring of compliance was also given contemplated for a screening method, are summarised in in Decisions 93/256/EEC and 93/257/EEC. Since the implementation of these Directives, a clear decrease in the use of growth promoting agents, including b-agonists, especially when dealing with solid foods like most of those of animal origin, are also getting increased More recently, the Commission attention due to the miniaturisation of commercial tests Decision 2002/657/EC implemented Council Directive and kits. These sample preparation ensures better Table 1. Lists of veterinary drugs and substances with anabolic effect, with some examples (Council Directive 96/23/EC) Group A: substances having anabolic effect Group B: veterinary drugs 1. Stilbenes (diethylstilbestrol) 1. Antibacterial substances 2. Antithyroid agents (thiouracils) Sulfonamides and quinolones 2. Other veterinary drugs Androgens (trenbolone acetate) a) Antihelmintics Gestagens (melengestrol acetate) b) Anticoccidials, including nitroimidazoles Estrogens (17-b estradiol) c) Carbamates and pyrethroids 4. Resorcycilic acid lactones (zeranol) 5. Beta-agonists (clenbuterol) e) Non-steroideal anti-inflammatory drugs 6. Other compounds (nitrofurans) f) Other pharmacologically active substances (dexamethasone) F. Toldra´, M. Reig / Trends in Food Science & Technology 17 (2006) 482–489 Then, a second antibody labelled with an enzyme such as Table 2. Main requirements for a screening methodology peroxidase is added to the well followed by a new wash.
The quantity of conjugate bound to the plate is detected after incubation with a specific substrate. Colour is developed during incubation and measured with a microplate reader, which is proportional to the amount Reduced time and low running costs for results of analyte in the sample.
Sensitivity (no positives are lost)Specificity (minimum number of false positives) In direct competitive ELISA tests, a primary antibody is coated onto the plate wells and incubated with thesample extract containing the antigens. Once theequilibrium is reached, an enzyme-labelled antigen is sensitivity of the screening tests. Different extraction added. This conjugate will bind to the free binding sites techniques are usually based on solid phase extraction for of the primary antibody. Thus, the more antigen in the sample clean-up. Different types of cartridges may be sample, the lower amount of enzyme-labelled antigen used depending on the analytes, ensuring the elimination bound. Appropriate specific substrate is added and the of potential interferents present in foods ( plate is incubated for colour development. In this case, there is an inverse relationship between the colour There are different techniques available for the developed and the concentration of the analyte in the screening of residues in animal foods as shown in In general, the limits of detection will depend Radioimmunoassay (RIA) implies the measure of on the previous extraction and clean-up of the sample.
radioactivity of immunological complex using a counter The immunological methods mainly consist of ELISA test kits. There are many kits commercially available.
possibilities include the measure of chemiluminiscence Other immunological methods are based on radio- with a luminometer when a chemiluminiscent compound immunoassay and, more recently, several methods using is bound to the antibody or fluorescence with a biosensors are commercially available. The chromato- fluorimeter when a fluorescent compound is used. They graphic methods mainly consist in two types, HPTLC allow an enhanced detectability in relation to conven- and HPLC, coupled to different detection systems.
tional colorimetry ().
Main advantages and disadvantages of immunological kits are compiled in These kits offer important Immunological techniques advantages like the large number of samples to be Antigen and antibody reaction has been used for many analysed per kit, fast to operate and its high specificity years to detect a wide variety of food constituents and sensitivity in comparison to conventional detection including substances responsible for adulterations and methods. Another advantage is the possibility to use the contaminations. The interaction antigen–antibody is very kit within the food-processing facility without the need to specific and useful for the detection of residues of transport the sample to the laboratory. Many diagnostic chemical and veterinary drugs in animal foods. The most companies have marketed ELISA test kits for the usual technique consists in the enzyme-linked-immuno- detection of such residues. Thus, ELISA kits are sorbent assay (ELISA) and the detection system is available for a large number of substances within each usually based on enzyme-labelled reagents. There are group listed in like b-agonists, corticoids, different formats for antigen quantification. In double steroids, stilbenes, resorcylic acid lactones and several antibody or sandwich ELISA tests, a primary antibody is antibiotics. Research continues for the development of bound to the plate well. The antigen of the sample new ELISA tests for other substances like sedatives and extract added to the well complexes with the bound the b-blocker carazolol ( antibody and remains bound to the plate after washing.
Regarding antibiotics, ELISA kits haveshown good performance for analysing antibiotic residueslike tylosin and tetracyclin in water, meat and fish Table 3. List of main techniques available for screening ), chloramphenicol in milk and meat Chromatography methods nitroimidazoles in eggs High performance thin-layer and chicken (gentamicin in milk ( chromatography (HPTLC) High performance liquid chromatography dihydrostreptomycin and colistin in milk (bacitracin, spiramycin, tylosin, olaquindox and virgiamycin in feedstuffs (). In general, these F. Toldra´, M. Reig / Trends in Food Science & Technology 17 (2006) 482–489 Table 4. Main advantages and disadvantages of ELISA test kits Increased costs since 2002 (more than V650 per kit) Available kits for a good number of specific compounds (i.e.
Limited storage (few months) under refrigeration clenbuterol, zeranol, etc.)Availability of kits for families of compounds (i.e. agonists, stilbenes, Expensive in the case of RIA and need for waste disposal sulphonamides, etc.)Large number of samples (42) per kit for a single analyte Interferences giving some false positives Reduced time (few hours) to obtain the results: about 2–2.5 h for Only one kit per residue searched most kitsHigh sensitivityHigh specificityPossibility to use within the food-processing facility methods require some time of manual operation for the are changes in the mass concentration of molecules in addition of sample, incubation, washing and discarding of liquids, reagents for colour development, etc. This has The target residue is covalently immobilised onto the prompted the development of automated ELISA tests by sensor chip surface. This technology is applied by some companies.
Biacore AB (Uppsala, Sweden) to analyse different Another recent approach to screen animal products for veterinary drug residues. Some recent applications veterinary drugs, ensuring quality and safety of meat and include progesterone in milk (and dairy products consists in the development of biosensors.
tylosine in honey ( These instruments comprise two elements: a biological Other biosensors are based on the use of biochip recognition element, usually an antibody, and a signal arrays that allow a real time monitoring of the interaction transduction element which is in close contact and between the recognition molecule and the analyte. The connected to data acquisition and processing systems recognition signal is converted into a quantifiable signal.
This technology is applied by Randox Laboratories Ltd Biosensors are getting expanded applications in food (Antrim, UK). However, the number of residues ready to analysis. In general, there are several elements. The use in an array is still commercially limited. Several target analyte contacts the biological receptor (antibody) factors like sensor surface ligand density, active antibody and the biochemical signal is converted by a transducer concentration and biosensor flow rate affect the assay into an electronic signal. Then, these signals are performance . Enzymic processed by a microprocessor that gives the final result biosensors use a specific enzyme for the capture and The construction of biosensors requires a catalytic generation of the product. For instance, good knowledge of the basic principles of immuno- penicillin V and G can be detected with penicillinase chemical reactions, pathways for receptor-based signal immobilised on a surface, either a membrane or porous amplification and the interfacial behaviour of biocom- glass, that produces penicilloic acid and thus a reduction pounds at the artificial transducer surface ( in pH and either a decrease in the fluorescence intensity Biosensors are designed to operate in of the dye or an increase in the electrical conductivity real time and be able for the simultaneous detection of (). Other type of biosensors are based on single or multiple veterinary drugs residues in a sample antibiotic sensor protein which are sensible to specific at a time. Some authors have reported no need for classes of antibiotics. These sensors are high-throughput sample clean-up (). The biomolecular and compatible with the ELISA-type format. The interaction analysis is based on surface plasmon biosensor protein is chemically linked to the solid resonance that measures variations in the refractive surface of the well in a microtitre plate which, in the index of the solution close to the sensor when there absence of antibiotic, remains bound to the operator Table 5. Main advantages and disadvantages of biochip array biosensors High initial investment (equipment) Results available in short time High operative costs (chips) Multiples residues analysed in one shot (as many as chips in an array) Analysis restricted to available chips Full automatisation: higher productivityHigh through-put technique: up to 120 samples per hour and array F. Toldra´, M. Reig / Trends in Food Science & Technology 17 (2006) 482–489 Table 6. Main advantages and disadvantages of HPTLC High number of samples for a single analyte Expertise required Reduced time (few hours) to obtain the results Need of sample preparation (extraction, filtration, etc.) Possibility of automatisation for higher productivity Interferences giving some false positives Only one thin-layer plate per residue searched Specificity depending on the detection techniqueSeparated sample can be recovered for further confirmatory analysis sequence and after detection by antibodies and coupling different residues like thyreostatic drugs ( to peroxidase produces a colour readout. However, the biosensor is unable to bind the operator when the clenbuterol and other agonists ( antibiotic is present and, depending on its amount, this operator is more or less lost in the washing steps. The ), nitroimidazol loss of colour gives a readout that is proportional to the and sulfonamides ( antibiotic concentration (). These sensors have shown successful detection of tetracycline, in animal tissues. It streptogramin and macrolide antibiotics at nanogram per has also been applied to the analysis of corticosteroids millilitre concentrations in milk and serum (Weber et al., Main advantages and disadvantages of biosensors are and antibiotics in milk ( compiled in These new technologies are getting good reception in control laboratories due to the The spots can be A variation, named TLC- reduction in total time and possibility to analyse bioautography, consists in the combination of thin-layer simultaneously multiple residues in short time for a chromatography with microbiological detection directly large number of samples In on the plate resulting in enhanced sensitivity. It has been fact, these technologies are fully automated (injection, applied to the detection of flumequine in milk ( addition of reagents, washing, incubation and output) and The main advantages and disadvantages of HPTLC are compiled in The use of high performance liquid chromatography (HPLC) expanded during the 1990s and the availability of automation somehow facilitated its use as a screening (HPTLC) has been applied successfully for the qualitat- technique. HPLC is a separative technique and its ability ive and quantitative detection of multi-residues in food to detect compounds depends on the type of detector samples even though its use has rapidly decreased during used. The choice of the detection system is very the last decade. Visualisation of the components can be important for selectivity and sensitivity. Some analytes performed either by spraying an appropriate chromogenic not detected by absorbance, refractive index or fluor- reagent or under UV light. Quantitative determination is escence may require chemical modifications to render possible through the relative intensity of the spot in the chromophore, fluorescent or UV-absorbing compounds plate, which is measured against that of the internal ). Usually, the detection of standard by scanning densitometry. Recent developments multi-residues is based on a solid-phase extraction clean- allow for automation in a similar way to HPLC with the up followed by filtration and injection into a reverse- appropriate equipment. HPTLC has been applied to phase HPLC with UV-diode array detection. It has been Table 7. Main advantages and disadvantages of HPLC Short time (few min/sample) to obtain the results Expertise required Need of sample preparation (extraction and filtration, addition ofinternal standard, etc.) Specificity depending on detector High initial investment (equipment) Automatisation leading to higher productivity Possibility to find more information from spectra when using diodearray detector F. Toldra´, M. Reig / Trends in Food Science & Technology 17 (2006) 482–489 applied for detection of antibiotics in meat, kidney and formulations administered to livestock ( veterinary drugs in eggs,milk, fish and meat There are several arising problems in this field such as thiouracils in urine the increased number of new substances in the ‘black anabolic steroids in nutritional supplements and market'. Every year, new substances with anabolic properties and used as growth promoters are being ) and corticosteroids like dexa- detected. An example of the evolution of this type of methasone in water, feed and meat ( substances may be observed in the high competitive sports. Another important problem is an extended practice consisting in the mixture of low amounts ofseveral substances, like a ‘cocktail' that exerts a ). A good number of synergistic effect giving similar efficiency to the use of substances with anabolic properties, that can be con- a single substance at higher and, thus, detectable sidered as growth promoters, have been successfully amounts. Finally, the development of interfering sub- separated and identified for screening purposes in urine stances to mask immunoassay detection systems also ). HPLC with fluor- complicates the efficient detection of the illegal escence detection has also been used for the simul- taneous determination of 10 quinolone antibacterial In addition to these problems, control laboratories face residues in multispecies animal tissues ( more strict requirements for the performance of analytical The main advantages and methods according to new Directives. This situation is disadvantages are compiled in creating some problems to control laboratories because of HPLC is getting expanded use in control laboratories due the large number of samples to analyse, the great variety in to the possibility to analyse simultaneously multiple samples and residues to be analysed, the need to adapt residues in a sample in relatively short time. Recent analytical methodologies to new Directives with strict developments of high speed HPLC can reduce sample guidelines, the increased costs in developing such new treatment and analysis time. In addition, this technology is methodologies, the increased number of residues to search fully automated (injection, elution, washing of column, per sample and the need to invest on powerful new detection) and computer-controlled, facilitating its use as a The availability of screening methodologies facilitates The next step after initial screening with HPLC is the the control of chemical and veterinary drugs in foods of injection of the presumed positive samples in a system animal origin, reducing the number of samples to be combining HPLC with mass spectrometry detection. In confirmed through tedious and costly confirmatory this sense, the coupling of high speed HPLC with MS– analysis. Recent new developments, already available in MS can substantially reduce the analysis time. 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ozcoasts.gov.au

management arrangements for victorian mangroves and coastal saltmarsh Management arrangements for mangroves and coastal saltmarsh are complex because of the occurrence of these vegetation types within the coastal zone, an area covered by a multiplicity of policies, statutes and by-laws and with diverse international, national, state and local influences. That complexity is further enhanced by the number of government agencies and non-government organisations that play a role in the planning and management of the coastal zone which encompasses mangrove and coastal saltmarsh environments.

Minutes of regular meeting, august 18, 2004

PORT COSTA SANITARY COMMISSION MINUTES OF REGULAR MEETING, MARCH 9, 2016 1. CALL TO ORDER: The meeting was called to order at 7:00 PM by Chairperson Surges. Present were Commissioners Cusack, Mann and alt. Barassi, along with Dept. Manager Barnhill and General Manager McDonald. Mr. Guarnieri arrived at 7:05 PM. Mr. English was absent. 2. AGENDA ORDER: There were no requests to hear agenda items out of order.