VOL. 67_2016 ISSN 2545-4315
International Scientific Journal INTERNATIONAL SCIENTIFIC JOURNAL JOURNAL OF AGRICULTURAL, FOOD AND ENVIRONMENTAL SCIENCES http://www.fznh.ukim.edu.mk/jafes/ The JAFES is an International scientific peerreviewed Open Access Journal published twice а yearly JAFES On line (e-ISSN 2545-4315) offers free access to all articles at http://www.fznh.ukim.edu.mk/jafes/
Published by: Издава: “Ss. Cyril and Methodius" University in Skopje, Универзитет „Св. Кирил и Методиј“ во Скопје, Faculty of Agricultural sciences and food-Skopje Факултет за земјоделски науки и храна Скопје
Advisory board Издавачки совет
EDITORIAL BOARD УРЕДУВАЧКИ ОДБОР
Еditor in Chief Главен уредник
Vjekoslav Tanaskovikj, Skopje, Macedonia Вјекослав Танасковиќ, Скопје, Македонија Kocho Porchu, Skopje, Macedonia Кочо Порчу, Скопје, Македонија
Associate Editor Уредници
Snezana Jovanovic, Belgrade, Serbia Снежана Јованович, Белград, Србија Jovica Vasin, Novi Sad, Serbia Јовица Васин, Нови Сад, Србија Radmila Stikić, Belgrade, Serbia Радмила Стикич, Белград, Србија Biljana Škrbić, Novi Sad, Serbia Билјана Шкрбич, Нови Сад, Србија Ana Marjanovic Jeromel¸ Novi Sad, Serbia Ана Марјанович Јеромел, Нови Сад, Србија Bojan Srdljević, Novi Sad, Serbia Бојан Срдљевич, Нови Сад, Србија Zoran Rajić, Belgrade, Serbia Зоран Рајич, Белград, Србија Jasmina Havranek, Zagreb, Croatia Јасмина Хавранек, Загреб, Хрватска Mirjana Herak Ćustić, Zagreb, Croatia Мирјана ХеракЧустич, Загреб, Хрватска Vlasta Piližota, Osijek, Croatia Иво Туршич, Осијек, Хрватска Ivo Tursich, Zagreb, Croatia Власта Пилижота, Загреб, Хрватска Darko Vončina, Zagreb, Croatia Дарко Вончина, Загреб, Хрватска Zlatan Sarić, Sarajevo, B&H Златан Сарич, Сарајево, БиХ Josip Ćolo, Sarajevo, B&H Јосип Чоло, Сарајево, БиХ Muhamed Brka, Sarajevo, B&H Мухамед Брка, Сарајево, БиХ Velibor Spalević, Podgorica, Montenegro Велибор Спалевич, Подгорица, Црна Гора Bozidarka Marković, Podgorica, Montenegro Божидарка Маркович, Подгорица, Црна Гора Nazim Gruda, Bonn, Germany Назим Груда, Бон, Германија Venelin Roychev, Plovdiv, Bulgaria Венелин Ројчев, Пловдив, Бугарија Nasya Tomlekova, Plovdiv, Bulgaria Насиа Томлекова, Пловдив, Бугарија Irena Rogelj, Ljubljana, Slovenia Ирена Рогељ, Љубљана, Словенија Drago Kompan, Ljubljana, Slovenia Драго Компан, Љубљана, Словенија Michael Murković, Graz, Austria Михаел Муркович, Грац, Австрија Hristaq Kume, Tirana, Albania Христаќ Куме, Тирана, Албаниа Sonja Srbinovska, Skopje, Macedonia Соња Србиновска, Скопје, Македонија Marjan Kiprijanovski, Skopje, Macedonia Марјан Кипријановски, Скопје, Македонија Marina Stojanova, Skopje, Macedonia Марина Стојанова, Скопје, Македонија Biljana Kuzmanovska, Skopje, Macedonia Билјана Кузмановска, Скопје, Македонија Mirjana Jankulovska, Skopje, Macedonia Мирјана Јанкуловска, Скопје, Македонија Dragi Dimitrievski, Skopje, Macedonia Драги Димитриевски, Скопје, Македонија
JOURNAL OF AGRICULTURAL, FOOD AND ENVIRONMENTAL SCIENCES Address Адреса (Editorial Board) (Редакција) “Ss. Cyril and Methodius" University in Skopje Универзитет „Св. Кирил и Методиј“ во Скопје Faculty of Agricultural sciences and food-Skopje Факултет за земјоделски науки и храна Скопје P.O. Box 297, MK-1000 Skopje, п. фах. 297, МК1000 Скопје, Republic of Macedonia Република Македонија E-mail: [email protected]
*THE AUTHORS ARE RESPONSIBLE FOR THE CONTENT AND FOR THE LANGUAGE OF THEIR CONTRIBUTION Journal of Agricultural, Food and Environmental Sciences
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TABLE OF CONTENTS – СОДРЖИНА
T. Eftimova - Tashevska, B. Petanovska - Ilievska, E. Dimitrieska - Stojkovik, L. Velkoska - Markovska COMMON NONCONFORMITIES DURING PROCEDURE FOR ACCREDITATION OF THE FOOD TESTING LABORATORIES IN THE R.MACEDONIA 1-10
L. Velkoska-Markovska, B. Petanovska-Ilievska, I. Stojanovska, M. S. Jankulovska SPE-RRLC DETERMINATION OF SOME PESTICIDE RESIDUES IN APPLE JUICE 11-17
M. S. Jankulovska , L. Velkoska-Markovska, B. Petanovska-Ilievska, S. Trpkovska HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETERMINATION OF PRESERVATIVES IN BEVERAGES 18-25
B. Petanovska-Ilievska , L.Velkoska-Markovska , I. Stojanovska , M. S. Jankulovska DEVELOPMENT OF HIGH SPEED RRLC METHOD FOR QUANTITATIVE DETERMINATION OF SOME PESTICIDE RESIDUES IN APPLE JUICE 26-32
A.Najdenoska DEVELOPMENT OF HPLC METHOD FOR ANALYSIS OF NITRITE AND NITRATE IN VEGETABLE 33-39
A. I. M. Loredana, T. T. Ioan, S. Daniela, R. Bogdan NUTRITIONAL VALUES OF PSEUDOCEREALS FLOUR MIXTURES USED IN GLUTEN-FREE SWEET BAKERY PRODUCTS 40-45 40-45
M. H. Elgammal, M. S.M. Hasanin ESTIMATION OF FURAN LEVELS IN SOME CANNED FOODS IN EGYPT AND HOW TO REDUCE IT 46-52
S. Sikder, P. Biswas, A. Chattopadhyay, P. Hazra ISOLATION OF PROMISING MUTANT OF ENHANCED FRUIT QUALITY IN TOMATO 53-58
D. Sylejmani, A. Hamidi INCIDENCE OF STAPHYLOCOCCUS AUREUS AND COAGULASE POSITIVE STAPH. AUREUS IN RAW MILK IN KOSOVO 59-61 JAFES, Vol 67 (2016)
Journal of Agricultural, Food and Environmental Sciences
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L. Velkoska-Markovska , B. Petanovska-Ilievska QUANTITATIVE DETERMINATION OF 2,4-D IN PESTICIDES MONOSAN HERBI AND DMA-6 62-67
C. Gözüaçık, A. Yiğit THE ALTERNATİVE HOSTS OF TRİSSOLCUS SPECİES, EGG PARASİTOİDS OF SUNN PEST AND HOST-PARAZİTOİD İNTERACTİONS İN SOUTH EASTERN ANATOLİA REGİON, TURKEY 68-74
M. Güllü, C. Gözüaçik, A. Konuksal, H. Hekimhan, H. Fidan THE DISTRIBUTION AND POPULATION DENSITY OF THE CEREAL WEEVIL, PACHYTYCHIUSHORDEI (BRULLÉ) (COLEOPTERA: CURCULIONIDAE) IN CEREAL FIELDS IN NORTHERN CYPRUS 75-81
JAFES, Vol 67 (2016)
Journal of Agricultural, Food and Environmental Sciences UDC 338.246.027:006.83/.87:542.2(047.31) Original scientific paper ______
COMMON NONCONFORMITIES DURING PROCEDURE FOR ACCREDITATION OF THE FOOD TESTING LABORATORIES IN THE R.MACEDONIA
T. Eftimova - Tashevska1*, B. Petanovska - Ilievska2, E. Dimitrieska - Stojkovik3, L. Velkoska - Markovska2
1Institute for Accreditation of the Republic of Macedonia 2Faculty of Agricultural Sciences and food, “Ss. Cyril and Methodius” University, Skopje, Republic of Macedonia 3Faculty of Veterinary Medicine, “Ss. Cyril and Methodius” University, Skopje, Republic of Macedonia
*corresponding author: [email protected]
Abstract During laboratory assessment in the frame of the accreditation procedure, they faced with number of nonconformities which are challenge for receiving the Certificate for accreditation and appropriate corrective measures shall be undertaken. Requirements that should be met by the food testing laboratories in the Republic of Macedonia, in order to gain accreditation certificate, contained in the standard МКС ENISO/IEC 17025:2006, ILAC (International Laboratory Accreditation Cooperation) document, ЕА (European Cooperation for Accreditation) document, Regulations and Procedures of the Institute for Accreditation of the Republic of Macedonia. In this paper the analysis is made and different proposals are given for the different ways of fulfilling of those requirements. The aim of this study is to emphasis the common nonconformities which are observed during realization of the Procedure of Accreditation of food testing laboratories and corrective measures undertaken. This investigation is of huge meaning for food testing laboratories which are already accredited and those which are planning to be accredited in the Republic of Macedonia. Furthermore, it is a very important for the Institute for accreditation of the Republic of Macedonia to come to conclusions for the weakest sides of the food testing laboratories and the assessment thereof. Such conclusions should initiate undertaking appropriate measures for improvement the Institute’s lead and technical assessors work towards nonconformities interpretation, identification and acceptance of the most suitable corrective measures. Key words: accreditation of food testing laboratories, common nonconformities, ISO/IEC 17025
Introduction proposals are given for the difеrent ways of During laboratory assessment in the frame of fullfiling of those requirements. the accreditation procedure, they faced with If a country’s industry could enter on the number of nonconformities which are global market, that country shall establish challenge for receving the Certificate for standards, technical regulations, metrology, accreditation and appropriate corrective testing, conformity assessment, certification measures shall be undertaken. Requirements and accreditation. All of these elements that should be met by the food testing consists the national quality infrastructure. laboratories in the Republic of Macedonia, in National quality infrastructure should provide order to gain accreditation certificate, approach to the international standards and contained in the standard МКС EN ISO/IEC technical regulation, to guarantee reliable 17025:2006, ILAC measures and to establish system for (InternationalLaboratoryAccreditationCoopera accreditation of the testing bodies and tion) document, ЕА (Europeanco- certification on the way that they are operationforAccreditation) document, international recognize. Regulations and Procedures of the Institute for The national quality infrastructure in the Accreditation of the Republic of Macedonia. Republic of Macedonia is consisting of: In this paper the analysis is made and difеrent Institute for Accreditation of the Republic of
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Macedonia, Institute for Standardization of the nonconformities and which measures can be Republic of Macedonia and Biro of accepted as corrective measures. Metrology. Accreditation is third party attestation of the III. Short history for accreditation conformity assessment body, which is The first world’s accreditation body for officially show it’s competence for performing laboratories is (National Association of the specific tasks from the conformity Testing Authorities, Australia). NATA is assessment. Attestation is issue of a statement, established 1947 and it’s a model for many based on a decision following review that similar world organization. It is formed during fulfillment of specified requirement has been Second World War when Australia had a need demonstrated [1]. to provide a ammunition production with high National body for accreditation in the Republic quality. The idea of assessment of the testing of Macedonia is Institute for Accreditation of standard has been very unusual. The second the Republic of Macedonia (IARM) which is laboratory accreditation system is form 1972 working according the Low of Accreditation in New Zeeland. Next year is formed the first [2]. accreditation body in Europe – it has been The main fields for performing the procedures Denmark accreditation body. The first of accreditation are: Testing laboratories, international conference has been held in 1977 Medical laboratories, Calibration laboratories, in Copenhagen which has leaded to forming a Inspection bodies and Certification bodies. first International Laboratory Accreditation The accreditation can be mandatory or Cooperation - ILAC [3]. voluntary. The laboratories for testing and calibration are IV. Food testing laboratories. accredited according the standard МКС Food testing laboratory is a laboratory which ISO/IEC 17025:2006, ILAC (International performs food testing on every phase of the Laboratory Accreditation Cooperation), process chain (from raw materials to the final documents, ЕА (European co-operation for products), food contact materials or Accreditation) documents and according environmental samples which has influence to regulations and procedures of IARM. If IARM the food [4]. intend to grant accreditation for testing Food testing laboratories belong to the next laboratory, IARM organize assessment for fields [4]: collecting the dates regarding the fulfillment the requirements of the documents which are Food chemistry, mention above. Food microbiology, In the framework of the procedure for Food reology and other physical accreditation, during assessment the testing, laboratories faced the number of Food toxicology, nonconformities which are challenge for Functional testing, taking the appropriate corrective action and Molecular biology (including GMO’s receiving the accreditation certificate. testing) The aim of this investigation is to receive an Sensor testing. awareness of the observed nonconformities during performing the procedure of With food testing, the food quality and/or food accreditation of food testing laboratories, to safety is determinate. The food testing make analyze of undertaken corrective laboratories can be accredited according measures as well. This investigation has a international standard for accreditation of huge meaning for the food testing laboratories testing and calibration laboratories МКС EN which are already accredited, the food testing ISO/IEC 17025:2006 an according GLP, Good laboratories which are in the process of Laboratory Practice - OECD (Organization for accreditation and for the IARM also to take the Economic Co-operation and Development) conclusion which are the weakest sides of the standard. The international food market often food testing laboratories from one side and to requires accreditation of food testing take some measures for improving the IARM’s laboratories according ISO/IEC 17025:2005. assessors work and IARM’s assessors competence regarding the interpretation of
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Also, IARM accredit testing laboratories only F05-57) and is checked on during the with this standard. surveillance visit. The CAB should the latest of three months inform the IARM on V. Nonconformity and grading of corrective actions that have been taken on nonconformity in IARM without enclosing proof for enforcement When during assessment it has been asserted thereof. that some of the requirements of the Standard - Observations are comments by the have not been met, non-conformities in the assessment team on potential operation of the CAB shall be identified and improvements of the quality system of the appropriate corrective actions prescribed. The conformity assessment body, though if not CAB’s lack of enforcement of its procedures applied do not represent a danger to a in accordance with prescribed procedures shall successful operation of the quality system. be considered as non-conformity. Non- [5]. conformity might be also identified in cases when the organization does not abide by the VI. МКС EN ISO/IEC 17025: 2006 – What rules of the IARM, EA or ILAC. [5]. does it contain? 1. Grading of non-conformities 1. Scope; Non-conformities are as follows: 2. Normative references; - Critical non-conformity: If the non- 3. Terms and definitions; conformity is severe to the extent that it 4. Management requirements; endangers the credibility of the granting or granted accreditation, accreditation is not 4.1 Organization, granted or a suspension or withdrawal of 4.2 Management system, the full scope or part thereof is granted. 4.3 Document control, - Non-conformity: At a particular time a 4.4 Review of request, tenders and corrective action must be taken on in order contracts to avoid suspension. There might be a 4.5 Subcontracting of testing and need for the corrective actions of such calibration, non-conformities to be examined on site in 4.6 Purchasing services and supplies, order to confirm successful 4.7 Service to the customer, implementation, especially in cases when 4.8 Complaints, the validity of results or integrity of the 4.9 Control of nonconforming testing accreditation body has been compromised. and/or calibration work, If the Assessment Commission agrees that 4.10 Improvement, the organization recognizes the problem at 4.11 Corrective action, hand, for elimination of non-conformity a 4.12 Preventive action, written confirmation can be accepted 4.13 Control of records, enclosing enforced corrective actions and 4.14 Internal audits, an objective proof of undertaken 4.15 Management reviews. measures. The maximum deadline for elimination of a non-conformity is as 5. Technical requirements; follows: 3 months for initial assessment; 5.1 General, 2 months for surveillance; 5.2 Personnel, 3 months in the procedure for 5.3 Accommodation and extension of scope irrespective of environmental conditions, whether the assessment addresses 5.4 Test and calibration methods and the scope extension only or method validation, extension during surveillance. 5.5 Equipment, - Remarks: If the non-conformity is a 5.6 Measurement traceability, minor one and does not influence the 5.7 Sampling, result of the activities, the non-conformity 5.8 Handling of test and calibration is noted down in the Report of the leading items, assessor (F05-51, F05-19, F05-48, F05-49, 5.9 Assuring the quality of test and calibration results,
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5.10 Reporting the results. There’s differences between Quality policy which is public available and Annex A (informative) Nominal cross- that which is in the Quality manual. references to ISO 9001:2000, There are problems regarding Annex B (informative) Guidelines for terminology in the laboratories establishing applications for specific fields documentation from the quality [6]. system and terms and definitions in the standard ISO/IEC 17000 VII Common nonconformities during Quality Policy is not transfer to the procedure for accreditation of the food employee. testing laboratories in the R. Macedonia The laboratory staff it’s not acquainted For this investigation the reports are review with the procedures from the quality from preliminary, initial assessments, system. surveillance visits and assessments for There’s no description of the reaccreditation from 20 food testing responsibilities of the laboratory laboratories and their nonconformities reports. management in the Quality Manuel. There are no observed critical nonconformities in all reviewed reports. The nonconformities 4.3 are grouped according the ISO/IEC 17025 Not respecting the Procedure for standard’s requirement. document control during changing the 4.1 old document or creating the new one. • Not appropriate organization scheme Document control procedure doesn't • Differences between the company‘s include external document Organization scheme and its management. Regulation for working pleases Master list of the documentation organization and systematization. doesn’t contain the Guidelines • There’s no written decision for the documents. management and the quality manager. There’s no record for the documents • There’s no evidence for appointed distribution for all or part of them. deputies for key management External documents are not in the positions. master – list of the documentation. • There’s no list of sighs. Procedures and Guideline are not • There’s no evidence of the laboratory indexed. legal responsibility. Page number from the total pages is • There’s problem regarding not marked. confidentiality, impartiality, and Some of the documents don’t contain releasing the personal from any kind data as data of making the document, of internal, external, financial and the person which made the document other kinds of pressure. and the person which approved it. • There’s no record of the laboratory’s Methods for testing and sampling are staff meetings out of the quality system, they are not • There’s no procedure for the on the master-list, they are not introducing the new staff with work. uniquely identified and they are not • There’s no record for the personal approved. supervision. Procedures and guidelines which are connected with the technical 4.2 requirements from the standard are not Quality policy doesn't contain the updated according to the application management engagement with the of the accreditation for the new requirements from the standard methods. ISO/IEC 17025 and its continuing The master – list of the improvement. documentations is not updated. Quality policy it’s not issued from the There’s no record of withdrawing of top management. not valid document, withdrawing
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documents are not marked and they the laboratory has that kind of are not withdraw for use. procedure there are no records. There’s no evidence or there’s no Luck of procedure for returning the records for documentation review. sample to its owner after testing There’s no procedure for electronic performed. records control, there approach, There are no records from the contact responsibilities for their chancheies and the meetings with the costumer. and their protection. The analyzes of the questionnaires is not made, and the frequency of the 4.4 contact with the clients is not defined. Laboratory is missing some data There is no record from the regarding date of accepting the sample questionnaires analysis from the or methods there’s going to be use for clients regarding the quality of the testing are not defined and they are not services they offer. agreed with the costumer. 4.8 4.5 There is no record from the complies The laboratory doesn’t have clear which are receive verbal. policy for subcontracting. 4.9 It’s not clear does laboratory inform The laboratory doesn’t inform the the costumer for the subcontractor clients for appearing of nonconformity used. during testing activities and there is no The laboratory doesn’t maintain the evidence for undertaken corrective subcontractor register. measure immediately together with the There’s no evidence for decision for accepting the subcontracting laboratory that is nonconformity. compliance with the international There is no prescribing evidence standard МКС EN ISO/IEC when the client is informed for the 17025:2006. nonconformity appearing during the 4.6 testing. There’s no procedure for services and There are no records for identified supplier evaluation. nonconformities during performing The laboratory doesn’t have a list of the everyday activities of testing. approved suppliers. There is no evidence for appointing There is no evidence for the the personal who is responsible for characteristic and supplied product informing the client concerning the quality as technical specification of appeared nonconformity. the supplied equipment, chemicals, The procedure for nonconformity reagents and services. management doesn’t assumed There is no procedure and records for nonconformity from the PT chemicals, reagents storage, especially participation. for those which is after validity date. 4.10 Records for verification for supplied The laboratory doesn’t have records products and services are not complete for improving during analyzes of the and they don’t give information for quality goals, results from the data verification of the specification analyzes, corrective measures and required. management review. 4.7 The analyze of the given goals from There is no evidence for possessing of the previous year is not performed. feedback from the clients. The laboratory doesn’t assign value Luck of the procedure for other measurable goals. client’s data protection when some From some nonconformity from the client participate on the testing of its IARM assessment the corrective sample or protect the data from the measures are not undertaken. other visitors in the laboratory, or if
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4.11 There is no procedure for electronic There is no record for cause analyzes records control, their protection, for appearing nonconformity, for determination of responsibilities for which the corrective measure is records back-up and its frequency. undertaken or documentation from the The record’s keeping time is not management system doesn’t assumed defined. that kind of procedure. The records list is not updated. Measures which arise from the 4.14 nonconformities during the testing, Internal audit plan is not respected and complies or management review are internal audit are not perform with the not recognize as corrective measures. require frequency. There are corrective measures only There is no program for internal audit. from internal audit. There is no evidence that director of The laboratory doesn’t fulfill the laboratory organization is corrective measure form from the informed for the date for internal IARM assessment remarks. audit. There is no evidence for measuring the Not undertaking the corrective corrective measure efficiency. measures for the nonconformities from 4.12 the internal audit. Not recognizing of some activities as There is no evidence for the internal preventive measures. auditor’s competence. There is no record and there is no Not respecting the internal audit initiative of the employee for procedure and not respecting the plan undertaking the preventive measures. of internal audit. Records from the undertaken There is no internal auditors list. preventive measures are not clear, The internal audit team member is a some dates are missing or they quality manager who is not existing but they are not logic, so the independent. date of undertaken preventive measure There is no record from the meeting is not comply with the date of when the decision is made for the measuring the efficiency of the internal audit dates and its team preventive measure or the dates are member. not record. From the internal audit program is not Some of the activities for improving clear which part of the laboratory the working conditions are not activity is covered from whom of the recorded as preventive measure. audit team. There is misunderstanding regarding There is no evidence that during the meaning of the “preventive internal audit the test method and measure” and there is no difference other technical requirements from the between corrective and preventive standard are checked. measure. There is no evidence for the internal 4.13 audit team member’s competence. The most common nonconformity 4.15 from this part of the standard is wrong The laboratory’s procedure doesn’t dates in the records or they are not contain or management review doesn’t written. cover all inputs elements from the The page number and the total page standard. number is not marked in the technical Records from the management review records and the records mistakes are don’t contain proposed measures, due not treated according standard dates and assign responsibilities. requirement. Part of the records is not marked and 5.2 they are not part of the system. The defined responsibilities are not The records are not kept on secure and complete for each of the personal. safety place. Not updates personal files.
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The laboratory doesn’t have procedure 5.4 and plan for training and there is no There is no records for standard record for evaluation of the training method verification, validation of efficiency. nonstandard method and records for There is no procedure and record for calculation of measurement surveillance of the new employee. uncertainty. There is not assigned personnel for Luck of records for validation of performing the specific testing, nonstandard methods or validation is handling with the equipment and sign not complete and some parameters are the test reports. missing as limit of detection, Luck of records for the personal reproductyvity, robustness, sensitivity. competence for specific activities. The method is not performing During test methods witnessing the according to the standard method laboratory staff shows insufficient requirements which laboratory apply knowledge for testing activities or for accreditation. equipment handling. The records of calculation of The realization plan analysis for the measurement uncertainty don’t training plan and analysis of the contain all components which training needs are not made. influence of the test result. The competence matrix is not clear There is no records and data for the and complete. testing activities. 5.3 There is no statement that method is There is no procedure for hygiene appropriate for intending use. maintaining and glass cleaning. Method guidelines are not according There is no evidence for performed with the standard method. cleaning of the premises. 5.5 There is no evidence for temperature Laboratory equipment for methods control and other environmental performing is not calibrated; the condition which influence of the test calibration certificates are from not results in the laboratory premises for accredited laboratory and don’t testing, chemicals, reagents and contain explanation, measurement samples storage, refrigerators and uncertainty and data for deviation. other devices or warehouses. There is no guidelines and records for Inappropriate placement of analytical equipment intermediate checks. scales, inappropriate bases of the scale The equipment which is not in use or which has vibration influence, scale defective it’s not clearly marked. which is placed in the premises with There is no appropriate equipment for evaporating of chemicals and presents the specific testing method or there is of humidity. no appropriate chemicals, reagents, Risk of cross contamination between dissolvers, distillated water with sterilization and decontamination in appropriate purity, indicators which the microbiological laboratories and are declare with certificate or made storage of the reference strains in the according appropriate procedure. reception unit. Water bath, driers, incubators, The laboratory doesn’t have limits for thermostats, refrigerators, stoves environmental control as temperature, ignition are not calibrated. humidity, biological sterility. There is no record for the temperature There is no record for air pollution and in the water bath, incubators (this kind working surfaces pollution. of nonconformity can apply on 5.3 There is no procedure for undertaking from the standard also). the activities when environmental The equipment is calibrated only in condition are not according method one point from the working range or is requirement and the staff doesn’t calibrated in the interval of values know how to react and which are their which its not use or calibrated outside responsibility. working range.
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Equipment list is not complete and 5.8 doesn’t contain all laboratory There is no time limit for storage the equipment. sample, when additional testing is The glass from “A” class is mixed needed. together with the glass of lower There is no procedure for receipt, classes. handling, storage and disposal of the There is no records of regularly samples and their not appropriate maintains of the equipment. storage in uncontrolled conditions There is no calibration plan. together with chemicals and dissolvers There is no evidence for quality. which can lead to cross contamination. The calculations in the laboratory are The laboratory doesn’t respect the performing with the calculator. procedure for record, marking and The calibration label contains due date identification of the testing samples. of calibration. There is no procedure, record and There is no matrix of competence for define responsibilities for treating the the equipment. sample which is not appropriate for 5.6 testing. The equipment is not calibrated or the During receipting the sample, critical plan of calibration is not respect. parameters for sample conditions are The laboratory doesn’t use certified not defined . reference materials. There is no procedure for treating the The equipment is calibrated in the sample after testing. accredited laboratory, but 5.9 accreditation is issued from the The laboratories don’t use referent accreditation body which is not EA materials or certified referent MLA signature. materials, or there is no record for PT The Certified reference material which participation. is use for equipment calibration There is no evidence for participation doesn’t contain all parameters which on PT or ILC for the new methods influence of the equipment applied or there is no 4 year plan for performance. PT participation and the sub discipline Guidelines for internal calibration are not determinate for PT or ILC don’t contain the assign participation. responsibilities for internal calibration, The laboratory doesn’t take any responsibilities for data calculation measures when the negative result and the competence of the personnel. from PT participation has arrived. Calibration of the water bath, drier or There is no stated frequency or there is similar equipment is made in one no plan for controlling the testing space point, which is not evidence of results with control material and there then deviation from other space points. is no procedure for undertaken There is no calibration plan or some measures after the diagrams analyzes. data is missing for the frequency of There is no use of statistical methods the calibration or the plan of of Shewhart diagrams data review or if calibration doesn’t contain the they use it there is no stated frequency calibration frequency for all for that activity. equipment. The results from ILC participation are 5.7 not analyzed and there is no defined There is no statement that laboratory criteria for accepting the results. doesn’t perform sampling activities. The causes from the deviation from There is luck of data for pH and the mean value are not analyzed. conductivity of water, the amount of 5.10 tacking food sample. The data from the test reports doesn’t contain requirements from the article 5.10.2 and 5.10.3 from the standard.
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The test report is not clear and precise, requested value”. Sometimes the doesn’t possessed appropriate title, nonconformities are related with requirements and clear identification, the method which don’t exist in the standard or used are not stated, the amount of the appropriate mandatory documents, as: “The tested sample, and the measurable unit equipment is not calibrated from the which related to the test result are not competent calibration laboratory, only internal stated. calibration is performed”, “The personal files There is no explanation for the marks don’t contain the statements of confidentiality used, name, surname and function of and impartiality”, “There is no list of obsolete the person who sigh the test result. documents”, “Microbiological control of the The non accredited methods are not working surfaces is not performed according clearly marked. EA document EA 4/10” [1, 7]. There are Opinion and interpretation of the test nonconformity reports which contain more result is not marked according IARM than one nonconformity. For example: “There Regulation on Requirements for use of is no calibration certificate, calibration plan the accreditation mark, text reference doesn’t contain all laboratory equipment and to accreditation and reference to the calibration frequency is not defined”. IARM’s EA MLA Signatory status. Sometimes the assessors connect the There is no statement that test results nonconformity with one part of the standard, refer to the delivered sample. but actually it is related with another part. The The test report doesn’t contain nonconformity for lucking the referent information that sampling is material the assessor connects it with 5.6 accredited activity. (Measurement traceability), actually it is There is no logo or reference to the related with 5.5 from the standard accreditation status on the test report. (Equipment). The same nonconformity is The end of the test report is not clearly grading different with different assessors. marked. Conclusion VII.1 Improving the work of a assessment The most frequent nonconformity from team preliminary visits or from initial assessment, The team of regular and extraordinary surveillance visit or reassessment is not assessments, which IARM form for calibrated equipment or Calibration Certificate conformity assessment bodies, consists of the doesn’t fulfilled standard’s requirements. This lead assessor and technical assessors or experts nonconformity is connected with technical from the assessed field. Improving the requirements from the standard ISO/IEC assessors work from the food testing field has 17025 (clausal 5) [6]. From the technical aim to increase the awareness and requirements the most frequent remark is understanding of the assessors for ISO/IEC related with “Opinion and interpretation of the 17025’s requirements from one side and test result”. They are located on the same side harmonization of the assessment process with the test result and they are not marked which lead to equivalent treatment to all food according Regulation on Requirements for testing laboratories, to all laboratories also and use of the accreditation mark, text reference to to other conformity assessment bodies. One accreditation and reference to IARM’s EA of the measures for improving is the way of MLA Signatory status– Р 05 [43]. The most expressing the nonconformity. The assessors frequent remark from the management shouldn’t formulate the nonconformity as requirements is related with treating the suggestion or consultation how laboratory mistakes in the technical records, thus they are could act, but to state what is missing or for not corrected according standard’s what has a lack of evidence. For example, it requirements. From the preliminary visits the noted the following: “The test report should most frequent nonconformities are follow: In contain the information from the 5.10.2 from the the Quality Policy is luck a commitment the standard” or “The sterilization activity and of the management to comply to the standard decontamination should be divided” or “The and lack of a written appointment of members laboratory must issue a procedure for acting in of the top management and quality manager the case when the temperature is above [8].
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References ISO/IEC 17000 : 2004 Conformity assessment – Vocabulary and general principles Low of Accreditation (Official Gazette of R.Macedonia No 120/09) http/www.nata.com.au APLAC TC007 APLAC Guidelines for Food Testing Laboratories http://www.iarm.gov.mk/dokumenti/en/Direct or%27s%20Procedures/PR%2005- 06%20Grading%20of%20nonconformities.pdf ISO/IEC 17025 : 2005 General requirements for the competence of testing and calibration laboratories EA 4/10 Accreditation of Microbiological laboratories http://www.iarm.gov.mk/dokumenti/en/Docu ments- IARM%27s_Council/R_05_Regulation_on_R equir_for_use_of_the_accred_mark_text_refer _to_accr_and_refer_to_IARM's_EA_MLA_Si gnat_EN.pdf
JAFES, Vol 67, (2016) Journal of Agricultural, Food and Environmental Sciences UDC 663.813:634.11]:632.95.028:534.544.5.068.7 Original scientific paper ______
SPE-RRLC DETERMINATION OF SOME PESTICIDE RESIDUES IN APPLE JUICE
L. Velkoska-Markovska*, B. Petanovska-Ilievska, I. Stojanovska, M. S. Jankulovska
Faculty of Agricultural Sciences and food, “Ss. Cyril and Methodius” University, Skopje, Republic of Macedonia
*corresponding author: [email protected]; [email protected]
Abstract This paper presents the development of RRLC (Rapid Resolution Liquid Chromatography) method for simultaneous determination of pesticides methomyl, methidathion and propiconazole in different clear apple juice samples. The experiments are performed using rapid resolution liquid chromatography system coupled with UV-VIS diode array detector. The developed high speed reversed-phase (RP) liquid chromatography method is carried out on the Purospher® Star RP-18 endcapped (30 mm × 4 mm; 3 μm) column, mobile phase consists of acetonitrille and water (50/50, V/V), flow rate of 1 mL/min, column temperature at 25 ºC and UV detection at 220 nm and 235 nm. Prior to RRLC analysis, the samples are cleaned up and concentrated using a solid-phase extraction (SPE). To assess the validity of the developed method, the following parameters are examined: selectivity, linearity, repeatability (precision), limit of detection, limit of quantification and accuracy. Key words: RRLC determination, SPE, UV-DAD, pesticide residues, apple juice
Introduction approved for use as pesticides. Both the The mass production of apple fruit requires European Union and the USA have adopted extensive use of plant protection chemicals, regulations concerning the MRLs (Maximum some of them being the pesticides methomyl, Residue Levels/Limits) for pesticides present methidathion and propiconazole. in food. The MRLs of pesticides contained in Methomyl, S-methyl N-(methylcarbamoyloxy) apple are laid down by the EU (Regulation thioacetimidate (IUPAC) is a mixture of (Z)- (EC) No 396/2005), and they are estimated at: and (E)-isomers, which controls a wide range 0.02 mg/kg (for methomyl), 0.03 mg/kg (for of insects and spider mites which attack fruits, methidathion) and 0.15 mg/kg (for vines, vegetables etc. Methidathion, 3- propiconazole). dimethoxyphosphinothioylthiomethyl-5- Apple juices are one of the most used from the methoxy-1,3,4-thiadiazol-2(3H)-one (IUPAC) whole population, especially children. These is used to control a wide range of sucking and juices are among the first non-dairy products chewing insects and spider mites in many that are given to infants, but also they are a crops, e.g. pome fruit, stone fruit, citrus fruit, favourite among older children. Therefore, the vines etc. Propiconazole, (±)-1-[2-(2,4- safety of these products is of particular dichlorophenyl)-4-propyl-1,3-dioxolan-2- importance. Children, especially infants fall ylmethyl]-1H-1,2,4-triazole (IUPAC) exhibits into high risk groups in terms of the possible both protective and curative action towards toxicity of pesticides. diseases caused by fungi which attack fruits, In order to monitor food safety, it is highly cereals, maize, coffee and peanuts (Tomlin et necessary to develop and employ reliable al., 1997). methods for determination of pesticide All three substances are registered for use in residues. the USA. Methomyl and methidathion are Gas Chromatography (GC) and Liquid classified by EPA as Restricted Use Pesticides Chromatography (LC) are commonly used (RUP, may be used only by certified methods for the determination of pesticide applicators), unlike propiconazole which is a residues in various matrixes (Nollet et al., General Use Pesticide (GUP), (EPA, 1998; 2000; Stoytcheva et al., 2011) using the EPA 2006a; EPA 2006b). In the European following detectors: Flame Ionization Union, only methomyl and propiconazole are Detector, FID (Kadifkova-Panovska et al.,
12 L. Velkoska-Markovska, B. Petanovska-Ilievska, I. Stojanovska, M. S. Jankulovska ______
2000), Nitrogen Phosphorous Detector, NPD The Pestanal grade analytical standards of (Ay et al., 2007), Mass Spectrometry, MS (He methomyl, methidathion and propiconazole, Qiang et al., 2010), Tandem Mass K2HPO4 (p.a.), H3PO4 (85.5 %) and HPLC- Spectrometry, MS/MS (Guo et al., 2012), Ion grade acetonitrile are purchased by Sigma- Trap Mass Spectrometry, ITMS (Sannino et Aldrich (Germany). Ultrapure water is al., 1999), Fluorescent detector, FD (Sánchez- produced by TKA Smart2 Pure 12 UV/UF Brunete et al., 2004), Electrospray Ionization water purification system (Germany). Mass/Tandem Mass Spectrometry, ESI-MS The juice samples are filtered by 0.45 µm (Schermerhorn et al., 2005) or ESI-MS/MS Nitrocellulose membrane filters (Millipore, (Zamora et al., 2004). Also, HPLC combined Ireland), and the final extracts are filtered with ultraviolet, UV detector (Yu et al., 2011) through 0.45 µm Iso-Disc PTFE syringe filters or Diode Array Detector, DAD (Carbo et al., (Supelco), just before application. 2008; Lagana et al., 1997; Jeannot et al., 2000) Various commercial 100 % clear apple juice is used for the determination of these samples made from different producers (A, B, components. C, D and E) are purchased in Macedonian Pretreatment of samples usually use the supermarkets. following procedures: liquid-liquid extraction, LLE (Jeannot et al., 2000), Solid Phase Preparation of Standard Solutions Extraction, SPE (Lagana et al., 1997), Solid Stock solutions methomyl, methidathion and Phase Microextraction, SPME (Hercegová et propiconazole are prepared by dissolving al., 2011) and Matrix Solid-Phase Dispersion, 0.0186 g, 0.0187 g and 0.0132 g, respectively, MSPD (Albero et al., 2003). of the pure analytical standards in acetonitrile However, the HPLC or RRLC method for in 10 mL volumetric flasks. The solutions are simultaneous determination of pesticides degassed for 15 min in an ultrasonic bath and methomyl, methidathion and propiconazole stored in a refrigerator at 4 oC. Stock solutions residues is not found. Hence, the aim of this are used to prepare standard mixtures with paper is to develop a reversed-phase RRLC different pesticide concentrations (10 – 200 method for simultaneous determination of mg/L for methomyl, 15 – 300 mg/L for methomyl, methidathion and propiconazole methidathion and 75 – 1500 mg/L for residues in apple juices using SPE and UV- propiconazole) in 10 mL volumetric flask by DAD. dilution with the mixture of acetonitrile/water (50/50, V/V). In order to construct the Materials and methods calibration plots, 5 μL of each working Equipment and Materials solution are injected in the chromatograph The development of the RRLC method for three times. The obtained chromatograms are simultaneous determination of methomyl, analyzed considering areas and heights of the methidathion and propiconazole in clear apple peaks. juice samples is performed using an Agilent 1260 Infinity Rapid Resolution Liquid Solid phase extraction Chromatography (RRLC) system equipped For recovery experiment, 1 kg apple juice with: vacuum degasser (G1322A), binary samples are spiked with concentrations pump (G1312B), autosampler (G1329B), a corresponding to MRL: 0.02 mg/kg (for thermostatted column compartment (G1316A), methomyl), 0.03 mg/kg (for methidathion) and UV-VIS diode array detector (G1316B) and 0.15 mg/kg (for propiconazole). Unspiked ChemStation software. The investigations are samples are used for blanks. After column carried out on a Purospher STAR RP-18e (30 conditioning the samples are passed through mm x 4 mm, 3 µm, Merck) analytical column. the cartridges. The retained pesticides are For the better dissolving of the stock solutions eluted, twice with 2 mL 100 % acetonitrile. an ultrasonic bath “Elma” is used. For the The eluates are evaporated to dryness. The clean-up and concentration of apple juice residues are redissolved with 1 mL of samples a system for solid phase extraction is acetonitrile, than filtered through 0.45 µm Iso- employed (Supelco, Sigma Aldrich) with Disc PTFE syringe filters and transferred into Supelclean™ Envi-18 SPE columns (6 mL; 500 vials for RRLC analysis. 5 μL of each sample mg). For vortexing of samples, an IKA Vortex are injected in the chromatograph three times. Genius 3 (Germany) is used.
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This procedure concentrated the amount of of detection (LOD) and limit of quantification pesticides in the samples 1000 times. (LOQ) are tested. To confirm the specificity of the developed Results and discussion method, UV-diode array detection is used to The UV spectra of investigated pesticides in check the peak purity and analyte peak acetonitrile/water mixture (50/50, V/V) show identity. The purity index for all analytes is that methomyl has an absorption maximum at greater than 999, which means that the 235 nm, while methidathion and propiconazole chromatographic peak is not affected by any have their absorption maxima at 220 nm. other compound. Therefore, RRLC analysis for simultaneous The obtained values for retention times of determination of these substances is performed components (tR) under these chromatographic at 235 nm (for methomyl) and 220 nm (for conditions are given in Table 1. methidathion and propiconazole). The linearity of the method is tested by In preliminary experiments, different flow-rate triplicate injections (5 µL) of five mixtures (0.9 – 2 mL/min) and different volume ratio of containing various concentrations of the three acetonitrile (80 - 45 %) and water in the compounds: methomyl in the range 10 - 200 mobile phase on Purospher® Star RP-18 mg/L, methidathion in the range 15 - 300 endcapped analytical column are tested. The mg/L and propiconazole in the range 75 -1500 best resolution with sharp and symmetrical mg/L. For these concentration ranges and peaks and satisfy purity indexes for the three using data for the peak areas and peak heights, analytes is achieved with the mobile phase calibration curves are constructed and the consisted of acetonitrile/water (50/50, V/V), correlation coefficients (R2) are calculated flow-rate of 1mL/min and column temperature (Table 1). The curves followed Lambert- of 25 ºC (Figure 1). Beer’s law and the R2 values obtained For the method validation, specificity and indicated the peak area as a preferable variable selectivity, linearity, precision, accuracy, limit for further accuracy testing.
I I
Figure 1. Chromatogram obtained from a standard mixture of methomyl (I), methidathion (II) and propiconazole (III)at 220 nm (a) and 235 nm (b)on Purospher® Star RP-18 endcapped column with acetonitrile/water (50/50, V/V) as a mobile phase, flow1 mL/min and temperature 25 ºC
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14 L. Velkoska-Markovska, B. Petanovska-Ilievska, I. Stojanovska, M. S. Jankulovska ______
Table 1. Data from experiments of investigated pesticides
LOD LOQ Compound t /min Regression equation R2 R (μg/L) (μg/L) 1y = 4473,8x + 24,217 0,9993 Methomyl (235 nm) 0.30 0.12 0.4 2y = 1933,3x + 134,67 0,9878 1y = 2175,8x + 22,005 0,9999 Methidathion (220 nm) 1.25 4.5 15 2y = 417,4x + 38,832 0,9868 1y = 2006,8x + 152,53 0,9999 Propiconazole (220 nm) 2.13 6 20 2y = 248,27x + 53,249 0,9980 1y = peak area; 2y =peak height
The precision is expressed as repeatability of obtained results (Table 2) indicated a very obtained results which is evaluated for peak good precision of peak area, height and areas peak, heights and retention times of the retention time under the conditions used in the analytes from eight successive injections (5 tested method. μL) of the mixture containing 20 mg/L The limits of detection (LOD) is defined as the methomyl, 30 mg/L methidathion and 150 amount of analyte for which the signal-to- mg/L propiconazole within 3 days. The noise ratio (S/N) is 3, whereas the limits of percent of relative standard deviation (RSD) quantification (LOQ) is defined as the amount for intra-day repeatability is less than 0.65 %, of analyte for which S/N =10. The LOD and while RSD values for inter-day repeatability is LOQ for each compound are listed in Table 1. less than 2.55 % for each component. The
Table 2. Precision data for investigated pesticides
Intra-day repeatability Inter-day repeatability
(n = 8) (n = 3)
x SD RSD (%) SD RSD (%) Methomyl 0.30 0.005 0.15 0.30 0.003 0.44 Retention Methidathion 1.24 0.003 0.22 1.23 0.02 1.93 time Propiconazole 2.13 0.005 0.25 2.10 0.05 2.18 Methomyl 526.17 0.58 0.11 536.10 7.22 1.35 Peak Methidathion 339.32 1.46 0.43 334.82 7.90 2.36 area Propiconazole 1671.71 1.80 0.11 1694.11 33.54 1.98 Methomyl 320.67 1.04 0.32 316.65 6.05 1.91 Peak Methidathion 94.03 0.31 0.33 92.36 2.33 2.53 height Propiconazole 244.45 1.55 0.64 248.67 6.21 2.50
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15 L. Velkoska-Markovska, B. Petanovska-Ilievska, I. Stojanovska, M. S. Jankulovska ______
Figure 2. Chromatogram obtained from unspiked apple juice (a) and sample of apple juice fortified at the concentration equal to MRL for methomyl (I), methidathion (II) and propiconazole (III) (b) on Purospher® Star RP-18 endcapped column with acetonitrile/water (50/50, V/V) as mobile phase, flow1 mL/min, temperature 25 ºC and UV detection 220 nm
The developed method based on solid-phase methidathion and 121.48 % for propiconazole. extraction is applied for the determination of In cases where pesticide residues in food are investigated pesticide residues from apple analyzed recoveries in the range 70 - 120 % juice samples. The chromatograms obtained and even outside this range are acceptable in under the same conditions from the cleaned-up cases of multiresidue methods (European and concentrated apple juice, after SPE, show Commission, 2010; SANCO, 2011). The a number of tall peaks (originating from the methidathion and propiconazole residues in apple juice matrix) in the area around 0.3 concentrations which are correspond to MRLs minutes, where the methomyl peak appears or higher are not detected in none of the five (Figure 2a). So we continued investigations samples of apple juice. aimed at determining only methidathion and propiconazole residues. Conclusion The values for the retention time and match The developed RRLC method with UV-DAD factor obtained by overlaid UV spectra of a is selective for two of the three pesticides pure analytical standard and absorption spectra examined, them being methidathion and of the same analyte in the spiked apple juice propiconazole. The matrix contains interfering samples are used for the identity of the compounds that disable the determination of analytes. The estimated values for the match methomyl. The proposed method is simple, factor are 994.092 for methidathion and linear, precise and sufficiently accurate for 999.899 for propiconazole. Being high above qualitative and quantitative determination of 990, these values confirm the identity of methidathion and propiconazole residues in methidathion and propiconazole. On Figure 2 apple juices after the SPE procedure. are presented chromatograms of matrix blank This method can be used for determination (unspiked apple juice that is apple juice free of pesticide residues in laboratories for control of investigated pesticides) (a) and sample of chemical hazards in food which will contribute apple juice fortified at the concentration equal to food safety. to MRL for each analyte (b). The efficiency of the extraction method using ENVI-18 SPE References cartridges expressed as recovery. The obtained Albero, B., Sánchez-Brunete, C., Tadeo, J. L. values for recovery are 114.46 % for (2003). Determination of organophosphorus
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16 L. Velkoska-Markovska, B. Petanovska-Ilievska, I. Stojanovska, M. S. Jankulovska ______pesticides in fruit juices by matrix solid-phase surface waters. Journal of chromatography A. dispersion and gas chromatography. Journal of No. 879, pp. 51-71. agricultural and food chemistry. No. 51(24), Kadifkova-Panovska, T., Kavrakovski, Z., pp. 6915-6921. Bauer, S. (2000). Determination of Ay, R., Yasar, B., Demirözer, O., Aslan, B., propiconazole residues in tomatoes by gas Yorulmaz, S., Kaya, M., Karaca, I. (2007). chromatography. Bulletin of the Chemists and The determination of the residue levels of Technologists of Macedonia. Vol. 19. No.1, some commonly used pesticides in Isparta pp. 27-23. apple orchards. Turkish Journal of Lagana, A., D'Ascenzo, G., Fago, G., Marino, Entomology. No. 31(4), pp. 297-306. A. (1997). Determination of Carbo, L., Souza, V., Dores, E. F. G. C., organophosphorus pesticides and metabolites Ribeiro, M. L. (2008). Determination of in crops by solid-phase extraction followed by pesticide multiresidues in shallow liquid chromatography/diode array detection. groundwater in a cotton-growing region of Chromatographia. No. 46(5/6), pp. 256–264. Mato Grosso, Brazil. Journal of the Brazilian Nollet, L. (2000). Food Analysis by HPLC, Chemical Society, Vol.19. No. 6, pp.1111- Marcel Dekker Inc., New York, Basel, pp. 1117. 693-715. European Commission, Directorate General Regulation (EC) No 396/2005 of the European Health and Consumer Protection. (2010). Parliament and of the Council of 23 February Guidance document on pesticide residue 2005 on maximum residue levels of pesticides analytical methods. pp. 9-11. in or on food and feed of plant and animal EPA. Registration Eligibility Decision (RED) origin and amending Council Directive for Methomyl. (1998). pp.14. 91/414/EEC Text with EEA relevance. EPA. Registration Eligibility Decision (RED) Sánchez-Brunete, C., Albero, B., Tadeo, J. L., for Methidathion. (2006a). pp.5. (2004). HPLC multiresidue method for the EPA. Registration Eligibility Decision (RED) determination of N-methyl carbamates in fruit for Propiconazole. (2006b). pp. 5. and vegetable juices. Journal of food Guo, M., Tan, Z., Wu, H., Li, J., Wang, Y., protection No. 67 (11), pp. 2565-9. Shi, J., Li, J., Jiang, L., Yao, X., Fang, L. SANCO. (2011). Method validation and (2012). Determination of 11 triazole quality control procedures for pesticide fungicides in fruits using solid phase residues analysis in food and feed, Document extraction and gas chromatography-tandem Nº SANCO/12495/2011 (implemented by mass spectrometry. Chinese Journal of 01/01/2012). pp. 13. Chromatography. Vol. 30. No. 3, pp. 262-266. Sannino, A., Bandini, M., Bolzoni, L. (1999). He, Q., Li, J., Kong, X., Yue, A., Dong, H., Multiresidue Determination of 19 Fungicides Zhao, J. (2010). GC-MS Determination of in Processed Fruits and Vegetables by Residual Amount of Propiconazole in Capillary Gas Chromatography after Gel Foodstuffs with Separation by Solid Phase Permeation Chromatography. Journal of Extraction. Physical Testing and Chemical AOAC International. Vol. 82. No. 5, pp. 1229- Analysis. No. 2, pp. 178-183. 1238. Hercegova, A., Mőder M. (2011). Schermerhorn, P., Golden, P. E., Krynitsky, A. Determination of some selected pesticide J., Leimkuehler, W. M. (2005). Determination residues in apple juice by solid-phase of 22 triazole compounds including parent microextraction coupled to gas fungicides and metabolites in apples, peaches, chromatography – mass spectrometry. Acta flour and water by Liquid universitatis agriculturae et silviculturae Chromatography/Tandem Mass Spectrometry. mendelianae brunensis. Vol. LIX. No. 1, pp. Journal of AOAC International. Vol. 88. No. 121-127. 5, pp. 1491-1502. http://ec.europa.eu/sanco_pesticides/public/ind Stoytcheva, M., Zlatev, R. (2011). Pesticides ex.cfm?event=activesubstance.selection in the Modern World - Trends in Pesticides Jeannot, R., Sabik, H., Sauvard, E., Genin, E. Analysis. In Tech. pp. 1-526 (2000). Application of liquid chromatography Tomlin, C. (1997). The Pesticide Manual with mass spectrometry combined with Incorporating the Agrochemicals Handbook. photodiode array detection and tandem mass 11th Edition. Crop Protection Publications. pp. spectrometry for monitoring pesticides in 675-676, 679-680, 855-857.
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Yu, X., Wang, B. (2011). Rapid HPLC Analysis Method for Prochloraz and Propiconazole Content in Bactericidal Suspension Emulsion. Agrochemicals. No. 10, pp. 734-736. Zamora, T., Pozo, O. J., López, F. J., Hernández, F. (2004). Determination of tridemorph and other fungicide residues in fruit samples by liquid chromatography- electrospray tandem mass spectrometry. Journal of Chromatography A. No. 1045(1-2), pp. 137-143.
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Journal of Agricultural, Food and Environmental Sciences UDC 664.8.022.3(497.2) Original scientific paper ______
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETERMINATION OF PRESERVATIVES IN BEVERAGES
M. S. Jankulovska 1*, L. Velkoska-Markovska 1, B. Petanovska-Ilievska1, S. Trpkovska
1Faculty of Agricultural Sciences and Food, Ss. Cyril and Methodius University, Skopje, Republic of Macedonia
*corresponding author: [email protected]; [email protected]
Abstract Benzoic and sorbic acids, and their sodium, potassium and calcium salts are widely used as preservatives in acidic foods and beverages. They inhibit the growth of molds and yeasts, and are also effective against wide range of bacteria which explains the benefit of their usage. The health effects have led to limitation on the concentrations that can be used in food and beverages. Because of that, the analytical determination of these preservatives is important for consumer interest and protection. Therefore, a new reversed-phased HPLC method for a fast, simple, accurate and precise determination of potassium sorbate and sodium benzoate in different beverages is developed. In this purpose, a HPLC system equipped with UV diode array detection is employed. Separation and determination of investigated preservatives are performed using Purospher STAR RP-18 (30 mm x 4 mm; 3 m) analytical column. Methanol/phosphate buffer (pH = 3.70) (20/80, V/V) is used as a mobile phase, with flow rate of 1 mL/min, constant column temperature of 25°C and UV detection at 225 nm and 255 nm. Successful separation conditions are obtained using an isocratic elution within 6 min. Accuracy, precision, limit of detection, limit of quantification, and linearity range are evaluated. The separation factor value of 1.128 showed that this method can be successfully used for simultaneous determination of benzoates and sorbates. The obtained data indicated that the concentration of investigated preservatives found in the analyzed beverages available on the local Macedonian markets is in accordance with Macedonian maximum levels for food safety. Key words: benzoic acid, benzoates, sorbic acid, sorbates, beverages, determination, HPLC.
Introduction inhibition of the growth of microbes. The type Preservatives are natural or synthetic of chemical preservative that can be used in chemicals that are added to different products beverages depends on the chemical and such as food, pharmaceuticals, cosmetics and physical properties of both, the preservative biological samples to prevent spoilage from and the beverage. In addition, the pH of the microbial growth and undesirable chemical beverages, the presence of vitamins, the type reaction (Ranken et al., 2005). Chemical of packaging and the storage conditions will preservation has become an increasingly determine whether preservatives are necessary important practice in modern food technology and what type should be used to prevent with the increase in production of processed microbial growth (Tfouni et al., 2002). and convenience foods. In order to enhance The main preservatives that can be used and preservative effectiveness combination of two are allowed in beverages are sorbic acid or more preservatives often is used (Russell et (E200) and benzoic acid (E210), as well as al., 2003). their corresponding salts. Sodium benzoate Beverages are rich in vitamins and minerals (E211) is the sodium salt of benzoic acid and and as a result of that they are an attractive is widely used in acidic media to inhibit environment for microbes. Furthermore, the yeasts, molds, and bacterial growth. Potassium water activity is also important factor that has sorbate (E202) is the potassium salt of sorbic significant influence of the microbial activity. acid and generally is used to inhibit mold However, the usually low pH of beverages due growth in juices. The salts are used in to carbonation, the sugar content in some of carbonated, non-alcoholic and juice beverages them, and the addition of preservatives help in because they work best between pH levels of 2 19 M. S. Jankulovska, L. Velkoska-Markovska , B. Petanovska-Ilievska, S. Trpkovska ______and 4 (Ferrand et al., 2000). Although these The present work deals with the development compounds are generally recognized as safe and characterization of rapid, economic and due to their low risk of acute and chronic simple RP-HPLC method for simultaneous toxicity, they have limitations of usage in determination of potassium sorbate and protecting human health (Pylypiw et al., 2000; sodium benzoate in beverages. The developed Mamur et al., 2010). method is applied to the analysis of these Safety for the human consumption of these preservatives in seven samples available on the products is regulated in each country by local Macedonian markets. national regulations based on codes and standards derived by the Codex Alimentarius Materials and methods Commission. European Union (EU) member Instrumentation states use European legislation for The chromatographic analyze is carried out microbiological criteria, food additives and with Agilent 1260 Infinity Rapid Resolution general hygiene requirements for the Liquid Chromatography (RRLC) system production, storage and trade of food products, equipped with: vacuum degasser (G1322A), as well as specific requirements for quality and binary pump (G1312B), auto sampler safety of beverages (Directive No 95/2/EC, (G1329B), a thermostatted column 2005). The Republic of Macedonia has compartment (G1316A), UV/Vis diode array developed national legislation for food safety, detector (G1316B) and ChemStation software. primarily based on European legislation and For optimization of the method following three Codex standards. According to these analytical columns are used: Hypersil ODS (25 regulations maximum permitted cm x 0.46 cm; 5 µm), LiChrospher 60RP- concentrations are 150 mg/L for sodium select B (12.5 cm x 0.4 cm; 5 µm) and HS benzoate and 300 mg/L for potassium sorbate (High Speed) type LiChroCART® 30-4 HPLC- if they are used separately, and 150 mg/L for Cartridge Purospher® STAR RP-18 (30 mm x sodium benzoate and 250 mg/L for potassium 4 mm; 3 µm). The pH measurements are sorbate, if they are used in combination. These performed with a Radiometer pH-meter maximum levels are expressed as benzoic and (Copenhagen, DK). An ultrasonic bath “Elma” sorbic acid, not the salts. is used for degasification of solutions. The analytical determination of these preservatives is not only important for quality Chemicals assurance purposes, but also for consumer Sodium benzoate (99.9%) and potassium interest and protection. In the literature there sorbates (99.9%) are produced from Supelco, are several analytical methods that can be acetonitrile (ACN) and methanol (MeOH) applied for determination of preservatives. The used for the preparation of the mobile phase most common analytical method for the (Sigma Aldrich-Germany) are HPLC grade. determination of benzoic acid (BA), sorbic Ultra pure water used for chromatography acid (SA), sodium benzoate (SB) and processing is obtained from Water Purification potassium sorbate (PS) are UV System TKA Smart 2 Pure 12 UV/UF. The spectrophotometry, gas-liquid chromatography components of phosphate buffer (H3PO4 (85.5 (GLC), thin-layer chromatography (TLC) and %) and K2HPO4 (99.0 %)) are from Sigma high performance liquid chromatography Aldrich, Germany. Phosphate buffer is (HPLC) with different detection (Wood et al., prepared dissolving 0.5 g K2HPO4 in 500 mL 2004; Dong et al., 2006; Bahremand et al., ultra pure water and adjusted to the pH 3.70 2013). Many of the reported methods use with H3PO4. complicated pre-treatment procedures such as solvent extraction method, while HPLC offers Chromatographic conditions simple sample preparation limited to filtration, The mobile phase composed of methanol and degasification and dilution. Furthermore, this phosphate buffer (pH 3.70) (20:80, V/V) is method is widely used because of the used. The optimal wavelength for detection is possibility to analyse preservatives with high set at 225 nm and 255 nm for sodium benzoate precision, and with low limit of detection. This and potassium sorbate, respectively. The shows that HPLC is a powerful technique in chromatographic separation is achieved with the area of preservative analysis (Isabel et al., isocratic elution (2.5 L of sample are injected 2001; Küçükçetin et al., 2008). into chromatographic system) at a flow rate of
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1.0 mL/min and column temperature of 25 oC. the system without clean-up procedures prior The peaks of the determined preservatives are to determination by HPLC (Veerabhadrarao identified by their UV spectrum and by 1987). comparing the retention time with that of the standard. Results and discussion Determination of food preservatives sodium Preparation of standard and sample solutions benzoate and potassium sorbate in beverages is The stock solutions are prepared by dissolving performed using a rapid, economic and simple 0.0365 g sodium benzoate and 0.0774 g RP HPLC method. The UV-Vis spectra using potassium sorbate in methanol in 10 mL photodiode array detector of sodium benzoate volumetric flasks. Then they are degassed in and potassium sorbate in 50/50 (V/V) an ultrasonic bath for 15 minutes. All prepared methanol/water solution show that sodium stock solutions are stored in a refrigerator at 4 benzoate has an absorption maximum at 225 ºC. The stock solutions are used to prepare nm, while potassium sorbate has an absorption sample solutions with different concentration maximum at 225 nm. Therefore, the detection of SB (30 mg/L, 60 mg/L, 90 mg/L, 120 mg/L of sodium benzoate and potassium sorbate is and 180 mg/L) and PS (62.6 mg/L, 125 mg/L, carried out at these wavelengths, respectively. 187.1 mg/L, 250 mg/L and 374 mg/L) In order to separate preservatives an attempt is dissolved in methanol/water 50/50 (V/V) in 10 made using the Hypersil ODS (25 cm x 0.46 mL volumetric flasks. In order to obtain cm; 5 µm) column with a mobile phase calibration curves, 2.5 μL of each working consisted of acetonitrile/water 80/20 (V/V), solution are injected in the chromatograph flow-rate of 1 mL/min, and column three times. The obtained chromatograms are temperature of 25 ºC. Unfortunately, the peaks analyzed considering area and height of the of benzoic acid (I) and potassium sorbate (II) peaks. The developed method was used for are not separated. There is only one peak with determination of benzoate and sorbate contents fronting and retention time of 2.5 min (Figure in seven different samples taken from 1). The changes in the composition of the Macedonian market. They are prepared by mobile phase (till 55 % ACN and 45 % water) filtering through a 0.45 µm membrane filter did not produce satisfactory separation. (T-Spartan) and after that they are injected into I + II
Figure 1. Chromatogram of benzoic acid (I) and potassium sorbate (II) obtained on Hypersil ODS (25 cm x 0.46 cm; 5 µm) column; mobile phase acetonitrile/water 80/20 (V/V), flow rate 1 mL/min, column temperature 25 ºC and UV detection at 220 nm.
Similarly, when LiChrospher 60 RP-select B temperature of 25 ºC. The peak shapes of (12.5 cm x 0.4 cm; 5 µm) column is used a benzoic acid (I) and potassium sorbate (II) are good separation is not obtained with the not acceptable for quantitative determination mobile phase consisted of acetonitrile/water of these two compounds (Figure 2). 50/50 (V/V), flow-rate of 1mL/min and column
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I II
Figure 2. Chromatogram of benzoic acid (I) and potassium sorbate (II) obtained on LiChrospher 60 RP-select B (12.5 cm x 0.4 cm; 5 µm) column; mobile phase acetonitrile/water 50/50 (V/V), flow rate 1 mL/min, column temperature 25 ºC and UV detection at 220 nm.
The previous results indicated that the changes pKa ± 1.5 (pKa = 4.19 for benzoic acid and in the stationary phase are necessary to be pKa = 4.76 for sorbic acid). The pH done. Hence, in the further investigation the dependence of the retention values can serve HS (High Speed) type LiChroCART® 30-4 as a guide to select the optimum pH value for HPLC-Cartridge Purospher® STAR RP-18 (30 the isocratic separation of a particular mixture. mm x 4 mm; 3 µm) is employed. In order to As expected, retention time decreases when obtain better separation the changes in the dissociation of the solutes increases, probably mobile phase are also made. The change of the as a result of interactions with the stationary solvent is a potentially useful variable for phase. If the sample is consisted only of acidic optimizing the separation. ACN is a stronger compounds the pH of the solution should be solvent than MeOH and can typically elute low to suppress dissociation and to obtain analytes faster in RP HPLC at similar stronger retardation and better separation concentration. On the other hand, MeOH may (Snyder et al., 1997; Meyer et al., 2010; Dong be preferred compared to ACN for separation et al., 2006; Davidson et al., 2005). of some ionic samples. Furthermore, the In order to obtain satisfactory separation buffers have the greater solubility in methanol- mobile phase consisted of methanol/phosphate water mixtures compared to mobile phases that buffer (pH 3.70) with different volume ratio contain ACN. Taking into consideration these are tested. The obtained chromatograms facts, methanol is used as a best choice of showed that the best separation with sharp and organic solvent in the mobile phase. symmetrical peaks for sodium benzoate (I) and The investigated preservatives can exist either potassium sorbate (II) is achieved with the into the nondissociated and ionized form, mobile phase composed of depending on the mode of separation. Hence, methanol/phosphate buffer (pH 3.70) 20/80 the chromatography process is important to be (V/V), flow-rate of 1 mL/min, injection volume performed at a well defined pH for separation of 2.5 µL, constant column temperature of 25 of ionizable compounds. For this purpose ºC and UV detection at 225.4 nm and 255.4 buffers are suitable choice because they can nm (Figure 3). The retention time under these control the pH of the mobile phase and chromatographic conditions is 4.823 min for neutralize the charge on the silica surface of benzoic acid and 5.407 min for potassium the stationary phase. Additionally, the buffers sorbate. The values of the retention factor (k) can act as ion pairing agents which can are 17.915 and 20.203 for sodium benzoate neutralize analyte charge. Inorganic buffers are and potassium sorbate, respectively. The usually relatively stable and because of that separation factor (α) between the adjacent they are often used. In this investigation the peaks of sodium benzoate and potassium phosphate buffer (pH 3.70) is used because its sorbate is 1.128. ionization occurs over pH range defined by
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I II
II
(a) (b)
Figure 3. Chromatogram of benzoic acid (I) and potassium sorbate (II) obtained on HS (High Speed) type LiChroCART® 30-4 HPLC-Cartridge Purospher® STAR RP-18 (30 mm x 4 mm; 3 µm) column; mobile phase methanol/phosphate buffer (pH 3.70) 20/80 (V/V), flow rate 1 mL/min, column temperature 25 ºC and UV detection at 225.4 nm (a) and 255.4 nm (b).
The calibration curve for sodium benzoate and indicated that the peak area is a preferable potassium sorbate are plotted on the basis of variable for further accuracy testing (Table 1). peak areas of chromatograms obtained for The limit of detection (LOD) is determined as different concentrations of sample solutions. signal to noise ratio 3:1, while the limit of The curves followed Lambert-Beer’s law in quantification (LOQ) is determined as signal the concentration range of 30 mg/L, 60 mg/L, to noise ratio 10:1 (Saad et al., 2005). LOQ is 90 mg/L, 120 mg/L and 180 mg/L for sodium referred to the lowest level of the compound benzoate, and 62.6 mg/L, 125 mg/L, 187.1 which can be determined with an acceptable mg/L, 250 mg/L and 374 mg/L for potassium degree of confidence. The obtained values for sorbate. Peak area/peak heights versus LOD and LOQ are given in table 1. According concentration are used to construct calibration to these results, the developed method allows curves. The correlation coefficients (R2) to identify and quantify the preservatives in the obtained concentration range.
Table 1. Statistical evaluation of calibration curves for sodium benzoate and potassium sorbate linearity range LOD LOQ compound regression equation R2 (mg/L) (mg/L) (mg/L) SB y = 8466.90x + 234.04 0.9906 30-180 0.0038 0.0128 (225.4 nm) y = 343.40x + 62.21 0.8893 PS y = 17619.46x + 1407.11 0.9869 62.6-374 0.0030 0.0099 (225.4 nm) y = 1105.81x + 125.91 0.9885
Precision of the method is calculated from the The inter-day (n = 3) and intraday (n = 8) values of retention time, area and height of the repeatability is evaluated, by eight successive peaks present in the chromatograms of the injection of the mixture containing 120 mg/L standard solutions containing both sodium sodium benzoate and 250 mg/L potassium benzoate and potassium sorbate in one day and sorbate (Meyer et al., 1994). three consecutive days (Lough et al., 1996).
Table 2. Relative standard deviation values (RSD, %) of the inter-day and intraday precision sodium benzoate potassium sorbate parameter RSD % RSD % RSD % RSD % inter-day (n = 3) intraday (n = 8) inter-day (n = 3) intraday (n = 8) peak area 0.17 0.22 0.18 0.22 peak height 0.08 1.78 0.06 1.28 retention time 0.09 1.06 0.16 1.36
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Presented data of RSD % calculated using the amounts spiked to a selected sample of retention time, peak area and peak height beverage which contained both analyzed values demonstrate excellent inter-day preservatives (Snyder et al., 1997). Recoveries precision of the used method (RSD 1 %), are evaluated at three different levels of while the intraday precision is satisfactory concentration for SB (18.75 mg/L, 37.50 mg/L when the data of the peak area are used. and 75.00 mg/L) and PS (31.30 mg/L, 62.60 In order to verify accuracy of the analytical mg/L and 125.00 mg/L), corresponding to the procedure, the recovery studies are carried out. minimum, the maximum and an intermediate The accuracy of the procedure is tested using value of the linearity range (Table 3). In cases method of standard additions and expressed as where preservatives in food are analyzed the deviation between calculated mean value recoveries in the range 80 - 110 % are of analyte amounts obtained by the analysis acceptable (AOAC, 2011). and the true (known) value of the analyte
Table 3. Results from recovery studies conducted on three concentration levels (n = 3) mass of analyte pure analyte total analyte recovery RSD compound (g) added (g) found (g) (%) (%) 0.1258 0.0469 0.1715 99.32 0.09 SB 0.1258 0.0938 0.1987 90.50 0.06 (225.4 nm) 0.1258 0.1875 0.2892 92.31 0.15 0.0272 0.0783 0.0960 91.00 1.75 PS 0.0272 0.1565 0.1764 96.05 0.09 (255.4 nm) 0.0272 0.3125 0.2938 86.46 0.16
The presented data suggested that the proposed and injected (2.5 µL) into the HPLC system method is characterized with high repeatability without any pre-treatment steps. Each analysis and it can be used for determination of is repeated in triplicate. The identity of SB and preservatives in beverages. PS is confirmed using the values of the match The developed RP-HPLC method is applied to factor obtained by comparison of the peaks of determine the sodium benzoate and potassium SB (999.929) and PS (999.974) of pure sorbate in real samples under the defined analytical standards with those of the sample. experimental conditions. Seven different The results for determination of sodium samples marked as: A, B, C, D, E, F, G and H benzoate (benzoic acid) and potassium sorbate are purchased randomly from Macedonian (sorbic acid) in seven different samples of market. Samples are prepared simply by beverages are presented in Table 3. filtering through 0.45 µm filters (T-Spartan)
Table 4. Na-benzoate (mg/L) and K-sorbate (mg/L) in different beverages Preservative content Referent values Sample SB PS Total labeled on product [mg/L] [mg/L] [mg/L] [mg/L] A 53.64 26.48 80.12 SB and PS are present* B 50.32 10.88 61.2 SB and PS are present* C 45.64 **ND 45.64 SB max 120 mg/L D 44.88 52.72 97.6 SB and PS max 250 mg/L E 59.44 103.48 162.92 SB and PS max 400 mg/L F 57.16 89.32 146.48 SB and PS max 400 mg/L G 50.60 12.88 63.48 SB (0.12 g/L) and PS (0.036 g/L) *the correct content of SB and PS is not labeled; **ND-not detected
From the results presented in the table 3 it can be seen that the mean concentration of SB and Conclusion PS found in the analyzed beverages is below The developed RP HPLC method is adequate the maximum permitted level. for the determination of sodium benzoate and
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24 M. S. Jankulovska, L. Velkoska-Markovska , B. Petanovska-Ilievska, S. Trpkovska ______potassium sorbate in different beverages with Lough, W. J., Wainer, I. W. (1996). High good analytical performances under the Performance Liquid Chromatography optimized experimental parameters. It is (Fundamental Principles and Practice), Blackie simple, rapid, reliable and allows good Academic and Professional, Chapman and recoveries of benzoates and sorbates in the Hall, Glasgow, UK, Vol. 50, No. 47: pp. 156- concentration range usually present in the 163. beverages. The obtained results showed that Mamur, S., Yüzbaşıoğlu, D., Ünal, F., Yılmaz, the concentration of SB (or BA) and PS (or S. (2010). Does potassium sorbate induce SA) varied between different kinds of genotoxic or mutagenic effects in beverages samples, with levels lower than the lymphocytes? Vol. 3, Toxicology in vitro, No. maximum values established by national and 24, pp. 790-794. international legislation. Meyer, V. (2010). Practical High-Performance Liquid Chromatography, Fifth Edition, John References Wiley & Sons, Ltd. Publication. Bahremand, N., Soheil, E. (2013). Pylypiw, J. H. M., Grether, M. T., (2000). Determination of Potassium Sorbate and Rapid high-performance liquid Sodium Benzoate in "Doogh" by HPLC and chromatography method for the analysis of Comparison with Spectrophotometry, Vol. 3, benzoate and potassium sorbate in foods, J of Int. J. Bio-Inorg. Hybd. Nanomat., No. 2, pp. Chromatogr. A, No. 883, pp. 299-304. 429-435. Ranken, M. D., Kill, R. C., Baker, C. G. J. Dong, C., Mei, Y., Chen, L. (2006). (2005). Food Industries Manual (24th Edition), Simultaneous determination of sorbic and Springer – Verlag. benzoic acids in food dressing by headspace Russell, N. J., Gould, G. W. (2003). Food solid-phase microextraction and gas preservatives, Kluwer Academic/Plenum chromatography, J. Chromatogr. A, No. 1117, Publishers, New York. pp. 109-114. Republic of Macedonia Ministry of Health Dong, M. W. (2006). Modern HPLC for (2005). Regulation on additives that can be Practicing Scientists, Synomics used for food production (in Macedonian). Pharmaceutical Services, LLC, Wareham, Official Gazette of Republic of Macedonia, Massachusetts, Published by John Wiley& 118 LXI/2005. Sons, Inc., Hoboken, New Jersey, published Snyder, R. L., Kirkland, J. J., Glajch, L. J. simultaneously in Canada. (1997). Practical HPLC Method Development, Davidson, P. M., Sofos, J. N., Branen, A. L. 2nd Ed., John Wiley & Sons, Inc., New York, (2005). Food Antimicrobials Third Edition, pp. 688-689. Published by CRC Press, Taylor & Francis Saad, B., Bari, M. F., Saleh, M. I., Ahmad, K. Group, LLC. Talib, M. K. (2005). Simultaneous European parliament and council directive No determination of preservatives (benzoic acid, 95/2/EC on food additives other than colours sorbic acid, methylparaben and propylparaben) and sweeteners, (2005). (OJ No L 61, 18. 3. in food staffs using high-permormance liquid 1995, pp. 1). chromatography, J. Chromatogr. A, No. 1073, Ferrand, C., Marc, F., Fritsch, P., Cassand, P., pp. 393-397. Blanquat, G. S. (2000). Genotoxicity study of Snyder, L. R., Kirkland, J. J., Glajch, J. L. reaction products of sorbic acid, J. Agric. Food (1997). Practical HPLC Method Development, Chem., No. 48, pp. 3605-3610. 2nd Ed., John Wiley & Sons, Inc., New York. Isabel, M. F., Eula, Â. M., Paula, B., Tfouni, S. A. V., Toledo, M. C. F. (2002). Margarida, A. F. (2001). Simultaneous Determination of benzoic and sorbic acids in determination of benzoic and sorbic acids in Brazilian food, Vol. 2. Food Control No. 13, quince jam by HPLC, Food Res. Inter., No. 33, pp. 117-123. pp. 113 - 7. Veerabhadrarao, M., Narayan, M. S., Kapur, Küçükçetin, A., Şik, B., Demir, M. (2008). O., Sastry, C. S. (1987). Reverse phase liquid Determination of sodium benzoate, potassium chromatographic determination of some food sorbate, nitrate and nitrite in some commercial additives, Vol. 3, J. Assoc. Off. Anal. Chem., dairy products, Vol. 4, GIDA, No. 33, pp. 159- No. 70, pp. 578-582. 164. World Health Organization, (2000). Benzoic acid and sodium benzoate. Concise
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25 M. S. Jankulovska, L. Velkoska-Markovska , B. Petanovska-Ilievska, S. Trpkovska ______international chemical assessment, Document No. 26, International Programme on Chemical Safety. Wood, R., Foster, L., Damant, A., Key, P. (2004). Analytical Methods for Food Additives, Published by Woodhead Publishing Limited, Abington Hall, Abington Cambridge CB1 6AH, England.
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Journal of Agricultural, Food and Environmental Sciences UDC 663.813:634.11]:632.95.028:543.544.5.068.7 Original scientific paper ______
DEVELOPMENT OF HIGH SPEED RRLC METHOD FOR QUANTITATIVE DETERMINATION OF SOME PESTICIDE RESIDUES IN APPLE JUICE
B. Petanovska-Ilievska 1*, L.Velkoska-Markovska 1, I. Stojanovska 1, M. S. Jankulovska1
1 Faculty of Agricultural Sciences and food, “Ss. Cyril and Methodius” University, Skopje, Republic of Macedonia
*corresponding author: [email protected], [email protected]
Abstract Possibilities of normal-phase (NP) and reversed-phase (RP) liquid chromatography methods for determination of methomyl, methidathion and propiconazole residues in apple juice were studied. The investigations were carried out on various analytical columns using RRLC (Rapid Resolution Liquid Chromatography) system coupled with UV-Vis diode array detector. The best conditions for separation and quantitative determination of tested pesticides were obtained using reversed-phase mode and Purospher® Star RP-18 endcapped (30 mm × 4 mm; 3 μm) column. Fast and simple method for direct determination of methidathion and propiconazole in different apple juice matrix was developed. In accordance to European Commission regulation the tested parameters for method validation (selectivity, linearity, precision, limit of detection, limit of quantification and accuracy) were satisfied. Key words: direct determination, methidathion, propiconazole, high speed RRLC, apple juice.
Introduction Different technique and detectors are also used Pesticides are highly effective substances used with various sensitivity and repeatability as it in control of pests and vectors of human is ultra-HPLC (UHPLC)/ESI quadrupole (Qq)- diseases. Their application in agriculture time-of-flight (TOF) MS (Wang et al., 2011), enabled increased crops yields and UHPLC-MS/MS (Moreno-González et al., manufacturing of high quality products in 2015), Fourier transform infrared (FTIR), order to satisfy the increasing food demands visible/near-infrared spectroscopy (Vis/NIR) over the world. On the other side the use of (Jamshidi et al., 2015; Guangdong et al., pesticides had caused concerns about their 2015) or HPLC equipped with ultraviolet effects on human health and the environment detector (UVD) (Wenbi et al., 2015; Tuzimski (Jokanović et al., 2012), they have been linked et al., 2016). to a wide range of human health hazards, Because of very low contaminants quantity ranging from short-term impacts such as and complexity of sample matrix, preliminary headaches and nausea to chronic impacts like sample preparation prior GS or LC analyses cancer, reproductive harm, and endocrine are often need. The widely used analytical disruption. methodology combining the As food safety is among the first priorities in extraction/isolation of pesticides from food many countries, there is a need for matrices with extract cleanup are QuEChERS determination of pesticide residues in various (quick, easy, cheap, effective, rugged, and food commodities. Pesticides residues are safe) (Zhao et al., 2012; AOAC Official usually determined by gas chromatography Method 2007.01). Supercritical fluid (GC) or liquid chromatography (LC) (Nollet et extraction (SFE) (Fernandes et al., 2011; al., 2004; Słowik-Borowiec et al., 2015) Boulaid et al., 2007), matrix solid coupled with mass spectrometry (MS) or phase dispersion (MSPD) (Zhou et al., 2015; tandem mass spectrometry (MS/MS) using a Liu et al., 2015), solid-phase microextraction triple quadrupole (QqQ) analyzer (Rosenblum (Blasco et al., 2008) and magnetic solid phase et al., 2001; Hernández et al., 2013; Martínez- extraction (SPE) (del Castillo et al., 2012) are Domínguez et al., 2015; Xiaoli et al., 2015; also used. Páleníková et al., 2015, Takatori et al., 2011;). 27 B. Petanovska-Ilievska, L.Velkoska-Markovska, I. Stojanovska, M. S. Jankulovska ______
The most widely used pesticides over the respectively, of the pure analytical standards in world for apple protection are methidathion acetonitrile in 10 mL volumetric flasks. The (Moura1 et al., 2013), methomyl and solutions were degassed for 15 min in an propionazole, therefore their residues are wary ultrasonic bath and stored in a refrigerator at often present in apples or apple juice. The 4°C. Stock solutions were used to prepare a current status of methidathion under EU series of 6 working solutions with different Regulation (EC) No 1107/2009 is not pesticide concentrations (6 – 30 µg/L for approved for use, against methomyl and methomyl, 9 – 45 µg/L for methidathion and propionazole which have opposite status. 45 – 225 µg/L for propiconazole) in 10 mL However, studies for simultaneous volumetric flask by dilution with the mixture determinations of methomyl, methidathion and of acetonitrile/water (50/50, V/V). In order to propiconazole in apple juice using RRLC construct the calibration plots, 20 μL of each system coupled with UV-Vis diode array working solution were injected in the detector are not available. Therefore the chromatograph three times. The obtained purpose of this paper is to develop a simple chromatograms were analyzed considering and easy for use method for determination of areas and heights of the peaks. all three pesticides in apple juices. Preparation of Sample Solutions Before the analysis, apple juice samples were Materials and methods filtered through 0.45 µm Iso-Disc PTFE The development of the RRLC method for syringe filters (Supelco). Unspiked apple juice quantitative determination of methomyl, sample was used as blank. methidathion and propiconazole residues in The solutions for recovery experiment were apple juice samples were provided on Agilent prepared in three 10 mL volumetric flasks. In 1260 Infinity Rapid Resolution Liquid each volumetric flask was added a known Chromatography (RRLC) system equipped amount of analytes which were corresponding with: vacuum degasser (G1322A), binary to the MRLs, 0.03 mg/L (600 pg) of pump (G1312B), autosampler (G1329B), a methidathion and 0.15 mg/L (3000 pg) of thermostatted column compartment (G1316A), propiconazole) and filled to volume with apple UV-VIS diode array detector (G1316B) and juice. 20µL of each of these solutions was ChemStation software. The separation of injected three times into the LC system. analytes were tested on different analytical columns: HS Pecosphere 3×3 Silica (3.3 cm × Results and discussion 0.46 cm; 3 μm) produced by Perkin-Elmer, In order to achieve the best separation of Hypersil ODS (25 cm × 0.46 cm; 5 μm) the investigated compounds few different produced by Sigma–Aldrich, and Purospher® analytical columns for normal-phase and Star RP-18 endcapped (3 cm × 4 mm; 3 μm) reverse-phase chromatography were tested. produced by Merck. An ultrasonic bath For normal-phase chromatography the polar “Elma” was used for better dissolving of the HS Pecosphere 3×3 Silica (3.3 cm × 0.46 cm; stock solutions. 3 μm) was used. The mobile phase was The Pestanal grade analytical standards of consisted n-hexane/dichloromethane in methomyl, methidathion and propiconazole different volume ratio, and different flow rate. and HPLC-grade acetonitrile were purchased The UV spectra obtained from these by Sigma-Aldrich (Germany). Ultrapure water chromatographic conditions showed maximum was produced by TKA Smart2 Pure 12 UV/UF at 235 nm for methomyl, and 225 nm for water purification system (Germany). methidathion and propiconazole respectively, Various commercial 100 % clear apple juice therefore the investigations were performed at samples from different producers (A, B, C, D both wavelengths. and E) were purchased in Macedonian When HS Pecosphere 3×3 Silica was used supermarkets. with mobile phase in ratio of 40/60 V/V to Preparation of Standard Solutions 0/100 V/V, and flow-rate of 1.2 mL/min to 1.8 Stock solutions of methomyl, methidathion mL/min, at column temperature of 25 ºC, only and propiconazole were prepared by methidathion eluted in a 6 minutes run (Fig. dissolving 0.0030 g, 0.0033 g and 0.0034 g, 1).
JAFES, Vol 67, (2016) 28 B. Petanovska-Ilievska, L.Velkoska-Markovska, I. Stojanovska, M. S. Jankulovska ______
Figure 1. Chromatogram obtained on HS Pecosphere 3×3 Silica (3.3 cm × 0.46 cm; 3 μm) column of a standard mixture containing methidathion (I) methomyl (II), and propiconazole (III); mobile phase n- hexane/dichloromethane 30/70 (V/V), flow rate 1.6 mL/min, temperature 25 ºC and UV detection at 230 nm
Because of methomyl and propiconazole Therefore the further investigations were precipitation on the HS Pecosphere 3×3 Silica providing on both wavelengths. column, the possibility for reverse-phase The best resolution of analytes using Hypersil chromatography was tested on Hypersil ODS ODS column was achieved with a mobile and Purospher® Star RP-18 endcapped phase acetonitrile/water 80/20 (V/V), flow-rate columns. The UV spectra of methomyl, of 1 mL/min and column temperature of 25 ºC. methidathion and propiconazole in Under using chromatography conditions the acetonitrile/water (50/50 V/V) show that tested column shoved unsatisfied peak methomyl has an absorption maximum at 235 symmetry, and peak splitting for methidathion nm, while methidathion and propiconazole (Figure 3). have their absorption maxima at 220 nm.
II
I
III
Figure 2. Chromatogram obtained on Hypersil ODS (25 cm × 0.46 cm; 5 μm) column from a standard mixture containing methomyl (I), methidathion (II) and propiconazole (III); mobile phase acetonitrile/water 80/20 (V/V), flow1 mL/min, temperature 25 ºC and UV detection at 220 nm
The best resolution with sharp and rate of 1mL/min and column temperature of 25 symmetrical peaks for all three pesticides was ºC (Figure 3). achieved on Purospher® Star RP-18 endcapped (30 mm × 4 mm; 3 μm) column, mobile phase consisting acetonitrile/water 50/50 (V/V) flow-
JAFES, Vol 67, (2016) 29 B. Petanovska-Ilievska, L.Velkoska-Markovska, I. Stojanovska, M. S. Jankulovska ______
I
II
III
Figure 3. Chromatogram obtained on Purospher® Star RP-18 endcapped (3 cm × 4 mm; 3 μm) column from a standard mixture containing methomyl (I), methidathion (II) and propiconazole (III); mobile phase acetonitrile/water 50/50 (V/V), flow1 mL/min, temperature 25 ºC and UV detection at 220 nm
The mean value of retention times obtained propiconazole. Because of high sensitivity of under describes chromatography conditions develop method in relation to investigated were: 0.302 min, 1.248 min and 2.130 min for analytes, the tested apples juice were used methomyl, methidathion and propiconazole, without concentration and cleanup procedures. respectively. The chromatogram obtained from apple juice Using the chromatography condition set on spiked with 0.02 mg/L (400 pg) methomyl, Purospher® Star RP-18 column (Fig. 7), the 0.03 mg/L (600 pg) methidathion and 0.15 estimated values for limit of detection (LOD) mg/L (3000 pg) propiconazole (detrmined as signal to noise ratio 3:1) was 0.6 (concentrations corresponding to their EU pg (0.12 μg/L) for methomyl, 22.5 pg (4.5 MRLs)8 showed that methomyl peak μg/L) for methidathion and 30 pg (6 μg/L) for overlap with the coeluting peaks from apple propiconazole. The obtained values for the juice matrix. (Figure 5.). This confirmed that limit of quantification (LOQ) (detrmined as under the given chromatographic conditions, signal to noise ratio 10:1) were: 2 pg (0.4 methomyl can not be determined in this μg/L) for methomyl, 75 pg (15 μg/L) for matrix. methidathion and 100 pg (20 μg/L) for
Figure 4. Chromatogram obtained on Purospher® Star RP-18 endcapped column from apple juice; mobile phase acetonitrile/water 50/50 (V/V), flow1 mL/min, temperature 25 ºC and UV detection at 220 nm
The linearity of the method was tested in methidathion and 0.9898 for propiconazole for different concentrations level up and below to the peak areas and 0.9556 for methomyl, analytes MRL’s (metholmyl in the range 6-30 0.9640 for methidathion and 0.9892 for μg/L, methidathion in the range 9-45 μg/L and propiconazole for the peak heights. These R2 propiconazole in the range 45-225 μg/L) values indicated the peak area as preferable revealed lower values of the correlation variable for methidathion and the peak height coefficients (R2). The calculated R2 values as preferable for propiconazole during were: 0.8180 for methomyl, 0.9528 for linearity testing.
JAFES, Vol 67, (2016) 30 B. Petanovska-Ilievska, L.Velkoska-Markovska, I. Stojanovska, M. S. Jankulovska ______
II
III
Figure 5. Chromatogram obtained on Purospher® Star RP-18 endcapped column from spiked apple juice; methidathion (II) and propiconazole (III); mobile phase acetonitrile/water 50/50 (V/V), flow 1 mL/min, temperature 25 ºC and UV detection at 220 nm
Table 1. Results for linearity of the method
Compound Regression equation R2
1y = 0.0017x + 0.5331 0.8180 Methomyl (235 nm) 2y = 0.0007x + 0.1193 0.9556 1y = 0.0015x + 0.0612 0.9528 Methidathion (220 nm) 2y = 0.0004x + 0.0113 0.9640 1y = 0.0007x - 0.2216 0.9898 Propiconazole (220 nm) 2y = 9E-05x - 0.0143 0.9892 1y = peak area; 2y = peak height
The precision was expressed as repeatability of μL) of the mixture containing 400 pg obtained results which was evaluated for peak methomyl, 600 pg methidathion and 3000 pg areas, peak heights and retention times of the propiconazole. analytes from eight successive injections (20
Table 2. Statistical data for precision of the method Intra-day repeatability (n = 8)
Retention time Peak height Peak area RSD RSD RSD Compound x SD SD SD (%) (%) (%) Methomyl 0.9595 0.0190 1.98 0.3426 0.0057 1.65 0.3049 0.0004 0.12 Methidathion 1.1644 0.0473 4.06 0.2912 0.0062 2.13 1.22 0.0019 0.15 Propiconazole 1.7255 0.0873 5.06 0.2244 0.0052 2.31 2.0815 0.0061 0.29
The accuracy was tested by the method of SANCO Guidance document on analytical standard additions. Three apple juice samples quality control and validation procedures for were spiked with 600 pg methidathion and pesticide residues analysis in food and feed. 3000 pg for propiconazole. The calculated Using developed method no residues of values for analyte recovery ware 98.82 % with methidathion and propiconazole were found in 2.51 % of RSD and 76.03 with 6.36 % of all tested clear apple juice samples (A, B, C, D RSD. The obtained values for recovery are in and E) taken from Macedonian markets. accordance with method performance Conclusions acceptability criteria (70-120 %) given by
JAFES, Vol 67, (2016) 31 B. Petanovska-Ilievska, L.Velkoska-Markovska, I. Stojanovska, M. S. Jankulovska ______
The develop method described in this paper is residue analysis in food and the environment, suitable for direct qualitative and quantitative Anal. Methods, Vol. 5, pp. 5875-5894. determination of methidathion and Guidance document on analytical quality propiconazole in 100 % clear apples juices. control and validation procedures for pesticide Due to strong matrix effect under developed residues analysis in food and feed. chromatography condition there was no SANCO/12571/ 2013 pp. 20-26. possibility for determination of methomyl in Jamshidi, B., Mohajerani, E., Jamshidi, J., apples sample. The proposed method is fast, Minaei, S., Sharifi, A., (2015). Non- simple, linear, precise and sufficiently accurate destructive detection of pesticide residues in for quantitative and qualitative determination cucumber using visible/near-infrared of methidathion and propiconazole in apple spectroscopy, Food Additives & juice. The obtained values for all tested Contaminants: Part A, Vol. 32, No. 6, pp. 857- parameters are in accordance with SANCO 863. Guidance document on analytical quality Jokanović, M., (2012). The Impact of control and validation procedures for pesticide Pesticides Published First Edition by residues analysis in food and feed. Academy Publish. org, USA pp.19. Liu, H., Kong, W., Gong, B., Miao, Q., Qi, Y., References Yang, M., (2015). Rapid analysis of multi- Blasco, C., Font G., Picó, Y., (2008). Solid- pesticides in Morinda officinalis by GC-ECD phase microextraction-liquid chromatography- with accelerated solvent extraction mass spectrometry applied to the analysis of assisted matrix solid phase dispersion and insecticides in honey, Food Additives & positive confirmation by GC-MS. J Contaminants: Part A, Vol. 25, No. 1, pp. 59- Chromatogr B Analyt Technol Biomed Life 69. Sci., Vol. 1, No. 974, pp. 65-74. Boulaid, M., Aguilera, A., Busonera, Martínez-Domínguez, G., Nieto-García, A. J., V., Camacho, F., Monterreal, A. V., Valverde, Romero-González, R., Garrido Frenich, A., A., (2007). Assessing supercritical fluid (2015). Application of QuEChERS based extraction for the analysis of fipronil, method for the determination of pesticides in kresoxim-methyl, acrinathrin, and nutraceutical products (Camellia sinensis) by pyridabenresidues in melon. J Environ Sci liquid chromatography coupled to triple Health B., Vol. 42, No. 7, pp. 809-815. quadrupole tandem mass spectrometry, Food del Castillo, M. L., Rodriguez-Valenciano, Chemistry, Vol. 177, No. 15, pp. 182–190. M., de la Peña, Moreno, F., Blanch, G. P., Moreno-González, D., Huertas-Pérez, F. (2012). Evaluation of pesticide residue J., Gámiz-Gracia, L., García-Campañ, A. M., contents in fruit juice by solid- (2015). High-Throughput Methodology for the phase microextraction and multidimensional Determination of 33 Carbamates in Herbal gas chromatography coupled with mass Products by UHPLC–MS/MS, Food spectrometry. Talanta, Vol. 30, No. 89, pp. Analytical Methods, Vol. 8, No. 8, pp. 2059- 77-83. 2068. Fernandes, V. C., Domingues, V. F., Mateus, Moura1, P., Alexandre, C., Geraldo, A., N., Delerue-Matos, C., (2011). Determination Botton M., (2012). Residual effect of of Pesticides in Fruit and Fruit Juices by pesticides used in integrated apple production Chromatographic Methods. An Overview, on chrysoperlaexterna (hagen) (neuroptera: Journal of Chromatographic Science, Vol. 49, chrysopidae) larvae, chilean journal of pp. 715-730. agricultural research 72(2) april-june, Guangdong, X., Daming, D., Tongqing, L., http://www.scielo.cl/scielo.php?script=sci_artt Yang L., Ling Z., Dongyan Z., Chunjiang Z., ext&pid = S0718-58392012000200009. (2015). Detection of Pesticide (Chlorpyrifos) Nollet, Leo M. L., 2004, Handbook of Food Residues on Fruit Peels Through Spectra of Analysis: Residues and other food component Volatiles by FTIR, Food Analytical Methods, analysis, Marcel Dekker, Inc, New York, May, Vol. 8, No. 5, pp. 1341-1346 USA, pp. 1187 Hernández, F., Cervera, M. I., Portolés, T., Páleníková, A., Martínez-Domínguez, G., Beltrán, J., Pitarch E., (2013). The role of GC- Javier Arrebola, F., Romero-González, R., MS/MS with triple quadrupole in pesticide Hrouzková, S., Garrido Frenich, A., (2015). Occurrence of pesticide residues and
JAFES, Vol 67, (2016) 32 B. Petanovska-Ilievska, L.Velkoska-Markovska, I. Stojanovska, M. S. Jankulovska ______transformation products in different types of Samples, Food Analytical Methods, Vol. 8, dietary supplements, Food Additives & No. 9, pp. 2202-2210. Contaminants: Part A, Vol. 32, No. 6, pp. 849- Xiaoli, C., Suhua, L., Xiaoqian, Y., Zhong, L., 856. Lanzheng, L., (2015). A Modified QuEChERS Pesticide Residues in Foods by Acetonitrile Sample Preparation Method for Simultaneous Extraction and Partitioning with Magnesium Determination of 62 Pesticide Residues in Sulfate Gas Chromatography/Mass Edible Fungi Using Gas Chromatography– Spectrometry and Liquid Triple Quadrupole Mass Spectrometry, Food Chromatography/Tandem Mass Spectrometry, Analytical Methods, Vol. 14, pp 1-12 AOAC Official Method 2007.01, Zhao, L., Shultz, D., Stevens, J., (2012). p://www.weber.hu/PDFs/QuEChERS/AOAC_ Analysis of pesticide residues in apple using 2007_01.pdf Agilent bond elut QuEChERS AOAC kits by Rosenblum, L., Hieber, T., Morgan, J., (2001). GC/MS, Agilent technologies, pp. 1-13, Determination of pesticides in composite https://www.agilent.com/cs/library/application dietary samples by gas chromatography/mass s/5990-4068EN.pdf spectrometry in the selected ion monitoring Zhou, M., Hu, N., Shu, S., Wang, M., (2015). mode by using a temperature-programmable Molecularly Imprinted Nanomicrospheres as large volume injector with preseparation Matrix Solid-Phase Dispersant Combined with column. J AOAC Int., Vol. 84, No. 3 pp. 891- Gas Chromatography for Determination of 900. Four Phosphorothioate Pesticides in Carrot Słowik-Borowiec, M., (2015). Validation of a and Yacon J Anal Methods Chem., Published QuEChERS-Based Gas Chromatographic online, Method for Multiresidue Pesticide Analysis in http://www.ncbi.nlm.nih.gov/pmc/articles/PM Fresh Peppermint Including Studies of Matrix C4411505/ Effects, Food Analytical Methods, Vol. 8, No. 6, pp. 1413-1424. Takatori, S., Okihashi, M., Kitagawa, Y., Fukui, N., Kakimoto, O., Obana, H., (2011). Rapid and Easy Multiresidue Method for Determination of Pesticide Residues in Foods Using Gas or Liquid Chromatography– Tandem Mass Spectrometry, Pesticides - Strategies for Pesticides Analysis, chapter 8, 197-214, Edited by Prof. Margarita Stoytcheva Publisher: InTech http://www.intechopen.com/books/pesticides- strategies-for-pesticides-analysis/rapid-and- easy-multiresidue-method-for-determination- of-pesticide-residues-in-foods-using-gas-or-liq Tuzimski, T., Rejczak, T., (2016). Application of HPLC–DAD after SPE/QuEChERS with ZrO2-based sorbent in d-SPE clean-up step for pesticide analysis in edible oils, Food Chemistry, Vol. 190, pp. 71–79. Wang J., Chow W., Leung D., (2011). Applications of LC/ESI-MS/MS and UHPLC/Qq-TOF-MS for the determination of 141 pesticides in tea, J AOAC Int., Vol. 94, No. 6, pp. 1685-714. Wenbi, G., Zhen, L., Jiangyu, L., Yifan, H., Yongqiang, M., Hongyan, Z., (2015). Unique Graphitic Carbon Nitride Nanovessels as Recyclable Adsorbent for Solid Phase Extraction of Benzoylurea Pesticides in Juices
JAFES, Vol 67, (2016) Journal of Agricultural, Food and Environmental Sciences UDC 635.546.173/.175]:543.544.5.068.7 Original scientific paper ______
DEVELOPMENT OF HPLC METHOD FOR ANALYSIS OF NITRITE AND NITRATE IN VEGETABLE
A. Najdenkoska*
Institute of Public Health, 50 Divisia 6, 1000, Skopje, Republic of Macedonia
*corresponding author: [email protected]
Abstract The presence of nitrates and nitrites in food is associated with an increased risk of gastrointestinal cancer and, in infants, methemoglobinemia. Approximately 80% of dietary nitrates are derived from vegetable consumption; sources of nitrites include vegetables, fruit, and processed meats. Nitrate may be reduced into cancerogenic nitrite under various condition. In this work development of a simple HPLC method for simultaneous analysis of nitrite and nitrate was performed. The retention of nitrate and nitrite on the sorbent RP- 18 endcapped was studied under the conditions of ion-pair high-performance liquid chromatography, depending on the concentration of organic solvent (methanol) in the mobile phase, pH of the phosphate buffer solution and flow of mobile phase. The optimum conditions were selected for the separation of nitrite and nitrate in the isocratic elution mode within 15 min. A procedure was developed for their determination in vegetable; the detection limit was 50 mg/L for nitrite and 25 mg/L for nitrate. The method is relatively simple, sensitive, selective and can be applied to the monitoring of nitrite and nitrate in vegetable products. Key words: HPLC, vegetables, nitrate, nitrite
Introduction spectrophotometry, high performance The simultaneous determination of nitrite liquid chromatography, ion and nitrate in different matrices is of vital chromatography, polarographic methods significance due to their toxicity to and capillary electrophoresis (S-S. Chou, infants and causing methemogloninemia. 2003). Considering the physicochemical Highly cancerogenic N-nitrozamines properties of nitrite and nitrate there is could be formed by the reaction of nitrite possibility to use liquid chromatography with secondary amines and amides, for their accurate and precise which are generally present in food determination. In this work Reverse items, such as meat and fish, where Phase Ion Pair Chromatography and nitrite is used as preservatives (Bilal Butt method of suppression of ion is used. at al., 2001). Other potential sources of Because stationary phase is non-polar it nitrite contamination are wastewater, could not be used for separation of vegetables and processed food. A variety charged analytes, so their separation is of analytical methods for determination possible by introducing ion pair reagent of nitrite and nitrate have been developed to obtain molecular form of analytes, and applied to the analysis of food. shown on Figure 1. These methods include 34 A. Najdenkoska ______
Figure1. Interaction of Ion Pair Reagent with analyte
Materials and methods (aqueous phase is 0.01M octylamine Chemicals prepared by dissolving of 1.29 g in 900 All solutions were prepared with mL deionized water and adjust pH to 7 distillated deionized water and all with 10 % phosphoric acid and fill up to chemicals were of analytical reagent 1L). grade. Octylamine used as ion-pair Sample preparation reagent was obtained from Supelco. Samples of spinach and lettuce were Methanol and standards sodium nitrite homogenized in electrical blender. 5g of and nitrate were supplied from Merck. samples was diluted with 100 mL Standard stock solution was 1000 mg/L extraction solution of methanol: water (1 for each anion and was prepared together : 1, V/V), (J Salomez and G Hofman by dissolving appropriate amount of (2002)). It was ultrasonically these compounds in deionized water. The homogenized. Extract was passed working solutions were prepared by through a 0.45 m Milipore syringe and serial dilution of stock solution. then through a RP-18e column for Instrumentation separation and quantification of nitrite A Perkin-Elmer HPLC system employed and nitrate. for this work was fitted with detector UV–Diode Array 235C, pump, Results and discussion autosampler and column oven Series Nitrates and nitrites are ionized 200. Analytical balance Sartorius, compounds. As using ion-pair reagent ultrasonic bath Fisherbrand FB 11021, which increase retention time and pH meter WTW 330 Weilheim and ultra- improve separation of charged analytes ware KONTES system for filtration of isocratic elution mode was adopted. At mobile phase were used. Brand the beginning of this work different transferpettor (1 - 5 mL) and Socorex (10 stationary phases were employed (RP-8, – 100 L) were used for precise RP select B and RP-18). When stationary measurement. phase RP-8 is used 22 minutes for Mobile phase determination of nitrates was needed The mobile phase was consisted from (Fig 2). aqueous 10% methanol phase (V/V),
JAFES, Vol 67, (2016) 35 A. Najdenkoska ______
Figure 2. Chromatogram of nitrate standard (50 mg/L) on RP-8 stationary phase.
As shown on Figure 3 under the same condition there is not precise answer for nitrite, two peaks were appeared.
Figure 3. Chromatogram of nitrite standard (50 mg/L) on RP-8 stationary phase.
Further analyses were towards changing phase where the shape of the nitrite peak of pH of mobile phase in range from 4 to was the same when RP-8 column was 7. Different retention time for nitrate was used. Retention time for nitrate was achieved by changing of pH. There was shorter on RP select B stationary phase. not precise peak for nitrite, so octyl As a result of decreasing of pH, retention phase RP-8 is not suitable for time was increased shown on Figure 4. determination of nitrite. It was confirmed Considering the retention time neutral by employed of RP select B stationary pH was selected as optimal.
JAFES, Vol 67, (2016) 36 A. Najdenkoska ______
25
20
15
tr (min) tr 10
5
0 3 4 5 6 7 8 pH
Figure 4. Effects of pH of mobile phase on retention time for nitrate
In the next step of this work octadecil (S-S. Chou, 2003). Under these RP-18 column was used. It is more conditions chromatogram shown on hydrophobic phase compared with RP-8 Figure 5 was obtained, where peaks of and RP select B. Applied ratio of nitrite and nitrate were overlapped. aqueous/organic phase was 70:30, V/V
Figure 5. Chromatogram of mixture of nitrite and nitrate standard C = 50 mg/L (aqueous 30 % methanol phase, V/V).
Unstable retention time was because of organic and water phase was changed. presence of free silanol groups as well as There is possibility to use high of neutral pH of mobile phase and high concentration of water in mobile phase percent of water phase in mobile phase. when ion-pair chromatography is used То achieve stable retention time in and from the other side to obtain stable further examination octadecil RP-18 retention time. Increasing the endcapped column was used. With aim concentration of water phase lead to to obtain better separation, ratio of better separation, shown on Figure 6.
JAFES, Vol 67, (2016) 37 A. Najdenkoska ______
Figure 6. Chromatogram of mixture of nitrite and nitrate standards (50 mg/L) (aqueous 20% methanol phase, V/V)
Optimal condition of mobile phase for % methanol, (V:V), Flow 0.8 mL/min. separation of nitrite and nitrate were Under these conditions chromatogram selected and they are: aqueous phase 10 shown on Figure 7 was obtained.
Figure 7. Chromatogram of mixture of standards nitrite and nitrate (50 mg/L) (aqueous 10 % methanol phase, V/V).
Total retention time was 14.34 minutes next Figure an increase of flow causes for both compounds. As shown on the decrease of the retention time.
JAFES, Vol 67, (2016) 38 A. Najdenkoska ______
Figure 8. Effects of flow of mobile phase on retention time
With further increasing of water phase conditions for separation of nitrate and up to 95 % better resolution was nitrite are selected and presented on obtained, but retention time of nitrate Table 1. was longer for four minutes. Optimal
Table 1. HPLC conditions for simultaneous separation of nitrite and nitrate Stacionary Phase Purospher RP -18 endcapped, (250 x 4.6mm), 5µm Mobile Phase methanol (10 %) - 0.01M octylamine (90 %) , isocratic mode pH 7 Injection V 10L Wavelengh 210 nm Flow 1mL/min T 30 0C
UV spectra of nitrite and nitrate shown presence in vegetables. The detection on Figure 9 were recorded for further limit was 50 mg/L for nitrite and 25 identification and confirmation of their mg/L for nitrate.
JAFES, Vol 67, (2016) 39 A. Najdenkoska ______
Figure 9. Wavelength scans of standards of nitrite and nitrate
Conclusion S. De Martin, P.Restani, (2003) The method reported herein is relatively "Determination of nitrates by a novel ion simple, sensitive, selective, quick and chromatographic method: occurrence in can be applied to the monitoring of leafy vegetables organic and nitrite and nitrate in vegetable products. conventional and exposure assessment Higher percent of water in mobile phase for Italian consumers”, Food Additives and more hydrophobic stationary phase and Contaminants, Vol.20,No.9, pp.787- were adopted for separation of these 792 components. Considering the retention R.Walker, (1990),“Nitrates, nitrites time neutral pH was selected as optimal. and N-nitrozocompounds: a rewiew of the occurrence in food and diet and the References toxicological implications”, food Addit. S. Bilal Butt, M Riaz, M. Zafar Iqbal, Contam. pp. 717-768; (2001), Simultaneous determination of A. Najdenkoska, (2005) Development of nitrite and nitrate by normal ion-pair HPLC method for determination of liquid chromatography, Talanta 55, 789- nitrite and nitrate, Specialist thesis. 797; S. S. Chou, J-C Chung, (2003), A High Performance Liquid Chromatography Method for Determining Nitrite and Nitrate Levels in Vegetable, Journal of Food and Drug Analysis, Vol 11, No 3, p 233-238; J. Salomez, G.Hofman, (2002) "Nitrate Extraction from Fresh Plant Material by Means of a Methanol : Water Extraction Solution”, Communications in soil science and plant analysis, Vol.33, pp.3397-3404;
JAFES, Vol 67, (2016) Journal of Agricultural, Food and Environmental Sciences UDC 664.664:664.236 Original scientific paper ______
NUTRITIONAL VALUES OF PSEUDOCEREALS FLOUR MIXTURES USED IN GLUTEN-FREE SWEET BAKERY PRODUCTS
I. M. Loredana *, T. T. Ioan, S. Daniela, R. Bogdan
Faculty of Food Processing Technology, Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I of Romania”, 119 Calea Aradului, 300645 Timisoara, Romania
*corresponding author: [email protected]
Abstract Coeliac disease is an autoimmune disorder caused by a reaction of the body’s immune system to prolamins found in wheat (gliadin), rye (secalin), barley (hordein) and their crossbreeds. At present, the only effective treatment for coeliac people is a lifelong gluten-free diet, but it is difficult to follow such a diet, due to the many gluten containing products excluded. Starting from these considerations, this study aimed to investigate some nutritional parameters, in order to support the use of pseudocereal gluten-free flour mixtures (amaranth, buckwheat and quinoa, respectively) along with rice flour, for obtaining gluten-free sweet bakery products, with a high nutritional value and acceptability. Thus, each sort of flour was used in various amounts of 10%, 20% and 30%, respectively, along with rice flour, in order to obtain the best gluten-free sweet products (muffins), regarding its nutritional value and acceptability score. Gluten-free sweet products were prepared by the same recipe, in three trials each, in which rice flour and amaranth, buckwheat and quinoa flour, respectively, varied: T1-RF:AF(90:10), T2-RF:AF(80:20), T3-RF:AF(70:30), T4-RF:BWF(90:10), T5-RF:BWF(80:20), T6-RF:BWF(70:30), T7-RF:QF(90:10), T8-RF:QF(80:20), T9-RF:QF(70:30). A blank sample of the sweet gluten-free bakery product was prepared by using rice flour only, as basis. Key words: Gluten-free sweet bakery products, pseudocereals flours, quality indices.
Introduction gluten-free bakery products suitable to Celiac disease known as gluten sensitive consumer’s needs, in order to increase his enteropathy, is a chronic disorder of the small dietary choices and improve quality of life in intestine caused by the exposure to gluten general (Kupper C., 2005). proteins from wheat, barley, rye and oat (Tye- Manufacturing bakery products without gluten Din et al., 2010 ), to genetically predisposed is a great challenge for science people and a individuals, both children and adults (Fasano major problem for bakers nowadays. In recent et al., 2013). In susceptible individuals, the years, the interest on gluten-free bakery ingestion of gluten leads to the damage of the products has been increasing. Thus, science mucosal surface of the small intestine (Catassi people have searched an alternative to classic C., Fasano A, 2008), which causes the wheat or other gluten containing flours, by malabsorbtion of nutrients in the human body. using pseudocereals or legume flours that are Celiac disease remains largely unrecognized, gluten-free and rich in proteins, for the affecting 1% of most populations, in spite of nutritional quality of the product (Alvarez- the advances made in it’s diagnosis (Rubio- Jubete et al., 2009). Tapia A., Murray J.A., 2010). Once diagnosed, Rice flour is among the most used raw a celiac patient must follow a life-long gluten- material for gluten-free bakery products free diet, which prevents morbidity and manufacturing, low in fat and protein content, reduces the incidence of the associated it is recommended for soft, doughs for gastrointestinal affections (Kupper C., 2005). muffins, cakes or biscuits. Bakery industry occupies an important place Pseudocereals, that is amaranth (Amaranthus in the framework of consumer goods cruentus L.), buckwheat (Fagopyrum manufacturing, due to the fact that bread is a esculentum Moench.) and quinoa daily consumed staple food, which results in (Chenopodium quinoa Willd.), exhibit a high an emerging need for the development of new quality nutritional profile, being an important 41 I. M. Loredana, T. T. Ioan, S. Daniela, R. Bogdan ______source of protein and amino acids, vitamins, Three trials were performed, for each sort of starch and fibre (Steadman KJ et al, 2001; pseudocereal flour, in which various blends of Alvarez-Jubete et al., 2009). gluten-free flours were used, that is: (RF:AF): Starting from these considerations, this paper T1-90:10, T2-80:20, T3-70:30; (RF:BWF) : aims to investigate some nutritional T4-90:10, T5-80:20, T6-70:30; (RF:QF): T7- parameters, in order to support the use of 90:10, T8-80:20, T9-70:30. Similarly, control pseudocereal gluten-free flour mixtures gluten-free muffins samples were prepared (amaranth, buckwheat and quinoa, from rice flour only. respectively) along with rice flour, for After being kneaded, the dough was placed in obtaining gluten-free sweet bakery products, small paper cups for baking. The optimum with a high nutritional value and acceptability. parameters of the technological process were: kneading – 15 minutes at high speed, baking Materials and methods for 20 minutes/180ºC. After baking, the Materials muffins were cooled at room temperature, and All raw materials used in these experiments put into paper bags. have been purchased from specialized stores. Sensory and physical-chemical evaluation of gluten-free muffins Methods Gluten-free muffins samples prepared Analytical methods applied to flours according to the protocol described in The analytical methods used in experiments paragraph 2.2.2. were subjected to sensory and for determinating the quality indices of rice physico-chemical evaluation, aiming: aspect and pseudocereals flours have been the and shape, colour, taste, flavour and softness following: Moisture content (%) SR-877- (according to STAS 91-2007: “Bread, loaf 1996; Protein content (%)- STAS 90-2007; products and bakery specialities. Analysis Lipids (%) STAS -90-2007; Water absorption methods”). The sensory analysis was (%) and Ash content (%) STAS-90-2007, performed by using the Romanian scoring respectively. scheme.
Technological process for obtaining gluten- Results and discussion free muffins The common technological process was used Quality of rice flour, pseudocereals flours and for gluten-free muffins manufacture. The their blends recipe used was the following one: gluten free The nutritional parameters of gluten-free flour blend 400 g, corn starch 100 g, xanthan flours and their mixtures, used in the gum 10 g, milk 450 mL, oil 350 mL, eggs 300 experiments, are shown in Table 1. g, sugar 250 g, baking powder 10 g and salt 5 g.
Table 1. Quality indices of gluten-free flours and their blends used in gluten-free muffins manufacture Experimental values Quality indices Moisture Lipids Protein Ash Water (%) (%) (%) (%) absorption (%) RF100% 12.88 2.46 5.29 1.47 50.55 AF100% 9.94 4.98 13.92 3.14 48.44 BWF100% 11.77 2.40 11.21 1.89 49.20 QF100% 11.90 6.22 14.19 2.35 48.05 RF90%AF10% 12.91 2.50 6.21 2.13 49.35 RF80%AF20% 12.22 3.05 6.23 2.64 49.09 RF70%AF30% 11.96 3.57 7.46 1.82 48.82 RF90%BWF10% 12.36 2.24 6.05 1.58 50.25 RF80%BWF20% 12.46 2.39 6.11 1.59 50.02 RF705BWF30% 12.50 2.45 7.4 1.71 49.22 RF90%QF10% 12.69 2.12 6.17 1.62 49.45 RF80%QF20% 12.60 3.07 6.22 1.69 49.12 RF70%QF30% 12.44 3.61 7.49 1.73 48.62
JAFES, Vol 67, (2016) 42 I. M. Loredana, T. T. Ioan, S. Daniela, R. Bogdan ______
By comparing the values obtained for the Quinoa flour may be characterized analysed samples (RF 100%, AF 100%, BWF with having the highest protein 100%, QF 100%, RF90%AF10%, content (14.19%) and lipids content RF80%AF20%, RF70%AF30% (6.22%), respectively; RF90%BWF10%, RF80%BWF20%, Rice flour exhibits the highest water RF70%BWF30%, RF90%QF10%, absorption (50.55%) and the lowest RF80%QF20%, RF70%QF30%) it can be ash content (1.47%) amongst the stated that these flours can be used in gluten- gluten-free flours. free baking, due to the fact that they are gluten-free mainly, but also for their Sensory evaluation of gluten-free muffins nutritional value. The use of well-proportioned blends of gluten- Summarizing the data presented in Table 1, free flours (rice flour and pseudocereals flours, regarding the quality indices and chemical respectively) led to the obtaining of final composition of rice and buckwheat flour and products with optimum sensory characteristics, their blends, the following assessments can be in accordance with STAS 1227-3/1990. made: Sensory evaluation of the assortments of The water absorption of the flour gluten-free muffins obtained in the “Milling blends increased with increasing and Baking Technology” Laboratory of protein contents; Faculty of Food Processing Technology of The moisture content of flour blends Banat’s University of Agricultural Sciences decreased with rice flour proportion and Veterinary Medicine “King Michael I of increasing; Romania” from Timisoara, Romania, was Amaranth flour is rich in protein performed using the points scale method, (13.92%) exhibiting the highest ash which accordingly, they have obtained the content (3.14%) among the gluten-free following qualifications, presented in Tables 2, flour blends; 3, and 4, respectively. The evaluation was Buckwheat flour, with a low lipids carried out by 10 tasters, untrained and not content (2.40%) comes second celiac people. regarding it’s protein content (11.21%);
Table 2. Scores assigned to gluten free amaranth flour added muffins Sample Maximum Scores obtained score Control muffins AF10% Muffins AF20% Muffins AF30% Muffins Aspect and 6 6 5.80 5.50 5.75 shape Colour 2 2 2 1.80 1.70 Taste 7 6.50 6.75 6.60 6.40 Flavour 3 3 3 2.70 2.60 Softness 2 2 2 1.90 1.80 Maximum 20 19.50 19.60 19.20 19.00 score average
Table 3. Scores assigned to gluten free buckwheat flour added muffins Sample Maximum Scores obtained score Control muffins BWF10% Muffins BWF20% Muffins BWF30% Muffins Aspect and 6 6 5.5 5.80 5.70 shape Colour 2 2 1.70 2 1.70 Taste 7 6.50 6.15 6.70 6.40 Flavour 3 3 2.60 2.70 2.60 Softness 2 2 1.70 1.80 1.90 Maximum 20 19.50 17.90 19.15 18.50 score average
JAFES, Vol 67, (2016) 43 I. M. Loredana, T. T. Ioan, S. Daniela, R. Bogdan ______
Table 4. Scores assigned to gluten free quinoa flour added muffins Sample Maximum Scores obtained score Control muffins QF10% Muffins QF20% Muffins QF30% Muffins Aspect and 6 6 5 5.70 5.60 shape Colour 2 2 1.80 2 2 Taste 7 6.50 7 7 6 Flavour 3 3 3 3 2.60 Softness 2 2 2 2 1.90 Maximum 20 19.50 18.80 19.7 19.6 score average
Summarizing the data presented in Table 2-4, Among the studied samples, the 10% and 20% it can be said that the samples sensory quinoa flour added muffins reached maxium evaluated (the ten assortments of gluten-free score regarding taste, flavour and colour muffins) fit in the first two categories of (Table 4), while 10% amaranth gluten-free quality (“very good” and “good”), but in terms muffins reached the highest score (of 2 points) of sensory analysis, the 20% quinoa flour for their softness (Table 2) and the 20% muffins, 10% amaranth flour muffins and 20% buckwheat flour added muffins gained buckwheat flour muffins, respectively, are maximum score for their colour - 2 points out best, by reaching a score of 19.70, 19.60 and of 2 (Table 3) . 19.15 points, respectively.
Control muffins
Aspect and shape 10 10%AF Softness Colour Muffins 0 Flavour Taste 20%AF Muffins 30%AF Muffins
Figure 1. Spider diagram for sensory evaluation of gluten-free amaranth flour added muffins
Aspect and shape 10
5 Control muffins Softness Colour 10%BWF Muffins 0 20%BWF Muffins 30%BWF Muffins Flavour Taste
Figure 2. Spider diagram for sensory evaluation of gluten-free buckwheat flour added muffins
JAFES, Vol 67, (2016) 44 I. M. Loredana, T. T. Ioan, S. Daniela, R. Bogdan ______
Aspect and shape 10
5 Control muffins Softness Colour 10%QF Muffins 0 20%QF Muffins 30%QF Muffins Flavour Taste
Figure 3. Spider diagram for sensory evaluation of gluten-free quinoa flour added muffins
Physical-chemical evaluation of gluten-free samples were subjected to physico-chemical muffins with pseudocereals flour added analysis. The experimental results obtained in After the sensory evaluation, the gluten free this study, are given in figure 4. muffins with pseudocereals flour added
45 40 35 Moisture (%) 30 25 Acidity (degrees/100g product) 20 Protein (%) 15 10 Lipids (%) 5 0
RF100%
RF90%AF10%RF80%AF20%RF70%AF30% RF90%QF10%RF80%QF20%RF70%QF30% RF90%BWF10%RF80%BWF20%RF70%BWF30%
Figure 4. Gluten-free muffins with pseudocereals flour added physical-chemical features variation
From data presented in Figure 4, it can be seen Figure 4 reveals that sample RF70%QF30% that gluten-free muffins moist is lowest in the muffins exhibited the highest lipids content buckwheat flour added samples (24.85% - (22.14%), while RF90%BWF10% sample had RF90%BWF10%), increasing gradually in the lowest lipids content (13.42%). control samples (32.61%), quinoa flour added muffins (33% - RF70%QF30%) and reaching Conclusions the maximum score in amaranth flour added The results obtained in this study indicate that muffins (41.06% - RF70%AF30%). the addition of pseudocereals flours can be The protein content of the gluten-free successfully used in gluten free baking, in pseudocereals flour added muffins increases order to obtain an end product – gluten free with the acidity decreasing, reaching it’s muffins in this case, with an enhanced maximum score in RF70%BWF30% sample nutritional value and a high acceptance by the (8.89%), while the minimum acidity of consumer. Thus, according to this study 1grades was recorded in RF90%BWF10%. obtained data, the optimum amount to be This increased acidity in gluten free QF added added in order to obtain good gluten-free muffins, leads to a higher instability to storage muffins, is: 20% quinoa flour, 10% amaranth comparative to the other assortments of gluten flour and 20% buckwheat flour, respectively. free muffins (Figure 4). The recommended recipe, following the results obtained in this study, is: gluten-free
JAFES, Vol 67, (2016) 45 I. M. Loredana, T. T. Ioan, S. Daniela, R. Bogdan ______flour blends 400 g, corn starch 100 g, xanthan Tie-Din JA, Stewart JA, Dromey JA, gum 10 g, milk 450 mL, oil 350 mL, eggs 300 Beissbarth T, van Heel DA, Tatham A, g, sugar 250 g, baking powder 10 g and salt 5 Henderson K, Anderson RP (2010). g; kneading – 15 minutes at high speed, baking Comprehensive, quantitative mapping of T for 20 minutes/180ºC. cell epitopes in Gluten in celiac disease. Sci Transl Med 2(41):41-51. Acknowledgements: This study was carried out with financial support as part of the project being co-financed from the European Social Fund through Sectoral operational programme human resources development 2007-2013. Contract POSDRU/159/1.5/S/132765.
References Alvarez-Jubete L, Arendt EK, Gallagher E (2009) Nutritive value and chemical composition of pseudocereals as gluten-free ingredients. Int J Food Sci Nutr 60(suppl 4):240–257 Alvarez-Jubete L, Holse M, Hansen A, Arendt EK, Gallagher E (2009). Impact of baking on the vitamin E content of the pseudocereals amaranth, quinoa and buckwheat. Cereal Chem 86(5):511–515 Alvarez-Jubete L, Wijngaard HH, Arendt EK, Gallagher E (2010). Polyphenol composition and in vitro antioxidant activity of amaranth, quinoa and buckwheat as aVected by sprouting and bread baking. Food Chem 119:770–778 Catassi C., Fasano A. (2008). Celiac Disease, Gluten Free Cerel Products and Beverages,; pp. 1-27 Fasano, A., Berti, I., Gerarduzzi, T., Al., E. (2013). Prevalence of Celiac Disease in At- Risk and Not-At-Risk Groups in the United States. Arch. Intern. Med. 163, 286–292. Kupper C., Dietary guidelines and implementation for celiac disease Gastroenterology. 2005 Apr; 128(4 Suppl 1):S121-7 Mandala I., Kapsokefalou M. (2011). Gluten- Free Bread: Sensory, Physicochemical and nutritional aspects. Flour and Breads and their Fortifications in Health and Disease Prevention, DOI: 10.1016/B978-0-12-380886- 8.10015-7 Rubio-Tapia A., Murray J.A. (2010). Celiac Disease, Curr. Opin. Gastroenterol.26(2):116- 122. Steadman KJ et al. (2001). Buckwheat seed milling fractions: Description, macronutrient composition and dietary fibre, Journal of Cereal Science, 33: 271-278
JAFES, Vol 67, (2016) Journal of Agricultural, Food and Environmental Sciences UDC 664.8.036:547.722(620) Original scientific paper ______
ESTIMATION OF FURAN LEVELS IN SOME CANNED FOODS IN EGYPT AND HOW TO REDUCE IT
M. H. Elgammal*, M. S.M. Hasanin Regional Center for Food and Feed, Agriculture Research Centre, Egypt
*corresponding author: [email protected]
Abstract Furan is naturally occurring compound found at low levels in many foods. Furan has been formed in a wide range of thermally treated foods especially heated foods sealed in cans and jars. The primary source of furan in food is thermal degradation and rearrangement of organic compounds. Furan is a five-membered ring which can induce tumors and liver toxicity in experimental animals and is classified as ‘possibly carcinogenic to humans’ by the International Agency for Research on Cancer. In this study furan levels in several kinds of canned and jarred foods from Egyptian markets have been estimated. Also, several heat treatments have been applied for some food samples aiming to reduce furan content in these samples. Noticed levels of furan have been shown in tested samples. Coffee samples had higher content of furan in comparison to other products. After thermal treatments the obtained results show that, furan content reduced to about 38-50% of the original content for 50 o C at 5 min and the loss percent raised to about 55-71% at 20 min. At 60 o C, furan content reduced to about 47-56% of the original content at 5 min and the loss percent raised to about 62-78% at 20 min. And at 70o C, furan content reduced to about 53-59% at 5 min and the loss percent raised to about 70- 89% at 20 min. Key words: Furan, canned foods, thermal treated, Egypt
Introduction Furan is a heterocyclic, lipophilic compound degradation of vitamins including ascorbic which can induce tumors and liver toxicity in acid. Based on studies in model systems of experimental animals. Furan is clearly thermally induced furan formation, Perez and carcinogenic to rats and mice, showing a dose- Yaylayan 2004 proposed a series of formation dependent increase in hepatocellular adenomas pathways for furan in food from sugars, amino and carcinomas in both sexes. Furan is rapidly acids, ascorbic acid and its derivatives. and extensively absorbed from the intestine Ascorbic acid had the highest furan formation and the lung. It can pass through biological potential of the precursors examined in simple membranes and enter various organs and is model systems. Mark et al. 2006 and Limacher classified as “reasonably anticipated to be a et al. 2007 elaborated further on probable human carcinogen” by the U.S. Department of reaction pathways from ascorbic acid to furan. Health and Human Services and as “possibly The US Food and Drug Administration (US carcinogenic to humans” by the International FDA) published a report on the occurrence of Agency for Research on Cancer (IARC) of the furan in a number of foods that undergo WHO (IARC 1995). thermal treatment, especially canned and Maga, 1979 reviewed early work of the jarred foods with levels ranging from non formation of furan in thermally treated food, detectable to 174 µg/kg. Very similar results identifying the primary source of furans in were published by the researchers from the food as thermal degradation and Swiss Federal Office of Public Health. Parent rearrangement of organic compounds, furan was identified in a small number of heat- particularly carbohydrates such as glucose, treated foods, such as coffee, canned meat and lactose, and fructose. Maga identified a cooked chicken. Other investigations focused number of experimental systems known to on heated foods sealed in cans and jars, such produce furan in food, including heating of as baby foods, infant formulas, canned sugars, heating of sugars in the presence of vegetables, baked beans, soups, sauces, stews, amino acids or protein, and thermal and canned meats and fishes. When consumed
47 M. H. Elgammal, M. S.M. Hasanin ______in high concentrations (2 mg/kg body weight and analyzed using FDA/CFSAN, 2004 and day) furan proved to be carcinogenic and method as describe below. mutagenic in animal testing. The International Gas chromatography-mass spectrometry Agency for Research on Cancer (IARC) has (GC/MS) with headspace sampling was used therefore classified furan as carcinogenic in to detect furan in selected-ion monitoring animal testing and presumably carcinogenic mode (SIM) using ions: m/z 39 and 68 for for humans. According to a preliminary furan. recommendation of the National Toxicology Reagents: Program (NTP), the acceptable daily intake 1- Furan standard minimum purity 99% (ADI) of 1 µg/kg body weight should not be (Fluka), store in -20°C freezer. exceeded (US FDA 2004). The toxicity of 2- Water, purified by water purification system orally administered furan has been extensively (Milli-Q) studied in mice and rats over a wide dose 3- Methanol, HPLC grade range. The primary site of toxicity of furan is Apparatus: the liver, although the kidneys and lungs are 1. Refrigerator at 4°C. also affected at high doses (>30 mg/kg-bw per 2. Freezer at -20°C. day). In addition, changes in some 3. Analytical balance capable of weighing to haematological and hormonal parameters nearest 0.1 mg. occur at doses as low as 0.12 mg/kg-bw per 4. GC/MS/MS (Agilent 7890N GC with day administered 5 days/week. In the latest Agilent 7000 MSD with Dynamic risk assessment on furan carried out by the headspace auto-sampler). joint Food and Agriculture Organization of the 5. GC column: HP-5, 15 m, 0.32 mm I.D., 20 United Nations (FAO)/World Health μm film. Organization (WHO) Expert Committee on Preparation of standards: Food Additives (JECFA) it was concluded that 10 µL of the stock solution was diluted to 10 the margin of exposure for furan indicates a mL in methanol to give a standard solution of human health concern for a carcinogenic 1 µg/mL. From this stock solution 10, 50 and compound that might act via a DNA-reactive 100 ppb furan concentrations were prepared as genotoxic metabolite. Mean exposures for working standard solutions and sealed in adults ranged between 0.25 and 1.17 μg/kg- headspace vials. The stock solution should be bw/d and upper percentile exposures ranged stored in a 4°C refrigerator to minimize loss of from 0.60 and 2.22 μg/kg-bw/d. JECFA chose furan by evaporation. an exposure of 1 μg/kg-bw/d for the average Samples preparation: consumer and 2 μg/kg-bw/d for the high consumer as the basis of its MOE calculation Test portions of 5 g were used, each diluted (JECFA, 2010). with 5 g of water. For foods that are not A great concern is given to the analysis of this homogeneous, samples were homogenized as substance naturally occurring in food. This follows: the unopened container was chilled at study aims first, to estimate furan levels in 4 °C in a refrigerator for approx. 4 h. The several kinds of canned and jarred food. sample was transferred to a beaker immersed Second, study reducing furan concentration in in an ice bath, and the sample was some food samples using different treatments. homogenized with a hand blender. Portions of Fortunately, furan is a volatile substance, so 5 g of the homogenates were used diluted with several heat treatments may affect furan 5 g of water. All samples were capped content in test samples. immediately with Teflon-lined crimp seals.
Materials and methods Operating Conditions: Samples of canned and jarred foods which Headspace operating conditions: equilibration undergo thermal treatment while temperature 60°C, equilibration time 15 min, manufacturing were collected from the local volume of headspace gas sampled 500 μl. market. The samples included: bean, sweet GC-MS operating conditions: carrier gas corn, white beans, sauces, beef, sausages, helium, constant flow 1.0 ml min-1, oven infant formula, baby food, snacks and coffee. temperature profile: initial 50 °C (1 min), rate Four cans or jars of each kind of food products 10 °C min-1 to 230 °C; 150 °C injector; have been collected. Samples were prepared injection mode splitless, purge 0.25 min; MS
JAFES, Vol 67, (2016) 48 M. H. Elgammal, M. S.M. Hasanin ______ionization mode 75 eV EI+; source All samples have been analyzed twice and the temperature 230 °C; scan mode: selected ion means were calculated. monitoring (SIM); ions (m/z): 39, 68 (furan); dwell time 100 ms each ion. Samples treatments: For studying the effect of different heat Identification and quantification: treatments on furan content in samples, some Identification of furan was based on the samples were heated to three degrees (50o, 60o relative retention time (RRT) and the presence & 70o C) with stirring and for four different of diagnostic ions. For MS confirmatory periods (5, 10, 15 & 20 min). The treated purposes, the response ratio for the test samples were also analyzed as described portions should agree with the average of the above. response ratios for the calibration standards by ± 10 percent, and the retention time (RT) for Results and discussion the test portions should agree with the average The obtained chromatograms for furan RTs for the calibration standards by ± 2 standards are shown in Fig. (1). Whereas, Fig. percent, considering m/z 39/68 according to (2) shows the obtained MS chromatogram of the acceptance criteria as stipulated in furan compound. By integrating the peak areas European Commission Decision, 2002. The of the standards chromatograms, furan quantification of furan in samples proceeded standard curve was estimated. The standard by extrapolation from a linear analytical curve. curve is viewed in Fig. (3) with its linear equation and correlation coefficient.
Fig. (1): GC/MC chromatograms for furan standards
JAFES, Vol 67, (2016) 49 M. H. Elgammal, M. S.M. Hasanin ______
x10 4 +EI SIM 4.2 68.0 4 3.8 3.6 3.4 3.2 3 39.0 2.8 2.6 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 Counts vs. Mass-to-Charge (m/z)
Fig. (2): The obtained MS chromatogram of peak at RT~ 4.5 min (m/z of furan compound)
Fig. (3): Furan Standard Curve Table (1) displays the different levels of furan the daily intake of each kind of samples which in different food samples. It is cleared that all have been calculated according to Codex chosen samples are shown positive results for Committee on Food Additives and furan concentration. Coffee samples showed Contaminants (CCFAC, 1989). This revealed high content of furan in comparison to other that, coffee is the major contributor to furan products and that may due to coffee beans are exposure in the adult population. Morehouse et rich in carbohydrates and amino acids and al., (2008) also identified coffee as the major therefore when subjected to the high source of furan in the adult diet in the USA. temperature roasting process, higher levels of furan are formed. Furan levels in food depend It is worth to mention that JECFA, 2010 on heat treatments and vary depending on the reported the following ranges for national product being processed. For example, beans mean levels of furan in foods with the highest are generally fried at a lower temperature and contamination levels: roasted coffee (powder), for a shorter time compared to roasting of 814–4590 μg/kg; instant coffee (powder), 90– coffee beans which involves higher 783 μg/kg; brewed roasted coffee, 34–113 temperatures (e.g. > 200ºC) and comparatively μg/kg; jarred baby foods, 19–96 μg/kg; soy longer periods of time depending on whether a sauce, 16–52 μg/kg; canned fish, 6–76 μg/kg; medium roast, medium to dark roast or dark and baked beans, 27–581 μg/kg. roast coffee is required. Also, Table (1) show Great concern should be taken to avoid consumption a lot of food which contains
JAFES, Vol 67, (2016) 50 M. H. Elgammal, M. S.M. Hasanin ______some levels of furan especially babies and opening the container may also evaporate over infant foods whereas - according to a time. As furan is a volatile substance, it preliminary recommendation of the National appears that furan levels can be reduced in Toxicology Program (NTP) - the acceptable some foods through volatilization e.g. by daily intake (ADI) of 1 µg/kg body weight a heating and stirring canned/jarred foods in an day should not be exceeded. However once open saucepan. So, five samples have been furan formed it cannot evaporate from chosen for heat treatments after opening the processed food contained in an airtight sealed container at three degrees (50o, 60o & 70o C) pack until the container is opened. Residual and for four different periods (5, 10, 15 & 20 furan that may be present in the food after first min).
Table (1): Furan content in different samples with its relative standard deviation and calculated daily intake Furan Furan Furan Furan Furan Sample conc.(ppb) conc.(ppb) conc.(ppb) after conc.(ppb) conc.(ppb) before treat. after 5 min. 10 min. after 15 min. after 20 min. bean 13.7 8.8 7.3 6.6 6.3 white beans 14.8 8.7 7.5 6.8 6.1 sauces 7.4 3.7 2.9 2 1.7 sausages 13.4 8.3 6.9 6.2 5.9 beef 21.1 11.8 9.4 8.5 7.1
The results of treatment at 50o, 60o & 70o C are of the original content for 50 o C at 5 min and illustrated in Tables (2, 3 & 4) respectively. the loss percent raised to about 55-71% at 20 From these results, it is clear that heating min. At 60 o C, furan content reduced to about reduced the level of furan in the tested food 47-56% of the original content at 5 min and samples in all treatment and that attributed to the loss percent raised to about 62-78% at 20 heating food samples with stirring allow min. And at 70o C, furan content reduced to partial volatilization and dissipation of about 53-59% at 5 min and the loss percent furan.Furan content reduced to about 38-50% raised to about 70-89% at 20 min. Table (2): Furan content in some samples after heat treatment at 50oC and at different period Range of Sample + RSD Daily intake furan concentration (ppb) Bean 9.55 – 13.7 0.18 0.87-1.02 sweet corn 18 – 23.2 0.17 0.64-0.83 white beans 12.1 – 14.8 0.15 0.94-1.16 sauces 4.8 – 7.4 0.3 0.47-0.73 infant formula 13.1 – 16.6 0.16 0.12-0.15 sausages 8.5 – 13.4 0.31 0.83-1.21 beef 17 – 21.1 0.15 1.6-2.03 baby food 25.7 – 34.7 0.21 0.23-0.33 snacks 6.4 – 8.3 0.18 0.53-0.69 Light-colored coffee 482.7 – 512.6 0.05 2.0-2.2 Dark coffee 702 - 789.2 0.1 2.8-3.2
Table (3): Furan content in some samples after heat treatment at 60oC and at different period Furan conc.(ppb) Furan conc.(ppb) Furan conc.(ppb) Furan conc.(ppb) Furan conc.(ppb) Sample before treat. after 5 min. after 10 min. after 15 min. after 20 min. Bean 13.7 8.4 7.1 6.2 5.3 white 14.8 7.9 7 6 4.9 beans Sauces 7.4 3.2 2.1 1.5 0.9 Sausages 13.4 8.4 7 5.9 5 Beef 21.1 10.2 8.9 7.7 6.1
JAFES, Vol 67, (2016) 51 M. H. Elgammal, M. S.M. Hasanin ______
Table (4): Furan content in some samples after heat treatment at 70oC and at different period Furan Furan Furan Furan Furan conc.(ppb) conc.(ppb) conc.(ppb) conc.(ppb) conc.(ppb) Sample before after 5 min. after 10 after 15 after 20 treat. min. min. min. bean 13.7 7.3 6 5.1 4.3 white 14.8 6.9 5.8 5 4.4 beans sauces 7.4 3 2 1.3 0.8 sausages 13.4 8.1 7 5.8 5 beef 21.1 9.8 8.1 7.1 6
Above results revealed the decreasing of furan FDA/ CFSAN, FDA/Center for Food Safety & content for all heat treatment and as long as Applied Nutrition (2004): Determination of time of heat exposure increases the furan Furan in Foods. CFSAN/Office of Plant and content decrease through volatilize tion. Then, Dairy Foods. May 7. the extent of furan loss is related to the Goldmann, T., Perisset, A., Scanlan, F. and temperature of the product and time of Stadler, R.H. (2005): Rapid determination of exposure to heat which agrees with Goldmann furan in heated foodstuffs by isotope dilution et al. (2005) who reported the decrease of solid phase microextraction-gas furan concentrations over time upon heating in chromatography-mass spectrometry open jars, thus leading to 85% loss. While, (SPMEGC-MS). Analyst, 130,878-883. Kim et al. (2009) found that heating canned International Agency for Research on Cancer meats to 50-70 ºC reduced furan levels 26 to (IARC) (1995): IARC Monographs on the 46 percent. Evaluation of Carcinogenic Risks to Humans, Volume 63, pp. 394-407. Conclusion JECFA ( 2010): Joint FAO/WHO Expert More concern should be taken for exhausting Committee on Food Additives Report of the canned or jarred foods, which undergo high seventy-second meeting (final edited), Rome, thermal treatment while manufacturing, 16–25 February 2010. especially baby and infant foods because of its Kim TK, Lee YK, Park YS and Lee KG furan content. Also, this study may be helpful (2009): Effect of cooking or handling in establishing approaches to lower the conditions on the furan levels of processed exposure of the population to furan from the foods. Food Additives & Contaminants: Part A consumption of coffee which contains the 26:767-775. highest furan levels in comparison to other Limacher A, Kerler J, Conde-Petit B and products. Furan is not stable in foods after or Blank I (2007): Formation of furan and opening the commercial products then it is methylfuran from ascorbic acid in model recommended to heat canned or jarred foods systems and food. Food Additives & before consumption. Heating the food after Contaminants 24, S1: 122-135. opening the container at about 60-70 ºC for Maga JA. (1979): Furans in foods. CRC few minutes with stirring would be enough for Critical Reviews in Food Science and evaporate most of furan content. Nutrition, 355-400. Mark J, Pollien P , Lindinger C , Blank I and References Märk T (2006): Quantitation of furan and CCFAC the Codex Committee on Food methylfuran formed in different precursor Additives and Contaminants (1989): systems by proton transfer reaction mass Guidelines for simple evaluation of food spectrometry. J Agric Food Chem 54: 2786- additive intake. CAC/GL 03. Codex 2793. Alimentarius international food standards. Morehouse K, Nyman P, McNeal T, Dinovi European Commission Decision (2002): EC MJ and Perfetti G, (2008): Survey of furan in concerning the performance of analytical heat processed foods by headspace gas methods and the interpretation of results. chromatography-mass spectrometry and Official Journal of European Communities, L 221:8-36.
JAFES, Vol 67, (2016) 52 M. H. Elgammal, M. S.M. Hasanin ______estimated adult exposure. Food Additives and Contaminants, 25(3), 259-264. Perez L C and Yaylayan V A (2004): Origin and mechanistic pathways of formation of the parent furan—a food toxicant. J Agric Food Chem 52: 6830-6836. US FDA (2004): United States Food and Drug Administration Furan in Food, Thermal Treatment; Request for Data and Information. Federal Register 69: 25911–25913.
JAFES, Vol 67, (2016) Journal of Agricultural, Food and Environmental Sciences UDC 635.64-152.86 Original scientific paper ______
ISOLATION OF PROMISING MUTANT OF ENHANCED FRUIT QUALITY IN TOMATO
S. Sikder1, P. Biswas2, A. Chattopadhyay2, P. Hazra2*
1Department of Vegetable and Spice crops, Uttar Banga Krishi Viswavidyalaya, Pundibari-736165, Coochbehar, West Bengal, India 2Department of Vegetable crops, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur-741252, West Bengal, India
* corresponding author: [email protected]
Abstract Induction of mutation by gamma rays, ethyl methane sulfonate (EMS) and their combined treatments was studied in three widely divergent genotypes of tomato, EC-620176, EC-620177 and Patharkutchi. Combination of gamma radiation and EMS caused more damage followed by EMS treatment and gamma radiation, mainly in M1 generation. Gamma irradiation (50-150 Gy) was most efficient followed by 0.05- 0.10 % EMS and their combination treatment in inducing wide array of macro- mutation in tomato. One mutant “Dark green fruit” was isolated from the M2 population of Patharkutchi treated by 150 Gy gamma radiation. Chlorophyll ‘a’, Chlorophyll ‘b’ and total chlorophyll content in the leaf and immature fruit of this mutant was significantly higher compared to the parental genotype. Average total chlorophyll content in the leaf and immature fruit of the mutant over M3 and M4 generation was 318.52 mg/100 g fresh and 21.93 mg/100 g fresh, respectively in sharp contrast to 198.25 mg/100 g and 12.21 mg/100 g fresh, respectively in the leaf and immature fruit of the parental line. This “Dark green fruit” mutant with higher average lycopene (7.49 mg/100 g fresh) and ascorbic acid (35.86 mg/100 g fresh) contents in the ripe fruits emerged as a promising genetic resource for further utilization in tomato breeding for enhancement of lycopene and ascorbic acid content in the fruits. Key words: Mutation frequency, Gamma rays, EMS, Dark green fruit, Tomato
Introduction The cultivated tomato (Solanum lycopersicum Materials and methods L.) is the second most consumed vegetable The present investigation was undertaken in after potato and contributes greatly to agro- the Department of Vegetable Crops, Bidhan based industry in the world. Spontaneous or Chandra Krishi Viswavidyalaya, Mohanpur, induced mutants, with desirable changes in West Bengal during 2010-2014. The gamma particular characters have been a key material radiation was applied to dry seeds at National for gene discovery, mapping, functional Botanical Research Institute, Lucknow, India genomics and breeding in many crops where Cobalt- 60 served as source of gamma including tomato. Mutant alleles in tomato are rays. Solutions of ethyl methane sulphonate only currently known for an insignificant (Sigma Chemical Company, USA) were made fraction of the about 35,000 genes in the with freshly prepared phosphate buffer (pH tomato genome hence, large scale mutagenesis 7.0) for treating the seeds. Dry seeds of three and introgression of natural genetic variation widely divergent genotypes of tomato viz., ps- can be useful to fill this gap. The present 2 functional male sterile line (EC-620176), investigation was undertaken to study the Berika (EC-620177), a variety from Bulgaria frequency and spectrum of macro-mutations of and Patharkutchi, the highly adaptable and gamma rays, ethyl methane sulphonate (EMS) popular variety of West Bengal were irradiated and their combinations in three genotypes of with 50, 100, 150, 200 and 250 Gy gamma tomato. rays. Pre-soaked seeds of these genotypes (6 h, in water) were treated with 0.05, 0.10, 0.15, 0.20 and 0.25% ethyl methane sulphonate
JAFES, Vol 67, (2016) 54 S. Sikder, P. Biswas, A. Chattopadhyay, P. Hazra ______
o (EMS) for 8 h at 25 ± 2 C. Gamma irradiated mutant bred true in both M3 and M4 generation (50, 100, 150, 200 and 250 Gy) seeds of these suggesting the involvement of single mutated three genotypes were also pre-soaked (6 h, in gene for manifestation of the basic character water) before treating with 0.15 % EMS “Dark green fruit” in this genotype. This solution for 8 h at 25 ± 2oC as combination putative mutant was characterized taking a treatment. The EMS treated seeds were number of morphological and fruit quality washed thoroughly in running water at least characters in the M4 generation. The data was for an hour before sowing. statistically compared with that of the parental Treated seeds of three genotypes were sown genotype “Patharkutchi”. separately in the trial using 100 seeds each in three replications along with parental controls Results and discussion (non-treated seeds) in well prepared seed beds. Damage in M1 generation Seeds from all plants of each of the three The impact and tolerance level of the tomato genotypes from respective treatment in M1 genotypes to the mutagen were manifested in generation were bulked to raise the M2 M1 generation itself in terms of lethality generation. Total of maximum 180 plants of (reduction in seed germination), injury the M2 generation in each treatment were (reduction in length of seedlings) and sterility grown keeping 60 plants at 60 x 50 cm spacing (reduction in pollen viability). Considering (5.0 x 3.6 m bed) in each replication along three genotypes together, percent reduction in with the parents in well prepared field. A germination over control was maximum in range of 150 to 173 M2 plants from 3 combination treatment (42.50%) followed by genotypes and all mutagenic treatments was sole EMS treatment (31.91%) and least examined for segregation. The spectrum of occurred due to gamma radiation (29.82%). mutation in the M2 progeny comprising Spectrum of mutation chlorophyll deficient mutations (both viable The spectrum of mutation is essentially a and non-viable) and other macro mutations parameter for the index of mutation frequency. could be detected based on altered plant Spectrum of mutation varied within genotypes stature, leaf morphology, inflorescence type, and it was the highest of twelve in Patharkutch floral morphology, pedicel character, fruit followed by eight in EC-620177 and the least morphology, color and chlorophyll content of five in EC-620176 indicating variation in over the set of characters specific for the three allelic mutability of different genotypes. genotypes. Non-viable chlorophyll deficient Chlorophyll deficient mutants were mostly mutants did not continue beyond 30 days after “Albino” type which perished within 30 days planting. after planting. The only viable chlorophyll Chlorophyll mutation frequency was mutant was isolated from EC-620176 in 0.1% determined as percentage of mutated M2 EMS treatment. The spectrum of mutation as a progenies for chlorophyll deficiency (both whole considering both chlorophyll deficient viable and non-viable). Total mutation and other macro mutations together decreased frequency (Mf) was determined as % of with the increasing doses of both gamma mutated M2 progenies for both chlorophyll radiation and EMS concentration in two deficient and other viable macro-mutants. A genotypes of Europe, but it did so in number of viable macro-mutants with Patharkutchi. Spectrum of mutation varied discernable morphological characters like, with the mutagen and dose. Widest mutation multiparous cyme, dwarf plant with pyriform spectrum was obtained in EC-620176 (four) fruit, irregular shaped fruit, and dark green with 100Gy gamma radiation and 50 Gy fruit could be isolated in the M2 generation gamma radiation + 0.15% EMS treatment; in from all three genotypes exposed to mutation. EC-620177 (four) with 50 Gy gamma All macro-mutants were self-pollinated to radiation and in Patharkutchi (four) with 50, produce the seeds and advanced up to M4 200 and 250 Gy gamma radiation and 0.05% generation. EMS treatment. The “Dark green fruit” mutant could be Among the macro-mutants, fruit mutants isolated from the M2 population of the (shape, size, high chlorophyll content, etc.) tropicalized and popular cultivar of West were more frequently occurring followed by Bengal, Patharkutchi treated by 150 Gy leaf mutants. Higher doses of gamma radiation gamma radiation. Selfed population of this produced more non-viable chlorophyll
JAFES, Vol 62, (2016) 55 S. Sikder, P. Biswas, A. Chattopadhyay, P. Hazra ______deficient mutants whereas EMS at higher EMS treatment. Combined treatment induced concentration produced comparatively lesser intermediate mutation frequency showing in number and in combination treatments. It additivity because of independent action of was clearly evident that the physical and two mutagens. Total mutation frequency also chemical mutagens induced different mutation did not necessarily reflect the spectrum of spectrum and the type of mutant depended not mutation and for this reason, the highest of only on the type of mutagen but also on the 6.96 % mutation frequency in EC-620176 with genotype used as recorded in several earlier 250 Gy gamma radiation emerged from only studies (Walter et al. 1987; Sakin and Senkar, two mutant types. Hence, both mutation 2002; Prem et al. 2011). spectrum and frequency are important to ascertain the genetic variation that is available Total mutation frequency for selection in M2 or M3 generations. Average total mutation frequency combining Of the different putative macro-mutants that three genotypes together differed significantly was isolated in the three genotypes in M2 among the mutagens. The highest mutation generation, five (exerted stigma flower (50 Gy frequency of 4.33 % resulted by gamma gamma rays), dark green fruit (150 Gy gamma radiation followed by 3.52% in combination rays), dwarf plant having pyriform fruit (200 treatment and the least of 3.19 % in EMS Gy gamma rays) from Patharkutchi; treatment (Table 1). Maximum efficiency of multiparous cyme (50 Gy gamma rays) from gamma radiation in inducing the highest EC-620177 and viable chlorophyll deficient mutation frequency might have been due to its mutant (0.10%EMS) from EC-620176 hold high penetrating power of causing more immense promise for their utilization in chromosomal aberration as compared to sole tomato breeding programme.
Table 1.Total mutation frequency in M2 generation of three tomato genotypes Total mutants Chlorophyll Total M2 plants Viable Non-viable Viable mutation mutation Mutagenic treatments examined chlorophyll chlorophyll macro- frequency frequency mutants mutants mutants (%) (Mf) % Positional sterile-2 line (EC-620176) 50 Gy γ rays 153 0 3 3 1.96 3.92 100 Gy γ rays 157 0 3 4 1.91 4.46 150 Gy γ rays 159 0 5 3 3.14 5.03 200 Gy γ rays 167 0 7 3 4.19 5.98 250 Gy γ rays 158 0 10 1 6.33 6.96 0.05%EMS 162 0 3 1 1.85 2.47 0.1% 167 1 2 2 1.79 2.99 0.15% 157 0 4 0 2.54 2.54 0.20% 158 0 5 0 3.16 3.16 0.25% 151 0 7 0 4.63 4.63 50 Gy + 0.15% EMS 172 0 2 3 1.16 2.91 100 Gy + 0.15% EMS 152 0 4 2 2.63 3.94 150 Gy + 0.15% EMS 153 0 3 2 1.96 3.26 200 Gy + 0.15% EMS 158 0 6 0 3.79 3.79 250 Gy + 0.15% EMS 164 0 5 2 3.05 4.26 Berika (EC-620177) 50 Gyγ rays 154 0 2 3 1.30 3.25 100 Gyγ rays 156 0 5 1 3.21 3.84 150 Gyγ rays 171 0 5 2 2.92 4.09 200 Gyγ rays 155 0 5 1 3.23 3.87 250 Gyγ rays 163 0 6 1 3.68 4.29 0.05%EMS 154 0 2 1 1.30 1.94 0.1% 153 0 2 2 1.31 2.61 0.15% 154 0 4 1 2.60 3.25 0.20% 151 0 5 0 3.31 3.31 0.25% 167 0 7 0 4.19 4.19 50 Gy + 0.15% EMS 168 0 4 1 2.38 2.98 100 Gy + 0.15% EMS 159 0 3 2 1.89 3.14 150 Gy + 0.15% EMS 165 0 6 0 3.63 3.63 200 Gy + 0.15% EMS 152 0 5 0 3.28 3.28
JAFES, Vol 62, (2016) 56 S. Sikder, P. Biswas, A. Chattopadhyay, P. Hazra ______
250 Gy + 0.15% EMS 166 0 6 1 3.61 4.21 Patharkutchi 50 Gyγ rays 172 0 2 3 1.16 2.91 100 Gyγ rays 152 0 4 1 2.63 3.29 150 Gyγ rays 157 0 4 2 2.54 3.82 200 Gyγ rays 173 0 5 3 2.89 4.04 250 Gyγ rays 153 0 5 3 3.27 5.22 0.05%EMS 172 0 1 3 0.58 2.32 0.1% 164 0 3 1 1.83 2.43 0.15% 154 0 3 3 1.95 3.89 0.20% 151 0 4 2 2.65 3.72 0.25% 161 0 6 1 3.73 4.34 50 Gy + 0.15% EMS 154 0 3 1 1.95 2.59 100 Gy + 0.15% EMS 151 0 4 0 2.65 2.65 150 Gy + 0.15% EMS 167 0 4 2 2.39 3.59 200 Gy + 0.15% EMS 154 0 6 0 3.89 3.89 250 Gy + 0.15% EMS 166 0 6 2 3.61 4.82
Dark green fruit mutant induced mutant appeared to be allelic to some Selfed population of this mutant bred true in spontaneous mutations already known in both M3 and M4 generation suggesting the tomato, such as dark green fruit (Kendrick et involvement of single mutated gene for al., 1997) and jointless pedicel (Mao et al., manifestation of the basic character “Dark 2000). This induced mutant dark green fruit green fruit” in this genotype. resembled the already identified spontaneous Different qualitative characters as documented mutant dark green fruit locus dg located in in this mutant and its parental genotype (Table chromosome 1 (Levin et al., 2003) which 2) indicated deviation in 3 characters viz., twig enhanced fruit carotenoid content (Van Tuinen color (dark green vis a vis medium green in the et al., 1997). Average lycopene content in the parent), leaf color (very dark green vis a vis ripe fruits of this mutant was much higher dark green in the parent), and unripe fruit color (7.49 mg/100 g fresh) compared to moderate (dark green vis a vis green in the parent). of 4.37 mg/100 g fresh in the parental Chlorophyll ‘a’, Chlorophyll ‘b’ and total genotype (Table 3) which supported the chlorophyll content in the leaf and immature proposition that exaggerated photo- fruit of this mutant was markedly high responsiveness as manifested by high compared to the parental genotype. Average chlorophyll content in fruit was responsible for total chlorophyll content in the leaf and enhanced carotenoid particularly lycopene immature fruit of the mutant over M3 and M4 content in the ripe fruits. generation was 318.52 mg/100 g fresh and Different quantitative characters that markedly 21.93 mg/100 g fresh, respectively in sharp altered in this mutant as recorded in Table 3 contrast to 198.25 mg/100 g and 12.21 mg/100 for both M3 and M4 generation was fruit g fresh, respectively in the leaf and immature number per plant (average 43.40 fruits less fruit of the parental line (Table 3). Isolation of than the parent), fruit weight (average 32.38 g high chlorophyll mutant in tomato was also more than the parent), total phenol content of reported earlier by 100 Gy gamma radiation immature fruit (average 4.85 mg/100 g fresh (El-Sayed et al., 1994) and 24 Gy gamma higher than the parent) and ascorbic acid radiation (Asmahan and Al-Twaty, 2006) content (average 8.19 mg/100 g fresh higher which correspond to the present findings. than the parent). Earlier reports also suggested that some useful
JAFES, Vol 62, (2016) 57 S. Sikder, P. Biswas, A. Chattopadhyay, P. Hazra ______
Table 2. Different qualitative characters of Patharkutchi and “Dark green fruit” mutant Character Patharkutchi Dark green fruit mutant Growth habit Semi-determinate Semi-determinate Branching pattern Upright Upright Twig colour Medium green Dark green Leaf orientation Pinnately compound Pinnately compound Leaf type Serrated Serrated Leaf size Narrow Narrow Leaf colour Dark green Very dark green Inflorescence Monoparous cyme Monoparous cyme Flower size Medium Medium Sepal size Medium Medium Anther dehiscence Consistent Consistent Male sterility/fertility Male fertile Male fertile Fruit shape Flattish-round Flat round to heart Green shoulder High High Pedicel attachment Jointed Jointed Fruit pubescence No No Fruit ribbing High High Blossom end Indented Indented Unripe fruit colour Green Dark green
Table 3. Different quantitative characters of Patharkutchi and “Dark green fruit” mutant in M3 and M4 generation
plant
Character Plant (cm) height Primary branch/ plant Days to flowering first Flower cluster per Fruitper cluster Fruitper Fruit(g) weight Polar diameter (mm) Equatorial (mm) diameter Pericarp (mm) thickness Locule No. Test weight of (g)seed
M3 141.23 9.13 31.25 7.87 6.87 51.67 77.33 50.16 54.34 6.52 4.51 3.31 Patharkutchi M4 123.16 9.67 33.72 8.16 6.33 56.33 82.91 49.52 50.26 5.87 4.38 3.35 Dark green fruit M3 93.44 9.31 47.24 6.67 3.17 9.33 107.45 58.71 61.25 7.16 4.83 3.31 mutant M4 98.33 10.33 46.91 5.87 2.87 11.87 117.56 53.48 55.52 6.82 5.25 3.28 M3 0.85 0.53 0.60 0.23 0.24 0.35 0.67 1.04 0.85 0.31 0.16 0.16 S.Em.(±) M4 0.71 2.07 2.35 0.29 0.31 0.71 0.89 1.06 1.02 0.39 0.18 0.20 M3 3.32 0.51 0.94 0.91 0.96 1.35 2.61 4.06 3.33 1.19 0.62 NS C.D.(5%) M4 2.78 1.96 3.71 1.15 1.19 2.76 3.51 4.15 4.00 1.53 0.73 NS
Character Chlorophyll a (mg/100g)leaf of Chlorophyll b (mg/100g)leaf of Total Chlorophyll of (mg/100g) leaf Total phenol of leaf (mg/100g) Total Chlorophyll of immature (mg/100g) fruit Total phenol immature of (mg/100g) fruit TSS (°B) Lycopene (mg/100g) β (mg/100g) Reducing (%) sugar Carotene Total (%) sugar Ascorbic (mg/100g) acid Acidity (%) Seed content(mg/100g) protein
M3 95.47 105.43 210.93 22.16 12.57 7.13 6.21 4.27 0.78 2.79 3.48 26.27 0.57 23.08 Patharkutchi M4 105.26 95.23 185.57 27.22 11.86 6.65 6.35 4.48 0.65 2.58 3.26 28.35 0.64 21.56 Dark green M3 170.19 159.54 329.73 37.02 23.23 12.11 5.86 7.73 0.84 3.14 3.85 34.85 0.58 23.17 fruit mutant M4 157.16 162.24 307.42 34.25 20.63 11.37 5.73 7.26 0.86 3.08 3.64 36.16 0.66 20.82 Dwarf plant M3 107.22 121.71 226.93 28.78 14.79 8.37 5.83 4.26 0.76 3.69 4.17 28.24 0.73 27.96 mutant M4 111.42 118.35 216.43 31.57 13.28 7.51 5.66 4.18 0.69 3.28 3.84 27.57 0.78 26.71 M3 1.42 2.23 2.09 0.94 0.67 0.62 0.33 0.29 0.27 0.15 0.06 0.49 0.20 0.24 S.Em.(±) M4 1.52 1.91 2.16 1.27 0.81 0.51 0.37 0.23 0.29 0.19 0.05 0.44 0.22 0.16 C.D. (5%) M3 5.57 8.71 8.17 3.69 2.61 2.43 NS 1.15 NS 0.57 0.23 1.94 0.78 0.96 M4 5.94 7.48 8.46 4.98 3.14 1.97 NS 0.91 NS 0.74 0.19 1.72 0.84 0.64
JAFES, Vol 62, (2016) 58 S. Sikder, P. Biswas, A. Chattopadhyay, P. Hazra ______
Conclusion phytochrome genes and photomorphogenic It emerged conclusively from the present mutants of tomato. Theor. Appl. Genet. 94, pp. investigation on applied mutagenesis of 115-122. tomato that lower doses of gamma radiation Walter R.F., Elinor L.F. and Holly J.J. 1987. (50 – 250 Gy) was the most effective Mutation Breeding Principles of Cultivar mutagenic treatment for inducing broad Development, Theory and Technique. spectrum of viable mutation in tomato and Macmillan Publishing Co. New York, pp. 287- induced mutation could alter a number of both 303. qualitative and quantitative characters. The “dark green fruit” mutant emerged as a promising genetic resource for further utilization in tomato breeding for enhancement of lycopene and ascorbic acid content in the fruits.
References Asmahan, A.A. and Al-Twaty, N. (2006). Effect of irradiation and sodium azide on some economic traits in tomato. Saudi J. Biol. Sci. 13, pp. 44-49. El-Sayed, H.H., Abd El-Tawab, F.M. and El- Souedy, A., Sharabash, M.T and Asmahan, A.M. (1994). Effect of gamma irradiation on growth, yield and chemical constituents for three tomato varieties and their crosses. Second Arab Conference on the Peaceful Uses Energy, Cairo5-9 Nov, pp. 913-923. Kendrick, R.E., Kerckhoffs, L.H.J., Van Tuinen, A. and Koornneef, M. (1997). Photomorphogenic mutants of tomato. Plant Cell Environ. 20, pp.746-751. Levin, I., Frankel, P., Gilboa, N., Tanny, S. and Lalazar, A. (2003). The tomato dark green mutation is a novel allele of the tomato homolog of the DEETIOLATED1 gene. Theor. Appl. Genet. 106, pp. 454-460. Mao, L., Begum, D., Chuang, H.W., Budiman, M.A., Szymkowiak, E.J., Irish, E.E. and Wing, R.A. (2000). Jointless is a MADS-box gene controlling tomato flower abscission zone development. Nature 406, pp.910-913. Prem D., Gupta K. and Agnihotri A. 2011. Can we predict mutagen-induced damage in plant systems mathematically? Insights from zygotic embryo and haploid mutagenesis in Indian mustard (Brassica juncea). Botanica Serbica 35, pp. 137-143. Sakin, M.A. and Sencar, O. 2002. The effects of different doses of gamma ray and EMS on formation of chlorophyll mutations in durum wheat (Triticum durum Desf.).Tarim Bilimleri Dergisi 8, pp. 15-21. Van Tuinen, A., Cordonnier-Pratt, M.M., Pratt, L.H., Verkerk, P., Zabel, P. and Koorneef, M. (1997). The mapping of
JAFES, Vol 62, (2016) Journal of Agricultural, Food and Environmental Sciences UDC 637.12.065(497.115) Original scientific paper ______
INCIDENCE OF STAPHYLOCOCCUS AUREUS AND COAGULASE POSITIVE STAPH. AUREUS IN RAW MILK IN KOSOVO
D. Sylejmani*, A. Hamidi
Faculty of Agriculture and Veterinary, University of Prishtina, Boul. ’’Bill Clinton’’, 10000, Kosovo
*corresponding author: [email protected]
Abstract The presence study was conducted to determine the presence of Staphylococcus aureus and coagulase positive S.aureus in raw milk collected from different localities in Kosovo. We have examined 55 samples of raw milk cow taken from cattle farms. The Staphylococcus aureus was isolated in Baird Parker agar where typical and atypical colonies were selected and submitted to coagulase and complementary tests. Out of 55 samples of raw milk studied, 17 showed contamination by Staphylococcus aureus corresponding to 31% of the samples being contaminated. Out of 17 samples showed contamination by S. aureus, 5 of them were coagulase positive S. aureus or 29 % (or 9 % of total samples analyzed). Results clearly suggested a possibility of potential public health threat of Staphylococcus aureus resulting from contamination of milk with pathogenic bacteria is mainly due to unhygienic processing, handling and unhygienic environment. Key-words: Staph.aureus, coagulase positive, incidence, raw milk.
Introduction of foodborne disease outbreaks from 1992- Raw milk produced by cows at the temperature 1997 found that S. aureus was the most of cow’s body is considered as one of the best frequent pathogen associated with milk related mediums for growth of Staphylococcus aureus outbreaks (De Buyser et al., 2001). Food (Zecconi et al, 2000). Mammary glands intoxications caused by using raw milk are infected by Staphylococcus aureus are the well known and the majority holds S. aureus or main cause of milk contamination (Jayarao et its enterotoxins as responsible agents (J.H.Lee, al, 2004). The presence of Staphylococcus 2003). aureus in raw milk generally comes from cows The aim of this study was to verify the with mastitis, from handlers or from deficient presence of Staphylococcus aureus and hygiene. The capacity to coagulate plasma, the coagulase positive S.aureus in raw milk. principal characteristic of S. aureus, is highly correlated to the capacity to produce Material and Methods enterotoxins harmful to the tissues of the Sample collection contaminated host (Murray PR et al, 2006). A total of 55 raw milk samples were collected Not all coagulase positive S. aureus produce from different regions in Kosovo. The samples toxin. In Norway, 48% of isolates from bovine collected aseptically were transferred to sterile raw milk and raw milk products were recipients and transported to the laboratory identified as SE producers (Loncarevic et al, under cold conditions, and analyzed within 24 2005). hours. Milk and its products are common vehicles of Staphylococcus aureus transmission to humans Staphylococcus aureus research (Lowy FD, 1998). The presence of To carry out the laboratory test were used ISO Staphylococcus aureus shows up unsanitary method 6888. Ten mililitres of each cheese conditions in the cattle herd and counts above sample were placed aseptically into a sterile 3 plastic bags and 90ml peptone water were 10 cfu in milk increase the risk of 0 staphylococcal toxin production more resistant added to make 10 dilutions. The bags were homoginezed in stomacher for 1 min. Then, to the heat processes of pasteurization. It’s 0 -2 -4 presence in foods can be a risk to human 0.1ml from each dilutions (10 , 10 , 10 …) health, causing a public health problem, as were inoculated on the surface of Baird Parker these bacteria produce toxins that can cause agar and spread with a Drigalski spatula. The toxic food infections (1-4). In France, a study incubation was done at a temperature of 37 ºC
60 D. Sylejmani, A. Hamidi ______for 24-48 hrs. Appearance of shiny black The microbiological analysis of raw milk colonies with an opaque ring, surrounded by a samples for presence of S. aureus and clear halo were considered to be presumptive coagulase positive S. aureus are presented in S. aureus. The suspected S. aureus colonies Tables 1 and 2. were submitted for Gram staining, oxidase test, Out of 55 samples of raw milk analyzed, 17 of catalase test and also were further identified them were contaminated by Staphylococcus biochemical profile, using api Staph. aureus, corresponding to 31% of samples Coagulase test contaminated, at a range of 2.5x102cfu/ml to Five typical colonies were selected for seeding 4.6x105cfu/ml. Out of the 17 contaminated in tubes containing BHI and the incubation samples of raw milk, 4 had levels of was done at a temperature of 35 ºC for 24 Staphylococcus aureus corresponding to 102 hours. From each tube cultivation in BHI, cfu/ml; 3 had levels of 103cfu/ml; 7 counts of 0.3ml was transferred to sterile tubes 104cfu/ml and 3 samples of 105cfu/ml (Tables containing 0.5ml of rabbit plasma. The 1, 2). incubation was done at a temperature of 35 ºC Out of 17 samples showed contamination by S. for 6 hours. aureus, 5 of them were coagulase positive S. aureus or 29 % (or 9% of total samples Results and discussion analyzed). Table 1. Staph.aureus and coagulase positive Staphylococcus aureus in the examined raw milk samples Sample type No. of samples Staph. aureus Coagulase positive Staph. aureus
Rawmilk (cow) 55 17 (31%) 5 (29%)
Table 2. Counts of S. aureus and coagulase positive S. aureus of positive analyzed raw milk samples Sample No S.aureus(cfu/ml) Coag.positive S. Sample No. S.aureus Coag.positive aureus (cfu/ml) S.aureus 1. 3.2x102 + 10. 2.5x102 2. 2.5x103 + 11. 2.4x104 3. 1.3x104 12. 5.1x104 + 4. 4.5x105 13. 4.6x105 5. 1.2x103 + 14. 1.5x103 6. 1.6x104 15. 5.1x102 7. 5.1x104 16. 1.2x104 + 8. 3.5x102 17. 1.5x105 9. 4.7x104
Of the 5 contaminated samples of raw milk, sample were coagulase positive S. aureus, the one had coagulase positive Staph. aureus raw milksamples analyzed may be a serious counts corresponding to 3.2x102cfu/ml; 2 with risk to the health of the population (Fig 1.). It 103cfu/ml (2.5x103cfu/ml and1.2x103cfu/ml) is generally considered that the numbers of S. and 2 with 104cfu/ml (1.2x104cfu/ml and aureus need to be >105 cfu/g or ml product for 5.1x104cfu/ml, Table. 2). the production of sufficient toxin to cause The incidence of S. aureus in raw milk illness (Le Loir et al. 2003, Pinchuk et al. depending on the health status of the milking 2010). cows and other factors (Adesiyun et al, 1998). S. aureus contamination can occur from raw The high incidence of Staphylococcus aureus milk produced from cows suffering from is indicative of poor hygienic measures during mastitis, food handlers who are carriers of S. production, handling and distribution. aureus, or poor hygiene practices (O’Brien et Considering that three of samples had counts al., 2008). of S. aureus 105cfu/ml, andin other hand five
JAFES, Vol 67, (2016) 61 D. Sylejmani, A. Hamidi ______
Fig.1 Staphylococcus aureus colonies on BPA Fig.2 Coagulase positive test
Negligence of hygienic condition such as Guidelines for monitoring bulk tank milk improper cleaning of bulk tank, dirty udders, somatic cell and bacterial counts. J. DairySci., milking equipments, cooking temperature, 87: 3561-3573. milk handling technique and improper storage Le Loir, Y., Baron,F., Gautier,M. : 2003. will increase the proportion Gram positive and Staphylococcus aureus and food Gram negative bacteria in the bulk tank milk poisoning.Genet.Mol.Res. 2:7-28. (Vasavada, 1988). Loncarevic S, Jørgensen HJ, Løvseth A, Mathisen T, Rørvik LM (2005). Diversity of Conclusion Staphylococcus aureus enterotoxin types Coagulase positive Staphylococcus aureus was within single samples of raw milk and raw identified in 29% of raw cow milk samples milk products. J ApplMicrobiol 98:344-350. collected and analyzed from different regions Lowy FD.(1998). Staphylococcus aureus of Kosovo. Contamination of raw milk infection. North England Journal of Medicine produced by cattle farms in Kosovo remains a 339:520-532. possible source of food intoxication because of Murray PR, Rosenthal KS, Pfaller MA (2006). the high number of S. aureus in milk. Medical Microbiology, Elsevier: 979. O’Brien, M., Hunt, K., McSweeney, S., References Jordan, K.: 2008. Occurrence of foodborne Adesiyun AA, Webb LA, Romain HT. (1998). pathogens in Irish farmhouse cheese. Food Prevalence and characteristics of Microbiol., 26: 910-14. Staphylococcus aureus strains isolated from Pinchuk IV, Beswick EJ, Rejes VE. 2010. bulk and composite milk and cattle handlers, Staphylococal enterotoxins. Toxins 2:2177- Jorunal of Food Protection 6:629-632. 2197. De Buyser M-L, Dufour B, Maire M, Lafarge Vasavada PC. (1988). Pathogenic bacteria in V (2001). Implication of milk and milk milk.A review. J. Dairy Sci., 71:2809-2816. products in foodborne diseases in France and Zecconi A, Hahn G. (2000). Staphylococcus in different industrialized countries.Int J Food aureus in raw milk and human health risk. Microbiol 67:1-17. Bulletin of International Diary Federation 345, Jayarao, B.M., Pillai, S.R., Sawant, A.A., 15-18. Wofgang, D.R., Hedge, N.V. (2004).
JAFES, Vol 67, (2016) Journal of Agricultural, Food and Environmental Sciences UDC 632.95:543.544.5.068.7 Original scientific paper ______
QUANTITATIVE DETERMINATION OF 2,4-D IN PESTICIDES MONOSAN HERBI AND DMA-6
L. Velkoska-Markovska 1*, B. Petanovska-Ilievska1
1Faculty of Agricultural Sciences and food, “Ss. Cyril and Methodius” University in Skopje, Republic of Macedonia
*corresponding author:[email protected], [email protected]
Abstract A rapid and reliable method for determination of active ingredient 2,4-D ((2,4-dichlorophenoxy)acetic acid) in the pesticide formulations Monosan herbi and DMA-6 is presented. The procedure utilizes high-performance liquid chromatography (HPLC) followed by UV diode array detection and two analytical columns with different stationary phases and dimensions. The better results for identification and quantitation of the active ingredient in two pesticides are achieved using LiChrospher 60 RP-select B (250 x 4 mm, 5 µm) column, UV detection at 220 nm, temperature at 25 0C, mobile phase consisted of acetonitrile and water (60/40; V/V) and flow rate of 1 mL/min. The method is validated by testing linearity, precision, recovery, LOD and LOQ. The values for multiple correlation coefficient (R2 > 0.999), relative standard deviation (RSD) of retention time and peak area (RSD ≤ 1.18 %), recoveries ranged from 98.16 % - 101.38 %, with RSD of 0.10 % - 1.96 %, revealed that the developed method has a good linearity, precision and accuracy. The proposed method is applicable for fast and accurate determination of active ingredient 2,4-D in the pesticides Monosan herbi and DMA-6. Key words: HPLC-method, UV-detection, 2,4-D, Monosan herbi, DMA-6.
Introduction weeds in cereals, maize, sorghum, grassland, 2,4-D, (2,4-dichlorophenoxy)acetic acid established turf, grass seed crops, orchards (IUPAC) belongs to aryloxyalkanoic acid (pome fruit and stone fruit), cranberries, (phenoxy carboxylic) acid group of herbicides asparagus, sugar cane, rice, forestry, and non- (Figure 1a) that is used post-emergence for crop land (Tomlin, 1997). control of annual and perennial broad-leaved
O O
Cl O CH2 C Cl O CH2 C CH3
OH O NH2 Cl CH Cl 3 (a) (b) Figure 1. Chemical structure of 2,4-D (a) and 2,4-D-dimethyl ammonium salt (b) 2,4-D is an ingredient in approximately 660 Several products containing 2,4-D as an active agricultural and home use products, as a sole substance, including Monosan herbi and active ingredient and in conjunction with other DMA-6, which are in the form of a liquid active ingredients. 2,4-D is formulated solution concentrate (SL) are registered in R. primarily as an amine salt in an aqueous Macedonia. solution or as an ester in an emulsifiable The actual CIPAC (Collaborative International concentrate (EC), but, also exists in the form Pesticides Analytical Council) handbook of granular, soluble concentrate/solid, water (1985) referee method for determination of dispersible granules, and wettable powder 2,4-D is by reversed-phase HPLC, using 4- (EPA, 2005). bromophenol as an internal standard and UV detection at 280 nm. 63 L. Velkoska-Markovska , B. Petanovska-Ilievska ______
А chromatography is a widely used analytical Monosan herbi is 1.15 g/mL which is very method for the determination of 2,4-D and its close to the experimentally determined value residues in different matrices. For example, for of 1.16 g/mL. determination of 13 phenoxy acid herbicide The pesticide formulation DMA-6 (in the form residues in soybean is used a gas of a soluble concentrate (SL)) contains 67 % chromatography with an electron capture of 2,4-D as an active ingredient, in the form of detector (Huaet al., 2006). Ion dimethyl ammonium salt, manufactured by chromatography is employed for analysis of “Dow AgroSciences”, France. some pesticides, including 2,4-D in Equipment agrochemicals (Gangalet al., 2000). The The chromatographic analysis are performed determination of chlorophenoxy herbicides on an Agilent 1260 Infinity Rapid Resolution (2,4-D and related compounds) in biological Liquid Chromatography (RRLC) system specimens is performed by HPLC and UV equipped with: vacuum degasser (G1322A), detection at 240 nm (Flanagan and Ruprah, binary pump (G1312B), autosampler 1989). For determination of 2,4-D in (G1329B), a thermostatted column environmental water samples are used liquid compartment (G1316A), UV-VIS diode array chromatography-tandem mass spectrometry detector (G1316B) and ChemStation software. (LC-MS/MS) (Laganaet al., 2002; Rainaet al., For better dissolving of the stock solutions an 2010), HPLC and UV detection (Jafari and ultrasonic bath “Elma” is used. The Marofi, 2005) or HPLC-UV DAD investigations are carried out on a Purospher (Nestorovska-Krsteska et al., 2008). Velkoska- STAR RP-18e (30 mm x 4 mm, 3 m, Merck) Markovska and Petanovska-Ilievska (2013) and LiChrospher 60 RP-select B (250 mm x 4 have been developed RP-HPLC method for mm, 5 m, Merck) analytical columns. quantitative determination of 2,4-D in Preparation of Standard Solutions pesticide formulations by UV-DA detection. Stock solution of 2,4-D is prepared by Although, there are analytical methods for dissolving 0.0253 g of the pure analytical determination of 2,4-D in pesticide standard with acetonitrile in a 25 mL formulations and other matrices, constantly volumetric flask. The prepared solution is thinking about their improvement, or to create ultrasonicated for 15 min, and stored in a new analytical methods. For these reasons, the refrigerator at 4 oC. Stock solution is used to purpose of this paper is to investigate new prepare a series of 5 working solutions with opportunities for developing a suitable, simple different analyte concentrations (1.82 g/mL – and fast HPLC-method for determination of a 14.59 g/mL) in 10 mL volumetric flask by content of active ingredient 2,4-D in pesticide dilution with the mixture of acetonitrile/water formulations Monosan herbi and DMA-6 (50/50, V/V). using reverse-phase liquid chromatography Preparation of Sample Solutions (RP-HPLC) and UV diode array detector (UV- Sample solutions of pesticide formulations DAD). Monosan herbi and DMA-6 are prepared in 10 mL volumetric flasks by dissolving the Material and methods weighed amounts of 0.0096 g and 0.0072 g, Reagents and Chemicals respectively, in the mixture of equal volumes The Pestanal analytical standard of 2,4-D (98.6 of acetonitrile and water. The samples are % purity) and HPLC-grade acetonitrile and degassed for 15 min in an ultrasonic bath. methanol are purchased by Sigma-Aldrich From each sample solution 0.1 mL is (Germany). Ultrapure water is produced by transferred in a 10 mL volumetric flask and TKA Smart2 Pure 12 UV/UF water dissolved with the mixture of purification system (Germany). acetonitrile/water (50/50, V/V), and four The pesticide formulation Monosan herbi in injections are performed with5L each. The form of a soluble concentrate (SL) is procured sample solutionsare clear, therefore filtering is free of charge from Galenika-fitofarmacija not necessary. (Serbia). It is declared as containing 464 ± The solutions for recovery experiment are 23.2 g/L of 2,4-D (corresponding to the prepared by dissolving 0.1 mL from each concentration of 2,4-D-dimethyl ammonium sample solution in a 10 mL volumetric flask. salt of 588 ± 23.2 g/L). The declared value for In each solution is added a known amount of the density of the pesticide formulation
JAFES, Vol 67, (2016) 64 L. Velkoska-Markovska , B. Petanovska-Ilievska ______analyte (0.91 mg/mL, 1.82 mg/mL and 3.65 1994). Therefore, the further investigations are mg/mL) and diluted to volume with the same performed on LiChrospher 60 RP-select B solvent mixture. 5L of each of these (250 x 4 mm, 5 µm). LiChrospher 60 RP- solutions is injected four times. select B is a versatile reversed-phase sorbent based on spherical silica particles with Results and discussion excellent properties for the determination of Two analytical columns with different basic, neutral and acidic substances stationary phases and dimensions, such as (Chrombook, 2011). Purospher STAR RP-18e (30 x 4 mm, 3 µm) It is found that the optimum separation and and LiChrospher 60 RP-select B (250 x 4 symmetrical peak shape of the investigated mm, 5 µm), different mixtures of pesticide is achieved with mobile phase methanol/water (10 – 90 % methanol) and consisted of acetonitrile/water (60/40, V/V) in acetonitrile/water (10 – 90 % acetonitrile) as isocratic elution with flow rate of 1.0 mL/min mobile phases, at different column temperature and column temperature at 25 0C (Figure 3a). (20 – 30 0C) are used for identification and UV detection is performed at 220 nm. Under quantitation of the active ingredient 2,4-D in these chromatographic conditions, the two pesticides Monosan herbi and DMA-6. obtained values for column dead time is 1.09 Under the conditions tested on the Purospher min and the retention time of 2,4-D is 1.31 STAR RP-18e column the obtained min, so the calculated values for the retention chromatographic peak of 2,4-D is asymmetric, factor (k’) is 0.20. i.e. with tailing (Figure 2). There are many Specificity, selectivity, linearity, precision reasons for tailing phenomenon, such as expressed as repeatability of retention time and unsuitable choice of mobile or stationary peak area, limit of detection (LOD), limit of phases which can be remedied by change the quantification (LOQ) and accuracy are tested mobile and/or stationary phases (Meyer, for the method validation.
Figure 2. Chromatogram of 2,4-D obtained on the Purospher STAR RP-18e column
(a) (b)
JAFES, Vol 67, (2016) 65 L. Velkoska-Markovska , B. Petanovska-Ilievska ______
(c) Figure 3. Chromatograms of 2,4-D obtained from standard solution (a), pesticide formulation Monosan herbi (b) and pesticide formulation DMA-6 (c)
In addition, to confirm the specificity and there are no other coeluted peaks that interfere selectivity of the developed method, the UV on its determination. Moreover, the value of diode array detection is used to check the peak the match factor obtained by overlaid spectra purity and analyte peak identity (Jenkie, 1996). is 999.008 (for Monosan herbi) and 999.293 The specificity and selectivity of the (for DMA-6), indicating that the peak is of the developed method are estimated by identifying same substance. the peak of interest and value for the index of The calibration curve of 2,4-D is obtained with peak purity. triplicate injections (5 μL each) of working The identification of the analyte is performed solutions. The area and height of by comparing its retention time in the standard chromatographic peak and the corresponding solution and the sample and confirmed by amount of 2,4-D are used to construct the overlaid spectra of pure analytical standard of standard curve using the least-squares method. the active substance and the absorption spectra The curve followed Beer’s law in the of the same substance in pesticide mentioned range. The obtained results for formulations (Jenkie, 1996). multiple correlation coefficients (R2 ≥ 0.9995) As can be seen from the chromatograms of the indicated that the method has an excellent pesticides (Fig. 3b and c) besides the linearity. The results are given in Table 1. chromatographic peak of the active ingredient
Table 1. Results for linearity and sensitivity of the method Linearity range LOD LOQ Regression equation R2 (μg/mL) (ng/mL) (ng/mL) Area y = 3.983x +1.1818 0.9999 1,82 - 14,59 2.56 7.68 Height y = 0.3143x + 5.4309 0.9995
The limits of detection (LOD) is defined as the times and peak areas of the analyte from eight amount of analyte for which the signal-to- successive injections with concentration 7.30 noise ratio (S/N) is 3 whereas the limits of g/mL within 3 days (Table 2). The results are quantification (LOQ) is defined as the amount tested according to the criteria laid down in of analyte for which S/N =10. The LOD and CIPAC Document 3807 (2011). The obtained LOQ are listed in Table 1. values of RSD for retention times ranged from The precision is expressed as repeatability of 0.11 to 0.59 % and from 0.43 to 1.18 % for obtained results (Meyer, 1994; Lough and peak areas indicated a very good precision of Wainer, 1996) which is evaluated for retention the tested method.
JAFES, Vol 67, (2016) 66 L. Velkoska-Markovska , B. Petanovska-Ilievska ______
Table 2.Statistical data for repeatability Inter-day Intra-day repeatability (n = 8) repeatability (n = 3) I day II day III day R R R RSD x SD SD RSD SD RSD SD RSD (%) (%) (%) (%) Retention 1.31 0.005 0.41 1.30 0.003 0.23 1.31 0.001 0.11 1.31 0.008 0.59 time Peak 146.811.74 0.18 146.28 1.12 0.76 146.36 0.62 0.43 146.48 1.27 0.86 area
The accuracy of the method is confirmed by obtained values for recovery are within the standard additions (CIPAC, 2011; Snyder et following ranges (98.16 – 99.68 % for the al., 1997). Accuracy of the method is pesticide Monosan herbi, and 100.58 – 101.38 expressed as the deviation between the % for the DMA-6) which are according to calculated mean value obtained by CIPAC criteria (CIPAC, 2011). Consequently, examination and the true value of the spiked it is concluded that the proposed method is amounts of the analyte into a sample matrix accurate enough for determination of active that already contains some quantity of the ingredient in the pesticide formulations analyte (Table 3). As it is shown in Table 3 the Monosan herbi and DMA-6.
Table 3. Results from recovery (n = 4) m (analyte) before m (analyte) m (analyte) Recovery RSD addition added after addition (%) (%) (μg) (μg) (μg) (±SD) 17.46 4.56 21.70 ± 0.27 98.54 1.25 Monosan 17.46 9.12 26.50 ± 0.52 99.68 1.96 herbi 17.46 18.24 35.05 ± 0.35 98.16 0.99 18.70 4.56 23.39 ± 0.28 100.58 1.18 DMA-6 18.70 9.12 28.20 ± 0.28 101.38 0.98 18.70 18.24 37.16 ± 0.04 100.61 0.10
The obtained mean concentrations of 2,4-D in equation and repeatability of retention time the pesticide formulation Monosan herbi are and peak area. The developed method is 448.92 g/L (n = 4, RSD = 0.76 %), which is simple, fast, precise and accurate for a routine corresponding to the concentration of 2,4-D- analysis of active ingredient 2,4-D in the dimethyl ammonium salt of 568.89 g/L) and pesticide formulations Monosan herbi and 54.10 % (n = 4, RSD = 0.63 %), which is DMA-6 according to CIPAC rules. corresponding to the concentration of 2,4-D- dimethyl ammonium salt of 68.56 %. These References values corresponded to the values declared by CIPAC Document 3807. (2011). Guidelines on the manufacturer. method validation to be performed in support of analytical methods for agrochemical Conclusion formulations. http://www.cipac.org (01.10.11). This study shows the new possibility for CIPAC Method Handbook C (2.4-D). (1985). identification and quantitation of the active pp. 2062-2063. ingredient 2,4-D in the pesticides Monosan ChromBook. (2011). Your guide to a herbi and DMA-6 by the reversed-phase fascinating world of chromatography, Merck. HPLC-DAD method using LiChrospher 60 pp. 226-227 RP-select B column (250 x 4 mm, 5 µm). The EPA. Reregistration Eligibility Decision for proposed method showed high value of 2,4-D. (2005). multiple correlation coefficient for calibration
JAFES, Vol 67, (2016) 67 L. Velkoska-Markovska , B. Petanovska-Ilievska ______
Flanagan, R. J., Ruprah, M. (1989). HPLC Meyer, V. R. (1994). Practical High- Measurement of chlorophenoxy herbicides, Performance Liquid Chromatography. 2nd Ed. bromoxynil, loxynil, in biological specimens John Wiley & Sons. Chichester. England. pp. to aid diagnosis of acute poisoning. Clin. 1-267. Chem. No. 35/7, pp. 1342-1347. Nestorovska-Krsteska, A., Mirčeska, M., Gangal, N. D., Bondre, S. S., Ramanathan, P. Aaron, J. J., Zdravkovski, Z. (2008). S. (2000). Determination of some pesticides Determination of dimethoate, 2,4- and intermediates by ion chromatography. dichlorophenoxy acetic acid, mecoprop and Journal of Chromatography A. No. 884, pp. linuron pesticides inenvironmental waters in 243-249. Republic of Macedonia by high performance Hua, K., Xiao-gang, C., Yu-xia, H., Chuan-lai, liquid chromatography. Maced. J. Chem. X. (2006). Simultaneous determination of 13 Chem. Eng., Vol. 27, No. 1, pp. 25-33. phenoxy acid herbicide residues in soybean by Raina, R., Etter, M. L. (2010). Liquid GC-ECD. Analytical letters. Vol. 39. No. 13, chromatography with post-column reagent pp. 2617-2627. addition of ammonia in methanol coupled to Jafari, A. J., Marofi, S. (2005). Photo-chemical negative ion electrospray ionization tandem degradation of 2,4-Dichlorophenoxyacetic acid mass spectrometry for determination of (2,4-D) in the effluent. J. Res. Health Sci. Vol. phenoxyacid herbicides and their degradation 5, No. 2, pp. 27-31. products in surface water. Analytical Jenkie, D. R. (1996). Chromatographic Chemistry Insights. No. 5, pp. 1-14. Method Validation: A Review of Current Snyder, R. L., Kirkland, J.J., Glajch, L. J. Practices and Procedures. I General Concepts (1997). Practical HPLC Method Development. and Guidelines. J. Liq. Chrom. & Rel. 2nd Ed. John Wiley & Sons. Inc. New York. Technol. No. 19 (5), pp. 737-757. pp. 688-689. Lagana, A., Bacaloni, A., De Leva, I., Faberi, Tomlin, C. (1997). The Pesticide Manual A., Fago, G., Marino, A. (2002). Occurrence Incorporating the Agrochemicals Handbook. and determination of herbicides and their 11th Edition. Crop Protection Publications. pp. major transformation products in 271-274. environmental waters. Analytica Chimica Velkoska-Markovska, L., Petanovska-Ilievska, Acta. No. 462, pp. 187–198. B. (2013). RP-HPLC method development for Lough, W. J., Wainer, I. W. (1996). High quantitative determination of 2,4-D in Performance Liquid Chromatography. pesticide formulations. Plant Protection. No. Fundamental Principles and Practice. Blackie 26/27 (XXIV). pp. 145-151 Academic & Professional. Chapman & Hall. Glasgow. UK. pp. 1-167.
JAFES, Vol 67, (2016) Journal of Agricultural, Food and Environmental Sciences UDC 632.79(560) Original scientific paper ______
THE ALTERNATİVE HOSTS OF TRİSSOLCUS SPECİES, EGG PARASİTOİDS OF SUNN PEST AND HOST-PARAZİTOİD İNTERACTİONS İN SOUTH EASTERN ANATOLİA REGİON, TURKEY
C. Gözüaçık1*, A. Yiğit2*
1 Igdir University Faculty of Agriculture Deparment of Plant Protection, Iğdır-Turkey 2 Mustafa Kemal University, Faculty of Agriculture, Deparment of Plant Protection, Hatay-Turkey
*corresponding author: [email protected]; [email protected]
Abstract Trissolcus species (Hymenoptera, Scelionidae) are important egg parasitoids of Sunn pest, Eurygaster integriceps Put. (Heteroptera, Scutelleridae). They need alternative host eggs to survive in the nature, at the end of the wheat growing season. This study was carried out to determine the alternative hosts of Trissolcus species and host-parasitoid interactions in laboratory and different ecosystems in Adıyaman, Diyarbakır and Şanlıurfa provinces during 2007-2009. The parasitoid adults were reared from Scutelleridae and Pentatomidae (Heteroptera) eggs, collected from wild plants and kept in tubes in the laboratory at 25±1ºC and 65±5% r.h. In this study, it was determined that 23 species belonging to Pentatomidae and Scutelleridae are alternative egg-hosts of Trissolcus spp. 13 species of which have been identified as the first time for alternative hosts of Trissolcus species. Key words: Trissolcus species, alternative hosts, Southeastern Anatolia Region, Turkey.
Introduction Öncüer & Kıvan, 1995; Koçak and Kılınçer One of the factors limiting the wheat 2002). Common and dominant ones of these production which is an essential source for species are T. semistriatus, T. grandisandT. human nutrition is phytophagous insects. Vassilievi (Yüksel, 1968; Kıvan, 1992; Şimşek Among these insects Sunn pest, Eurygaster et al.,1994; Melan, 1994; Memişoğlu et al., spp. (Heteroptera: Scutelleridae) is the most 1994; Tarla, 1997; Doğanlar, 1999; Koçak and injurious crop pest in Turkey. Considerable Kılınçer 2001; Kodan, 2007; Gözüaçık and yield losses occur every year in case of this Yiğit, 2011). It has been reported that T. pest is not controlled. Egg parasitoids from semistriatus reproduces approximately 3 times Scelionidae (Hymenoptera), adult and nymph during oviposition of Sunn pest in wheat fields parasitoids belonging to Tachinidae (Diptera), (Lodos, 1961). In Thrace Region, the and numerous polyphagous predators are parasitoid coming from winter quarters to reported within the natural enemy complex of wheat fields reproduces 9 times during the Sunn pest (Waage, 1998). The egg parasitoids, oviposition period of Sunn pest in the field Trissolcus species, are the most effective ones conditions and laboratory (Kıvan, 1998). among the natural enemies of the pest(Brown, Lazarov et al., (1969) stated that the parasitoid 1962; Safavi, 1968; Yüksel, 1968; Barbulescu, reproduced 7 times in nature and 8 times in 1971; Popov and Paulian, 1971; Semyanov et. laboratory during summer season. Alternative al., 1981; Lodos, 1982; Novozhilov and hosts of Trissolcus have a quite significant Dzyuba, 1983). T. anitus, T. basalis, T. place in maintaining their life-span in nature choaspes, T. culturatus, T. festivae, T. grandis, during the period when eggs of Sunn pest do T. histani, T. rufiventris, T. pseudoturesis, T. not exist in nature. Numerous Scutelleridae rungsi, T. reticulatus, T. scutellaris, T. and Pentatomidae (Hemiptera) species of are semistriatus, T. simoni, and T. vassilievi are known to be alternative hosts for T. identified as egg parasitoids of the pest in semistriatus (Kılıç et al., 1980; Kıvan 1998; Turkey (Brown,1962; Yüksel, 1968; Kılıç et Tarla & Doğanlar 1999; Kodan et al., 2007; al., 1980; Dikyar, 1981; Şimşek & Sezer, Çetin et al., 2009; Gözüaçık & Yiğit, 2011). 1985; Akıncı and Soysal, 1992; Melan, 1994; This study was conducted at different Memişoğlu & Özer, 1994; Şimşek et al., 1994; ecosystems and laboratories in order to
JAFES, Vol 67, (2016) 69 C. Gözüaçık, A. Yiğit ______determine Pentatomidae and Scutelleridae the incubator adjusted to 25±1ºC and %65±5 (Heteroptera) species, which are the alternative RH. Emergedadult parasitoids were kept in hosts of Trissolcus, in terms of contributing absolute alcohol (96%) for identification. the egg parasitoids of Sunn pest, T. Trissolcus species were identified according to semistriatus, T. grandis and T. vassilievi to Doğanlar (2001) and Koçak & Kılınçer maintain their existence in nature during the (2003). seasons after wheat harvesting in Adıyaman, Scutelleridae and Pentatomidae adults, Diyarbakır, and Şanlıurfa provinces of collected from field and/or reared from Southeastern Anatolia Region during the years nymphs were killed with ethyl acetate for of 2007-2009. identification. The samples were identified by Assoc. Prof. Dr. Meral Fent (Trakya Material and methods University, Faculty of Science, Department of Field studies Biology, Edirne, Turkey). Adult individuals, nymphs, and eggs of Laboratory studies Scutelleridae and Pentatomidae species were In order to determine the alternative hosts of collected from weed/wild plants, trees, and Trissolcus species, Scutelleridae and cultivated plants present in lands neighboring Pentatomidae adults collected from different to wheat fields in the provinces of Adıyaman, ecosystems were cultured with their native host Diyarbakır, and Şanlıurfaat intervals of 4-7 plants in plastic culture containers (23x23x30 days during May-November in 2007-2009. cm) in the laboratory (Table1). Foods in plates The eggs were cultured in the glass tubes were changed daily. (2x10cm) reserved with cotton and labeled, in
Table1. Some Pentatomidae and Scutelleridae species to be alternative hosts for Trissolcus and their host plants in laboratory Pentatomidae and Scutelleridae Host Plants species Carpocoris pudicus Centaurea solstitialis L. Anchyrosoma leucogrammes Daucus carota L. Eurydema ornate Sinapis arvensisL.+Brassica oleracea L. Dolycoris baccarum S. arvensis + B. oleracea Piezedorus lituratus Lens culinaris M.+Trifolium spp. Holcostethus vernalis Helianthus annuus + Trifolium spp. Codophila varia Centaurea calcitrapa L.+ Carduus pycnocephalus L. Graphosoma semipunctatum D. carota + Conium maculatum L. Acrosternumbreviceps Capparis spp. Bagrada abeillei S. arvensis + Sysmbrium sp. Odontotarsus obseletus C. calcitrapa + C. pycnocephalus Pasias martini Morus spp. Staria lunata C. pycnocephalus
Egg packs obtained daily from cultures were Trissolcus semistriatus Nees, T. vassilievi attached to cardboards (1x5 cm) and put in test Mayr and T. grandis Thomson (Hymenoptera: glass tubes (2x10 cm). Females of T. Scelionidae), the egg parasitoids of Sunn pest, semistriatus, T. grandis, and T. vassilievi were Eurygaster integriceps Put. (Hemiptera: confined with Sunn pest egg packs within the Scutelleridae)in laboratory and in non- tubes separately for parasitization. They also agricultural lands of Adıyaman, Diyarbakır are provided as food with honey droplets on a and Şanlıurfa during the years of 2007-2009 small strip of wax paper. Studies were (Table 2 and 3). conducted as two repeats in an incubator In order to determine the alternative hosts of adjusted to 25±1ºCand %65±5 R.H. Trissolcus semistriatus, T. grandisand T. vassilievi, a total of 1019 eggs belonging to C. Results and discussion pudicus (n=48), A. leucogrammes (n=60), E. Twenty three species belonging to ornata (n=232), D. baccarum (n=97), P. Pentatomidae and Scutelleridae (Heteroptera) lituratus (n=60), H. vernalis (n=60), C. varia were identified as alternative hosts of (n=100), G. semipunctatum (n=107), A.
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70 C. Gözüaçık, A. Yiğit ______breviceps (n=120), B. abeillei (n=24), O. C. purpureipennis, C. pudicus, Aelia rostrata, obseletus (n=42), P. martini (n=57), and S. Eurydema ornata, E. blandum, E. festivum, E. lunata (n=12) from Pentatomidae and oleracearum, E. ventrale, D. baccarum, N. Scutelleridae were used in the laboratory. As a viridula, R. nebulosa, P. lituratus, N. viridula, result of experiments, T. semistriatus and T. Codophila pusia, Graphosoma lineatum, G. grandis females parasitized the eggs belonging stali, G. semipunctatum and H.vernalis were to 12 species, and T. vassilievi parasitized the reported as alternative host of egg parasitoids eggs belonging to 11 species from of Sunn pest in Turkey (Memişoğlu and Melan Pentatomidae and Scutelleridae(Table 2). T. 1998; Tarla and Doğanlar 1999; Kıvan and semistriatus parasitized the eggs of all the Kılıç 2002, 2003; Kodan, 2007; Çetin et al., species except for C. iranus, A. lunata and R. 2014). nebulosa;T. grandis parasitized the eggs of all In the studies conducted in nature, totally the species except for C. pudicus,C. iranus,E. 20838 eggs from C. pudicus (n=28),C. ornata, D. flavoguttata, A. lunata, H. vernalis, fuscispinus (n=14), C. iranus (n=14), A. A.heegeri, B. amoenula,R. nebulosa, O. leucogrammes (n=7680), E. ornata (n=2112), plicatulus, O. robustus, S. lunata and V. Derula flavoguttata (n=12), D. baccarum horvathi; and T. vassilievi parasitized the eggs (n=2200), P. lituratus (n=66), A. lunata of all the speciesexcept for C. fuscispinus, E. (n=96), H. vernalis (n=60), C. varia (n=240), ornata, D. flavoguttata, A. lunata,H. vernalis, G. semipunctatum (n=588), A. breviceps G. semipunctatum, B. abeillei, B. amoenula, R. (n=5796), A.heegeri(n=240), B. abeillei nebulosa, O. obseletus, O. plicatulus, O. (n=1056), B. amoenula (n=60), R. nebulosa robustus, S. lunata, and V. horvathi (Table 3). (n=14),O. obseletus (n=224), O. plicatulus The reason behind why Trissolcusspecies did (n=56), O. robustus (n=42), P. martini not parasitize some Pentatomidae and (n=168), S. lunata (n=12), and V. horvathi Scutelleridae eggs were thought to be the eggs (n=60) were found on host plants belonging to was not suitable for development of parasitoid Poaceae, Asteraceae, Brassicaceae, and the number of eggs collected failed to Scrophulariaceae, Apiaceae, Caryopyllaceae, satisfy. In related studies conducted in Ranunculaceae, Verbenaceae, Lamiaceae, different countries, Troilus luridus (Fabricius), Euphorbiaceae, and Boraginaceae (Table 4). T. A. acuminata (Linnaeus), A. cognata Fieber, semistriatus, T. grandis and T. vassilievi were A. furcula Fieber, A. germari Küster, A. reared from eggs of 20, 11 and 9 Pentatomidae rostrata Boheman, A. virgata (Herrich- and Scutelleridae species, respectively. Schäffer), Carpocoris pudicus (Poda), Regarding the combined results of field and Dolycoris baccarum (Linnaeus), Holcostethus laboratory A. breviceps, A.heegeri, A.millerei, strictus vernalis (Wolff), Palomena prasina A. lunulata, A. leucogrammes, B. abeillei, B. (Linnaeus), Eurydema ornata (Linnaeus), E. amoenula, C. varia, C. pudicus,C. fuscispinus, ventralis Kolenati, Graphosoma lineatum C. iranus, D. baccarum, E. ornatatum, H. (Linnaeus), Ventocoris fischeri (Herrich- strictus vernalis, S. lunata,P. martini, P. Schäffer) and Odontotarsus impictus Jakovlev lituratus, R. nebulosa,D. flavoguttata, and V. were reported as hosts of T. semistriatus Aelia horvathi from Pentatomidae and O. plicatulus, cognata Fieber, A. germari, C. pudicus, D. O. robustus, and O. obsoletus from baccarum, D. indicus Stål, Chlorochroa Scutelleridae were found to be as alternative pinicola (Mulsant and Rey), Palomena prasina hosts of T. semistriatus, T. grandis and T. (Linnaeus), A. acuminata, E. ornata, E. vassilievi. Among these species A. breviceps, ventralis, G. lineatumas hosts of T. grandis; A.heegeri, A. lunulata, B. abeillei,C. varia, S. and A. acuminata, A. furcula, A. rostrata, C. lunata, P. martini,A. leucogrammes, D. pudicus and D. baccarum as hosts of T. flavoguttata, V. horvathi O. plicatulus, O. vassilievi (NDSU, 2015). Furthermore, A. robustus and O. obsoletus weredetermined as acuminata, C. fuscispinus, C. mediterraneus, new alternative hosts for Trissolcus species.
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Table2.Parasitization status of Trissolcus semistriatus, T. grandis andT. Vassilievi on eggs of some Scutelleridae and Pentatomidae species under laboratory conditions Family Species T. semistriatus T. grandis T. vassilievi Pentatomidae Acrosternumbreviceps + + + Anchyrosoma leucogrammes + + + Bagrada abeillei - + Ω Carpocoris pudicus + + + Codophila varia + + + Dolycoris baccarum + + + Eurydema ornata + + + Graphosoma semipunctatum + + + Holcostethus strictus vernalis + + + Pausias martini + + + Piezedorus lituratus + + + Staria lunata + Ω Ω Scutelleridae Odontotarsusobseletus + + + +: Parasitizing, -: Not parasitizing, Ω: No given eggs
Table 3.Parasitizing status of Trissolcus semistriatus, T. grandis andT. vassilievi species on eggs of some Scutelleridae and Pentatomidae species in non-agricultural lands of Adıyaman, Diyarbakır and Şanlıurfa during the years of 2007-2009 Family Species T. semistriatus T. grandis T. vassilievi Acrosternumbreviceps + + + A.heegeri + - + Anchyrosoma leucogrammes + + + Antheminia lunulata - - - Bagrada abeillei + + - B. amoenula + - - Carpocoris pudicus + - + C. fuscispinus + + - C. iranus - - + Codophila varia + + + Pentatomidae Derula flavoguttata + - - Dolycoris baccarum + + + Eurydema ornata + - - Graphosoma semipunctatum + + - Holcostethus strictus vernalis + - - Pausias martini + + + Piezedorus lituratus + + + Raphigaster nebulosa - + - Staria lunata + - - Venthocoris horvathi + - - Scutelleridae Odontotarsusobseletus + + - O. plicatulus + - - O. robustus + - - +: Parasitizing, -: Not parasitizing
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Table 4. Pentatomidae and Scutelleridae species, alternative hosts of Trissolcus spp. and their host plants found in Adıyaman, Diyarbakır, and Şanlıurfa Pentatomidae and Scutelleridae species Host plants Punica granatum L., Rosa canina L., Lycoperscium esculentum L., Morus sp., Acrosternum breviceps Phaseolus vulgaris L., Capparis spinosa L., Medicago sp. P. granatum, R. canina, Lycoperscium esculentum L., Morus sp., P. vulgaris,C. Acrosternumheegeri spinosa, Medicago sp. Acrosternummilleri P. granatum, R. canina, L. esculentum, Morus sp., P. vulgaris, C. spinosa Bagrada abeillei Brassica napus var. Oleifera D.C., Sysmbrium officinale L. C.Spinosa,EryngiumcampestreL., Sinapis arvensis L. Bagrada amoenula B. napus., S. officinale, C. spinosa, E. campestre, S. arvensis Helianthus annuus L.,E. creticum, E. campestre, Centaurea calcitrapa L., Carpocoris pudicus Echinops microcephalus Sibth and Sm., Centaurea iberica Trever ex Sprengel, C. solstitialis L., Carduus pycnocephalus L., Trifolium spp. H. annuus, E. creticum, E. campestre, C. calcitrapa, E. microcephalus, C. iberica, Carpocoris fuscippinus C. solstitialis, C. pycnocephalus, Trifolium spp. Helianthus annuus L., Sesamum indicum L., E. creticum, E. campestre, Echinops Codophila varia ritro L., Scolymus sp. C. iberica, C. solstitialis,C. pycnocephalus Antheminia lunulata Euphorbia helioscopia L. Lens culinaris Medik., B. napus, S. indicum, Ervum ervilia L., Silene colorata Dolycoris baccarum Poir., Onopordum acanthium L., Onopordum carduchorum, Verbascum thapsus L., S. arvensis,S. officinale,Gundelia sp. Piezedorus lituratus L. culinaris, M. sativa, E. ervilia, Trifolium spp. Eurydema ornate B. napus, S. arvensis, Lepidum sativum L.,Cardaria draba (l.) Desv., Crambe orientalis L., S. officinale Holcostethus vernalis Morus sp., M. sativa, Trifolium sp. Anchyrosoma leucogrammes Daucus carota L.,E. creticum, E. campestre Derula flavoguttata Verbena officinalis L. Graphosoma semipunctatum E. creticum, E. campestre,D. carota, Conium maculatum L. Staria lunata C. pycnocephalus Pausias martini Morus spp. Ventocoris horvathi Chondrilla sp. Odontotarsus plicatulus C. calcitrapa, C. iberica, C. pycnocephalus, E. creticum, E. campestre Odontotarsus robustus C. calcitrapa, C. iberica, C. pycnocephalus, E. creticum, E. campestre Odontotarsus obsoletus S. indicum, C. calcitrapa, C. iberica, C. pycnocephalus, E.creticum,E. campestre
Conclusions alternative host species for Trissolcus spp. As a result of three-year studies, 23 species belong to Pentatomidae and Scutelleridae and belong to Pentatomidae and Scutelleridae were their host plants which they feed from and found in Adıyaman, Diyarbakır, and Şanlıurfa shelter on. as alternative hosts for T. semistriatus, T. It is considered that taking precautions grandis, and T. vassilievi, egg parasitoids of regarding to form a green zone is necessary by Sunn pest. Thirteen of these species were composed of the host plants above determined determined as newhosts of Trissolcus spp. species, afforest and protect the wild plants As a result of the prohibition of aerial pesticide around the wheat fields for the mentioned applications for Sunn pest control in recent parasitoid species to over winter, shelter, feed, years in Turkey, biological control approach is and find their alternative hosts to realize the emphasized for control of the pest. In order to natural biological control of Sunn pest increase the efficiency of egg parasitoids in throughout the year. nature bio-diversity attempts came into prominence to support the surviving and References increasing the efficacy of the egg parasitoids, Akıncı, A. R. & Soysal, A.(1992).Trakya Trissolcus spp. throughout the year under field Bölgesi’nde Süne (Eurygaster spp.)'nin conditions. Due to these reasons it has become yumurta parazitoitleri ve etkinlikleri üzerinde gradually more important to determine the araştırmalar. Uluslararası Entegre Zirai
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Mücadele Sempozyumu Bildirileri (15-17 araştırılması.Diyarbakır Bölge Zirai Mücadele Ekim 1992, İzmir/Türkiye), Gen Matb. ve Araştırma Enstitüsü.14 s. Rek. Tic. Ltd. Şti.,Ankara, 258 pp. Kıvan, M.(1992).Tekirdağ ve çevresinde Barbulescu, A. (1971). The role of egg Eurygaster spp. (Heteroptera: Scutelleridae) parasites in the populationdynamics of cereal türleri, tanınmaları ve yayılışları ile bunlardan bugs (Eurygaster sp.). Analee-Instutului-de- Eurygaster integriceps Put.‘in biyolojisi ve Cercetari-pentru-Protectia-Plantelor, 7: 159- doğal düşmanları üzerinde araştırmalar. Ege 164 (CAB, 1971). Üniv. Fen Bilimleri Enst. Bitki Koruma Brown, E., S. (1962).Notes on parasites of Anabilim dalı, (Ph.D. thesis, Unpublished). Pentatomidae and Scutelleridae (Hemiptera- İzmir,106pp. Heteroptera) in Middle East Countries, with Kıvan, M.(1998).Eurygaster integriceps Put. observations on biological control. Bulletin of (Heterotera, Scutellerdae)’nin yumurta Entomology Research, 53: 241–256. parazitoiti Trissolcus semistriatus Nees Çetin, G., Koçak E.& Hantaş C. (2009).Güney (Hymenoptera: Scelionidae)’un biyolojisi Marmara Bölgesi Hububat Ekosistemindeki üzerinde araştırmalar. Türk.entomol.derg., 22 Hemipterler ve Yumurta Parazitoitleri Üzerine (4): 243-257. Bir Çalışma. Türkiye III. Bitki Koruma Kıvan M. & Kılıç, N.(2002).Host preference: Kongresi (15-18 Temmuz 2009, Van)p. 323 parasitism, emergence and development of Çetin,G., KoçakE.&Hantaş C.(2014). Bursa ve Trissolcus semistriatus (Hym., Scelonidae) in Yalova illeri buğday tarlaları ve ağaçlık various host eggs. J. Appl. Ent. 126: 395–399. alanlardaki süne yumurta parazitoit Kıvan, M. & Kılıç, N. (2003). Influence of (Hymenoptera: Scelonidae) türleri, bulunma host species and their ages on host oranları ve popülasyon takibi. Derim, 31(1): repreference of Trissolcus semistriatus. 35-49. BioControl, 49 (5): 1-10. Doğanlar, F.(1999). Hatay ilinde bulunan Koçak, E., &Kılınçer N. (2001). Türkiye Süne Proctotrupoidea üst familyasına bağlı [Eurygaster spp. (Het.:Scutelleridae)] yumurta Scelionidae familyasına giren türler ve parazitoidi Trissolcus (Hym.:Scelionidae) kısa biyolojileri (Insecta: Hymenoptera). türleri. Bitki Koruma Bülteni, 41 (3-4): 167 Mustafa Kemal Üniversitesi, Fen Bilimleri 181. Enstitüsü, MSc Thesis, Antakya, 58 pp. Koçak, E. & Kılınçer, N. (2002). The Doğanlar, M.(2001). Parasitic Hymenoptera: taxonomy and morphology of Trissolcus spp. Taxonomy and biological control International (Hymenoptera: Scelionidae) parasitoids on the Symposium (14-17 May 2001, Kőszeg, eggs of Sunn pest (Eurygaster spp., Hungary).p. (?) Scutelleridae, Hemiptera), across Turkey.Eggs Gözüaçık, C. & Yiğit A. (2011).Süne, Parasitoids 6th International Symposium, 15 Eurygaster integriceps Put.yumurta 18 September, p 38, Perugia, Italy. parazitoitleri, Trissolcus spp.’nin iki Koçak, E. & Kılınçer, N.(2003). Taxonomic ekosistemde etkilerinin karşılaştırılması. on studies on Trissolcus sp. (Hym.: Türkiye IV. Bitki Koruma Kongresi Bildirileri Scelionidae) egg parasitoids of Sunn Pest (28–30 Haziran 2011, Kahramanmaraş, (Hem.; Scutelleridae, Eurygaster sp.), in Türkiye),p.118 Turkey.Turk. J. Zool. 27: 301-317. Gözüaçık, C. (2011). Güneydoğu Anadolu Kodan, M., Koçak, E., Candan, S. & Yıldırım, Bölgesi’nde Süne, Eurygaster integriceps Put. A.(2007).Orta Anadolu Bölgesi’nde yabancı (Heteroptera: Scutelleridae)’in Yumurta otlarda bulunan Pentatomidae türleri ve Parazitoitleri, Trissolcus spp. (Hymenoptera: yumurta parazitoitleri.VII.Ulusal Ekoloji ve Scelionidae)’nin Pentatomid ve Scutellerid Çevre Kongresi (10-13 Eylül 2007, Malatya). konukçuları, doğada parazitoit/konukçu Lazarov, A., Grigorov, S., Popov, V., ilişkileri ve bunun Süne populasyonları ve Bogradov, V., Abaciev, D., Kontev, H., zararı üzerine etkileri.Mustafa Kemal Kayzatov, H., Gospodinov, H., Fitanov, H.& Üniversitesi, Fen Bilimleri Enstitüsü, PhD Duçevski, D. (1969). Bulgaristan'da Thesis, Antakya (Türkiye), 255 pp. buğdaygillerde zarar yapan Scutelleridae ve Kılıç, A. U., Çatalpınar, A.& Adıgüzel, N. Pentatomidae (Hemiptera) familyalarına bağlı 1980. Güneydoğu Anadolu Bölgesi’nde türlerin biyo-ekolojisi ve mücadelesi üzerine Süne(Eurygaster integriceps Put.) üzerinde çalışmalar (Çeviri: Musa Altay),144 pp. entegre mücadele imkanlarının
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Lodos, N., (1961).Türkiye, Irak, İran ve Noveishiedostizheniya- Suriye’de Süne ( Eurygaster integriceps Put ) sel'skokhozyaistvennoi-entomologii-po- problemi üzerinde incelemeler. Ege materialam-Ush-sezda-VEO-Vilnyus,9-13- Üniversitesi Ziraat Fakültesi Yayınları, No:51, oktyabrya-1979-g, 184-186 115 pp. Şimşek, N. & Sezer A.C.(1985).Hatay ilinde Lodos, N.(1982). Türkiye Entomolojisi II buğdayda Süne (Eurygaster (Genel, Uygulamalı ve Faunistik) E.Ü. Ziraat integricepsPut.)'nin yumurta ve nimf Fak.Yay., No: 429, 591 pp. popülasyonu ile zararı üzerinde ön çalışmalar. Melan, K.(1994). Trakya Bölgesi’nde Süne Bitki Koruma Bülteni, 25(1-2): 30-48. türleri ve Süne yumurta parazitoitleri, Türkiye Şimşek, Z., Yılmaz, T.& Yaşarakıncı, N. III. Biyolojik Mücadele Kongresi (25-28 Ocak (1994). Güneydoğu Anadolu Bölgesinde Süne 1994, İzmir), Ege Üniversitesi Basımevi, pp. (Eurygaster integriceps Put.) ile yumurta 147-154. parazitoiti (Trissolcus semistriatus Nees)’nin Memişoğlu, H., Özer, M. (1994). Ankara populasyon gelişmeleri üzerinde araştırmalar. İlinde Avrupa Sünesi (Eurygaster maura L., Türkiye III. Biyolojik Mücadele Kongresi (25- (Hemiptera: Scutelleridae)'nin doğal 28 Ocak 1994, İzmir),Ege Üniversitesi düşmanları ve etkinlikleri. Türkiye III. Basımevi, pp. 165-175. Biyolojik Mücadele Kongresi(25-28 Ocak Tarla, Ş.(1997).Antakya ve çevresinde Süne, 1994, İzmir), Ege Üniversitesi Basımevi, pp. (Eurygaster integriceps Put.) yumurta 175-186. parazitoitlerinin tespiti ve bunların kitle üretim Memişoğlu, H.&Melan K. (1998). Türkiye’de olanakları üzerinde araştırmalar.Mustafa Kemal Süne’nin doğal düşmanları.I. Üniversitesi, Fen Bilimleri Enstitüsü,Bitki WorkshopRaporu, (6-9 Ocak 1998, Ankara). Koruma Anabilim Dalı, (M.Sc. thesis), 57 pp. Zirai Mücadele Merkez Araştırma Enstitüsü, Tarla, Ş. & Doğanlar, M.(1999). Hatay ilinde pp. 85-92. Süne, Eurygaster integriceps Put.(Heteroptera: Novozhilov, K.V. &Dzyuba Scutelleridae) yumurta parazitoitleri, bunlara Z.A.(1983).Effectiveness of field populations alternatif konukçu olan pentatomid türleri ve bu of natural enemies of the Sunn pest türlerin konukçu bitkileri. Türkiye IV. (Eurygaster integriceps Put) in the steppe zone Biyolojik Mücadele Kongresi Bildirileri, (26- of the Krasnodar region. Biotsenoticheskoe 29 Ocak 1999, Adana) Ç. Ü. Zir.Fak. Bitki obosnovanie kriteriev effektivnosti prirodnykh Koruma Böl. Ç. Ü. Basımevi, pp. 97-106. Entomofagov, 51-55 Waage, J.K. (1998). Prospects for NDSU, (2015). Augmentation of Egg Parasitoids for http://www.ndsu.nodak.edu/ndsu/rider/Pentato Management of Sunn Pest, Eurygaster moidea/Natural_Enemies/parasitoid_Hymen_P integriceps and related species.Integrated Sunn ent.htm Pest Control. Ankara Plant Protection Central (Access to: 20.6.2015) Research Institute (6-9 January 1998, Ankara). Öncüer, C. &Kıvan, M. (1995). Tekirdağ ve Yüksel, M.(l968). Güney ve Güneydoğu çevresinde Eurygaster (Het.: Scutelleridae) Anadoluda Süne(Eurygaster integriceps türleri, tanımları, yayılışları ve bunlardan Put.)'nin yayılışı, biyolojisi, ekolojisi, Eurygaster integriceps Put.’in biyolojisi ve epidemiyolojisi ve zararı üzerinde doğal düşmanları üzerinde araştırmalar. araştırmalar. T.C. Tarım Bakanlığı, Zir. Türkiye Tarım ve Orman Dergisi, 19(4): 223- Müc.ve Zir. Kar. Gn. Md. yayınları No: 46, 230. Teknik Bülten, Ankara, 255 pp. Popov, C.& Papulian, F. (1971). Present possibilities of using parasites in the control of cereal bugs. Probleme-Agricole, 23: 53-61. Safavi, M. (1968). Etude biologique et ecologique des hymenopteres parasites des eufs des punasies des cereals.Entomophaga,13 (5): 381-495. Semyanov, V.P., Tilmenbaev A.T., Beksultanov S.Z. & Sarbaev, A.T. (1981).The main elements of integrated control of the Sunn pest in Kazakhstan.
JAFES, Vol 62, (2016)
Journal of Agricultural, Food and Environmental Sciences UDC 633.1-276(564.3-17) 632.76:633.1(564.3-17) Original scientific paper ______
THE DISTRIBUTION AND POPULATION DENSITY OF THE CEREAL WEEVIL, PACHYTYCHIUSHORDEI (BRULLÉ) (COLEOPTERA: CURCULIONIDAE) IN CEREAL FIELDS IN NORTHERN CYPRUS
M. Güllü1*, C. Gözüaçik2, A. Konuksal3, H. Hekimhan4, H. Fidan1
1Biological Control Research Institute, Department of Entomology, P. O. BOX: 21, 01321 Adana/Turkey 2Iğdır University, Faculty of Agriculture, Department of Plant Protection Iğdır/Turkey 3Agricultural Research Institute, Lefkoşa (Nicosia)/TRNC 4Egean Agricultural Research Institute, Menemen-İzmir/Turkey
*corresponding author: [email protected]
Abstract
The Cereal weevil, Pachytychiushordei (Brulle) (Coleoptera: Curculionidae) is an important pest of barley and wheat. This study was conducted to determine the distribution areas and the population density of P. hordeiin Northern Cyprus. The sampling were conducted in 57 cereal fields of 46 villages in Lefkoşa, Girne, Güzelyurt, Gazimağusa and İskele districts in 2012-2013 years. Sweep net were used in samplings. At the end of the study, P. horde ihas been found common all in regions and the highest population densities were 379 adults/100 sweepnet and 247 adults/100 sweep net in Hisarköy/Girne in the years 2012-2013 respectively. In addition, damage states of P. hordeiwas evaluated. Key words: Cereal, Cereal weevil, Pachytychiushordei, distribution, population density, Northern Cyprus.
Introduction exist in Cyprus fauna and spread across the Cereals are the leading agricultural products in whole island; in addition Pachytychius Northern Cyprus agriculture with 84,163 basimaculatus Voss, 1964 sub-species exists hectares of cultivated land. Barley is cultivated as well, albeit limited to Paphos and Larnaca in 92.7% of the total cereal fields, whereas regions (Alziar, 1995; Alziar, 2010). Cereal wheat is cultivated in 6.4% and oat and weevil, P. hordei adults are 2-4 mm long, triticale are cultivated in 0.9%, which in total bright black or brown insects with whitish- yield 134,149 tons of grain (Anonymous, cream hairs or scales on its elytra 2011). Most of the cultivated barley is used as (Anonymous, 2008; Alziar, 2010) (Fig. 1a). feed in animal farming industry, and wheat is Adult individuals exit from the soil during the utilized as human nutrition. Thus, cereal days when weather temperature is 12-14⁰C production is of utmost importance for North and relative humidity is 57-80% in barley and Cypriot farmers, especially the barley. As in wheat fields and feed on the cereal stem and any product, there are various species of pests leaves (Şimşek, 1993). Three adjacent that affect cereals both in pre-harvest and post- characteristic small round holes similar to harvest periods, causing different levels of staple holes occur on the leaf surface as a harm. One of these pests is the Cereal weevil, result of the feeding of P. Hordeiadults (Fig. Pachytychius hordei (Brullé, 1832) 1b). It was reported that adults also feed on (Coleoptera: Curculionidae) that harms the flowers during heading stage, destroying them fields before the harvest. It was reported that and preventing fertilization in barley and the Cereal weevil, P. hordei which is a wheat by 4.4% and 2.7%, respectively prevalent and significant pest in Palearctic (Şimşek, 1998a). While male and female region, has two sub-species: Pachytychius adults feed, they also mate (Fig. 1c) and leave hordei hordei (Brullé, 1835) and P. hordei their whitish translucent eggs (Fig.1d) between squamosus (Waltl, 1836) (Caldara, 2010). It spikelet husks in the spikes (Fig. 1e). Each was reported that P. hordeihordei sub-species female could leave a total of 40-50 eggs, one
76 M. Güllü, C. Gözüaçik, A. Konuksal, H. Hekimhan, H. Fidan ______per spikelet (Anonymous, 2008). It was population density of Cereal weevil, P. hordei reported that young larvae that get out of the were identified in the study (Figure 2, Table eggs in a short period of time gnaw on the 1). Figure 2 demonstrates that this insect is barley and wheat grains in milk and dough common in all cereal fields in the villages in development stages, harming the crops worse assessed regions of Lefkoşa (Nicosia), than the adults and the yield loss in barley is Gazimağusa (Famagusta), Girne (Kyrenia), more than the wheat (Şimşek (1991; Şimşek, Güzelyurt (Morphou), and İskele (Trikomo). 1998a). As a result, decreases are observed in In a previous study conducted in Northern number of grain, grain weight and germination Cyprus cereal fields, it was also reported that power in the spikes harmed by P. P. hordei was among the significant pests of hordei(Anonymous, 2008). the island (Güllü et al., 2014). Alziar (1995; This study was conducted to determine the 2010) reported that area of distribution and adult population Pachytychiushordeisubsp.hordei (Brullé, density of Cereal weevil, P. hordei, a cereal 1832), a sub-species of P. hordei (Brullé, pest, in the cereals of Turkish Republic of 1832) and Pachytychiusbasimaculatus Voss, Northern Cyprus during 2012 and 2013. 1964 existed in Cyprus fauna and P. hordei hordei sub-species was common in all Cyprus Material and methods Island, while P. basimaculatus species was The study was conducted during 2012-2013 observed only in Paphos and Larnaca districts. years at the end of stem elongation and Literature review demonstrates that in addition heading stages of cereals in Lefkoşa (Nicosia), to Cyprus, these species and sub-species are Gazimağusa (Famagusta), Girne (Kyrenia), also common in Greece (Central Macedonia, Güzelyurt (Morphou), and İskele (Trikomo) Crete, Ionian Islands, North Aegean, Regions of Northern Cyprus using survey Peloponnese, South Aegean, Thessaly, West method. Materials used were cereal (wheat, Greece) (Bahr et al., 2011), Middle East barley, oat, rye, and triticale) fields, sweep (Alziar, 2010), Syria (Weill et al., 2011), nets and plastic bags. The survey was Turkey (Karman et al., 1971; Zümreoğlu, conducted in 46villages and a total of 57 1972; Lodos et al., 1978;Kavut and Kaya, fields, 1-3 fields in each village to determine 1979; Şimşek, 1991; Şimşek et al., 1996; the area of distribution. To determine adult Şimşek, 1998a,b; Şimşek, 2000; Lodos et al., population density, studies were conducted in 2003; Avgın and Colonnelli, 2011), Italy 49 fields selected randomly in 36 villages, 1-3 (Abbazzi and Maggini 2009; Abbazzi and fields per village, and averages were taken for Zinetti, 2013), France (Deliry, 2011), Spain the fields studied in each village. Standard (Alonso-Zarazaga, 2002; Alonso-Zarazaga et sweep nets (38 cm Ø) were utilized in the al., 2006), Portugal (Zipcodezoo, 2011), Malta study. A total of 100 sweep nets were cast in (Mifsud and Colonnelli, 2010), Sardinia the form of 10 sweep nets in 10 different (Colonnelli et al., 2011), Sicily (Stejskal, points in each field. Cereal weevil, P. hordei 2004), and Egypt (Alziar, 2010). adult individuals caught by the sweep nets The assessment of adult population densities were transferred to closed plastic bags with showed that the highest population density for location labels, and brought to the lab in was found in Girne Region and in the villages iceboxes. In the laboratory, adult individuals in on the southwestern slopes of the Kyrenia the plastic bags for each village were counted MountainsRenge. Among these villages, it was and recorded. Areas of distribution of P. observed that there were 379 adults/100 sweep hordei in the fields were determined as region nets and 247 adults/100 sweep nets in average and village based on the label information and in 2012 and 2013, respectively in the barley + adult population densities were determined as oat field in Hisarköy; and 174 adults/100 adult individual count/100 sweep nets. sweep nets and 132 adults/100 sweep nets in average in barley fields in Ağırdağ village. Results and discussion Another region where the population density Barley cultivation areas have a high share of was high was Lefkoşa region. In this region, total cereal cultivation in Northern Cyprus; adult population densities of P. hordei were 99 92.7% (Anonymous, 2011). Thus, the surveys adults/100 sweep nets and 111 adults/100 conducted in 2012 and 2013 included mostly sweep nets in average in 2012 and 2013, barley fields. Distribution areas and adult respectively in Serhatköy. These determined
JAFES, Vol 67, (2016) 77 M. Güllü, C. Gözüaçik, A. Konuksal, H. Hekimhan, H. Fidan ______adult population densities are at a level that would cause a high possibility of yield losses could cause economic damage. Thus, these in extensive amounts. Hence, Şimşek (1998a) areas are considered risky villages and fields. reported that in a barley field that contains Villages located in the north and south of 61.3 -103.3 P. hordei adult density in 10 Lefkoşa-Güzelyurt highway neighbor this sweep nets during the heading stage average risky area and are considered to have potential yield loss of 20.8% occurs due to adult + risk. Chemical pest control is conducted larvae harm; while in a wheat field with 60.3 - against Cereal weevil, P. hordei in Turkey and 127.5 adult density, an average yield loss of the recommended economic threshold level 14.9% was observed. It was also stated that P. was reported as 5 adult/m2 or 15 adult/10 hordei adults feed on flowers during heading sweep nets (Anonymous, 2008). In the fields stage, destroying the heads and preventing that are above the economic threshold in fertilization, and thus causing an unfertilized Northern Cyprus and in those that carry a grain rate of 4.4% in barley and 2.7% in wheat potential risk, the required pest control should (Şimşek, 1998). be initiated. Otherwise, the lack of pest control
M. Güllü
Figure 1.Cereal weevil, Pachytychiushordei: a) Adult b) Injury on leaf c)Adult mating d)Adults in spikelets e) Egg in spikelets
JAFES, Vol 67, (2016) 78 M. Güllü, C. Gözüaçik, A. Konuksal, H. Hekimhan, H. Fidan ______
Table1.Population density of the Cereal weevil,Pachytychius hordei in NorthernCyprus cereal fields. Districts Villages Crops Pachytychiushordei Adults number/100 Sweep nets
2012 2013 Güzelyurt Yukarı Bostancı Barley 31 Morphou AşağıBostancı Barley 9 Taşpınar Wheat+Barley 22 14 Akçay Wheat 11 4 Gazimağusa Akdoğan Barley 45 Famagusta Mağosa Barley 7 Geçitkale Barley 2 Çayönü Barley 4 6 Dörtyol Barley 61 Ulukışla Barley 14 İnönü Barley 40 Serdarlı Barley +Vetch 5 Paşaköy barley 5 17 Boğaziçi Barley 7 5 İskele Kalecik Barley 17 Trikomo Tuzluca Barley 24 11 İskele Barley 10 Dipkarpaz Barley 8 5 Yeşilköy Wheat 7 Yeni Erenköy Barley 6 Girne Hisarköy Barley +Oat 379 247 Kyrenia Tepebaşı Barley 7 11
Şirinevler Barley 35
Göçeri Barley +Wheat 10 Ağırdağ Barley 174 132 Dağyolu Barley 135 89 Lapta Wheat 9 Lefkoşa Balıkesir Barley 10 3 Nicosia Hamitköy Barley 12 26 Gaziköy Triticale + Oat 8 Erdemli Barley 3 Yılmazköy Barley 10 Serhatköy Barley +Wheat 99 111 Gönyeli Barley 10 Meriç Barley 9 Alayköy Barley 6 19
JAFES, Vol 67, (2016) 79 M. Güllü, C. Gözüaçik, A. Konuksal, H. Hekimhan, H. Fidan ______
Figure 2. Distribution of the Cereal Weevil, Pachytychius hordei in Northern Cyprus.
Conclusions References In studies conducted during the years of 2012 Abbazzi, P., F. Zinetti, 2013. and 2013 in Northern Cyprus, it was Elencosistematico- determined that Cereal weevil, P. hordei was faunisticodeiCurculionoideaitaliani, Scolytidae common in all regions in different population Platypodidaeesclusi (Insecta, Coleoptera). 2. densities. The region with the highest Addenda e corrigenda. Memorie Soc. population density was the Girne region, and Entomol. Ital., 90 (2): 89-104. especially the fields at the southwestern slopes Abbazzi P. &Maggini L., of Kyrenia Mountain Range, and this region 2009.Elencosistematico- was considered high-risk areas. The villages in faunisticodeiCurculionoideaitaliani, Scolytidae the north and the south of Lefkoşa - Güzelyurt e Platypodidaeesclusi (Insecta, highway, neighboring the risky areas, were Coleoptera).Aldrovandia, 5: 29–216. considered as potential risk areas. Thus, the Alonso-Zarazaga, M. A., M. Sánchez-Ruiz y farmers in these villages with P. hordei risk, T. Domingo-Quero, 2006. ListaPreliminar De should be alert and should be warned Los Curculionoidea (Coleoptera) De La whenever necessary. However, it was Comunidad De Madrid (España).Graellsia, observed that no studies were conducted 62(númeroextraordinario): 43-52. previously on Cereal weevil, P. hordei and Alonso-Zarazaga, M. A. 2002. Listapreliminar several farmers were not informed about this de los ColeopteraCurculionoidea del insect and its harms. Thus, the need for áreaibero-balear, con descripción de Melicius introduction of this insect and providing gen. nov.ynuevascitas. information about its control to the farmers is a BoletínSociedadEntomólogicaAragonesa, significant matter. To design and implement (31): 9-33. pest control programs against Cereal weevil, Alziar, G., 2010.TheCurculionoidea-Fauna of P. hordei it is necessary to determine the Cyprus. - Le Charançon: Catalogues &Keys, fundamental biological criteria for pest control No.3, CURCULIO- such as the time of exit of adults from the soil, Institute,Mönchengladbach. time of feeding on spikes and the time the http://www.curci.de/illustrated_catalogue/curc larvae feeding on spikes move from the spikes ulionoidea-fauna_of_cyprus/ to the soil, pest control methodology, and the Alziar, G., 1995. Contribution à la state of yield loss. In brief, it is of utmost connaissance de l’histoire naturelle de l’île de significance to conduct studies on P. hordei Chypre. Coleoptera: Curculionidae I. bio-ecology, state of crop loss and control. Biocosme Mésogéen, 12(2/3): 65-82. Anonymous, 2011. Tarımsal Yapı ve Üretim (Agricultural Structure and Production) 2010 KKTC Tarım ve Doğal Kaynaklar Bakanlığı
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