Nitrofurans Case Study: Thailand's experience^[89]

Sujittra Phongvivat, Bangkok, Thailand

1 Introduction

Thailand exports agricultural commodities i.e. animal and aquaculture products to Europe, Asia, Middle-east, Africa; she is world largest exporter of farmed shrimp and the fourth largest exporter of poultry. Production and quality assurance system were mainly implemented by the Department of Livestock Development (DLD) and the Department of Fisheries (DoF), both under the Ministry of Agriculture and Cooperatives. Other Ministries were involved partially (Ministry of Health, Ministry of Commerce).

Late in 2001 animal products from some countries (Vietnam, China and Brazil) were found to contain residues related to nitrofurans and chloramphenicol, veterinary drugs which had been banned in many countries due to their genotoxic and carcinogenic properties. In March 2002, first cases of similar residues in Thai chicken and shrimp were reported, findings that affected soon all consignments of poultry and shrimp imported from Thailand. Originally, the product sampling and testing was done in order to demonstrate their wholesomeness. Value of exports during 2002 dropped causing significant economic losses.

The Thai government addressed this problem immediately and seriously by revising the existing legislation and regulations including import restrictions of 16 chemicals that were prohibited according to Annex IV of EU Council regulation (EEC) No 2377/90 (MRL regulation) and to the USFDA list of drugs prohibited from extra-label use in food animals by Import and Export Act B.E. 2522 (1979). The objective was to ensure traceability of any imports and restriction of uses for industrial and medical purpose only. Further steps included:

o Development of a regulation to control use of veterinary drugs and a proposal to improve Drug Act B.E. 2510 (1967).

o Development of legislation and regulations to control importation and use of animal feed and premix containing prohibited veterinary drugs including nitrofurans and chloramphenicol under the Feed Quality Control Act B.E. 2522 (1979).

o Development of a regulation to control standard of broiler farm by promotion Good Animal Health and Husbandry Practices (GAHP) concept at farm level.

o Implementation of the International Code of Practice for control the use of veterinary drugs by veterinarian in standard farm accredited by DLD and DoF.

o Development of legislation to regulate veterinary profession and veterinarian practice with morality including the possibility of suspension of veterinary license if prohibited drugs are prescribed.

o Development of a regulation to facilitate safe production of food producing animals by inspection of products during the slaughtering process allowing sampling for laboratory test.

In addition an efficient nitrofurans testing laboratory for product exit control was intended. An emergency budget of 288 million Baht (~ 7.2 million US.D.) by government allowed DLD and DOF to purchase of six units of ultra-high sensitive and considerably expensive LC-MS-MS system including toll-synthesis of some costly, but commercially unavailable deuterated internal standards, the recruitment of additional personnel and the training of technical staff. The challenge was that this laboratory would be able to perform the most recent methodology (as in the EU) with an expertise that would allow the operation of such a high technology instrument LC-MS-MS and the interpretation of the results obtained from it. It should be noted that expertise as described could not be created overnight. (Ref: 51).

The analysis of every lot before export was an extremely heavy and costly burden, but it is recognized that it was necessary in order to guarantee the quality of and to protect the markets for Thai goods. A significant decrease of the number of positive samples and a reduction of trace levels of residues was observed after 21 September 2002. Thailand could prove that her stringent measures and approach guarantee the soundness of animal products. EU Commission issued accordingly two decisions of 2003/477/EC 24 June 2003 and 2003/895/EC of 19 December 2003 for amending Decision 2002/251/EC of 27 March 2002 which revoked the protective measures with regard to the fishery and aquaculture products and certain consignments of poultry meat imported from Thailand.

Meanwhile the modern technology of nitrofurans metabolite residues analysis was made available and training provided by RIKILT to involved regulatory laboratories' personnel from Europe in July 2002 and from third countries in November 2002. This new nitrofurans metabolites analytical method with increased sensitivity was introduced in the residue monitoring schemes of many countries during late year 2002 and 2003. As a consequence, a metabolite (AMOZ) was found in Portuguese poultry in October 2002 and in March 2003. (Ref: 45 http://www.foodlaw.rdg.ac.uk/news/eu-03029.htm, Ref: 46). The first round of investigation uncovered of positive results of nitrofurans in 47 farms (36 chicken, 5 turkey, 4 quail, 1 rabbit and 1 swine farms) in Portugal. The second round investigation 12 farms were confirmed positive, 9 were negative and 34 remain under restriction (Ref: http://www.fsai.ie/alerts/archive/fa20030327.asp). Nitrofurans were identified in pork in 10 cases from Portugal and one case each from Italy and Greece, respectively. (Ref: 47) AMOZ is the metabolite most frequently found in Brazilian chicken (Ref: 48). Another metabolite (AOZ) was found in shrimps from India (Ref: 49) and in fish from Taiwan. (Ref: 50). SEM and AOZ were found in egg powder from India. (Ref: 64) These initial observations showed that nitrofurans residues resulting from possible illegal use were not limited to developing countries.

Although raw poultry meat free of nitrofuran-related residues had been produced and exported successfully from Thailand, such residues were still found in cooked or further processed products. Levels of 1.1 µg/kg of semicarbazide (SEM) in cooked frozen chicken breast meat onion stick, 13 µg/kg SEM in frozen fully cooked chicken oriental dim sum mix, and AOZ and SEM in spring roll selection (frozen fully cooked chicken product) were detected (Ref: 52). Additional investigation revealed the origin of the metabolites found in such products: AOZ was found in egg powder, SEM in a broad range of ingredients derived from animal and non-animal such as egg powder, milk, bread crumb, spice, flour and carrageen have been analyzed. Recently a research project of EU concluded that SEM is present as an impurity in the chemical azodicarbonamide (ADA), a substance used as a plastic blowing in o-ring gasket producing of metal lid glass jar. (Ref: 38)

Using ultra-high sensitive machines and methods can increase the detection of undesirable substances; not only nitrofurans at trace levels but sometimes also other banned substances were found in European food e.g. chloramphenicol 2.7 µg/l in Spanish white wine and even in environmental samples e.g. chloramphenicol at 0.56 µg/l in the effluent of a sewage treatment plant in the south of Germany. (Ref: 53, 54)

These findings of trace levels of residues of banned veterinary drug in some food products raise the more general question how foods may become contaminated? Besides the misuse of veterinary drugs other possible sources could be the contamination from historical use, from human medical use, by a genuine environmental source, contamination in the laboratory caused by inappropriate practice and, the worst case like in the case of SEM detected from the use of ADA, a source regularly used in food manufacture. Any of these low levels of residues will, if found in food, violate the EU regulation leading to the destruction of large amount of food products worth billion of dollars.

Thailand and other export countries have confronted with a variety of issues during the nitrofurans crisis. This paper is presented as a case study to learn from the Thai experience in order to propose means to resolve or prevent similar problems which might arise in future from the presence of residues of other substances for which the ADI and/or MRL are not allocated. The existing different approaches to the theoretical zero tolerance limits, to any other limits or parameters of analytical methods need to be harmonized in order to assure fair trade.

2 Nitrofurans Residues in Food of Animal Products

2.1 Chemical Structures and Metabolites

The nitrofurans are synthetic antibiotics characterised by their basic chemical structure i.e. a nitrofuran ring:

o Furazolidone or, 3-([(5-nitro-2-furanyl)methylene]amino)-2-oxazolidinone

o Furaltadone or 5-(morpholinomethyl)-3-[(5-nitrofurfurylidene)amilno]-2-oxazolidinone,

o Nitrofurantoin or 1-[(5-nitrofurfurylidene) amino]hydantoin

o Nitrofurazone or 2-[(5-nitro-2-furanyl)methylene]-hydrazinecarboxamide.

They are cheap, effective and continued to be manufactured and used for companion animals, aquarium fish and human medicine in many countries. In the past they were widely used in feed and medication of food producing animal as well, however, due to evidence that they have genotoxic and carcinogenic properties (ref WHO 832TRS) this use was terminated most countries.

Figure 1: Chemical structures of 4 nitrofurans and their correspond metabolites

For furazolidone it was shown that the detection of the parent drug is not feasible (McCracken et al. 1995 Nouws and Laurensen 1990) because residues of the parent drug are highly unstable in vitro and exhibit a very short half-life in vivo and in post-mortem tissues. The drug is therefore not found as a residue or only at very low concentrations at zero withdrawal time. (WHO TRS 832). Despite the research has proved that tissue-bound metabolites are formed, the free side chain of furazolidone, the molecule 3-amino-2-oxazolidinone (AOZ) is more stable. It can be detected and measured in tissues of pigs for up to 7 weeks after withdrawal of drug (McCracken et al. 1997) thus being a more suitable marker residue for furazolidone. (Ref 7-9). By analogy, side-chain moieties of other metabolites of furaltadone, nitrofurantoin and nitrofurazone are the molecules 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) respectively and serve as marker residues for these drugs.

2.2 Properties of nitrofurans metabolites (AOZ, AMOZ, AHD and SEM)

AOZ: Hoogenboom et al, (ref) showed that free AOZ could be detected in the blood of pigs with treated with furazolidone at levels up to 0.3 µg/ml. However, the liver of such treated pig when given to rats, leads itself to the presence of free AOZ in the blood of rats. This was evidence that the side-chain AOZ was bound to proteins and could be released not only from the parent drug but also from tissue containing chemically bound AOZ. Further studies showed that AOZ could be released from both the parent drug and protein-bound residues under mild acidic condition (like in the stomach) by cleavage of the azomethine bond (15-25% bound residues in pig liver could be released). AOZ possibly metabolises into the mutagen and carcinogen 2-hydroxyethylhydrazine (HEH). In addition to the inhibition of the enzyme monoamine oxidase and the formation of protein-adducts, AOZ gave a dose related positive response in the Salmonella/microsome mutagenicity test in tester strains TA 1535 and TA 100, especially in the presence of rat liver homogenate. Furthermore, a positive response was obtained in the chromosome aberration test with human lymphocytes and in bone marrow micronucleus test with mice treated intraperitoneally with AOZ. Based on these data it cannot be excluded that AOZ is also involved in the formation of tumours as observed in rats and mice treated with furazolidone. These data lead to the conclusion that ingestion of protein-bound residues of furazolidone may result in the release and absorption of AOZ, a compound with potential genotoxic properties. (Ref: 33).

AMOZ: Pig hepatocytes, incubated with the AMOZ side-chain of furaltadone, showed a decreased monoamine oxidase activity at high dose levels (IC50 3.7 mM), whereas exposure to AOZ resulted in a clear inhibition at 10,000-fold lower concentrations (IC50 0.5 µM). This AMOZ residue might therefore be of less toxicological concern than AOZ. (Ref: 34; Xenobiotica. 1994 Aug; 24(8): 713-27.)

AHD: 1- Aminohydantoine, is the moiety of nitrofurantoin which has structural relationships to the identified carcinogenic 5-nitrofuran compounds. There were no toxicological data available for AHD but toxicological data from a study of nitrofurantoin led to the conclusion that there was some evidence of carcinogenic activity of the parent drug for male F344/N rats (increased incidences of uncommon kidney tubular cell neoplasms). Uncommon osteosarcomas of the bone and neoplasms of the subcutaneous tissue were observed in dosed male rats. There was clear evidence of carcinogenic activity of nitrofurantoin for female B6C3F1 mice as shown by increased incidences of tubular adenomas, benign mixed tumors, and granulose cell tumors of the ovary. (Ref TR-341 No. 38)

SEM: Semicarbazide hydrochloride (or hydrazinecarboxamide monohydrochloride) belongs to a family of hydrazines which are known to cause cancer in laboratory animals. SEM itself demonstrated weak genotoxic activity in vitro and weak carcinogenic activity in female but not male mice when given via diet or via drinking water. The types of tumours found with semicarbazide were lung and vascular tumours, which have also been found with other hydrazines. However SEM was found to be one of the least potent carcinogens among several hydrazines. (Ref: 58, 59, 60).

The European Food Safety Authority (EFSA) concluded that there is limited evidence that SEM at high levels may be carcinogenic. There is no scientific evidence that SEM is carcinogenic to humans, it is therefore not possible to conclude whether SEM may pose a carcinogenic risk to humans. There is no risk of immediate illness to adults, children or infants from consumption of foods containing semicarbazide. The concern relates to health in the long term because of the possibility that semicarbazide may cause cancer.

Recent research showed that semicarbazide detected in foods is not an artefact of the analytical method but originates from azodicarbonamide (ADA), a reagent used in the production of o-rings/gaskets. (Ref 38) The levels of semicarbazide found in foods packed in glass jars closed with metal lids that are sealed with such plastic gaskets are variable, in the range from not detectable and up to 25 µg/kg and in the range 1-7 µg/kg in gaskets themselves. For some baby food samples also higher concentrations have been reported. The exposure estimate evaluated a scenario of a hypothetic worst case of a 6-month old baby with 7.5 kg body weight, eating 700 g of food that contains a level of 25µg/kg: the potential intake of semicarbazide would be 2.3µg/kg body weight/day. Taking into consideration the types of food packaged in glass jars and bottles in which semicarbazide had been found it could be concluded that the estimated intake by adults and children on a body weight basis will be many times lower than the preliminary estimate of the intake for babies. EFSA has concluded and recommended that taking into account the information available to date, on the levels in food, intake and toxicology, the risk, if any, to consumers eating products containing semicarbazide is likely to be very small (Ref 8, 22).

2.3 Regulatory Status

The European Union banned the use of nitrofurans in food producing animals by classifying it in ANNEX IV (list of pharmacologically active substances for which no maximum residue limits can be fixed) of the Council Regulation 2377/90. The Food and Drug Administration (FDA) of the United States of America prohibited furaltadone since February 1985 and withdrew the approval for the other nitrofuran drugs (except some topical uses) in January 1992. The topical use of furazolidone and nitrofurazone was prohibited in 2002. Australia prohibited the use of nitrofurans in food production in late 1992. Japan did not allocate MRLs for nitrofurans leading to the implementation of a "zero tolerance or no residue standard". In Thailand, the Ministry of Health issued in 2001 Proclamation No. 231 MRL of veterinary drug in food which did not allocate MRL for nitrofurans. The Ministry of Agriculture and Cooperatives had already prohibited importation and use of furazolidone and nitrofurazone in animal feed in 1999 which was extended to all nitrofurans in 2002. Furazolidone and nitrofurazone were withdrawn from the list of veterinary drug formulations in 2002.

JECFA, on request from Codex, evaluated furazolidone and nitrofurazone at the 40th meeting in 1992 (WHO TRS 832 and FAO 41/5). No ADI was established for furazolidone because of its genotoxic and carcinogen, and the insufficiency of the residue data of both drugs presented. The Committee could not identify marker residues and no information was available on the quantity and nature of the total residues.

In most of the countries that recognize nitrofurans as toxic substances, the prohibition of their use in food producing animal was implemented consequently. Food containing residues of these drugs at any concentration is considered "not fit for human consumption". Nitrofurans continue to be manufactured as drugs of choice for treatment of urinary infections in humans.

2.4 Method of Analysis

2.4.1 Previous methods

During the last two decades furazolidone drug use was monitored in most countries by detecting the residues of the parent drug. Analytical methods for nitrofurans up to 1984 were reviewed by Kalim (1985). The methods were all based on chromatography coupled with various detectors i.e. HPLC with UV, diode array or electrochemical detection or GC with electron capture detection system. The methods detect residues in parent form and have limits of detection (LOD) around 0.2 - 2.0 µg/kg. In Thailand, the Veterinary Public Health Laboratory of the Department of Livestock Development used for the monitoring of residues of the four parent forms (furazolidone, furaltadone, nitrofurazone and nitrofurantoin) the HPLC-UV method described by Winterlin et al, 1981 (limit of quantification, LOQ, of 10 µg/kg) in the annual residue monitoring plan. Up to year 2001, the results were reported to the EU Commission.

Since the parent drugs are metabolized in the body and disappear rapidly, these methods are not used anymore.

2.4.2 Current methods

Due to the instability of the parent drugs and the difficulties of their detection, researchers had to study and look for other suitable markers that can replace the parent drugs.

The development of a method to use AOZ as a marker residue started at RIKILT (Netherlands) in the late 1980's. It involves the following steps: homogenization of the liver, incubation with weak hydrochloric acid to hydrolyse or cut the side-chain (AOZ) from the metabolite and subsequent derivatisation with 2-nitrobenzaldehyde, extraction of the derivative NPAOZ with ethyl acetate and determination with HPLC-UV at 275 nm detection. The derivatisation was necessary since the metabolites of nitrofurans are very small molecules which do not absorb at UV wave length. Using nitrobenzaldehyde as a derivatising agent yields a nitrophenyl chromophore derivative with intense UV absorption in the molecule and hence being detectable by an LC-UV detector.

Furthermore the FoodBRAND (Bound Residues and Nitrofurans Detection) project funded by European Commission was launched in January 2000. The project consortium includes four National Reference Laboratories (NRLs), one research laboratory, one SME and consumer representatives from UK, Ireland, the Netherlands, Belgium, Hungary and the Czech Republic. Objectives were (1) to control abuse of the nitrofurans antibiotics by improving the methods; (2) to detect 4 marker residues (side chain) of AOZ, AMOZ, AHD and SEM the metabolite forms of furazolidone, furaltadone, nitrofurantoin and nitrofurazone respectively which are more stable; (3) to develop two different types of screening test and a confirmatory method and to improve the ability of control laboratories across the EU to be able to detect residues of these drugs.

2.4.2.1 Screening tests

ELISA immunological screening test kits were developed for nitrofurans metabolites under the FoodBRAND project. Late in 2003 there were only two kits for AOZ and AMOZ commercially available (by FoodBRAND partner r-biopharm AG: RIDASCREEN). The detection limits of the ELISA test kits claimed for AOZ are approximately 0.1 µg/kg and 0.2 µg/kg in meat, fish, whole egg, milk and liver, shrimp respectively, for AMOZ is approximately 0.2 µg/kg in shrimp, meat, fish, liver and whole egg. At present no other ELISA screening test kits or other immunochemical methods have been successfully developed for the detection of all four metabolites.

2.4.2.2 Confirmatory Method

The FoodBRAND project developed a confirmatory method that detects the four main metabolites (AOZ, AMOZ, AHD and SEM) of nitrofurans residues using very sophisticated and high technology equipment of HPLC coupled with tandem mass spectrometry (LC-MS-MS) because the previously used HPLC-UV method gave high interference with matrix compounds and had a low sensitivity.

Although such an alternative LC-MS has proven to be a valuable tool, this technique, even with careful optimization of separating conditions may be suitable for screening purpose at Minimum Required Performance Limit of 1 µg/kg, but is insufficient for identification according to EU requirements (Decision 2002/657/EC).

The extraction and derivatisation steps are the same as described above (2.4.2.) and yield the nitrophenyl derivative forms (NPAOZ, NPAMOZ, NPAHD and NPSEM) which are more stable than the parent drugs. Due to the increased molecular mass, the mass spectra contain more characteristic ions that are better suitable for the identification of the analytes. The derivatives are extracted with ethyl acetate; the extracts are dried and reconstituted in the solution of methanol/water which is analysed using HPLC, the substances are identified and quantified by tandem mass spectrometer (LC-MS-MS). At present this LC-MS-MS is the only technique accepted for nitrofurans metabolite residues regulatory testing and in general is considered to be acceptable in legal procedures as reliable and unequivocal.

2.4.3 Extraction procedures of metabolites

Before derivatisation and extraction, two different approaches, one without washing the other involving additional washing steps are applied (Diagram 1). The first technique aims to quantify total metabolite residue which comprises protein/tissue-bound and free/unbound residue. The second one, with washing steps, removes unbound metabolites and yields only tissue-bound residues. The question to be answered now is whether both extraction procedures lead to the same results with respect to the proof of illegal use of nitrofurans.

The Department of Agriculture and Rural Development in Northern Ireland (DARDNI), United Kingdom, used the technique including washing steps before extraction and has validated the method for testing tissue-bound metabolites in animal tissues. This tissue-bound metabolite method is confirmed to test appropriately for the use of nitrofurans in food producing animal; it is also recommended to test only the meat part not the whole processed product.

Diagram 1: Nitrofurans metabolite residues extraction flow chart

Whereas RIKILT, Netherlands, when conducting training courses for analysts from EU member states laboratories in July 2002 and from third countries in November 2002, introduced a standard operating procedure without washing steps to determine and confirm the analysis of the sum of residues of free and tissue-bound metabolites of nitrofurans drugs in fresh muscle of poultry, rabbit and aquaculture (like shrimp, prawn and fish) products (cooked, battered, ready-to-eat). (Ref 57)

Analysts from the Veterinary Public Health Laboratory (VPHL) of the Department of Livestock Development (DLD) in Thailand were trained at DARDNI in April 2002 and adopted therefore the same technique and validated it for tissue-bound residue for the control of raw poultry meat and liver. Products for export are tested with this method since May 2002. Analysts from the DOF were advised and trained to test shrimp and shrimp products using the total residue method from the beginning (2002) to February 2003 when further advice was made by DARDNI to continue with the total residue method for screening and to re-test using the tissue-bound method for confirmation (Se diagram 1).

VPHL decided to test each lot of poultry meat for tissue-bound residues, and to allow only meat with negative results to enter the food chain and to be used as raw material for further processing. Surprisingly, Thai animal products continued to be alerted for finding dangerous substances, in most cases SEM was detected in processed poultry and shrimp products as well.

From mid of 2002 to 2003, EU member states' laboratories continued to detect nitrofuran related residues in samples from consignments of imported poultry and aquaculture products that ranged from frozen raw meat to various already cooked products (e.g. battered chicken, fried products, chicken essence, spring roll, dim sum, etc). Such processed products contain not only meat but also various ingredients that originate from animal and non- animal sources e.g. vegetables, flour, bread crumb, spices like pepper, salt, sugar, soy sauce, oil and carrageenan. The method employed analysed samples as a whole which were homogenized and extracted without washing technique yielding total residues (sum of free and tissue bound).

Confronted with this confusing situation the question arose one could be sure that the residues found in finished products did originate from the meat part only and not from other ingredients? Could the detected residues be the result of some unidentified contamination? Was there the possibility of false positive results which were not related to illegal use of nitrofurans? In order to clarify this confusion, the Thai government decided to find out how laboratories in UK, the Netherlands, Germany and France actually perform their analysis. Three examples illustrate that there is no harmonisation of sample preparation between the official laboratories of one single EU member state:

Example 1: Reply from the BVL (Mr. Bernhard Kuhnle, 21 November 2003) which referred to an investigation of the 16 German state control laboratories.

Answer to the question "Do all laboratories examine only for bound residues?" Determination can either be done for total residues (sum of the bound and the free residues) or for the so-called bound residues of a sample only. This does not depend exclusively on the related reconditioning method or the nature of the sample (exclusively animal matrix or animal matrix plus processing contingent, suspending other materials as well as breadcrumbs or sheets of dough, sauce etc.).

Answer to the question "Do the laboratories examine exclusively the animal matrix or finished product?" A survey of the official laboratories of the states by the national reference laboratory was carried out in the past month. 16 answers out of 12 states were included into the analysis. 14 out of 15 laboratories indicated that they undertake a separation of the animal matrix from other components. This separation is achieved using different methods (washing, mechanical separation).

Evidence is available that sample preparation without washing steps continued to be performed for residue testing of imported products:

Example 2: RASFF Notification No. 2003/BBG, dated 15.05.2003: 0.7 µg/kg of SEM was found in a sample of pooled Frozen Soft Shell Crab. The analytical method used was SOP BIO 220 V 2 "Determination of Total Nitrofuran Residues in Tissue using LC-MS-MS" and was carried out by Institut fur Hygiene und Umwelt, Marckmannstr, 129a/b, 20539 Hamburg. (Ref: 32)

Example 3: The report on results of Nitrofurans in Shrimps and Chicken Meat, Interlaboratory Study NIFU06/03 proficiency testing scheme provided by European Reference Laboratory for Residues of Veterinary Drugs CRL/NRL Berlin, 25 November 2003 has shown that the laboratory participants perform the routine test which mostly used no-washing (total residue) method. (Ref: 40)

Table 1 Experimental analysis of total and tissue bound SEM in processed chicken products and ingredients. Decision limit (CCá) of SEM for chicken tissue is 0.15 µg/kg

The different approach in sample preparation prior to the extraction can distinguishably result in amounts of residues. Especially SEM could originate from the substance called ADA which is used in some countries to improve the characteristic of dough and in gasket production. The fact that cooked products' ingredients cannot be removed completely may lead to the wrong assumption that the contamination resulted from the illegal use of nitrofurans.

In 2003, VPHL carried out the 2^nd tissue-bound analysis of 82 processed food and various ingredient samples were they found SEM (range 0.16 - 2.56 µg/kg) at first time in total residue analysis. The results are shown in Table 1. Sixty three out of 82 samples were found to contain no tissue-bound SEM and 19 of 82 samples were found having tissue-bound SEM at concentration range 0.22 - 1.09 µg/kg. The ratio of bound SEM and total SEM, the frequency and concentration of bound-SEM and total SEM are displayed in Figure 2 to 5. As the experimental results of 2^nd tissue bound analysis found SEM at maximum of 1.09 µg/kg it could be discussed whether of SEM originated from contamination of the irremovable ingredient/substances during heat treatment of food or from Nitrofurazone abuse.

The FAPAS Proficiency Testing of Series 2 Round 48, March - May 2004, report No. 0248 can give information of concentration of total and tissue-bound of 2 metabolites, AHD and AMOZ analyzed by different techniques. The consensus assigned values were statistically calculated from valid participants' data. The values for total AHD, total AMOZ, bound AHD, and bound AMOZ were 1.51, 3.51, 0.58 and 0.68 µg/kg, respectively. (Ref: 65, Table 3)

Figure 2: Bound-SEM and total SEM concentration found in processed food and ingredients.

Figure 3: Bound-SEM in processed food sample is calculated in percentage.

Figure 4:

Figure 5:

Analytical method without washing steps measures total residues and does not discriminate between free and tissue-bound residues; total method certainly gives higher amount of residue, such method should not be used for regulatory purposes to determine illegal presence of nitrofuran related residues resulting from drug use. They may be useful for screening purposes, but for confirmation samples need to be washed and any non-meat part should be mechanically removed as much as possible before extraction. This recommendation was made and communicated by the French Community Reference Laboratory (AFSSA) only in December 2003; the implementation would harmonize analytical procedures and reduce unfair difficulties. (Ref: 61)

2.4.4 Analytical method performance and validation

Method Performance: The LC-MS-MS analytical method for nitrofurans metabolites were developed only recently and are performed only by a limited number of laboratories. The detection limits (LOD) or decision limits (CCá) of this method are at very low levels, less than one part per billion. The performance of historic and current analytical methods for the determination of nitrofurans is summarized in Table 2.

Table2. Performance of nitrofurans analytical method

Table 3: Description of confirmatory methods for the determination of nitrofuran metabolites

a =limit of detection, b = limit of quantification, c = preliminary figures, validation not yet completed d = validation not yet completed, e = only shrimps were examined, f = limit of detection derived from additional examinations concerning the blank matrix, g = limit of quantification derived from additional examinations concerning the blank matrix, h = lowest point of calibration curve was fixed as limit of detection and limit of quantification, i = only chicken meat was examined

Method validation: In May 2002 the Veterinary Public Health Laboratory of Department of Livestock Development started the nitrofurans residue analysis of export poultry products with the first LC-MS-MS unit. In accordance with Council Directive 96/23/EC (Ref 41) and Commission Decision 2002/657/EC (Ref 42) VPHL, a competent authorized laboratory accredited according to international standards had completed the validation for the determination of tissue-bound nitrofurans metabolite residues in chicken tissues by December 2002. In June 2003 VPHL participated in proficiency testing scheme NIFU06/03 "Nitrofurans in Shrimps and Chicken Meat" which was organized by the German Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (BVL), one of the European Reference Laboratory for Residues of Veterinary Drugs. The aim of NIFU06/03 was to promote the residue analysis of nitrofurans in shrimp and chicken and to enable the participants to check their routine methods which allowed a comparison between 2 national reference labs (RIKILT, the Netherlands and BVL, Germany, 5 German routine laboratories and two Thai labs (DLD and DOF). The results of analysis of each lab participant were sent to BVL for evaluation. The report on result of NIFU06/03 "Nitrofurans in Shrimps and Chicken Meat" had been made and submitted back to participants in November 2003. A summary of the confirmatory methods used is in table 3 (ref: 40).

All participants received a questionnaire concerning the quality assurance measures performed during the confirmatory analysis. What had to be indicated were the performance parameters in accordance with point 3 of the Annex of Commission Decision 2002/657/EC. Only three (laboratory 0010, 0012 and 0028) of the nine participating laboratories had already finished the method validation and thus had given full details as regards the performance parameters. The other laboratories answered the questions only partially. One laboratory did not submit data for this result form.

Only three laboratories (laboratory 0010, 0012 and 0028) indicated the decision limit (CCá) and the detection capability (CCâ) in accordance with Commission Decision 2002/657/EC. Laboratory 0019 stated preliminary data. The remaining laboratories made indications concerning "limits of detection and limit of quantification" which were still used but base on different calculations. The values indicated for AOZ, AMOZ, and SEM generally lie significantly below the Minimum Required Performance Limit (MRPL) value (1.0 µg/kg) for these substances. The AHD values handed in by laboratory 0006, 0021 and 0028 did not lie below MRPL.

The additional SOP for aquaculture products - on the detection, identification and quantitation of residues of metabolites of furazolidone, furaltadone, nitrofurantoin and nitrofurazone in muscle of poultry, rabbit and aquaculture products by LC-MS/MS confirmatory analysis, draft 2002-11-01, provided by RIKILT for the training course in November 2002 stated in chapter 2 that the described method is experimental and not yet validated. (Ref: 57)

VPHL has received only recently the report (No. 0248) of the latest FAPAS Proficiency Testing Scheme for Veterinary Drug Residue (Nitrofurans metabolite) Series 2, Round 48, March - May 2004, on date 11August 2004 which gives the following information. Instruction was given to participants to treat the test material as if it was a sample for routine analysis. 1^st un-cleared proforma result was provided to participant. The 2^nd proforma result was sent again asking participants to confirm what kind of residue (total or bound) they analysed. The participants were asked to specify the form of residue and return the answer of the 2^nd proforma before 28 May 2004. There were 39 participants' results submitted before closing date (7 May 2004) for this round. Two participants submitted their 2 sets of results for total (bound plus free) and tissue-bound. Thirty one participants submitted results for only total residue. Seven participants (including VPHL) submitted results for only tissue-bound but results of VPHL were mistakably assessed as total residue and unsatisfactorily results (Z-score >-3) were given to VPHL. These figures could give an idea that around 3/4 of the participants prefer to test total residue for nitrofurans metabolites as their routine methods. The test sample was incurred pig kidney which AHD and AMOZ should be detected. The L.O.Q.s for total AHD, total AMOZ, bound AHD and bound AMOZ stated by (not all) participants are all below 1 µg/kg. (Ref: 65)

This chapter demonstrates that a number of laboratories have not yet finished the validation of nitrofurans analytical methods. With their workload of routine testing most of the laboratories seem to apply rather a policy of "test first, validate later" which raises the question whether the test results from non-validated methods may be used when controlling for implementation of legal analytical methods?

2.4.5 Reporting limits and Minimum Required Performance Limits (MRPL)

For the European Union, the Commission Decision 2002/657/EC established so-called Minimum Required Performance Limits (MRPL) for analytical methods to be used for substances for which no permitted limit has been established. A later decision (March 2003) set the MRPL for nitrofuran related residues detected in Poultry meat or aquaculture products at 1.0 µg/kg for each metabolite. Table 4 illustrates the differences between European countries in setting value of reporting limits which are equal to or less than MRPL. These reporting limits are used to judge/report the positive or negative finding status of products. In other words the products are judged to be condemned or to be released by the reporting limits.

Table 4: Reporting limits of Nitrofurans metabolites expressed in µg/kg

One of the reasons for the UK to change the reporting limit, were clear indications that importers had started to divert consignments for import to ports in other Member States subsequently transporting them after import to the UK (without any additional testing requirements). Since nitrofurans are genotoxic carcinogens, the Agency consulted the Chairman of the Committee on Carcinogenicity of Chemical in Food, Consumer Products and the Environment (COC). His judgement was that changing the UK reporting level from 0.3 ppb to 1 ppb for nitrofurans would not result in a meaningful change in risk to consumers.

When deciding to harmonise the reporting limit for nitrofurans it was acknowledged that it would not possible to quantify the risks associated with low level exposure to nitrofuran residues and that levels should be kept as low as reasonably practicable. The agency accepted the opinion of the COC chairman that changing the UK reporting level (from the present level of 0.3 ppb to 1.0 ppb) for nitrofurans would not result in meaningful change in risk to consumers.(Ref: 37)

The member states of EU have implemented their own zero tolerance policy with different criteria for establishing reporting and decision limits, thereby rather creating technical barrier to trade.

2.5 Sources of residues in foods and food ingredients

The presence of residues of veterinary drugs in food products may not be caused by the veterinary use as drugs. Sources of residues of prohibited substances could be:

o Historical veterinary use before a ban.
o Illegal veterinary use after ban or drug abuse or extra label use.
o Cross contamination from feed
o Environmental contamination from human or pet/aquarium fish medical use
o Contamination in the laboratory
o From other chemicals i.e. SEM originated from ADA, hypochlorite etc.

2.5.1 Investigation of possible source of nitrofurans metabolites found in foods

After Thailand had submitted her action plan to the EU Commission and had implemented stringent measures in order to solve the nitrofuran residues crises it was expected that significant improvement should be observed after 21September 2002. However, exported products from Thailand continued to be notified by the weekly Rapid Alert System for Food and Feed (RASFF) due to presence of SEM and AOZ (Ref 63). These astonishing findings prompted a vast investigation in order to identify the sources of these residues which were mostly SEM but to a lesser extent also AOZ and which were found mainly in cooked/processed food. The investigation tested therefore beyond raw poultry meat/shrimp a rather bizarre range of ingredients from both animal and non-animal sources (egg/milk powder, starch, wheat flour, pre-dust, pepper, salt, soy sauce, sea weed derived carrageen etc.). SEM was found from traces to high concentration of hundreds ppb, AOZ was also detected in egg and milk products. It should be noted that the mentioned ingredients were from local or foreign production. (Table 5)

Being informed by the European food industry (CIAA) that an independent laboratory during routine analysis of nitrofuran antibiotic had identified SEM in food from non-animal sources, the European Food Standard Agency (EFSA) started in July 2003 her own investigations. Almost in parallel in April 2003, an increase in samples of Brazilian chicken containing semicarbazide was reported. Further investigation showed that most of the chicken products containing semicarbazide had been coated with flour, salt and spices. (Ref: 20 & http://www.foodproductiondaily.com/news/printnews-NG.asp?id=49643). These events in different countries confirmed our finding that SEM originates from food other than animal products.

Table 5: Nitrofurans metabolites residues in food ingredient from local and imported product

Remarks: (unpublished data of analysis carried out by VPH lab, DLD, Thailand)

2.5.2 SEM resulting from other sources

Azodicarbonamide (ADA), 1,1'-Azobisformamide; is a yellow to orange red crystalline solid, odourless substance with a molecular weight of 116.08.

The chemical is used as

o Aging and bleaching ingredient (FDA § 172.806)

o Dough conditioner in bread baking (FDA § 172.806)

o Exothermic foaming agent for the plastic gaskets sealed of metal lids of glass containers.

ADA has been extensively studied and the possible presence of unconverted ADA was studied in experiments using flour treated above technological levels and bread made from such flour. The evidence strongly supports the view that ADA is rapidly and completely converted to biurea on wetting and that this substance is stable in bread. Biurea itself is metabolically inert, has low toxicity and does not present any carcinogenic hazard. (Ref: 19, 31) Considering the resemblance of the molecular structure of biurea with semicarbazide, it has been suggested that upon acid treatment, semicarbazide residues could be formed. A mechanism of two hydrolysis reactions has been proposed for conversion of biurea to SEM. (Figure 6).

By products of ADA released at decomposed temperature (195 to 202?C) are Urazol (UR), Biurea (BU), Isocyanuric acid and Cyamelide approximately 36%, 24%, 15% and 7% respectively (Figure 7).

Figure 6: A proposed mechanism for conversion of biurea in SEM by two hydrolysis reactions (Ref: 20)

Figure 7: ADA and its by-products (solids and sublime substances)

ADA has been adequately studied in several species and is similarly free from carcinogenic hazard. Long-term studies in mice are in progress (Frazer, 1966).

Semicarbazide (SEM) is a known metabolite of the drug "nitrofurazone" and is monitored as a marker for nitrofurazone abuse in food animal origin, but SEM has also been reported in seaweed derived products, which are widely used as food additives (Ref: 62). SEM was found in food products packaged in glass jar with metal lids that have foamed plastic seals.

An investigation conducted by the National Food Agency Finland found SEM in 15 baby foods and five other food stuffs at similar levels than European average level.(SEM ranged from 1 µg/kg to 28.4 µg/kg, average was 11.3 µg/kg).

It was also discussed whether SEM could possibly originate at high concentrations from reactions between hypochlorite and some amino acids. Hypochlorite is used for the bleaching of ingredients such as carrageenan, egg powder. (Ref: 53 Mad Science Discussion Board www.sciencemadness.org/talk/viewthread.php?tid=148&page=2#pid.

An investigation of SEM found in chicken essence soup packed in glass jar and comparison results of bleached and un-bleached pepper analysed for nitrofurans metabolite were carried out by VPHL, DLD in 2003 has shown in table below.

Table 6: SEM found in glass jar product and bleaching pepper

The EU research project A03037 concluded that SEM is present as impurity in all three chemicals ADA, Biurea and Urazol. (Ref: 38)

Based on current information, the only likely source of positive results involving SEM originates from its occasional presence in ingredients of finished products other than meat. This is of concern if total residues are determined for the whole product. Experts from the EU therefore agreed and advised that the most appropriate methods try to determine tissue-bound residues in animal tissue which minimize the risks of false positive results and will remove any criticism that the methods are being applied to sample matrices for which they have not been appropriately validated. (Ref: 61)

3 Socio-economical impact

Agricultural products and processed foods are Thailand's second highest export income next to industrial goods. Thailand is the 7^th largest poultry producer and ranks at No. 4 among poultry exporters following U.S.A., Brazil and the EU. Most important Thai export markets are Japan and the EU. Livestock products constitute 10% of agro-food products for export.

The figures in Table 5 reflect the decrease of amount and value of poultry exports from Thailand to EU in year 2002 and to a lesser extent in year 2003. If we assume an annual increase of poultry exports from Thailand to Europe of 10 %, the actual value dropped by approximately 20% in 2002 and still by 7% in year 2003 (Figures 10 and 11).

Table 7: Demonstration figure in metric tons (MT) and price of chicken raw meat and products export to Europe of year 2001, 2002 and 2003

In addition to the government's emergency budget of approximately 7.2 Million US Dollars that were provided to address the residue problem, the decrease of country's income as shown in table 7 and Figure 8 to 11, and the value of products destroyed due to nitrofurans residues found in them, add up to significant losses. Costs results also from the expenses paid for retesting products and the renting of cold storage for longer periods to keep the products fresh while waiting for the analytical results. However, all such amounts of money do not include the social impact and chain reaction to the grass root farmers and their household when chicken is tested to contain residues, product is not exported, and the company would not pay or pay less to farmers. They are directly or indirectly punished if the residue found in their chicken even in the case of false positives (see SEM!). Some of them had to face hard circumstances and tragedies in life e.g. bankruptcy, no sufficient income for daily life expense.

Figure 8: The Export Quantity of Poultry Products from Thailand to EU display in graph

Figure 9: The Export Value (in million US $) of Poultry Products from Thailand to EU.

Figure 10: Forecast and Actual Value in Thousand Metric Tonnes of Poultry Export to EU

Figure 11: The Forecast and Actual Value in Million US $ of Poultry Export to EU.

Examples of trade barriers by different reporting limits, un-unified analytical method and SEM at trace level below 25 µg/kg found in glass jar packaged product, resulting in product destroyed could be given below.

o RASFF Notification No. 2003/BBG, dated 15.05.2003: SEM 0.7 µg/kg was found in sample of pooled Frozen Soft Shell Crab taken from three consignments of 800 kg, 200 kg and 1500 kg Products were rejected and destroyed. With the reporting limit of 0.5 µg/kg and SOP BIO 220 V 2, "Determination of Total Nitrofuran Residues in Tissue using LC-MSMS", the analysis was carried out by Institut fur Hygiene und Umwelt, Marckmannstr. 129a/b, 20539 Hamburg.

o RASFF 2002/AXS, 30 July 2002, Sweden, Metabolites of Nitrofurazone (SEM) 2.4 µg/kg, Crab Paste with Soya Bean Oil (CPB: ACDCF 200 g x 24 Jars/Carton, 50 Cartons, Net weight 240 Kg). (Place of test: RIKILT)

o RASFF 2002/AXS, 30 July 2002, Sweden, Metabolites of Nitrofurazone (SEM) 2.8 µg/kg, Shrimp Paste with Soya Bean Oil (SPB: ACDCF 200 g x 24 Jars/Carton, 50 Cartons, Net weight 240 Kg). (Place of test: RIKILT)

o RASFF 2002/274, 1 August 2002, Germany, Nitrofuran metabolite AOZ 0.5 ug/kg, Thailand Black Tiger Shrimps 26/30 Raw, Minus Shells, Deep-frozen (Penaeus spp.).

o RASFF 2002/BLI, 12 November 2002, Germany, Nitrofurans metabolite SEM 0.8 ug/kg, Bottled Shrimp in Brine, Pan Asia (1981) Ref Nr./BEZUGS-NR.:P>A>/050702/595 DATE: 20.09.2002

o RASFF 2002/BPF, 16 December 2002, Germany, AOZ 0.9 ug/kg, Frozen Shrimp Ball Breaded Black Tiger Shrimp

Example of discrimination of same residues of SEM found in carrageen (2003/086 add 43) and AOZ found in egg powder (2003/086 add 57) products which were placed under temporary seizure and were later released.

o RASFF 2003/086, Brussels,12 December 2003, Subject: Nitrofuran (metabolite)-Furazolidone (AOZ) in egg products from India, Measures taken in Belgium, Release of batches of carrageenan and egg powder. (Ref: 65)

4 Conclusion

Thailand's stringent measures have shown her sincerity to solve the drug residue problem because it is most important to produce safe and sound food for the consumers of the world not only for export markets but for local consumer as well.

The "nitrofurans crisis" was a severe problem not only in developing countries but impacted on worldwide trade especially developing countries. The nitrofurans case is an example from which many lessons can be learnt and can prevent occurrence of similar problems in the future:

o Analytical method for substances without ADI/MRL substances should be discussed, harmonized internationally and approved as a regulatory method.

o Criteria or policies of setting critical parameters which would affect trade such as "reporting limit" should be discussed and be based on risk assessments rather than be based on the sensitivity of technology or instruments.

o Substances should be banned on the basis of their toxicity risk to human not because of insufficient data or unfinished evaluation.

o Marker residues should be appropriate and valid and not create false positive.

o Residue of veterinary drug should be test from target tissues or organs of animal e.g. liver, kidney, muscle, fat, skin, egg, milk, etc, not from processed or finished product.

o Terms or parameters involved in regulatory aspects and analytical aspects i.e. MRPL, Zero Tolerance, L.O.D., L.O.Q., CCa, CCb etc. lead to confusion and need to be clarified, defined and harmonized.

o A consultation of experts would be necessary to solve scientifically the problems and unify/harmonize different approaching/implementation of countries which should be transparency and stop or minimize conflict and unfair trade.

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34. L.A.P. Hoogenboom, T.H. Polman, A. Lommen, M.B.M. Huveneers-Oorsprong, J.A. van Rhijn. 1994. Biotransformation of furazolidone by pig hepatocytes and Salmonella typhimurium TA 100 becteria, and the formation of protein-bound metabolites. Xenobiotica, 24(8), p 713-727.

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38. W.A. Read and L. Castle, Central Science Laboratory, York & L. Coulier, E. K. Zonzervan-van Beuken, M. A. H. Rijk, TNO Nutrition and Food Research, The Netherlands, December 2003. Project A03037: LC-MS Method Development for the Screening of Non-Volatile and Polar Compounds Present in Paper and Board or Plastic Food Contact Materials and Articles: Testing for SEMICARBAZIDE without Derivatisation or Acid Hydrolysis: Final Report. http://www.foodstandards.gov.uk/multimedia/pdfs/semicarbazide.pdf

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49. Information Notification reference no. 2003.BHF, dated 25/06/03, RASFF reference no. 2003.193, dated 28/07/2003, RASFF no. 2003.194, date 28/07/2003.

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57. J.A. van Rhijn, P. P. J. Mulder; (9-10-2002) Muscle of poultry, rabbit, and aquaculture products- Detection and identification of residues of metabolites of furazolidone, furaltadone, nitrofurantoin and nitrofurazone - LC-MS/MS confirmatory analysis. Final 9-10-2002, p2, 3, 13, 15 and 20 of 25. and additional SOP on the detection, identification and quantitation of residues of metabolites of furazolidone in muscle of poultry, rabbit and aquaculture products by LC-MS/MS confirmatory analysis, draft 2002-11-01, chapter 2 Field of application.

58. IARC (1976), IARC Monographs on the evaluation of the carcinogenic Risk of Chemicals to Man: Some Aromatic Amines, Hydrazine and Related Substances, N-Nitroso Compounds and Miscellaneous Alkylating Agents. Volume 4. International Agency for Research on Cancer, Lyon.

59. Parodi S, Flora SD, Cavanna M, Pino A, Robbiano L, Bennicelli C & Brambilla G (1981). DNA-damaging activity in vivo and bacterial mutagenicity of sixteen hydrazine derivatives as related quantitatively to their carcinogenicity. Cancer Research 41, 1469-1481.

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62. RASFF week 2003/31, Ref: 2003.201, date 31.07.2003, SEM found in carrageen, derived from sea weed, products of Indonesia, Chile, Canada, Tanzania, notified by Denmark.

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64. RASFF week 21, Ref: 2003.BBU, date 21.05.2003, SEM and AOZ found in egg powder of India, notified by Belgium.

65. RASFF 2003/086-add58 Additional information, Brussels, 12 December 2003, SUBJECT: NITROFURAN (METABOLITE) - FURAZOLIDONE (AOZ) IN EGG PRODUCTS FROM INDIA

66. FAPAS Proficiency Testing, VETERINARY DRUG RESIDUES, FAPAS^® Series 2 Round 48, March-May 2004, Report No. 0248.

Relevant web-pages and other documents available on-line

http://www.afsni.ac.uk/foodbrand/

http://www.efsa.eu.int/pdf/p_afc_doc_01.pdf (Advice on the possible occurrence of Semicarbazide in packaged foods, 28 July 2003 AFC/adhoc SEM/

http://www.efsa.eu.int/pdf/p_afc_doc_02_en.pdf (Statement of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food. Updating the advice available on Semicarbazide in Packaged foods, Adopted on 1 October 2003.

http://www.europa.eu.int/comm/food/fs/sc/scf/out181_en.pdf Opinion of the SCF on the 21st additional list of monomers and additives for food contact materials

http://www.naturalovens.com/lib/pdf/betterhealth/Toxin_Additives_in_Food_and_Drink.PDF ADDITIVES PERMITTED IN U.S. FOR BAKED FOODS (F.D.A.)

http://www.crl.fougeres.afssa.fr/publicdoc/Microsoft%20Word%20-%2003-09PS-SEM-L7349-NoteCRL211103.pdf

http://www.foodstandards.gov.uk/multimedia/pdfs/semicarbazide.pdf

http://www.rikilt.dlo.nl/Services/Info%20Nitrofuran.htm (screening test)

http://www.efsa.eu.int/pdf/pressrel20031015_en.pdf (SEM advice by EFSA)

http://www.efsa.eu.int/pdf/p_afc_doc_01.pdf

http://www.efsa.eu.int/p_foodadd_en.html

http://www.nzfsa.govt.nz/dairy

http://www.rikilt.wur.nl/Services/Asorption_of_a_genotoxic_metabo.htm

http://www.efsa.eu.int/pdf/p_afc_doc_01.pdf

http://www.efsa.eu.int/p_foodadd_en.html

http://www.efsa.eu.int/pdf/pressrel20031015_en.pdf)

(http://www.rikilt.dlo.nl?Services/Info%20Nitrofuran.htm)

(Ref: r-biopharm AG: leaflet, http://www.rikilt.dlo.nl/Services/Info%20Nitrofuran.htm)

http://www.rikilt.dlo.nl/Services/Info%20Nitrofuran.htm