Sulfamethoxazole Residues Research Articles

Hypersensitive electrochemical sensor based on thermally annealed gold–silver alloy nanoporous matrices for the simultaneous determination of sulfathiazole and sulfamethoxazole residues in food samples

In this study, we have developed a novel, hypersensitive, and ultraselective electrochemical sensor containing thermally annealed gold–silver alloy nanoporous matrices (TA-Au-Ag-ANpM) integrated with f-MWCNTs-CPE and poly(l-serine) nanocomposites for the simultaneous detection of sulfathiazole (SFT) and sulfamethoxazole (SFM) residues in honey, beef, and egg samples. TA-Au-Ag-ANpM/f-MWCNTs-CPE/poly(l-serine) was characterized using an extensive array of analytical (UV–Vis, FT-IR, XRD, SEM, and EDX), and electrochemical (EIS, CV and SWV) techniques. It exhibited outstanding performance over a wide linear range, from 4.0 pM to 490 μM for SFT and 4.0 pM to 520 μM for SFM, with picomolar detection and quantification limits (0.53 pM and 1.75 pM for SFT, 0.41 pM and 1.35 pM for SFM, respectively). The sensor demonstrated exceptional repeatability, reproducibility, and anti-interference capability, with percentage recovery of 95.6–102.4% in food samples and RSD below 5%. Therefore, the developed sensor is an ideal tool to address the current antibiotic residue crisis in food sources.

Epigallocatechin-3-gallate attenuates the sulfamethoxazole-induced immunotoxicity and reduces SMZ residues in Procambarus clarkii

Sulfamethoxazole (SMZ) is a commonly used antibiotic in aquaculture, and its residues in water bodies pose a significant threat to aquatic organisms in the water environment. In the present study, epigallocatechin-3-gallate (EGCG), a catecholamine, was used to mitigate the immunotoxicity caused by SMZ exposure in Procambarus clarkii. EGCG reduced the apoptosis rate, which was elevated by SMZ exposure, and increased the total hemocyte count. Simultaneously, EGCG enhanced the activities of enzymes related to antibacterial and antioxidant activities, such as superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM), acid phosphatase (ACP), and GSH, which were decreased following SMZ exposure. Hepatopancreatic histology confirmed that EGCG ameliorated SMZ-induced tissue damage caused by SMZ exposure. In addition to EGCG attenuating SMZ-induced immunotoxicity in crayfish, we determined that EGCG can effectively reduce SMZ residues in crayfish exposed to SMZ. In addition, at the genetic level, the expression levels of genes related to the immune response in hemocytes were disrupted after SMZ exposure, and EGCG promoted their recovery and stimulated an increase in the expression levels of metabolism-related transcripts in hemocytes. The transcriptome analysis was conducted, and “phagosome” and “apoptosis” pathways were shown to be highlighted using Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. To the best of our knowledge, this is the first study to confirm that EGCG attenuates SMZ-induced immunotoxicity in aquatic animals and reduces SMZ residues in aquatic animals exposed to SMZ. Our study contributes to the understanding of the mechanisms by which EGCG reduces the immunotoxicity of antibiotic residues in aquatic animals.

Silver Nanoparticle-Embedded Hydrogels for Electrochemical Sensing of Sulfamethoxazole Residues in Meat.

A disposable electrochemical sensor based on silver nanoparticle-embedded cellulose hydrogel composites was developed for sensitive detection of sulfamethoxazole residues in meat samples. Scanning electron microscopy confirmed the porous structure of the cellulose matrix anchored with 20-50 nm silver nanoparticles (AgNPs). Fourier transform infrared spectroscopy and X-ray diffraction verified that the metallic AgNPs coordinated with the amorphous cellulose chains. At an optimum 0.5% loading, the nanocomposite sensor showed a peak-to-peak separation of 150 mV, diffusion-controlled charge transfer kinetics, and an electron transfer coefficient of 0.6 using a ferro/ferricyanide redox probe. Square-wave voltammetry was applied for sensing sulfamethoxazole based on its two-electron oxidation peak at 0.72 V vs. Ag/AgCl in Britton-Robinson buffer of pH 7.0. A linear detection range of 0.1-100 μM sulfamethoxazole was obtained with a sensitivity of 0.752 μA/μM and limit of detection of 0.04 μM. Successful recovery between 86 and 92% and less than 6% RSD was achieved from spiked meat samples. The key benefits of the proposed disposable sensor include facile fabrication, an antifouling surface, and a reliable quantification ability, meeting regulatory limits. This research demonstrates the potential of novel cellulose-silver nanocomposite materials towards developing rapid, low-cost electroanalytical devices for decentralized on-site screening of veterinary drug residues to ensure food safety.

Bifunctional Solar Evaporator with Co/N-Doped Graphene Oxide for Synergistic Steam Generation and Organic Pollutant Degradation.

Solar-driven interfacial steam generation (SISG) is a promising technology for alleviating freshwater shortage. However, when the SISG technology is applied to wastewater treatment, the contaminant would be enriched in residual bulk water. Herein, a dual-functional evaporator was constructed via tactfully decorating Co/N-doped graphene oxide (GO) on melamine foam (MF), which can simultaneously achieve efficient vapor production and source water purification. N-doped carbon nanotubes (NCNTs) endowed evaporators with powerful light absorption and water transport performance, guaranteeing an evaporation rate of 2.02 kg m-2 h-1 under 1 sun irradiation. Meanwhile, the catalytic activity of the carbon layer was adjusted by the N dopant and embedded Co particles, providing abundant active sites to activate peroxymonosulfate (PMS). When treating the solution containing sulfamethoxazole (SMX), no SMX residues were detected in the remaining bulk water (up to 100% SMX degradation efficiency within 60 min), demonstrating that reactive oxygen species (ROS) were generated to attack SMX in the source water. The bifunctional evaporator successfully combined SISG and advanced oxidation processes (AOPs), providing an ingenious strategy for solving the problem of wastewater enrichment during SISG.

Efficient Adsorption of a Sulfonamide Antibiotic in Aqueous Solutions with N-doped Magnetic Biochar: Performance, Mechanism, and Reusability.

Conventional biochar has limited effectiveness in the adsorption of sulfonamide antibiotics, while modified biochar exhibits greater adsorption potential. Residues of sulfamethoxazole (SMX) in the aquatic environment can threaten the safety of microbial populations as well as humans. In this study, iron-nitrogen co-doped modified biochar (Fe-N-BC) was prepared from palm fibers and doped with Fe and urea via synthesis at 500 °C. Fe-N-BC has a richer surface functional group based on elemental content, X-ray photoelectron spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The Brunauer-Emmett-Teller (BET) specific surface area test exhibited Fe-N-BC, which possessed a greater surface area (318.203 m2/g) and a better developed pore structure (0.149 cm3/g). The results of the hysteresis loop and the Raman spectrum show that Fe-N-BC has a higher degree of magnetization and graphitization. Fe-N-BC showed a remarkable adsorption capacity for SMX (42.9 mg/g), which could maintain 93.4% adsorption effect after four cycles, and 82.8% adsorption capacity in simulated piggery wastewater. The adsorption mechanism involves pore filling, surface complexation, electrostatic interactions, hydrogen bonding, and π-π EDA interactions. The results of this study show that Fe-N-BC prepared from palm fibers can be a stable, excellent adsorbent for SMX removal from wastewater and has promise in terms of practical applications.

The occurrence of antimicrobial residues and antimicrobial resistance genes in urban drinking water and sewage in Southern Brazil.

Antimicrobial resistance (AMR) is currently discussed as an important issue worldwide, and the presence of antimicrobial residues (ARs) and antimicrobial resistance genes (ARGs) in the environment, especially in the water sources, is a challenge for public health. This study was conducted to evaluate the occurrence and diversity of AR and ARG in water sources from an urban center, in Southern Brazil. A total of thirty-two water samples from drinking water treatment plants (24) and sewage systems (8) were collected during two annual samplings, winter and summer. The PCR was performed by 18 ARGs, and the detection of 47 ARs was performed by LC-MS/MS. All sewage samples presented carbapenemases, ESBL, and mcr-1 genes as well as quinolones and sulfamethoxazole residues. In drinking water, we just detected blaTEM and tetB genes and doxycycline residues in samples before treatment. This study provides data about AR and ARG in drinking water and sewage systems showing that these sources are important reservoirs of both. The limited effectiveness of wastewater treatment processes to remove mainly AR demonstrates the need to implement better protocols of disinfection, in order to limit the spread of AMR in the environment.

Biodegradation and metabolic pathway of sulfamethoxazole by Sphingobacterium mizutaii

Sulfamethoxazole (SMX) is the most commonly used antibiotic in worldwide for inhibiting aquatic animal diseases. However, the residues of SMX are difficult to eliminate and may enter the food chain, leading to considerable threats on human health. The bacterial strain Sphingobacterium mizutaii LLE5 was isolated from activated sludge. This strain could utilize SMX as its sole carbon source and degrade it efficiently. Under optimal degradation conditions (30.8 °C, pH 7.2, and inoculum amount of 3.5 × 107 cfu/mL), S. mizutaii LLE5 could degrade 93.87% of 50 mg/L SMX within 7 days. Four intermediate products from the degradation of SMX were identified and a possible degradation pathway based on these findings was proposed. Furthermore, S. mizutaii LLE5 could also degrade other sulfonamides. This study is the first report on (1) degradation of SMX and other sulfonamides by S. mizutaii, (2) optimization of biodegradation conditions via response surface methodology, and (3) identification of sulfanilamide, 4-aminothiophenol, 5-amino-3-methylisoxazole, and aniline as metabolites in the degradation pathway of SMX in a microorganism. This strain might be useful for the bioremediation of SMX-contaminated environment.

Fate of sulfamethoxazole in compost, manure and soil amended with previously stored organic wastes

Application of organic wastes as soil fertilizers represents an important route of agricultural soil contamination by antibiotics such as sulfamethoxazole (SMX). Soil contamination may be influenced by the storage time of organic wastes before soil spreading. The objective of this work was to study the fate of SMX in two organic wastes, a co-compost of green waste and sewage sludge and a bovine manure, which were stored between 0 and 28 days, then incorporated in an agricultural soil that has never received organic waste and monitored for 28 days under laboratory conditions. Organic wastes were spiked with 14C-labelled SMX at two concentrations (4.77 and 48.03 mg kg−1 dry organic waste). The fate of SMX in organic wastes and soil-organic waste mixtures was monitored through the distribution of radioactivity in the mineralised, available (2-hydroxypropyl-β-cyclodextrin extracts), extractable (acetonitrile extracts) and non-extractable fractions. SMX dissipation in organic wastes, although partial, was due to i) incomplete degradation, which led to the formation of metabolites detected by high performance liquid chromatography, ii) weak adsorption and iii) formation of non-extractable residues. Such processes varied with the organic wastes, the manure promoting non-extractable residues, and the compost leading to an increase in extractable and non-extractable residues. Short storage does not lead to complete SMX elimination; thus, environmental contamination may occur after incorporating organic wastes into soil. After addition of organic wastes to the soil, SMX residues in the available fraction decreased quickly and were transferred to the extractable and mostly non-extractable fractions. The fate of SMX in the soil also depended on the organic wastes and on the prior storage time for manure. However the fate of SMX in the organic wastes and soil-organic waste mixtures was independent on the initial spiked concentration.

Egg residue and depletion in Rhode Island Red hens (Gallus gallus domesticus) following multiple oral doses of trimethoprim-sulfamethoxazole

Sulfamethoxazole-Trimethoprim residues in eggs can cause risks to human health. The most common cause of residues in eggs results from failure to meet an appropriate withdrawal interval. The aim of this study was to determine the quantity and duration of sulfamethoxazole-trimethoprim residues in eggs and evaluate the drug elimination parameters in egg components and whole egg to better estimate the withdrawal interval of sulfamethoxazole and trimethoprim following oral administration for 7 days at a purposed dosage regimen (time average 46 mg kg−1 day−1 for sulfamethoxazole, time average 25 mg kg−1 day−1 for trimethoprim). Residues of sulfamethoxazole and trimethoprim in albumen and yolk were analyzed by ultra-performance liquid chromatography mass spectrometry. A greater percentage of sulfamethoxazole was distributed into the albumen (91.53–96.74%) and a greater percentage of trimethoprim was distributed into yolk (63.92–77.36%) during treatment. The residues levels in whole egg declined below or reached the limit of quantification until 13 days for SMZ and TMP respectively. The withdrawal interval for SMZ and TMP were 43 days and 17 days respectively using the FDA tolerance method.

Stress responses and biological residues of sulfanilamide antibiotics in Arabidopsis thaliana.

Sulfonamides (SAs) are antibiotics widely used in clinical practice, livestock and poultry production, and the aquaculture industry. The compounds enter the soil environment largely through livestock and poultry manure application to farmland. SAs not only affect plant growth, but also pose a potential threat to human health through SA residues in plant tissues. In particular, sulfamethoxazole (SMZ) has been classified as a Category 3 carcinogen by the World Health Organization, and thus its soil ecological toxicity and possible health risks are of concern. Using A. thaliana as a model plant, stress responses and biological residues of sulfadiazine (SD), sulfametoxydiazine (SMD), and SMZ were investigated in the present study. Root length and aboveground plant biomass were significantly inhibited by the three types of SA, whereas lateral roots exposed to SMD grew vigorously. The contents of chlorophyll a and chlorophyll b and photosystem II maximum photochemical quantum yield declined with increase in drug concentration, which indicated that exposure to SAs affected photosynthesis and inhibited chlorophyll synthesis in A. thaliana. With increase in drug concentration, reactive oxygen species (ROS) accumulation in the leaves increased significantly. Activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were activated at low SA concentrations, but increased lipid peroxidation occurred with increase in SA concentration. Of the three compounds, SMZ was the most toxic to A. thaliana, followed by SD, and SMD was the least toxic. The results indicated that the risk of SMD entering an organism through the food chain is greater than that for SMZ and SD.

The investigation of antibiotic residues, antibiotic resistance genes and antibiotic-resistant organisms in a drinking water reservoir system in Germany.

Between August 2018 and June 2019, a river system in Germany that supplies a drinking water reservoir and is subject to the discharge from two sewage treatment plants was monitored for antibiotic residues via liquid chromatography-tandem mass spectrometry, antibiotic resistance genes (including blaNDM, blaVIM, blaOXA-48, blaKPC, blaGIM, blaSME, blaIMI, blaIMP, blaSPM, blaSIM, blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58, mcr) via qualitative real-time PCR and antibiotic-resistant bacteria [belonging to the ESKAPE-group (Enterococcus faecium, Staphyhlococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter ssp.; with resistance against Carbapenemases, Cephalosporines and Colistin) and Escherichia coli] based on cultivation methods followed by a characterization via MALDI-TOF MS and susceptibility testing applying microdilution. Residues of macrolide antibiotics such as clarithromycin (up to 0.60μg/L) and residues of sulfamethoxazole (up to 0.40μg/L) and trimethoprim (up to 0.39μg/L) were detected downstream of the sewage treatment plants. In addition, no antibiotic residues were detected upstream the respective sewage treatment plants, except for anhydroerythromycin (n=1, <LOQ). In total, blaOXA-58 was found in 6, and mcr in one water sample throughout the whole sample period. No MRSA, extensively drug-resistant or pan-resistant bacteria, according to Magiorakos et al. (2011), could be isolated. In contrast, multidrug-resistant bacteria, especially E. coli and Enterococcus faecium were found. In conclusion, sewage treatment plant effluents are point sources for antibiotic residues (p=0.0000 and p=0.0001) and antibiotic-resistant bacteria (p=0.0021 and p=0.0060) since their concentrations increased significantly after the discharge of treated wastewater into the investigated rivers. In this specific catchment area, which lacked clinical, hospitals, and intensive livestock farming, no significant bacterial or analyzed chemical (antibiotic residues) influences of the sewage treatment plants were observed in the river downstream of the drinking water reservoir during the study. Furthermore, no increased selection pressure could be expected as no measured antibiotic residues exceeded the predicted no effect concentration for antibiotic resistance selection, according to Bengtsson-Palme and Larsson (2016).

Seasonal Variations in Water-Quality, Antibiotic Residues, Resistant Bacteria and Antibiotic Resistance Genes of Escherichia coli Isolates from Water and Sediments of the Kshipra River in Central India.

Objectives: To characterize the seasonal variation, over one year, in water-quality, antibiotic residue levels, antibiotic resistance genes and antibiotic resistance in Escherichia coli isolates from water and sediment of the Kshipra River in Central India. Methods: Water and sediment samples were collected from seven selected points from the Kshipra River in the Indian city of Ujjain in the summer, rainy season, autumn and winter seasons in 2014. Water quality parameters (physical, chemical and microbiological) were analyzed using standard methods. High-performance liquid chromatography–tandem mass spectrometry was used to determine the concentrations of antibiotic residues. In river water and sediment samples, antibiotic resistance and multidrug resistance patterns of isolated E. coli to 17 antibiotics were tested and genes coding for resistance and phylogenetic groups were detected using multiplex polymerase chain reaction. One-way analysis of variance (ANOVA) and Fisher tests were applied to determine seasonal variation. Results: In river water, seasonal variation was significantly associated with various water quality parameters, presence of sulfamethoxazole residues, bacteria resistant to ampicillin, cefepime, meropenem, amikacin, gentamicin, tigecycline, multidrug resistance and CTX-M-1 gene. The majority of the Extended Spectrum Beta-Lactamase (ESBL)-producing E. coli isolates from river water and sediment in all different seasons belonged to phylogenetic group A or B1. Conclusions: Antibiotic pollution, resistance and resistance genes in the Kshipra River showed significant seasonal variation. Guidelines and regulatory standards are needed to control environmental dissemination of these “pollutants” in this holy river.

Impact of sludge treatments on the extractability and fate of acetyl sulfamethoxazole residues in amended soils

Sludge recycled in agriculture may bring antibiotics into cropped soils. The nature, total amount, and availability of the antibiotics in soil partly depend on the sludge treatments. Our paper compares the fate of N-acetyl sulfamethoxazole (AC-SMX) residues between soils incubated with the same sludge but submitted to different processes before being added in soil.The fate of 14C-AC-SMX residues was studied in mixtures of soil and sludges at different treatment levels: 1) activated and 2) centrifuged sludges, both enriched with 14C-AC-SMX, and 3) limed and 4) heat-dried sludges obtained by treating the previously contaminated centrifuged sludge. The evolution of the extractability of 14C residues (CaCl2, methanol) and their mineralization were followed during 119 days.More than 80% of the initial 14C-activity was no longer extractable after 14 days, except in soil with limed sludge. Liming and drying the centrifuged sludge decreased the mineralized 14C fraction from 5.7-6.4% to 1.2–1.8% and consequently, the corresponding soils contained more 14C residues after 119 days. Although 14C residues were more CaCl2-extractable in soil with limed sludge, they seemed to be poorly bioavailable for biodegradation. For all solid sludges, the mineralization rate of 14C-AC-SMX residues was strongly correlated to that of sludge organic carbon, with a coefficient three times lower for the limed and dried sludges than for the centrifuged sludge after 14 days.

Water metagenomic analysis reveals low bacterial diversity and the presence of antimicrobial residues and resistance genes in a river containing wastewater from backyard aquacultures in the Mekong Delta, Vietnam

The environmental pathways for the dissemination of antibiotic resistance have recently received increased attention. Aquatic environments act as reservoirs or sources of antimicrobial-resistant bacteria, antimicrobial residues, and antimicrobial resistance genes (ARGs). Therefore, it is imperative to identify the role of polluted water in the dissemination of antimicrobial resistance. The aim of this study was to evaluate the antimicrobial residues, ARGs, and microbiota in the freshwater systems of the Mekong Delta. We selected 12 freshwater sites from aquacultures and rivers in Can Tho, Vietnam and analyzed them for 45 antimicrobial residues and 8 ARGs by LC/MS/MS and real-time PCR, respectively. A 16S rDNA-based metagenomic analysis was conducted to characterize the water microbiota. Residues of sulfamethoxazole (10/12) and sulfadimidine (7/12) were widely detected, together with the sulfa-resistance genes sul1 (11/12) and sul2 (9/12). Additionally, sulfamethoxazole residues and the β-lactamase-resistance gene blaCTX-M-1 were detected in eight freshwater systems (8/12), suggesting that these freshwater systems may have been polluted by human activity. The metagenomic analysis showed that all the tested freshwater systems contained the phyla Proteobacteria, Actinobacteria, and Bacteroidetes, representing 64% of the total microbiota. Moreover, the Cai Rang River site (Ri-E), which is located at the merge point of wastewaters from backyard-based aquacultures, contained the genera Polynucleobacter, Variovorax, and Limnohabitans, representing more than 78.4% of the total microbiota. Bacterial diversity analysis showed that the Ri-E exhibited the lowest diversity compared with other regions. Principal coordinate analysis showed that the differences among water microbiotas in backyard-based aquacultures could be explained by the farmers' aquaculture techniques.In conclusion, this study demonstrated a collapse of bacterial diversity at the merge point of wastewaters from backyard-based aquacultures in the Mekong Delta.

Development of immunoassays to determinate sulfamethoxazole residues in wastewaters

Different immunoassays have been developed to monitor sulfamethoxazole (SMX), an antibiotic frequently detected in water. The immunoassays were developed and optimized in two different formats: ELISA in polystyrene 96-well plate and microarray on compact disc (CD) support. Competitive microimmunoassays were performed by direct adsorption of immunoreagents on the polycarbonate surface of a low-reflectivity CD. Results were displayed using nanogold-labeled immunoglobulins and silver staining developer. High sensitivity was achieved with both formats: LD 0.001 ng mL(-1) in the ELISA-plate, and 0.09 ng mL(-1) in the CD format. The novel CD methodology presents advantages such as simplicity, sensitivity, portability, analytical capacity (2560 spots per disc), in addition to reductions in immunoreagents required, costs and time for analysis. Both proposed methods were applied to determine SMX at nanograms per litre level in wastewater samples without previous preconcentration. Finally, the determination of several fortified wastewater samples using the CD format protocol showed a mean recovery of 87%, which confirms that this assay format is a good screening tool to detect sulfamethoxazole in water samples rapidly and reliably.

Application of a multi-sulfonamide biosensor immunoassay for the detection of sulfadiazine and sulfamethoxazole residues in broiler serum and its use as a predictor of the levels in edible tissue

A multi-sulfonamide biosensor immunoassay (BIA), based on a previously developed mutant antibody (A.3.5) in an optical biosensor (Biacore 3000), was applied to analyse the serum and plasma samples obtained from the broilers treated with sulfamethoxazole and sulfadiazine. The assay was fast (5 min per sample), the sample preparation easy (dilution in antibody containing buffer only) and an equal sensitivity for the two sulfonamides was obtained with limits of detection (LOD) in serum and plasma below 10 ng ml −1. The concentrations found with the BIA in serum and plasma of the treated broilers were comparable and higher than the concentrations found in the tissue by LC–MS/MS. The average serum/tissue ratios for sulfamethoxazole were 6.2 (leg meat), 2.5 (liver) and 1.3 (skin + fat) and for sulfadiazine 8.7 (leg meat), 3.1 (liver) and 2.2 (skin + fat). To predict the concentrations of the two sulfonamides below the maximum residue limit (MRL) of 100 ng g −1 in the tissue with the highest level (skin + fat), the proposed action level of the multi-sulfonamide BIA in serum is 130 ng ml −1. A later developed mutant antibody (M.3.4), with a better sensitivity towards more sulfonamides, was applied during a survey. Serum samples ( n = 300) of broilers from 30 different flocks were found negative. Concentrations between <5 and 152 ng ml −1 (sulfamethoxazole equivalents) were found in the serum samples of one flock ( n = 160) with an average of 25 ± 21 ng ml −1. The sulfonamide identified by LC–MS/MS in these samples was sulfamethoxazole.

Usefulness of Enzyme Immunoassay (EIA) as an Analytical Method of Sulfamethoxazole Residues in Animal Tissues

Applicability of enzyme immunoassay (EIA) for determination of drug residues in animal tissues was examined by using sulfamethoxazole (SMX) as a test drug. EIA of SMX was specific for SMX, though N4-acetyl SMX showed a higher reactivity in the system. Control swine serum and plasma spiked with 8ng/ml SMX were negative. Control liver or other tissues showed reactivity consistent with that expected when spiked with up to 25ng/g (ml) SMX. It seems necessary for the accurate determination of SMX in tissues to use standard curves prepared by adding SMX to control tissues, and in some cases by use of HPLC as well. EIA was, however, applicable as a screening method to detect SMX in chicken muscle without using control muscle when an adequate cut-off value was used.