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Primary validation of Charm II tests for the detection of antimicrobial residues in a range of aquaculture fish

Primary validation of Charm II tests for the detection of antimicrobial residues in a range of... The study carried out a primary validation of Charm II tests for the detection of antimicrobial residues in aquaculture fish. The validation was performed according to European Commission Decision 2002/657/EC and the parameters determined included: detection capability, repeatability, reproducibility, specificity and robustness for the detection of antimicrobial residues in fish. Fish materials from different species including cat fish, trout, salmon, sea bass, tilapia, lingue and pangasius, were spiked with varying concentrations of selected antimicrobials including sulfonamides, β-lactams, macrolides, tetracyclines and aminoglycosides to determine the detection capabilities and other validation parameters of the Charm II tests. Results of the validation showed that the detection capabilities for the tetracyclines ranged from 25 to 100 µg/kg, while the sulfonamides and aminoglycosides were detected at 25 µg/kg for all species under study. The detection capabilities for the beta-lactams ranged from 25 to 300 µg/kg; and was 100 µg/kg for the tested macrolides. Results of the study showed that there was no significant difference between counts for samples read immediately after addition of the scintillation liquid and those read 14 h after addition of the scintillation liquid, provided that there was good vortexing before analysis. There was also no significant difference between counts for the same samples analyzed in different runs under repeatability and reproducibility conditions at the same spiking concentrations for the different fish species analyzed. The relative standard deviation for both repeatability and repro - ducibility ranged from 1.2 to 15.1%. The Charm II tests were found to be 100% group specific, as none of the antimi- crobials kits, gave false positive results when testing non-target antimicrobial drugs. Results of this study demonstrate the suitability of the Charm II technique as a rapid screening tool for detection of antimicrobial residues in a variety of fish species at maximum residue limits (MRL) established in the EU guidelines, with the exception of tilmicosin which was detected at 2 MRL. The results also prove the robustness, specificity, reliability and precision of the Charm II assay in the detection of various antimicrobial residuals in fish and its applicability for the rapid evaluation of the quality of aquaculture fish for safety and trade purposes. Keywords: Charm II tests, Antimicrobial residues, Rapid screening, Method validation, Aquaculture fish, Maximum residue limit Introduction Fish farming is a fast emerging industry that besides creating employment, is a source of good quality animal protein and essential macronutrients in the diet. Fish and *Correspondence: emmanuel@cns.mak.ac.ug fish related products provide income and livelihoods for Department of Chemistry, College of Natural Sciences, Makerere University, P.O. Box, 7062, Kampala, Uganda numerous communities across the world besides playing Full list of author information is available at the end of the article © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mukota et al. BMC Chemistry (2020) 14:32 Page 2 of 15 a crucial role in assuring sufficient availability of safe and of animal origin [12]. The safety concerns regarding drug healthy food [1, 2]. The increased demand for fish for residues in various food products, calls for develop- the growing international population, especially in the ment and validation of rapid and reliable techniques for developing world, has continued to deplete the sustain- detection of these compounds. Such rapid techniques able yields from lakes, rivers, swamps, seas and other can facilitate fast decision making to minimize technical natural water bodies. Aquaculture is growing rapidly and barriers to trade and also enhance routine monitoring in is seen as a remedy to address and supplement the dwin- order to protect consumer health. dling quantities and shortfall in wild catch [3]. However, The Charm II radio receptor assay technique devel - big numbers of fish in a confined volume of space tend to oped by Charm Sciences Inc, is one of the rapid screen- increase incidences of bacterial infections and other dis- ing techniques for detection of residues of antimicrobials eases; which greatly affects yield in the aquaculture busi - such as beta-lactams, sulfonamides, tetracyclines, chlo- ness. Productivity in aquaculture may be enhanced by ramphenicol, quinolones, macrolides and aminoglyco- use of antimicrobials such as tetracyclines, macrolides, sides in various food products including fish, meat, eggs, beta-lactams, sulfonamides, and streptomycins, for the honey, and milk, as well as non-food matrices including prevention and treatment of opportunistic infections in water, feed and urine. This technique utilizes a microbial fish [ 4, 5]. cell with receptor sites that bind the specific antimicro - Antimicrobials are used to control ectoparasitic, fungal bial drug. The analytical process involves a binder being and bacterial diseases of the body and gills of fish [6–8]. added to a sample extract along with an amount of H Tetracyclines in particular are frequently employed in or C labeled antimicrobial tracer. Any antimicrobial in aquaculture due to their broad spectrum of activity as the sample extract competes for the binding sites with well as their low cost, compared to other antibiotics. the tracer. The amount of tracer that binds to the recep - The tetracyclines are used to combat bacterial hemor - tor sites is measured and compared to a previously deter- rhagic septicemia in catfish as well as diseases caused mined control point. Therefore, the more radiolabelled by Pseudomonas liquefaciens [9]. Currently, there are antimicrobial detected in the mixture, the lower the con- over 20 tetracyclines available; although, tetracycline, centration of antimicrobial in the sample. The smaller chlortetracycline, oxytetracycline, and doxycycline are the amount of tracer measured, the greater the drug the most common ones in veterinary medicine and aqua- concentration in the sample [21, 22]. The Charm II tech - culture [10, 11]. The aforementioned antibiotics are the nique has very limited validation data for the detection only tetracyclines with registration within the European of antimicrobials in different fish species. Thus, this study Union (EU) for use as veterinary medicinal products in conducted a primary validation of the Charm II tests in food producing animals; with established maximum resi- order to generate comprehensive analytical data to prove due limits (MRLs) in different food matrices [12]. Other the validity, applicability and also address potential limi- antimicrobials such as sulfonamides, beta-lactams, mac- tations of the Cham II assays particularly for the screen- rolides and aminoglycosides also have a wide spectrum of ing of antimicrobials in different aquaculture fish species. activities against most Gram positive and Gram negative organisms and are used for the prevention and treatment of bacterial infections in livestock and aquaculture. The Materials and methods antimicrobials are typically administered in the water, Reagents, materials and equipment often as components of fish feed, and are occasionally The antimicrobial test assay kit was obtained from Charm injected [13, 14]. Sciences Inc., Lawrence, MA; and included items for the The extensive use and misuse of antimicrobials in farm detection of beta-lactams (PMSU-050A); sulfonamides animals as growth promoters or as nonspecific means of (SMMSU-022C), macrolides (EMSU-023A); tetracyclines infection prevention has been reported to lead to accu- (TMSU-025); and streptomycin (STMSU-023A). Con- mulation of residues in edible tissue [7, 15, 16]; which sumables and equipment used for the tests included: M2 may cause allergic and toxic effects in consumers as Buffer, zero and positive control standards, MSU extrac - well as contributing to the development of antimicro- tion buffer, radioactive labelled tablets; scintillation fluid bial resistant bacteria [17–20]. In this respect, residues (Opti-Fluor O, PerkinElmer), Intronic incubator (Charm in foodstuffs create public health concerns, consumer Sciences Inc.), Wallac 1409 scintillator counter, refriger- perception problems and trade disputes that have enor- ated centrifuge Sigma 4K15c (Sigma-Aldrich), R2 blender mous negative impacts on the food industry. In order to (Robot-Coupe) and a water bath (Julabo MB13). In addi- protect human health, regulatory authorities like the EU, tion, scintillation vials, AES mix masticator stomacher established maximum residue limits (MRLs) for some and IEC Centra CL-3 centrifuge were also used. pharmaceutical compounds in fish and other foodstuffs M ukota et al. BMC Chemistry (2020) 14:32 Page 3 of 15 Preparation of standard reference material and stock residues was detected. Absence of residual antibiotics solutions in the fish samples was confirmed through evaluation The multi antimicrobial concentrate standard (MSU, of their counts per minute in comparison with results Charm Sciences Inc.) was prepared fresh on the day of obtained using the negative control extraction buffers use and diluted with 10  ml of deionized water, shaken supplied with the Charm II kits. The control buffers are well and allowed to stand on ice for 15 min. The reconsti - contaminant free and are used to qualify the matrix as tuted stock solution contained; penicillin G, 1000  µg/kg; negative when a known negative is not available. The erythromycin A, 10,000  µg/kg; sulfamethazine, 1000  µg/ tolerance considered for the fish matrix to qualify as kg; chlortetracycline, 4000  µg/kg; and streptomycin, negative and selected for use in subsequent test was for 10,000 µg/kg. Other analytical standards were purchased counts within ± 20% of the average result obtained with from Sigma Aldrich, Pfizer Inc., US Pharmacopeia Con - the respective negative control extraction buffer. Sam - vention and Acros Organics (Additional file 1: Table S1a). ples with counts beyond the tolerance limits were dis- These standards were appropriately diluted with deion - carded while those meeting the criteria were selected ized water to make working standard solutions of the for the study. The selected blank fish materials after respective antimicrobial, and kept below 4 °C. The work - blending, were spiked with antimicrobial standards of ing standards were used for spiking fish samples at dif - known concentrations and used as control samples for ferent concentration levels ranging from 25 to 300 µg/kg. the establishment of the control point counts per min- ute (cpm). A list of control standards used in the study is shown in Additional file 1 : Table S1a. Methods The study carried out a primary validation of the Charm II tests for the detection of antimicrobial resi- Extraction of drugs from the fish materials dues in aquaculture fish. The validation was performed The MSU extraction buffer (30 ml) was added to blended according to Commission Decision 2002/657/EC [23] fish material (10  g) in a polypropylene centrifuge tube. and all methods of analysis used were adopted from the The mixture was homogenized using a stomacher for general Charm II protocols [21]. 2 min and returned to the centrifuge tube. The homoge - nate was incubated in water bath at 80  °C for 30  min, during the determination of streptomycin, macrolides, Fish samples selected for the study or beta-lactams; and 45  min, when determining tetra- The fish materials used in the study were obtained from cyclines or sulfa drugs. After incubation, the tube was dead fish purchased from Melle and Ghent fish shops cooled on ice water for 10  min and then centrifuged at and supermarkets in Belgium. Aquaculture fish species 3300  rpm for 10  min, using a refrigerated centrifuge including cat fish (Siluriformes), trout (Oncorhynchus 4K15C (Sigma-Aldrich). The resulting supernatant solu - mykiss), salmon (Salmo salar), seabass (Dicentrar- tion was collected and used for the required tests. The chus labrax), tilapia (Oreochromis niloticus), lingue pH of the supernatant was where necessary adjusted to (Molva molva), dorade (Sparus aurata) and pangasius pH 7.5 using reconstituted Charm II kit M2 buffer for (Pangasius bocourti), were selected for the study. Fish low pH, or 0.1 M hydrochloric acid for high pH. sample materials were taken by carefully removing the muscle tissue from the side of each fish taking precau - tion to exclude scales and skin. The fish samples that Determination of tetracyclines in the fish samples were not used immediately were stored below − 18  °C In the detection of tetracycline, the white tablet from for a maximum of 2 months. the kit containing the binding reagent (TMSU-025) was introduced into a test tube, and water (300  μl) was Sample preparation added. The contents of the tube were mixed for at least The fresh fish sample was weighed in a centrifuge tube 10  s to ensure breakup of the tablet. The sample extract and stored at − 18 °C until further processing. The frozen or control sample (4 ml) was added to the tube, followed fish samples were thawed at 4  °C overnight and cut into by addition of the orange tablet containing the tracer small pieces before blending in a high speed blender. The reagent from the kit (TMSU-025). The resultant solu - blended fish material (10  g) was transferred into a poly - tion was mixed for about 10 s and the mixture was incu- propylene centrifuge tube and used immediately. bated at 35 °C for 5 min; and then centrifuged for another 5  min on a IEC Centra CL-3 centrifuge. The superna - tant was poured off carefully, deterring the formed pel - Preparation of control samples let from sliding out of test tube. Deionized water (300 μl) All fish samples were first tested with the different was added to the tube and the contents mixed thoroughly Charm II kits and only used in case no veterinary drug to break up the pellet. After suspension of the pellet in Mukota et al. BMC Chemistry (2020) 14:32 Page 4 of 15 water, the scintillation liquid (3.0 ml) was added and test capped. The tube was shaken until the mixture had a uni - tube capped. The tube was shaken until the mixture had form cloudy appearance. The glass tube contents were a uniform cloudy appearance. The glass tube contents transferred completely into a scintillation vial and the were transferred completely into a scintillation vial and mixture counted using a Wallac liquid scintillation coun- the mixture counted using a Wallac liquid scintillation ter for 60  s on the [ H] channel. The cpm results of the counter for 60  s on the [ H] channel. The results for the sample were compared with the control point. sample was compared with the control point counts per minute. Determination of aminoglycoside‑streptomycin in the fish samples Determination of macrolides in the fish samples In the determination of streptomycin, the white tablet During the detection of macrolides, the white tablet from from the kit containing the binding reagent (STMSU- the Charm II kit containing the binding reagent (EMSU- 023A) was introduced into a test tube, and water (300 μl) 023A) was introduced into a test tube, and water (300 μl) added. The contents of the tube were mixed for at least was added. The contents of the tube were mixed for at 10 s to ensure breakup of the tablet. The sample extract or least 10  s to ensure breakup of the tablet. The sample control sample (2 ml) was added to the tube and mixed. extract or control sample (4  ml) was added to the tube This was followed by addition of the green tablet contain - and the contents mixed on a vortex for 10  s. The result - ing the tracer reagent (STMSU-023A). The resultant was ant was incubated at 55  °C for 2  min, followed by addi- mixed by swirling the contents up and down for about tion of a green tablet containing the tracer reagent from 10 s. The mixture was incubated at 35 °C for 2 min, and the kit (EMSU-023A). The resultant was mixed on a then centrifuged for another 3 min. The supernatant was vortex for 10  s. The mixture was incubated at 55  °C for poured off carefully and the edge of tube was blotted with 2  min, and then centrifuged for 5  min. The supernatant absorbent paper. Deionized water (300  μl) was added to was poured off carefully and the edge of tube blotted on the tube and the contents mixed thoroughly. After sus- absorbent paper. Deionized water (300  μl) was added to pension of the pellet in water, the scintillation liquid the tube and the contents mixed thoroughly to break up (3.0  ml) was added and test tube capped. The tube was the formed pellet. After suspension of the pellet in water, shaken until the mixture had a uniform cloudy appear- the scintillation liquid (3.0  ml) was added and the test ance. The glass tube contents were transferred com - tube capped. The contents were mixed on a vortex until pletely into a scintillation vial and the mixture counted the mixture had a uniform cloudy appearance. The con - using a Wallac liquid scintillation counter for 60 s on the tent of the glass tube was transferred completely into a [ H] channel. The cpm results for the sample were com - scintillation vial and the mixture counted using a Wallac pared with the control point. liquid scintillation counter for 60 s on the [ C] channel. The counts per minute (cpm) of the sample was com - Determination of β‑lactams in the fish samples pared with the control point. In the determination of β-lactams, the green tablet from the Charm II kit containing the binding reagent (PMSU- Determination of sulfa drugs in the fish samples 050A) was introduced into a test tube, and water (300 μl) In the detection of sulfa drugs, the white tablet from the was added. The contents of the tube were mixed to Charm II kit containing the binding reagent (SMMSU- ensure breakup of the tablet. The sample extract or con - 022C) was introduced into a test tube, and water (300 μl) trol (2 ml) was added to the tube and the contents mixed added. The contents of the tube were mixed for at least on a vortex for 10 s. The resultant was incubated at 55 C 10  s to ensure breakup of the tablet. The sample extract for 2 min, followed by addition of a yellow tablet contain- or control sample (4 ml) was added to the tube, followed ing the tracer reagent (PMSU-050A) from the kit. The by addition of the pink tablet containing the tracer rea- resultant was mixed on a vortex for 10 s. The mixture was gent (SMMSU-022C) from the kit. The resultant solu - incubated at 55  °C for 2  min, and then centrifuged for tion was mixed by swirling the contents up and down for 5  min at 1750 G. The supernatant was poured off care - about 15 s. The mixture was incubated at 65 °C for 3 min, fully and the edge of tube blotted on absorbent paper. and then centrifuged for another 3 min. The supernatant Deionized water (300  μl) was added to the tube and the was poured off carefully, deterring the formed pellet from contents mixed thoroughly to break up the pellet. After sliding out of test tube; and the edge of tube was blotted suspension of the pellet in water, the scintillation liquid on absorbent paper. Deionized water (300 μl) was added (3.0  ml) was added and test tube capped. The contents to the tube and the contents mixed thoroughly to break were mixed on a vortex until the mixture had a uniform up the pellet. After suspension of the pellet in water, cloudy appearance. The mixture was transferred com - the scintillation liquid (3.0  ml) was added and test tube pletely into a scintillation vial and counted using a Wallac M ukota et al. BMC Chemistry (2020) 14:32 Page 5 of 15 liquid scintillation counter for 60 s on the [ C] channel. researchers using the same method and a scintillation The cpm of the sample was compared with the control fluid counter equipment. point. Robustness The robustness of the techniques was tested by delib - Method validation erately varying the experimental time indicated in the The method validation was done according to the cri - Charm II analytical protocol. This was intended to study teria of the European Commission Decision 2002/657/ the effect of variation in reading time interval for a large EC [23]. The validation parameters performed included; batch of processed samples. Reading of the cpm for detection capability (CCβ), repeatability, reproducibility, the samples spiked with 50  µg/kg amoxicillin was done robustness and cross reaction activity. immediately after the addition of the scintillation liquid and then after 14 h on the same batch of extracted sam- ple. The samples after the first reading were stored over - Detection capability night in the fridge at 4 °C, removed and allowed to attain The CCβ was examined by spiking blank fish matrices room temperature, and then read the second time after with different antimicrobials including tetracyclines, vortexing. macrolides, β-lactams, aminoglycosides, and sulfona- mides. The number of samples analyzed for each individ - ual antimicrobial agent ranged from 20 to 30 as indicated Cross reaction activity in Table 3. The spiking concentrations varied around the Cross reactivity was investigated by spiking residue-free recommended maximum residue limit (MRL), including blank fish samples with high concentrations (up to 10 0.05 MRL, 0.25 MRL, 0.5 MRL, 0.75 MRL and MRL, for MRL) of the respective antimicrobial belonging to other the respective antimicrobial. The CCβ was then deter - antimicrobial groups and the samples run on targeted mined as the lowest concentration of the antimicrobial channels to investigate false identification. that could be detected in the sample giving at least 95% positive results. Data Analysis All data generated was statistically analyzed using one- way analysis of variance (ANOVA) to examine any sig- Repeatability nificant differences between the observed results under The repeatability of the technique was studied by analy - different experimental setups. sis of selected fish samples spiked with different antimi - crobials including tetracyclines, macrolides, β-lactams, Results and discussion aminoglycosides, and sulfonamides. The total number Counts per minute for blank samples of samples analyzed for each individual antimicrobial The blank samples used in the study were those fish tis - compound ranged from 20 to 30, and n ≥ 6 for the same sue matrices which were carried through the complete fish species. The spiking concentrations varied around analytical procedure, and no antimicrobial residues were the MRL, including 0.05 MRL, 0.25 MRL, 0.5 MRL, 0.75 detected in them using the respective Charm II assay MRL and MRL, for the respective antimicrobial. The kits [21]. The blank fish samples to which the binder and analysis was performed within a short interval, by a sin- tracer had been added but without addition of an anti- gle researcher using the same method and scintillation microbial agent were extracted with the different kits and fluid counter equipment. read on the respective channels. The results of the cpm for the blank fish samples are summarized in Table 1. Reproducibility From Table  1, the cpm for tilapia, trout, salmon, pan- The reproducibility of the method was studied by repeat gasius, seabass, dorate, catfish, and lingue fish species analysis of selected fish samples spiked with differ - were statistically evaluated using ANOVA and it was ent antimicrobials including tetracyclines, macrolides, found that the overall F-calculated (0.22) was less than β-lactams, aminoglycosides, and sulfonamides. The num - F-critical (2.5), which implied that there was no signifi - ber of samples analyzed for each individual antimicrobial cant difference between results for the blank fish samples ranged from 20 to 30, with n ≥ 6 for the same fish species. of the aforementioned species when using antimicro- The spiking concentrations varied around the recom - bial test kits for β-lactams, tetracyclines, macrolides and mended MRL, including 0.05 MRL, 0.25 MRL, 0.5 MRL, streptomycins. However significant difference in cpm 0.75 MRL and MRL, for the respective antimicrobial. The values was observed with the sulfonamides extraction kit analysis was performed on different days by two different while testing catfish, lingue and pangasius. The cpm for Mukota et al. BMC Chemistry (2020) 14:32 Page 6 of 15 Table 1 Blank counts per minute for the different fish species obtained using the Charm II technique Scintillation counter results (cpm) Charm II test β‑lactams kit Sulfonamides kit Tetracyclines kit Macrolides kit Streptomycins kit Fish species Mean SD Mean SD Mean SD Mean SD Mean SD Tilapia 2704 0.9 2596 736 3027 0.7 2448 191.1 5103 346 Trout 2506 192.0 2332 1184 2830 260 2799 87.1 4799 259.9 Salmon 2571 207.4 2472 541.2 2939 165.0 2110 117.2 3085 133.4 Pangasius 2469 195.7 5625 1254 2931 221.4 2893 110 4796 437.7 Seabass 2432 232.1 2144 672.1 2971 252.2 2700 153.6 4805 594.7 Dorate 2512 171.1 1977 621.4 2864 93.4 2803 167.3 4967 485.8 Catfish 2493 312.7 5872 774.3 Lingue 4454 650.1 cpm counts per minute, SD standard deviation these species were almost double those of the other types In this respect, the CP for the β-lactams was evaluated of fish and their F-calculated (15.1) was greater than from averaging the results of 6 negative samples spiked F-critical (2.4). The big variation in cpm for the catfish, with penicillin G at 25 µg/kg (0.5 MRL) and adding 20% of the obtained average cpm value. Whereas, for the sul lingue and pangasius fish species as compared to the rest - could be attributed to the high fish fat content extracted fonamides, the CP was evaluated by averaging results of by the sulfonamide kit protocol. In this respect, the three negative samples spiked at 50  µg/kg with sulfamethaz- fish species (catfish, lingue and pangasius) need to be ine and adding 30% of the average obtained cpm value. handled separately when calculating control points to A control point of 1530 was calculated for the β-lactams. minimize chances of getting false negative or false posi- On the other hand, the CP for tetracyclines was calcu- tive results. For the rest of the fish species, the blank cpm lated by averaging cpm results of negative control stand- results were used to derive the respective control points ards provided in the tetracyclines test kit and subtracting for the different residues. 40% of the obtained average cpm value (Table 2). For macrolides, the CP was derived from averaging the Evaluation of the Control Points for the different drug results of 6 negative samples spiked with erythromycin A residues at 100  µg/kg (0.5 MRL) and adding 20% of the obtained The control point (CP) of a sample is the cut-off point average cpm value. Using a similar approach, the CP for between a negative or positive result. Any antimicrobial streptomycin was derived from averaging results of nega- agent present in the sample extract competes for the tive samples spiked at 25  µg/kg with streptomycin and binding sites with the tracer, thus, the greater the cpm adding 30% of the average obtained cpm value. measured, the lower the antimicrobial drug concentra- During the analysis of antimicrobial residues in fish tion in the sample and vice versa. Samples with high samples, results less than or equal to each respective CP counts are considered negative (tracer antimicrobials are were interpreted as positive while those greater than the largely bound to the binder) while those with low counts CP, as negative. Blank sample readings below the set CP are considered positive (tracer antimicrobials are largely were considered false positive. The results in Table  2, free in solution). The CP for the different antimicrobials show that the false positive rate was 0% for tetracyclines, were determined independently; and with the exception β-lactams and sulfonamides; 3.6% for macrolides, and 5% of tetracyclines, the MRL value for each drug was spiked for streptomycin; this proved the validity of the obtained to the respective blank fish sample. In order to cater for data since it met the acceptance criteria of being within the deviations in the different fish matrices, a percentage 5%. A comparison of the CP for the different antimicro - tolerance was added to or subtracted from the obtained bials obtained using the Charm II assay with the corre- average cpm value of the spiked blank fish sample. The sponding cut-off points (Fm) and technical threshold (T) CP evaluation was performed according to the Charm II values, calculated following Annex II of the EU guideline protocol, and the percentages added to the mean value of for Community Reference Laboratories Residues for vali- spiked samples at detection capability or subtracted from dation of screening methods [26], is shown in Table 3. the mean value of blanks serve to minimise occurrence of According to the EU guideline, the cut off factor false positive or negative readings [21, 24, 25]. (Fm), refers to the response or signal from a screening M ukota et al. BMC Chemistry (2020) 14:32 Page 7 of 15 Table 2 Control points for the different antimicrobials in blank fish samples Antimicrobial Spiked samples Blank samples family Level of analyte Mean cpm Allowance Control Mean blank Range of blank No. of false False spiking (µg/kg) of spiked for matrix point cpm cpm readings positives/no. positive samples effect cpm of samples rate (%) β-lactams 25 µg/kg penicil- 1275 Spiked 1530 2502 2160–2907 0/30 0 lin G cpm + 20% Sulfonamides 50 µg/kg sul- 1096 Spiked 1424 3162 1431–6995 0/30 0 famethazine cpm + 30% Tetracyclines 0 µg/kg tetracy- 2524 Blank cpm 1514 2524 2451–3269 0/30 0 cline − 40% Macrolides 100 µg/kg eryth- 1765 Spiked 2118 2587 1906–2952 1/28 3.6 romycin A cpm + 20% Streptomycin 25 µg/kg strep- 2574 Spiked 3346 4605 2942–5488 1/20 5.0 tomycin cpm + 30% Number of samples used per parameter Ns ≥ 20 Table 3 Comparison of  control points by  Charm II protocol, cut-off points and  technical threshold values calculated according to the EU guideline [26, 27] Antimicrobial Compound Spiked B average Calculated T value Calculated Fm Calculated family concentration response of blank as per EU guideline value as per EU control point CP (μg/kg) samples T = B − 1.64 * SDb guideline as per Charm II assay [26, 27] Fm = M + 1.64 * SDs [26, 27] Tetracyclines Tetracycline 25 2958 2776 816 1514 Chlortetracycline 25 3050 2780 1417 Oxytetracycline 100 2891 2579 1427 Macrolides Erythromycin A 100 2814 2564 1904 2118 Tilmicosin 100 2486 2164 2002 Tylosin A 100 2512 2115 1740 β-Lactams Penicillin G 25 2523 2176 1438 1530 Ampicillin 50 2455 2042 1341 Amoxicillin 50 2702 2536 1487 Oxacillin 300 2398 2171 1478 Dicloxacillin 300 2524 2384 1489 Cloxacillin 300 2500 2368 1413 Aminoglycosides Streptomycin 25 4822 3867 2592 3346 Sulfonamides Sulfamethazine 25 2593 1428 1379 1424 Sulfadimethoxine 25 2266 1129 972 Sulfamerazine 25 2210 1095 930 Sulfadiazine 25 2297 1296 1184 Sulfathiazole 25 2266 1643 1485 Cut-off factor (Fm) = M + 1.64 * SDs; Technical threshold (T ) = B − 1.64 * SDb; M, mean response of spiked samples; B, mean response of blank samples; SDs, standard deviation of the spiked sample readings; SDb, standard deviation of blank readings test which indicates that a sample contains an analyte of the blank samples are greater than those for spiked at or above the screening target concentration [26], samples, because the responses are inversely propor- while the Charm II protocol CP is the cut-off point tional to concentrations of the antimicrobials. In this between a negative or positive result [21]. On the other respect, the assay is considered valid only when Fm < T hand, the technical threshold (T), refers to the limit for and the CCβ is validated when Fm < B. Accordingly, the positivity [26]. For the Charm II technique the readings number of spiked samples with mean responses below Mukota et al. BMC Chemistry (2020) 14:32 Page 8 of 15 Detection capability for the different antimicrobials the cut-off level (deemed positive) is identified and the in selected fish species false positive rate determined. If T < Fm < B, the false- The CCβ is the lowest concentration of the analyte that positive rate is greater than 5%. In the case Fm < T the could be detected in the sample giving at least 95% posi- false positive rate is below 5%. If more than 5% of the tive results. In the CCβ studies, blank negative fish tis - spiked samples at the screening target concentration sue samples were spiked with different antimicrobials at gave a response greater than the cut-off level (deemed various concentrations. Spiked samples that exhibited false negative), the concentration chosen for the spiking readings above the set CP value, were interpreted as false is considered too low for validation and a higher con- negatives. In case more than 5% of the spiked samples at a centration is tested [26, 27]. target concentration gave false negative readings, the con- From the results presented in Table  3, the Fm val- cerned concentration was deemed too low for validation ues obtained using the EU guideline and the respec- and a higher concentration was considered. A summary tive calculated CP according to the Charm II protocol of the CCβ for the different drugs involved in the study is are comparable. For all antimicrobials, the respective presented in Table 4. Results show that the Charm II tech- CCβ, presented in Table  3 are valid since in all cases nique can detect tetracycline and chlortetracycline spiked the Fm < B. In addition, for all antibiotics involved in at 25  µg/kg (0.25 MRL) and oxytetracycline at 100  µg/kg the study the Fm < T, which implies that the Charm II (MRL) for the different fish species (cat fish, trout, techniques is validated for the detection of antimicro- salmon, seabass, tilapia, lingue, dorade, and pangasius) bial residues in fish matrix, with a false positive rate with 100% detection. However, the batch of the multi- of less than 5%. In comparison with the Charm II pro- antimicrobial standard, provided in the Charm II kit was tocol, it should be noted that in all cases the CP value not sensitive enough for chlortetracycline to be detected for a particular family of antibiotics is slightly higher at 100 µg/kg (MRL) level. This could be attributed to the the corresponding Fm readings, with the exception of deterioration of the chlortetracycline in the standard due sulfathiazole. This suggests that there will be less inci - to poor handling, probably during transportation. In this dences of false negative readings in the detection of the respect, a Sigma Aldrich standard was used and chlortet- different antimicrobial compounds in fish matrix based racycline detected at a concentration as low as 0.25 MRL. on CP values, although this may increase incidences of Interestingly, it was observed that the technique is also false positive readings. capable of detecting other antimicrobials belonging to the Table 4 Detection capability for the selected antimicrobials Family Compound EU‑MRL CCβ (μg/kg) No of samples No Counter results % Detection (μg/kg) of positive (cpm) of each samples antimicrobial Mean Min Max Tetracyclines (CP = 1514 cpm) Tetracycline 100 25 20 20 724 650 825 100 Chlortetracycline 100 25 21 21 1200 942 1421 100 Oxytetracycline 100 100 31 31 1269 1074 1460 100 Macrolides (CP = 2118 cpm) Erythromycin A 200 100 30 30 1669 954 1955 100 Tilmicosin 50 100 21 21 1565 1221 2078 100 Tylosin A 100 100 21 21 1440 1103 1742 100 β-Lactams (CP = 1530 cpm) Penicillin G 50 25 22 22 1175 921 1421 100 Ampicillin 50 50 21 21 1055 837 1451 100 Amoxicillin 50 50 22 22 1132 908 1409 100 Oxacillin 300 300 24 24 1286 1082 1459 100 Dicloxacillin 300 300 22 21 1186 827 1839 95.5 Cloxacillin 300 300 20 19 1143 681 1547 95.0 Aminoglycosides (CP = 3346 cpm) Streptomycin 500 25 22 22 2424 1642 3074 100 Sulfonamides (CP = 1424 cpm) Sulfamethazine 100 25 29 28 1240 813 1831 96.6 Sulfadimethoxine 100 25 20 20 968 737 923 100 Sulfamerazine 100 25 21 21 842 716 960 100 Sulfadiazine 100 25 20 20 948 735 1361 100 Sulfathiazole 100 25 20 19 989 698 1782 95.0 M ukota et al. BMC Chemistry (2020) 14:32 Page 9 of 15 tetracycline family (tetracycline, oxytetracycline) and not penicillin G, ampicillin, amoxicillin, oxacillin, dicloxacil- limited to the chlortetracycline provided for in the Charm lin and cloxacillin were detected at 25  µg/kg, 50  µg/kg, II test kit. 50  µg/kg, 300  µg/kg, 300  µg/kg and 300  µg/kg respec- The sulfa drugs including, sulfadimethoxine, sulfadia - tively, for all fish species involved in the study. Thus, zine, sulfamerazine were detected at 25 µg/kg (0.25 MRL) penicillin G is detected at 0.5 MRL, whereas ampicillin, for the different fish species (trout, salmon, seabass, tila - amoxicillin, oxacillin, dicloxacillin and cloxacillin are pia and dorade) at 100% detection; sulfamethazine was all detected at their respective MRL. However, 4.5 and detected at 25 µg/kg (0.25 MRL) at 96.6% detection (3.4% 5% of the results for dicloxacillin and cloxacillin respec- false negatives), and sulfathiazole was detected at 25 µg/ tively, were false negatives (Table  4). Further more, the kg (0.25 MRL) at 95.0% detection (5.0% false negatives). Charm II technique is capable of detecting streptomycin The results also show that the technique can detect other at 25 µg/kg (0.05 MRL) for all fish species involved in the antimicrobials belonging to the sulfonamides group study at 100% detection. (sulfamethazine, sulfadimethoxine, sulfamerazine, sul- A comparison of the CCβ and MRL for the different fadiazine and sulfathiazole), which are not included in antimicrobials is shown in Fig.  1. The results show that, the MSU multi-antimicrobial standard mix, provided in CCβ for the validated antimicrobials were below or equal the Charm II test kit. For the macrolides; erythromycin to the MRL for all drug residues in this study, with the A, tilmicosin, and tylosin A were detected at 100  µg/kg, exception of tilmicosin which was detected at 2 MRL. for the different fish species (cat fish, trout, salmon, sea - Most of the drug residues exhibited CCβ in the range bass, tilapia, lingue, dorade, and pangasius) with 100% 0.05 MRL to 0.5 MRL, with 100% detection. Moreover, detection. Whereas, results for the β-lactams show that the incidences of false negative results observed for all Fig. 1 The detection capabilities and maximum residue limits for the different antimicrobials Mukota et al. BMC Chemistry (2020) 14:32 Page 10 of 15 antimicrobials involved in the study were within the 5% unlike some of the chemical techniques that can simulta- requirement of the EU decision 2002/657, and therefore neously detect numerous antimicrobials [30, 31]. the validation results are satisfactory. The Charm II tech - nique exhibited better CCβ for tetracyclines at 25  ppb Repeatability of the method (0.25 MRL) compared to other rapid screening tech- Repeatability analysis was performed using the same niques such as the ELISA kit of R-Biopharm for screening Charm II protocol for a specific antimicrobial on differ - tetracycline antibiotic residues in the muscle of chicken, ent fish species performed by the same researcher. The beef, and shrimp, which detected the same at 100  ppb analysis was evaluated by means of the intra-day coef- (MRL) [27]. In another study, results of the revolution- ficient of variations and the results are presented in ary Biochip Array Technology showed better detectabil- Table  5. Results of the repeatability study characterized ity for tylosin A and oxytetracycline at 0.10 and 0.5 of the by the relative standard deviation (%RSD) were satisfac- respective MRL in samples [28]. tory with a precision of less than 12% for the different The limits of detection (LOD) obtained using the antimicrobial drugs including tetracyclines, macrolides, Charm Test II assays, and the limits of quantitation β-lactamss, aminoglycosides, and sulfonamides; spiked (LOQ) for selected literature chemical methods are pre- in blank fish samples at MRL, 0.5 MRL or concentra - sented in Additional file  1: Table S1b. The LOD results for tion less than 0.5 MRL and analysed under repeatability fish matrix obtained in this validation using the Charm II conditions (n ≥ 6). The coefficient of variation expressed kits, are comparable to the manufacturer’s claims for the as percentage relative standard deviation (R SD ) ranged tissue matrix. However, some antimicrobial compounds from 7.8 to 9.8% for tetracyclines (chlortetracycline and could be detected in fish tissue at levels lower than the oxytetracycline), 2.8 to 6.3% for macrolides (erythromy- manufacturer’s claim (Additional file  1: Table  S1b). The cin A), 6.9 to 9.7% for β-lactams (penicillin G), 10.01 to LOD results were also compared with the LC–MS/MS 11.5% for aminoglycosides (streptomycin); and for sul- analysis of sulfadimethoxine [29], HPLC–MS/MS analy- fonamides (sulfathiazole) it was from 1.2 to 8.7%. These ses of tetracyclines, chlortetracycline, oxytetracycline, results, ably demonstrate the protocol’s repeatability sulfadimethoxine, sulfamerazine and sulfadiazine [30]; when used for testing different antimicrobial residues in and LC–ESI–MS/MS analyses of a range of tetracyclines, fish tissue matrix. β-lactams, aminoglycosides and sulfonamides [31]. Gen- A closer look at results obtained under repeatability erally, the rigorous chemical techniques, as expected, conditions in the analysis of different fish samples spiked offer lower LOQ values compared to the respective with 25  µg/kg sulfathiazole is presented in Table  6. The LOD obtained with the Charm II tests. Nonetheless, results showed that there was no significant difference in the Charm II test demonstrated ability to detect a wider cpm readings for the same fish species, and amongst dif - range of antimicrobials belonging to different classes ferent fish species including dorade, salmon and seabass, including tetracyclines, macrolides, β-lactams, aminogly- spiked with sulfathiazole at the same concentration level cosides and sulfonamides at MRL or lower levels, but it (ANOVA, overall F-critical 3.35 > F-calculated 1.99) with requires use of different antimicrobial test kits in parallel; RSD < 10%. Similar observations were made for the other Table 5 Repeatability study at MRL, 0.5 MRL or concentration < 0.5 MRL Family Compound Spiking concentration (µg/kg) Mean cpm SD RSD (%) r r Tetracyclines Chlortetracycline 25 µg/kg (0.25 MRL) 1207.0 118.2 9.8 Oxytetracycline 100 µg/kg (MRL) 1270.06 98.41 7.75 Macrolides Erythromycin A 100 µg/kg (0.5 MRL) 1762.4 110.4 6.3 200 µg/kg (MRL) 1478.1 41.2 2.8 β-Lactams Penicillin G 25 µg/kg (0.5 MRL) 1285.6 89.3 6.9 50 µg/kg (MRL) 648.5 62.7 9.7 Aminoglycosides Streptomycin 250 µg/kg (0.5 MRL) 1125.8 112.7 10.01 500 µg/kg (MRL) 1110.5 127.2 11.5 Sulfonamides Sulfathiazole 25 µg/kg (0.25 MRL) 922.2 80.1 8.7 100 µg/kg (MRL) 706.5 8.6 1.2 SD , standard deviation under repeatability conditions, RSD , relative standard deviation under repeatability conditions, Mean cpm average of counts per minute under r r reproducibility conditions M ukota et al. BMC Chemistry (2020) 14:32 Page 11 of 15 Table 6 Repeatability in  the  detection of  sulfathiazole tetracyclines (chlortetracycline and oxytetracycline) was at 25 µg/kg for selected fish samples 7.2 to 11.4%; macrolides (erythromycin A) ranged from 5.8 to 8.9%; β-lactams (penicillin G) from 10.4 to 11.2%; Parameter Fish samples spiked with sulfathiazole at 25 µg/kg, cpm aminoglycosides from 8.9 to 15.1% and sulfonamides (sulfathiazole) from 2.8 to 8.3% as indicated in Table 7. Dorade Salmon Seabass An elaborate presentation of some results of the 989 914 735 reproducibility studies performed by two different 815 969 780 researchers following the same Charm II protocol 862 886 976 on selected fish species, spiked with oxytetracycline 876 1015 896 at a concentration level of 100  µg/kg, is presented in 782 932 890 Table  8. A comparison of the results obtained by the 934 1075 978 two researchers for the same fish species showed no 985 930 976 significant difference; and the overall analysis showed 877 1070 898 no significant difference in the cpm results for the dif - 863 935 975 ferent fish species including seabass, pangasius and 986 890 976 salmon (ANOVA, F-critical 4.1 > F-calculated 0.64), Average 896.9 961.6 908 with RSD < 10%, which further demonstrates the tech- SD 73.6 69.3 88.3 nique’s reproducibility with little matrices interference. RSD 0.082 0.07 0.097 Similar observations were made for the other antimi- crobial compounds, whose summarized results are pre- sented in Table 7. antimicrobial agents, whose summarized results are pre- sented in Table 5. Robustness of the method Reproducibility of the method Analysis of batches of many samples often require a The reproducibility studies were performed by two differ - couple of hours before completion; and there is likely to ent researchers following the same Charm II protocol on be a time interval between the first and last analysis of selected fish species, spiked with different antimicrobial the processed samples. In the robustness testing of the agents and evaluated by means of intra-day and inter-day Charm II assay, the effect of variation in reading time coefficient of variations. The reproducibility study char - interval for processed samples was studied. Robustness acterized by the relative standard deviation (%RSD) was testing was performed on samples spiked with 50  µg/ satisfactory with a precision of less than 15.3% for the kg amoxicillin and analysed on the β-lactams channel different antimicrobial drugs (tetracyclines, macrolides, immediately after mixing (0 h) and after 14 h. The con - β-lactams, aminoglycosides, and sulfonamides) spiked trol point for β-lactams was set at 1530, and the robust- in blank fish samples at MRL, 0.5 MRL or concentra - ness results are presented in Table 9. tion less than 0.5 MRL and studied under reproducibility From Table  9, it is evident that there was no signifi - conditions (n ≥ 6). The coefficient of variation calculated cant difference in the cpm for both pangasius and dorade as percentage relative standard deviation (%RSD) for Table 7 Reproducibility study at MRL, 0.5 MRL or concentration < 0.5 MRL Family Compound Spiking concentration (µg/kg) Mean cpm SD RSD (%) r r Tetracyclines Chlortetracycline 25 µg/kg (0.25 MRL) 1224.5 139.2 11.4 Oxytetracycline 100 µg/kg (MRL) 1277 92.6 7.2 Macrolides Erythromycin A 100 µg/kg (0.5 MRL) 1748.5 156.1 8.9 200 µg/kg (MRL) 1456.5 83.9 5.8 β-Lactams Penicillin G 25 µg/kg (0.5 MRL) 1204.9 135.0 11.2 50 µg/kg (MRL) 702.1 73.0 10.4 Aminoglycosides Streptomycin 250 µg/kg (0.5 MRL) 1110.6 98.7 8.9 500 µg/kg (MRL) 1132.6 171.4 15.1 Sulfonamides Sulfathiazole 25 µg/kg (0.25 MRL) 943.7 78.1 8.3 100 µg/kg (MRL) 647.1 18.1 2.8 SD standard deviation under reproducibility conditions, RSD relative standard deviation under reproducibility conditions r r Mukota et al. BMC Chemistry (2020) 14:32 Page 12 of 15 Table 8 Reproducibility in the detection of oxytetracycline spiked with 50 µg/kg of beta-lactams and read after 0 or at 100 µg/kg for selected fish samples 14 h (ANOVA, F-critical 4.3 > F- calculated 0.2) confirm - ing the robustness of the method, in regard to variation in Fish sp. Researcher 1, cpm Researcher 2, cpm reading time intervals of the processed samples. A com- Seabass 1176 1296 parison of cpm for blank fish samples for both pangasius 1177 1265 and dorade after 0 and 14  h, also showed that there was 1295 1177 no significant difference between counts since ANOVA 1334 1281 F-critical 4.9 > F-calculated 0.2. The combined results of 1127 1166 these studies demonstrate that the Charm II technique is 1341 1417 quite robust for the analysis of antimicrobials in fish. 1170 1460 1261 1074 Specificity and cross reactivity of the technique 1371 1361 The cross reactivity analysis was carried out in order to 1201 1335 determine whether the presence of non-target drugs may Pangasius 1225 1185 lead to false identification of the target drug; or whether 1166 1223 the identification of the target analyte may be hindered 1094 1224 by the presence of one or more interferences. Repre- 1408 1407 sentative blank fish samples were spiked with different 1408 1405 antimicrobial drugs at known concentration levels higher Salmon 1307 1307 than those likely to interfere with the identification of the 1378 1299 analyte of interest, and then analysed using the respec- 1298 1310 tive Charm II protocol for the target drug. The aminogly - 1274 1311 cosides (spectinomycin, neomycin B and paromomycin) 1313 1279 were analysed using the macrolide channel (meant for 1266.2 1289.1 erythromycin A, tilmicosin and tylosin A). A standard Average 1176 1296 mix containing aminoglycosides (spectinomycin, neomy- Standard deviation 94.6 96.2 cin B and paromomycin) was used to spike different fish RSD 0.07 0.07 samples at 150, 300 and 500  µg/kg level, and the results are presented in Table 10. Results show that although the macrolides which were the targeted antimicrobials tested positive (sam- ples spiked with erythromycin A at 200  µg/kg, gave Table 9 Robustness testing using amoxicillin spiked at 50 µg/kg for selected fish samples Time Run Spiked at 50 µg/kg Non spiked Pangasius, cpm Dorade, cpm Blanks, cpm Fish species Results at 0 h 1 1019 1069 2433 Pangasius 2 1024 959 2398 Pangasius 3 1017 1019 2399 Pangasius 4 1201 1069 2064 Dorade 5 1155 1033 2200 Dorade 6 1020 1067 2109 Dorade Results after 14 h 7 1120 1120 2399 Pangasius 8 1059 1080 2064 Pangasius 9 1260 1195 2399 Pangasius 10 1011 1113 2068 Dorade 11 1089 1089 2210 Dorade 12 1099 1092 2205 Dorade Average 1089.5 1075.4 2245.7 SD 81.6 57.9 150.8 RSD 0.07 0.05 0.07 M ukota et al. BMC Chemistry (2020) 14:32 Page 13 of 15 Table 10 Specificity and cross reactivity tests using the Macrolides kit Blanks cpm AMGL spiked AMGL spiked AMGL spiked AMGL spiked AMGL spiked AMGL spiked at 150 µg/kg, at 300 µg/kg, at 500 µg/kg, at 150 µg/kg, at 300 µg/kg, at 500 µg/kg, Trout Salmon cpm Salmon cpm Pangasius cpm Catfish cpm Catfish cpm cpm 5377 5097 5239 5239 5536 5511 4694 5538 4860 4931 4931 5224 5393 5262 5538 5106 4800 5034 5223 5803 5412 5076 4703 4900 5351 4966 5558 5686 5571 5039 4950 5121 5236 5807 5728 Macrolide calculated control point cpm = 2118 AMGL aminoglycosides standard mix containing spectinomycin, neomycin B and paromomycin Table 11 Specificity and cross reactivity tests with mixed standards of different antimicrobial using the Aminoglycosides kit Antibiotics used and their respective MRL Mixed standard and respective spiking level, Fish species Spiked fish µg/kg samples cpm Tetracycline (MRL 100 µg/kg) Tetracycline spiked at 1000 µg/kg Cat fish 5742 Cat fish 6286 Penicillin G (MRL 50 µg/kg) Penicillin G spiked at 500 µg/kg Cat fish 5780 Cat fish 5776 Sulfamethazine (MRL 100 µg/kg) Sulfamethazine spiked at 1000 µg/kg Salmon 5418 Salmon 5700 Salmon 5584 Tilmicosin (50 MRL µg/kg) Tilmicosin spiked at 500 µg/kg Trout 5776 Trout 5584 Trout 6155 Trout 5962 Trout 5777 Trout 5800 Trout 5671 Trout 6010 Trout 5699 Trout 5700 Trout 5800 Trout 5810 Trout 5156 Aminoglycosides calculated control point cpm = 3346 1478 cpm), the non-target aminoglycosides intentionally The results of these studies also showed that no resi - analyzed on the same channel, tested negative since in all dues of the non-target drugs (tetracycline, penicillin G, cases the observed cpm were above the set control point sulfamethazine and tilmicosin) could be detected using of the macrolides of 2118. In similar studies, cross reac- the aminoglycosides channel as shown in Table  11; tivity was further investigated by spiking residue-free, whereas samples spiked with spectinomycin at 500 µg/ blank fish samples with high concentrations (10 MRL) of kg tested positive with 1110 cpm when analysed under antimicrobial substances belonging to other groups (sul- the same channel. All spiked samples tested negative fonamides, β-lactams, macrolides, and tetracyclines) and and the non-target compounds could not be detected were analysed on the aminoglycosides channel; and the even at high concentration (10 MRL). Similar obser- results are presented in Table 11. vations were made when utilizing the Biochip Array Mukota et al. BMC Chemistry (2020) 14:32 Page 14 of 15 Technology assay, where none of the tested antimicro- detection of antimicrobial residues in a variety of fish bials could be detected under cross-reactivity studies species; and its applicability for the rapid evaluation of [28]. the quality of aquaculture products for safety and trade purposes. Conclusions The Charm II radio receptor assay technique was suc - Supplementary information cessfully validated for screening residues of tetra- Supplementary information accompanies this paper at https ://doi. org/10.1186/s1306 5-020-00684 -4. cyclines, sulfonamides, β-lactams, aminoglycosides and macrolides in different aquaculture fish species. Additional file 1: Table S1a. List of analytical standards used for spiking. The Charm II technique can detect tetracycline and Table S1b. Limits of quantification and detection from Charm II assays chlortetracycline at 25  µg/kg (0.25 MRL) and oxytet- and published results. racycline at 100  µg/kg (MRL) for different fish spe - cies including, cat fish, trout, salmon, seabass, tilapia, Abbreviations lingue, dorade, and pangasius, with 100% detection. ANOVA: One-way analysis of variance; CCβ: Detection capability; CP: Control The sulfonamides including sulfadimethoxine, sulfam - point; CPM: Counts per minute; EU: European Union; FAO: Food and Agricul- ture Organization; IAEA: International Atomic Energy Agency; ILVO: Flanders erazine, sulfadiazine, sulfathiazole could be detected Research Institute for Agriculture, Fisheries and Food; MRL: Maximum residue at 25  µg/kg (0.25 MRL) for all fish species involved in limit; MSU: Multi antimicrobial standard; RSD: Relative standard deviation; SD: the study, with the exception of catfish, pangasius, and Standard deviation; UNBS: Uganda National Bureau of Standards. lingue, which gave high counts for the blank samples. Acknowledgements Results for the macrolides analysis, showed that eryth- The study was performed with support from the International Atomic Energy romycin A, tilmicosin, and tylosin A, could be detected Agency (IAEA) program on Development and Strengthening of Radio-Analyt- ical and Complementary Techniques to Control Residues of Veterinary Drugs at 100 µg/kg (0.5 MRL), 100 µg/kg (2 MRL) and 100 µg/ and Related Chemicals in Aquaculture Products, Vienna Austria. The authors kg (MRL), respectively, for the different fish species. would also like to acknowledge support from the Uganda National Bureau Whereas, the β-lactams including penicillin G, ampi- of Standards, Kampala-Uganda; Institute of Medical Research and Medicinal Plant Studies, Yaounde, Cameroon; Flanders Research Institute for Agriculture, cillin, amoxicillin, oxacillin, dicloxacillin and cloxa- Fisheries and Food, Melle-Belgium and Department of Chemistry, Makerere cillin were detected at 25  µg/kg (0.5 MRL), 50  µg/kg University Kampala, Uganda. We are also very grateful to Charm Sciences Inc. (MRL), 50  µg/kg (MRL), 300  µg/kg (MRL), 300  µg/kg for providing the Charm II test reagents and kits. (MRL) and 300  µg/kg (MRL), respectively. Under the Authors’ contributions aminoglycosides analysis, streptomycin was detected at AKM, WR, MFKG and JS participated in experimental design and coordination 25 µg/kg (0.05 MRL) for all fish species involved in the of laboratory activities. AKM, MFKG and WR carried out the sampling, sample preparation and analysis. WR, JJTT, AKM and ET participated in data analysis. study. Interestingly, the technique can detect a broader AKM, ET, MH and SAN wrote the manuscript. All authors participated in manu- range of antimicrobials other than only the compounds script review. All authors read and approved the final manuscript. specified in the Charm II assay kit. In addition, all anti - Funding microbial compounds involved in the study could be The work was financially supported by the International Atomic Energy successfully detected using the Charm II assay at 100% Agency (IAEA) program on Development and Strengthening of Radio- rate, with the exception of dicloxacillin, cloxacillin, Analytical and Complementary Techniques to Control Residues of Veterinary Drugs and Related Chemicals in Aquaculture Products. The work was partially sulfamethazine and sulfathiazole that exhibited false supported by Charm Sciences Inc. who provided all the Charm II test reagents negative rates of 4.5, 5.0, 3.4 and 5.0%, respectively. and kits used in the study. Moreover, these false negative rates fall within the 5% Availability of data and materials requirement of the EU decision 2002/657, and there- All supporting information including table of results and detailed methods is fore, the validation results are satisfactory. available upon request. Robustness studies showed that there was no signifi - Competing interests cant difference between results for counts of the same The authors declare that they have no competing interests. samples read immediately or after 14  h of addition of the scintillation fluid. In addition, no evidence of cross- Author details Uganda National Bureau of Standards, Headquarters. Plot 2–12 Bypass Link, reactivity was observed among the targeted antimicro- Industrial & Business Park, Kyaliwajala Road, P.O. Box 6329, Kampala, Uganda. bial compounds on interchanging the antimicrobial 2 Institute of Medical Research and Medicinal Plant Studies, P.O. Box 6163, analysis channels. The results of this validation study Yaounde, Cameroon. International Atomic Energy Agency (IAEA), Vienna International Centre, P. O. Box 100, 1400 Vienna, Austria. Flanders Research prove the robustness, specificity, reliability and preci - Institute for Agriculture, Fisheries and Food (ILVO), Technology and Food sion of the Charm II radio receptor assay technique in 5 Science Unit, Brusselsesteenweg 370, 9090 Melle, Belgium. Department the detection of various antimicrobials residues in dif- of Chemistry, College of Natural Sciences, Makerere University, P.O. Box, 7062, Kampala, Uganda. ferent fish species. 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Granados CF, Rodriguez C (2017) Tetracyclines in food and feeding tuffs: mination of antimicrobials in fish tissues by HPLC–ESI-MS/MS method. J from regulation to analytical methods, bacterial resistance, and environ- Chromatogr B 978–979:103–110 mental and health implications. J Anal Methods Chem. 2017:1315497 30. Cháfer-Pericás C, Maquieira Á, Puchades R, Company B, Miralles J, Moreno 12. Commission European (2010) Commission Regulation (EU) No 29 A (2010) Multiresidue determination of antibiotics in aquaculture fish 37/2010 of 22 December 2009 on pharmacologically active substances samples by HPLC–MS/MS. Aquac Res 41:217–225 and their classification regarding maximum residue limits in foodstuffs of 31. Guidi RL, Santos AF, Robeiro SRAC, Fernandes C, Silva HML, Gloria BAM animal origin. Off J Eur Union L 15:1–72 (2017) A simple, fast and sensitive screening LC-ESI-MS/MS method for 13. Marshall BM, Levy SB (2011) Food animals and antimicrobials: impacts on antibiotics in fish. 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Primary validation of Charm II tests for the detection of antimicrobial residues in a range of aquaculture fish

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Abstract

The study carried out a primary validation of Charm II tests for the detection of antimicrobial residues in aquaculture fish. The validation was performed according to European Commission Decision 2002/657/EC and the parameters determined included: detection capability, repeatability, reproducibility, specificity and robustness for the detection of antimicrobial residues in fish. Fish materials from different species including cat fish, trout, salmon, sea bass, tilapia, lingue and pangasius, were spiked with varying concentrations of selected antimicrobials including sulfonamides, β-lactams, macrolides, tetracyclines and aminoglycosides to determine the detection capabilities and other validation parameters of the Charm II tests. Results of the validation showed that the detection capabilities for the tetracyclines ranged from 25 to 100 µg/kg, while the sulfonamides and aminoglycosides were detected at 25 µg/kg for all species under study. The detection capabilities for the beta-lactams ranged from 25 to 300 µg/kg; and was 100 µg/kg for the tested macrolides. Results of the study showed that there was no significant difference between counts for samples read immediately after addition of the scintillation liquid and those read 14 h after addition of the scintillation liquid, provided that there was good vortexing before analysis. There was also no significant difference between counts for the same samples analyzed in different runs under repeatability and reproducibility conditions at the same spiking concentrations for the different fish species analyzed. The relative standard deviation for both repeatability and repro - ducibility ranged from 1.2 to 15.1%. The Charm II tests were found to be 100% group specific, as none of the antimi- crobials kits, gave false positive results when testing non-target antimicrobial drugs. Results of this study demonstrate the suitability of the Charm II technique as a rapid screening tool for detection of antimicrobial residues in a variety of fish species at maximum residue limits (MRL) established in the EU guidelines, with the exception of tilmicosin which was detected at 2 MRL. The results also prove the robustness, specificity, reliability and precision of the Charm II assay in the detection of various antimicrobial residuals in fish and its applicability for the rapid evaluation of the quality of aquaculture fish for safety and trade purposes. Keywords: Charm II tests, Antimicrobial residues, Rapid screening, Method validation, Aquaculture fish, Maximum residue limit Introduction Fish farming is a fast emerging industry that besides creating employment, is a source of good quality animal protein and essential macronutrients in the diet. Fish and *Correspondence: emmanuel@cns.mak.ac.ug fish related products provide income and livelihoods for Department of Chemistry, College of Natural Sciences, Makerere University, P.O. Box, 7062, Kampala, Uganda numerous communities across the world besides playing Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mukota et al. BMC Chemistry (2020) 14:32 Page 2 of 15 a crucial role in assuring sufficient availability of safe and of animal origin [12]. The safety concerns regarding drug healthy food [1, 2]. The increased demand for fish for residues in various food products, calls for develop- the growing international population, especially in the ment and validation of rapid and reliable techniques for developing world, has continued to deplete the sustain- detection of these compounds. Such rapid techniques able yields from lakes, rivers, swamps, seas and other can facilitate fast decision making to minimize technical natural water bodies. Aquaculture is growing rapidly and barriers to trade and also enhance routine monitoring in is seen as a remedy to address and supplement the dwin- order to protect consumer health. dling quantities and shortfall in wild catch [3]. However, The Charm II radio receptor assay technique devel - big numbers of fish in a confined volume of space tend to oped by Charm Sciences Inc, is one of the rapid screen- increase incidences of bacterial infections and other dis- ing techniques for detection of residues of antimicrobials eases; which greatly affects yield in the aquaculture busi - such as beta-lactams, sulfonamides, tetracyclines, chlo- ness. Productivity in aquaculture may be enhanced by ramphenicol, quinolones, macrolides and aminoglyco- use of antimicrobials such as tetracyclines, macrolides, sides in various food products including fish, meat, eggs, beta-lactams, sulfonamides, and streptomycins, for the honey, and milk, as well as non-food matrices including prevention and treatment of opportunistic infections in water, feed and urine. This technique utilizes a microbial fish [ 4, 5]. cell with receptor sites that bind the specific antimicro - Antimicrobials are used to control ectoparasitic, fungal bial drug. The analytical process involves a binder being and bacterial diseases of the body and gills of fish [6–8]. added to a sample extract along with an amount of H Tetracyclines in particular are frequently employed in or C labeled antimicrobial tracer. Any antimicrobial in aquaculture due to their broad spectrum of activity as the sample extract competes for the binding sites with well as their low cost, compared to other antibiotics. the tracer. The amount of tracer that binds to the recep - The tetracyclines are used to combat bacterial hemor - tor sites is measured and compared to a previously deter- rhagic septicemia in catfish as well as diseases caused mined control point. Therefore, the more radiolabelled by Pseudomonas liquefaciens [9]. Currently, there are antimicrobial detected in the mixture, the lower the con- over 20 tetracyclines available; although, tetracycline, centration of antimicrobial in the sample. The smaller chlortetracycline, oxytetracycline, and doxycycline are the amount of tracer measured, the greater the drug the most common ones in veterinary medicine and aqua- concentration in the sample [21, 22]. The Charm II tech - culture [10, 11]. The aforementioned antibiotics are the nique has very limited validation data for the detection only tetracyclines with registration within the European of antimicrobials in different fish species. Thus, this study Union (EU) for use as veterinary medicinal products in conducted a primary validation of the Charm II tests in food producing animals; with established maximum resi- order to generate comprehensive analytical data to prove due limits (MRLs) in different food matrices [12]. Other the validity, applicability and also address potential limi- antimicrobials such as sulfonamides, beta-lactams, mac- tations of the Cham II assays particularly for the screen- rolides and aminoglycosides also have a wide spectrum of ing of antimicrobials in different aquaculture fish species. activities against most Gram positive and Gram negative organisms and are used for the prevention and treatment of bacterial infections in livestock and aquaculture. The Materials and methods antimicrobials are typically administered in the water, Reagents, materials and equipment often as components of fish feed, and are occasionally The antimicrobial test assay kit was obtained from Charm injected [13, 14]. Sciences Inc., Lawrence, MA; and included items for the The extensive use and misuse of antimicrobials in farm detection of beta-lactams (PMSU-050A); sulfonamides animals as growth promoters or as nonspecific means of (SMMSU-022C), macrolides (EMSU-023A); tetracyclines infection prevention has been reported to lead to accu- (TMSU-025); and streptomycin (STMSU-023A). Con- mulation of residues in edible tissue [7, 15, 16]; which sumables and equipment used for the tests included: M2 may cause allergic and toxic effects in consumers as Buffer, zero and positive control standards, MSU extrac - well as contributing to the development of antimicro- tion buffer, radioactive labelled tablets; scintillation fluid bial resistant bacteria [17–20]. In this respect, residues (Opti-Fluor O, PerkinElmer), Intronic incubator (Charm in foodstuffs create public health concerns, consumer Sciences Inc.), Wallac 1409 scintillator counter, refriger- perception problems and trade disputes that have enor- ated centrifuge Sigma 4K15c (Sigma-Aldrich), R2 blender mous negative impacts on the food industry. In order to (Robot-Coupe) and a water bath (Julabo MB13). In addi- protect human health, regulatory authorities like the EU, tion, scintillation vials, AES mix masticator stomacher established maximum residue limits (MRLs) for some and IEC Centra CL-3 centrifuge were also used. pharmaceutical compounds in fish and other foodstuffs M ukota et al. BMC Chemistry (2020) 14:32 Page 3 of 15 Preparation of standard reference material and stock residues was detected. Absence of residual antibiotics solutions in the fish samples was confirmed through evaluation The multi antimicrobial concentrate standard (MSU, of their counts per minute in comparison with results Charm Sciences Inc.) was prepared fresh on the day of obtained using the negative control extraction buffers use and diluted with 10  ml of deionized water, shaken supplied with the Charm II kits. The control buffers are well and allowed to stand on ice for 15 min. The reconsti - contaminant free and are used to qualify the matrix as tuted stock solution contained; penicillin G, 1000  µg/kg; negative when a known negative is not available. The erythromycin A, 10,000  µg/kg; sulfamethazine, 1000  µg/ tolerance considered for the fish matrix to qualify as kg; chlortetracycline, 4000  µg/kg; and streptomycin, negative and selected for use in subsequent test was for 10,000 µg/kg. Other analytical standards were purchased counts within ± 20% of the average result obtained with from Sigma Aldrich, Pfizer Inc., US Pharmacopeia Con - the respective negative control extraction buffer. Sam - vention and Acros Organics (Additional file 1: Table S1a). ples with counts beyond the tolerance limits were dis- These standards were appropriately diluted with deion - carded while those meeting the criteria were selected ized water to make working standard solutions of the for the study. The selected blank fish materials after respective antimicrobial, and kept below 4 °C. The work - blending, were spiked with antimicrobial standards of ing standards were used for spiking fish samples at dif - known concentrations and used as control samples for ferent concentration levels ranging from 25 to 300 µg/kg. the establishment of the control point counts per min- ute (cpm). A list of control standards used in the study is shown in Additional file 1 : Table S1a. Methods The study carried out a primary validation of the Charm II tests for the detection of antimicrobial resi- Extraction of drugs from the fish materials dues in aquaculture fish. The validation was performed The MSU extraction buffer (30 ml) was added to blended according to Commission Decision 2002/657/EC [23] fish material (10  g) in a polypropylene centrifuge tube. and all methods of analysis used were adopted from the The mixture was homogenized using a stomacher for general Charm II protocols [21]. 2 min and returned to the centrifuge tube. The homoge - nate was incubated in water bath at 80  °C for 30  min, during the determination of streptomycin, macrolides, Fish samples selected for the study or beta-lactams; and 45  min, when determining tetra- The fish materials used in the study were obtained from cyclines or sulfa drugs. After incubation, the tube was dead fish purchased from Melle and Ghent fish shops cooled on ice water for 10  min and then centrifuged at and supermarkets in Belgium. Aquaculture fish species 3300  rpm for 10  min, using a refrigerated centrifuge including cat fish (Siluriformes), trout (Oncorhynchus 4K15C (Sigma-Aldrich). The resulting supernatant solu - mykiss), salmon (Salmo salar), seabass (Dicentrar- tion was collected and used for the required tests. The chus labrax), tilapia (Oreochromis niloticus), lingue pH of the supernatant was where necessary adjusted to (Molva molva), dorade (Sparus aurata) and pangasius pH 7.5 using reconstituted Charm II kit M2 buffer for (Pangasius bocourti), were selected for the study. Fish low pH, or 0.1 M hydrochloric acid for high pH. sample materials were taken by carefully removing the muscle tissue from the side of each fish taking precau - tion to exclude scales and skin. The fish samples that Determination of tetracyclines in the fish samples were not used immediately were stored below − 18  °C In the detection of tetracycline, the white tablet from for a maximum of 2 months. the kit containing the binding reagent (TMSU-025) was introduced into a test tube, and water (300  μl) was Sample preparation added. The contents of the tube were mixed for at least The fresh fish sample was weighed in a centrifuge tube 10  s to ensure breakup of the tablet. The sample extract and stored at − 18 °C until further processing. The frozen or control sample (4 ml) was added to the tube, followed fish samples were thawed at 4  °C overnight and cut into by addition of the orange tablet containing the tracer small pieces before blending in a high speed blender. The reagent from the kit (TMSU-025). The resultant solu - blended fish material (10  g) was transferred into a poly - tion was mixed for about 10 s and the mixture was incu- propylene centrifuge tube and used immediately. bated at 35 °C for 5 min; and then centrifuged for another 5  min on a IEC Centra CL-3 centrifuge. The superna - tant was poured off carefully, deterring the formed pel - Preparation of control samples let from sliding out of test tube. Deionized water (300 μl) All fish samples were first tested with the different was added to the tube and the contents mixed thoroughly Charm II kits and only used in case no veterinary drug to break up the pellet. After suspension of the pellet in Mukota et al. BMC Chemistry (2020) 14:32 Page 4 of 15 water, the scintillation liquid (3.0 ml) was added and test capped. The tube was shaken until the mixture had a uni - tube capped. The tube was shaken until the mixture had form cloudy appearance. The glass tube contents were a uniform cloudy appearance. The glass tube contents transferred completely into a scintillation vial and the were transferred completely into a scintillation vial and mixture counted using a Wallac liquid scintillation coun- the mixture counted using a Wallac liquid scintillation ter for 60  s on the [ H] channel. The cpm results of the counter for 60  s on the [ H] channel. The results for the sample were compared with the control point. sample was compared with the control point counts per minute. Determination of aminoglycoside‑streptomycin in the fish samples Determination of macrolides in the fish samples In the determination of streptomycin, the white tablet During the detection of macrolides, the white tablet from from the kit containing the binding reagent (STMSU- the Charm II kit containing the binding reagent (EMSU- 023A) was introduced into a test tube, and water (300 μl) 023A) was introduced into a test tube, and water (300 μl) added. The contents of the tube were mixed for at least was added. The contents of the tube were mixed for at 10 s to ensure breakup of the tablet. The sample extract or least 10  s to ensure breakup of the tablet. The sample control sample (2 ml) was added to the tube and mixed. extract or control sample (4  ml) was added to the tube This was followed by addition of the green tablet contain - and the contents mixed on a vortex for 10  s. The result - ing the tracer reagent (STMSU-023A). The resultant was ant was incubated at 55  °C for 2  min, followed by addi- mixed by swirling the contents up and down for about tion of a green tablet containing the tracer reagent from 10 s. The mixture was incubated at 35 °C for 2 min, and the kit (EMSU-023A). The resultant was mixed on a then centrifuged for another 3 min. The supernatant was vortex for 10  s. The mixture was incubated at 55  °C for poured off carefully and the edge of tube was blotted with 2  min, and then centrifuged for 5  min. The supernatant absorbent paper. Deionized water (300  μl) was added to was poured off carefully and the edge of tube blotted on the tube and the contents mixed thoroughly. After sus- absorbent paper. Deionized water (300  μl) was added to pension of the pellet in water, the scintillation liquid the tube and the contents mixed thoroughly to break up (3.0  ml) was added and test tube capped. The tube was the formed pellet. After suspension of the pellet in water, shaken until the mixture had a uniform cloudy appear- the scintillation liquid (3.0  ml) was added and the test ance. The glass tube contents were transferred com - tube capped. The contents were mixed on a vortex until pletely into a scintillation vial and the mixture counted the mixture had a uniform cloudy appearance. The con - using a Wallac liquid scintillation counter for 60 s on the tent of the glass tube was transferred completely into a [ H] channel. The cpm results for the sample were com - scintillation vial and the mixture counted using a Wallac pared with the control point. liquid scintillation counter for 60 s on the [ C] channel. The counts per minute (cpm) of the sample was com - Determination of β‑lactams in the fish samples pared with the control point. In the determination of β-lactams, the green tablet from the Charm II kit containing the binding reagent (PMSU- Determination of sulfa drugs in the fish samples 050A) was introduced into a test tube, and water (300 μl) In the detection of sulfa drugs, the white tablet from the was added. The contents of the tube were mixed to Charm II kit containing the binding reagent (SMMSU- ensure breakup of the tablet. The sample extract or con - 022C) was introduced into a test tube, and water (300 μl) trol (2 ml) was added to the tube and the contents mixed added. The contents of the tube were mixed for at least on a vortex for 10 s. The resultant was incubated at 55 C 10  s to ensure breakup of the tablet. The sample extract for 2 min, followed by addition of a yellow tablet contain- or control sample (4 ml) was added to the tube, followed ing the tracer reagent (PMSU-050A) from the kit. The by addition of the pink tablet containing the tracer rea- resultant was mixed on a vortex for 10 s. The mixture was gent (SMMSU-022C) from the kit. The resultant solu - incubated at 55  °C for 2  min, and then centrifuged for tion was mixed by swirling the contents up and down for 5  min at 1750 G. The supernatant was poured off care - about 15 s. The mixture was incubated at 65 °C for 3 min, fully and the edge of tube blotted on absorbent paper. and then centrifuged for another 3 min. The supernatant Deionized water (300  μl) was added to the tube and the was poured off carefully, deterring the formed pellet from contents mixed thoroughly to break up the pellet. After sliding out of test tube; and the edge of tube was blotted suspension of the pellet in water, the scintillation liquid on absorbent paper. Deionized water (300 μl) was added (3.0  ml) was added and test tube capped. The contents to the tube and the contents mixed thoroughly to break were mixed on a vortex until the mixture had a uniform up the pellet. After suspension of the pellet in water, cloudy appearance. The mixture was transferred com - the scintillation liquid (3.0  ml) was added and test tube pletely into a scintillation vial and counted using a Wallac M ukota et al. BMC Chemistry (2020) 14:32 Page 5 of 15 liquid scintillation counter for 60 s on the [ C] channel. researchers using the same method and a scintillation The cpm of the sample was compared with the control fluid counter equipment. point. Robustness The robustness of the techniques was tested by delib - Method validation erately varying the experimental time indicated in the The method validation was done according to the cri - Charm II analytical protocol. This was intended to study teria of the European Commission Decision 2002/657/ the effect of variation in reading time interval for a large EC [23]. The validation parameters performed included; batch of processed samples. Reading of the cpm for detection capability (CCβ), repeatability, reproducibility, the samples spiked with 50  µg/kg amoxicillin was done robustness and cross reaction activity. immediately after the addition of the scintillation liquid and then after 14 h on the same batch of extracted sam- ple. The samples after the first reading were stored over - Detection capability night in the fridge at 4 °C, removed and allowed to attain The CCβ was examined by spiking blank fish matrices room temperature, and then read the second time after with different antimicrobials including tetracyclines, vortexing. macrolides, β-lactams, aminoglycosides, and sulfona- mides. The number of samples analyzed for each individ - ual antimicrobial agent ranged from 20 to 30 as indicated Cross reaction activity in Table 3. The spiking concentrations varied around the Cross reactivity was investigated by spiking residue-free recommended maximum residue limit (MRL), including blank fish samples with high concentrations (up to 10 0.05 MRL, 0.25 MRL, 0.5 MRL, 0.75 MRL and MRL, for MRL) of the respective antimicrobial belonging to other the respective antimicrobial. The CCβ was then deter - antimicrobial groups and the samples run on targeted mined as the lowest concentration of the antimicrobial channels to investigate false identification. that could be detected in the sample giving at least 95% positive results. Data Analysis All data generated was statistically analyzed using one- way analysis of variance (ANOVA) to examine any sig- Repeatability nificant differences between the observed results under The repeatability of the technique was studied by analy - different experimental setups. sis of selected fish samples spiked with different antimi - crobials including tetracyclines, macrolides, β-lactams, Results and discussion aminoglycosides, and sulfonamides. The total number Counts per minute for blank samples of samples analyzed for each individual antimicrobial The blank samples used in the study were those fish tis - compound ranged from 20 to 30, and n ≥ 6 for the same sue matrices which were carried through the complete fish species. The spiking concentrations varied around analytical procedure, and no antimicrobial residues were the MRL, including 0.05 MRL, 0.25 MRL, 0.5 MRL, 0.75 detected in them using the respective Charm II assay MRL and MRL, for the respective antimicrobial. The kits [21]. The blank fish samples to which the binder and analysis was performed within a short interval, by a sin- tracer had been added but without addition of an anti- gle researcher using the same method and scintillation microbial agent were extracted with the different kits and fluid counter equipment. read on the respective channels. The results of the cpm for the blank fish samples are summarized in Table 1. Reproducibility From Table  1, the cpm for tilapia, trout, salmon, pan- The reproducibility of the method was studied by repeat gasius, seabass, dorate, catfish, and lingue fish species analysis of selected fish samples spiked with differ - were statistically evaluated using ANOVA and it was ent antimicrobials including tetracyclines, macrolides, found that the overall F-calculated (0.22) was less than β-lactams, aminoglycosides, and sulfonamides. The num - F-critical (2.5), which implied that there was no signifi - ber of samples analyzed for each individual antimicrobial cant difference between results for the blank fish samples ranged from 20 to 30, with n ≥ 6 for the same fish species. of the aforementioned species when using antimicro- The spiking concentrations varied around the recom - bial test kits for β-lactams, tetracyclines, macrolides and mended MRL, including 0.05 MRL, 0.25 MRL, 0.5 MRL, streptomycins. However significant difference in cpm 0.75 MRL and MRL, for the respective antimicrobial. The values was observed with the sulfonamides extraction kit analysis was performed on different days by two different while testing catfish, lingue and pangasius. The cpm for Mukota et al. BMC Chemistry (2020) 14:32 Page 6 of 15 Table 1 Blank counts per minute for the different fish species obtained using the Charm II technique Scintillation counter results (cpm) Charm II test β‑lactams kit Sulfonamides kit Tetracyclines kit Macrolides kit Streptomycins kit Fish species Mean SD Mean SD Mean SD Mean SD Mean SD Tilapia 2704 0.9 2596 736 3027 0.7 2448 191.1 5103 346 Trout 2506 192.0 2332 1184 2830 260 2799 87.1 4799 259.9 Salmon 2571 207.4 2472 541.2 2939 165.0 2110 117.2 3085 133.4 Pangasius 2469 195.7 5625 1254 2931 221.4 2893 110 4796 437.7 Seabass 2432 232.1 2144 672.1 2971 252.2 2700 153.6 4805 594.7 Dorate 2512 171.1 1977 621.4 2864 93.4 2803 167.3 4967 485.8 Catfish 2493 312.7 5872 774.3 Lingue 4454 650.1 cpm counts per minute, SD standard deviation these species were almost double those of the other types In this respect, the CP for the β-lactams was evaluated of fish and their F-calculated (15.1) was greater than from averaging the results of 6 negative samples spiked F-critical (2.4). The big variation in cpm for the catfish, with penicillin G at 25 µg/kg (0.5 MRL) and adding 20% of the obtained average cpm value. Whereas, for the sul lingue and pangasius fish species as compared to the rest - could be attributed to the high fish fat content extracted fonamides, the CP was evaluated by averaging results of by the sulfonamide kit protocol. In this respect, the three negative samples spiked at 50  µg/kg with sulfamethaz- fish species (catfish, lingue and pangasius) need to be ine and adding 30% of the average obtained cpm value. handled separately when calculating control points to A control point of 1530 was calculated for the β-lactams. minimize chances of getting false negative or false posi- On the other hand, the CP for tetracyclines was calcu- tive results. For the rest of the fish species, the blank cpm lated by averaging cpm results of negative control stand- results were used to derive the respective control points ards provided in the tetracyclines test kit and subtracting for the different residues. 40% of the obtained average cpm value (Table 2). For macrolides, the CP was derived from averaging the Evaluation of the Control Points for the different drug results of 6 negative samples spiked with erythromycin A residues at 100  µg/kg (0.5 MRL) and adding 20% of the obtained The control point (CP) of a sample is the cut-off point average cpm value. Using a similar approach, the CP for between a negative or positive result. Any antimicrobial streptomycin was derived from averaging results of nega- agent present in the sample extract competes for the tive samples spiked at 25  µg/kg with streptomycin and binding sites with the tracer, thus, the greater the cpm adding 30% of the average obtained cpm value. measured, the lower the antimicrobial drug concentra- During the analysis of antimicrobial residues in fish tion in the sample and vice versa. Samples with high samples, results less than or equal to each respective CP counts are considered negative (tracer antimicrobials are were interpreted as positive while those greater than the largely bound to the binder) while those with low counts CP, as negative. Blank sample readings below the set CP are considered positive (tracer antimicrobials are largely were considered false positive. The results in Table  2, free in solution). The CP for the different antimicrobials show that the false positive rate was 0% for tetracyclines, were determined independently; and with the exception β-lactams and sulfonamides; 3.6% for macrolides, and 5% of tetracyclines, the MRL value for each drug was spiked for streptomycin; this proved the validity of the obtained to the respective blank fish sample. In order to cater for data since it met the acceptance criteria of being within the deviations in the different fish matrices, a percentage 5%. A comparison of the CP for the different antimicro - tolerance was added to or subtracted from the obtained bials obtained using the Charm II assay with the corre- average cpm value of the spiked blank fish sample. The sponding cut-off points (Fm) and technical threshold (T) CP evaluation was performed according to the Charm II values, calculated following Annex II of the EU guideline protocol, and the percentages added to the mean value of for Community Reference Laboratories Residues for vali- spiked samples at detection capability or subtracted from dation of screening methods [26], is shown in Table 3. the mean value of blanks serve to minimise occurrence of According to the EU guideline, the cut off factor false positive or negative readings [21, 24, 25]. (Fm), refers to the response or signal from a screening M ukota et al. BMC Chemistry (2020) 14:32 Page 7 of 15 Table 2 Control points for the different antimicrobials in blank fish samples Antimicrobial Spiked samples Blank samples family Level of analyte Mean cpm Allowance Control Mean blank Range of blank No. of false False spiking (µg/kg) of spiked for matrix point cpm cpm readings positives/no. positive samples effect cpm of samples rate (%) β-lactams 25 µg/kg penicil- 1275 Spiked 1530 2502 2160–2907 0/30 0 lin G cpm + 20% Sulfonamides 50 µg/kg sul- 1096 Spiked 1424 3162 1431–6995 0/30 0 famethazine cpm + 30% Tetracyclines 0 µg/kg tetracy- 2524 Blank cpm 1514 2524 2451–3269 0/30 0 cline − 40% Macrolides 100 µg/kg eryth- 1765 Spiked 2118 2587 1906–2952 1/28 3.6 romycin A cpm + 20% Streptomycin 25 µg/kg strep- 2574 Spiked 3346 4605 2942–5488 1/20 5.0 tomycin cpm + 30% Number of samples used per parameter Ns ≥ 20 Table 3 Comparison of  control points by  Charm II protocol, cut-off points and  technical threshold values calculated according to the EU guideline [26, 27] Antimicrobial Compound Spiked B average Calculated T value Calculated Fm Calculated family concentration response of blank as per EU guideline value as per EU control point CP (μg/kg) samples T = B − 1.64 * SDb guideline as per Charm II assay [26, 27] Fm = M + 1.64 * SDs [26, 27] Tetracyclines Tetracycline 25 2958 2776 816 1514 Chlortetracycline 25 3050 2780 1417 Oxytetracycline 100 2891 2579 1427 Macrolides Erythromycin A 100 2814 2564 1904 2118 Tilmicosin 100 2486 2164 2002 Tylosin A 100 2512 2115 1740 β-Lactams Penicillin G 25 2523 2176 1438 1530 Ampicillin 50 2455 2042 1341 Amoxicillin 50 2702 2536 1487 Oxacillin 300 2398 2171 1478 Dicloxacillin 300 2524 2384 1489 Cloxacillin 300 2500 2368 1413 Aminoglycosides Streptomycin 25 4822 3867 2592 3346 Sulfonamides Sulfamethazine 25 2593 1428 1379 1424 Sulfadimethoxine 25 2266 1129 972 Sulfamerazine 25 2210 1095 930 Sulfadiazine 25 2297 1296 1184 Sulfathiazole 25 2266 1643 1485 Cut-off factor (Fm) = M + 1.64 * SDs; Technical threshold (T ) = B − 1.64 * SDb; M, mean response of spiked samples; B, mean response of blank samples; SDs, standard deviation of the spiked sample readings; SDb, standard deviation of blank readings test which indicates that a sample contains an analyte of the blank samples are greater than those for spiked at or above the screening target concentration [26], samples, because the responses are inversely propor- while the Charm II protocol CP is the cut-off point tional to concentrations of the antimicrobials. In this between a negative or positive result [21]. On the other respect, the assay is considered valid only when Fm < T hand, the technical threshold (T), refers to the limit for and the CCβ is validated when Fm < B. Accordingly, the positivity [26]. For the Charm II technique the readings number of spiked samples with mean responses below Mukota et al. BMC Chemistry (2020) 14:32 Page 8 of 15 Detection capability for the different antimicrobials the cut-off level (deemed positive) is identified and the in selected fish species false positive rate determined. If T < Fm < B, the false- The CCβ is the lowest concentration of the analyte that positive rate is greater than 5%. In the case Fm < T the could be detected in the sample giving at least 95% posi- false positive rate is below 5%. If more than 5% of the tive results. In the CCβ studies, blank negative fish tis - spiked samples at the screening target concentration sue samples were spiked with different antimicrobials at gave a response greater than the cut-off level (deemed various concentrations. Spiked samples that exhibited false negative), the concentration chosen for the spiking readings above the set CP value, were interpreted as false is considered too low for validation and a higher con- negatives. In case more than 5% of the spiked samples at a centration is tested [26, 27]. target concentration gave false negative readings, the con- From the results presented in Table  3, the Fm val- cerned concentration was deemed too low for validation ues obtained using the EU guideline and the respec- and a higher concentration was considered. A summary tive calculated CP according to the Charm II protocol of the CCβ for the different drugs involved in the study is are comparable. For all antimicrobials, the respective presented in Table 4. Results show that the Charm II tech- CCβ, presented in Table  3 are valid since in all cases nique can detect tetracycline and chlortetracycline spiked the Fm < B. In addition, for all antibiotics involved in at 25  µg/kg (0.25 MRL) and oxytetracycline at 100  µg/kg the study the Fm < T, which implies that the Charm II (MRL) for the different fish species (cat fish, trout, techniques is validated for the detection of antimicro- salmon, seabass, tilapia, lingue, dorade, and pangasius) bial residues in fish matrix, with a false positive rate with 100% detection. However, the batch of the multi- of less than 5%. In comparison with the Charm II pro- antimicrobial standard, provided in the Charm II kit was tocol, it should be noted that in all cases the CP value not sensitive enough for chlortetracycline to be detected for a particular family of antibiotics is slightly higher at 100 µg/kg (MRL) level. This could be attributed to the the corresponding Fm readings, with the exception of deterioration of the chlortetracycline in the standard due sulfathiazole. This suggests that there will be less inci - to poor handling, probably during transportation. In this dences of false negative readings in the detection of the respect, a Sigma Aldrich standard was used and chlortet- different antimicrobial compounds in fish matrix based racycline detected at a concentration as low as 0.25 MRL. on CP values, although this may increase incidences of Interestingly, it was observed that the technique is also false positive readings. capable of detecting other antimicrobials belonging to the Table 4 Detection capability for the selected antimicrobials Family Compound EU‑MRL CCβ (μg/kg) No of samples No Counter results % Detection (μg/kg) of positive (cpm) of each samples antimicrobial Mean Min Max Tetracyclines (CP = 1514 cpm) Tetracycline 100 25 20 20 724 650 825 100 Chlortetracycline 100 25 21 21 1200 942 1421 100 Oxytetracycline 100 100 31 31 1269 1074 1460 100 Macrolides (CP = 2118 cpm) Erythromycin A 200 100 30 30 1669 954 1955 100 Tilmicosin 50 100 21 21 1565 1221 2078 100 Tylosin A 100 100 21 21 1440 1103 1742 100 β-Lactams (CP = 1530 cpm) Penicillin G 50 25 22 22 1175 921 1421 100 Ampicillin 50 50 21 21 1055 837 1451 100 Amoxicillin 50 50 22 22 1132 908 1409 100 Oxacillin 300 300 24 24 1286 1082 1459 100 Dicloxacillin 300 300 22 21 1186 827 1839 95.5 Cloxacillin 300 300 20 19 1143 681 1547 95.0 Aminoglycosides (CP = 3346 cpm) Streptomycin 500 25 22 22 2424 1642 3074 100 Sulfonamides (CP = 1424 cpm) Sulfamethazine 100 25 29 28 1240 813 1831 96.6 Sulfadimethoxine 100 25 20 20 968 737 923 100 Sulfamerazine 100 25 21 21 842 716 960 100 Sulfadiazine 100 25 20 20 948 735 1361 100 Sulfathiazole 100 25 20 19 989 698 1782 95.0 M ukota et al. BMC Chemistry (2020) 14:32 Page 9 of 15 tetracycline family (tetracycline, oxytetracycline) and not penicillin G, ampicillin, amoxicillin, oxacillin, dicloxacil- limited to the chlortetracycline provided for in the Charm lin and cloxacillin were detected at 25  µg/kg, 50  µg/kg, II test kit. 50  µg/kg, 300  µg/kg, 300  µg/kg and 300  µg/kg respec- The sulfa drugs including, sulfadimethoxine, sulfadia - tively, for all fish species involved in the study. Thus, zine, sulfamerazine were detected at 25 µg/kg (0.25 MRL) penicillin G is detected at 0.5 MRL, whereas ampicillin, for the different fish species (trout, salmon, seabass, tila - amoxicillin, oxacillin, dicloxacillin and cloxacillin are pia and dorade) at 100% detection; sulfamethazine was all detected at their respective MRL. However, 4.5 and detected at 25 µg/kg (0.25 MRL) at 96.6% detection (3.4% 5% of the results for dicloxacillin and cloxacillin respec- false negatives), and sulfathiazole was detected at 25 µg/ tively, were false negatives (Table  4). Further more, the kg (0.25 MRL) at 95.0% detection (5.0% false negatives). Charm II technique is capable of detecting streptomycin The results also show that the technique can detect other at 25 µg/kg (0.05 MRL) for all fish species involved in the antimicrobials belonging to the sulfonamides group study at 100% detection. (sulfamethazine, sulfadimethoxine, sulfamerazine, sul- A comparison of the CCβ and MRL for the different fadiazine and sulfathiazole), which are not included in antimicrobials is shown in Fig.  1. The results show that, the MSU multi-antimicrobial standard mix, provided in CCβ for the validated antimicrobials were below or equal the Charm II test kit. For the macrolides; erythromycin to the MRL for all drug residues in this study, with the A, tilmicosin, and tylosin A were detected at 100  µg/kg, exception of tilmicosin which was detected at 2 MRL. for the different fish species (cat fish, trout, salmon, sea - Most of the drug residues exhibited CCβ in the range bass, tilapia, lingue, dorade, and pangasius) with 100% 0.05 MRL to 0.5 MRL, with 100% detection. Moreover, detection. Whereas, results for the β-lactams show that the incidences of false negative results observed for all Fig. 1 The detection capabilities and maximum residue limits for the different antimicrobials Mukota et al. BMC Chemistry (2020) 14:32 Page 10 of 15 antimicrobials involved in the study were within the 5% unlike some of the chemical techniques that can simulta- requirement of the EU decision 2002/657, and therefore neously detect numerous antimicrobials [30, 31]. the validation results are satisfactory. The Charm II tech - nique exhibited better CCβ for tetracyclines at 25  ppb Repeatability of the method (0.25 MRL) compared to other rapid screening tech- Repeatability analysis was performed using the same niques such as the ELISA kit of R-Biopharm for screening Charm II protocol for a specific antimicrobial on differ - tetracycline antibiotic residues in the muscle of chicken, ent fish species performed by the same researcher. The beef, and shrimp, which detected the same at 100  ppb analysis was evaluated by means of the intra-day coef- (MRL) [27]. In another study, results of the revolution- ficient of variations and the results are presented in ary Biochip Array Technology showed better detectabil- Table  5. Results of the repeatability study characterized ity for tylosin A and oxytetracycline at 0.10 and 0.5 of the by the relative standard deviation (%RSD) were satisfac- respective MRL in samples [28]. tory with a precision of less than 12% for the different The limits of detection (LOD) obtained using the antimicrobial drugs including tetracyclines, macrolides, Charm Test II assays, and the limits of quantitation β-lactamss, aminoglycosides, and sulfonamides; spiked (LOQ) for selected literature chemical methods are pre- in blank fish samples at MRL, 0.5 MRL or concentra - sented in Additional file  1: Table S1b. The LOD results for tion less than 0.5 MRL and analysed under repeatability fish matrix obtained in this validation using the Charm II conditions (n ≥ 6). The coefficient of variation expressed kits, are comparable to the manufacturer’s claims for the as percentage relative standard deviation (R SD ) ranged tissue matrix. However, some antimicrobial compounds from 7.8 to 9.8% for tetracyclines (chlortetracycline and could be detected in fish tissue at levels lower than the oxytetracycline), 2.8 to 6.3% for macrolides (erythromy- manufacturer’s claim (Additional file  1: Table  S1b). The cin A), 6.9 to 9.7% for β-lactams (penicillin G), 10.01 to LOD results were also compared with the LC–MS/MS 11.5% for aminoglycosides (streptomycin); and for sul- analysis of sulfadimethoxine [29], HPLC–MS/MS analy- fonamides (sulfathiazole) it was from 1.2 to 8.7%. These ses of tetracyclines, chlortetracycline, oxytetracycline, results, ably demonstrate the protocol’s repeatability sulfadimethoxine, sulfamerazine and sulfadiazine [30]; when used for testing different antimicrobial residues in and LC–ESI–MS/MS analyses of a range of tetracyclines, fish tissue matrix. β-lactams, aminoglycosides and sulfonamides [31]. Gen- A closer look at results obtained under repeatability erally, the rigorous chemical techniques, as expected, conditions in the analysis of different fish samples spiked offer lower LOQ values compared to the respective with 25  µg/kg sulfathiazole is presented in Table  6. The LOD obtained with the Charm II tests. Nonetheless, results showed that there was no significant difference in the Charm II test demonstrated ability to detect a wider cpm readings for the same fish species, and amongst dif - range of antimicrobials belonging to different classes ferent fish species including dorade, salmon and seabass, including tetracyclines, macrolides, β-lactams, aminogly- spiked with sulfathiazole at the same concentration level cosides and sulfonamides at MRL or lower levels, but it (ANOVA, overall F-critical 3.35 > F-calculated 1.99) with requires use of different antimicrobial test kits in parallel; RSD < 10%. Similar observations were made for the other Table 5 Repeatability study at MRL, 0.5 MRL or concentration < 0.5 MRL Family Compound Spiking concentration (µg/kg) Mean cpm SD RSD (%) r r Tetracyclines Chlortetracycline 25 µg/kg (0.25 MRL) 1207.0 118.2 9.8 Oxytetracycline 100 µg/kg (MRL) 1270.06 98.41 7.75 Macrolides Erythromycin A 100 µg/kg (0.5 MRL) 1762.4 110.4 6.3 200 µg/kg (MRL) 1478.1 41.2 2.8 β-Lactams Penicillin G 25 µg/kg (0.5 MRL) 1285.6 89.3 6.9 50 µg/kg (MRL) 648.5 62.7 9.7 Aminoglycosides Streptomycin 250 µg/kg (0.5 MRL) 1125.8 112.7 10.01 500 µg/kg (MRL) 1110.5 127.2 11.5 Sulfonamides Sulfathiazole 25 µg/kg (0.25 MRL) 922.2 80.1 8.7 100 µg/kg (MRL) 706.5 8.6 1.2 SD , standard deviation under repeatability conditions, RSD , relative standard deviation under repeatability conditions, Mean cpm average of counts per minute under r r reproducibility conditions M ukota et al. BMC Chemistry (2020) 14:32 Page 11 of 15 Table 6 Repeatability in  the  detection of  sulfathiazole tetracyclines (chlortetracycline and oxytetracycline) was at 25 µg/kg for selected fish samples 7.2 to 11.4%; macrolides (erythromycin A) ranged from 5.8 to 8.9%; β-lactams (penicillin G) from 10.4 to 11.2%; Parameter Fish samples spiked with sulfathiazole at 25 µg/kg, cpm aminoglycosides from 8.9 to 15.1% and sulfonamides (sulfathiazole) from 2.8 to 8.3% as indicated in Table 7. Dorade Salmon Seabass An elaborate presentation of some results of the 989 914 735 reproducibility studies performed by two different 815 969 780 researchers following the same Charm II protocol 862 886 976 on selected fish species, spiked with oxytetracycline 876 1015 896 at a concentration level of 100  µg/kg, is presented in 782 932 890 Table  8. A comparison of the results obtained by the 934 1075 978 two researchers for the same fish species showed no 985 930 976 significant difference; and the overall analysis showed 877 1070 898 no significant difference in the cpm results for the dif - 863 935 975 ferent fish species including seabass, pangasius and 986 890 976 salmon (ANOVA, F-critical 4.1 > F-calculated 0.64), Average 896.9 961.6 908 with RSD < 10%, which further demonstrates the tech- SD 73.6 69.3 88.3 nique’s reproducibility with little matrices interference. RSD 0.082 0.07 0.097 Similar observations were made for the other antimi- crobial compounds, whose summarized results are pre- sented in Table 7. antimicrobial agents, whose summarized results are pre- sented in Table 5. Robustness of the method Reproducibility of the method Analysis of batches of many samples often require a The reproducibility studies were performed by two differ - couple of hours before completion; and there is likely to ent researchers following the same Charm II protocol on be a time interval between the first and last analysis of selected fish species, spiked with different antimicrobial the processed samples. In the robustness testing of the agents and evaluated by means of intra-day and inter-day Charm II assay, the effect of variation in reading time coefficient of variations. The reproducibility study char - interval for processed samples was studied. Robustness acterized by the relative standard deviation (%RSD) was testing was performed on samples spiked with 50  µg/ satisfactory with a precision of less than 15.3% for the kg amoxicillin and analysed on the β-lactams channel different antimicrobial drugs (tetracyclines, macrolides, immediately after mixing (0 h) and after 14 h. The con - β-lactams, aminoglycosides, and sulfonamides) spiked trol point for β-lactams was set at 1530, and the robust- in blank fish samples at MRL, 0.5 MRL or concentra - ness results are presented in Table 9. tion less than 0.5 MRL and studied under reproducibility From Table  9, it is evident that there was no signifi - conditions (n ≥ 6). The coefficient of variation calculated cant difference in the cpm for both pangasius and dorade as percentage relative standard deviation (%RSD) for Table 7 Reproducibility study at MRL, 0.5 MRL or concentration < 0.5 MRL Family Compound Spiking concentration (µg/kg) Mean cpm SD RSD (%) r r Tetracyclines Chlortetracycline 25 µg/kg (0.25 MRL) 1224.5 139.2 11.4 Oxytetracycline 100 µg/kg (MRL) 1277 92.6 7.2 Macrolides Erythromycin A 100 µg/kg (0.5 MRL) 1748.5 156.1 8.9 200 µg/kg (MRL) 1456.5 83.9 5.8 β-Lactams Penicillin G 25 µg/kg (0.5 MRL) 1204.9 135.0 11.2 50 µg/kg (MRL) 702.1 73.0 10.4 Aminoglycosides Streptomycin 250 µg/kg (0.5 MRL) 1110.6 98.7 8.9 500 µg/kg (MRL) 1132.6 171.4 15.1 Sulfonamides Sulfathiazole 25 µg/kg (0.25 MRL) 943.7 78.1 8.3 100 µg/kg (MRL) 647.1 18.1 2.8 SD standard deviation under reproducibility conditions, RSD relative standard deviation under reproducibility conditions r r Mukota et al. BMC Chemistry (2020) 14:32 Page 12 of 15 Table 8 Reproducibility in the detection of oxytetracycline spiked with 50 µg/kg of beta-lactams and read after 0 or at 100 µg/kg for selected fish samples 14 h (ANOVA, F-critical 4.3 > F- calculated 0.2) confirm - ing the robustness of the method, in regard to variation in Fish sp. Researcher 1, cpm Researcher 2, cpm reading time intervals of the processed samples. A com- Seabass 1176 1296 parison of cpm for blank fish samples for both pangasius 1177 1265 and dorade after 0 and 14  h, also showed that there was 1295 1177 no significant difference between counts since ANOVA 1334 1281 F-critical 4.9 > F-calculated 0.2. The combined results of 1127 1166 these studies demonstrate that the Charm II technique is 1341 1417 quite robust for the analysis of antimicrobials in fish. 1170 1460 1261 1074 Specificity and cross reactivity of the technique 1371 1361 The cross reactivity analysis was carried out in order to 1201 1335 determine whether the presence of non-target drugs may Pangasius 1225 1185 lead to false identification of the target drug; or whether 1166 1223 the identification of the target analyte may be hindered 1094 1224 by the presence of one or more interferences. Repre- 1408 1407 sentative blank fish samples were spiked with different 1408 1405 antimicrobial drugs at known concentration levels higher Salmon 1307 1307 than those likely to interfere with the identification of the 1378 1299 analyte of interest, and then analysed using the respec- 1298 1310 tive Charm II protocol for the target drug. The aminogly - 1274 1311 cosides (spectinomycin, neomycin B and paromomycin) 1313 1279 were analysed using the macrolide channel (meant for 1266.2 1289.1 erythromycin A, tilmicosin and tylosin A). A standard Average 1176 1296 mix containing aminoglycosides (spectinomycin, neomy- Standard deviation 94.6 96.2 cin B and paromomycin) was used to spike different fish RSD 0.07 0.07 samples at 150, 300 and 500  µg/kg level, and the results are presented in Table 10. Results show that although the macrolides which were the targeted antimicrobials tested positive (sam- ples spiked with erythromycin A at 200  µg/kg, gave Table 9 Robustness testing using amoxicillin spiked at 50 µg/kg for selected fish samples Time Run Spiked at 50 µg/kg Non spiked Pangasius, cpm Dorade, cpm Blanks, cpm Fish species Results at 0 h 1 1019 1069 2433 Pangasius 2 1024 959 2398 Pangasius 3 1017 1019 2399 Pangasius 4 1201 1069 2064 Dorade 5 1155 1033 2200 Dorade 6 1020 1067 2109 Dorade Results after 14 h 7 1120 1120 2399 Pangasius 8 1059 1080 2064 Pangasius 9 1260 1195 2399 Pangasius 10 1011 1113 2068 Dorade 11 1089 1089 2210 Dorade 12 1099 1092 2205 Dorade Average 1089.5 1075.4 2245.7 SD 81.6 57.9 150.8 RSD 0.07 0.05 0.07 M ukota et al. BMC Chemistry (2020) 14:32 Page 13 of 15 Table 10 Specificity and cross reactivity tests using the Macrolides kit Blanks cpm AMGL spiked AMGL spiked AMGL spiked AMGL spiked AMGL spiked AMGL spiked at 150 µg/kg, at 300 µg/kg, at 500 µg/kg, at 150 µg/kg, at 300 µg/kg, at 500 µg/kg, Trout Salmon cpm Salmon cpm Pangasius cpm Catfish cpm Catfish cpm cpm 5377 5097 5239 5239 5536 5511 4694 5538 4860 4931 4931 5224 5393 5262 5538 5106 4800 5034 5223 5803 5412 5076 4703 4900 5351 4966 5558 5686 5571 5039 4950 5121 5236 5807 5728 Macrolide calculated control point cpm = 2118 AMGL aminoglycosides standard mix containing spectinomycin, neomycin B and paromomycin Table 11 Specificity and cross reactivity tests with mixed standards of different antimicrobial using the Aminoglycosides kit Antibiotics used and their respective MRL Mixed standard and respective spiking level, Fish species Spiked fish µg/kg samples cpm Tetracycline (MRL 100 µg/kg) Tetracycline spiked at 1000 µg/kg Cat fish 5742 Cat fish 6286 Penicillin G (MRL 50 µg/kg) Penicillin G spiked at 500 µg/kg Cat fish 5780 Cat fish 5776 Sulfamethazine (MRL 100 µg/kg) Sulfamethazine spiked at 1000 µg/kg Salmon 5418 Salmon 5700 Salmon 5584 Tilmicosin (50 MRL µg/kg) Tilmicosin spiked at 500 µg/kg Trout 5776 Trout 5584 Trout 6155 Trout 5962 Trout 5777 Trout 5800 Trout 5671 Trout 6010 Trout 5699 Trout 5700 Trout 5800 Trout 5810 Trout 5156 Aminoglycosides calculated control point cpm = 3346 1478 cpm), the non-target aminoglycosides intentionally The results of these studies also showed that no resi - analyzed on the same channel, tested negative since in all dues of the non-target drugs (tetracycline, penicillin G, cases the observed cpm were above the set control point sulfamethazine and tilmicosin) could be detected using of the macrolides of 2118. In similar studies, cross reac- the aminoglycosides channel as shown in Table  11; tivity was further investigated by spiking residue-free, whereas samples spiked with spectinomycin at 500 µg/ blank fish samples with high concentrations (10 MRL) of kg tested positive with 1110 cpm when analysed under antimicrobial substances belonging to other groups (sul- the same channel. All spiked samples tested negative fonamides, β-lactams, macrolides, and tetracyclines) and and the non-target compounds could not be detected were analysed on the aminoglycosides channel; and the even at high concentration (10 MRL). Similar obser- results are presented in Table 11. vations were made when utilizing the Biochip Array Mukota et al. BMC Chemistry (2020) 14:32 Page 14 of 15 Technology assay, where none of the tested antimicro- detection of antimicrobial residues in a variety of fish bials could be detected under cross-reactivity studies species; and its applicability for the rapid evaluation of [28]. the quality of aquaculture products for safety and trade purposes. Conclusions The Charm II radio receptor assay technique was suc - Supplementary information cessfully validated for screening residues of tetra- Supplementary information accompanies this paper at https ://doi. org/10.1186/s1306 5-020-00684 -4. cyclines, sulfonamides, β-lactams, aminoglycosides and macrolides in different aquaculture fish species. Additional file 1: Table S1a. List of analytical standards used for spiking. The Charm II technique can detect tetracycline and Table S1b. Limits of quantification and detection from Charm II assays chlortetracycline at 25  µg/kg (0.25 MRL) and oxytet- and published results. racycline at 100  µg/kg (MRL) for different fish spe - cies including, cat fish, trout, salmon, seabass, tilapia, Abbreviations lingue, dorade, and pangasius, with 100% detection. ANOVA: One-way analysis of variance; CCβ: Detection capability; CP: Control The sulfonamides including sulfadimethoxine, sulfam - point; CPM: Counts per minute; EU: European Union; FAO: Food and Agricul- ture Organization; IAEA: International Atomic Energy Agency; ILVO: Flanders erazine, sulfadiazine, sulfathiazole could be detected Research Institute for Agriculture, Fisheries and Food; MRL: Maximum residue at 25  µg/kg (0.25 MRL) for all fish species involved in limit; MSU: Multi antimicrobial standard; RSD: Relative standard deviation; SD: the study, with the exception of catfish, pangasius, and Standard deviation; UNBS: Uganda National Bureau of Standards. lingue, which gave high counts for the blank samples. Acknowledgements Results for the macrolides analysis, showed that eryth- The study was performed with support from the International Atomic Energy romycin A, tilmicosin, and tylosin A, could be detected Agency (IAEA) program on Development and Strengthening of Radio-Analyt- ical and Complementary Techniques to Control Residues of Veterinary Drugs at 100 µg/kg (0.5 MRL), 100 µg/kg (2 MRL) and 100 µg/ and Related Chemicals in Aquaculture Products, Vienna Austria. The authors kg (MRL), respectively, for the different fish species. would also like to acknowledge support from the Uganda National Bureau Whereas, the β-lactams including penicillin G, ampi- of Standards, Kampala-Uganda; Institute of Medical Research and Medicinal Plant Studies, Yaounde, Cameroon; Flanders Research Institute for Agriculture, cillin, amoxicillin, oxacillin, dicloxacillin and cloxa- Fisheries and Food, Melle-Belgium and Department of Chemistry, Makerere cillin were detected at 25  µg/kg (0.5 MRL), 50  µg/kg University Kampala, Uganda. We are also very grateful to Charm Sciences Inc. (MRL), 50  µg/kg (MRL), 300  µg/kg (MRL), 300  µg/kg for providing the Charm II test reagents and kits. (MRL) and 300  µg/kg (MRL), respectively. Under the Authors’ contributions aminoglycosides analysis, streptomycin was detected at AKM, WR, MFKG and JS participated in experimental design and coordination 25 µg/kg (0.05 MRL) for all fish species involved in the of laboratory activities. AKM, MFKG and WR carried out the sampling, sample preparation and analysis. WR, JJTT, AKM and ET participated in data analysis. study. Interestingly, the technique can detect a broader AKM, ET, MH and SAN wrote the manuscript. All authors participated in manu- range of antimicrobials other than only the compounds script review. All authors read and approved the final manuscript. specified in the Charm II assay kit. In addition, all anti - Funding microbial compounds involved in the study could be The work was financially supported by the International Atomic Energy successfully detected using the Charm II assay at 100% Agency (IAEA) program on Development and Strengthening of Radio- rate, with the exception of dicloxacillin, cloxacillin, Analytical and Complementary Techniques to Control Residues of Veterinary Drugs and Related Chemicals in Aquaculture Products. The work was partially sulfamethazine and sulfathiazole that exhibited false supported by Charm Sciences Inc. who provided all the Charm II test reagents negative rates of 4.5, 5.0, 3.4 and 5.0%, respectively. and kits used in the study. Moreover, these false negative rates fall within the 5% Availability of data and materials requirement of the EU decision 2002/657, and there- All supporting information including table of results and detailed methods is fore, the validation results are satisfactory. available upon request. Robustness studies showed that there was no signifi - Competing interests cant difference between results for counts of the same The authors declare that they have no competing interests. samples read immediately or after 14  h of addition of the scintillation fluid. In addition, no evidence of cross- Author details Uganda National Bureau of Standards, Headquarters. Plot 2–12 Bypass Link, reactivity was observed among the targeted antimicro- Industrial & Business Park, Kyaliwajala Road, P.O. Box 6329, Kampala, Uganda. bial compounds on interchanging the antimicrobial 2 Institute of Medical Research and Medicinal Plant Studies, P.O. Box 6163, analysis channels. The results of this validation study Yaounde, Cameroon. International Atomic Energy Agency (IAEA), Vienna International Centre, P. O. Box 100, 1400 Vienna, Austria. Flanders Research prove the robustness, specificity, reliability and preci - Institute for Agriculture, Fisheries and Food (ILVO), Technology and Food sion of the Charm II radio receptor assay technique in 5 Science Unit, Brusselsesteenweg 370, 9090 Melle, Belgium. Department the detection of various antimicrobials residues in dif- of Chemistry, College of Natural Sciences, Makerere University, P.O. Box, 7062, Kampala, Uganda. ferent fish species. 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