Environ Sci Pollut Res (2014) 21:3427–3434 DOI 10.1007/s11356-013-2286-5
RESEARCH ARTICLE
Daily ingestion of tetracycline residue present in pasteurized milk: a public health problem Sergio Augusto de Albuquerque Fernandes & Ana Prudencia Assis Magnavita & Sibelli Passini Barbosa Ferrao & Simone Andrade Gualberto & Amanda Santos Faleiro & Abdias Jose Figueiredo & Soraia Vanessa Matarazzo
Received: 21 June 2013 / Accepted: 24 October 2013 / Published online: 17 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract The objective of this study was to evaluate (qualitatively and quantitatively) the occurrence of antibiotic residue in pasteurized milk in Brazil. Pasteurized milk samples (n = 252) were collected monthly from Nov. 2010–Oct. 2011 from 21 commercial establishments (brands). A screening test (Delvotest® SP-NT) was applied to those samples. In positive (n =19) and/or suspect samples (n =24), we quantified oxytetracycline (OTC) and tetracycline (TC) by highperformance liquid chromatography with diode array detector (HPLC-DAD). OTCs were detected in all positive samples and TCs in six. In the 24suspected samples, OTCs were detected in 23 and TCs were not found in 8. Of the milk brands evaluated (n =21), the presence of antibiotic residue was not detected in 4; in the other brands, both positive and suspect samples were verified. Results indicate the presence of antibiotic residue above legal limits. According to actual milk consumption in Brazil (441 mL/kg BW/day), in only 9 of the 17 brands of milk with antibiotic residue, the estimated daily intake was at or less than the maximum recommended by the European Union. The screening test used was effective to identify the presence of antibiotic residue (OTC and TC), confirmed by HPLC-DAD. The OTC is the predominant antimicrobial used by dairy farmers. Ingestion of contaminated milk by OTC and TC can increase the resistance of microorganisms to antibiotics. Responsible editor: Philippe Garrigues S. A. de Albuquerque Fernandes (*) : A. P. A. Magnavita : S. P. B. Ferrao : S. A. Gualberto : A. S. Faleiro : A. J. Figueiredo : S. V. Matarazzo Southwest State University of Bahia, Itapetinga, Bahia, Brazil e-mail: [email protected] S. V. Matarazzo State University of Santa Cruz, Ilheus, Bahia, Brazil
Keywords Antibiotic residue ingestion . Delvotest® SP-NT . Oxytetracycline . Tetracycline
Introduction In Brazil, monitoring antibiotic residue in milk is a requirement according to the official norms for production, processing, and commercialization of milk (BRASIL 2003, 2011), and meeting the international norms (CODEX ALIMENTARIUS 2012) is one of the technological concerns of companies in the dairy sector and international public health. Milk is a food of great social and economic importance, being produced in different edaphoclimatic conditions around the world in diverse production systems, from highly to barely technical. Its average composition consists of 87.4 % water, 12.6 % total solids, 3.9 % fat, 3.2 % crude protein, 4.6 % lactose, and 0.9 % minerals. It also includes other solids, which vary according to species, breed, diet, and other factors. These features indicate its strategic importance in human nutrition, especially for children and the elderly. The somatic cell count (SCC) in milk is indicative of the health of the mammary gland (MG). Thus, health of the MG provides milk with less than 105 somatic cells/mL−1 on average, considering 2×105 cells/mL−1 as the upper limit for a health of the MG (Dohoo and Leslie 1991). Higher values of the SCC in the MG indicate mastitis. Mastitis negatively influences the composition of the milk, which in turn affects its processing (Barbano et al. 1991). In Brazil, the SCC varies between 1.5×105 and 106 (Mazal et al. 2007; Costa Sobrinho et al. 2012), demonstrating high rates of mastitis infection in cow herds. These results indicate
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the use of medications for treatment of cows and may result in residue and antibiotics in the milk. According to Fonseca et al. (2009) and Oliveira et al. (2012), samples positive for antibiotics residue in milk based on a screening test range from 0 to 4 % in Brazil. Bando et al. (2009) observed that 41.5 % of samples were positive using enzymatic tests. Freitas et al. (2013) found 20 % using the LCIT-TOF MS/QuEChERS; Martins-Júnior et al. (2007) reported positives samples at 8.0 %, using LC-ESI/MS/MS. Antibiotics are used prophylactically or therapeutically in dairy cattle, especially to control mastitis. This is the principal use of antibiotics in lactating cows as it is the most prevalent disease in these animals (Bansal et al. 2011). The maximum residue level (MRL) for tetracyclines classes (oxytetracyclines, tetracyclines, and chlortetracyclines) is 100 ng mL−1, single or in combination (Codex Alimentarius 2012). The assessment of antimicrobial residue in milk is performed using qualitative screening tests in the industry. The most common test is the inhibition of microbial growth (Hennart and Faragher 2012). Several methods are marketed, including the Delvotest® SP-NT, CMT COPAN®, Eclipse® 100, Delvotest® Accelerator, and Charm Cowside®. The Delvotest® SP-NT is reliable, with its broad spectrum of microbial inhibition in the detection of antibiotic residue; it is also easy to use and generates results in a short period of time (3 h), which is essential in the dairy industry (Linage et al. 2007). The Delvotest® SP-NT (DSM Food Specialties Ingredients, Netherlands) allows for detection of five classes of antimicrobial substances as follows: beta-lactam antibiotics, sulfonamides, tetracyclines, macrolides, aminoglycosides, and others such as trimethoprim and dapsone. For identification and quantification of antimicrobial residue present in animal-derived foods, more sensitive and specific analytical techniques are recommended, such as highperformance liquid chromatography (HPLC) (Mamani et al. 2009). HPLC is a fast and reliable technique, with high sensitivity for the analysis of antibiotic residue like tetracyclines. It permits the confirmation, identification, and quantification of antimicrobial compounds present in the sample (Goto et al. 2005). However, some methodologies, such as liquid chromatography-tandem mass spectrometry (Freitas et al. 2013) and ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (Kennedy et al. 1998; Martins-Júnior et al. 2007; Stockler et al. 2009), are more efficient to identify and quantify the veterinary drugs in milk rather than liquid chromatography–diode array detector (Mamani et al. 2009). The presence of antibiotic residue in milk may trigger allergic phenomena, such as toxic effects (Stolker and Brinkman 2005) and the development of resistance of microorganisms to antibiotics (Gao et al. 2012), making dairy products a source of risk to the population (Can and Çelik 2012). In the industry, the presence of antibiotics in milk
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causes losses in the processing of yogurt, butter, and cheese due to inhibition of lactic bacteria activity (Knappstein et al. 2003), which makes it necessary to monitor the presence of antibiotic residue in milk (Ilić et al. 2012). Although Brazil is the fifth largest milk producer in the world (33.2 million tons) (FAO 2013), the rate of dairy products for exportation is low. Consequently, improving the milk quality and offering a safer product is important in increasing export. The aim of this study is to qualitatively (Delvotest ® SP-NT) and quantitatively (high-performance liquid chromatography with diode array detector; HPLC-DAD) evaluate the presence of antibiotic residue (oxytetracycline, OTC and tetracycline, TC) in pasteurized milk samples to estimate the average daily ingestion of this residue by humans.
Material and methods Sampling Analyses were performed in duplicate on 21 brands of pasteurized milk. Milk samples (n =252) were collected monthly in commercial establishments from November 2010 to October 2011 in the south and southwest regions of Bahia State, the largest producer in the States. These samples were collected in the commercial market (packaged in 1,000 mL). After collection, the samples were frozen and sent to the laboratory for processing of milk and dairy products at the State University of Southwest Bahia (Universidade Estadual do Sudoeste da Bahia), where they were identified and remained frozen until analysis. Screening test The Delvotest® SP-NT kit (DSM Food Specialties) was used as a screening test, and three experts performed this measurement visually for the final determination. Samples with positive and/or suspect results were also analyzed by HPLC-DAD. Sample preparation for extraction Extractions of TCs were performed according to the validated methodology described by Denobile and Nascimento (2004). Aliquots of 1.0 mL were taken from each milk sample, followed by adding 0.35 mL of a trichloroacetic acid solution in acetonitrile at 80 % (w/v). The mixture was agitated (Ika®, Werkw Gmbh & Co., Germany) for 5 min with subsequent centrifugation for 15 min at 15,000 rpm. After centrifugation, the supernatant was collected and filtered with the aid of a syringe through a Durapore membrane filter (0.45 μm pore), and this was stored in an amber vial under refrigeration until the time of analysis.
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Equipment For identification and quantification of tetracyclines in the samples, a model SPD20A liquid chromatography was used (Shimadzu, Kyoto, Japan) with a quaternary solvent injection valve and a sample loop of 20 μL, column oven, and diode array detector (DAD). The antimicrobial compounds TC and OTC were separated in a C 8 analytical column (15 cm×4 mm di × 5 μm). A solution of 0.01 M oxalic acid, acetonitrile, and 0.1 % triethylamine (70:20:10 v/v/v) was used as the mobile phase at a flow rate of 1 mL min−1, and the temperature of the oven was adjusted to 40 °C. The analysis time was 10 min. The solvents used were chromatographic grade (JC Baker) and were filtered and degassed before use. The chromatograms were processed at 363 nm. Identification of OTC and TC was tentatively performed by comparing the retention time of the peaks with the standard samples, oxytetracycline hydrochloride (95 %) and tetracycline (88 %), both acquired from Sigma-Aldrich® Chemical Co. (St. Louis, MO, USA). Evaluation of the presence of antibiotic residue in the milk samples analyzed was performed by observing the results of screening tests (descriptive analysis) and determining the percentages of positive, suspect, and negative samples expressed in nanograms per milliliter. The general characteristics of HPLC-DAD of the OTC and TC proved to be effective for the identification and quantifi-
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cation by CLAE of these substances in pasteurized milk samples were (1) linearity data showed a positive correlation between the concentrations of tetracyclines (OTC, y=25.2x+ 49.16 and R2 =0.999; TC, y=31.8x+518.8 and R2 =0.999) in the ranges of 100, 200, 400, 800, and 1,600 ng mL−1. The linear correlation coefficients (0.999) found for the curves are in compliance with those set by ANVISA (2003); (2) the coefficients of variation (CV) obtained in the study of intra/ interday precision, of the milk samples added with the standards of OTC and TC, in concentrations of 200, 400 and 800 ng mL−1, respectively, were 1.7, 2.5, and 1.9 (intraday) and 1.7, 3.6, and 4.5 % (interday) for oxytetracycline and 1.7, 2.6, and 1.7 (intraday) and 6.8, 7.5, and 8.1 % (interday) for tetracycline; (3) the detection limits and quantification were of 50 ng mL−1 for OTC and TC, with CV 6.8 and 8.7 %, respectively, and (4) the recovery rate values of tetracyclines in the studied concentrations (100, 200, and 400 ng mL−1) were 64, 64, and 67 % (OTC) and 46, 54, and 61 % (TC) for each concentration, respectively Calculation of the estimated daily intake The concentration was determined using the average content of total tetracyclines observed, and the estimated daily intake (EDI) was calculated according to Juan et al. (2010).
EDI ¼ ½ðmean in μg of the antibiotic residue per Kg of foodÞ ðMean consumption of the foodÞ Live adult weight of 60 Kg
For determination of the EDI, the estimated milk consumption values used were for Brazil in 2012 (441 mL/inhabitant/ day), and this was compared to the recommended consumption by the Brazilian Ministry of Health, which is 575 mL/ inhabitant/day (Embrapa 2013). This evaluated the impact of increased milk consumption on the estimate.
Results Among the 252 pasteurized milk samples analyzed by the Delvotest® SP-NT in this study, 207 (82.0 %) were negative (no antibiotic residue), and 20 presented positive results, revealing a contamination frequency of 8.0 %. On the other hand, 25 samples (10 %) were defined as suspect. In most of the milk samples studied, the presence of antibiotic residue (AM) was not detected by the screening test. Distribution of the results throughout the year indicated that there was a period in which the number of positive and
suspected samples was greater. Of the 20 positive milk samples, 13 (65 %) were detected between January and June. The total number of suspected samples observed during the year was 25, where 22 (91.7 %) were obtained between January and June (Fig. 1). These results indicate that season likely had an effect on the occurrence of positive and suspected milk samples, as was observed by Kaya and Filazi (2010). Of the 21 pasteurized milk brands, only four (D, E, P, and T) showed no positive results over the year of study. All other brands (17) tested positive or suspect at some point (Fig. 2). Brand N showed the highest number of samples with positive (25 %) and suspect (25 %) results, whereas L and O both presented 17 % positive samples and 8 % suspect samples, respectively. For brands B, C, and M, 17, 17, and 25 % of the samples were reported as positive, respectively. Brands A, F, H, Q, S, and V presented positive samples (8.3 % each) with variations in the percentage of suspect samples. However, brands G, I, J, R, and U exhibited only suspect samples (25, 17, 8, 16.7, and 17 %, respectively).
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21 19
Dairy industry numbers (n = 21)
Fig. 1 Monthly distribution of negative, positive, and suspected pasteurized milk samples by screening test in dairy industry (Nov. 2010 to Oct. 2011)
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19
21
21
Positives
19
18
Suspecteds
Negatives
17
15
14
13 10 10
5 2
Jan
44
4 2
Feb
2
Mar
2
Apr
The positive and suspect samples discovered during the screening test were subjected to HPLC-DAD for confirmation of the presence of tetracyclines. In samples with positive results (n =19), the highest concentration observed individually for OTC was 2,782 ng mL−1 (Fig. 3) in sample I-Jan. In the same sample, the concentration of TC was 1,145 ng mL−1. The lowest concentrations above the maximum residue limit (MRL=100 ng mL−1) were observed in samples F-Apr (125 ng mL−1) for OTC and sample S-Mar (107 ng mL−1) for TC. In 12 samples, no TC residue was detected, and in one (P-Sept), the TC concentration was below the MRL (73 ng mL−1), in which no false positive was detected. In suspect samples, the lowest OTC concentration was observed in sample U-Mar (67 ng mL−1), and the highest was 2,072 ng mL−1 (S-Feb). With respect to TC, the highest was 2,297 ng mL−1 (R-Feb), and the lowest was 78 ng mL−1
1 May
4 2
2 1 Jun Jul Months
Aug
Sept
Oct
Nov
Dec
(N-Mar). In 16 samples, the presence of TC was not detected; only 1 sample (N-mar) showed no OTC (Fig. 4). The antibiotic concentrations observed in most samples with positive or suspect results exceed the MRL permitted for tetracyclines (100 ng mL−1, singly or in combination). Of the 19 positive samples in the screening tests, all presented OTC concentrations above the MRL (Table 1); in six, the TC concentration was excessive; and in one, it was below the legal limit. In 12 samples, TC was not detected. Among the samples considered suspect in the screening test, in 21, the OTC concentration was greater than that permitted by legislation; in 2, the concentration was lower; and in 1, no OTC was detected. TC was not detected in 16 samples; only in 1 did it not exceed the MRL; and in 7, it exceeded the MRL. The “total tetracycline concentration” in suspect samples was below the MRL in only three samples.
Sample percentage
Fig. 2 Percentage of negative, positive, and suspect pasteurized milk samples by screening test per dairy industry (Nov. 2010 to Oct. 2011)
Dairy industry
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2800
OTC
2600
TC 2354
2400
Concentração em ng.mL-1
Fig. 3 Antibiotics residues concentration (OTC oxytetracycline, TC tetracycline) by HPLC-DAD in the positives milk samples in the screening test (Delvotest® SP-NT). MRL maximum residue limit=100 ng mL−1
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2200 1926
2000 1800 1600 1400
1145
1200 1000 800
848
875
855
938
1051
929
885
828
775
736
701
685 597
506
600 282
400 125 200
73
359
316 135
107
194 0
0 A-nov B-sept B-nov C-aug C-nov E-sept P-sept F-apr H-jun H-feb I-jan I-feb I-jun Q-jun Q-jul S-may N-feb N-apr N-may
Dairy industry-month
The EDI was based on the average concentration of tetracycline residue in milk, per dairy facility, observed in this study. Average daily milk consumption in Brazil in 2011 was 441 mL/inhabitant/day, but the desired average milk consumption is 575 mL/inhabitant/day (Embrapa 2013). When evaluating the EDI of antibiotics (tetracycline) by means of Fig. 4 Concentration (ng/mL−1) of antibiotics residues (OTC oxytetracycline, TC tetracycline) by HPLC-DAD in the suspects milk samples by screening test (Delvotest® SP-NT). MRL maximum residue limit=100 ng mL−1
the actual milk consumption in Brazil (Fig. 5), it was observed that of the 16 brands, in 9, the EDI was in accordance with that recommended by the European Union (EMEA 1995), with variations between 1.3 and 3 μg/kg BW/day. In the other 7 brands, these estimates varied between 3.2 and 8.8 μg/kg BW/ day, where the greatest value was observed in brand I. The
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Table 1 Number of positive and suspect pasteurized milk samples sold according to the screening test for antibiotic residues, confirmed by HPLC-DAD MRL/milk
Greater than Less than Not detected
Positive (n =19)
milk). Bando et al. (2009), using immunoassay, found that 41.5 % of samples were positive. Freitas et al. (2013) found 20 % (LC-IT-TOF MS/QuEChERS), and Martins-Júnior et al. (2007) reported 8 % (LC-ESI/MS/MS). In our study, all positive samples and most suspect samples (87.5 %) exceeded the MRL (Table 1). Screening tests are quick and easy to use, but they are subjective, so we need more precise and technical measures, such as liquid chromatography, to find the dynamic of the contamination of antibiotics in dairy cattle in Brazil. It is necessary improve the sampling number and submit it to liquid chromatography. The Delvotest SP-NT has low sensitivity to OTC (Le Breton et al. 2007), but in this study, it was effective even at low concentrations because it detected even a minimal presence of antibiotic residue in milk (67 ng mL−1) (Fig. 4), showing its sensitivity to the analyte (Hennart and Faragher 2012; Sierra et al. 2009). Rainfall in the studied area is concentrated during the summer season, from December to March (Moscati and Gan 2007). The high humidity could facilitate the development of microorganisms that cause mastitis, increasing its incidence. This higher mastitis incidence may increase the detection of antibiotic residue (Fig. 1) because mastitis is the most common cause of antibiotic use in lactating cows (Bansal et al. 2011). Milk production systems in the region involve minimal technology and milking hygiene measures are of little use, making these environments fertile for mastitis. Our results indicate that within the class of tetracyclines, OTC is most commonly used among the dairy products of the region studied as well as in other countries (Schlegelova et al. 2002).
Suspect (n =24)
OTC
TC
TTC
OTC
TC
TTC
19 – –
06 01 12
19 – –
21 02 01
07 01 16
21 03 –
OTC oxytetracycline, TC tetracycline, TTC total tetracyclines
simulation that utilized values of the recommended milk consumption of 575 mL/inhabitant/day indicated that only 3 of the 16 brands met the maximum recommended by the European Union for EDI of antibiotics (3 μg/kg BW/day), with a variation between 1.7 and 2.9 μg/kg BW/day. In 13, the EDI exceeded the recommendations (3.3 to 11.5 μg/kg BW/ day).
Discussion Sampling from large volumes can lack analytical precision (dilution effect) (FAO 2000), especially during screening tests. In our study, the samples were collected from large volume industries, but the percentage of positive samples was still close to 20 %. Distinct percentages of pasteurized milk samples positive for antibiotics after a screening test were reported in Brazil. Oliveira et al. (2012) observed no antibiotic residue in milk samples (raw and pasteurized) evaluated. Fonseca et al. (2009) found 0–4 % noncompliance of samples evaluated (UHT
BR actual (441 mL/inhabitant/day)
12.0
11.5
BR/MS (575 mL/inhabitant/day) 10.4
EDI (µg/Kg Body Weight/day)
Fig. 5 Estimated daily intake EDI simulation (μg/kg body weight/day) of the antimicrobial residue (tetracyclines) in milk. Recommendation of the EMEA (1995) of average daily consumption of tetracyclines=3 μg/ kg BW/day
8.8
9.0
7.9 7.4
7.4
6.2 5.7
6.0
6.2
5.7 4.7
3.3 3.0
3.5 3.0
2.5
4.7
4.1
3.9 2.7
3.7
3.5
3.3 3.2
2.9
2.7
2.5
2.2
2.9 2.2 1.7
1.7
1.3
0.0 A
B
C
E
F
G
H
I
L
Dairy industry
M
N
P
Q
R
S
U
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The results obtained in this study indicate the need for greater monitoring and control by health surveillance agencies of the production and consumption of milk. One of the major causes of antibiotic residue in milk is animal treatment with antibiotics; such animals should be milked separately for a specified period. These substances may be excreted with the milk even after finalizing treatment, making it necessary that such milk be discarded during this period, which may vary according to the recommendations of the manufacturer (Knappstein et al. 2003; Stockler et al. 2009; Bansal et al. 2011). In addition, it is important for quality control at the dairies to reduce the risks to public health (e.g., toxic effects and development of resistance to pathogenic microorganisms in humans) (Gao et al. 2012; Can and Çelik 2012) or to animals. Soil contamination is also a problem (Carvalho et al. 2013). In the dairy industry, antibiotics inhibit lactic culture and affect the quality and identity of cheeses by altering their composition and structure (Knappstein et al. 2003). Adjustments to the Brazilian milk production chain are imperative; a new development model is fundamental and should be supported by better quality and safety of products (Farina et al. 2005; Ilić et al. 2012). The consumer contamination risk is in a direct relationship with the danger of the chemical product and exposure to the same (risk = hazard × exposure) (Davies et al. 2003). Antibiotic residue in milk products presents a danger to the consumer, so these results indicate that the inappropriate use of antibiotics in dairy farming (high dosages, not observing the grace periods) may pose health risks to the public as well as lead to economic losses in the dairy industry.
Conclusions The results obtained indicate the presence of antibiotic residue above the legal limits within the class of tetracyclines. OTCs are the most commonly found in dairy products in the region studied. Further studies are required as follows: (1) antimicrobials resistance, (2) epidemiological status, (3) effects on the ground water, especially near the dairy industry, and (4) screening tests followed by HPLC with more sensitive detectors, such as mass spectrometry. It is recommended that training and guidance, particularly on the use of proper practices of hygiene during milking, be offered to the owners and their workers involved in the dairy industry. Acknowledgments This research was funded by Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB) and Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (Capes) for the support to the research. Conflict of interest None of the author has any support or relationship with other people that could inappropriately influence.
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Environ Sci Pollut Res (2014) 21:3427–3434 for antimicrobial detection in ovine milk. Jour Dairy Sci 90:5374– 5379 Mamani MCV, Reyes FGR, Rath S (2009) Multiresidue determination of tetracyclines, sulfonamides, and chloramphenicol in bovine milk using HPLC-DAD. Food Chem 117:545–552 Martins-Júnior HA, Kussumi TA, Wang AY, Lebre DT (2007) A rapid method to determine antibiotic residues in milk using Liquid Chromatography Coupled to Electrospray Tandem Mass Spectrometry. J Braz Chem Soc 18(2)397–405 Mazal G, Vianna PCB, Santos MV, Gigante ML (2007) Effect of somatic cell count on Prato cheese composition. J Dairy Sci 90:630–636 Moscati MCL, Gan MA (2007) Rainfall variability in the rainy season of semiarid zone of Northeast Brazil (NEB) and its relation to wind regime. Int J Climatol 27:493–512 Oliveira LP, Barros LSS, Silva VC, Cirqueira MG (2012) Microbiological quality and detection of antibiotic residue in raw and pasteurized milk consumed in the Reconcavo Area of the State of Bahia, Brazil. Food Process Technol 3(1):2–5. doi:10.4172/21577110.1000137 Schlegelova J, Babák V, Klímová E, Lukásová J, Navrátilová P, Sústácková A, Sédivá I, Rysánek D (2002) Prevalence of and resistance to anti-microbial drugs in selected microbial species isolated from bulk milk samples. J Vet Med B 49:216–225 Sierra D, Contreras A, Sánches A (2009) Short-communication: detection limits of non-beta-lactam antibiotics in goat's milk by microbiological residues screening tests. J Dairy Sci 92: 4.200–4.206 Stockler RM, Morin DE, Lantz RK, Constable PD (2009) Effect of milking frequency and dosing interval on the pharmacokinects of cephapirin after intramammary infusion in lactating cows. J Dairy Sci 92:4262–4275 Stolker AAM, Brinkman UA (2005) Analytical strategies for residue analysis of veterinary drugs and growth-promoting agents in foodproducing animals—a review. J Chromatogr Anal 1067:15–53
RESEARCH ARTICLE
Daily ingestion of tetracycline residue present in pasteurized milk: a public health problem Sergio Augusto de Albuquerque Fernandes & Ana Prudencia Assis Magnavita & Sibelli Passini Barbosa Ferrao & Simone Andrade Gualberto & Amanda Santos Faleiro & Abdias Jose Figueiredo & Soraia Vanessa Matarazzo
Received: 21 June 2013 / Accepted: 24 October 2013 / Published online: 17 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract The objective of this study was to evaluate (qualitatively and quantitatively) the occurrence of antibiotic residue in pasteurized milk in Brazil. Pasteurized milk samples (n = 252) were collected monthly from Nov. 2010–Oct. 2011 from 21 commercial establishments (brands). A screening test (Delvotest® SP-NT) was applied to those samples. In positive (n =19) and/or suspect samples (n =24), we quantified oxytetracycline (OTC) and tetracycline (TC) by highperformance liquid chromatography with diode array detector (HPLC-DAD). OTCs were detected in all positive samples and TCs in six. In the 24suspected samples, OTCs were detected in 23 and TCs were not found in 8. Of the milk brands evaluated (n =21), the presence of antibiotic residue was not detected in 4; in the other brands, both positive and suspect samples were verified. Results indicate the presence of antibiotic residue above legal limits. According to actual milk consumption in Brazil (441 mL/kg BW/day), in only 9 of the 17 brands of milk with antibiotic residue, the estimated daily intake was at or less than the maximum recommended by the European Union. The screening test used was effective to identify the presence of antibiotic residue (OTC and TC), confirmed by HPLC-DAD. The OTC is the predominant antimicrobial used by dairy farmers. Ingestion of contaminated milk by OTC and TC can increase the resistance of microorganisms to antibiotics. Responsible editor: Philippe Garrigues S. A. de Albuquerque Fernandes (*) : A. P. A. Magnavita : S. P. B. Ferrao : S. A. Gualberto : A. S. Faleiro : A. J. Figueiredo : S. V. Matarazzo Southwest State University of Bahia, Itapetinga, Bahia, Brazil e-mail: [email protected] S. V. Matarazzo State University of Santa Cruz, Ilheus, Bahia, Brazil
Keywords Antibiotic residue ingestion . Delvotest® SP-NT . Oxytetracycline . Tetracycline
Introduction In Brazil, monitoring antibiotic residue in milk is a requirement according to the official norms for production, processing, and commercialization of milk (BRASIL 2003, 2011), and meeting the international norms (CODEX ALIMENTARIUS 2012) is one of the technological concerns of companies in the dairy sector and international public health. Milk is a food of great social and economic importance, being produced in different edaphoclimatic conditions around the world in diverse production systems, from highly to barely technical. Its average composition consists of 87.4 % water, 12.6 % total solids, 3.9 % fat, 3.2 % crude protein, 4.6 % lactose, and 0.9 % minerals. It also includes other solids, which vary according to species, breed, diet, and other factors. These features indicate its strategic importance in human nutrition, especially for children and the elderly. The somatic cell count (SCC) in milk is indicative of the health of the mammary gland (MG). Thus, health of the MG provides milk with less than 105 somatic cells/mL−1 on average, considering 2×105 cells/mL−1 as the upper limit for a health of the MG (Dohoo and Leslie 1991). Higher values of the SCC in the MG indicate mastitis. Mastitis negatively influences the composition of the milk, which in turn affects its processing (Barbano et al. 1991). In Brazil, the SCC varies between 1.5×105 and 106 (Mazal et al. 2007; Costa Sobrinho et al. 2012), demonstrating high rates of mastitis infection in cow herds. These results indicate
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the use of medications for treatment of cows and may result in residue and antibiotics in the milk. According to Fonseca et al. (2009) and Oliveira et al. (2012), samples positive for antibiotics residue in milk based on a screening test range from 0 to 4 % in Brazil. Bando et al. (2009) observed that 41.5 % of samples were positive using enzymatic tests. Freitas et al. (2013) found 20 % using the LCIT-TOF MS/QuEChERS; Martins-Júnior et al. (2007) reported positives samples at 8.0 %, using LC-ESI/MS/MS. Antibiotics are used prophylactically or therapeutically in dairy cattle, especially to control mastitis. This is the principal use of antibiotics in lactating cows as it is the most prevalent disease in these animals (Bansal et al. 2011). The maximum residue level (MRL) for tetracyclines classes (oxytetracyclines, tetracyclines, and chlortetracyclines) is 100 ng mL−1, single or in combination (Codex Alimentarius 2012). The assessment of antimicrobial residue in milk is performed using qualitative screening tests in the industry. The most common test is the inhibition of microbial growth (Hennart and Faragher 2012). Several methods are marketed, including the Delvotest® SP-NT, CMT COPAN®, Eclipse® 100, Delvotest® Accelerator, and Charm Cowside®. The Delvotest® SP-NT is reliable, with its broad spectrum of microbial inhibition in the detection of antibiotic residue; it is also easy to use and generates results in a short period of time (3 h), which is essential in the dairy industry (Linage et al. 2007). The Delvotest® SP-NT (DSM Food Specialties Ingredients, Netherlands) allows for detection of five classes of antimicrobial substances as follows: beta-lactam antibiotics, sulfonamides, tetracyclines, macrolides, aminoglycosides, and others such as trimethoprim and dapsone. For identification and quantification of antimicrobial residue present in animal-derived foods, more sensitive and specific analytical techniques are recommended, such as highperformance liquid chromatography (HPLC) (Mamani et al. 2009). HPLC is a fast and reliable technique, with high sensitivity for the analysis of antibiotic residue like tetracyclines. It permits the confirmation, identification, and quantification of antimicrobial compounds present in the sample (Goto et al. 2005). However, some methodologies, such as liquid chromatography-tandem mass spectrometry (Freitas et al. 2013) and ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (Kennedy et al. 1998; Martins-Júnior et al. 2007; Stockler et al. 2009), are more efficient to identify and quantify the veterinary drugs in milk rather than liquid chromatography–diode array detector (Mamani et al. 2009). The presence of antibiotic residue in milk may trigger allergic phenomena, such as toxic effects (Stolker and Brinkman 2005) and the development of resistance of microorganisms to antibiotics (Gao et al. 2012), making dairy products a source of risk to the population (Can and Çelik 2012). In the industry, the presence of antibiotics in milk
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causes losses in the processing of yogurt, butter, and cheese due to inhibition of lactic bacteria activity (Knappstein et al. 2003), which makes it necessary to monitor the presence of antibiotic residue in milk (Ilić et al. 2012). Although Brazil is the fifth largest milk producer in the world (33.2 million tons) (FAO 2013), the rate of dairy products for exportation is low. Consequently, improving the milk quality and offering a safer product is important in increasing export. The aim of this study is to qualitatively (Delvotest ® SP-NT) and quantitatively (high-performance liquid chromatography with diode array detector; HPLC-DAD) evaluate the presence of antibiotic residue (oxytetracycline, OTC and tetracycline, TC) in pasteurized milk samples to estimate the average daily ingestion of this residue by humans.
Material and methods Sampling Analyses were performed in duplicate on 21 brands of pasteurized milk. Milk samples (n =252) were collected monthly in commercial establishments from November 2010 to October 2011 in the south and southwest regions of Bahia State, the largest producer in the States. These samples were collected in the commercial market (packaged in 1,000 mL). After collection, the samples were frozen and sent to the laboratory for processing of milk and dairy products at the State University of Southwest Bahia (Universidade Estadual do Sudoeste da Bahia), where they were identified and remained frozen until analysis. Screening test The Delvotest® SP-NT kit (DSM Food Specialties) was used as a screening test, and three experts performed this measurement visually for the final determination. Samples with positive and/or suspect results were also analyzed by HPLC-DAD. Sample preparation for extraction Extractions of TCs were performed according to the validated methodology described by Denobile and Nascimento (2004). Aliquots of 1.0 mL were taken from each milk sample, followed by adding 0.35 mL of a trichloroacetic acid solution in acetonitrile at 80 % (w/v). The mixture was agitated (Ika®, Werkw Gmbh & Co., Germany) for 5 min with subsequent centrifugation for 15 min at 15,000 rpm. After centrifugation, the supernatant was collected and filtered with the aid of a syringe through a Durapore membrane filter (0.45 μm pore), and this was stored in an amber vial under refrigeration until the time of analysis.
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Equipment For identification and quantification of tetracyclines in the samples, a model SPD20A liquid chromatography was used (Shimadzu, Kyoto, Japan) with a quaternary solvent injection valve and a sample loop of 20 μL, column oven, and diode array detector (DAD). The antimicrobial compounds TC and OTC were separated in a C 8 analytical column (15 cm×4 mm di × 5 μm). A solution of 0.01 M oxalic acid, acetonitrile, and 0.1 % triethylamine (70:20:10 v/v/v) was used as the mobile phase at a flow rate of 1 mL min−1, and the temperature of the oven was adjusted to 40 °C. The analysis time was 10 min. The solvents used were chromatographic grade (JC Baker) and were filtered and degassed before use. The chromatograms were processed at 363 nm. Identification of OTC and TC was tentatively performed by comparing the retention time of the peaks with the standard samples, oxytetracycline hydrochloride (95 %) and tetracycline (88 %), both acquired from Sigma-Aldrich® Chemical Co. (St. Louis, MO, USA). Evaluation of the presence of antibiotic residue in the milk samples analyzed was performed by observing the results of screening tests (descriptive analysis) and determining the percentages of positive, suspect, and negative samples expressed in nanograms per milliliter. The general characteristics of HPLC-DAD of the OTC and TC proved to be effective for the identification and quantifi-
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cation by CLAE of these substances in pasteurized milk samples were (1) linearity data showed a positive correlation between the concentrations of tetracyclines (OTC, y=25.2x+ 49.16 and R2 =0.999; TC, y=31.8x+518.8 and R2 =0.999) in the ranges of 100, 200, 400, 800, and 1,600 ng mL−1. The linear correlation coefficients (0.999) found for the curves are in compliance with those set by ANVISA (2003); (2) the coefficients of variation (CV) obtained in the study of intra/ interday precision, of the milk samples added with the standards of OTC and TC, in concentrations of 200, 400 and 800 ng mL−1, respectively, were 1.7, 2.5, and 1.9 (intraday) and 1.7, 3.6, and 4.5 % (interday) for oxytetracycline and 1.7, 2.6, and 1.7 (intraday) and 6.8, 7.5, and 8.1 % (interday) for tetracycline; (3) the detection limits and quantification were of 50 ng mL−1 for OTC and TC, with CV 6.8 and 8.7 %, respectively, and (4) the recovery rate values of tetracyclines in the studied concentrations (100, 200, and 400 ng mL−1) were 64, 64, and 67 % (OTC) and 46, 54, and 61 % (TC) for each concentration, respectively Calculation of the estimated daily intake The concentration was determined using the average content of total tetracyclines observed, and the estimated daily intake (EDI) was calculated according to Juan et al. (2010).
EDI ¼ ½ðmean in μg of the antibiotic residue per Kg of foodÞ ðMean consumption of the foodÞ Live adult weight of 60 Kg
For determination of the EDI, the estimated milk consumption values used were for Brazil in 2012 (441 mL/inhabitant/ day), and this was compared to the recommended consumption by the Brazilian Ministry of Health, which is 575 mL/ inhabitant/day (Embrapa 2013). This evaluated the impact of increased milk consumption on the estimate.
Results Among the 252 pasteurized milk samples analyzed by the Delvotest® SP-NT in this study, 207 (82.0 %) were negative (no antibiotic residue), and 20 presented positive results, revealing a contamination frequency of 8.0 %. On the other hand, 25 samples (10 %) were defined as suspect. In most of the milk samples studied, the presence of antibiotic residue (AM) was not detected by the screening test. Distribution of the results throughout the year indicated that there was a period in which the number of positive and
suspected samples was greater. Of the 20 positive milk samples, 13 (65 %) were detected between January and June. The total number of suspected samples observed during the year was 25, where 22 (91.7 %) were obtained between January and June (Fig. 1). These results indicate that season likely had an effect on the occurrence of positive and suspected milk samples, as was observed by Kaya and Filazi (2010). Of the 21 pasteurized milk brands, only four (D, E, P, and T) showed no positive results over the year of study. All other brands (17) tested positive or suspect at some point (Fig. 2). Brand N showed the highest number of samples with positive (25 %) and suspect (25 %) results, whereas L and O both presented 17 % positive samples and 8 % suspect samples, respectively. For brands B, C, and M, 17, 17, and 25 % of the samples were reported as positive, respectively. Brands A, F, H, Q, S, and V presented positive samples (8.3 % each) with variations in the percentage of suspect samples. However, brands G, I, J, R, and U exhibited only suspect samples (25, 17, 8, 16.7, and 17 %, respectively).
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21 19
Dairy industry numbers (n = 21)
Fig. 1 Monthly distribution of negative, positive, and suspected pasteurized milk samples by screening test in dairy industry (Nov. 2010 to Oct. 2011)
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The positive and suspect samples discovered during the screening test were subjected to HPLC-DAD for confirmation of the presence of tetracyclines. In samples with positive results (n =19), the highest concentration observed individually for OTC was 2,782 ng mL−1 (Fig. 3) in sample I-Jan. In the same sample, the concentration of TC was 1,145 ng mL−1. The lowest concentrations above the maximum residue limit (MRL=100 ng mL−1) were observed in samples F-Apr (125 ng mL−1) for OTC and sample S-Mar (107 ng mL−1) for TC. In 12 samples, no TC residue was detected, and in one (P-Sept), the TC concentration was below the MRL (73 ng mL−1), in which no false positive was detected. In suspect samples, the lowest OTC concentration was observed in sample U-Mar (67 ng mL−1), and the highest was 2,072 ng mL−1 (S-Feb). With respect to TC, the highest was 2,297 ng mL−1 (R-Feb), and the lowest was 78 ng mL−1
1 May
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(N-Mar). In 16 samples, the presence of TC was not detected; only 1 sample (N-mar) showed no OTC (Fig. 4). The antibiotic concentrations observed in most samples with positive or suspect results exceed the MRL permitted for tetracyclines (100 ng mL−1, singly or in combination). Of the 19 positive samples in the screening tests, all presented OTC concentrations above the MRL (Table 1); in six, the TC concentration was excessive; and in one, it was below the legal limit. In 12 samples, TC was not detected. Among the samples considered suspect in the screening test, in 21, the OTC concentration was greater than that permitted by legislation; in 2, the concentration was lower; and in 1, no OTC was detected. TC was not detected in 16 samples; only in 1 did it not exceed the MRL; and in 7, it exceeded the MRL. The “total tetracycline concentration” in suspect samples was below the MRL in only three samples.
Sample percentage
Fig. 2 Percentage of negative, positive, and suspect pasteurized milk samples by screening test per dairy industry (Nov. 2010 to Oct. 2011)
Dairy industry
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2800
OTC
2600
TC 2354
2400
Concentração em ng.mL-1
Fig. 3 Antibiotics residues concentration (OTC oxytetracycline, TC tetracycline) by HPLC-DAD in the positives milk samples in the screening test (Delvotest® SP-NT). MRL maximum residue limit=100 ng mL−1
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2200 1926
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Dairy industry-month
The EDI was based on the average concentration of tetracycline residue in milk, per dairy facility, observed in this study. Average daily milk consumption in Brazil in 2011 was 441 mL/inhabitant/day, but the desired average milk consumption is 575 mL/inhabitant/day (Embrapa 2013). When evaluating the EDI of antibiotics (tetracycline) by means of Fig. 4 Concentration (ng/mL−1) of antibiotics residues (OTC oxytetracycline, TC tetracycline) by HPLC-DAD in the suspects milk samples by screening test (Delvotest® SP-NT). MRL maximum residue limit=100 ng mL−1
the actual milk consumption in Brazil (Fig. 5), it was observed that of the 16 brands, in 9, the EDI was in accordance with that recommended by the European Union (EMEA 1995), with variations between 1.3 and 3 μg/kg BW/day. In the other 7 brands, these estimates varied between 3.2 and 8.8 μg/kg BW/ day, where the greatest value was observed in brand I. The
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Table 1 Number of positive and suspect pasteurized milk samples sold according to the screening test for antibiotic residues, confirmed by HPLC-DAD MRL/milk
Greater than Less than Not detected
Positive (n =19)
milk). Bando et al. (2009), using immunoassay, found that 41.5 % of samples were positive. Freitas et al. (2013) found 20 % (LC-IT-TOF MS/QuEChERS), and Martins-Júnior et al. (2007) reported 8 % (LC-ESI/MS/MS). In our study, all positive samples and most suspect samples (87.5 %) exceeded the MRL (Table 1). Screening tests are quick and easy to use, but they are subjective, so we need more precise and technical measures, such as liquid chromatography, to find the dynamic of the contamination of antibiotics in dairy cattle in Brazil. It is necessary improve the sampling number and submit it to liquid chromatography. The Delvotest SP-NT has low sensitivity to OTC (Le Breton et al. 2007), but in this study, it was effective even at low concentrations because it detected even a minimal presence of antibiotic residue in milk (67 ng mL−1) (Fig. 4), showing its sensitivity to the analyte (Hennart and Faragher 2012; Sierra et al. 2009). Rainfall in the studied area is concentrated during the summer season, from December to March (Moscati and Gan 2007). The high humidity could facilitate the development of microorganisms that cause mastitis, increasing its incidence. This higher mastitis incidence may increase the detection of antibiotic residue (Fig. 1) because mastitis is the most common cause of antibiotic use in lactating cows (Bansal et al. 2011). Milk production systems in the region involve minimal technology and milking hygiene measures are of little use, making these environments fertile for mastitis. Our results indicate that within the class of tetracyclines, OTC is most commonly used among the dairy products of the region studied as well as in other countries (Schlegelova et al. 2002).
Suspect (n =24)
OTC
TC
TTC
OTC
TC
TTC
19 – –
06 01 12
19 – –
21 02 01
07 01 16
21 03 –
OTC oxytetracycline, TC tetracycline, TTC total tetracyclines
simulation that utilized values of the recommended milk consumption of 575 mL/inhabitant/day indicated that only 3 of the 16 brands met the maximum recommended by the European Union for EDI of antibiotics (3 μg/kg BW/day), with a variation between 1.7 and 2.9 μg/kg BW/day. In 13, the EDI exceeded the recommendations (3.3 to 11.5 μg/kg BW/ day).
Discussion Sampling from large volumes can lack analytical precision (dilution effect) (FAO 2000), especially during screening tests. In our study, the samples were collected from large volume industries, but the percentage of positive samples was still close to 20 %. Distinct percentages of pasteurized milk samples positive for antibiotics after a screening test were reported in Brazil. Oliveira et al. (2012) observed no antibiotic residue in milk samples (raw and pasteurized) evaluated. Fonseca et al. (2009) found 0–4 % noncompliance of samples evaluated (UHT
BR actual (441 mL/inhabitant/day)
12.0
11.5
BR/MS (575 mL/inhabitant/day) 10.4
EDI (µg/Kg Body Weight/day)
Fig. 5 Estimated daily intake EDI simulation (μg/kg body weight/day) of the antimicrobial residue (tetracyclines) in milk. Recommendation of the EMEA (1995) of average daily consumption of tetracyclines=3 μg/ kg BW/day
8.8
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The results obtained in this study indicate the need for greater monitoring and control by health surveillance agencies of the production and consumption of milk. One of the major causes of antibiotic residue in milk is animal treatment with antibiotics; such animals should be milked separately for a specified period. These substances may be excreted with the milk even after finalizing treatment, making it necessary that such milk be discarded during this period, which may vary according to the recommendations of the manufacturer (Knappstein et al. 2003; Stockler et al. 2009; Bansal et al. 2011). In addition, it is important for quality control at the dairies to reduce the risks to public health (e.g., toxic effects and development of resistance to pathogenic microorganisms in humans) (Gao et al. 2012; Can and Çelik 2012) or to animals. Soil contamination is also a problem (Carvalho et al. 2013). In the dairy industry, antibiotics inhibit lactic culture and affect the quality and identity of cheeses by altering their composition and structure (Knappstein et al. 2003). Adjustments to the Brazilian milk production chain are imperative; a new development model is fundamental and should be supported by better quality and safety of products (Farina et al. 2005; Ilić et al. 2012). The consumer contamination risk is in a direct relationship with the danger of the chemical product and exposure to the same (risk = hazard × exposure) (Davies et al. 2003). Antibiotic residue in milk products presents a danger to the consumer, so these results indicate that the inappropriate use of antibiotics in dairy farming (high dosages, not observing the grace periods) may pose health risks to the public as well as lead to economic losses in the dairy industry.
Conclusions The results obtained indicate the presence of antibiotic residue above the legal limits within the class of tetracyclines. OTCs are the most commonly found in dairy products in the region studied. Further studies are required as follows: (1) antimicrobials resistance, (2) epidemiological status, (3) effects on the ground water, especially near the dairy industry, and (4) screening tests followed by HPLC with more sensitive detectors, such as mass spectrometry. It is recommended that training and guidance, particularly on the use of proper practices of hygiene during milking, be offered to the owners and their workers involved in the dairy industry. Acknowledgments This research was funded by Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB) and Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (Capes) for the support to the research. Conflict of interest None of the author has any support or relationship with other people that could inappropriately influence.
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