A simple and economic method for simultaneous

A simple and economic method for simultaneous determination of 11 antibiotics in manure by solid-phase extraction and high-performance liquid chromatography Yao Feng, Chaojun Wei, Wenjuan Zhang, Yuanwang Liu, Zhaojun Li, Haiyan Hu, Jianming Xue & Murray Davis Journal of Soils and Sediments ISSN 1439-0108 J Soils Sediments DOI 10.1007/s11368-016-1414-5

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Author's personal copy J Soils Sediments DOI 10.1007/s11368-016-1414-5

SOILS, SEC 3 • REMEDIATION AND MANAGEMENT OF CONTAMINATED OR DEGRADED LANDS • RESEARCH ARTICLE

A simple and economic method for simultaneous determination of 11 antibiotics in manure by solid-phase extraction and high-performance liquid chromatography Yao Feng 1 & Chaojun Wei 2 & Wenjuan Zhang 1,3 & Yuanwang Liu 1 & Zhaojun Li 1 & Haiyan Hu 1 & Jianming Xue 4 & Murray Davis 5

Received: 25 December 2015 / Accepted: 22 March 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract Purpose A simple and highly efficient economic method for the analysis of 11 antibacterial drugs including two tetracyclines, three quinolones, four sulfonamides, chloramphenicol and tylosin, in livestock manure, was developed using solidphase extraction (SPE) and high-performance liquid chromatography (HPLC). Materials and methods The analytes were successively extracted by EDTA-McIlvaine solution and organic solvent mixture. The extracts were degreased with n-hexane and cleaned through SPE on a hydrophile-lipophile balance (HLB) cartridge. All compounds were determined on a C18 reverse phase column with gradient elution. Results and discussion Recoveries calculated from spiked samples of animal manures ranged from 62.65 to 99.16 % for 11 antibiotics with relative standard deviations of less than Responsible editor: Jan Schwarzbauer Yao Feng, Chaojun Wei and Wenjuan Zhang contributed equally to this work. * Zhaojun Li [email protected]

1

Key Laboratory of Plant Nutrition and Fertilizer, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, Peoples Republic China

2

Key Laboratory of Urban Agricultural (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, Peoples Republic China

3

College of Urban and Environment Sciences, Shanxi Normal University, Linfen, Shanxi 041000, Peoples Republic China

4

Scion, Christchurch 29-237, New Zealand

5

Rangiora, New Zealand

10.0 %. Limits of detection ranged from 0.1 to 1.9 μg kg−1, and limits of quantification ranged from 0.3 to 5.9 μg kg−1. Conclusions The results show that SPE-HPLC is an inexpensive and practical method for rapid detection of multiple antibiotics in animal manure. Keywords Antibiotics . Determination . High-performance liquid chromatography . Manures . Solid-phase extraction

1 Introduction Antibiotics, a group of chemicals of environmental concern, are widely used in human medicine and veterinary applications (Marcela et al. 2009). In the latter field, they are given to disease-free animals for disease prevention and growth promotion (Martínez-Carballo et al. 2007; Gbylik-Sikorska et al. 2013, 2015; Kemper 2008). One study estimates that approximately 2300 tons of antibiotics are consumed in veterinary medicine in European countries (Hirsch et al. 1999). In China, 52 % of all antibiotics (approximately 162,000 tons) were used for veterinary medicine in 2013 (Zhang et al. 2015). Antibiotics are not completely absorbed by the animal body, with most being excreted in urine or faeces, either unaltered or as metabolites (Hartmann et al. 1998). Early researchers have found antibiotic residues in water and soil environments (Chitescu et al. 2012; Hu et al. 2010; Braschi et al. 2013; Li et al. 2013; Sun et al. 2013), which may be due to the frequent application of excrement containing antibiotic residues to agricultural fields (Pino et al. 2015). Antibiotic residues may cause serious environmental problems and human health damage. It has been found that antibiotic residues have negative impacts on non-target organisms such as wheat and maize (Li et al. 2011a, b; Zhao et al. 2013). As the main route by which antibiotics enter soil and water

Author's personal copy J Soils Sediments

environments, antibiotic residues in animal manures have received increasing attention, especially in regard to their determination (Ye et al. 2006). Although methods have been developed for the determination of multiple antibiotic residues in samples, most have focused on detection of residues in liquid matrices such as milk, honey, sewage water and animal urine, and only a few have been developed for use in solid matrices such as soils and manures (Table 1). Manure contains more complex components than some liquid matrices such as milk, honey, urine or some sewage waters, and simultaneous extraction of antibiotic residues from manure is therefore more difficult than from liquid samples. A method for simultaneous determination of 11 antibiotics in manure has been developed with detection limits ranging from 2.7 to 32.1 μg kg−1; however, it uses high-cost liquid chromatography tandem mass spectrometry equipment (Jacobsen and Halling-Sorensen 2006) (Table 1), which can lead to high determination costs. An efficient low-cost method for simultaneous determination of multiple antibiotics in manure is lacking. The present study focuses on development of a rapid method for the simultaneous determination of 11 antibiotics (Table 1) in animal manure, using relatively low-cost solid-

Table 1

phase extraction (SPE) followed by determination using highperformance liquid chromatography (HPLC). Different extracting agents and solvents for sample extraction and several SPE cartridges for clean-up were compared. The method was applied to manure samples from different large-scale livestock and poultry farms. We believe this method is simpler, more effective and has lower costs than existing methods and also gives low limits of detection and quantification.

2 Materials and methods 2.1 Chemicals and standards Oxytetracycline (OTC), chlortetracycline (CTC), norfloxacin (NOR), ciprofloxacin (CIP), enrofloxacin (ENR), sulfathiazole (ST), sulfamethazine (SDMe), sulfamethoxazole (SMZ), chloramphenicol (CAP) and tylosin (TYL) (Table 2) were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). One further antibiotic, sulfamonomethoxine (SMN), was purchased from Sigma-Aldrich (Israel). Methanol (MeOH), acetonitrile (ACN) and n-hexane were

Comparison of existing methods for determination of antibiotic residues in different matrix media

Matrix media

Methods

Total number of different types of antibiotics determined

Detection limits

References

Soils

PLE-SPE-LC-MS/MS

4 (tetracyclines) 5 (tetracyclines, macrolides and sulfonamides) 8 (macrolides, ionophores and tiamulin)

1–3 μg kg−1 0.6–5.6 μg kg−1 0.2–1.6 μg kg−1

Andreu et al. 2009 Jacobsen et al. 2004 Schlüsener et al. 2003

LLE–HPLC

3 (tetracyclines)

8–15 μg kg−1

LLE-LTP–LC-MS/MS CZE-MS/MS SPE-UPLC–MS/MS

1 (chloramphenicol) 8 (quinolones) 38 (beta-lactams, sulfonamides, quinolones, tetracyclines, macrolides and lincosamide) 104 (aminoglycosides, endectocides, fluoroquinolones, ionophores, β-Lactams, macrolides, NSAIDs, phenicols, sulfonamides and tetracyclines) 43 (sulfonamides, tetracyclines, macrolides, aminoglycosides, beta-lactams, amphenicols and flumequine) 11 (tetracyclines, sulfonamides and tylosin)

0.015 μg kg−1 4.0–6.0 μg kg−1 0.1–5.0 μg kg−1

Li et al. 2010 Rego et al. 2015 Lara et al. 2006 Han et al. 2015

–

Wang and Leung 2012

0.1–80 μg kg−1

Kaufmann et al. 2002; Hammel et al. 2008

2.7–32.1 μg kg−1

3 (tetracyclines, quinolones and sulfadimidine) 2 (oxytetracycline and tylosin) 5 (florfenicol, lincomycin, oxytetracycline, tylosin and valnemulin) 16 (fluoroquinolones, sulfonamides, trimethoprim, beta-lactams, nitroimidazoles and tetracyclines) 1 (amoxicillin)

0.04–0.25 mg kg−1 10 μg kg−1 0.01–0.1 μg L−1

Jacobsen and Halling-Sorensen 2006 Wang et al. 2013 Marco et al. 2003 Tagiri-Endo et al. 2009

0.1–3.6 μg L−1

Lindberg et al. 2004

2.0 μg mL−1

Beltran et al. 2008

Milk or honey

SPE-UHPLC QqTOF MS

SPE-LC-MS/MS

Manure

LLE-SPE-LC-MS/MS

Sewage water

SPE-HPLC-MS/MS HPLC SPE-LC–MS/MS

Animal urine

SPE-MIP

PLE pressurized liquid extraction, SPE solid-phase extraction, LC-MS/MS liquid chromatography tandem mass spectrometry, LLE liquid-liquid extraction, HPLC high-performance liquid chromatography, LTP low-temperature partitioning, CZE capillary zone electrophoresis, MS mass spectrometry, UPLC ultra-high-performance liquid chromatography, UHPLC QqTOF MS ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry, MIP molecularly imprinted polymer

Author's personal copy J Soils Sediments Table 2

Properties and primary usage of selected antibiotics in this study

Class

Antibiotic

Acronym

Tetracyclines (TCs)

Oxytetracycline

OTC

Structure OH

N

HO

pKa

LogKow

Primary usage

3.3/7.3/9.1

− 0.90, − 1.22

Human

OH

Cow, sheep, pig, chicken

NH2

Chlortetracycline

O

O

OH

O

Norfloxacin

OH

HO

O

OH

O

OH

CTC

NH2 O

3.3/7.4/9.3

− 0.62, – 0.36

Human Cow, sheep, pig, chicken

OH

H

H

HO

Cl

Fluoroquinolones (FQs)

OH

N

O

NOR

OH

F

6.22/8.51

− 1.0, –1.7

Human

O

fish, cow, sheep, pig, chicken N

N

N

N

NH

Ciprofloxacin

CIP

6.43/8.49

NH

0.28

Human fish, cow, sheep, pig, chicken

OH F O

O

Enrofloxacin

ENR

6.27/8.3

N N

1.1

N

Human fish, cow, sheep, pig

OH F O

O

Sulfonamides (SAs)

Sulfathiazole

O

ST H2N

2.65/7.65

0.26

O H N

H3C N

Sulfamonomethoxine

S

SMN

Fish, cow, sheep, pig, chicken

NH2

N

H N

S

NH2

6.05

0.18

Human Fish, cow, sheep, pig, chicken

O

N

O

SMZ

Human

O

N

Sulfamethoxazole

Human Fish, cow, sheep, pig, chicken

S

CH3

SDMe

0.02

NH

O

Sulfamethazine

7.10

N

S

NH2

1.4/5.8

0.89

Human

NH S O

Chloramphenicols

Chloramphenicol

Fish, cow, sheep, pig, chicken

O

OH

CAP

9.5

1.14

Cow, chicken, shrimp

7.1

3.5

Cow, sheep, pig, chicken

OH O

O

HN N O

Cl

Cl

O

Macrolides (MAs)

Tylosin

TYL

O

O O

OH

OH O

O

N

O O O

OH

O

O O

OH HO

all HPLC grade from Fisher (Geel, Belgium). All other chemicals such as acetone, disodium hydrogen orthophosphate anhydrous (Na 2 HPO 4 ), EDTA disodium salt (Na 2 EDTA), citric acid, formic acid, phosphoric acid (H3PO4) and sodium hydroxide (NaOH) were analytical grade

from XL (Shantou, Guangdong, China). Deionized water (DI) used in the experiments was prepared with a Milli-Q plus water system (Millipore, Billerica, MA, USA). The extract buffer (EDTA-McIlvaine buffer) was prepared following Storey et al. (2014). MeOH, ACN and acetone were mixed


(2:2:1 by volume) to form an organic extractant which was adjusted to pH 4.0 with H3PO4. Each of the primary standards was accurately weighed (10 mg) into individual 10-mL amber volumetric flasks, dissolved and made up to the mark with MeOH, to give individual stock standard solutions in MeOH with the concentration of 1 mg mL−1. To obtain working standard solutions, 1-, 5-, 10-, 50-, 100- and 500-μL aliquots of the individual stock standard solutions were added into individual 10-mL brown volumetric flasks. All solutions were stored at 4 °C.

1 g lyophilized manure EDTA-Mcllvaine buffer pH=4 (2 × 10 mL) MeOH-ACN-Acetone (40+40+20, v/v/v) (2 × 5 mL) Extraction: Homogenization for 30 s Ultrasound for 15 min Centrifugation for 15 min

Degreased with n-hexane (2 × 5 mL) Filtration with 0.45 µm filters Rotary evaporator (70 rpm, 40 °C) Concentration to 5 mL

2.2 Manure samples For method optimization, manure samples were collected from a pig eco-farm which did not use antibiotics in Shanxi province. For method proof, cow, pig and chicken manures were each collected from five large-scale animal farms which used antibiotics near Beijing in July 2014. All samples were put into dark plastic bags and kept in a cooler with ice until transported to the laboratory where all samples were stored at −80 °C before analysis.

SPE-HLB cartridge

Pretreated with: - 5 mL MeOH - 10 mL H2O

Elution:

5 mL MeOH-H2O (25+75, v/v)

10 mL MeOH-H2O (65+35, v/v)

Evaporation to dryness

Re-dissolved to 1 mL of ACN: H2O (20:80, v/v) Filtration with 0.22 µm filters

Injection into HPLC

2.3 Sample preparation During method validation and optimization, different extract agents and SPE cartridges were tested for analysis of the target antibiotics in animal manure. To determine antibiotic recoveries, final concentrations of 5.0, 10.0 and 50.0 μg g−1 in manure were obtained by adding 1.0 mL of mixed antibiotic methanol solution, with the above respective concentrations, to 1.0 g of lyophilized manure sample. Each sample was mixed well and placed in the fume hood at room temperature for 24 h for complete removal of the methanol by evaporation and for interaction of the analytes with the matrix in order to approximate real conditions. Each sample was extracted and analysed in triplicate (Li et al. 2005; Elena et al. 2007; Huang et al. 2012). 2.4 Sample extraction and clean-up In the present study, the manure samples were lyophilized using a vacuum freeze drier and sieved through a 2-mm sieve before further handling. Multiple extracting agents were used and extraction was assisted with ultrasound. Clean-up steps were performed using solid-phase extraction. The extraction scheme used to extract the target compounds is illustrated in Fig. 1. 2.5 Liquid chromatography spectrometry analysis The analysis was performed using a Waters Alliance 2695 HPLC system (Waters, Milford, USA) with a Waters 2998 Photo-Diode Array (PDA) detector. The chromatographic

Fig. 1 Procedure used for extraction of antibiotics. Optimized extraction, clean-up and elution procedures developed in the present study are given

separation was carried out with the use of a Waters Atlantis® T3 column (150 × 4.6 mm, 3 μm) at 40 °C (Liu et al. 2012). The flow rate was maintained at 1.0 mL min−1, the injection volume was 10 μL and the detection wavelength was 274 nm (Hu et al. 2008). The mobile phases A and B were 0.1 % formic acid in water (A) and acetonitrile (B), respectively. The mobile phase gradient elution procedure took 35 min, as follows: 0–20 min, 90–80 % A, 10–20 % B; 20–24 min, 80 % A, 20 % B; 24–25 min, 80–40 % A, 20–60 % B; 25–31 min, 40 % A, 60 % B; 31–32 min, 40–90 % A, 60–10 % B; 32– 35 min, 90 % A, 10 % B. A standard HPLC chromatogram (Fig. 2) was used to determine the 11 antibiotics in the present study. In order to guarantee the reliability of the results obtained for the selected antibiotics by HPLC, some samples were further measured by LC-MS/MS. The results obtained using LCMS/MS were the same as those from HPLC, indicating that the data obtained with HPLC were reliable. 2.6 Detection of antibiotics in manure Approximately 1.0 g of manure sample was accurately weighed into a 50-mL centrifuge tube and 10 mL of EDTAMcIlvaine buffer was added. The tubes were vortexed (Berlin Wiggens, Germany) for 30 s, sonicated (Ningbo Scientz, China) at 4 °C for 15 min and then centrifuged (Sartorius Sigma, Germany) at 6000 g for 15 min at 4 °C. The


Fig. 2 The standard HPLC chromatogram of 11 antibiotics assayed in the study. Antibiotic abbreviations: ST sulfathiazole, CIP ciprofloxacin, OTC oxytetracycline, NOR norfloxacin, SDMe sulfamethazine, ENR enrofloxacin, SMN sulfamonomethoxine, SMZ sulfamethoxazole, CTC chlortetracycline, CAP chloramphenicol, TYL tylosin

supernatant was then decanted into a 50-mL glass bottle. The pellets were repeatedly extracted once with the same procedure using 10 mL of McIlvaine buffer and twice using 5 mL of a solution with MeOH, ACN and acetone (with the volume ratio of 2:2:1). Approximately 30 mL total of each supernatant was then degreased with 10 mL of n-hexane and passed through a 0.45-μm PVDF syringe filter (Tianjin Jinteng, China) into a round-bottom flask. The extracts were concentrated to approximately 5 mL on a rotary evaporator (70 rpm, 40 °C) (Hydrographic Guelph, Germany), and the liquid was purified and concentrated using Oasis HLB (6 cm3, 500 mg) cartridges from Waters (Millford, MA), which were preconditioned with 5 mL of methanol and 10 mL of DI water. The analytes were passed through the cartridges at a flow rate of 1 mL min−1. After isolation, cartridges were rinsed with 5 mL of MeOH with DI water (25:75, v/v) and dried under vacuum for 5 min. The analytes were eluted using 10 mL of MeOH with DI water (65:35, v/v). The eluates were then evaporated to near dryness at 40 °C and redissolved in 1 mL of ACN with ultrapure water (20:80, v/v) for HPLC analysis (Fig. 1). Where concentrations of antibiotics exceeded the chromatogram peak heights (Fig. 2), samples were further diluted as required.

target antibiotics from pig manure varied with extracting agent (Fig. 3). Recoveries using extracting agent M1 ranged from 21 to 61 % with relative standard deviations (RSDs) of between 5 and 12 %. Extracting agent M2 gave wider recoveries which ranged from 17 to 81 % with RSDs of between 5 and 11 %. Tetracyclines produce strong fluorescence with metal ions or under basic conditions (Mitscher 1978; Kohn 1961). They form chelate complexes with metal ions and bind with proteins in the stationary phase (Zia and Price 1976; Oka et al. 2000). EDTA is chosen as a chelating agent as it competes strongly for metal ions. McIlvaine buffer effectively reduces bonding of tetracyclines with proteins (Liu et al. 2007). The extracting agent M3 could more thoroughly extract antibiotics from manure; however, it could also simultaneously extract large amounts of impurities which impact negatively on the extraction of the target antibiotics. The combined extracting agent (M4) was much stronger than the single extracting agents (M2 and M3) for extraction of antibiotics from the manure. The mixture (M4) was the best of all the extracting agents and could simultaneously extract the 11 target antibiotics from animal manure with exceptionally good recoveries ranging from 48 to 99 %, with RSDs of between 4 and 13 %. 3.2 Optimization of SPE cartridges Selective adsorption is the basis for ensuring efficient extraction of antibiotics from manures (Chen et al. 2014). Three kinds of SPE cartridges including Oasis HLB (Waters, US), C18 (Agela, US) and NH2 (Agela, US) were used to get efficient clean-up of the sample. Both the C18 and NH2 cartridges had lower recoveries than the HLB cartridge (Fig. 4). The recoveries ranged from 5.2 to 71.7 % and from 3.7 to 59.2 % for the C18 and NH2 cartridges respectively, while those for the HLB cartridge ranged from 38.3 to 97.2 %. The difference may be due to both the C18 and NH2 cartridges having high selectivity of the target compounds. The HLB cartridge contained lipotropic divinyl benzene and hydrophilic N-vinyl pyrrolidone as the adsorbents in contrast to the C18 and NH2 cartridges, which contained silica gel. The HLB cartridge has higher adsorption capacity for antibiotics with high polarity (Guo et al. 2014).

3 Results and discussion 3.3 Eluent optimization 3.1 Validation of extracting agents A solution with MeOH, acetic acid and water with the volume ratio of 6:3:1 (M1), EDTA-McIlvaine buffer with the concentration of 0.1 mol L−1 (M2), a solution with MeOH, ACN, and acetone with the volume ratio of 2:2:1 (M3), and the group (M4) of M2 and M3 were chosen as the extracting agents. The recoveries of the

To evaluate appropriate eluent strength, 1.0 g of lyophilized control samples was spiked with 10 μg mL−1 of all the selected antibiotics (resulting in a concentration of 10 μg g−1 in manure for each target antibiotic). Antibiotics were then extracted as described in section 2.6. After the concentrated sample extracts were passed through the HLB cartridges, the analytes were eluted using 10 mL of 15 %, 25 %, 35 %, 45 %,

Author's personal copy J Soils Sediments 140

Fig. 3 Recoveries of 11 antibiotics from spiked manures with four different extraction agents. Bars show residual standard deviations. See Fig. 2 for antibiotic abbreviations

M1: MeOH+acetic acid+water (6:3:1, v/v/v) M2: EDTA-McIlvaine buffer 0.1M pH=4 M3: MeOH+ACN+acetone (2:2:1, v/v/v) M4: EDTA-McIlvaine buffer and MeOH+ACN+acetone (2:2:1, v/v/v)

120

Recoveries (%)

100

80

60

40

20

TY L

CA P

CT C

SM Z

SM N

EN R

N OR SD M e

OT C

CI P

ST

0

Substances

55 %, 65 %, 75 % and 85 % of MeOH in DI water, respectively. Recoveries of the 11 target compounds in the eluents are shown in Table 3. Low concentrations (15 % and 25 % of MeOH in DI water) of eluent failed to recover the target antibiotics. When the proportion of MeOH was 35 % or greater,

120

Fig. 4 Recovery of 11 antibiotics from spiked manure with three types of SPE cartridge. Bars show residual standard deviations. See Fig. 2 for antibiotic abbreviations

the recoveries of eluted target antibiotics increased with the increasing proportion of MeOH up to 65 %, when recoveries generally either levelled off or declined. Recoveries of three antibiotics (CTC, ENR and SMN) were marginally greater (0.9, 2.9 and 1.0 % respectively) at higher concentrations of

HLB C18 NH2

100

60

40

20

Substances

TY L

CA P

CT C

SM Z

SM N

EN R

N OR SD M e

OT C

CI P

0

ST

Recoveries (%)

80


MeOH. Large amounts of impurities were also eluted by the aqueous MeOH when its proportion was 75 % or more. These impurities had negative impacts on the isolation of target antibiotics. Therefore, cartridges were rinsed with 25 % of MeOH in DI water and the analytes were eluted using 65 % of MeOH in DI water.

(Blackwell et al. 2004). Although Schlüsener et al. (2003) found low LOD and LOQ values with similar antibiotic concentrations using liquid chromatography tandem-mass spectrometry, the method developed in the present study is recommended because of the low economic and time costs.

3.5 Analysis of samples from animals from farms 3.4 Method efficiency and practicability In order to test the efficiency of the method, 1.0 g of lyophilized samples of pig manure were spiked with different concentrations of the selected antibiotics (5, 10 and 50 μg g−1), and the antibiotics were then determined in these samples following the protocol described in step 2.6. The limits of detection (LOD) and the limits of quantification (LOQ) were calculated as three times and ten times the standard deviation of the measurement of control samples divided by the slope of the calibration curve, respectively. Recoveries, RSDs, LODs and LOQs are shown in Table 4. On average, antibiotic recoveries increased with the amount of antibiotics added to the manure. Recovery rates when the antibiotics were added at 5, 10 and 50 μg g−1 were 62.7–90.4 %, 72.4–97.1 % and 74.1–99.2 %, respectively. The recovery RSDs were not more than 10 %, which are similar to the results of Wang et al. (2013) for seven antibiotics simultaneously determined. It is noted that the present method gave very low LOD and LOQ values, with values ranging from 0.1 to 1.9 μg kg−1 and from 0.3 to 5.9 μg kg−1, respectively. These data were substantially lower than those (70 to 140 μg L−1) obtained using SPE cleanup and HPLC–UV analysis of the three veterinary antibiotics (OTC, TYL and sulfachloropyridazine) in pig slurry

Table 3

Once the analytical methodology was validated, 15 different animal manures were analysed by the optimum procedures as described in section 2.6. The concentrations varied greatly amongst different classes of antibiotics (Table 5). Tetracyclines were the predominant antibiotics in the three different animal manures and on average contributed 98.4 % of the total concentration of antibiotics. In contrast, quinolones, sulphonamides, CAP and TYL contributed little to the total antibiotic concentrations (0.6, 0.7, 0.4, and