The fate, occurrence and extraction of antibiotics in solid matrices
Jamie Harrower
Glasgow Caledonian University/James Hutton Institute
[email protected]
ECG Bulletin January 2020
Glasgow Caledonian University/James Hutton Institute
[email protected]
ECG Bulletin January 2020
Advances in analysis and sample preparation have allowed the detection of antibiotics in surface water in the range ngL-1 to µgL-1 (1). This has drawn interest due to current concerns surrounding antimicrobial resistance (AMR) and the occurrence of antibiotic resistance genes (ARGs) in the aquatic environment (2).
Overview of antibiotics
The discovery of antibiotics is considered one of the most significant scientific achievements of the 20th century. Antibiotics can be broadly divided into two different groups: bactericidal antibiotics (beta-lactams, cephalosporins, fluoroquinolones and sulfonamides) which destroy the bacteria directly, and bacteriostatic antibiotics (macrolides and tetracyclines) which prevent bacteria from dividing and multiplying (3).
The misuse and over prescription of antibiotics has aided the emergence of bacteria carrying ARGs. Scientific evidence suggests that many factors play a role in the development and spread of antibiotic resistance within the environment, such as antibiotics and ARGs accumulating within wastewater treatment plants (WWTPs) (2), the release of biocides, use of antibiotics in agriculture, and the direct animal to human transmission of resistant bacteria (4).
The World Health Organisation (WHO) and its Global Action Plan broadly outlines five strategic objectives to tackle AMR, which can be described as improving awareness and understanding of AMR, strengthening knowledge through surveillance and research, reducing the incidence of infection, optimising the use of antimicrobial agents, and ensuring sustainable investment in combating AMR (5).
Sources and fate in the environment
Antibiotics are only partially metabolised within the human body, and the fraction that is excreted by humans will enter the WWTP and follow one of three fates;
The physicochemical properties of antibiotics heavily influence their chemical fate and behaviour at different aqueous pHs and within differing soil compositions. Metabolites, in turn, are able to partition and migrate into other phases in the environment, making it challenging to predict their behaviour and chemical fate (13).
The discovery of antibiotics is considered one of the most significant scientific achievements of the 20th century. Antibiotics can be broadly divided into two different groups: bactericidal antibiotics (beta-lactams, cephalosporins, fluoroquinolones and sulfonamides) which destroy the bacteria directly, and bacteriostatic antibiotics (macrolides and tetracyclines) which prevent bacteria from dividing and multiplying (3).
The misuse and over prescription of antibiotics has aided the emergence of bacteria carrying ARGs. Scientific evidence suggests that many factors play a role in the development and spread of antibiotic resistance within the environment, such as antibiotics and ARGs accumulating within wastewater treatment plants (WWTPs) (2), the release of biocides, use of antibiotics in agriculture, and the direct animal to human transmission of resistant bacteria (4).
The World Health Organisation (WHO) and its Global Action Plan broadly outlines five strategic objectives to tackle AMR, which can be described as improving awareness and understanding of AMR, strengthening knowledge through surveillance and research, reducing the incidence of infection, optimising the use of antimicrobial agents, and ensuring sustainable investment in combating AMR (5).
Sources and fate in the environment
Antibiotics are only partially metabolised within the human body, and the fraction that is excreted by humans will enter the WWTP and follow one of three fates;
- biodegradation (2, 6),
- adsorption onto sewage sludge (7, 8),
- exit the effluent as the unchanged drug (9, 10).
The physicochemical properties of antibiotics heavily influence their chemical fate and behaviour at different aqueous pHs and within differing soil compositions. Metabolites, in turn, are able to partition and migrate into other phases in the environment, making it challenging to predict their behaviour and chemical fate (13).
Antibiotics usually have more than one acid dissociation constant (Ka) and can therefore form ionised and zwitterion structures (14). Unlike other hydrophobic contaminants, such as the polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs) or pesticides such as dichlorodiphenyltrichloroethane (DDT), the value logKow (log octanol/water partition coefficient) is not enough to measure chemical fate and distribution of antibiotics. It needs to be modified to take into account the ionised fraction. Even this approach may not be sufficient as the neutral species are highly polar. A study conducted by Tolls (15), demonstrated that antibiotics, despite being quite hydrophilic compounds, displayed a wide range of mobilities within soil based on their logKow values. This illustrates that other interactions, such as electrostatic, are potentially occurring, indicating that charged antibiotic structures are influential in chemical fate.
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Occurrence in river sediment, sludge and soil
River sediments have been identified as a major sink for antibiotics, as the concentrations of antibiotics detected in sediments are often much higher than those detected in the water column (16). Using extraction techniques such as accelerated solvent extraction (ASE), quick, easy, cheap, effective, rugged and safe extraction (QuEChERS), and ultrasonic-assisted extraction (further described below) antibiotics can be extracted from solid phases such as soil and sediment. Understanding what drives the interactions of antibiotics between phases is very important to determine compound fate and assess the risk they pose. In a study by Chen (17), a variety of pharmaceutical compounds, including antibiotics, were detected in water and sediment. Using the data, they were able to calculate logKow and logKoc (organic carbon to water partition coefficient) (18). They concluded from the study that there was a correlation between logKow vs logKoc, and logKoc vs molecular weight (MW) of compounds, indicating the importance of these parameters when considering inter-phase behaviour. This study detected five different classes of antibiotics: chloramphenicols, sulfonamides, fluoroquinolones, tetracyclines and macrolides. Sulfonamides demonstrated the highest concentrations in water samples (34-859 ngL-1). Tetracyclines (average concentration 18 µgkg-1 dry weight) and macrolides (12 µgkg-1 dry weight) dominated sediment samples. Similarly, Zhou et al. (19) investigated sediment-water interactions in organic contaminants and concluded that there was also a positive relationship between logKoc and MW, suggesting that partitioning is driven by the physical properties of the contaminants.
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Aerial View of of storage tanks in sewage water
treatment plant. Josekubes via Shutterstock.
Solvent extraction equipment used by the author
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Selective Pressurised Liquid Extraction
Selective Pressurised Liquid Extraction (SPLE) is an analytical technique used to extract organic contaminants from solid matrices at elevated temperatures and pressures. The sample cell for extraction consists of the sample, retainer/sorbent, Na2SO4 to remove moisture and filter papers (20).
Selective Pressurised Liquid Extraction (SPLE) is an analytical technique used to extract organic contaminants from solid matrices at elevated temperatures and pressures. The sample cell for extraction consists of the sample, retainer/sorbent, Na2SO4 to remove moisture and filter papers (20).
Studies such as Vazquez Roig et al. (21) have employed SPLE followed by solid phase extraction (SPE) as a clean-up step and liquid chromatography – mass spectrometry (LC-MS) to detect pharmaceuticals in soils and sediments, including several antibiotics (Ciprofloxacin, Norfloxacin, Ofloxacin, Oxytetracycline, Sulfamethoxazole and Tetracycline). The advantage of using SPLE is that it can be very selective at targeting specific compounds, but the drawbacks are that it requires a large amount of sample preparation and high solvent usage.
QuEChERS Extraction
QuEChERS was originally developed for extracting and recovering pesticide residues in fruits and vegetables (22). It requires the addition of Milli Q water and acetonitrile (15 mL) to a glass tub containing the soil sample. The sample is then agitated, and a buffer solution (AOC buffer – sodium acetate/magnesium sulphate) is added, and further agitated with a vortex mixer. The sample is placed in a sample homogeniser and centrifuged at 5000 rpm for 2 minutes. The organic fraction (acetonitrile) is then transferred to a glass vial and evaporated to dryness and analysed for the target analytes (23). Sample clean up by SPE after the extraction step is required. By utilising the QuEChERS Salvia et al. (23) successfully extracted a number of antibiotics, including sulfonamides, macrolides and penicillins. The QuEChERS method is still a fairly new technique for extracting antibiotics in solid matrices, however, it is rapid, easily set up, and low cost.
Ultrasonication
Ultrasonic-assisted extraction (UAE) is another technique that has been used to obtain antibiotics from solid matrices (16, 19). The extraction step involves accurately weighing the sample (soil/sediment) into a glass tube, followed by the addition of appropriate working standards. Organic solvent (acetonitrile) and citric acid buffer are added, and samples then placed on a vortex mixer, followed by ultrasonication and centrifugation for 10 minutes. The supernatant liquor is then pipetted off and the organic fraction evaporated at 55°C to remove the organic solvent, and finally diluted to 200 mL using water. The extract is then further purified using two SPE steps involving a strong anion exchange (SAX) and a reverse phase HLB cartridge. Using the above technique, the authors (24) were able to detect and extract 17 commonly used antibiotics of four classes. The investigation concluded that river sediment plays an important role in acting as a sink for antibiotics, particularly tetracyclines and fluoroquinolones. Recoveries using this method were reported as being >75% for all antibiotics detected.
QuEChERS Extraction
QuEChERS was originally developed for extracting and recovering pesticide residues in fruits and vegetables (22). It requires the addition of Milli Q water and acetonitrile (15 mL) to a glass tub containing the soil sample. The sample is then agitated, and a buffer solution (AOC buffer – sodium acetate/magnesium sulphate) is added, and further agitated with a vortex mixer. The sample is placed in a sample homogeniser and centrifuged at 5000 rpm for 2 minutes. The organic fraction (acetonitrile) is then transferred to a glass vial and evaporated to dryness and analysed for the target analytes (23). Sample clean up by SPE after the extraction step is required. By utilising the QuEChERS Salvia et al. (23) successfully extracted a number of antibiotics, including sulfonamides, macrolides and penicillins. The QuEChERS method is still a fairly new technique for extracting antibiotics in solid matrices, however, it is rapid, easily set up, and low cost.
Ultrasonication
Ultrasonic-assisted extraction (UAE) is another technique that has been used to obtain antibiotics from solid matrices (16, 19). The extraction step involves accurately weighing the sample (soil/sediment) into a glass tube, followed by the addition of appropriate working standards. Organic solvent (acetonitrile) and citric acid buffer are added, and samples then placed on a vortex mixer, followed by ultrasonication and centrifugation for 10 minutes. The supernatant liquor is then pipetted off and the organic fraction evaporated at 55°C to remove the organic solvent, and finally diluted to 200 mL using water. The extract is then further purified using two SPE steps involving a strong anion exchange (SAX) and a reverse phase HLB cartridge. Using the above technique, the authors (24) were able to detect and extract 17 commonly used antibiotics of four classes. The investigation concluded that river sediment plays an important role in acting as a sink for antibiotics, particularly tetracyclines and fluoroquinolones. Recoveries using this method were reported as being >75% for all antibiotics detected.
Sample preparation and analytical procedures
GC-MS and LC-MS are the most commonly applied techniques for detecting antibiotics within solid environmental matrices. Many studies have utilised the triple quadrupole mass detector (MS/MS), which offers the ability for enhanced mass fragment analysis of micropollutants. Sample pre-treatment is crucial when analysing trace contaminants at concentrations in the mg L-1 or µg L-1 scale in environmental samples, but is particularly important for extracting solid mixtures.
GC-MS and LC-MS are the most commonly applied techniques for detecting antibiotics within solid environmental matrices. Many studies have utilised the triple quadrupole mass detector (MS/MS), which offers the ability for enhanced mass fragment analysis of micropollutants. Sample pre-treatment is crucial when analysing trace contaminants at concentrations in the mg L-1 or µg L-1 scale in environmental samples, but is particularly important for extracting solid mixtures.
Legislation
The release of antibiotics into the environment will continue as humans and animals still rely on antibiotics. Therefore, the challenge to remove and prevent antibiotics entering the environment remains an international task.
The Water Framework Directive (Directive 2000/60/EC) aims to meet environmental quality standards in all surface and ground waters (rivers, lakes, transitional waters and coastal waters), with a focus to protect the ecology and wildlife. The directive 2013/39/EU, amending the Environmental Quality Standards Directive 2008/105/EC under the European Water Framework Directive (WFD) has introduced a ‘Watch-List’ monitoring mechanism to collect high-quality EU-wide monitoring data of potentially polluting substances in the aquatic environment. The Watch-List contains a number of organic pollutants, which should be carefully monitored by EU Member States. The first Watch-List was published in 2015, and now contains the antibiotics Erythromycin, Clarithromycin, Azithromycin (macrolides) and Ciprofloxacin (fluoroquinolone), all of which are regularly prescribed by the National Health Service (NHS).
References
The release of antibiotics into the environment will continue as humans and animals still rely on antibiotics. Therefore, the challenge to remove and prevent antibiotics entering the environment remains an international task.
The Water Framework Directive (Directive 2000/60/EC) aims to meet environmental quality standards in all surface and ground waters (rivers, lakes, transitional waters and coastal waters), with a focus to protect the ecology and wildlife. The directive 2013/39/EU, amending the Environmental Quality Standards Directive 2008/105/EC under the European Water Framework Directive (WFD) has introduced a ‘Watch-List’ monitoring mechanism to collect high-quality EU-wide monitoring data of potentially polluting substances in the aquatic environment. The Watch-List contains a number of organic pollutants, which should be carefully monitored by EU Member States. The first Watch-List was published in 2015, and now contains the antibiotics Erythromycin, Clarithromycin, Azithromycin (macrolides) and Ciprofloxacin (fluoroquinolone), all of which are regularly prescribed by the National Health Service (NHS).
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