Anxiety in plants: influence of benzodiazepines
Environmental consciousness has been gaining popularity globally, and research in this field is blooming. More and more studies regarding pharmaceutics in the environment are published yearly, considering different active pharmaceutical substances and their occurrence and fate in the environment. The most frequently discussed medicines are antibiotics, anti-inflammatory drugs, antihypertensives, lipid regulators, antidiabetics, antiepileptics, cancer therapeutics, antidepressants, steroids, hormones and others 1. Also, substances of abuse are not neglected 2,3. The scope is to study the eventual adverse effects of these substances on aquatic environments, assess routes of entering the environment, and see how they can be eliminated. Regarding benzodiazepines, their occurrence is well mapped, but the effect on plants is still uncovered.
Benzodiazepines, such as diazepam (figure 1.1), are a commonly used group of anti anxiety medicines, and their use is widely spread. Benzodiazepines are used to release acute stress but are often abused as recreational drugs. Worldwide, benzodiazepines and their metabolites have been detected in many surfaces and ground waters, including seas, ponds, lakes, and rivers, and are marked as highly persistent in the sediment 4,5 .
Several studies focusing on the effect of benzodiazepines on aquatic organisms show adverse effects, such as modulating the activity, sociality, boldness and feeding rates in fish, mussels, and other invertebrates 6,7. When it comes to the studies focusing on plants, it has been reported that some plants can metabolise benzodiazepines into the same metabolites as the human body 8 Not only does the metabolic pathway of diazepam (figure 1) decomposition in plants seem to be comparable to the human metabolic pathway, but laboratory experiments also show that the effect of diazepam in plants can be very similar to the one in humans. Diazepam seems to modulate stress response in Lemna minor (figure 2), the model aquatic plant for acute toxicity tests. It reduces the stress response by lowering superoxide dismutase levels and increasing levels of catalase and stimulates both frond division and biomass growth. Biochemical processes suggest that internal plant communication related to stress uses signalling similar to one in humans and can be modulated by benzodiazepines 9. However, further molecular studies need to be carried out
Figure 1.1 The chemical structure of Diazepam
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It is often discussed whether chemicals pose an environmental threat. From the current studies, one can judge that photosynthesis is not affected by benzodiazepines and that plants can metabolise many chemicals and act as active bioremediation. Indeed, bioremediation ponds are used as an efficient water treatment, relying on plants and aerobic processes to remove residues of pollutants. Lemna minor (figure 1), Vallisneria spiralis, Eichhornia crassipes, Hydrilla verticillata, Pistia stratiotes, and Cladophora glomerata are used in bioremediation ponds and seem to have the capacity to extract heavy metals and decrease the concentration of ammonia cations10,11 However, we often only assess the acute effect of pharmaceutics on organisms. It is important to realise that the pollutants are chronically present in the aquatic environment, continuously replenished, and are not found separately but in a broad spectrum and concentration ranges. For this reason, studies need to address the so called “chemical mixtures” and the way chemicals potentiate the effects of each other. Only then can we see pharmaceutical substances' actual impact on the environment.
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EPSA European Pharmaceutical Students’ Association
Adela Lamaczova
EPSA Alumna and PhD student
Figure 1.2 Lemna minor, model aquatic plant for acute toxicity tests
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How medicines enter the environment
Both the pharmacokinetics and pharmacodynamics of each pharmaceutical substance must be well studied and described before its market launch. However, the fate of the substance after it leaves the human body often remains unknown. Therefore, it is necessary to consider how the drug is metabolised, in which form it leaves the human body and whether or not it will enter the environment 1,2
After elimination, drug molecules leave the patient’s body in either their original or metabolised form. Both forms can still be active and influence if they enter the aquatic system. After entering the sewage via the toilet, drug metabolites follow the wastewater path to wastewater treatment plants, where most pollutants get removed 3,4 .
Another way pharmaceutics enter the environment in larger quantities is by improperly disposing expired medications by flushing them down the toilet. Other chemical molecules also enter the environment by being flushed out of the surfaces by rain, such as paints, pesticides, insecticides, large area disinfectants and others.
Most households in Europe are connected to wastewater treatment systems. However, many households still dispose of the wastewater directly into the rivers nearby. When it comes to specific facilities, such as hospitals or factories, it is often the case that they have their pre treatment and only release the wastewater that has already undergone several processes to remove the pollutants.
When reaching the wastewater treatment plant, water runs through several chemical phases, during which the molecules are inactivated and broken into inactive parts. However, wastewater treatment is not 100 % reliable. Therefore, many pharmaceutics can still be detected at wastewater treatment plant outlets. This way, even with an effort from society, active pharmaceutical substances enter the aquatic environment and influence a whole range of organisms, starting with plankton and plants, ending with large marine animals and eventually humans 5,6
Another important aspect is the bioaccumulation of chemicals in fish that are then consumed either by other animals or humans. Also, it can be the case when the water is used for crop irrigation 7. Even though the aquatic organisms intensively metabolise all the pollutants and the concentration that could eventually reach the human due to the food chain is negligible, it can have a negative effect on the behaviour of animals, their feeding rates, mating activity and survival 8,9
Water pollution by emerging contaminants has been in the scope of many different organisations, such as The Organisation for Economic Co-operation and Development
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Figure 2.1 Drugs on their way to the environment
(OECD) and the European Commission, which have issued policies and recommendations for freshwater pharmaceutical residues, aiming to keep the water clean and accessible. They call to improve the knowledge, understanding, and reporting, to ensure a source directed approach, to use a use orientated approach, apply end of pipe measures, and request several policy sectors to collaborate 10,11
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EPSA European Pharmaceutical Students’ Association
Adela Lamaczova
EPSA Alumna and PhD student
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Measuring drugs in the environment using chromatography coupled with mass spectroscopy
Different analytical methods are employed to precisely define the amount of pharmaceutical substances present in the aquatic environment. One of the most common instrumental methods is liquid chromatography coupled with mass spectroscopy (LC MS/MS) analysis, where the samples undergo a careful extraction procedure, and a very exact concentration is determined using chromatography separation and mass spectroscopy.
Before the whole procedure is started, it is essential to do a thorough planning of the experiment. One of the first things to consider is sampling. It is vital to decide on the sampling places, which are often around the wastewater treatment plant it can be upstream or downstream from the effluent point, where the treated water enters the aquatic environment. It can be directly inside the treatment plant at different points of water treatment. It can also be unrelated to the water treatment, and samples can be grabbed from the sediments or sludge in lakes, seas, rivers, and underground water. Another perspective on location selection is the density of the population. Different results will be obtained in a city, a village or outside inhabited areas.
When it comes to sampling itself, it is necessary to maintain the same conditions for each of the sampling spots, which means the same depth from which the sample was grabbed and making sure that the place is still the same in case of multiple analyses in time (differences between seasons, years, etc.). There are also different methods for collecting the samples samples can be either grabbed at a specific time only once or continuous sampling, making a grab sample every hour and then analysing the mix. Continuous sampling can answer intra daily differences but are less time effective. Therefore it strongly depends on the purpose of the study.
During sampling, the water's temperature, pH and chemical composition is analysed and noted together with a place (GPS coordinates), time and date on the sampling bottle. These can be either plastic or glass bottles, depending on the investigated compound, its characteristics, sorption capacity, etc. Usually, a photograph of the place is taken.
Samples are then transferred to the laboratory in pre set conditions based on the molecule of interest it can require darkness or specific storage conditions due to the stability. In the laboratory, samples are filtered to remove unwanted particles, and pH can be adjusted if needed.
To perform the analysis, we need to isolate the compound of interest. This is done mainly by separation on columns or by liquid liquid extraction. It makes the compound of interest migrate from our sample into a matrix that is further analysed so that we only run a small but concentrated volume of sample through the instruments, making it more efficient. When we use separation on column, the target molecule gets adsorbed on silica or carbon inside the column (which looks quite like a syringe), and then it is eluted with the selected solvent. Liquid liquid extraction is based on the affinity of the target molecule towards the chosen solvent. By mainly adding an organic solvent that doesn’t mix with water, the molecule can be extracted into the organic phase and further separate these two liquid phases, one with our target molecule and the rest of the water solution. The sample's exact amount (typically a litre or half a litre) is extracted. Finally, the solvent is dried out using an inert gas, and then, the leftover
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is reconstituted by a precisely defined amount of medium and analysed by chromatography.
LC-MS/MS stands for liquid chromatography-mass spectroscopy. The other MS means that two mass spectroscopy analysers are built into one instrument, increasing the method's selectivity. LC MS/MS is so sensitive that it can identify hundreds of molecules in parallel, saving time and money. The mobile phase is carrying the dissolved sample through the chromatographic column, separating individual compounds, which are then assessed by mass spectroscopy. Thanks to the internal standard that is artificially added to each sample, compounds can be identified, and their concentrations precisely calculated. Knowing how much has been artificially added and comparing it with the signal output helps to re-calculate the initial concentration in the sample.
Mathematics comes. Next, it is necessary to calculate the final concentration in the sampling place, considering the starting amount and dilution used. And connected to that statistics are performed to exclude errors and to estimate the effect, reliability, repeatability, and other parameters.
The last part is making sense of the data, connecting the dots, and drawing conclusions that need to be compounded into a scientific article.
Adela Lamaczova EPSA Alumna and PhD student
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EPSA European Pharmaceutical Students’ Association
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Antimicrobials in the environment! A special concern
Alarming numbers of antimicrobials in the environment
Infectious diseases were the leading cause of death before the discovery of antimicrobials. Back then, life expectancy was less than 50 years old1. Antimicrobials are very important in eliminating infections, but can those health-friendly agents become health enemies? The answer is yes. Even though the resistance to antimicrobials is a naturally occurring process, abusing antimicrobials and their irresponsible disposal can be a big responsibility for the emergence of resistant microorganisms, especially bacteria, leading to the so called superbugs, microbes resistant to more than one drug class.
According to statistics divulged by the United Nations (UN), there was a 36% increase in antibiotics usage in humans from 2000 to 2010 2,3. Studies have shown that antibiotic consumption in animals is higher than that in humans, estimated to be 63,101 tons in 2010 and predicted to increase by 67% in 20303. Despite being the most important, health consequences of antimicrobial resistance (AMR) are not the only ones, as it was estimated that, in the European Union (UE), AMR costs hit 1.5 billion/year4 . According to those statistics, 700000 people die annually due to infections of microbial resistant to antimicrobials (AMR infections), and many animals turned irresponsive to treatments1
China is considered the principal consumer of antibiotics, as the consumption was estimated to be approximately 162 million kg of antibiotics in 2013, half of which are for animal use. On the other hand, the use of antibiotics to induce animal growth in the EU was banned in 2006, and the EU is working on making those medicines accessible only over a prescription3 .
Like other drugs and pharmaceuticals, antimicrobials access the environment through different ways, such as untreated or unproperly treated effluent water, human and animal wastes containing antimicrobials such as toilets wastewater (40-90% of antibiotics are excreted in their active form), medications applied to plants, incorrect disposal of antimicrobials such as disposing them into the trash, also from industrial leftovers5,6, as it was found that that the concentration of antibiotics in effluents from antibiotics synthesising pharmaceutical industries is remarkably high. For example, a study found 45 Kg of ciprofloxacin (an antibiotic) released in rivers from approximately 90 drug industries in India7. Nevertheless, effluent from hospitals is one of the most critical sources of antibiotics6
Besides that, improperly informed patients and the lack of communication with clinicians and health professionals can lead to less awareness of the correct disposal of antibiotics6 .
Antimicrobials versus Antibiotics:
It is essential to understand the difference between antimicrobials and antibiotics. Antibiotics are drugs used to defeat bacterial infections. Meanwhile, antimicrobials is a term used to categorise drugs used to combat a more comprehensive range of infections such as bacteria, viruses, fungi or protozoa8
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How do antimicrobials end up in the environment?
Environmental antibiotics and the risk of resistance
Bearing in mind that the resistance to antimicrobials, especially antibiotics, is turning into a huge global problem, this issue has been studied on different levels, including the genetic level. It is referred to as the pool of all resistant genes to antibiotics in the environment as “resistome”. Resistant bacteria present in the environment can transfer their resistance genes to the pathogenic bacteria from sewage. This process occurs preferentially in wastewater plants where bacteria from humans and the environment flock together. Furthermore, studies have proven that some resistance genes from bacteria found in soil are very similar to resistance bacteria in humans7 .
Figure 4.1 The chemical structure of Tetracycline a water soluble antibiotic
Tetracyclines, such as tetracycline (figure 4), doxycycline, minocycline, and tigecycline, are a class of antibiotics that inhibit the synthesis of proteins in bacteria9. Tetracyclines are considered broad spectrum antibiotics. They are water soluble and persist in soil for long periods. Therefore, they are highly delectated in soils3
Other consequences of antimicrobials in the environment:
1. In plants, antibiotics can be toxic and alter plants' growth and photosynthesis. For example, tetracyclines have phytotoxic effects that can cause chromosomal aberrations and inhibit plant growth, reducing the content of photosynthetic chlorophyll and carotenoid pigments in plants. However, antibiotics such as tetracycline can still improve plant growth at low concentrations6 .
2. Causing toxicities or allergies in humans, as some plants fertilised with antibiotics-contaminated animal manure can reach humans through the food chain6 .
The role of regulations6: Studies have shown that stricter regulations are related to less consumption of antibiotics10. Also, to mitigate resistance and avoid future disasters, some big pharma companies signed an antimicrobial-resistance roadmap aiming mainly a better environmental management of antibiotics production. Furthermore, the European Commission has admitted that environmental contamination with pharmaceuticals significantly threatens public health.
Limitations on studying antibiotics in the environment6:
1. There is still no consensus on the accepted concentration of antibiotics in the environment.
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2. Antibiotics are chemicals and change according to the characteristics of the environment of their disposal. Therefore, judging if they are eliminated in their original form isn't easy.
3. Antibiotics are degraded in the environment by microorganisms or other chemical reactions.
4. Formation of other compounds of difficult analysis, such as binding to other compounds or adsorption to matrices like soil particles.
Although those environmental alterations can limit access to the actual concentration of the original compound, they indicate some harmful degradation products.
Conclusion:
Despite all the technological and industrial advancements, only a few antimicrobials have been developed in the last 30 years1. What that means is that no near alternative to the already existing antimicrobials is available yet. That is a big responsibility of health care professionals, mainly pharmacists, to raise awareness and instruct people about the correct consumption and disposal of those medicines.
Science Coordinator 2022 2023
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Rahaf Alsayyed
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EPSA European Pharmaceutical Students’ Association
References:
Article and infographic 1: Anxiety in plants: influence of benzodiazepines
1. Nikolaou, A.; Meric, S.; Fatta, D. Occurrence patterns of pharmaceuticals in water and wastewater environments. Analytical and Bioanalytical Chemistry 2007, 387, 1225 1234, doi:10.1007/s00216 006 1035 8.
2. Bijlsma, L.; Emke, E.; Hernandez, F.; de Voogt, P. Investigation of drugs of abuse and relevant metabolites in Dutch sewage water by liquid chromatography coupled to high resolution mass spectrometry. Chemosphere 2012, 89, 1399 1406, doi:10.1016/j.chemosphere.2012.05.110.
3. Jurado, A.; Mastroianni, N.; Vazquez Sune, E.; Carrera, J.; Tubau, I.; Pujades, E.; Postigo, C.; de Alda, M.L.; Barcelo, D. Drugs of abuse in urban groundwater. A case study: Barcelona. Science of the Total Environment 2012, 424, 280-288, doi:10.1016/j.scitotenv.2012.02.074.
4. Loffler, D.; Rombke, J.; Meller, M.; Ternes, T.A. Environmental fate of pharmaceuticals in water/sediment systems. Environmental Science & Technology 2005, 39, 5209 5218, doi:10.1021/es0484146.
5. Fernández Rubio, J.; Rodríguez Gil, J.L.; Postigo, C.; Mastroianni, N.; López de Alda, M.; Barceló, D.; Valcárcel, Y. Psychoactive pharmaceuticals and illicit drugs in coastal waters of North-Western Spain: Environmental exposure and risk assessment. Chemosphere 2019, 224, 379 389, doi:https://doi.org/10.1016/j.chemosphere.2019.02.041
6. Cerveny, D.; Brodin, T.; Cisar, P.; McCallum, E.S.; Fick, J. Bioconcentration and behavioral effects of four benzodiazepines and their environmentally relevant mixture in wild fish. Science of The Total Environment 2020, 702, 134780, doi:https://doi.org/10.1016/j.scitotenv.2019.134780.
7. Vossen, L.E.; Cerveny, D.; Sen Sarma, O.; Thornqvist, P.O.; Jutfelt, F.; Fick, J.; Brodin, T.; Winberg, S. Low concentrations of the benzodiazepine drug oxazepam induce anxiolytic effects in wild caught but not in laboratory zebrafish. Science of the Total Environment 2020, 703, 9, doi:10.1016/j.scitotenv.2019.134701.
8. Carter, L.J.; Williams, M.; Martin, S.; Kamaludeen, S.P.B.; Kookana, R.S. Sorption, plant uptake and metabolism of benzodiazepines. Science of the Total Environment 2018, 628 629, 18 25, doi:10.1016/j.scitotenv.2018.01.337.
9. Lamaczova, A.; Malina, T.; Marsalkova, E.; Odehnalova, K.; Opatrilova, R.; Pribilova, P.; Zezulka, S.; Marsalek, B. Anxiety in Duckweed Metabolism and Effect of Diazepam on Lemna minor. Water 2022, 14, 12, doi:10.3390/w14091484.
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10. Malschi, D.; Muntean, L.; Oprea, I.; Roba, C.; Popita, G.; Stefanescu, L.; Florian, B.M.; Rinba, E. RESEARCH ON WASTEWATERS BIOREMEDIATION WITH AQUATIC SPECIES FOR CONSTRUCTED WETLANDS. Environmental Engineering and Management Journal 2018, 17, 1753 1764, doi:10.30638/eemj.2018.174.
11. Shammout, M.W.; Zakaria, H. Water lentils (duckweed) in Jordan irrigation ponds as a natural water bioremediation agent and protein source for broilers. Ecological Engineering 2015, 83, 71-77, doi:10.1016/j.ecoleng.2015.05.041.
Article and infographic 2: How medicines enter the environment
1. Barreto, E.F.; Larson, T.R.; Koubek, E.J. Drug Excretion. In Reference Module in Biomedical Sciences; Elsevier: 2021.
2. Nikolaou, A.; Meric, S.; Fatta, D. Occurrence patterns of pharmaceuticals in water and wastewater environments. Analytical and Bioanalytical Chemistry 2007, 387, 1225-1234, doi:10.1007/s00216-006-1035-8.
3. Acuña, V.; von Schiller, D.; García-Galán, M.J.; Rodríguez-Mozaz, S.; Corominas, L.; Petrovic, M.; Poch, M.; Barceló, D.; Sabater, S. Occurrence and in stream attenuation of wastewater derived pharmaceuticals in Iberian rivers. Science of The Total Environment 2015, 503 504, 133 141, doi:https://doi.org/10.1016/j.scitotenv.2014.05.067
4. Alygizakis, N.A.; Gago Ferrero, P.; Borova, V.L.; Pavlidou, A.; Hatzianestis, I.; Thomaidis, N.S. Occurrence and spatial distribution of 158 pharmaceuticals, drugs of abuse and related metabolites in offshore seawater. Science of The Total Environment 2016, 541, 1097 1105, doi:https://doi.org/10.1016/j.scitotenv.2015.09.145.
5. Bjorklund, E.; Svahn, O.; Bak, S.; Bekoe, S.O.; Hansen, M. Pharmaceutical Residues Affecting the UNESCO Biosphere Reserve Kristianstads Vattenrike Wetlands: Sources and Sinks. Archives of Environmental Contamination and Toxicology 2016, 71, 423-436, doi:10.1007/s00244-016-0303-7.
6. Baltazar Estrada Arriaga, E.; Cortes Munoz, J.E.; Gonzalez Herrera, A.; Calderon Molgora, C.G.; Rivera-Huerta, M.D.; Ramirez-Camperos, E.; Montellano-Palacios, L.; Gelover Santiago, S.L.; Perez Castrejon, S.; Cardoso Vigueros, L.; et al. Assessment of full-scale biological nutrient removal systems upgraded with physicochemical processes for the removal of emerging pollutants present in wastewaters from Mexico. Science of the Total Environment 2016, 571, 1172 1182, doi:10.1016/j.scitotenv.2016.07.118.
7. Malchi, T.; Maor, Y.; Tadmor, G.; Shenker, M.; Chefetz, B. Irrigation of Root Vegetables with Treated Wastewater: Evaluating Uptake of Pharmaceuticals and the Associated Human Health Risks. Environmental Science & Technology 2014, 48, 9325-9333, doi:10.1021/es5017894.
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8. Cerveny, D.; Brodin, T.; Cisar, P.; McCallum, E.S.; Fick, J. Bioconcentration and behavioral effects of four benzodiazepines and their environmentally relevant mixture in wild fish. Science of The Total Environment 2020, 702, 134780, doi:https://doi.org/10.1016/j.scitotenv.2019.134780.
9. Brodin, T.; Nordling, J.; Lagesson, A.; Klaminder, J.; Hellstrom, G.; Christensen, B.; Fick, J. Environmental relevant levels of a benzodiazepine (oxazepam) alters important behavioral traits in a common planktivorous fish, (Rutilus rutilus). Journal of Toxicology and Environmental Health-Part a-Current Issues 2017, 80, 963-970, doi:10.1080/15287394.2017.1352214.
10. OECD. Pharmaceutical Residues in Freshwater Hazards and Policy Responses. 2019.
11.Lockwood, S.; Saïdi, N.; Morgan, V.A. Options for a strategic approach to pharmaceuticals in the environment. 2016.
Article and infographic 3: Measuring drugs in the environment using chromatography coupled with mass spectroscopy
No references. The information is retrieved from the author’s work in the laboratory.
Article and infographic 4: Antimicrobials in the environment! A special concern
1. The costs and risks of AMR water pollution. (2020). https://www.weforum.org/reports/the costs and risks of amr water pollution/
2. Https://www.unep.org/news-and-stories/press-release/antimicrobial-resistanceenvironmental pollution among biggest. (2020, November 25). UN Environment Programme. https://www.unep.org/news and stories/story/healthy environment key-antibiotics-work
3. Wang, X., Ryu, D., Houtkooper, R. H., & Auwerx, J. (2015). Antibiotic use and abuse: A threat to mitochondria and chloroplasts with impact on research, health, and environment. BioEssays, 37(10), 1045 1053. https://doi.org/10.1002/bies.201500071
4. Antimicrobial resistance is a serious threat to human and planetary health. (2021, August 24). UN Environment Programme. https://www.unep.org/news-andstories/speech/antimicrobial resistance serious threat human and planetary health
5. Antibiotics and the Environment: What You Should Know. (2018). Minnesota One Health Antibiotic Stewardship Collaborative. https://www.pca.state.mn.us/featured/antibiotics and environment
6. Polianciuc, S. I., Gurzău, A. E., Kiss, B., Ștefan, M. G., & Loghin, F. (2020). Antibiotics in the environment: Causes and consequences. Medicine and Pharmacy Reports. https://doi.org/10.15386/mpr-1742
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7. Antibiotics in the environment UNDERSTAND HOW DID WE END UP HERE? (n.d.). ReAct. Retrieved 22 September 2022, from https://www.reactgroup.org/toolbox/understand/how did we end up here/antibiotics in the environment/
8. What is the difference between antibiotic and antimicrobial resistance? (n.d.). WHO. Retrieved 22 September 2022, from https://www.emro.who.int/health topics/drug resistance/what is the difference between antibiotic and antimicrobial-resistance.html
9. Mollie C. Shutter; Hossein Akhondi. (n.d.). Tetracycline. NIH National Library of Medicine. Retrieved 8 September 2022, from https://www.ncbi.nlm.nih.gov/books/NBK549905/
10. Mueller, T., & ÿstergren, P. O. (2016). The correlation between regulatory conditions and antibiotic consumption within the WHO European Region. Health Policy, 120(8), 882 889. https://doi.org/10.1016/j.healthpol.2016.07.004
Images:
Figure 1.1 https://pubchem.ncbi.nlm.nih.gov/compound/Diazepam colour edited on Canva.
Figure 1.2 Retrieved from Canva
Figure 2.2 Retrieved from Canva
Figure 4.1 https://pubchem.ncbi.nlm.nih.gov/compound/Tetracycline colour edited on Canva.
Front page image: designed on Canva
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