Accepted Manuscript Title: Pharmaceuticals in the environment: Biodegradation and effects on natural microbial communities. A Review Author: Anna Barra Caracciolo Edward Topp Paola Grenni PII: DOI: Reference:
S0731-7085(14)00576-7 http://dx.doi.org/doi:10.1016/j.jpba.2014.11.040 PBA 9827
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
7-8-2014 20-11-2014 22-11-2014
Please cite this article as: A.B. Caracciolo, E. Topp, P. Grenni, Pharmaceuticals in the environment: Biodegradation and effects on natural microbial communities. A Review, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.11.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical abstract
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Pharmaceuticals in the environment: Biodegradation and effects on natural microbial
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communities. A Review
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Anna Barra Caracciolo1*, Edward Topp2, Paola Grenni1
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*Corresponding Author
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00015 Monterotondo (Rome), Italy, tel +39 0690672786, Fax +39 0690672787 e.mail:
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[email protected]
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Agriculture and Agri-Food Canada, 1391 Sandford Street, London ON Canada, N5V 4T3
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Istituto di Ricerca sulle Acque, Consiglio Nazionale delle Ricerche, Via Salaria km 29.300 -
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Abstract
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Environmental microorganisms play a key role in fundamental ecological processes such as
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biogeochemical cycling and organic contaminant degradation. Microorganisms comprise a large
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unexplored reservoir of genetic diversity and metabolic capability providing several ecosystem
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services, most importantly the maintenance of soil and water quality. Pharmaceutical occurrence in
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the environment can compromise microbial community structure and activities in different ways.
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The fate of a pharmaceutical in soil or water depends on numerous factors, including its inherent
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physic-chemical properties (e.g. water solubility, lipophilicity, vapour pressure), environmental
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factors and climate conditions (e.g. temperature, incident radiation, pH) and most importantly the
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presence and activity of microorganisms that possess the ability to biodegrade it. The presence of a
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natural microbial community is a necessary prerequisite for an effective response to the various
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chemicals that can contaminate an ecosystem. The recovery from contamination is only possible if
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toxicity does not hamper microbial activity. This review presents current knowledge on the effects
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on natural microbial communities of some pharmaceuticals and of some biocides commonly found
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as environmental microcontaminants.
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Highlights
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Biodegradation is the main process for eliminating the majority of pharmaceuticals
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Pharmaceuticals can inhibit microbial activity
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Evidence of pharmaceutical effects on natural microbial processes is scarce
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Environmental microorganisms comprise an enormous reservoir of antibiotic resistance and
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biodegradative capability
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Keywords: anti-inflammatories; analgesic; blood lipid regulators; antidepressant, antiepileptic,
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antibiotic, microbiocides 2 Page 2 of 34
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1.
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Pharmaceuticals are indispensable for the maintenance of public health and the quality of life.
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Rapid advances in drug therapies to meet health challenges and their timely availability are
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essential for a healthy society. Veterinary pharmaceuticals prevent and treat disease and increase
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the efficiency of food production. Thousands of different active compounds are currently in use in
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large quantities to treat or to prevent diseases [1,2]. Recently, the widespread detection of
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pharmaceuticals in terrestrial and aquatic systems has engendered significant scientific and
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regulatory concern [3,4]. Following administration, a great portion of human pharmaceuticals is
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excreted unaltered or as an active metabolite, and ends up in wastewater treatment plants (WWTPs)
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[5]. Effluents from WWTPs represent an important source point for aquatic exposure to
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pharmaceuticals if they are not efficiently removed during the wastewater treatment process
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[3,5,6,7]. Moreover, hospital and industrial wastewaters [8], uncontrolled and illegal drug disposal
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[9] and aquaculture [10] can also be a significant source of aquatic contamination [11,12]. Many
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drugs, owing to their widespread human and veterinary usage, are being continuously added to
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ecosystems and can exhibit pseudo-persistence [13]. The use of sewage sludge (biosolids) and
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manure as a crop fertilizer and irrigation with reclaimed water will entrain human and veterinarian
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pharmaceuticals into agricultural soil [14,15,16,17]. Pharmaceuticals can be than leached from soil
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to groundwater [8,18,19,20], and they are also detected in coastal marine waters [8,21,22,23,24].
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Different classes of pharmaceuticals and biocides are widespread in the environment including anti-
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inflammatories, analgesic, blood lipid regulators, antidepressants, antiepileptics and antimicrobials
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[25,26,27,28,29].
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There are concerns for both ecosystem health and a potential health risk for humans through the
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consumption of food and water containing pharmaceuticals residues. Ecological studies generally
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report that human pharmaceuticals are not acutely toxic to aquatic organisms, because their
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environmental concentrations are typically very low [29,30]. A few studies have revealed evidence
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for chronic long-term negative impacts of environmental pharmaceutical exposure on living
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organisms and populations [29,30,31]. Pharmaceuticals are distinct from conventional pollutants
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with respect to their potential ecotoxicology since they are specifically designed to be bioactive at
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low concentrations. Moreover, they frequently act on specific targets that can be widely conserved
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across multiple life forms. Environmental concentrations (in impacted aquatic systems typically
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ranging from ng to µg per litre) may therefore be of concern, and sub-lethal effects on non-target
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organism have in some cases been found [31,32, 33].
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Overall, knowledge concerning the ecotoxicology and sub-lethal effects in water and soil
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organisms is scarce, but some experimental studies show that some pharmaceuticals can disrupt or
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alter the endocrine system [34], induce pathogen resistance, as in the case of several antibiotics
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[35,36], and also have detrimental effects on natural microbial communities and their key functions
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[37,38].
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Key factors in determining environmental exposure concentrations include volume of usage, the
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degree of release to the environment, and persistence once in the environment. Environmental
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persistence depends on a variety of factors, such as a pharmaceuticals inherent physicochemical
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properties, environmental factors (e.g. light, temperature, pH) and most importantly the presence
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and activity of microorganisms with the capability to degrade it via metabolic and/or co-metabolic
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pathways [39,40]. The biodegradation by metabolic pathways refers to the use of a chemical as a
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source of energy, carbon, nitrogen or other nutrients. Co-metabolism is the fortuitous breakdown of
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a contaminant by an enzyme or cofactor that is produced during microbial metabolism of another
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compound.
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Microorganisms are important degraders of organic matter and of xenobiotics, including
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pharmaceuticals, and provide products as nutrients to other organisms in the food web [41].
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Microbial communities are therefore clearly vital for maintaining ecosystem functioning. Changes
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within microbial functional groups are correlated to changes in ecosystem processes [42,43,44].
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Consequently, they are essential in the overall processes that contribute to the quality state of
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natural ecosystem.
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2. Biodegradation of pharmaceuticals and effects on natural microbial communities
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Natural microbial communities in soil and water are key players in several processes controlling
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the quality of ecosystems and regulating the fate of pollution released into the environment and in
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this sense, they provide the Ecosystem Service termed “Regulation” [45]. Microorganisms are
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involved in ecosystem self-purification processes since they can degrade contaminants by
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metabolic and/or co-metabolic pathways. Biodegradation is considered the most important process
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for eliminating the majority of xenobiotics, including pharmaceuticals [28,43,45]. Recovery from
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contamination is possible only if the toxicity of the molecules does not inhibit microbial activity.
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However, knowledge on biodegradation of drugs and their effects on ecological processes driven
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by microorganisms is quite scarce, so far.
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Pharmaceuticals include hundreds of substances with widely varying chemical-physical properties,
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environmental behaviour and biochemical activities [8]. Pharmaceuticals typically enter the
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environment in complex effluents, and thus natural microbial communities are exposed to a
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mixture of active substances. In this review we report some studies regarding the effects of some
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antibiotics (i.e. tylosin, sulfamethazine, chlortetracycline), other pharmaceuticals (naproxen, 4 Page 4 of 34
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ibuprofen, diclofenac, gemfibrozil, paracetamol, clofibric acid, fluoxetine, carbamazepine), and
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biocides (triclosan and triclocarban) on natural microbial communities. These compounds were
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selected because they are among the most commonly found as environmental microcontaminants
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[46,47,48,49,50]. In Table 1 the list of the selected compounds is reported.
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2.1 Non-steroidal anti-inflammatory drugs
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Analgesics are pain-relief drugs that include narcotic analgesics, non-narcotic analgesics and non-
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steroidal anti-inflammatory drugs (NSAID) used to alleviate the pain present in almost all diseases.
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These compounds work by blocking cyclooxygenase (COX) enzymes, which catalyze the synthesis
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of different prostaglandins from arachidonic acid [51]. NSAIDs include naproxen, ibuprofen and
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diclofenac, which are among the most frequently detected pain killers in surface water, at
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concentrations ranging from tens of ng/L to hundreds of µg/L [26,46,47].
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Naproxen is a polar pharmaceutical commonly found as a microcontaminant of rivers [25,52,53]
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and also of groundwater from aquifers recharged with reclaimed water [54].
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Naproxen persistence in surface water has been reported in some studies [25,53,55]. Photolytic
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degradation was found to be an important abiotic degradation process, although it is not able to
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degrade naproxen thoroughly [56]. Moreover, the photoproducts were found to be significantly
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more toxic than the parent compound [56].
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In recent microcosm studies naproxen (100 µg/L initial concentration) was found to be
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biodegraded by natural bacterial populations from river water, belonging to Alpha- and Gamma-
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Proteobacteria [25,57] with half-lives of about 20-30 days. In another recent work a Gamma-
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Proteobacteria, Stenotrophomonas maltophilia KB2 strain, an aromatic compound degrader, was
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found to metabolically transform 28% of initial naproxen concentration in culture media (6 mg/L;
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30°C) within 35 days. However, the naproxen degradation was accompanied by a decrease in the
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number of bacteria cells. Most likely, this compound was not a sufficient carbon and energy source
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for strain KB2. At the same time, it was able to co-metabolically degrade, with addition of glucose
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or phenol, 78% and 40% of naproxen, respectively [58].
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Naproxen biodegradation and mineralization by aerobic population was found in agricultural soils
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[15]. Three different agricultural soils (sandy loam, loam and silt loam), which have never been
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exposed to the drug, were treated with 5 µg naproxen/g soil and incubated in the dark at 30°C.
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During the 27 days of incubations, [O-14CH3]naproxen was rapidly mineralized to 14CO2 with very
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comparable kinetics in the three soils. In the loam soil naproxen mineralization was hastened by the
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addition of liquid municipal biosolids, and when the experiment was performed at 4°C, a 35 day
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lag-phase was observed. An enrichment culture obtained from aerobically digested liquid
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municipal biosolid in a mineral salts medium with naproxen as the sole carbon source was able to
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convert the parent compound to the corresponding naphthol, O-desmethyl naproxen [15]. Lin and
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Gan [59], comparing aerobic and anaerobic degradation of naproxen (40 µg/kg; 20°C) in soil
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microcosms found that it was biodegraded aerobically with different half-lives values depending on
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specific soil characteristics (from 17 to 69 days for the very gravelly fine loamy sand soil and for
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loam soil, respectively), while it was persistent in anaerobic conditions.
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A concentration of 50 ng/g of naproxen was biodegraded co-metabolically in another soil
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microcosm experiment using reclaimed water with a half-life of 9 to 18 days [60].
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The different half-life values reported in the various studies above described may be explained by
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the initial biotic an abiotic characteristics of the sample (microbial community composition, soil
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type, moisture content, temperature etc.) that may affect degradation, together with the specific
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laboratory experimental conditions.
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Several studies pointed out the role of fungi in naproxen transformation [43,61,62]. Marco-Urrea et
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al. [62] found that the fungus Trametes versicolor was able to degrade naproxen in a few hours in
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degradation experiments performed at two different concentrations (10 mg/L and 55 μg/L).
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Moreover, Rodarte-Morales et al. [43] reported the complete removal of naproxen (1 mg/L) in 7
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days by the fungi Bjerkandera sp. R1 and Bjerkandera adusta, and in 4 days by Phanerochaete
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chrysosporium.
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The overall results of these degradation experiments suggest that naproxen is not an intrinsically
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persistent compound and it is biodegradable in aerobic conditions. However, since it is chronically
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discharged into surface water from waste water treatment plants, owing to large volume and daily
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use among the human population, it is not only constantly found in surface water, but also at
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concentrations higher than most other, more intrinsically persistent drugs. Consequently, naproxen
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can be considered a pseudo-persistent compound in surface waters and its effects on ecosystem
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functioning warrants investigation. Regarding this aspect some detrimental effects on microbial
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communities were recently found. In surface water microcosms, acute effects on the river microbial
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community were observed three hours after addition of 100 μg/L naproxen. The microbial cell
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viability and the Beta-Proteobacteria group decreased significantly [57] and a toxic effect on the
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bacterial populations involved in key ecosystem functioning was not excluded, because Beta-
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Proteobacteria include the Ammonia Oxidizing Bacteria (AOB) involved in the nitrogen cycle
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[63]. A toxic effect on AOB was also found in a waste water treatment plant when naproxen was
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present [64].
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Ibuprofen, like naproxen, is a commonly used over the counter drug and it is also used in veterinary
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medicine. It is one of the most abundant polar pharmaceuticals detected in river waters [26,30,52].
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Moreover, it was also found in groundwater at concentrations up to 12 µg/L [54].
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Ibuprofen is a chiral pharmaceutical and two different isomers can be detected in the environment:
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S-(+)-ibuprofen, which is the pharmacologically active isomer, and R-(−)-enantiomer which is the
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inactive enantiomer [65]. Ibuprofen was found to be degraded in wastewaters and surface waters in
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both field observations and laboratory incubations, with the S-enantiomer biodegraded faster,
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because microorganisms mainly utilize this enantiomer [66]. However, it is not clear to what extent
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the change in enantiomer composition was due to biodegradation versus enantiomerization, as
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happens with the structurally similar phenoxyalkanoic herbicides [66]. Ibuprofen metabolites are
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also chiral and were found to be more toxic than the parent compound [61].
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Ibuprofen degradation was not observed in sterile river water (150 µg/L) [67] and in sterile soil
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(1.25 µg/g soil) experiments [68], suggesting that abiotic degradation has a negligible role in its
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transformation.
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Biodegradation of ibuprofen (20 µg/L) was observed in a few hours in river water including
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sediment microcosms [69]. Biodegradation of an initial concentration of 100 µg/L of ibuprofen was
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also observed in a laboratory study using microbial biofilms from river water. The pharmaceutical
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degradation occurred between 4-8 days after its initial addition [67]. In soil, Carr et al. [68] in a 14-
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day study reported ibuprofen persistence (initial concentration: 1.25 µg/g soil) to be highly variable
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depending on the moisture content. The calculated half-life ranged from 30 to 1,706 days in un-
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saturated and water-saturated soil, respectively [68]. In another soil microcosm experiments
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ibuprofen biodegradation was found to be quite rapid with the half-life ranging from 0.3-0.9 days
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with an initial concentration of 0.050 µg/g soil, and between 3-7 days with an initial concentration
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of 5 µg/g soil [59]. In a liquid medium, the bacterium Nocardia sp. (strain NRRL 5646) was found
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to be able to degrade the R(-)-ibuprofen isomer in two major metabolites, ibuprofenol and the
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corresponding acetate derivative [69]. The Alpha-Proteobacteria Sphingomonas sp., isolated from
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an enrichment culture (500 mg/L ibuprofen) using an inoculum from a wastewater treatment plant,
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was able to degrade ibuprofen as the sole carbon source [70]. Quintana et al. [71] found that when
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ibuprofen was the sole growth substrate, under aerobic conditions, the drug was not transformed
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after 28 days in a laboratory test using fresh sludge from a membrane bioreactor as inoculum.
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However, when an additional carbon source was added (powdered milk), co-metabolism of
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ibuprofen was completed at 22 days. Consequently, Ibuprofen may be more easily metabolized
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when other carbon sources are available.
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Ibuprofen is biodegraded by several fungi. Marco-Urrea et al. [62] found that the fungi Trametes
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versicolor, Irpex lacteus, Ganoderma lucidum and Phanerochaete chrysosporium degrade
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ibuprofen (10 mg/L) in liquid media within 7 days after its addition.
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Clearly microorganisms play an important role in ibuprofen degradation, although its
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transformation is a complex process and two isomers of it occur in the environment, consequently
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the degradation pathways and metabolite formation need to be clarified [72].
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There has been little research into ibuprofen’s effects on microorganisms. However, some studies
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report that ibuprofen has significant antibacterial activity against Gram-positive bacteria [73] and
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also an antifungal activity [74]. Ibuprofen was found to reduce the overall bacterial biomass of a
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riverine biofilm community originated from rotating annular bioreactors and exposed for 8 weeks
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to a concentration of 10 µg/L; moreover, the microbial biofilm composition, analyzed by the
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Fluorescence in Situ Hybridization method, changed during the experiment. In particular,
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Cyanobacteria decreased significantly together with Gamma-Proteobacteria and Gram-positive
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bacteria Firmicutes, while Alpha-, Beta-Proteobacteria, Cytophaga-Flavobacteria and sulfate-
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reducing bacteria increased, suggesting a role of these groups in Ibuprofen biodegradation [75].
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Ibuprofen was found to influence the growth of a river sediment microbial community in a liquid
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media with sucrose and glucose, at an environmental concentration of 50 ng/L [76].
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Corcol et al. [77] performed the Pollution-Induced Community Tolerance (PICT) short-term dose-
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response test in fluvial biofilm communities affected by wastewater treatment effluents. Biofilms
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were exposed for 48 h to increasing concentrations of ibuprofen, alone or in the co-presence of
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diclofenac. Two different endpoints were used to assess biofilm tolerance as Effective
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Concentration 50 (EC50): the photosynthetic efficiency of autotrophic organisms, and the beta-
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glucosidase activity of heterotrophic organisms. The EC50 values of photosynthetic efficiency and
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of beta-glucosidase activity indicated that the microbial communities were less tolerant to the
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mixture of the two pharmaceuticals.
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Despite ibuprofen has been classified as an easily degradable compound [78], an environmental
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risk assessment study indicates that it may represent a risk for the aquatic environment [79]. In fact
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the calculated PEC (Predicted Environmental Concentration) for water receiving wastewater
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effluent
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Concentration/Predicted No EffectConcentration) was >1 [79]. Ibuprofen has been proposed to be
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identified as a priority substance under the Water Frame Directive because it is suspected of
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influencing sex steroid hormones in both vertebrates and invertebrates and having cytogenetic
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effects in freshwater bivalves [80,81]. However, the inclusion of ibuprofen was rejected in January
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2012 owing to a lack of sufficient evidence of significant risks to aquatic environments [82].
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was
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(Predicted
Environmental
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Diclofenac is a polar pharmaceutical compound mostly used as the sodium salt diclofenac-Na in
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human and veterinary medicine to reduce inflammation and pain [83,84]. It is a non-steroidal anti-
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inflammatory drug and is included in a number of different formulations with generic names such
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as Diclometin, Diclomex and Voltaren. Diclofenac is one of the most commonly detected
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pharmaceuticals in European rivers [26,50]. It has also been detected in groundwater at a maximum
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concentration of about 4 µg/L [20,85] and in soil irrigated with reclaimed water at concentrations
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ranging from 0.35 to 1.16 µg/kg [86].
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Diclofenac (0.1 μg/g) was found to be rapidly mineralized in various agricultural soils with half-
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lives 25%) on nitrification
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and denitrification rates at concentrations ⩾250 mg/L
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2.3 Blood Lipid regulators
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Lipid abnormalities are among the key risk factors for cardiovascular disease and lowering low-
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density lipoprotein (LDL) cholesterol concentrations can significantly reduce the incidence of
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coronary heart disease. In the decade 1995-2005, the volume of cholesterol-lowering drugs
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prescribed almost tripled [95]. There are two main groups of blood lipid lowering agents, with
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different modes of action: statins and fibrates. Although statins (e.g. simvastatin, omeprazole or
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paroxetine) are among the pharmaceuticals most consumed without medical prescription [96], they
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are rarely found as environmental microcontaminants, because their excretion occurs mainly as
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metabolites [96]. Fibrates or fibric acid derivatives are a class of drugs which inhibit the production
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of very low-density lipoprotein (VLDL) and reduce plasma triglyceride level. Gemfibrozil and
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clofibric acid, the main metabolite of clofibrate, are among the pharmaceuticals most frequently
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detected in European waters [26,97]. 10 Page 10 of 34
Gemfibrozil is an acidic drug and a blood lipid-regulating agent, clinically prescribed since the
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early 1980s for patients with a high risk of coronary heart disease [13,97,98]. It is metabolised in
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the liver and approximately 70% is excreted through urine, mostly as a glucuronide conjugate [98].
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Glucuronides are fundamentally inactive, but are readily cleaved during sewage treatment releasing
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the active compound. Although the International Agency for Research on Cancer has classified it in
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group 3, i.e. not classifiable regarding its carcinogenicity for humans [99]. Gemfibrozil has been
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shown to induce peroxisome proliferation in rodents and in eels leading to liver cancer and it can
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also have some detrimental effects on non-target aquatic organisms [31,100]. Gemfibrozil is found
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in surface waters at concentrations ranging from ng/L to µg/L [26,50,101].
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Gemfibrozil was initially reported to be not biodegradable [101], but the fungus Cunninghamella
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elegans ATCC 9245 was found to degrade it in a liquid culture by hydroxylation processes [103].
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Several studies report gemfibrozil to be a quite persistent compound in water with half-lives ranging
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from 70 to 288 days in surface water [25,55]. In sterile river water incubated in the dark,
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gemfibrozil was not degraded, whereas in the same microbiologically active water a significant
315
decrease in concentration (half-life of about 70 days) was observed, suggesting the role of
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microorganisms in its biodegradation [25]. Moreover, FISH analysis of the microbial community,
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revealed that the Gamma-Proteobacteria group increased during gemfibrozil biodegradation,
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indicating that some populations of this group might be involved in its transformation [25].
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Although, Lin et al [102] reported gemfibrozil degradation with a half-life less than 1 day and Zhou
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et al [104] found that a strain of Bacillus sp., isolated from the sludge of a WWTP, rapidly degraded
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gemfibrozil in a liquid culture,
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persistence [25, 55, 105].
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Gemfibrozil has been shown to be inhibitory to natural microbial communities. For example, a
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detrimental effect was observed in natural river water microcosms three hours after pharmaceutical
325
addition (100 µg/L) [25]. Bacterial community viability significantly decreased, compared to
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control microcosms, and this was ascribed to an initial acute toxic effect from this drug on the
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overall bacterial community [25]. Similar concentrations were effective both with AOB from a
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wastewater treatment plant [64] and in aquatic toxicological tests [106].
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Clofibric acid is a polar compound and is the active metabolite of clofibrate lipid regulators (e.g.
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clofibrate, etofyllin). It has been detected in water, including drinking water, for at least twenty
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years. Buser et al. [88] found concentrations of clofibric acid of 7.8 ng/L in the North Sea, a higher
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concentration than that of mecoprop, a pesticide structurally related to clofibric acid. Buser’s
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conclusions were that 50-100 tons/year of clofibric acid have been entering the North Sea and that
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it was the drug most commonly found in open waters. Recent investigations report a maximum
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concentration in natural surface water of more than 240 ng/L [47].
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Clofibric acid is regarded as extremely persistent, with an estimated environmental residence time
337
of 21 years [95]. Clofibric acid was not biodegraded in laboratory reactors containing river biofilm
338
[71]. On the other hand, in an aerobic sequencing batch reactor clofibric acid was biodegraded co-
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metabolically by a mixed microbial culture, dominated by AOBs. This culture, originating from a
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WWTP was firstly acclimatized to a chlorinated aromatic molecule with a structure similar to
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clofibric acid and it was then used as an inoculum [107].
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2.4 Antidepressants
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Antidepressants are a largely prescribed group of pharmaceuticals. Selective Serotonin Reuptake
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Inhibitors (SSRIs) are the most commonly used class of antidepressants that act by modulating the
346
levels of the neurotransmitter serotonin [108]. They are used for treating depression, anxiety, panic
347
disorder, obsessive compulsive disorder, eating disorders and social phobia.
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There are several studies that report environmental concentration of antidepressants [109], but very
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few studies that report their biodegradation owing to natural microbial communities.
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Fluoxetine (Prozac) is considered among the most toxic SSRIs in acute tests. Toxicological tests on
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aquatic organisms report EC50 values (48 h, algae) of 24 µg/L and LC50 (Lethal Concentration to
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50% of the population after 48h of exposure) of 2 mg/L [110]. It also has the potential to affect sex
353
hormones and modulate genes involved in the reproductive function of fish [110]. Fluoxetine has
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been reported in surface river waters at concentrations similar to EC50 values found in literature
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[111]. Environmental studies on fluoxetine persistence and biodegradation are very scarce. At an
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initial concentration of 1 µg/L this drug had an estimated half-life of 6-10 days in natural surface
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water owing to natural microbial degradation [112,113].
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2.5 Antiepileptics
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Carbamazepine is an anti-seizure drug used worldwide, is very persistent and has frequently been
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detected in surface water and occasionally in groundwater [114]. It is one of the most frequently
362
detected pharmaceuticals in European natural waters, at concentrations of tens of μg/L [26,52]. It
363
has been found in groundwater [20] and detected in soils and sediments where treated wastewater
364
is used to recharge groundwater [115] and for this reason it was suggested as a molecular marker
365
for anthropogenic contamination of water bodies recharged with reclaimed water or contaminated
366
riverine, groundwater and coastal environments by sewage [116,117]. Carbamazepine was also
367
found to accumulate from year to year in soil [118] and other studies report carbamazepine to be 12 Page 12 of 34
highly recalcitrant to microbial degradation in soil [119]. However, several fungi were found to
369
biodegrade it, such as Cunninghamella elegans ATCC 9245 which transforms it in a liquid culture
370
[120], Trametes versicolor and Ganoderma lucidum [61]. Gauthier et al [121] report that the
371
bacterium Rhodococcus rhodochrous and the fungus Aspergillus niger can degrade by
372
cometabolism carbamazepine (100 ppm) in liquid media containing glucose.e
373
Overall the field data indicate that carbamazepine is environmentally persistent, even if some
374
microbial populations are able to partially transform it in controlled laboratory experiments;
375
however knowledge on its complete degradation is not available so far.
376
Carbamazepine was found to reduce the bacterial biomass of a riverine biofilm [75]. It can be
377
classified as potentially harmful to aquatic organisms, because most of the acute toxicity data
378
report effective concentrations below 100 mg/L [116] far below environmentally-relevant
379
concentrations.
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2.6 Antibiotics
382
Antimicrobial pharmaceuticals are chemical substances that kill or inhibit the growth of
383
microorganisms, namely bacteria, viruses, fungi, and parasites [122,123]. The term antibiotic
384
commonly refers to antimicrobial agents that specifically target bacteria. Antibiotics may be natural
385
products, typically secondary metabolites of fungal or bacterial origin, semi-synthetic derivatives
386
of natural products, or entirely synthetic. There are currently about 250 different antibiotics
387
registered for use in human and veterinary medicine [124,125].
388
There is widespread concern that environmental emissions of antibiotics through wastewater
389
effluents and agricultural effluents may be promoting the development of antibiotic resistance [8].
390
In the presence of antibiotics bacteria that develop or acquire resistance mechanisms have a
391
selective advantage. Genes conferring resistance are often amenable to horizontal transfer, and
392
should these be acquired by human pathogens clinically relevant antibiotics may be compromised.
393
Governments are now recognizing that antibiotic resistance is a priority public health concern.
394
The resistance to antibiotics and other anti-infective agents constitutes a major threat to public
395
health and ought to be recognized as such more widely than it is at present. Consequently, the
396
European Union (EU) recommends the prudent use of antimicrobial agents both in human and
397
veterinary medicine [126].
398
Moreover, another important issue associated with antibiotic occurrence can be the disappearance
399
or inhibition of organic matter and chemical degrading microorganisms in sewage treatment plants,
400
soil and water ecosystems. Antibiotic occurrence can simultaneously entail both effects.
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Antibiotics can be grouped according to either their chemical structure or mode of action. They are
402
a diverse group of chemicals that can be divided into different sub-groups such as macrolides,
403
sulphonamides, tetracycline and others. They are often complex and can possess different
404
functionalities within the same molecule.
405
In addition to water ecosystem contamination from waste water treatment plants [5], the application
406
of manure [124,127] or use of sewage sludge for land amendment are direct sources of soil
407
antibiotic contamination. Moreover, through water run-off or leaching, antibiotics can also from the
408
soil reach surface water and groundwater, respectively [16,17,18]. Effluents from intensive
409
aquaculture systems can contaminate water because antibiotics are the most used drugs in these
410
sites as well [8].
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2.6.1 Macrolide antibiotics
413
Macrolide antibiotics belong to the Macrolide–Lincosamide–Streptogramin B class (MLS) of
414
antibiotics, which includes structurally different but functionally similar drugs, which all bind to
415
the 50S ribosomal subunit, blocking the path by which nascent peptides exit the ribosome [128].
416
Macrolides inhibit bacterial protein synthesis via binding preferentially to the 23S rRNA of the 50S
417
subunit, which overlaps with the binding site of lincosamides and streptogramin B, but differs from
418
those of phenicols like chloramphenicol, and pleuromutilins. Owing to this similar mode of action
419
resistance is also often linked. Macrolides and Lincosamides are generally bacteriostatic, which is
420
mainly time-dependent [128,129].
421
Macrolides and lincosamides are used for treatment of diseases that are common in food producing
422
animals, including medication of large groups of animals. In addition, MLS were listed by the
423
World Health Organization (WHO) in the First Meeting of the WHO Advisory Group on
424
Integrated Surveillance of Antimicrobial Resistance [130] as critically important for the treatment
425
of certain zoonotic infections in humans and risk mitigation measures are needed in order to reduce
426
the risk of a spread of resistance between animals and humans.
427
Some macrolides have been used for group and herd/flock medication for several decades. Before
428
the authorisation of growth promoters expired in EU, these molecules were added in low doses in
429
animal feed to increase feed conversion. Such use has not been allowed in the EU since 2006 (Reg.
430
1831/2003 EC), but there have been numerous products approved for prophylaxis likewise using
431
continuous low dosing [131,132].
432
Tylosin is a macrolide veterinary antibiotic that is active mostly against Gram-positive bacteria and
433
Mycoplasmas [133] and is also used in non-European Countries as a growth promoter. Tylosin
434
fate, degradation and effects have been studied mainly in soil, because its occurrence in the
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environment is due to the application of manure as a fertilizer. A multi-year study is being
436
undertaken in Ontario Canada consisting of a set of experimental plots treated each year, with
437
several annual consecutive applications with Sulfamethazine mixed with Tylosin and
438
Chlortetracycline at different concentrations (0, 0.1, 1.0, and 10.0 mg of each drug per kilogram of
439
soil) to simulate soil exposure from a spring application of pig manure. The persistence in soil of
440
these antibiotics was evaluated after the annual applications in the field. Tylosin was dissipated
441
more rapidly (half-life of about 2 days) in soils chronically exposed to the highest concentration of
442
the antibiotic mixture than in the non-treated control soils (half-life about 10 days) [28].
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2.6.2 Sulfonamide antibiotics
445
Sulfonamides are a class of synthetic antibiotics that interrupt the bacterial synthesis of folic acid,
446
essential for the synthesis of bacterial DNA. They have a bacteriostatic effect, limiting bacterial
447
growth [134].
448
Sulfonamides have been detected not only in soil after applying manure, but also in groundwater
449
below animal waste lagoons, as well as shallow groundwater from areas where animal waste had
450
been applied to fields [19]. Sulfamethazine concentrations in groundwater (0.6 ng/L) were found to
451
be two orders of magnitude lower than below animal waste lagoons [134].These observations were
452
consistent with a greater dilution and attenuation potential and a lower total load in soil-applied
453
manure than in lagoons. In addition, it was observed that the different reducing condition below the
454
lagoons contrasted with the manure-applied soils the more aerobic manure-treated soil may also
455
strongly influenced potential dissipation pathway [134].
456
As with tylosin (described above), the persistence of sulfamethazine has been evaluated in the
457
multi-year study field plot experiment being undertaken in Ontario Canada [28]. The
458
mineralization in soil of sulfamethazine after more than 10 annual applications in the field utilizing
459
[U-phenyl-14C]-sulfamethazine
460
sulfamethazine was mineralized much more rapidly in soils repeatedly exposed to 10 mg/kg. From
461
the activated soils, a Gram positive Microbacterium sp. strain C448 was isolated that grow at the
462
expense of sulfamethazine as sole carbon source, mineralizing the benzylic portion of the molecule
463
and stoichiometrically excreting the pyrimidine portion as an end product [28].
464
Sulfamethazine in manure and soils may affect soil microbial and enzyme activities. For example it
465
was found to have significant effects on soil respiration with an effective concentration (EC10) of
466
13 mg/kg in the first 2 days of an experimental test [135].
467
Gutierrez et al. [133] studied the influence of sulfonamides on microbial community patterns in
468
different soils by measuring Phospholipid Fatty Acid (PLFA) profiles and performing a polymerase
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was
evaluated.
Compared
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soil,
15 Page 15 of 34
chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA. Changes
470
in microbial community patterns due to antibiotics were observed when accompanied by the input
471
of a carbon source like glucose, straw or manure which supported bacterial growth. Moreover, in a
472
long term study sulfonamides, including sulfamethazine, were found to affect both the functioning
473
(i.e. enzyme activities) and the structural diversity (evaluated by PLFA) of a soil microbial
474
community at relatively low antibiotic concentrations (1-900 µg/g) [133].
475
2.6.3 Tetracycline antibiotics
476
Tetracyclines are among the most heavily used antibiotics either in human or veterinary therapy,
477
both for therapeutic purposes and as growth promoters in husbandry of cattle, swine and poultry
478
[129]. Tetracyclines are a large group of broad spectrum antibiotics obtained in different ways: by
479
fermentation of specific bacteria such as Streptomyces aureofaciens and Streptomyces rimosus
480
(tetracycline, chlortetracycline and oxytetracycline) or through semisynthetic (demeclocycline,
481
rolitetracycline and methacycline) or synthetic production (doxycycline and minocycline).
482
Tetracyclines have a low molecular weight, good oral absorption and efficient hepatic excretion
483
[136] and act as an inhibitor of protein synthesis by preventing the binding of aminoacyl-tRNA to
484
the bacterial ribosome. Chlortetracycline is one several antibiotics authorized in the United States
485
as growth promoters for cattle [137].
486
Due to their widespread use in intensive animal farming and aquaculture and their low metabolism
487
in the animals treated, chlortetracycline and oxytetracycline have a high potential for
488
environmental dissemination [138].
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2.7 Antiseptics and disinfectants
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Although antiseptics and disinfectants are not in a strict sense antibiotics, they are biocides used
492
extensively to kill or prevent the growth of microorganisms. There is a concern that biocide
493
resistance, conferred by efflux pumps for example, can also provide cross-resistance to clinically-
494
relevant antibiotics [139]. Triclosan and triclocarban are widely used in consumer products such as
495
antiseptics, disinfectants or preservatives in clinical settings, cosmetics, household cleaning
496
products, plastic materials, toys, paints, etc. They have been detected in all environments such as
497
surface waters (1.4 ng/L-40,000 ng/L), sediments (
S0731-7085(14)00576-7 http://dx.doi.org/doi:10.1016/j.jpba.2014.11.040 PBA 9827
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
7-8-2014 20-11-2014 22-11-2014
Please cite this article as: A.B. Caracciolo, E. Topp, P. Grenni, Pharmaceuticals in the environment: Biodegradation and effects on natural microbial communities. A Review, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.11.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical abstract
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Pharmaceuticals in the environment: Biodegradation and effects on natural microbial
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communities. A Review
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Anna Barra Caracciolo1*, Edward Topp2, Paola Grenni1
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*Corresponding Author
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1
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00015 Monterotondo (Rome), Italy, tel +39 0690672786, Fax +39 0690672787 e.mail:
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[email protected]
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Agriculture and Agri-Food Canada, 1391 Sandford Street, London ON Canada, N5V 4T3
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Istituto di Ricerca sulle Acque, Consiglio Nazionale delle Ricerche, Via Salaria km 29.300 -
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Abstract
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Environmental microorganisms play a key role in fundamental ecological processes such as
14
biogeochemical cycling and organic contaminant degradation. Microorganisms comprise a large
15
unexplored reservoir of genetic diversity and metabolic capability providing several ecosystem
16
services, most importantly the maintenance of soil and water quality. Pharmaceutical occurrence in
17
the environment can compromise microbial community structure and activities in different ways.
18
The fate of a pharmaceutical in soil or water depends on numerous factors, including its inherent
19
physic-chemical properties (e.g. water solubility, lipophilicity, vapour pressure), environmental
20
factors and climate conditions (e.g. temperature, incident radiation, pH) and most importantly the
21
presence and activity of microorganisms that possess the ability to biodegrade it. The presence of a
22
natural microbial community is a necessary prerequisite for an effective response to the various
23
chemicals that can contaminate an ecosystem. The recovery from contamination is only possible if
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toxicity does not hamper microbial activity. This review presents current knowledge on the effects
25
on natural microbial communities of some pharmaceuticals and of some biocides commonly found
26
as environmental microcontaminants.
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Highlights
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Biodegradation is the main process for eliminating the majority of pharmaceuticals
30
Pharmaceuticals can inhibit microbial activity
31
Evidence of pharmaceutical effects on natural microbial processes is scarce
32
Environmental microorganisms comprise an enormous reservoir of antibiotic resistance and
33
biodegradative capability
34 35
Keywords: anti-inflammatories; analgesic; blood lipid regulators; antidepressant, antiepileptic,
36
antibiotic, microbiocides 2 Page 2 of 34
37
1.
38
Pharmaceuticals are indispensable for the maintenance of public health and the quality of life.
39
Rapid advances in drug therapies to meet health challenges and their timely availability are
40
essential for a healthy society. Veterinary pharmaceuticals prevent and treat disease and increase
41
the efficiency of food production. Thousands of different active compounds are currently in use in
42
large quantities to treat or to prevent diseases [1,2]. Recently, the widespread detection of
43
pharmaceuticals in terrestrial and aquatic systems has engendered significant scientific and
44
regulatory concern [3,4]. Following administration, a great portion of human pharmaceuticals is
45
excreted unaltered or as an active metabolite, and ends up in wastewater treatment plants (WWTPs)
46
[5]. Effluents from WWTPs represent an important source point for aquatic exposure to
47
pharmaceuticals if they are not efficiently removed during the wastewater treatment process
48
[3,5,6,7]. Moreover, hospital and industrial wastewaters [8], uncontrolled and illegal drug disposal
49
[9] and aquaculture [10] can also be a significant source of aquatic contamination [11,12]. Many
50
drugs, owing to their widespread human and veterinary usage, are being continuously added to
51
ecosystems and can exhibit pseudo-persistence [13]. The use of sewage sludge (biosolids) and
52
manure as a crop fertilizer and irrigation with reclaimed water will entrain human and veterinarian
53
pharmaceuticals into agricultural soil [14,15,16,17]. Pharmaceuticals can be than leached from soil
54
to groundwater [8,18,19,20], and they are also detected in coastal marine waters [8,21,22,23,24].
55
Different classes of pharmaceuticals and biocides are widespread in the environment including anti-
56
inflammatories, analgesic, blood lipid regulators, antidepressants, antiepileptics and antimicrobials
57
[25,26,27,28,29].
58
There are concerns for both ecosystem health and a potential health risk for humans through the
59
consumption of food and water containing pharmaceuticals residues. Ecological studies generally
60
report that human pharmaceuticals are not acutely toxic to aquatic organisms, because their
61
environmental concentrations are typically very low [29,30]. A few studies have revealed evidence
62
for chronic long-term negative impacts of environmental pharmaceutical exposure on living
63
organisms and populations [29,30,31]. Pharmaceuticals are distinct from conventional pollutants
64
with respect to their potential ecotoxicology since they are specifically designed to be bioactive at
65
low concentrations. Moreover, they frequently act on specific targets that can be widely conserved
66
across multiple life forms. Environmental concentrations (in impacted aquatic systems typically
67
ranging from ng to µg per litre) may therefore be of concern, and sub-lethal effects on non-target
68
organism have in some cases been found [31,32, 33].
69
Overall, knowledge concerning the ecotoxicology and sub-lethal effects in water and soil
70
organisms is scarce, but some experimental studies show that some pharmaceuticals can disrupt or
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alter the endocrine system [34], induce pathogen resistance, as in the case of several antibiotics
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[35,36], and also have detrimental effects on natural microbial communities and their key functions
73
[37,38].
74
Key factors in determining environmental exposure concentrations include volume of usage, the
75
degree of release to the environment, and persistence once in the environment. Environmental
76
persistence depends on a variety of factors, such as a pharmaceuticals inherent physicochemical
77
properties, environmental factors (e.g. light, temperature, pH) and most importantly the presence
78
and activity of microorganisms with the capability to degrade it via metabolic and/or co-metabolic
79
pathways [39,40]. The biodegradation by metabolic pathways refers to the use of a chemical as a
80
source of energy, carbon, nitrogen or other nutrients. Co-metabolism is the fortuitous breakdown of
81
a contaminant by an enzyme or cofactor that is produced during microbial metabolism of another
82
compound.
83
Microorganisms are important degraders of organic matter and of xenobiotics, including
84
pharmaceuticals, and provide products as nutrients to other organisms in the food web [41].
85
Microbial communities are therefore clearly vital for maintaining ecosystem functioning. Changes
86
within microbial functional groups are correlated to changes in ecosystem processes [42,43,44].
87
Consequently, they are essential in the overall processes that contribute to the quality state of
88
natural ecosystem.
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2. Biodegradation of pharmaceuticals and effects on natural microbial communities
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Natural microbial communities in soil and water are key players in several processes controlling
92
the quality of ecosystems and regulating the fate of pollution released into the environment and in
93
this sense, they provide the Ecosystem Service termed “Regulation” [45]. Microorganisms are
94
involved in ecosystem self-purification processes since they can degrade contaminants by
95
metabolic and/or co-metabolic pathways. Biodegradation is considered the most important process
96
for eliminating the majority of xenobiotics, including pharmaceuticals [28,43,45]. Recovery from
97
contamination is possible only if the toxicity of the molecules does not inhibit microbial activity.
98
However, knowledge on biodegradation of drugs and their effects on ecological processes driven
99
by microorganisms is quite scarce, so far.
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Pharmaceuticals include hundreds of substances with widely varying chemical-physical properties,
101
environmental behaviour and biochemical activities [8]. Pharmaceuticals typically enter the
102
environment in complex effluents, and thus natural microbial communities are exposed to a
103
mixture of active substances. In this review we report some studies regarding the effects of some
104
antibiotics (i.e. tylosin, sulfamethazine, chlortetracycline), other pharmaceuticals (naproxen, 4 Page 4 of 34
105
ibuprofen, diclofenac, gemfibrozil, paracetamol, clofibric acid, fluoxetine, carbamazepine), and
106
biocides (triclosan and triclocarban) on natural microbial communities. These compounds were
107
selected because they are among the most commonly found as environmental microcontaminants
108
[46,47,48,49,50]. In Table 1 the list of the selected compounds is reported.
109
2.1 Non-steroidal anti-inflammatory drugs
111
Analgesics are pain-relief drugs that include narcotic analgesics, non-narcotic analgesics and non-
112
steroidal anti-inflammatory drugs (NSAID) used to alleviate the pain present in almost all diseases.
113
These compounds work by blocking cyclooxygenase (COX) enzymes, which catalyze the synthesis
114
of different prostaglandins from arachidonic acid [51]. NSAIDs include naproxen, ibuprofen and
115
diclofenac, which are among the most frequently detected pain killers in surface water, at
116
concentrations ranging from tens of ng/L to hundreds of µg/L [26,46,47].
117
Naproxen is a polar pharmaceutical commonly found as a microcontaminant of rivers [25,52,53]
118
and also of groundwater from aquifers recharged with reclaimed water [54].
119
Naproxen persistence in surface water has been reported in some studies [25,53,55]. Photolytic
120
degradation was found to be an important abiotic degradation process, although it is not able to
121
degrade naproxen thoroughly [56]. Moreover, the photoproducts were found to be significantly
122
more toxic than the parent compound [56].
123
In recent microcosm studies naproxen (100 µg/L initial concentration) was found to be
124
biodegraded by natural bacterial populations from river water, belonging to Alpha- and Gamma-
125
Proteobacteria [25,57] with half-lives of about 20-30 days. In another recent work a Gamma-
126
Proteobacteria, Stenotrophomonas maltophilia KB2 strain, an aromatic compound degrader, was
127
found to metabolically transform 28% of initial naproxen concentration in culture media (6 mg/L;
128
30°C) within 35 days. However, the naproxen degradation was accompanied by a decrease in the
129
number of bacteria cells. Most likely, this compound was not a sufficient carbon and energy source
130
for strain KB2. At the same time, it was able to co-metabolically degrade, with addition of glucose
131
or phenol, 78% and 40% of naproxen, respectively [58].
132
Naproxen biodegradation and mineralization by aerobic population was found in agricultural soils
133
[15]. Three different agricultural soils (sandy loam, loam and silt loam), which have never been
134
exposed to the drug, were treated with 5 µg naproxen/g soil and incubated in the dark at 30°C.
135
During the 27 days of incubations, [O-14CH3]naproxen was rapidly mineralized to 14CO2 with very
136
comparable kinetics in the three soils. In the loam soil naproxen mineralization was hastened by the
137
addition of liquid municipal biosolids, and when the experiment was performed at 4°C, a 35 day
138
lag-phase was observed. An enrichment culture obtained from aerobically digested liquid
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5 Page 5 of 34
municipal biosolid in a mineral salts medium with naproxen as the sole carbon source was able to
140
convert the parent compound to the corresponding naphthol, O-desmethyl naproxen [15]. Lin and
141
Gan [59], comparing aerobic and anaerobic degradation of naproxen (40 µg/kg; 20°C) in soil
142
microcosms found that it was biodegraded aerobically with different half-lives values depending on
143
specific soil characteristics (from 17 to 69 days for the very gravelly fine loamy sand soil and for
144
loam soil, respectively), while it was persistent in anaerobic conditions.
145
A concentration of 50 ng/g of naproxen was biodegraded co-metabolically in another soil
146
microcosm experiment using reclaimed water with a half-life of 9 to 18 days [60].
147
The different half-life values reported in the various studies above described may be explained by
148
the initial biotic an abiotic characteristics of the sample (microbial community composition, soil
149
type, moisture content, temperature etc.) that may affect degradation, together with the specific
150
laboratory experimental conditions.
151
Several studies pointed out the role of fungi in naproxen transformation [43,61,62]. Marco-Urrea et
152
al. [62] found that the fungus Trametes versicolor was able to degrade naproxen in a few hours in
153
degradation experiments performed at two different concentrations (10 mg/L and 55 μg/L).
154
Moreover, Rodarte-Morales et al. [43] reported the complete removal of naproxen (1 mg/L) in 7
155
days by the fungi Bjerkandera sp. R1 and Bjerkandera adusta, and in 4 days by Phanerochaete
156
chrysosporium.
157
The overall results of these degradation experiments suggest that naproxen is not an intrinsically
158
persistent compound and it is biodegradable in aerobic conditions. However, since it is chronically
159
discharged into surface water from waste water treatment plants, owing to large volume and daily
160
use among the human population, it is not only constantly found in surface water, but also at
161
concentrations higher than most other, more intrinsically persistent drugs. Consequently, naproxen
162
can be considered a pseudo-persistent compound in surface waters and its effects on ecosystem
163
functioning warrants investigation. Regarding this aspect some detrimental effects on microbial
164
communities were recently found. In surface water microcosms, acute effects on the river microbial
165
community were observed three hours after addition of 100 μg/L naproxen. The microbial cell
166
viability and the Beta-Proteobacteria group decreased significantly [57] and a toxic effect on the
167
bacterial populations involved in key ecosystem functioning was not excluded, because Beta-
168
Proteobacteria include the Ammonia Oxidizing Bacteria (AOB) involved in the nitrogen cycle
169
[63]. A toxic effect on AOB was also found in a waste water treatment plant when naproxen was
170
present [64].
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6 Page 6 of 34
Ibuprofen, like naproxen, is a commonly used over the counter drug and it is also used in veterinary
172
medicine. It is one of the most abundant polar pharmaceuticals detected in river waters [26,30,52].
173
Moreover, it was also found in groundwater at concentrations up to 12 µg/L [54].
174
Ibuprofen is a chiral pharmaceutical and two different isomers can be detected in the environment:
175
S-(+)-ibuprofen, which is the pharmacologically active isomer, and R-(−)-enantiomer which is the
176
inactive enantiomer [65]. Ibuprofen was found to be degraded in wastewaters and surface waters in
177
both field observations and laboratory incubations, with the S-enantiomer biodegraded faster,
178
because microorganisms mainly utilize this enantiomer [66]. However, it is not clear to what extent
179
the change in enantiomer composition was due to biodegradation versus enantiomerization, as
180
happens with the structurally similar phenoxyalkanoic herbicides [66]. Ibuprofen metabolites are
181
also chiral and were found to be more toxic than the parent compound [61].
182
Ibuprofen degradation was not observed in sterile river water (150 µg/L) [67] and in sterile soil
183
(1.25 µg/g soil) experiments [68], suggesting that abiotic degradation has a negligible role in its
184
transformation.
185
Biodegradation of ibuprofen (20 µg/L) was observed in a few hours in river water including
186
sediment microcosms [69]. Biodegradation of an initial concentration of 100 µg/L of ibuprofen was
187
also observed in a laboratory study using microbial biofilms from river water. The pharmaceutical
188
degradation occurred between 4-8 days after its initial addition [67]. In soil, Carr et al. [68] in a 14-
189
day study reported ibuprofen persistence (initial concentration: 1.25 µg/g soil) to be highly variable
190
depending on the moisture content. The calculated half-life ranged from 30 to 1,706 days in un-
191
saturated and water-saturated soil, respectively [68]. In another soil microcosm experiments
192
ibuprofen biodegradation was found to be quite rapid with the half-life ranging from 0.3-0.9 days
193
with an initial concentration of 0.050 µg/g soil, and between 3-7 days with an initial concentration
194
of 5 µg/g soil [59]. In a liquid medium, the bacterium Nocardia sp. (strain NRRL 5646) was found
195
to be able to degrade the R(-)-ibuprofen isomer in two major metabolites, ibuprofenol and the
196
corresponding acetate derivative [69]. The Alpha-Proteobacteria Sphingomonas sp., isolated from
197
an enrichment culture (500 mg/L ibuprofen) using an inoculum from a wastewater treatment plant,
198
was able to degrade ibuprofen as the sole carbon source [70]. Quintana et al. [71] found that when
199
ibuprofen was the sole growth substrate, under aerobic conditions, the drug was not transformed
200
after 28 days in a laboratory test using fresh sludge from a membrane bioreactor as inoculum.
201
However, when an additional carbon source was added (powdered milk), co-metabolism of
202
ibuprofen was completed at 22 days. Consequently, Ibuprofen may be more easily metabolized
203
when other carbon sources are available.
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Ibuprofen is biodegraded by several fungi. Marco-Urrea et al. [62] found that the fungi Trametes
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versicolor, Irpex lacteus, Ganoderma lucidum and Phanerochaete chrysosporium degrade
206
ibuprofen (10 mg/L) in liquid media within 7 days after its addition.
207
Clearly microorganisms play an important role in ibuprofen degradation, although its
208
transformation is a complex process and two isomers of it occur in the environment, consequently
209
the degradation pathways and metabolite formation need to be clarified [72].
210
There has been little research into ibuprofen’s effects on microorganisms. However, some studies
211
report that ibuprofen has significant antibacterial activity against Gram-positive bacteria [73] and
212
also an antifungal activity [74]. Ibuprofen was found to reduce the overall bacterial biomass of a
213
riverine biofilm community originated from rotating annular bioreactors and exposed for 8 weeks
214
to a concentration of 10 µg/L; moreover, the microbial biofilm composition, analyzed by the
215
Fluorescence in Situ Hybridization method, changed during the experiment. In particular,
216
Cyanobacteria decreased significantly together with Gamma-Proteobacteria and Gram-positive
217
bacteria Firmicutes, while Alpha-, Beta-Proteobacteria, Cytophaga-Flavobacteria and sulfate-
218
reducing bacteria increased, suggesting a role of these groups in Ibuprofen biodegradation [75].
219
Ibuprofen was found to influence the growth of a river sediment microbial community in a liquid
220
media with sucrose and glucose, at an environmental concentration of 50 ng/L [76].
221
Corcol et al. [77] performed the Pollution-Induced Community Tolerance (PICT) short-term dose-
222
response test in fluvial biofilm communities affected by wastewater treatment effluents. Biofilms
223
were exposed for 48 h to increasing concentrations of ibuprofen, alone or in the co-presence of
224
diclofenac. Two different endpoints were used to assess biofilm tolerance as Effective
225
Concentration 50 (EC50): the photosynthetic efficiency of autotrophic organisms, and the beta-
226
glucosidase activity of heterotrophic organisms. The EC50 values of photosynthetic efficiency and
227
of beta-glucosidase activity indicated that the microbial communities were less tolerant to the
228
mixture of the two pharmaceuticals.
229
Despite ibuprofen has been classified as an easily degradable compound [78], an environmental
230
risk assessment study indicates that it may represent a risk for the aquatic environment [79]. In fact
231
the calculated PEC (Predicted Environmental Concentration) for water receiving wastewater
232
effluent
233
Concentration/Predicted No EffectConcentration) was >1 [79]. Ibuprofen has been proposed to be
234
identified as a priority substance under the Water Frame Directive because it is suspected of
235
influencing sex steroid hormones in both vertebrates and invertebrates and having cytogenetic
236
effects in freshwater bivalves [80,81]. However, the inclusion of ibuprofen was rejected in January
237
2012 owing to a lack of sufficient evidence of significant risks to aquatic environments [82].
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was
about
9
µg/L
and
the
ratio
PEC/PNEC
(Predicted
Environmental
8 Page 8 of 34
Diclofenac is a polar pharmaceutical compound mostly used as the sodium salt diclofenac-Na in
239
human and veterinary medicine to reduce inflammation and pain [83,84]. It is a non-steroidal anti-
240
inflammatory drug and is included in a number of different formulations with generic names such
241
as Diclometin, Diclomex and Voltaren. Diclofenac is one of the most commonly detected
242
pharmaceuticals in European rivers [26,50]. It has also been detected in groundwater at a maximum
243
concentration of about 4 µg/L [20,85] and in soil irrigated with reclaimed water at concentrations
244
ranging from 0.35 to 1.16 µg/kg [86].
245
Diclofenac (0.1 μg/g) was found to be rapidly mineralized in various agricultural soils with half-
246
lives 25%) on nitrification
287
and denitrification rates at concentrations ⩾250 mg/L
288
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2.3 Blood Lipid regulators
290
Lipid abnormalities are among the key risk factors for cardiovascular disease and lowering low-
291
density lipoprotein (LDL) cholesterol concentrations can significantly reduce the incidence of
292
coronary heart disease. In the decade 1995-2005, the volume of cholesterol-lowering drugs
293
prescribed almost tripled [95]. There are two main groups of blood lipid lowering agents, with
294
different modes of action: statins and fibrates. Although statins (e.g. simvastatin, omeprazole or
295
paroxetine) are among the pharmaceuticals most consumed without medical prescription [96], they
296
are rarely found as environmental microcontaminants, because their excretion occurs mainly as
297
metabolites [96]. Fibrates or fibric acid derivatives are a class of drugs which inhibit the production
298
of very low-density lipoprotein (VLDL) and reduce plasma triglyceride level. Gemfibrozil and
299
clofibric acid, the main metabolite of clofibrate, are among the pharmaceuticals most frequently
300
detected in European waters [26,97]. 10 Page 10 of 34
Gemfibrozil is an acidic drug and a blood lipid-regulating agent, clinically prescribed since the
302
early 1980s for patients with a high risk of coronary heart disease [13,97,98]. It is metabolised in
303
the liver and approximately 70% is excreted through urine, mostly as a glucuronide conjugate [98].
304
Glucuronides are fundamentally inactive, but are readily cleaved during sewage treatment releasing
305
the active compound. Although the International Agency for Research on Cancer has classified it in
306
group 3, i.e. not classifiable regarding its carcinogenicity for humans [99]. Gemfibrozil has been
307
shown to induce peroxisome proliferation in rodents and in eels leading to liver cancer and it can
308
also have some detrimental effects on non-target aquatic organisms [31,100]. Gemfibrozil is found
309
in surface waters at concentrations ranging from ng/L to µg/L [26,50,101].
310
Gemfibrozil was initially reported to be not biodegradable [101], but the fungus Cunninghamella
311
elegans ATCC 9245 was found to degrade it in a liquid culture by hydroxylation processes [103].
312
Several studies report gemfibrozil to be a quite persistent compound in water with half-lives ranging
313
from 70 to 288 days in surface water [25,55]. In sterile river water incubated in the dark,
314
gemfibrozil was not degraded, whereas in the same microbiologically active water a significant
315
decrease in concentration (half-life of about 70 days) was observed, suggesting the role of
316
microorganisms in its biodegradation [25]. Moreover, FISH analysis of the microbial community,
317
revealed that the Gamma-Proteobacteria group increased during gemfibrozil biodegradation,
318
indicating that some populations of this group might be involved in its transformation [25].
319
Although, Lin et al [102] reported gemfibrozil degradation with a half-life less than 1 day and Zhou
320
et al [104] found that a strain of Bacillus sp., isolated from the sludge of a WWTP, rapidly degraded
321
gemfibrozil in a liquid culture,
322
persistence [25, 55, 105].
323
Gemfibrozil has been shown to be inhibitory to natural microbial communities. For example, a
324
detrimental effect was observed in natural river water microcosms three hours after pharmaceutical
325
addition (100 µg/L) [25]. Bacterial community viability significantly decreased, compared to
326
control microcosms, and this was ascribed to an initial acute toxic effect from this drug on the
327
overall bacterial community [25]. Similar concentrations were effective both with AOB from a
328
wastewater treatment plant [64] and in aquatic toxicological tests [106].
329
Clofibric acid is a polar compound and is the active metabolite of clofibrate lipid regulators (e.g.
330
clofibrate, etofyllin). It has been detected in water, including drinking water, for at least twenty
331
years. Buser et al. [88] found concentrations of clofibric acid of 7.8 ng/L in the North Sea, a higher
332
concentration than that of mecoprop, a pesticide structurally related to clofibric acid. Buser’s
333
conclusions were that 50-100 tons/year of clofibric acid have been entering the North Sea and that
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11 Page 11 of 34
it was the drug most commonly found in open waters. Recent investigations report a maximum
335
concentration in natural surface water of more than 240 ng/L [47].
336
Clofibric acid is regarded as extremely persistent, with an estimated environmental residence time
337
of 21 years [95]. Clofibric acid was not biodegraded in laboratory reactors containing river biofilm
338
[71]. On the other hand, in an aerobic sequencing batch reactor clofibric acid was biodegraded co-
339
metabolically by a mixed microbial culture, dominated by AOBs. This culture, originating from a
340
WWTP was firstly acclimatized to a chlorinated aromatic molecule with a structure similar to
341
clofibric acid and it was then used as an inoculum [107].
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2.4 Antidepressants
344
Antidepressants are a largely prescribed group of pharmaceuticals. Selective Serotonin Reuptake
345
Inhibitors (SSRIs) are the most commonly used class of antidepressants that act by modulating the
346
levels of the neurotransmitter serotonin [108]. They are used for treating depression, anxiety, panic
347
disorder, obsessive compulsive disorder, eating disorders and social phobia.
348
There are several studies that report environmental concentration of antidepressants [109], but very
349
few studies that report their biodegradation owing to natural microbial communities.
350
Fluoxetine (Prozac) is considered among the most toxic SSRIs in acute tests. Toxicological tests on
351
aquatic organisms report EC50 values (48 h, algae) of 24 µg/L and LC50 (Lethal Concentration to
352
50% of the population after 48h of exposure) of 2 mg/L [110]. It also has the potential to affect sex
353
hormones and modulate genes involved in the reproductive function of fish [110]. Fluoxetine has
354
been reported in surface river waters at concentrations similar to EC50 values found in literature
355
[111]. Environmental studies on fluoxetine persistence and biodegradation are very scarce. At an
356
initial concentration of 1 µg/L this drug had an estimated half-life of 6-10 days in natural surface
357
water owing to natural microbial degradation [112,113].
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2.5 Antiepileptics
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Carbamazepine is an anti-seizure drug used worldwide, is very persistent and has frequently been
361
detected in surface water and occasionally in groundwater [114]. It is one of the most frequently
362
detected pharmaceuticals in European natural waters, at concentrations of tens of μg/L [26,52]. It
363
has been found in groundwater [20] and detected in soils and sediments where treated wastewater
364
is used to recharge groundwater [115] and for this reason it was suggested as a molecular marker
365
for anthropogenic contamination of water bodies recharged with reclaimed water or contaminated
366
riverine, groundwater and coastal environments by sewage [116,117]. Carbamazepine was also
367
found to accumulate from year to year in soil [118] and other studies report carbamazepine to be 12 Page 12 of 34
highly recalcitrant to microbial degradation in soil [119]. However, several fungi were found to
369
biodegrade it, such as Cunninghamella elegans ATCC 9245 which transforms it in a liquid culture
370
[120], Trametes versicolor and Ganoderma lucidum [61]. Gauthier et al [121] report that the
371
bacterium Rhodococcus rhodochrous and the fungus Aspergillus niger can degrade by
372
cometabolism carbamazepine (100 ppm) in liquid media containing glucose.e
373
Overall the field data indicate that carbamazepine is environmentally persistent, even if some
374
microbial populations are able to partially transform it in controlled laboratory experiments;
375
however knowledge on its complete degradation is not available so far.
376
Carbamazepine was found to reduce the bacterial biomass of a riverine biofilm [75]. It can be
377
classified as potentially harmful to aquatic organisms, because most of the acute toxicity data
378
report effective concentrations below 100 mg/L [116] far below environmentally-relevant
379
concentrations.
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2.6 Antibiotics
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Antimicrobial pharmaceuticals are chemical substances that kill or inhibit the growth of
383
microorganisms, namely bacteria, viruses, fungi, and parasites [122,123]. The term antibiotic
384
commonly refers to antimicrobial agents that specifically target bacteria. Antibiotics may be natural
385
products, typically secondary metabolites of fungal or bacterial origin, semi-synthetic derivatives
386
of natural products, or entirely synthetic. There are currently about 250 different antibiotics
387
registered for use in human and veterinary medicine [124,125].
388
There is widespread concern that environmental emissions of antibiotics through wastewater
389
effluents and agricultural effluents may be promoting the development of antibiotic resistance [8].
390
In the presence of antibiotics bacteria that develop or acquire resistance mechanisms have a
391
selective advantage. Genes conferring resistance are often amenable to horizontal transfer, and
392
should these be acquired by human pathogens clinically relevant antibiotics may be compromised.
393
Governments are now recognizing that antibiotic resistance is a priority public health concern.
394
The resistance to antibiotics and other anti-infective agents constitutes a major threat to public
395
health and ought to be recognized as such more widely than it is at present. Consequently, the
396
European Union (EU) recommends the prudent use of antimicrobial agents both in human and
397
veterinary medicine [126].
398
Moreover, another important issue associated with antibiotic occurrence can be the disappearance
399
or inhibition of organic matter and chemical degrading microorganisms in sewage treatment plants,
400
soil and water ecosystems. Antibiotic occurrence can simultaneously entail both effects.
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Antibiotics can be grouped according to either their chemical structure or mode of action. They are
402
a diverse group of chemicals that can be divided into different sub-groups such as macrolides,
403
sulphonamides, tetracycline and others. They are often complex and can possess different
404
functionalities within the same molecule.
405
In addition to water ecosystem contamination from waste water treatment plants [5], the application
406
of manure [124,127] or use of sewage sludge for land amendment are direct sources of soil
407
antibiotic contamination. Moreover, through water run-off or leaching, antibiotics can also from the
408
soil reach surface water and groundwater, respectively [16,17,18]. Effluents from intensive
409
aquaculture systems can contaminate water because antibiotics are the most used drugs in these
410
sites as well [8].
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2.6.1 Macrolide antibiotics
413
Macrolide antibiotics belong to the Macrolide–Lincosamide–Streptogramin B class (MLS) of
414
antibiotics, which includes structurally different but functionally similar drugs, which all bind to
415
the 50S ribosomal subunit, blocking the path by which nascent peptides exit the ribosome [128].
416
Macrolides inhibit bacterial protein synthesis via binding preferentially to the 23S rRNA of the 50S
417
subunit, which overlaps with the binding site of lincosamides and streptogramin B, but differs from
418
those of phenicols like chloramphenicol, and pleuromutilins. Owing to this similar mode of action
419
resistance is also often linked. Macrolides and Lincosamides are generally bacteriostatic, which is
420
mainly time-dependent [128,129].
421
Macrolides and lincosamides are used for treatment of diseases that are common in food producing
422
animals, including medication of large groups of animals. In addition, MLS were listed by the
423
World Health Organization (WHO) in the First Meeting of the WHO Advisory Group on
424
Integrated Surveillance of Antimicrobial Resistance [130] as critically important for the treatment
425
of certain zoonotic infections in humans and risk mitigation measures are needed in order to reduce
426
the risk of a spread of resistance between animals and humans.
427
Some macrolides have been used for group and herd/flock medication for several decades. Before
428
the authorisation of growth promoters expired in EU, these molecules were added in low doses in
429
animal feed to increase feed conversion. Such use has not been allowed in the EU since 2006 (Reg.
430
1831/2003 EC), but there have been numerous products approved for prophylaxis likewise using
431
continuous low dosing [131,132].
432
Tylosin is a macrolide veterinary antibiotic that is active mostly against Gram-positive bacteria and
433
Mycoplasmas [133] and is also used in non-European Countries as a growth promoter. Tylosin
434
fate, degradation and effects have been studied mainly in soil, because its occurrence in the
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14 Page 14 of 34
environment is due to the application of manure as a fertilizer. A multi-year study is being
436
undertaken in Ontario Canada consisting of a set of experimental plots treated each year, with
437
several annual consecutive applications with Sulfamethazine mixed with Tylosin and
438
Chlortetracycline at different concentrations (0, 0.1, 1.0, and 10.0 mg of each drug per kilogram of
439
soil) to simulate soil exposure from a spring application of pig manure. The persistence in soil of
440
these antibiotics was evaluated after the annual applications in the field. Tylosin was dissipated
441
more rapidly (half-life of about 2 days) in soils chronically exposed to the highest concentration of
442
the antibiotic mixture than in the non-treated control soils (half-life about 10 days) [28].
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443
2.6.2 Sulfonamide antibiotics
445
Sulfonamides are a class of synthetic antibiotics that interrupt the bacterial synthesis of folic acid,
446
essential for the synthesis of bacterial DNA. They have a bacteriostatic effect, limiting bacterial
447
growth [134].
448
Sulfonamides have been detected not only in soil after applying manure, but also in groundwater
449
below animal waste lagoons, as well as shallow groundwater from areas where animal waste had
450
been applied to fields [19]. Sulfamethazine concentrations in groundwater (0.6 ng/L) were found to
451
be two orders of magnitude lower than below animal waste lagoons [134].These observations were
452
consistent with a greater dilution and attenuation potential and a lower total load in soil-applied
453
manure than in lagoons. In addition, it was observed that the different reducing condition below the
454
lagoons contrasted with the manure-applied soils the more aerobic manure-treated soil may also
455
strongly influenced potential dissipation pathway [134].
456
As with tylosin (described above), the persistence of sulfamethazine has been evaluated in the
457
multi-year study field plot experiment being undertaken in Ontario Canada [28]. The
458
mineralization in soil of sulfamethazine after more than 10 annual applications in the field utilizing
459
[U-phenyl-14C]-sulfamethazine
460
sulfamethazine was mineralized much more rapidly in soils repeatedly exposed to 10 mg/kg. From
461
the activated soils, a Gram positive Microbacterium sp. strain C448 was isolated that grow at the
462
expense of sulfamethazine as sole carbon source, mineralizing the benzylic portion of the molecule
463
and stoichiometrically excreting the pyrimidine portion as an end product [28].
464
Sulfamethazine in manure and soils may affect soil microbial and enzyme activities. For example it
465
was found to have significant effects on soil respiration with an effective concentration (EC10) of
466
13 mg/kg in the first 2 days of an experimental test [135].
467
Gutierrez et al. [133] studied the influence of sulfonamides on microbial community patterns in
468
different soils by measuring Phospholipid Fatty Acid (PLFA) profiles and performing a polymerase
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was
evaluated.
Compared
to
historically-untreated
soil,
15 Page 15 of 34
chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA. Changes
470
in microbial community patterns due to antibiotics were observed when accompanied by the input
471
of a carbon source like glucose, straw or manure which supported bacterial growth. Moreover, in a
472
long term study sulfonamides, including sulfamethazine, were found to affect both the functioning
473
(i.e. enzyme activities) and the structural diversity (evaluated by PLFA) of a soil microbial
474
community at relatively low antibiotic concentrations (1-900 µg/g) [133].
475
2.6.3 Tetracycline antibiotics
476
Tetracyclines are among the most heavily used antibiotics either in human or veterinary therapy,
477
both for therapeutic purposes and as growth promoters in husbandry of cattle, swine and poultry
478
[129]. Tetracyclines are a large group of broad spectrum antibiotics obtained in different ways: by
479
fermentation of specific bacteria such as Streptomyces aureofaciens and Streptomyces rimosus
480
(tetracycline, chlortetracycline and oxytetracycline) or through semisynthetic (demeclocycline,
481
rolitetracycline and methacycline) or synthetic production (doxycycline and minocycline).
482
Tetracyclines have a low molecular weight, good oral absorption and efficient hepatic excretion
483
[136] and act as an inhibitor of protein synthesis by preventing the binding of aminoacyl-tRNA to
484
the bacterial ribosome. Chlortetracycline is one several antibiotics authorized in the United States
485
as growth promoters for cattle [137].
486
Due to their widespread use in intensive animal farming and aquaculture and their low metabolism
487
in the animals treated, chlortetracycline and oxytetracycline have a high potential for
488
environmental dissemination [138].
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490
2.7 Antiseptics and disinfectants
491
Although antiseptics and disinfectants are not in a strict sense antibiotics, they are biocides used
492
extensively to kill or prevent the growth of microorganisms. There is a concern that biocide
493
resistance, conferred by efflux pumps for example, can also provide cross-resistance to clinically-
494
relevant antibiotics [139]. Triclosan and triclocarban are widely used in consumer products such as
495
antiseptics, disinfectants or preservatives in clinical settings, cosmetics, household cleaning
496
products, plastic materials, toys, paints, etc. They have been detected in all environments such as
497
surface waters (1.4 ng/L-40,000 ng/L), sediments (
