Determination of Emerging Substances in the Danube

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Research Article Determination of Emerging Substances in the Danube and Potential Risk Evaluation† Nevena N. Grujic Letic 1, Maja Lj. Milanovic 1, Nataša B. Milic 1, Mirjana B. Vojinovic Miloradov 2, Jelena R. Radonic 2, Ivana J. Mihajlovic 2, *, and Maja M. Turk Sekulic 2 1

University of Novi Sad, Faculty of Medicine, Novi Sad, Serbia


University of Novi Sad, Faculty of Technical Sciences, Novi Sad, Serbia

Correspondence: I. J. Mihajlovic, University of Novi Sad, Faculty of Technical Sciences, Trg Dositeja Obradovica 6, 21000 Novi Sad, Serbia

E-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: [10.1002/clen.201400402]

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Received 08 June 2014; Revised 08 June 2014; Accepted 10 July 2014

Abstract Seasonal variations of caffeine and antibiotic concentrations were investigated along the selected sites of the Danube using solid phase extraction followed by high-performance liquid chromatography (HPLC). The potential risks on the aquatic organisms were evaluated. HPLC methods for caffeine and antibiotic determination were modified and validated showing good accuracy, repeatability, selectivity and robustness. The obtained results confirmed the presence of caffeine at four sampling points, near the wastewater discharges.The mean caffeine concentrations for summer, autumn, winter and spring were 24.78, 26.83, 24.61, and 86.29 ng/L, respectively. The analysed antibiotics (sulfamethoxazole, chloramphenicol and tiamuline) were under the limit of detection in all tested samples taken from the Danube. The maximum risk indexes (MaxRIs) were calculated for caffeine at each sampling site. Four out of seven selected sampling sites showed sublethal effects on the aquatic organisms with 10 < MaxRI < 100 (class II), while the other three had no potential risk for aquatic organisms. The highest potential risk was calculated for the sampling site in the vicinity of one of the bigest wastewater discharges.

Abbreviations: HPLC, high-performance liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; MaxRI, maximum RI; RI, risk index; SPE, solid phase extraction; T U, toxicity unit

Keywords: Antibiotics, Caffeine, Ecotoxicological impact, Pharmaceutical residues, Water quality



Surface water is often directly or indirectly used as a source for a drinking water production or for leisure and recreational activities. The safety of surface water systems should be an imperative for each society since the contamination of these systems can cause serious effects on human health [1--3]. Recently recognized environmental pollutants named as emerging substances are widely perceived as unregulated substances [4]. The emerging substances include global organic and some inorganic contaminants, such as pharmaceuticals, personal care products, flame retardants, endocrine-modulating compounds, industrial chemicals, nanoparticles, biological metabolites, toxins and many other chemicals [5, 6]. Today, over 750 most frequently discussed emerging substances are included in the open and dynamic NORMAN list of 27 different substance classes and subclasses ( The presence of the pharmaceutical residues in the environment has become the subject of growing concern in the past decade. Due to the continuous input that leads to the long-term adverse effects on the aquatic and terrestrial organisms, the special attention is being paid to their concentration levels in the aquatic environment [7, 8]. Most of the pharmaceuticals cannot be removed completely by the wastewater treatment plants and consequently, they are released into natural water. Pharmaceuticals are generally highly water soluble and poorly degradable. Therefore, they can pass through all the natural filtrations and reach groundwater and ultimately drinking water [9--11]. The major task in numerous studies [8, 12] was to find an appropriate indicator to locate the source of surface water pollution. Caffeine, as an emerging substance, is found to be a good indicator for human sewage because of its relatively high concentrations in surface water and its unambiguous anthropogenic origin [13--15]. Caffeine is a

methylxanthine derivative that can be naturally found in coffee and cocoa beans, tea leaves and other plants [16, 17]. It is considered to be one of the most commonly consumed drugs. More than 80 % of the world's population consume caffeine daily [18, 19]. The Europeans are found to be the world's largest consumers of caffeine of approximately 4.6 kg/person per year [20]. This pharmacologically active substance stimulates the central nervous system, increases heartbeat rate, dilates blood vessels and has weak diuretic effect [21--23]. The main caffeine paths entering the wastewater stream are either via urine or caffeine-containing products discharged through household pipelines or sewers [14]. Monitoring caffeine can be of fundamental issue in the stressed urban aquatic environments where frequent accidental ruptures of sewer lines and discharges of untreated effluents undermine water quality. Nowadays, antibiotics are extensively used and despite their positive effects, their serious overuse has become a new environmental problem. They are mainly excreted as glucuronide and sulphate conjugates that can easily hydrolyse in the environment. However, large amounts of antibiotics are eliminated without being metabolised and in an unchanged form are discharged into the water system. The antibiotics in the wastewater are frequently detected in ppb, ppt or lower concentrations in the aquatic environment. Although the growing resistance of the vast groups of bacteria is registered, there is still no scientific evidence about the negative consequences of the continuous input of the sub-low levels of antibiotics for the humans and the environment. The investigations of the antibiotic occurrence used in medicine in the aquatic environment have been carried out mainly in the developed countries so far. More than 30 antibiotics have been identified in the sewage influent and effluent samples in the surface, ground and drinking water samples [24, 25]. Besides, the worrying impact on the public health caused by the spread of bacteria resistant to the high doses of antibiotics, there is a direct influence on the aquatic organisms at very low doses and a potential toxicity via drinking water. Macrolide antibiotics and sulphonamides are the most prevalent antibiotics detected in the environment in the concentrations of about a few mg/L. For example, sulfamethoxazole was detected to be about 0.48 mg/L in the surface water, and the concentration in the sewage treatment plant effluents was 2 mg/L [26--28]. The most prescribed antibiotics, penicillins and cephalosporins, are not recognized as serious threat to the environment due to the poor stability of ßlactam-ring which can easily be hydrolysed, either chemically or microbiologically. However, sulfonamides and macrolides are more stable and present important environmental contaminants. Sulfamethoxazole is widely used in human medicine as well as in animal husbandry as feed additive. It is non-degradable in a sewage treatment. Tiamuline is a veterinary drug, frequently used in the treatment of pigs and poultry. Chloramphenicol is a broadspectrum antibiotic that was widely used in veterinary medicine. Beside the fact that the use of chloramphenicol has been banned in the EU since 1994 due to certain toxicological problems, such as aplastic anemia and the grey baby syndrome, the residues of chloramphenicol were detected at the concentrations of 0.56 and 0.06 µg/L in some sewage treatment plant effluents [10, 24]. The previous research, conducted in the vicinity of Novi Sad municipality, proved the presence of these antibiotics in a few samples of wastewater screening analysis. In order to examine the potential impact of discharged wastewater to the quality of the Danube water in the vicinity of Novi Sad, sulfamethoxazole, chloramphenicol and tiamuline were determined in this study. According to the literature data and our studies, the mentioned antibiotics were found to respond well in UV detections [29, 30]. Sulfamethoxazole, chloramphenicol and tiamuline are used in meat industry of Serbia; therefore, there is an objective requirement to

monitor these antibiotics in the water environment. The reports on antibiotic occurrence in water are still rare and limited on only few compounds and sampling sites in the Western Balkan region, especially in Serbia. Unfortunately, no data on most pharmaceutical levels in water are available from any monitoring or research activities in Serbia. The lack of experience in analytical procedures for qualitative and quantitative determination of emerging contaminants is actual and current. Novi Sad has joint collector for both industrial and municipal wastewaters, directly discharged into the Danube. The Novi Sad Municipality Water Supply System generally uses groundwater from alluvial aquifers of the Danube. The quantitative analyses conducted in this study could serve as a base for monitoring of ecological and chemical water status in order to establish a coherent and comprehensive quality overview of drinking water sources and the occurrence of “emerging” organic contaminants along the Danube in the vicinity of Novi Sad. Accordingly, the primary goal of the study was to monitor seasonal variations of caffeine and antibiotic (sulfamethoxazole, chloramphenicol and tiamuline) concentrations along the selected sites of the Danube.


Materials and methods

2.1. Chemicals The standards of caffeine, sulfamethoxazole, chloramphenicol and tiamuline (99 % purity), acetonitrile and methanol were obtained from Sigma (Deisenhofen, Germany). Chloroform and formic acid were purchased from Mallinckrodt Baker (Phillipsburg, NJ, USA). Acetone and ethyl acetate were obtained from Promochem (Wesel, Germany) or J.T. Baker (Deventer, Netherlands). Ultra pure water was used for the preparation of all solutions (Milli-Q-quality). All solvents and reagents were of analytical grade unless indicated otherwise.

2.2. Sample collection The samples were collected in summer (July 2011), autumn (November 2011), winter (March 2012) and spring (Jun 2012) in amber bottles from seven representative locations (GC1, GC2, RP, RO, DM, DL, DR) of the Danube (Table 1 and Fig. 1) around the teritory of Novi Sad, Serbia and stored at 4 °C until the analysis were done. The hydrometeorological data during sample collection were obtained from the Republic Hydrometeorological Service of Serbia and they are listed in Table 2. The water samples were taken at the depth of 50 cm in the central parts of the river and along the coast (three samples) and 100 m downstream of each sewage discharge (four samples). The sampling sites selection was conducted according to the recommendations of ICPDR’s experts (The International Commission for the Protection of the Danube ICPDR,, in order to estimate the level of violation of the ecologycal status of the Danube by the Novi Sad Municipality. The sites GC1 and GC2 were positioned 100 m downstream, i.e. transversely from the two biggest municipal wastewater discharges (with capacity of 400 L/s during the dry period). The sampling locations DM, DL and DR were chosen as the control sites in order to estimate the possible existance and impact intensity of the municipality on the river water quality after some period (estimation of the impact of dilution and filtration by the river bank layers).

2.3. Sample preparation All the samples were filtered through a 0.45 µm Nylon membrane filter prior the extraction. The SupelcleanTM LC18 solid phase extraction (SPE) cartridges, used for caffeine extraction, were conditioned with 2 × 6 mL of each solvent: chloroform, dichloromethane, methanol and high-performance liquid chromatography (HPLC) grade water. The Danube samples were passed through the SPE cartridges and the elutes were rejected. The extraction volume of each sample was 5 L. Caffeine was eluted from the SPE cartridges with 2 × 10 mL of chloroform into an evaporating flask. The solution was evaporated to dryness under nitrogen. The residue of all samples was reconstituted in 1 mL of water, pH 8. Prior to the analysis, the samples were filtered through a 0.45 µm Nylon filter and injected into HPLC system [31]. The samples were concentrated prior to the HPLC analysis. The extraction and cleanup procedure for antibiotics were performed according to the protocols set by Loos et al. [32]. The SupelcleanTM LC-18 SPE cartridges 6 mL (0.5 g) used for solid phase extraction of caffeine and SupelSelect HLB cartridges (200 mg/6 mL) for antibiotic extraction were obtained from Supelco (Bellefonte, PA, USA). The SPE was performed in a 12-position Vacuum Manifold (Supelco, Bellefonte, PA, USA). The cartridges were activated and conditioned with 5 mL of methanol and 5 mL of distilled water. 3 L of water samples were passed through the wet cartridges and washed with 2 mL of distilled water. The cartridges were dried for 5 min under vacuum. Elution was done with 6 mL of methanol/acetone/ethyl acetate (2:2:1, v/v) solvent mixture. After isolation, the extracts were evaporated to the final volume of 1 mL.

2.4. Instrumentation and operating conditions The determination of caffeine in the Danube samples was done by the modified and validated HPLC method [31]. The HPLC-diode array detection model Agilent HP 1100 system equipped with an autosampler (Waldbronn, Germany) was used. The analytical column was the Zorbax Eclipse XDB-C8 (4.6 × 150 mm, id, 5 µm particle size). The mobile phase was water (pH 8)/acetonitrile (85:15) with a flow rate of 0.9 mL/min. The HPLC mobile phase was prepared fresh daily and filtered through a 0.45 µm Nylon filter. The run time was 10 min and the column temperature 25 °C. The injection volume was 15 µL and the analyte was detected at 273 nm. The analytical column Zorbax Eclipse XDB-C18 (4.6 × 150 mm, id, 5 µm particle size) was used for antibiotic determination. The HPLC method for the separation of sulfamethoxazole and chloramp henicol [33] used a gradient elution program with 0.7 mL/min flow rate. The mobile phase consisted of a mixture of 0.1 % formic acid in water and acetonitrile. The antibiotics were detected at 280 nm with a retention time of 22 min for sulfamethoxazole and 29 min for chloramphenicol. A different method [34] was used for the tiamuline determination. Tiamuline was detected at 210 nm with a mixture of ammonium carbonate 1 % in water and methanol (30:70, v/v) as a mobile phase, used in isocratic mode with the flow rate of 0.8 mL/min. The retention time was 1.7 min. The injection volume for antibiotic determination was 15 µL.

2.5. Calibration The standard stock solution of caffeine was prepared by dissolving 1 mg of the standard substance in 10 mL water, pH 8 (adjusted with 0.1 M NaOH). The solution was stable approximately three days under refrigeration (4 °C). The

working solutions were prepared by diluting stock solution with water, pH 8, to obtain different concentrations of caffeine (0.015--0.1 mg/L). The standard solutions of antibiotics were obtained by dissolving 1 mg of the standard substance in 10 mL methanol (95 %, v/v). The working standard solutions (0.01--0.5 mg/L) were obtained by diluting standard stock solution with the same solvent. The working solutions were injected into the HPLC system and the peak area responses were obtained. The method of the external standard calibration was used. The linear standard curves for caffeine and antibiotics were determined by plotting the concentration versus the area response and each of six calibration points was obtained as an average of three injections.

2.6. Calculation of the maximum Risk Index (RI) Fernandez et al. [35] proposed the calculations for the determination of the RI in their research. The toxicity units (TU) for caffeine were calculated by dividing the measured environmental concentrations with the lowest acute toxicity value (covering three representative taxonomic groups for aquatic system: algae, invertebrates and fish) obtained from the available publications [36, 37]. Caffeine acute aquatic toxicity was found to be 87.5 mg/L for fish ( [36]. The total TUs for each sampling site were calculated by summarizing all TUs from each location. The inverse of the total TUs gave the margin of exposure. The maximum RI (MaxRI), as an expression of the identified level of risk, was obtained by dividing the margin of exposure by 10.

2.7. Statistical analysis The statistical analyses were done by MS Excel® 2010 software. The comparison of the mean values of the measured parameters was performed by one-way ANOVA (SPSS, version 17) using Duncan's multiple range tests for the level of significance p < 0.05. The correlations between hydrometeorological variables (hydrometeorological data, Table 2) and the caffeine concentration levels, as well as the correlations among caffeine concentrations at seven sampling sites, in different seasons (four sampling campaigns) were analysed using Pearson’s correlation coefficients by SPSS software (a significance threshold of p = 0.05 was retained).


Results and discussion

The HPLC method [31] was developed for methylxanthines determination in food and beverages. Some of the chromatography conditions were changed in this study (flow rate and mobile phase composition) in order to achieve a shorter time of analysis, so the method had to be validated and applied for caffeine determination in the river water samples. The effect of the flow rate and the mobile composition on the retention time (tR), the peak width (W 50) and the number of theoretical plates (N ) for caffeine were tested using working standard solution. According to the results (mean value of three injections) presented in Table 3 it can be concluded that the combination of 0.9 mL/min flow rate and mobile phase consisted of water (pH 8)/acetonitrile (85:15, v/v) compromises between the analyte retention time and the consumption of solvent. The procedures used for the validation are described in the available literature [37--40]. The linearity between the caffeine concentrations and the response was tested for the

concentration levels ranging from 1--100 µg/L. Under the determined HPLC conditions, working standard solutions of caffeine were injected into the HPLC system and according to the obtained areas under the curves a linear standard curve for caffeine was constructed by plotting the concentration versus the area (y = 12594 x - 196.3). The high value of the correlation coefficient r = 0.998 showed an excellent correlation between the concentrations and peak areas. The limit of detection (LOD – 3.3 S/a) and quantification (LOQ – 10 S/a) were determined based on the standard deviation of the response (S) and the slope of the calibration curve (a). The accuracy of this method was tested by comparing the measured and known values of the concentrations for standard solutions of caffeine. According to the recovery value of 100.22 ± 2.43%, the method showed acceptable accuracy. Repeatability of the method was tested by analysing three different concentrations of caffeine standards in six repetitions. The relative standard deviations ranged from 0.028 % to 0.063 % for the retention time and from 0.015 % to 0.65 % for the peak area showing excellent repeatability. As shown in Supporting Information Fig. S1, there was no interference in the HPLC results by impurities or matrix in the tested samples, which indicated that the developed method had a very good selectivity. The recovery for the clean-up procedure was calculated by adding 300 µL of the stock solution in the surface water samples subjected to the clean-up procedure described previously and the recovery value was 92.88 ± 1.17 %. Analysing the effects of slightly changed parameters of the used HPLC conditions, such as different column temperature (±1 °C), flow rate (±0.05 mL/min) and detection wavelength (±3 nm) led to the conclusion that there were no significant differences in the obtained results, proving the robustness of the method. When the HPLC method [31] was applied prior to the modification, the retention time for caffeine was 12.2 min. The modified and validated method shortened the time for caffeine analysis to 8.1 min. The statistical parameters for the validation of the HPLC method to determine caffeine were: linear range: 1 to 100 µg/L, slope: 12 594, intercept: 196.3, determination coefficient (r 2): 0.996, correlation coefficient (r): 0.998, LOD: 0.0066 mg/L, LOQ: 0.012 mg/L. Under











chloramphenicol and tiamuline were injected into the HPLC system and linear standard curves were obtained by plotting the concentration versus the area (sulfamethoxazole: y = 50 509 x + 70.66 r² = 0.999; chloramphenicol: y = 36 242 x + 50.55 r² = 0.999 and tiamuline: y = 15 745 x + 100.3 r² = 0.999). The LOD and the LOQ were calculated for each antibiotic and the obtained values were for sulfametoxazole: LOD = 0.005, LOQ = 0.016; chloramphenicol: LOD = 0.007, LOQ = 0.02 and tiamuline: LOD = 0.007, LOQ = 0.02 mg/L. The chromatograms of working stock solutions of all three antibiotics are presented in Supporting Information Fig. S2a, while calibration curves for caffeine and antibiotics are shown in Supporting Information Fig. S3. Sulfamethoxazole and chloramphenicol are used both in human and veterinary medicine and tiamuline is used as an antimicrobial agent in veterinary medicine only, to treat infections in farm animals. Some studies reported low concentrations of these antibiotics in surface water and wastewater [24, 29, 30] which was not the case with our research. The results of our investigation showed that the analysed antibiotics were under the limit of detection in all Danube tested samples therefore, no risk assessment for antibiotics needed to be done. The HPLC chromatogram of sulfamethoxazole and chloramphenicol determination in the sample collected at the sampling site GC1 is presented in Supporting Information Fig. S2b. The results of the seasonal variations in caffeine concentrations at seven selected sites along the Danube are

summarized in Table 4. The obtained results confirmed the presence of this emerging substance in four samples taken near the wastewater discharges and followed the prediction that caffeine could be present in the areas of the human impact. Caffeine is not only used as a stimulant in medicine, but it is also used in various food and beverages; hence, it is considered one of the primary indicators of anthropogenic contributions in the natural aquatic systems. The presence of this compound could be directly related to the discharge of sewerage into the watershed (Table 4). The lowest caffeine concentrations (under the limit of detection) were found in the middle of the river and on the left and right banks of the Danube, the sites far away from the direct human impact, which could indicate that anthropogenic activities in Novi Sad area does not have the global impact on the ecotoxicological status of the Danube. The mean caffeine concentrations for summer, autumn, winter and spring periods were 24.78, 26.83, 24.61, and 86.29 ng/L, respectively. The significantly higher mean caffeine concentration was detected in the samples collected during the spring period. This seasonal difference could be the result of the variations in the environmental conditions such as rainfall and temperature or different seasonal caffeine consumption. There was no significant difference between the mean caffeine concentrations detected for summer, autumn and winter in this study. The obtained results are in agreement with the study done in the United States (The Mississippi) where the highest values were determined in the samples collected during the spring period [41]. Similar results were obtained in another publication which evaluated the seasonal caffeine concent rations in surface water samples on the territory of Spain finding the highest concentrations in the samples collected in autumn and spring and the lowest in winter [35]. Another European study analysed the caffeine concentrations in surface water samples from January to May and detected the lowest concentrations in the samples collected during January and the highest during May [42]. Relatively high levels of caffeine about 200 ng/L were detected in the Han River in Korea; however, the seasonal variations were not presented [43]. On the contrary, Musollf et al. [44] documented that the loads of caffeine during the colder months increased when rainfall occurred. Rodriguez et al. [45] measured caffeine concentrations in the Oregon coastal waters (from 100 [35]. Thus, the lower value of MaxRI leads to the higher environmental potential risk. The sampling sites DM, DL and DR showed no potential risk for the aquatic organisms, while the sites GC1, GC2, RP and RO showed 10 < MaxRI < 100 (class II). The highest potential risk was calculated for the sampling site GC2. These results for the MaxRI showed that the potential risk for the chronic effects may occur in the resident organisms in the long-term period. The obtained results for the caffeine risk potential were lower than the one obtained in the similar research done in Spain, finding MaxRI < 10 for most of the tested sampling sites of the Henares--Jarama--Tajo river system [35].



This paper is representing the pioneering work on determination of ecotoxicological impact of emerging substances in surface water in the Balkans. The validated HPLC methods for caffeine and antibiotic determination showed good accuracy, repeatability, selectivity, robustness and provided a shorter time of analysis. The significant concentrations of caffeine were determined at the sampling sites near the wastewater discharges. The MaxRI showed that the potential risk for the chronic effects which might occur in the resident organisms in the long-term period. Caffeine was under the limit of detection at the sampling sites far away from wastewater discharges. There is black box in methodology as well as in the legislation of emerging contaminants for surface water. Therefore, the main aim of this paper was to share the data with more comprehensive European database. It is necessary to have the ecological information of each segment of the whole river stream to be able to define the Danube environmental status integrally. The quality of surface water is of great importance for risk management and health and safety environment of inhabitants in the city of Novi Sad. Although the results have strictly been focused on the area of the Novi Sad municipality and they seem only as locally important, the obtained data can contribute to the assessment of the “emerging” status of the Danube. Upon the conducted research, the NORMAN Association has expressed interest to include presented data in the EMPODAT or NORMAN MassBank databases. Sharing collected data within various research studies and presenting them in a joint database, such as NORMAN, would give a better overview of the pollutants of concern, facilitate the research and contribute to timely and adequate

policy measures. Also, the levels of emerging pharmaceuticals detected along the Danube midstream represent an argument for considering the prioritization of these compounds within the extended list of priority / priorityhazardous substances within WFD. The “local” national data for the surface water of Danube in the vicinity of Novi Sad conducted within the NATO Science for Peace and Security Project (ESP.EAP.SFP 984087) could be significant for international databases of EmS. Acknowledgement The work was financially supported by Ministry of Education, Science and Technological Development, Republic of Serbia (III46009), NATO Science for Peace Project (ESP.EAP.SFP 984087) and bilateral project No. 680-00140/2012-09/13.

The authors have declared no conflict of interest.

References 1.

A. P. Ferreira, Caffeine as an environmental indicator for assessing urban aquatic ecosystems, Cad. Saude Publica 2005, 21 (6), 1884--1892.


K. Kümmerer, Emerging Contaminants versus Micro-pollutants, Clean – Soil Air Water 2011, 39 (10), 889–890.


J. R. Dominguez-Vargas, T. Gonzalez, P. Palo, E. M. Cuerda-Correa, Removal of Carbamazepine, Naproxen, and Trimethoprim from Water by Amberlite XAD-7: A Kinetic Study, Clean – Soil Air Water 2013, 41 (11), 1052--1061.


S. M. Rodrigues, G. A. Glegg, M. E. Pereira, A. C. Duarte, Pollution Problems in the NE Atlantic: Lessons Learned for Emerging Pollutants such as the Platinum Group Elements, Ambio 2009, 38 (1), 17--23.


D. Calamari, E. Zuccato, S. Castiglioni, R. Bagnati, R. Fanelli, Strategic Survey of Therapeutic Drugs in the Rivers Po and Lambro in Northern Italy, Environ. Sci. Technol. 2003, 37, 1241--1248.


S. Grujic, T. Vasiljevic, M. Lauševic, Determination of multiple pharmaceutical classes in surface and ground waters by liquid chromatography-ion trap-tandem mass spectrometry, J. Chromatogr. A 2009, 1216 (25), 4989--5000.


Emerging Substances of Concern, Florida Department of Environmental Protection, Division of Environmental Assessment and Restoration, Tallahassee, FL 2008.


F. A. Caliman, M. Gavrilescu, Pharmaceuticals, Personal Care Products and Endocrine Disrupting Agents in the Environment – a Review, Clean – Soil Air Water 2009, 37 (4–5), 277–303.


J. Martin, D. Camacho-Munoz, J. L. Santos, I. Aparicio, E. Alonso, Monitoring of pharmaceutically active compounds on the Guadalquivir River basin (Spain): occurrence and risk assessment, J. Environ. Monit. 2011, 13 (7), 2042--2049.

10. N. Milic, M. Milanovic, N. Grujic Letic, M. Turk Sekulic, J. Radonic, I. Mihajlovic, M. Vojinovic Miloradov, Occurrence of antibiotics as emerging contaminant substances in aquatic environment, Int. J. Environ. Health Res. 2013, 23 (4), 296--310.

11. N. Grujic Letic, N. Milic, M. Turk Sekulic, J. Radonic, M. Milanovic, I. Mihajlovic, M. Vojinovic Miloradov, Quantification of emerging organic contaminants in the Danube River samples by HPLC, Chem. Listy 2012, 106, 264--266. 12. J. Cohen, H. I. Shuval, Coliforms, fecal coliforms, and fecal streptococci as indicators of water pollution, Water Air Soil Pollut. 1973, 2, 85--95. 13. E. Sanchez, M. F. Colmenarejo, J. Vicente, A. Rubio, M. G. Garcia, L. Travieso, R. Borja, Use of the Water Quality Index and Dissolved Oxygen Deficit as Simple Indicators of Watershed Pollution, Ecol. Indic. 2007, 7 (2), 315--328. 14. I. J. Buerge, T. Poiger, M. D. Müller, H. R. Buser, Caffeine, an anthropogenic marker for wastewater comtamination of surface waters, Environ. Sci. Technol. 2003, 37 (4), 691--700. 15. R. L. Seiler, S. D. Zaugg, J. M. Thomas, D. L. Howcroft, Caffeine and pharmaceuticals as indicators of waste water contamination in wells, Ground Water 1999, 37 (3), 405--410. 16. C. I. Heck, E. G. De Mejia, Yerba Mate Tea (Ilex paraguariensis): a comprehensive review on chemistry, health implications, and technological considerations, J. Food Sci. 2007, 72 (9), R138--R151. 17. S. Armenta, S. Garrigues, M. De La Guardia, Solid-phase FT-Raman determination of caffeine in energy drinks, Anal. Chim. Acta 2005, 547, 197--203. 18. R. M. Gilbert, J. A. Marshman, M. Schwieder, R. Berg, Caffeine content of beverages as consumed, Can. Med. Assoc. J. 1976, 114 (3), 205--208. 19. T. R. Norton, A. B. Lazev, M. J.Sullivan, The “Buzz” on Caffeine: Patterns of Caffeine Use in a Convenience Sample of College Students, J. Caffeine Res. 2011, 1 (1), 35--40. 20. J. A. Carrillo, J. Benitez, Clinically significant pharmacokinetic interactions between dietary caffeine and medications, Clin. Pharmacokinet. 2000, 39 (2), 127--153. 21. B. Abebe, T. Kassahun, R. Mesfin, A. Araya, Measurement of caffeine in coffee beans with UV/Vis spectrometer, Food Chem. 2008, 108 (1), 310--315. 22. S. M. Evans, R. R. Griffiths, Caffeine tolerance and choice in humans, Physchopharmacology 1992, 108, 51--59. 23. D. K. Singh, A. Sahu, Spectrophotometric determination of caffeine and theophylline in pure alkaloids and its application in pharmaceutical formulations, Anal. Biochem. 2006, 349, 176--180. 24. R. Hirsch, T. Ternes, K. Haberer, K. L. Kratz, Occurrence of antibiotics in the aquatic environment, Sci. Total Environ. 1999, 225, 109–118. 25. C. Brenner, C. Mallmann, D. Arsand, F. Mayer, A. Martins, Determination of Sulfamethoxazole and Trimethoprim and Their Metabolites in Hospital Ef ?uent, Clean – Soil Air Water 2011, 39 (1), 28–34. 26. A. Martins, C. Mallmann, D. Arsand, F. Mayer, C. Brenner, Occurrence of the Antimicrobials Sulfamethoxazole and Trimethoprim in Hospital Effluent and Study of Their Degradation Products after Electrocoagulation, CLEAN – Soil Air Water 2011, 39 (1), 21–27. 27. B. Halling-Sørensen, S. N. Nielsen, P. F. Lanzky, F. Ingerslev, H. C. Holten-Lutzhoft, S. E. Jørgensen, Occurrence, fate and effects of pharmaceutical substances in the environment – a review, Chemosphere

1998, 36, 357–393. 28. S. Yang, K. Carlson, Routine monitoring of antibiotics in water and wastewater with a radioimmunoassay technique, Water Res. 2004, 38, 3155–3166. 29. X. Peng, Z. Wang, W. Kuang, J. Tan, K. Li, A preliminary study on the occurrence and behavior of sulfonamides, ofloxacin and chloramphenicol antimicrobials in wastewaters of two sewage treatment plants in Guangzhou, China, Sci. Total Environ. 2006, 371, 314–322. 30. S. Babic, D. Asperger, D. Mutavdzic, A. Horvat, M. Kastelan-Macan, Solid phase extraction and HPLC determination of veterinary pharmaceuticals in wastewater, Talanta 2006, 70, 732–738. 31. B. Srdjenovic, V. Djordjevic-Milic, N. Grujic, R. Injac, Z. Lepojevic, Simultaneous HPLC determination of caffeine, theobromine, and theophylline in food, drinks, and herbal produts, J. Chromatogr. Sci. 2008, 46, 144--149. 32. R.












perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and nonylphenol and its carboxylates and ethoxylates in surface and tap waters around Lake Maggiore in Northern Italy, Anal. Bioanal. Chem. 2007, 387, 1469--1478. 33. R. Fernandez-Torres, M. Olías Consentino, M. A. Bello Lopez, M. Callejon Mochon, Simultaneous determination of 11 antibiotics and their main metabolites from four different groups by reversed-phase high-performance liquid chromatography–diode array –fluorescence (HPLC –DAD–FLD) in human urine samples, Talanta 2010, 81, 871–880. 34. M. J. Nozal, J. L. Bernal, M. T. Martín, J. J. Jimenez, J. Bernal, M. Higes, Trace analysis of tiamulin in honey by liquid chromatography–diode array–electrospray ionization mass spectrometry detection, J. Chromatogr. A 2006, 1116, 102–108. 35. C. Fernandez, M. Gonzales-Doncel, J. Pro, G. Carbonell, J. V. Tarazona, Occurrence of pharmaceutically active compounds in surface waters of the Henares--Jarama--Tajo river system (Madrid, Spain) and a potential risk characterization, Sci. Total Environ. 2010, 408 (3), 543--551. 36. J. Park, Pharmaceuticals in the environment and management approaches in Korea, Korea Environment Institute, Seoul 2005. 37. The United States Pharmacopoeia-NF 19, 24th Ed., United States Pharmacopoeial Convention, Rockville, MD 2002. 38. International Conference on Harmonization, Guideline for Industry, Q2A Text on validation of analytical procedures, Federal Register, US Food and Drug Administration, Silver Spring, MD 1995. 39. International Conference on Harmonization, Guideline for Industry, Q2B Validation of analytical procedures: methodology, Federal Register, US Food and Drug Administration, Silver Spring, MD 1997. 40. Y. V. Heyden, A. Nijhuis, J. Smeyers-Verbeke, B. G. M. Vandeginste, D. L. Massart, Guidance for robustness/ruggedness tests in method validation, Pharm. Biomed. Anal. 2001, 24, 723--753. 41. L. B. Barber, J. A. Leenheer, W. E. Pereira, T. I. Noyes, G. K. Brown, C. F. Tabor, J. H. Writer, in Contaminants in the Mississippi River, 1987--1992 (Ed.: R. H. Meade), USGS Water-Supply Circular

1133, Vol. 99, USGS, Reston, VA 1995, pp. 115–136. 42. I. J. Buerge, T. Poiger, M. D. Muller, H. R. Buser, Behavior of the polycyclic musks HHCB and AHTN in lakes, two potential anthropogenic markers for domestic wastewater in surface waters, Environ. Sci. Technol. 2003, 37 (4), 691--700. 43. C. Kyungho, K. Younghee, P. Jeongim, P. Chan Koo, K. MinYoung, K. Hyun Soo, K. Pangyi, Seasonal variations of several pharmaceutical residues in surface water and sewage treatment plants of Han River, Korea, Sci. Total Environ. 2008, 405, 120--128. 44. A. Musolff, S. Leschik, M. Moder, G. Strauch, F. Reinstorf, M. Schirmer, Temporal and spatial patterns of micropollutants in urban receiving waters, Environ. Pollut. 2009, 157, 3069–3077. 45. Z. Rodriguez del Rey, E. F. Granek, S. Sylvester, Occurrence and concentration of caffeine in Oregon coastal waters, Mar. Pollut. Bull. 2012, 64 (7), 1417--1424. 46. R. Loos, G. Locoro, S. Contini, Occurrence of polar organic contaminants in the dissolved water phase of the Danube River and its major tributaries using SPE-LC-MS2 analysis, Water Res. 2010, 44 (7), 2325-2335. 47. K. A. Peeler, S. P. Opsahl, J. P. Chanton, Tracking anthropogenic inputs using caffeine, indicator bacteria and nutrients in rural freshwater and urban marine systems, Environ. Sci. Technol. 2006, 40 (24), 7616-7622. 48. K. L. Knee, R. Gossett, A. B. Boehm, A. Paytan, Caffeine and agricultural pesticide concentrations in surface water and groundwater on the north shore of Kauai (Hawaii, USA), Mar. Pollut. Bull. 2010, 60 (8), 1376--1382.

Table 1. List of the sampling sites along the Danube banks of Novi Sad No.



Northern latitude

Eastern longitude







Beogradski kej





Rokov potok





Ratno ostrvo





Danube middle





Danube left





Danube right




Table 2. Hydrometeorological conditions of Novi Sad areas during the sampling obtained from the Republic Hydrometeorological Service of Serbia Parameter


July 2011

November 2011

March 2012

June 2012

Ambient air temperature












Wind direction






Wind speed












Hum. index






Water stage






Change of water stage






River flow






The amount of water in riverbed






Water temperature






Weekly range of water stage






Reporting station: Novi Sad, River: Dunav, Basin: Black Sea, Foundation year: 1819, Kota “0” (m n. J. m.): 71.73, Distance from the river mouth (km): 1254.98, Basin area (km2): 254 085

Table 3. Study of chromatographic variables for caffeine determination Chromatographic condition

tR (min)

W50 (min)


Flow rate (mL/min) Mobile phase: water (pH 8)/acetonitrile (85:15, v/v) 0.5







11 542.82




11 398.25

Mobile phase composition; flow rate 0.9 mL/min 80:20



10 963.55




11 535.72





Table 4. Seasonal monitoring of caffeine concentrations in the Danube samples

Sampling site

Caffeine (ng/L) Summer


(July 2011)


(November 2011) b



(March 2012) c

(May 2012)


27.78 ± 0.009

39.90 ± 0.006

42.80 ± 0.011

306.12 ± 0.082f


37.40 ± 0.001c

84.24 ± 0.033d

81.70 ± 0.028d

228.01 ± 0.045e


84.05 ± 0.030d

28.84 ± 0.009b

15.91 ± 0.009a

31.11 ± 0.023b


24.28 ± 0.007b

34.88 ± 0.007b

31.93 ± 0.013b

39.20 ± 0.011c
















Data are expressed as the averages ± standard deviations of triplicate measurements. Statistically significant differences are noted by different superscript letters (p < 0.05). n.d., not detected.

Figure 1. Map of the sampling areas showing sampling sites Figure 2. Stability test for caffeine. (n.d., not detected) Figure 3. MaxRI at sampling sites along the Danube River

Figure 1

Figure 2

Figure 3

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