![\"\"](\"https:\/\/www.vtei.cz\/wp-content\/uploads\/2025\/07\/Baudisova-obr-1.jpg\")
<\/a>\nFig. 1. View of the biological purifying zone<\/h6>\nINTRODUCTION<\/h2>\n
Bathing ponds, natural swimming pools, biologically filtered swimming pools, living pools, eco-pools\u00a0\u2013 these are terms used for public or private swimming facilities where the\u00a0primary role in maintaining water quality is played by living organisms. In the\u00a0Czech Republic, the\u00a0first public bathing pond was opened in 2007 in Kovalovice\u00a0[1], and by 2024, there were already 40 in operation (IS\u00a0PiVo,\u00a02025). Bathing ponds were incorporated into national legislation in 2011, along with the\u00a0definition of requirements for parameters, their monitoring, and evaluation. At that time, experience with bathing ponds in the\u00a0country was still limited, so the\u00a0requirements were inspired by German and Austrian regulations\u00a0[1].<\/span><\/p>\n At present, the\u00a0Public Health Protection Act\u00a0[2] defines a\u00a0bathing pond as \u201ca\u00a0facility approved for the\u00a0purpose of bathing, equipped with a\u00a0natural water purification system for bathing purposes.\u201d Decree No. 238\/2011 Coll. of the\u00a0Ministry of Health\u00a0[3] requires the\u00a0monitoring of indicators of faecal contamination, specifically Escherichia\u00a0coli<\/span> and intestinal enterococci, as well as water transparency. The\u00a0requirement to monitor Pseudomonas aeruginosa<\/span><\/em>\u00a0was removed in 2014 due to unresolved methodological reasons. The\u00a0current legislation relating to bathing ponds is certainly not perfect, and it would be appropriate to improve or update the\u00a0definitions and requirements during the\u00a0next revision of Czech legislation.<\/span><\/p>\n According to data\u00a0from the\u00a0IS PiVo system, the\u00a0numbers of E. coli<\/span> <\/em>and intestinal enterococci are generally well below the\u00a0limit values (i.e. below 100\u00a0and 50\u00a0CFU\/100 ml, respectively); however, occasional exceedances occur at most sites. At least once per season between 2018 and 2023, E. coli<\/span><\/em> limits were exceeded at 20\u201330\u00a0% of bathing ponds, and the\u00a0limit for intestinal enterococci at approximately 40\u00a0%\u00a0[1]. In 2023 and 2024, the\u00a0figures were lower: E.\u00a0coli<\/span> exceedances occurred at 11\u00a0% and 8\u00a0% of sites, and intestinal enterococci at\u00a026\u00a0% and 28\u00a0%, respectively. Unlike natural bathing sites, the\u00a0dominant source of contamination is bathers themselves.<\/span><\/p>\n Given the\u00a0predominantly anthropogenic nature of this type of pollution, the\u00a0use of additional indicators could be considered to help identify it, or to distinguish it from more \u201cnatural\u201d forms of contamination. Other potential sources include pollution introduced by birds (primarily mallards, which visit bathing ponds) and, occasionally, seepage into the\u00a0water body during periods of elevated groundwater levels following heavy rainfall.<\/span><\/p>\n The\u00a0article deals with the\u00a0assessment of additional possible chemical indicators of anthropogenic pollution (namely caffeine and urea) as well as the\u00a0results of total adenosine triphosphate (ATP) analysis, which reflects overall microbial activity.<\/span><\/p>\n Urea\u00a0CO(NH\u2082)\u2082 is found in the\u00a0urine of mammals, amphibians, and some fish. It is synthesised in the\u00a0liver through the\u00a0urea\u00a0cycle. In the\u00a0body, urea\u00a0serves as a\u00a0waste product through which excess nitrogen is excreted in urine, and to a\u00a0lesser extent also through the\u00a0skin during sweating. During the\u00a0urea\u00a0cycle, the\u00a0amino acid arginine is broken down into urea\u00a0and ornithine. Urea\u00a0is excreted from the\u00a0body in urine, while ornithine is reused as a\u00a0precursor in the\u00a0synthesis of arginine\u00a0[4]. Another source of urea\u00a0is the\u00a0decomposition of algal and cyanobacterial biomass, as well as the\u00a0breakdown of certain nitrogen-containing organic compounds and zooplankton excretion\u00a0[5]. Urea\u00a0concentrations have been monitored, for example, in Poland in the\u00a0Great Masurian Lakes, where values typically ranged from 30 to 48\u202f\u00b5g\/l, with the\u00a0highest levels (up to 1.5\u202fmg\/l) detected in spring, and the\u00a0lowest at the\u00a0end of summer during peak phytoplankton development\u00a0[6]. Urea\u00a0concentration was found to be inversely proportional to the\u00a0trophic status of the\u00a0studied lakes, and it was also shown that the\u00a0rate of enzymatic urea\u00a0degradation increases exponentially with water temperature\u00a0[6]. Given the\u00a0expected anthropogenic load in bathing ponds, the\u00a0results of urea\u00a0measurements in swimming pools, where significantly higher values are often detected, are particularly relevant for comparison. Public bathing and swimming in pools are associated with occasional unintentional (though unfortunately sometimes deliberate) urination in the\u00a0water. According to a\u00a0study\u00a0[7] conducted in Canada\u00a0in two pools with volumes of 840\u202fm\u00b3 and 420\u202fm\u00b3, where the\u00a0concentration of the\u00a0artificial sweetener acesulfame in pool water was compared over three weeks with its average concentration in human urine, the\u00a0volume of urine in the\u00a0pools reached just under 0.01\u00a0%. An earlier study\u00a0[8] reported an estimated input of 60 to 80\u202fml of urine per swimmer per day, based on changes in potassium concentration in the\u00a0pool. Karimi\u00a0[9] monitored water samples from ten swimming pools in Tehran, Iran, which were disinfected using various methods. The\u00a0average urea\u00a0concentration in pools disinfected with chlorine was 5.5\u202fmg\/l. In pools disinfected with ozone followed by chlorine, the\u00a0average concentration was 4\u202fmg\/l, and in those disinfected with UV light followed by chlorine, the\u00a0average urea\u00a0concentration was 3.5\u202fmg\/l. Zhang et al.\u00a0[10] report that urea\u00a0concentrations in Beijing (China) ranged from 0.07 to 18.73\u202fmg\/l. Zhou et al.\u00a0[11] found urea\u00a0concentrations ranging from 0.74 to 15.02\u202fmg\/l in their study.<\/span><\/p>\n Caffeine (1,3,7-trimethylxanthine, C\u2088<\/sub>H\u2081\u2080<\/sub>N\u2084<\/sub>O\u2082<\/sub>) is a\u00a0purine alkaloid found both in widely consumed beverages (coffee, Coca-Cola, tea, energy drinks) and in pharmaceutical products. It enters the\u00a0environment primarily through wastewater from human settlements and industry. Caffeine concentrations in beverages range from tens to hundreds of mg\/l; for example, coffee contains 36 to 804 mg\/l, tea\u00a0122 to 183 mg\/l, Coca-Cola\u00a0and similar drinks 41 to 132 mg\/l, and energy drinks 267 to 340 mg\/l\u00a0[12]. After consumption, caffeine is absorbed into the\u00a0bloodstream relatively quickly, with about one-fifth absorbed directly from the\u00a0stomach\u00a0[13]. Almost all ingested caffeine is metabolized in the\u00a0liver by demethylation into primary metabolites, paraxanthine (80\u00a0%), theobromine (11\u00a0%), and theophylline (4\u00a0%), which can undergo further demethylation and oxidation, resulting in uric acid salts and uracil derivatives\u00a0[13]. The\u00a0rate of caffeine metabolism depends on many factors, with its half-life in the\u00a0body ranging from 2 to 12 hours, most commonly 4 to 5 hours\u00a0[13]. Only a\u00a0small portion of caffeine is excreted unchanged in urine, with reported amounts varying between 0.5\u00a0% and 10\u00a0% according to different publications\u00a0[14]. Caffeine is primarily excreted from the\u00a0body through urine along with its main metabolite, paraxanthine, although traces can also be found in perspiration. Rybak et al.\u00a0[15] studied the\u00a0concentrations of caffeine and its metabolites in human urine, reporting median values of 3.39 \u00b5mol\/l for caffeine, 15.2 \u00b5mol\/l for paraxanthine, 20.3 \u00b5mol\/l for theobromine, and 1.63 \u00b5mol\/l for theophylline; when converted to mass concentration, the\u00a0median for caffeine is 658\u00a0\u00b5g\/l. The\u00a0detected concentrations vary mainly depending on age, with the\u00a0highest values observed in the\u00a040 to 59-year age group\u00a0[15]. Excretion rates differ between caffeine and its individual metabolites, ranging from 0.423\u00a0nmol\/min to 46.0\u00a0nmol\/min\u00a0[15].<\/span><\/p>\n The\u00a0average caffeine consumption per capita\u00a0per day was 288.58 mg in 2013, according to data\u00a0from Food Balance Sheet of the\u00a0United Nations<\/span>, with significant differences between individual countries\u00a0[16]. Numerous studies have investigated the\u00a0presence of caffeine in water; there is much more data\u00a0on its occurrence in wastewater and flowing waters than in bathing waters (including lakes). So far, no one has monitored the\u00a0presence of caffeine in bathing ponds. Buerge et al.\u00a0[14] propose caffeine as a\u00a0useful chemical marker for assessing wastewater contamination in watercourses. They determined its removal efficiency in Swiss wastewater treatment plants (WWTPs) to be between 81\u00a0and 99.9\u00a0%. These authors also commonly detected caffeine in Swiss lakes and rivers (6\u2013250 ng\/l), except in mountain lakes. The\u00a0proposal to include caffeine as an indicator of anthropogenic pollution is also supported by Portuguese researchers Paiga\u00a0et al.\u00a0[17], who reported caffeine concentrations in rivers ranging from 25.3 to 321 ng\/l. More recent studies have reported caffeine concentrations in lakes in Maine (USA), with average values ranging from 6 to 11 ng\/l and a\u00a0maximum of 21 ng\/l\u00a0[18]. In our previous research, maximum concentrations detected in standing bathing waters were 296 ng\/l for caffeine and 0.127\u00a0mg\/l for urea\u00a0[16]. In the\u00a0Vltava\u00a0River at Prague\u2013Podol\u00ed, the\u00a0average caffeine concentration recorded between 2005 and 2018 was 220 ng\/l, with a\u00a0maximum of 960 ng\/l and a\u00a0minimum of 100 ng\/l\u00a0[19]. Given the\u00a0relatively effective biodegradation of caffeine at WWTPs, the\u00a0presence of caffeine in surface waters is more indicative of recent contamination by raw sewage rather than pollution via\u00a0effluents from WWTPs. Caffeine degradation also occurs in natural waters, primarily through the\u00a0activity of bacteria\u00a0from genera\u00a0such as Pseudomonas<\/span><\/em>, Klebsiella<\/span><\/em>, Bacillus<\/span><\/em>, Rhodococcus<\/span><\/em>, and others\u00a0[20]. In 12 chlorinated swimming pools in Australia, caffeine concentrations of up to 1,540\u202fng\/l were detected, with significant fluctuations observed throughout the\u00a0day depending on visitor numbers\u00a0[21]. In thermal pools in Slovakia, caffeine was found at 44 out of 49\u00a0sites, with the\u00a0highest concentration reaching 69,000\u202fng\/l (median 310\u202fng\/l and arithmetic mean 1,140\u202fng\/l)\u00a0[22].<\/span><\/p>\n Urea\u00a0and caffeine are not as stable in the\u00a0aquatic environment as certain pharmaceuticals, for example. Urea\u00a0gradually breaks down in water into ammonium cyanate and subsequently into ammonia\u00a0and carbon dioxide. Both acidic and alkaline conditions accelerate this reaction, as does elevated temperature. Even minimal urea\u00a0decomposition causes an increase in pH, and this alkaline pH in turn catalyses further breakdown. The\u00a0optimal pH for urea\u00a0is 6.2\u00a0[23]. The\u00a0alkaline environment in bathing ponds would further accelerate the\u00a0breakdown of urea\u00a0(in our cases, the\u00a0pH ranged from 8.51 to 8.53\u00a0\u2013 see Results). Caffeine is readily broken down during wastewater treatment (81\u201399.9\u00a0% elimination according to\u00a0[14]), so its degradation can also be expected in bathing ponds. Other authors\u00a0[24] report complete biodegradation of caffeine by a\u00a0pure culture of Pseudomonas<\/span> spp. at an optimal pH of 6.0 within 24 hours, and also mention the\u00a0inhibitory effect of both organic and inorganic nitrogen compounds, with the\u00a0effect of urea\u00a0found to be stronger than that of ammonium salts. In another study\u00a0[25], also working with Pseudomonas<\/span> bacteria, the\u00a0maximum rate of caffeine biodegradation was quantified at 0.345 \u00b5mol\/min, with an optimal pH for biodegradation 8.0. It was also observed that among metal ions, Cu2+<\/sup> and Zn2+<\/sup> ions had a\u00a0strongly inhibitory effect on caffeine biodegradation, whereas Fe2+<\/sup>, Ca2+<\/sup> and Mg2+<\/sup> ions had a\u00a0stimulating effect\u00a0[25]. In the\u00a0aquatic environment, caffeine may undergo hydrolysis and, under suitable conditions, also photodegradation or biodegradation. Alhassen et al.\u00a0[26] investigated caffeine photodegradation induced by artificial light with a\u00a0wavelength of 400 to 500\u00a0nm simulating sunlight and recorded a\u00a0caffeine half-life of 2.3 to 16.2 hours depending on the\u00a0type of matrix (demineralised water, river water alone, and river water with added leaves). It was found that certain organic substances reduce the\u00a0rate of caffeine degradation, and that in addition to photodegradation, hydrolysis also contributes to breakdown, although it is significantly slower\u00a0[26]. In another study, a\u00a0half-life of six days was observed following inoculation with effluent from a\u00a0WWTP, or ten days in the\u00a0case of biodegradation by microorganisms from activated sludge\u00a0[27].<\/span><\/p>\n The\u00a0ATP test analyses the\u00a0presence of overall microbial contamination and is a\u00a0process that measures active microorganisms. The\u00a0test is based on the\u00a0detection of adenosine triphosphate (ATP), a\u00a0molecule that serves as the\u00a0primary energy carrier in and around living cells, thus providing a\u00a0direct measure of biological concentration in the\u00a0sample. ATP is detected by measuring light, and the\u00a0amount of light produced is directly proportional to the\u00a0amount of ATP present in a\u00a0given sample. The\u00a0measurement is expressed in relative light units (RLU). A\u00a0direct proportionality is always applied during measurement\u00a0\u2013 the\u00a0higher the\u00a0ATP level, the\u00a0higher the\u00a0RLU value. We discussed this indicator in detail in our previous article, which also cites relevant literature and examines the\u00a0issue of free (extracellular) and total ATP\u00a0[28].<\/span><\/p>\n Grab samples were analysed, collected in accordance with the\u00a0applicable sampling regulations at several bathing ponds in 2023. These were public bathing ponds located in Prague\u00a0\u2013 Radot\u00edn (hereinafter A), Prague\u00a0\u2013 Lhotka\u00a0(B), Kosmonosy near Mlad\u00e1 Boleslav (C), and Lipany (D), situated approximately 3\u00a0km west of \u0158\u00ed\u010dany. Site D is not a\u00a0typical bathing pond. It is an open-access reservoir with a\u00a0partial bypass; however, a\u00a0natural method of water treatment is also applied. The\u00a0capacity of bathing pond A\u00a0is 700 and that of pond B 1,000 visitors per day. The\u00a0design capacity of the\u00a0water area\u00a0at bathing pond C is 100 people at any one time, and 300\u2013500 persons per day. Fig.\u00a03<\/span><\/em> shows the maximum daily air temperature, precipitation in the study area (archive on the website https:\/\/www.in-pocasi.cz\/\u00a0[29]), and the\u00a0sampling days for the\u00a0individual sites. These data\u00a0allow at least partial inference of the\u00a0usage intensity of the\u00a0bathing ponds before sampling. Tropical days are defined as days with a\u00a0maximum daily temperature exceeding 30 \u00b0C; however, the\u00a0attendance at bathing ponds increases steadily from the\u00a0threshold for summer days (i.e., maximum daily temperature > 24 \u00b0C)\u00a0[30]. Precipitation, cloud cover, and other factors also have an influence. During sampling, air temperature and recent visitor numbers were always recorded. In terms of visitor attendance, and therefore the\u00a0expected loading of the\u00a0bathing pond, it is important to consider not only the\u00a0weather on the\u00a0day of sampling but also the\u00a0conditions over the\u00a0preceding few days. As\u00a0shown in Fig.\u00a03<\/span><\/em> (and supported by our records), the\u00a0days leading up to the\u00a0June and July sampling dates at sites A\u00a0and B were warm, and those before the\u00a0August and September sampling dates were very warm (site B was out of operation during July). At Site C, samples were taken on a\u00a0scheduled closing day; however, the\u00a0days preceding the\u00a0sampling were warm and without precipitation.<\/span><\/p>\n <\/p>\n At sites A\u2013C, two samples were taken from opposite sides of the\u00a0bathing pond (Fig.\u00a04<\/span><\/em>) for microbiological analysis and determination of urea\u00a0and caffeine. At site D, samples for these parameters were taken from a\u00a0single location. Samples for chlorophyll-a\u00a0were collected at sites A\u2013C from only one sampling point (Fig.\u00a04<\/span><\/em>). Samples for basic chemical analysis were taken at sites A and B: from one location at site A and from two locations at site B (Fig.\u00a04<\/span><\/em><\/span>). Total ATP was measured in situ<\/span> at the\u00a0above-mentioned sampling points.<\/span><\/p>\n Two bathing ponds (A\u00a0and B) were sampled repeatedly at four-week intervals throughout the\u00a0entire bathing season, while two sites (C and D) were sampled only once (Fig.\u00a03<\/span><\/em>).<\/span><\/p>\n Total number of samples collected at each site is shown in Tab.\u00a01<\/span><\/em>. Samples were transported under constant cooling to the\u00a0laboratory and processed immediately, except for caffeine determination, where samples were frozen at -18 \u00b0C and analysed collectively at the\u00a0end of the\u00a0bathing season. Possible changes in urea\u00a0and caffeine in the\u00a0samples during transport and storage were verified (considering the\u00a0effect of sample containers, duration, and storage method), using an internal standard (urea) or blank and duplicate samples (caffeine). Total ATP was measured on-site.<\/span><\/p>\n <\/p>\n Urea\u00a0was determined using a\u00a0method based on the\u00a0enzymatic breakdown of urea\u00a0by urease into ammonium ions, which were then measured spectrophotometrically (a\u00a0modification of the\u00a0method according to\u00a0[31]; for details, see\u00a0[16]). The\u00a0method\u2019s detection limit is 60 \u00b5g\/l and the\u00a0quantification limit is 110 \u00b5g\/l. Caffeine was determined by LC-MS\/MS with a\u00a0detection limit of 25 ng\/l and a\u00a0quantification limit of 50 ng\/l (a\u00a0modified procedure based on the\u00a0\u010cSN\u00a0ISO\u00a021676 standard\u00a0[32]).<\/span><\/p>\n The\u00a0indicators of faecal contamination, E. coli<\/span><\/em> and intestinal enterococci, were determined using standardized methods according to \u010cSN\u00a0EN\u00a0ISO\u00a09308-2\u00a0[33] and \u010cSN\u00a0EN\u00a0ISO\u00a07899-2\u00a0[34], respectively. Total ATP was measured luminometrically (Aquasnap, Hygiena) directly at the\u00a0site. During the\u00a0first sampling at\u00a0sites A\u00a0and B, we also measured bound ATP, but since these were revitalized sites, the\u00a0differences between total and bound ATP were small (up to 10\u00a0%), as opposed to other previously studied matrices\u00a0[28]. For this reason (and because it is simpler for potential operational measurements), we decided to measure only total ATP at the\u00a0bathing ponds.<\/span><\/p>\n P. aeruginosa<\/em>\u00a0<\/span><\/span>was analysed using an optimised method for bathing waters (i.e. for waters with a\u00a0high content of accompanying microflora)\u00a0[35]. Chlorophyll-a\u00a0was determined according to the\u00a0procedure specified in \u010cSN\u00a0ISO\u00a010260\u00a0[36].<\/span><\/p>\n Basic chemical analysis included organic carbon, total nitrogen, inorganic forms of nitrogen (nitrite, nitrate, and ammoniacal), orthophosphate phosphorus, total phosphorus, dissolved oxygen, and pH value. Water transparency was measured on site using a\u00a0Secchi disk, and the\u00a0concentration of dissolved oxygen (as well as oxygen saturation and water temperature) was measured with a\u00a0Hach LDO HQ 10 oximeter with an optical probe, at three depths: 30\u00a0cm, 1 m, and 2 m. As the\u00a0values did not vary significantly with depth, only the\u00a0\u2018surface\u2019 values (i.e. 30 cm below the\u00a0surface), where all samples for analysis were taken, are reported. pH value was measured immediately after transfer to the\u00a0laboratory using a\u00a0WTW inoLab\u00ae pH level 2 meter with a\u00a0combined THETA\u00a090 electrode. Organic carbon and total nitrogen were determined using a\u00a0Shimadzu TOC-V CPH analyser. Spectrophotometric methods in accordance with \u010cSN\u00a0EN\u00a026777\u00a0[37], \u010cSN\u00a0ISO\u00a07890-3\u00a0[38], and \u010cSN\u00a0ISO\u00a07150-1\u00a0[39] were used to determine nitrite, nitrate, and ammoniacal nitrogen. For the\u00a0determination of orthophosphate phosphorus and total phosphorus (after mineralisation with peroxodisulphate), the\u00a0spectrophotometric molybdenum blue method was used in accordance with \u010cSN\u00a0EN\u00a0ISO\u00a06878\u00a0[40]. Samples were filtered through Whatman GF\/C glass fibre filters. The\u00a0concentration of organically bound nitrogen was calculated as the\u00a0difference between the\u00a0total nitrogen (NT<\/span>) concentration and the\u00a0sum of the\u00a0inorganic forms of nitrogen (N-NO2<\/span>–<\/span>, N-NO3<\/span>–<\/span> a\u00a0Namo<\/span>).<\/span><\/p>\n The\u00a0following tables present the\u00a0results of all analyses carried out during the\u00a02023 bathing season. Tab.\u00a02<\/em> <\/span>shows the\u00a0basic chemical analysis at bathing ponds A\u00a0and B, while Tab.\u00a03<\/span><\/em> presents the\u00a0results for caffeine, urea, and microbiological and biological indicators (E. coli<\/span><\/em>, intestinal enterococci, P. aeruginosa<\/span><\/em>, total ATP, and chlorophyll) in all four bathing ponds.<\/span><\/p>\n <\/p>\n <\/p>\n The\u00a0values of chemical indicators in bathing ponds A\u00a0and B (Tab.\u00a02<\/span><\/em>) demonstrate a\u00a0very low level of pollution by both organic substances and nitrogen compounds. The\u00a0concentrations of orthophosphate and total phosphorus were below the\u00a0detection limits in all samples, which are 0.03 mg\/l and 0.04 mg\/l, respectively (not shown in the\u00a0table). The\u00a0water trophic potential is therefore very low, and any development of phytoplankton is strongly limited by phosphorus. This is reflected in the\u00a0low concentrations of chlorophyll-a\u00a0(Tab.\u00a02<\/span><\/em>). During the\u00a0bathing season, a\u00a0slight increase in the\u00a0concentration of organically bound nitrogen was observed at both sites, occurring as early as June at site A, and only during summer holidays at site B. The\u00a0highest recorded concentrations of Norg<\/span><\/sub> were 5.41 mg\/l at site A\u00a0and 4.27 mg\/l at site B. The\u00a0concentration of ammoniacal nitrogen fluctuated, with no clear consistent increase, trend, or correlation observed between the\u00a0concentrations of Namo<\/span><\/sub> and Norg<\/span><\/sub>. Nitrite nitrogen concentrations were usually below or just above the\u00a0detection limit (0.005\u00a0mg\/l), and nitrate nitrogen concentrations were also very low. Transparency at both monitored sites always reached the\u00a0bottom (3.25\u00a0m), except for the\u00a0August measurement at site B, when it was only 2 m (chlorophyll-a\u00a0was unfortunately not determined on that day). In terms of organic matter content, the\u00a0water was only minimally polluted, and during the\u00a0summer a\u00a0slight increase in the\u00a0concentration of Corg <\/span>was observed, which, together with the\u00a0temperature drop in September, fell back to the\u00a0June level. pH values at both sites ranged within the\u00a0mildly alkaline range. Changes in pH can be attributed to ongoing photosynthesis. The\u00a0concentration of dissolved oxygen remained around or slightly above the\u00a0equilibrium value throughout the\u00a0monitored period at both sites, except for the\u00a0August measurement at site B, where oxygen saturation rose to 122\u00a0%, possibly due to relatively intense photosynthetic activity of the\u00a0present producers. Apart from this isolated fluctuation, none of the\u00a0monitored indicators changed significantly over time. This suggests chemical stability of the\u00a0water and, considering the\u00a0low concentrations of nutrients and organic substances, also high efficiency of the\u00a0water\u2019s self-purification. Comparison of the\u00a0measured indicator values from two sampling points at site B shows that the\u00a0water in the\u00a0bathing area\u00a0is very well mixed.<\/span><\/p>\n The\u00a0chemical indicators included in the\u00a0basic analysis provide information on water quality and the\u00a0effectiveness of self-purification processes; however, some determinations, such as organic carbon, do not give information on the\u00a0nature of the\u00a0pollution. Therefore, they are mainly suitable as supplementary parameters. The\u00a0most promising results are those for total nitrogen\u00a0(NT<\/span><\/sub>), which show higher values from July onwards at site A, and from August (with the\u00a0site out of operation in July) at site B, compared to the\u00a0beginning of the\u00a0season.<\/span><\/p>\n Tab.\u00a03<\/span><\/span><\/em> presents microbiological and biological indicators for all four sites. Nearly all E. coli<\/span><\/em> and intestinal enterococci numbers complied with current legislative requirements (in accordance with Decree No. 238\/2011 Coll. of the\u00a0Ministry of Health), i.e. 100 CFU (MPN)\/100 ml and 50 CFU\/100 ml, respectively, and P. aeruginosa<\/span><\/em>\u00a0numbers exceeded the\u00a0former limit value of 10 CFU\/100 ml in only one sample. Total ATP values corresponded with our theoretical expectations: at site A, they were significantly higher in July and August during peak visitor numbers, and lower in June and September. At site B, they were lowest in the\u00a0July sample, when the\u00a0bathing pond was out of operation due to technical reasons. Despite high ATP values (above 200, and even exceeding 500 RLU), no elevated E. coli<\/span> <\/em>numbers were recorded at the\u00a0same time, in contrast with urban water features\u00a0[41]. This suggests the\u00a0presence of different, much more natural microbial communities, and in this case ATP did not demonstrate a\u00a0direct indicative value for faecal contamination. On the\u00a0other hand, the\u00a0presence of opportunistic pathogens associated with anthropogenic pollution cannot be entirely ruled out. As results of ATP determination in surface waters, including bathing and natural ponds, have not yet been published, we are unable to compare or discuss them further.<\/span><\/p>\n The\u00a0results of the\u00a0urea\u00a0and caffeine analyses are also presented in Tab 3<\/span><\/em>. The\u00a0measured values were predominantly above the\u00a0limit of quantification, with the\u00a0highest concentrations again recorded during the\u00a0summer period when visitor numbers peaked (with the\u00a0exception of site B, which was out of operation in July; the\u00a0values recorded at that time were therefore the\u00a0lowest). The\u00a0results for both indicators showed a\u00a0certain degree of correlation (R\u00b2 = 0.82). A\u00a0degree of correlation was also observed between caffeine and ATP (R\u00b2 = 0.65), and between ATP and urea\u00a0(R\u00b2 = 0.66). The\u00a0coefficients of variation for duplicate samples ranged from 9 to 26\u00a0% for caffeine determination and from 5 to 48\u00a0% for urea\u00a0determination. These correlations are also illustrated in Figs. 5a<\/span><\/em>\u00a0and 5b<\/span><\/em>.<\/span><\/p>\n <\/p>\n The work presented in this article builds upon our previous study of bathing waters [16], which, however, focused on significantly larger water bodies (ponds and sand pits) with relatively lower visitor numbers (i.e. a higher ratio of water volume to anthropogenic pollution input). As a result, caffeine and urea concentrations were considerably lower than those observed in the bathing ponds and only rarely exceeded the limit of quantification (for comparison, caffeine (in ng\/l): M\u011blice sand pit < 50\u2013170, \u0160eber\u00e1k pond < 50\u2013204, Pilsk\u00fd pond 84\u201393, Pod\u011bbrady sand pit < 50\u2013121, Eli\u0161ka pond < 50\u2013296).\u00a0 Measured values in lakes reported in the cited literature were also lower; for example, in Maine (USA), concentrations ranged from 6 to 11 ng\/l, with a maximum of 21 ng\/l [18]. The anthropogenic load of bathing ponds is therefore more comparable to that of artificial swimming pools, where average caffeine concentrations of up to 1,540 ng\/l [21] and 1,140 ng\/l [22] have been reported. High concentrations of urea have also been detected in swimming pools, namely 3.5\u20135.5 mg\/l [9], 0.07\u201318.73 mg\/l\u00a0[10], and 0.74\u201315.62 mg\/l\u00a0[11]. Zhang et al.\u00a0[10] also report that the\u00a0average volume of urine released per swimmer could be between 25 and 77 ml. Urea\u00a0is considered the\u00a0main nitrogen-based contaminant introduced into pool water by swimmers and is therefore an important indicator of water quality and hygiene in swimming pools. In China, it is regulated at 3.5 mg\/l (i.e.\u00a03,500\u00a0\u00b5g\/l; our highest recorded value was 871 \u00b5g\/l).<\/p>\n Relatively high caffeine concentrations in the\u00a0bathing ponds were recorded despite the\u00a0fact that only a\u00a0small proportion (0.5\u201310\u00a0%) of caffeine is excreted in unchanged form\u00a0[14], and metabolites were not analysed.<\/p>\n Among other (as yet unpublished) results, caffeine concentrations were as follows: wastewater outflow system (1,160 ng\/l), treatment pond (273 ng\/l), fishing pond (upper section 66 ng\/l, lower section < 50 ng\/l), and bathing pond (258 ng\/l). Urea\u00a0in these samples was mostly below the\u00a0limit of quantification, probably having already degraded into ammonium ions, with detected concentrations of 916, 471, 161, 250, and 185 \u00b5g\/l. These results also indicate that caffeine and urea\u00a0levels are higher in bathing ponds than in ponds of any other type.<\/p>\n The\u00a0indicators we monitored (particularly caffeine, urea, as well as ATP and total nitrogen) exhibited a\u00a0significant seasonal pattern in the\u00a0bathing ponds (Fig.\u00a05<\/em>). Therefore, results for the\u00a0entire season cannot be averaged; each individual measurement must be evaluated separately. At site B, high \u2018summer\u2019 caffeine and urea\u00a0values were already recorded during the\u00a0peak bathing season of 2022 (27 July at a\u00a0concentration of 993 ng\/l and 12 August at 432 ng\/l; results not previously published), which are comparable to the\u00a0values we measured during the\u00a0same period in 2023. Although duplicate samples (in 2023 always taken from two different locations within the\u00a0bathing area) generally showed only minor differences (mostly within 20\u00a0%) in some cases greater variation between duplicates was observed (caffeine 9\u201326\u00a0%, urea\u00a05\u201348\u00a0%). Therefore, especially for newly tested bathing ponds, it would be advisable to collect multiple samples or, preferably, to always take a\u00a0composite sample.<\/p>\n Confirmation of the\u00a0correlation between caffeine concentration and the\u00a0actual number of bathers can be found in the\u00a0work of Lempart et al.\u00a0[42], who conducted caffeine monitoring deliberately unaffected by current pool attendance (early morning). The\u00a0highest average caffeine concentration detected was only 12.81 ng\/l at the\u00a0waterslide, while the\u00a0average concentration in the\u00a0swimming zone of the\u00a0pool was 3.68 ng\/l.<\/p>\n Although this research has yielded many interesting insights, it has also raised a\u00a0number of further questions that would be advisable to address, especially in connection with anticipated future changes to Czech legislation. The\u00a0indicators currently used to assess water quality in bathing ponds are inadequate (in addition, results take three days to obtain) and do not fully capture the\u00a0main issues at the\u00a0sites. In contrast with natural swimming waters, the\u00a0situation in bathing ponds can be influenced, for example, by increasing the\u00a0intensity of purification processes. Furthermore, these findings can be used when establishing new bathing ponds. However, for the\u00a0actual operation of bathing ponds, there is a\u00a0particular lack of operational indicators that provide rapid results, which the\u00a0operator can determine independently and use to adjust management in a\u00a0timely manner. Unfortunately, the\u00a0determination of caffeine, urea, or total nitrogen does not meet this requirement, as these analyses must be carried out in a\u00a0laboratory. The\u00a0potential use of in situ total ATP measurement will require further study, particularly its variations in the\u00a0treatment section, which is highly biologically active. From a\u00a0hygiene perspective, it is important to consider the\u00a0long-term loading of bathing ponds. Prolonged and high loading of bathing ponds may also introduce numerous other substances (such as pharmaceuticals, hormones, cosmetic residues) and occasionally pathogenic microorganisms originating from the\u00a0skin or mucous membranes, even in cases where existing indicators of faecal contamination do not exceed prescribed limits. This was confirmed, for example, in the\u00a0case of the\u00a0occasional ear pathogen Pseudomonas<\/em> otitidis in bathing ponds during the\u00a0peak bathing season\u00a0[35], when the\u00a0highest counts were found at the\u00a0end of the\u00a0season. However, no correlation was observed with caffeine or urea. For monitoring long-term loading, caffeine, urea, and total nitrogen could find application. However, caffeine determination is expensive and is carried out in only a\u00a0limited number of laboratories. Similarly, urea\u00a0analysis is not commonly available in laboratories and is relatively labour-intensive. From this perspective, total nitrogen appears the\u00a0most promising, as it is measured by more laboratories and is more affordable.<\/p>\n Bathing ponds are unique systems in which living organisms play a\u00a0key role in maintaining water quality, while simultaneously a\u00a0large number of people bathe\u00a0in a\u00a0relatively small area\u00a0during the\u00a0peak season. Although bathing ponds have been studied for some time, the\u00a0knowledge gained remains limited and further questions continue to arise. The\u00a0prevailing pollution is of anthropogenic origin (with bathers representing the\u00a0main source of contamination), and the\u00a0monitored indicators of anthropogenic load (caffeine and urea) reach values of over 500 ng\/l and \u00b5g\/l, respectively, during the\u00a0peak summer season. The\u00a0indicators prescribed by current legislation (faecal contamination indicators E. coli<\/span> and intestinal enterococci) did not reflect the\u00a0increased anthropogenic load. The\u00a0results of total ATP determination proved to be of interest, but further research is needed, particularly in the\u00a0treatment zones, which are highly microbiologically active (including its relationship with overall biological activity, etc.). Total nitrogen could be a\u00a0suitable indicator for tracking the\u00a0gradual increase in anthropogenic load throughout the\u00a0season.<\/span><\/p>\n The\u00a0article was supported by the\u00a0Ministry of Health of the\u00a0Czech Republic\u00a0\u2013 RVO (National Institute of Public Health\u00a0\u2013 NIPH, Company ID 75010330). We thank the\u00a0operators of natural bathing ponds for allowing sampling and for providing valuable additional information.<\/span><\/span><\/em><\/p>\n ATP\u00a0\u2013 adenosine triphosphate<\/span><\/p>\n IS PiVo\u00a0\u2013 information system for drinking and bathing water <\/span><\/p>\n RLU\u00a0\u2013 relative light unit<\/span><\/p>\n CFU\u00a0\u2013 colony-forming unit<\/span><\/p>\n MPN\u00a0\u2013 most probable number<\/span><\/p>\n NT\u00a0<\/span>\u2013 total nitrogen<\/span><\/p>\n The\u00a0Czech version of\u00a0this article was peer-reviewed, the\u00a0English version was translated from the\u00a0Czech original by Environmental Translation Ltd.<\/p>\n","protected":false},"excerpt":{"rendered":" Bathing ponds represent a specific ecosystem where living organisms play a dominant role in maintaining the quality of water in the water body. At the same time, they are very frequently visited, so the biggest source of pollution is from bathers. The aim of this publication is to present and evaluate possible chemical indicators of anthropogenic load \u2013 caffeine and urea at four sites (two of which during the entire bath-ing season) in the summer of 2023. <\/p>\n","protected":false},"author":8,"featured_media":36019,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[86,92],"tags":[3897,3870,3896,3899,3898],"coauthors":[231,3871,1687,3872,3873],"class_list":["post-36280","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-hydraulics-hydrology-and-hydrogeology","category-main","tag-anthropogenic-pollution","tag-atp","tag-bathing-ponds","tag-caffeine","tag-urea"],"acf":[],"_links":{"self":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts\/36280","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/users\/8"}],"replies":[{"embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/comments?post=36280"}],"version-history":[{"count":4,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts\/36280\/revisions"}],"predecessor-version":[{"id":36284,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts\/36280\/revisions\/36284"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/media\/36019"}],"wp:attachment":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/media?parent=36280"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/categories?post=36280"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/tags?post=36280"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/coauthors?post=36280"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}BACKGROUND TO THE ISSUE<\/h2>\n
METHODOLOGY<\/h2>\n
<\/a><\/h2>\nFig. 2. Bathing pond Kosmonosy<\/h6>\n
<\/a>\nFig. 3. Maximum daily air temperature and daily precipitation at the Prague \u2013 Libu\u0161 station and sampling days in the summer season 2023<\/h6>\n
<\/a>\nFig. 4. Position of sampling points at bio-swimming pools A\u2013C; red marks sampling points for all indicators, orange marks microbiological indicators, chlorophyll-a, caffeine\u00a0and\u00a0urea, yellow marks microbiological indicators, caffeine and urea (source photomap: www.mapy.cz)<\/h6>\n
Tab. 1. Total number of samples collected at individual sites<\/h5>\n
<\/a>\nRESULTS<\/h2>\n
Tab. 2. Basic chemical analysis; at site A, only one sample was taken from the bathing area for basic chemical analysis<\/h5>\n
<\/a>\nTab. 3. Results of caffeine, urea, total ATP, microbiological and biological indicators detection (NA = not analyzed)<\/span><\/h5>\n
<\/a>\nBasic chemical indicators<\/h2>\n
Microbiological and biological indicators<\/span><\/h3>\n
Chemical indicators of anthropogenic pollution\u00a0\u2013 caffeine and urea<\/h3>\n
<\/a>\n<\/h6>\n
<\/a>\nFig. 5 a, b. Seasonal course of average concentrations (from sites A\u00a0and B) for\u00a0caffeine, urea, ATP, ammonia (Namo<\/sub>) and total nitrogen (NT<\/sub>)<\/h6>\n
DISCUSSION<\/h2>\n
CONCLUSION<\/h2>\n
Acknowledgements<\/h3>\n
ABBREVIATIONS<\/h3>\n