1. Background
Pharmaceuticals are chemical compounds prepared or dispensed in pharmacies and hospitals and used in the medical treatment of humans and animals. They come in the form of prescription, over the counter, veterinary, or therapeutic pharmaceuticals. Due to industrialized production, there are many pharmaceuticals that became relatively largely accessible worldwide on the free market, including in terms of prices [
1,
2]. In the last decade, the unintentional presence of pharmaceuticals in the aquatic ecosystems (water, sediment, and biota) has become increasingly apparent in concentrations that can have a negative impact on the aquatic organisms and ecological processes. Due to their presence in the environment, pharmaceuticals are starting to be considered emerging pollutants: compounds not yet included in water-quality regulations, with unknown or poorly understood effects, and pose a potential threat to the ecosystems and human safety and health [
3].
In the Lower Danube Basin, footprints of human presence go back in history to 180,000 BC, with noticeable increasing damaging effects on the environment throughout time [
4,
5,
6]. One of the large-scale second-order tributaries of the Danube is the Mureş River; its upper and middle sectors are located in the amphitheater-like Transylvanian depression, ringed by the South-Eastern Carpathians, and inhabited by over seven million people [
7], making it a zone containing important human activities causing adverse effects [
8].
The Mureș River is the largest tributary of the Tisza River (Danube Basin), with a length of 761 km and a watershed with an area of 28,319 km
2, located in the central and western part of Romania (longitude: 20°11′ west and 25°44′ east and latitude: 45°14′ south and 47°08′ north). This basin relief varies significantly; mountains cover 25% of the surface, while 55% of the surface consists of hills and plateaus, 15% valleys and meadows, and 5% plains [
9,
10].
The Mureş Basin was chosen for this study for several reasons: the relatively large surface/importance in the Danube Basin [
6], the relatively significant human population living in the basin area including in large cities [
9,
11], the important historical and present human impact including pollution problems in the basin [
12,
13], the presence of numerous WWTPs (wastewater treatment plants) along the river using similar technology (all 15 WWTPs included in this study have mechanical, biological, and chemical processes used for the treatment of wastewater), the diversity of habitats in the watershed, the total lack of pharmaceutical-aquatic ecosystems related data in this basin, etc.
Pharmaceutical compounds have been investigated and found in the sewerage system’s contaminated waters, permanently affected by effluents from hospitals, residential, office, and production areas of Mureş River and its main tributaries. These compounds come from different pharmaceutical classes, such as non-steroidal anti-inflammatory (ibuprofen), psychotropic (carbamazepine), cardiovascular (enalapril), and diuretic pharmaceuticals (furosemide). Besides their presence in water environments, these pharmaceuticals have been characterized according to their water solubility, predicted no-effect concentration (PNEC), and pKa or log K
ow (
Table 1). Alongside the parent pharmaceuticals, it is interesting to look at their metabolites. For example, enalapril and the ibuprofen metabolites (carboxyibuprofen, 1-hydroxyibuprofen, and 2-hydroxyibuprofen) can be found in the environment. Of the above-listed compounds, carbamazepine is identified as a future emerging pollutant priority candidate, while ibuprofen is a proposed addition to this list [
14]. One reason carbamazepine has been thoroughly investigated is its ubiquitous presence (94%) in analyzed rivers. This presence is not so much due to its high use but more likely due to its slow degradation rate and ability to be extracted efficiently from contaminated samples or efficient extraction methods [
15,
16].
1.1. Pollution Sources and Environmental Hazards of Studied Pharmaceutical Compounds
It has been reported that some pharmaceuticals that are present in surface waters, groundwater, and the discharge from WWTPs pose a severe environmental problem, since these compounds could affect non-target and susceptible species because they are biologically active [
23,
24]. Furthermore, these compounds have potentially toxic effects (or could determine behavioral alteration) in the aquatic trophic nets, affecting the food chain organisms such as phytoplankton [
25], amphipods [
26] and crustaceans [
27], fish [
28,
29], and finally, mammals [
30]. When looking specifically at the pharmaceuticals of interest, it is observed that carbamazepine induces a stress response in rainbow trout individuals (
Oncorhynchus mykiss) [
28]. At the same time, ibuprofen negatively affects the health of African catfish (
Clarias gariepinus) individuals [
29].
Due to their polar nature (
Table 1), these compounds stay in the solution and do not adhere to soil and particles; therefore, they are mobile in the environment. Another downside of pharmaceuticals is the continuous release into the aquatic environment, which gives them the characteristic of persistent organic pollutants (POPs) regarding their high detection rate. These characteristics make the studied pharmaceutical compounds likely to reach drinking water sources, posing a serious problem for human safety and health in places dependent on recycled water. The problem has been reported in France [
23], the United States [
31], and Australia [
32]. In Romania, the effluent from WWTPs is not reused as drinking water and is discharged back into rivers [
33]. This effluent mixes with the hyporheic water and can potentially influence other downstream water sources. When considering the dilution of the pharmaceuticals, the human risk is lowered, but there are still problems when mixtures are involved [
34], and new compounds are added to the mixture every day. On the other hand, the threat to the environment is problematic, and high interest is accorded to pharmaceutical’s presence and their effects on flora and fauna [
35]. From this point of view, the studied basin is a grey area, with few reports about pharmaceutical concentrations [
36,
37,
38], a relatively common situation, especially in the southeastern part of Europe, but not only.
The two primary biological sources of pharmaceuticals in the studied environment are derived from veterinary and medical uses, through animal and human excretion of active metabolites consisting of a mixture of metabolized and conjugated compounds and unmetabolized compounds [
3]. Humans excrete mainly 55 to 80% unmetabolized compounds (with few exceptions) through urine and partially through feces [
39,
40,
41]. The following can be different sources of aquatic environment contamination: WWTP discharges, hospital effluents, direct disposal of unused or expired pharmaceuticals, manufacturing, landfill leachates, livestock activities, aquaculture, and soil fertilization with sewage sludge and livestock waste [
42,
43,
44]. Among the mentioned sources, WWTPs are considered the most problematic source of contamination [
45]. This is because the fact that WWTPs do not effectively eliminate all the pharmaceutical compounds from the WWTP influents during the procedures of removing pollutants [
46,
47,
48,
49,
50,
51,
52,
53,
54,
55,
56,
57,
58]. Examples of inadequate removal of pharmaceuticals at the WWTP are carbamazepine, with a low (10–20%) to no removal efficiency [
53,
54], and furosemide, with a removal efficiency under 42% [
55]. Pharmaceuticals could have long-term effects on biota and could exhibit bioaccumulation, and the presence of different pharmaceutical mixtures, which could have additive and synergistic effects [
3]. In terms of avoiding pharmaceutical contamination of water, a series of methods have been proposed and implemented, such as physical adsorption processes, biological degradation processes, chemical processes, advanced oxidation processes, and various combined methods [
56]. Alongside these methods, a series of procedures limiting the disposal of surplus pharmaceuticals should also be considered.
Very few studies investigated the occurrence of pharmaceuticals in river waters in Romania [
57,
58,
59,
60,
61]; even fewer studies mention parts of the investigated area and pharmaceuticals [
36,
37,
38].
1.2. Aim
This study focuses on the occurrence, distribution, and fate of several pharmaceuticals that are on the emerging pollutants list or that are commonly used in the Mureș River Basin, where no large-scale data about them are widely available. This study aims to assess the potential risk of the investigated pharmaceuticals in the environment. One way of tackling this problem is using the hazard quotient (HQ: ≥1 indicates a potential for negative impact on the lotic ecosystem, <1 low ecological risk), i.e., the ratio between the measured environmental concentration (MEC) and predicted no-effect concentration (PNEC). HQ is the measure of the potential exposure to a substance for which no adverse effect is expected.
4. Discussion
In the Water Framework Directive (WFD) [
66], there are two types of water environmental quality standards (EQS) concerning pharmaceuticals: the annual average concentration (AA-EQS) based on chronic toxicity data and the maximum acceptable concentration (MAC-EQS) [
67]. These values are 0.5 µg/L (AA-EQS) and 1600 µg/L (MAC-EQS) for carbamazepine [
67] and 1 µg/L (AA-EQS) and 40 µg/L (MAC-EQS) for ibuprofen [
68]. There are no values calculated for enalapril, enalaprilat, furosemide, 1-hydroxyibuprofen, 2-hydroxyibuprofen, and carboxyibuprofen. We found the maximum concentration of carbamazepine in the downstream sample to be above the annual average environmental quality standard but lower than the maximum accepted. However, the situation worsens in the WWTP effluent, where the concentration average is higher than the AA-EQS for carbamazepine. Because the average and median concentrations for carbamazepine in the downstream sample are less than half of the average accepted value, these concentrations do not pose a threat as of yet. Still, they should be monitored closely so as to not rise above the annual accepted average through accumulation.
We found that the HQ is lower than one for all the pharmaceuticals tested, in all the sampling sites, both in the WWTP effluent and in the river. The values we found are similar to values already reported [
19] or slightly lower than those values. Ibuprofen has been found to have the highest HQ in the WWTP [
19], while in our study, furosemide and carbamazepine seem to have slightly higher HQs. The HQ values that range from 0.1 to 1 are considered low hazard with potential adverse effects; between 1 and 10, the adverse effects and hazard are probable, while for values higher than 10, hazard are anticipated [
19,
69,
70]. These results imply that for the pharmaceuticals studied, the measured levels pose a threat to the environment, especially for the effluent. Although WWTPs do not have designated methods for removal of the studies pharmaceuticals, it has been reported that some of them degrade during wastewater treatment. Using reported percentages of WWTP clean-up of pharmaceuticals, 42% for furosemide [
55], 20% for carbamazepine [
53], 80% for ibuprofen [
71], and 95% for enalapril, we calculated the putative influent concentration (
Figure 5). The results show much higher concentrations in the influent compared with the effluent (
Figure 5). These concentrations could be potentially hazardous, especially during heavy rain periods when WWTPs overflow and discharge the effluent at a higher rate than normal.
Moreover, we considered the impact of these high concentrations of pharmaceuticals on the bacteria contained in the activated sludge and the possibility that they would be killed or inhibited. When comparing the MECs of pharmaceuticals in the WWTP with known concentrations that impact bacterial survival, we find them to be at least three orders of magnitude lower than concentrations that would impact bacterial survival [
72]. Even if we extrapolate the putative influent concentrations, we do not obtain values over the risk concentrations. Enalapril, which had the highest degradation/removal rate, has a putative concentration in the influent lower than the rest of the pharmaceuticals investigated. Therefore, we do not anticipate a negative impact caused by this singular pharmaceutical’s presence on the biological processes taking place in the WWTP.
Our measurement that in the majority of cases the downstream contamination is lower than the effluent contamination, coupled with the correlations observed between downstream and effluent concentration, leads us to believe that the WWTPs are primary sources of river pollution with pharmaceuticals. The underlying concentration that appears in the river (upstream sites) is not high enough to hint at other sources of contamination that could topple the effect of the WWTPs. The fact that enalaprilat and furosemide concentrations are correlated between the upstream and downstream of WWTP sites could mean that these compounds could quickly traverse the river’s length between the WWTP sites before precipitating out of the solution or being degraded. This is backed up by their higher solubility in water compared to the other pharmaceuticals, calculated through the Log K
ow parameter (
Table 1). The fact that the concentrations of ibuprofen and 2-hydroxyibuprofen are positively correlated in all the investigated cases (upstream, downstream, and WWTP effluent), while concentrations of ibuprofen and carboxyibuprofen are positively correlated in two cases (upstream and downstream) could be due to the metabolization of ibuprofen in humans, for which 2-hydroxyibuprofen and carboxyibuprofen are final products and 1-hydroxyibuprofen is an intermediary step to carboxyibuprofen [
73]. Therefore, it is conceivable that 2-hydroxyibuprofen and carboxyibuprofen would have higher concentrations than 1-hydroxyibuprofen, which appears to be the case (
Table 4).
The frequency of detection was above 50% both in the WWTP effluent and downstream from it, reaching 100% for some of the pharmaceuticals (
Table 4). Enalapril and its metabolite enalaprilat had the lowest frequency of detection overall. The rest had low frequencies in the upstream samples, with the frequencies rising above 80% for most pharmaceuticals in the WWTP effluent and downstream from it. Carbamazepine had the highest detection level, with 93% upstream, 93% downstream, and 100% at the WWTP. This high frequency of detection points to several things: it shows the level of pollution with the tested pharmaceuticals, it confirms our ability to measure the presence of these pharmaceuticals in the river water, and it shows the slow degradation rate and persistence of this pharmaceutical in the environment [
74].
There is no apparent rise in concentrations from the source of the river towards the exit from Romania to Hungary, which could be the result of a high rate of precipitation out of the solution due to the low level of solubility of the investigated pharmaceuticals. Another reason could be the short half-life of these pharmaceuticals, which are hours to days for ibuprofen [
75] and 63 days [
76] or 38 days [
77] for carbamazepine; this could be an indication of the high degradation rate of these compounds. One way of identifying the accumulation of pharmaceuticals in the river is to look at the river sediment, which could be an interesting topic for future research.
In Romania, the concentration of ibuprofen has been reported between 61.3 and 115.2 ng/L in the Someș River in 2006 [
57], between 9 and 63 ng/L at different main localities in the Someș River in 2007 [
58], and between 16 and 63 ng/L at the WWTP effluent in the Someș River in 2008 [
59]. Concentrations were in the range of 1.65–71.85 ng/L for the upstream sites, 15.68–403.06 ng/L for WWTPs, and 8.84–117.14 ng/L for the downstream sites (
Table 4). For carbamazepine, it has been reported that the concentrations ranged from 67 to 75 ng/L in the Someș River in 2006 [
57] and 38 to 56 ng/L in 2008 [
58] and 20–49 ng/L in the Danube River, while a maximum concentration of 140 ng/L was detected in the Argeș River [
76]. In 2015, it was also reported that the concentration of carbamazepine was situated in the interval of 4 to 40 ng/L in the Danube River and some tributaries [
61] and the interval of 5 to 25 ng/L for some major Romanian rivers (Prahova, Timiș, Danube, Siret, Prut, and Jijia) [
60]. The previously reported concentrations have a maximum that is lower than the concentrations reported in this study, at all the investigated sites (upstream, WWTP, and downstream) (
Table 4). The only exception is for the concentration of ibuprofen from the Târgu Mureș WWTP, which was investigated in three studies [
36,
37,
38] and have identified high concentrations, up to 7600 ng/L. These results point to a problematic situation of the Mureș River as a hotspot, with levels of carbamazepine in the WWTP effluent reaching a maximum of 1992.43 ng/L. These high concentrations could be due to sampling the effluent, where higher concentrations are to be expected. Another explanation for these high MECs is the fact that Mureș River has a smaller volume of water than the Danube, which makes detecting higher concentrations of pharmaceuticals more likely, as already pointed out [
60]. The fact that we detected these pharmaceuticals in high concentrations does not come as a surprise, especially given that a European wide study in 2009 has detected ibuprofen and carbamazepine, among others, as compounds with the highest maximum concentrations in the range of µg/L [
77]. Taking into consideration the 90th percentile and the proposed indicative warning levels mentioned in 2009 [
77], we can see that the concentrations that we detect are well above the threshold for carbamazepine (limit of 100 ng/L) when looking at the average concentrations of all the substances detailed in this paper in the WWTP effluent and downstream of the WWTP, and ibuprofen (limit of 200 ng/L) in the WWTP effluent when looking at the maximum detected concentration. In this respect, the Mureș River Basin can be considered polluted, and these results warrant further investigation.
The potential synergic effects of these pharmaceutical compounds with other pollutants present in the basin, such as POPs [
12,
13,
78], can raise the area’s risk potential for natural and semi-natural ecosystems and human settlements health and welfare, which increases the importance of monitoring in the catchment basin of rivers receiving wastewater [
79,
80]. The problem with high levels of pharmaceuticals in the environment is tied directly to human exposure. In this case, the concentrations to which we are exposed could be higher, especially when the individuals are under treatment with the investigated pharmaceuticals.
The potential risk associated with these pharmaceuticals can rise in the present situation. The climate changes [
81] tend to reduce the minimum, average, and maximum dilution flow [
5] in the Danube Basin too, and the increasing human water consumption will put supplementary pressure on this respect as well.