## 1 Introduction  
In recent years, pharmaceuticals have become one of the most important environmental contaminants. Pharmaceuticals enter the aquatic environment as a consequence of their increasing use in human and veterinary medicine and incomplete removal during wastewater treatment processes [1; 2; 3; 4; 5]. Pharmaceutical residues and their metabolites are being reported to be present in surface water, as well as groundwater. In the aquatic environment, pharmaceuticals from many different classes (such as antibiotics, antidepressants, analgesics, antipeipletics, etc.) have been found worldwide, at concentration levels ranging from ng/L to ug/L [6; 7; 8; 9; 10; 11; 12]. Moreover, extremely high concentrations in the order of mg/L have also been reported in India, China, Korea and Israel [13]. Although residues of pharmaceuticals occur in water bodies mostly at minor levels, there is a risk that they may negatively affect a number of non-target organisms (both vertebrates and invertebrates), as these substances are designed to induce a biological effect in organisms at minor concentrations [14; 15]. Tramadol hydrochloride is a synthetic opioid analgesic drug used to relieve pain but also to treat anxiety and depression [15; 16]. This substance has become the most prescribed opioid worldwide [17]. Tramadol interacts with opioid, adrenergic and serotonin receptors, and with its metabolites, it is mainly excreted through kidneys, with a mean elimination half-life of about 6 h [18; 19]. The unchanged excretion of the tramadol parent compound in urine is about 15%-35% [7]. Tramadol hydrochloride belongs to the group of frequently detected substances in aquatic environments, with reported concentrations of tens of ng/L. Higher concentrations, reaching up to hundreds of ng/L, are found in locations with elevated anthropogenic activities or near an effluent of wastewater treatment plants [20; 21; 22]. [PERSON] et al. [11] determined an average tramadol concentration in water samples from Czech stream (Zivny stream--tributary of Bianice river) to be highly affected by wastewater-treatment-plant effluents; the measured values reached up to 1400 ng/L. However, this pharmaceutical was also reported in samples from the control site (upstream of the wastewater treatment plant), with concentrations ranging from 4 to 33 ng/L. [PERSON] et al. [23] reported tramadol at a concentration of 0.24 ug/L in sewage-treatment-plant influent and 0.23 ug/L in effluent (in Germany). Even higher levels were found by [PERSON] et al. [7] in raw sewage in the United Kingdom (average concentration > 30 ug/L). As pointed out by [PERSON] et al. [24], most psychoactive drugs are soluble in water, because they need to cross the blood-brain barrier in order to have their desired effects. Unfortunately, their hydrophobicity means that, once they enter aquatic environments, they will be in the form to be taken up by aquatic organisms. Moreover, various drugs might concentrate in invertebrates, resulting in exposing predators (including fish) to levels exceeding surface-water concentrations [25]. [PERSON] et al. [26] observed hypoactivity in zebrafish larvae after 144 h exposure to a range of various tramadol concentrations, with the lowest observed effect concentration at 320 ug/L. [PERSON] et al. [14] and [PERSON] et al. [15] found behavioral changes--in particular, increased shelter dwelling--induced by tramadol exposure at a concentration as low as 1 ug/L in crayfish. Apart from behavioral effects, tramadol has been proven to have significant effects on development retardation and cause inhibition of growth in _Cyprimus carpio_ early life stages after 32-days-long exposure to tramadol concentrations starting at 10 ug/L. Moreover, histopathological examination revealed gill hyperemia [27].