Multiclass determination of drug residues in water and fish for bioaccumulation potential assessment.

In this work, a wide-scope liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of environmental levels of multiclass drugs and their metabolites in water and fish samples was developed. The method allowed the reliable determination of 44 drugs, covering a rather wide range of chemistries and physicochemical characteristics. In order to obtain a reliable and robust analytical protocol, different combinations of extraction and cleanup techniques were systematically examined. Aqueous samples were extracted using a simple Oasis HLB SPE enrichment protocol with pH-optimized sample percolation (pH 3). The extraction of cryo-homogenized biota samples was performed using double extraction with MeOH basified with 0.5% NH3, which allowed high extraction recoveries for all target analytes. The problem of the coextracted lipid matrix, which is known to be the key obstacle for reliable biota analysis, was systematically examined in a series of model cleanup experiments. A combination of cryo-precipitation, filtration, and HLB SPE cleanup was proposed as a protocol, which allowed reliable and robust analysis of all target compounds at low ng/g levels. At the final conditions, the method which was validated at three concentration levels showed high extraction recoveries (68-97%), acceptable matrix effects (12 to -32%), accuracies (81-129%), and reproducibilities (3-32%) for all analytes. The developed method was used to determine drug concentrations in river water and in feral freshwater fish, including whole fish and muscle tissue, from the Sava River (Croatia), in order to estimate their corresponding bioaccumulation potential. With respect to bioaccumulation potential in whole fish and fish muscle, the most relevant drugs were lisinopril, sertraline, terbinafine, torsemide, diazepam, desloratadine, and loratadine with estimated bioaccumulation factors ranging from 20 to 838 and from 1 to 431, respectively.

Long-term monitoring of drug consumption patterns in a large-sized European city using wastewater-based epidemiology: Comparison of two sampling schemes for the assessment of multiannual trends.

A comprehensive study aimed at monitoring of temporal variability of illicit drugs (heroin, cocaine, amphetamine, MDMA, methamphetamine and cannabis) and therapeutic opiate methadone in a large-sized European city using wastewater-based epidemiology (WBE) was conducted in the city of Zagreb, Croatia, during an 8-year period (2009-2016). The study addressed the impact of different sampling schemes on the assessment of temporal drug consumption patterns, in particular multiannual consumption trends and documented the possible errors associated with the one-week sampling scheme. The highest drug consumption prevalence was determined for cannabis (from 59 ±â€¯18 to 156 ±â€¯37 doses/day/1000 inhabitants 15-64 years), followed by heroin (from 11 ±â€¯10 to 71 ±â€¯19 doses/day/1000 inhabitants 15-64 years), cocaine (from 8.3 ±â€¯0.9 to 23 ±â€¯4.0 doses/day/1000 inhabitants 15-64 years) and amphetamine (from 1.3 ±â€¯0.9 to 21 ±â€¯6.1 doses/day/1000 inhabitants 15-64 years) whereas the consumption of MDMA was comparatively lower (from 0.18 ±â€¯0.08 to 2.7 doses ±0.7 doses/day/1000 inhabitants 15-64 years). The drug consumption patterns were characterized by clearly enhanced weekend and Christmas season consumption of stimulating drugs (cocaine, MDMA and amphetamine) and somewhat lower summer consumption of almost all drugs. Pronounced multiannual consumption trends were determined for most of the illicit drugs. The investigated 8-year period was characterized by a marked increase of the consumption of pure cocaine (1.6-fold), THC (2.7-fold), amphetamine (16-fold) and MDMA (15-fold) and a concomitant decrease (2.3-fold) of the consumption of pure heroin. The heroin consumption decrease was associated with an increase of methadone consumption (1.4-fold), which can be linked to its use in the heroin substitution therapy. The estimated number of average methadone doses consumed in the city of Zagreb was in a good agreement with the prescription data on treated opioid addicts in Croatia.

Wastewater-based assessment of regional and temporal consumption patterns of illicit drugs and therapeutic opioids in Croatia.

A comprehensive study of spatial and temporal consumption patterns of the selected illicit drugs (heroin, cocaine, amphetamine, MDMA, methamphetamine, cannabis) and therapeutic opioids (codeine, methadone) has been performed in six Croatian cities by applying wastewater-based epidemiology. The investigated cities (Bjelovar, Vinkovci, Varazdin, Karlovac, Zadar and Zagreb) varied widely in the population size (27,000-688,000 inhabitants) as well as in the number of registered drug consumers included in compulsory and voluntary medical treatment and rehabilitation programs (30-513 persons/100,000 inhabitants of age 15-64). The most consumed illicit drugs were cannabis (10-70doses/day/1000 inhabitants), heroin (<0.2-10doses/day/1000 inhabitants) and cocaine (0.2-8.7doses/day/1000 inhabitants), while the consumption of amphetamine-type drugs was much lower (<0.01-4.4doses/day/1000 inhabitants). Enhanced consumption of illegal drugs was generally associated with larger urban centers (Zagreb and Zadar) however comparatively high consumption rate of cocaine, MDMA and methadone was determined in some smaller cities as well. The overall average dose number of 3 major illegal stimulants (cocaine, MDMA, amphetamine) was rather similar to the number of corresponding heroin doses, which is in disagreement with a comparatively much higher proportion of heroin users in the total number of registered drug users in Croatia. Furthermore, the illicit drug consumption pattern in the large continental city (Zagreb) was characterized by a significant enhancement of the consumption of all stimulants during the weekend, which could not be confirmed neither for the coastal city of Zadar nor for the remaining small continental cities. On the other hand, the city of Zadar exhibited a significant increase of stimulant drug usage during summer vacation period, as a result of pronounced seasonal changes of the population composition and lifestyle in coastal tourist centers. The obtained results represent a valuable complementary data source for the optimisation and implementation of strategies to combat drug abuse in Croatia.

Assessment of stability of drug biomarkers in municipal wastewater as a factor influencing the estimation of drug consumption using sewage epidemiology.

Stability of the selected urinary biomarkers of six illicit drugs and two therapeutic opioids in municipal wastewater was studied in order to determine errors associated with their possible transformation in the sewer. The stability was assessed in experiments conducted at 10°C and 20°C in order to simulate typical winter and summer temperature conditions in the sewer system. Among fourteen substances tested, the most unstable compounds were morphine-3-ß-D glucuronide (MG), 6-acetyl morphine (6-AM), cocaine (COC) and 6-acetyl codeine (6-AC), while all other investigated compounds appeared to be relatively stable over a period of 72 h. The transformation of all degradable compounds followed pseudo-first order kinetics with significantly longer half-times (t1/2) at winter conditions. At 20°C, t1/2 of MG, 6-AM, COC and 6-AC was 7h, 87 h, 35 h and 58 h, respectively, while the corresponding t1/2 values at 10°C were 18 h, 139 h, 173 h and 87 h. The main transformation mechanism of MG, 6-AM and 6-AC was most probably their enzymatic hydrolysis to morphine (MOR) and codeine (COD), while COC transformation to benzoylecgonine (BE) was primarily governed by chemical hydrolysis. The results indicate that the effect of the observed transformation of urinary biomarkers of COC and 6-AM on the estimates of COC and heroin consumption are relatively small (<10%) if the in-sewer hydraulic retention time is lower than 12h. Acidification of the wastewater samples proved to be the good way to stabilise the wastewater samples for the analysis of all selected compounds, except for 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH). This finding should be taken into account when selecting the preservation technique for multiresidual analyses of different groups of illicit drugs.

Assessment of toxicological profiles of the municipal wastewater effluents using chemical analyses and bioassays.

The hazardous chemical contamination of untreated wastewater and secondary effluent from the wastewater treatment plant (WWTP) of the city of Zagreb, Croatia was comprehensively characterized using large-volume solid-phase extraction (SPE) and silica gel fractionation, followed by a detailed analysis of the resulting extracts by a combination of chemical and bioassay methods. Over 100 individual contaminants or closely related-contaminant groups were identified by high-resolution gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-QTOF). Ecotoxicity profiling of the investigated samples, including cytotoxicity, chronic toxicity and EROD activity; inhibition of the multixenobiotic resistance (MXR), genotoxicity and estrogenic potential, revealed the most significant contribution of toxic compounds to be present in polar fractions. Wastewater treatment using conventional activated sludge process reduced the initial toxicity of raw wastewater to various extents, ranging from 28% for algal toxicity to 73.2% for an estrogenic activity. The most efficient toxicity removal was observed for the polar compounds.

Nontarget analysis of polar contaminants in freshwater sediments influenced by pharmaceutical industry using ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry.

A comprehensive analytical procedure for a reliable identification of nontarget polar contaminants in aquatic sediments was developed, based on the application of ultra-high-pressure liquid chromatography (UHPLC) coupled to hybrid quadrupole time-of-flight mass spectrometry (QTOFMS). The procedure was applied for the analysis of freshwater sediment that was highly impacted by wastewater discharges from the pharmaceutical industry. A number of different contaminants were successfully identified owing to the high mass accuracy of the QTOFMS system, used in combination with high chromatographic resolution of UHPLC. The major compounds, identified in investigated sediment, included a series of polypropylene glycols (n=3-16), alkylbenzene sulfonate and benzalkonium surfactants as well as a number of various pharmaceuticals (chlorthalidone, warfarin, terbinafine, torsemide, zolpidem and macrolide antibiotics). The particular advantage of the applied technique is its capability to detect less known pharmaceutical intermediates and/or transformation products, which have not been previously reported in freshwater sediments.