Polychlorinated biphenyls in mussels, small pelagic fish, tuna, turtles, and dolphins from the Croatian Adriatic Sea waters: an overview of the last two decades of monitoring.

This review summarises our two decades of polychlorinated biphenyl (PCB) monitoring in different marine organisms along the eastern Adriatic Sea. The aim was to gain an insight into the trends of PCB distribution in order to evaluate the effectiveness of past and current legislation and suggest further action. Here we mainly focus on PCB levels in wild and farmed Mediterranean mussels, wild and farmed bluefin tuna, loggerhead sea turtles, common bottlenose dolphins, and small pelagic fish. The use of artificial intelligence and advanced statistics enabled an insight into the influence of various variables on the uptake of PCBs in the investigated organisms as well as into their mutual dependence. Our findings suggest that PCBs in small pelagic fish and mussels reflect global pollution and that high levels in dolphins and wild tuna tissues raise particular concern, as they confirm their biomagnification up the food chain. Therefore, the ongoing PCB monitoring should focus on predatory species in particular to help us better understand PCB contamination in marine ecosystems in our efforts to protect the environment and human health.

Human health risk assessment based on direct and indirect exposure to endocrine disrupting herbicides in drinking, ground, and surface water in Croatia.

The recognition of certain herbicides as endocrine disrupting compounds has raised concerns due to their ability to interfere with the normal functioning of the endocrine system, which regulates various physiological processes in organisms. The objective of this study was to assess the possible human health risks associated with terbuthylazine and endocrine-disrupting herbicides atrazine, acetochlor, and metolachlor in the drinking, surface, and groundwater of the Zagreb city region, Croatia. We relied on advanced statistical methods and principal component analysis (PCA), which revealed higher levels of atrazine and acetochlor in drinking and groundwater samples and higher presence of metolachlor and terbuthylazine in surface waters. To evaluate the danger to human health, various exposure scenarios have been assessed. The risk of direct human exposure to analyzed herbicides through drinking or bathing with drinking (tap) or groundwater, as well as from recreational activities like swimming in rivers, streams, and lakes, has been quantified. In addition to these direct exposure scenarios, indirect ones based on consumer goods, fruits, and vegetables, treated with surface and groundwater for irrigation, were assessed to investigate the danger to human health. Judging by the reported herbicide levels there was no significant risk of carcinogenic (CR ≤ 1 × 10-6) or non-carcinogenic (HI < 1) diseases, not even when we assessed the so-called "cocktail effect" of combined the herbicide exposure in different waters.

Environmental Monitoring and Analysis of Persistent Organic Pollutants.

Persistent organic pollutants (POPs) are a group of 28 toxic compounds of different chemical classes listed in the Stockholm Convention on POPs, which aims to protect the environment and human health [...].

Seasonal distribution of multiclass pesticide residues in the surface waters of northwest Croatia.

As part of our OPENTOX project, we evaluated the incidence and mass concentrations of multiclass pesticide residues in 23 river/stream water samples collected in urban and agricultural areas of northwest Croatia at various points of the pesticide application season in 2015. The study included 16 compounds of five herbicide classes and seven compounds of three insecticide classes. Pesticide residues were accumulated from water by solid-phase extraction and analysed using high performance liquid chromatography with UV-diode array detection and/or gas chromatography-mass spectrometry. Herbicide residues were more common than the insecticide ones, and, as expected, they peaked in the middle of the application season. Metolachlor showed the highest concentrations and was found in 91 % of all samples, followed by terbuthylazine, found in 70 % of the samples. The highest total mass concentration of detected pesticides was measured in the water samples of the Krapina (3992 ng/L) and Sutla (3455 ng/L) collected in rural areas with intensive agriculture. Our findings strongly speak in favour of continued monitoring of surface waters and possibly extending the list of priority water pollutants.

Distribution of Organochlorine Pesticide and Polychlorinated Biphenyl Residues in Lake Sediment Cores from the Plitvice Lakes National Park (Croatia).

The occurrence and distribution of 11 organochlorine pesticides (OCPs) and their degradation products as well as of 17 polychlorinated biphenyl congeners (PCBs) were investigated for the first time in sediments of three (Lake Prosce, Lake Kozjak, Lake Kaluderovac) out of 16 cascading karst lakes within the protected area of the Plitvice Lakes National Park in central Croatia. The 15-cm-long sediment cores were divided for analysis into three 5-cm-long segments. The abundance and levels of sediment-associated OCPs and PCBs were evaluated with respect to sampling location and sediment depth, presumed age of deposition, and organic carbon content. The burden of sediments with OCPs and PCBs decreased downstream with the highest mass fractions measured in the uppermost Lake Prosce (total OCPs 2.72-5.86 µg kg-1 d.m., total PCBs 0.37-1.78 µg kg-1 d.m.) and the lowest in Lake Kaluderovac (total OCPs 0.30-0.58 µg kg-1 d.m., total PCBs 0.07-0.12 µg kg-1 d.m.). The predominant organochlorine pollutants were DDT-type compounds (total DDX, w = 0.30-5.72 µg kg-1 d.m.) with p,p'-DDE and p,p'-DDD accounting for up to 100% and 50%, respectively, of the total DDX. The findings indicated an old input of p,p'-DDT that largely converted to its degradation products. The OCP and PCB mass fractions in the Plitvice lake sediments were at trace levels characteristic for preserved pristine natural environments with no or limited anthropogenic impact wherein long-range atmospheric transport is considered as their main source.

Soil burden by persistent organochlorine compounds in the vicinity of a coal-fired power plant in Croatia: a comparison study with an urban-industrialized area.

The impact of a coal-fired Plomin Power Plant (PPP) in Croatia on PCB soil burden was examined by comparing the occurrence, levels, and profile of PCBs in soil from the PPP with the values determined in urban-industrialized soil (Varazdin, Croatia). Soil burden by organochlorine pesticides (OCPs) were also investigated at both locations. Topsoil samples were collected at five distances (100-800 m) along a downwind pollution gradient from the PPP and across the city. The total content of PCBs in 100-m soil was nearly 20-fold the levels found in 800-m soil, which pointed to the PPP as a local source of soil contamination. The PPP soils were dominated by indicator PCB congeners, particularly hexa-homologs. A different profile and mass fraction range of PCBs in soils from PPP and Varazdin area indicated the different sources of contamination. Levels of total PCBs in PPP soils (0.25-19.07 µg kg-1) were higher than PCB levels determined in soils from Varazdin (0.29-5.52 µg kg-1), partially as a result of higher OC content in PPP soils. PPP soil burden by PCBs corresponded to a lower end of PCB level ranges reported for cities with high population and heavy industry. OCPs were detected at significantly higher levels in Varazdin soils than in PPP soils, with the highest contribution of the DDT-like compounds (DDX) detected in soils affected by river deposits. The p,p'-DDE/p,p'-DDT ratio in Varazdin soils indicated a fresh atmospheric input of p,p'-DDT. The PPP soil analysis detected a presence of only p,p'-DDE and HCB at levels corresponding to their global environmental presence.

Urinary metabolites as biomarkers of human exposure to atrazine: atrazine mercapturate in agricultural workers.

Human exposure to atrazine and other triazine herbicides results in urinary excretion of traces of parent compounds and of their metabolites formed by N-dealkylation or conjugation with mercapturic acid. In contrast to N-dealkylated metabolites, which are not compound-specific, the measurement of atrazine mercapturate and unchanged atrazine provides an unambiguous confirmation of exposure to this herbicide. The aim of this study was to investigate the levels of these two compounds in a group of agricultural workers who may be considered representative for typical behaviour and procedures during the atrazine application in Croatia. The spot urine samples were collected at the beginning (samples A) and at the end (samples B) of a working day and 12h after exposure has ended (samples C). Atrazine and atrazine mercapturate were extracted from acidified urine samples (pH 2) with ethyl acetate and the extracts were analysed using high performance liquid chromatography-tandem mass spectrometry with a turbo ion spray (electrospray) ionization interface. The detection limits based on treatment of 2ml urine samples were 0.2ngml(-1) for both analytes. Atrazine was not detected in any of 27 analysed urine samples but traces of atrazine mercapturate were measured in about a third of pre-exposure and in all post-exposure urine samples in mass concentrations ranging from 0.3 to 10.4ngml(-1) (0.3 to 8.0µgg(-1) of creatinine). The metabolite concentrations in B and C group of post-exposure samples were not significantly different. The urinary atrazine mercapturate post-exposure concentrations were comparable to those reported for U.S. farmers engaged in a single field application of atrazine.

Effect of organic and mineral soil fractions on sorption behaviour of chlorophenol and triazine micropollutants.

This article compares the sorption behaviour of 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, pentachlorophenol, chlorotriazine atrazine, methylthiotriazine ametryn, methoxytriazine atratone, hydroxyatrazine, and didelakylated atrazine in a topsoil and an aquifer sediment before and after removal of sorbent organic matter and in humic acid. Freundlich isotherm coefficients Kf and 1/n and free energy change (deltaG degrees ) were calculated for all compounds in all sorbents. According to sorbent pH values, chlorophenolate anions and uncharged triazine species dominated in all sorption experiments with topsoil and aquifer sediment. In experiments with humic acid, chlorophenols, atrazine, and didealkylated atrazine existed almost completely as neutral species, whereas protonated species dominated for hydroxyatrazine, atratone, and ametryn. In addition to a hydrophobic partition, sorption of all compounds in native soil and sediment sorbents includes specific, more polar interactions, which greatly depend on sorbate acidity/basicity, specific properties of the sorbent organic matter and of mineral surface, as well as on the system pH. A significantly greater sorption intensity of all compounds in "organic-free" than in the native aquifer sediment confirmed the importance and possible dominance of mineral surface in the sorption process. Sorption intensity of chlorophenol and triazine compounds in humic acid was closely related to compound hydrophobicity. Greater sorption of almost completely protonated hydroxyatrazine than of the more hydropohobic but uncharged atrazine indicated different humic acid reaction sites for two compounds and consequently different sorption mechanisms.

Ion chromatography of azide in pharmaceutical protein samples with high chloride concentration using suppressed conductivity detection.

Methods based on reversed-phase liquid chromatography with UV detection of 4-nitrobenzoyl- or 3,5-dinitrobenzoyl azide derivatives lack in accuracy and stability of derivatives to be applied for azide determination in pharmaceutical protein samples with high sodium chloride concentrations. This paper describes a sensitive and selective ion chromatographic method, with simple sample preparation and suppressed conductivity detection, developed for trace determination of azide in protein samples containing sodium chloride in concentrations as high as 11.6 g L(-1). Anion exchange stationary phase with quaternary alkyl amine functional groups and gradient elution with sodium hydroxide enabled good resolution of anions with similar retention times: azide, bromide and nitrate, as well as chloride whose retention time was shorter than azide's. Anions with high affinity to stationary phase (phosphate and citrate) were also eluted within acceptable analysis time of 32 min. The stability of sample solutions and the method selectivity, accuracy, precision and sensitivity satisfied the validation criteria of international organizations competent for pharmaceutical industry. The detection and quantitation limit ranges of sodium azide in protein samples were 0.007-0.02 mg L(-1) and 0.02-0.06 mg L(-1), respectively. Both limits increased with the concentration of sodium chloride.

Partitioning of selected environmental pollutants into organic matter as determined by solid-phase microextraction.

Partitioning/sorption of selected environmental pollutants (PCBs, organochlorine insecticides, triazine and amide herbicides) into dissolved humic acids (HA), soil and mineral substances was evaluated by measuring their free concentrations by solid-phase microextraction (SPME). Compounds were chosen to cover a wide range of logK(ow) (2.2-7.6). Two different types of partitioning behaviour for dissolved HA were observed. Compounds with logK(ow)>5 partitioned almost instantly into HA fraction and the remaining free fraction remained rather constant. LogK(HA) and logK(DOC) were calculated and found to be similar for commercial HA, HA standard and isolated HA. The behaviour of these compounds in soil suspension was similar, but strong sorption on CaCO3 and Florisil was also noticed. For compounds with logK(ow)<5, we have not noticed significant changes in free concentrations in HA solutions over time. In soil suspension, however, some sorption/partitioning was observed over time for some compounds, but it was matching the sorption on CaCO3 and Florisil.

Evaluation of recombinant human interferon alpha-2b structure and stability by in-gel tryptic digestion, H/D exchange and mass spectrometry.

Stability and structure of recombinant interferon alpha-2b (rHuINF alpha-2b) was studied by mass spectrometry (MALDI-TOF and Q-TOF MS), chromatography (LC-UV-FLD-DAD, LC-MS) and CD spectroscopy. Besides analysis of the substance according to Ph. Eur. methods, two additional mass spectrometric methods were developed. The aim of both methods was to estimate structure-stability relationship connected to methionine oxidation or protein degradation. Preservation or degradation of protein structure was confirmed by H/D exchange in four separate experiments. Kinetics of deuterium incorporation into macromolecule was monitored over 2670 min. Isoforms of rHuINF alpha-2b were separated by 2D gel electrophoresis. In-gel digestion with trypsin and mass spectrometric analysis, performed on four separated isoforms at the mass corresponding to the mass of rHuINF alpha-2b with oxidized methionines, confirmed oxidation of all methionines to a different extent. Another four isoforms observed in 2D gel are most likely dimers of the same macromolecules with scrambled disulphide bridges. Oxidation and dimerisation are consequences of protein interaction with oxidizing reagents in polyacrilamide gel.