| Abstract | Onečišćenje antibioticima predstavlja veliki problem ako se radi o perzistentnom onečišćenju, iako su u pitanju male koncentracije. Porijeklo im je teško odrediti u blizini naseljenih mjesta s razvijenom poljoprivredom i stočarstvom. Istraživanje je provedeno u Republici Hrvatskoj, u slivu priobalnog krškog vodonosnika koji je ugrožen intruzijom slane morske vode, a analizirani uzorci su uzeti iz zdenaca uključenih u sustav vodoopskrbe stanovništva u čijoj vodozaštitnoj zoni se nalazi i farma goveda. Prikazan je cjelokupan postupak identifikacije i kvantifikacije najčešće korištenih antibiotika u humanoj i veterinarskoj medicini (amoksicilin, azitromicin, eritromicin i gentamicin) te povezivanje prisutnosti i otpornosti (stabilnosti) antibiotika ovisno o salinitetu podzemne vode. Uzorkovana je podzemna voda i morska voda na četiri lokacije u slivu Bokanjac–Poličnik periodično tijekom 36 mjeseci, a uz to su uzeti uzorci vodovodne vode na slavini za ne–ciljanu analizu organskih onečišćivala. Pripremljeni uzorci su najprije miješani s certificiranim standardima navedenih antibiotika i pohranjeni 10 mjeseci na različitim uvjetima, odnosno, na sobnoj temperaturi i prirodnom svijetlu te u hladnjaku. Miješani su uzroci podzemne vode sa 50 % i 75 % morske vode, kao i sa standardima navedenih antibiotika kako bi se vidjelo na koji način povećani salinitet koji simulira intruziju morske vode u podzemnu slatku, utječe na stabilnost (otpornost) antibiotika. Analize su odrađene tekućinskom kromatografijom uz detekciju spektrometrom mase na LC/MS QToF (eng. Liquid Chromatography/mass spectrometry quadrupole time of flight) uređaju. Korišteni su podaci o električnoj vodljivosti i pH mjereni na mjestima uzorkovanja, kao i u uzorcima iz simuliranih uvjeta. Izrađena je i karta opasnosti koja je obuhvatila stočarstvo, a prikazani su i podaci iz Registra onečišćivanja okoliša o ispustima onečišćivala u vodotoke i more na tom području. Prikazana je prirodna ranjivost područja i izrađena karta rizika te je odrađena ne–ciljana analiza organskih onečišćenja na uzorcima podzemne vode, vode iz slavine i morske vode. Detektirani su analizirani antibiotici u uzorcima podzemne vode i mora, prikazan je i objašnjen utjecaj različitih hidroloških uvjeta godišnjih doba na ponašanje antibiotika. Određenim kalibracijskim pravcima kvantificirane su vrijednosti koncentracije, a radi se o niskim koncentracijama koje prema trenutno važećim propisima, ne utječu na zdravstvenu ispravnost vode za piće. Potvrđeno je da stabilnost (otpornost) navedenih antibiotika ovisi o salinitetu, odnosno da veća količina morske vode u podzemnoj doprinosi duljoj stabilnosti ovih antibiotika. |
| Abstract (english) | Antibiotic pollution is a great problem if it is persistent, even though it is a question of small concentrations that are expected and found in the aquatic environment. It is difficult to determine their origin in the vicinity of populated areas with developed agriculture and animal husbandry, and they are mainly the sources of this pollution through urine and faeces from wastewater from settlements or farms. The antibiotics investigated in this study are the most used in human and veterinary medicine, namely Amoxicillin, Azithromycin, Erythromycin and Gentamicin.
The research was carried out in the basin of the karst coastal aquifer, which is threatened by the intrusion of salty seawater in the Republic of Croatia, and the analysed samples were taken from the wells included in the water supply system for city of Zadar and surrounding settlements. Sampling was done periodically within 36 months, once in each season to cover the entire hydrological year in order to show the influence of different seasonal conditions on the detection and quantification of named antibiotics. Also, groundwater and seawater were sampled to see if antibiotics were present in both matrices. Due to the expected low concentrations of the mentioned antibiotics in the samples, they were prepared by the Solid Phase Extraction method in order to concentrate the samples into the appropriate analyte for analysis. Analyses were performed by liquid chromatography in tandem with quadrupole time of flight mass spectrometry with high resolution and sensitivity (LC/MS QToF).
Optimization of the method was carried out to obtain appropriate chromatograms for each of the analysed antibiotics. With the use of the MassHunter Qualitative and Quantitative Analysis software, the results were presented, and with the help of the Isotope Distribution Calculator software, the normalized decay of each of the analysed antibiotics was obtained to confirm the obtained masses. The samples were also mixed with certified standards of analysed antibiotics and were stored under different conditions, i.e., at room temperature and natural light, as well as in a refrigerator at a constant temperature of 4° C and in the dark. In addition to mixed samples with antibiotic standards, groundwater samples were mixed with 50 % and 75 % sea water and antibiotic standards and stored in the same manner. After mixing, blank samples were taken and analysed immediately. The mixed samples stood for 10 months under the mentioned conditions and were then filtered and analysed using the same methodology as
the blank samples. All four antibiotics were detected in groundwater samples, but in all samples, they were detected only in the summer months, which indicates that hydrological conditions affect the detection of antibiotics in the groundwater of this karst aquifer. Also, all four antibiotics were quantified, and the concentrations were determined and the mean values per sampling location were shown.
The results from simulated conditions show for all four antibiotics that they are present and more stable (resistant) in colder conditions and in the dark than those stored at room temperature and in the light, as well as that in samples with a higher percentage of salty seawater, the analysed antibiotics are more stable. An analysis of the results of electrical conductivity and pH measured at the location and in simulated conditions is also presented, and an analysis of the measured chloride values in the area, as well as the amount of daily precipitation, was performed.
The natural vulnerability of the area is presented, and a hazard analysis related to the polluters of the area from the Register of Environmental Pollution and Animal Husbandry, i.e., farms in the area, is performed. Based on the impact and the size of the farms, a risk map was created that shows where the mentioned antibiotics were detected.
A non – targeted analysis of groundwater, seawater and tap water samples in the area was also performed and the identified pollutants according to the Agilent Water PCDL were presented.
The emphasis is on detected small concentrations that do not affect the healthiness of drinking water but can affect the development of bacteria in the water environment that become resistant and can be considered a threat to the environment. |
