Comparison of three systems for testing the toxicity of pesticides dimethoate, pirimiphos-methyl and deltamethrin using molecular biomarkers of earthworms (Lumbricidae)

U ovoj disertaciji istraživana je višestruka različitost u toksičnim učincima i načinu istraživanja toksičnih učinaka tri učestalo korištena insekticida, organofosfata dimetoata i pirimifos-metila i piretroida deltametrina, na različite ekološke kategorije gujavica primjenom dva standardizirana testa toksičnosti (kontaktni filter papir test i test s umjetnim tlom) i mikrokozmičkog sustava. Rezultati su pokazali da postoji mjerljiva razlika u odgovoru biljega gujavica između pokusa koji se provode u pretežno determinističkim uvjetima (standardizirani testovi toksičnosti) i pokusa koji se provode u pretežno stohastičkim uvjetima (mikrokozmički sustavi). Iz rezultata dobivenih primjenom okolišno relevantnih koncentracija istraživanih pesticida, može se zaključiti da primjena istraživanih organofosfata u poljoprivredi ima mjerljiv toksikološki učinak na jedinke gujavica, dok primjena deltametrina u poljoprivredi najvjerojatnije neće imati utjecaj na odgovor biokemijskih biljega gujavica u okolišu. Rezultati mjerenja su pokazali da toksičnom djelovanju pesticida na gujavice može znatno doprinijeti pojava oksidativnog stresa i promjene u aktivnosti transmembranskih crpki za izbacivanje ksenobiotika. Dobiveni podatci o oporavku biljega su pokazali da je vrijeme oporavka istraživanih biokemijskih biljega bilo relativno sporo, pa je stoga nužno pri dizajniranju monitoringa zagađenja okoliša pomoću biokemijskih biljega uzeti u obzir to vrijeme. Usporedba osjetljivosti vrsta pokazala je da se vrsta-specifična ovisnost istraživanih vrsta gujavica nakon izlaganja istraživanim organofosfatima razlikuje ovisno o primijenjenom sustavu testiranja toksičnosti, odnosno osjetljivost vrsta bila je različita u standardiziranim testovima toksičnosti u usporedbi s mikrokozmičkim sustavom. Te su razlike ukazale na potrebu razlikovanja dva tipa osjetljivosti, fiziološku i okolišnu osjetljivost vrste, koje obje treba uzimati u obzir pri interpretaciji rezultata toksikoloških i ekotoksikoloških testova.In the present thesis, the multiple differences in toxic effects and research methods of toxic effects of three commonly used insecticides, organophosphates dimethoate and pirimiphos-methyl and pyrethroid deltamethrin, on different ecological categories of earthworms by using two standardized toxicity tests (filter paper contact test and artificial soil test) and microcosmic system were investigated. The results showed measurable difference in responses between earthworm biomarkers obtained in experiments conducted mainly in deterministic conditions (standardized toxicity tests) and experiments conducted mainly in stochastic conditions (microcosmic systems). From the results obtained after applying environmentally relevant concentrations of investigated pesticides, it can be concluded that the application of the investigated organophosphates in agriculture has a measurable toxicological effects on earthworms, whereas application of deltamethrin in agriculture is unlikely to affect the response of biochemical biomarkers of earthworms in the environment. The measurements showed that the occurrence of oxidative stress and changes in the activity of efflux pumps might considerably contribute to the toxic effects of investigated pesticides on earthworms. The obtained data on the recovery of biomarkers showed that the recovery of investigated biochemical biomarkers was slow, so it is necessary to take into account the recovery time when designing the monitoring studies of environmental pollution using biochemical biomarkers. Comparison of the earthworm sensitivities showed that the species-specific sensitivity was different after exposure to organophosphates using standardized toxicity tests and microcosm. The obtained differences in sensitivities point to the need of distinguishing between two types of species sensitivity, physiological and environmental sensitivity, and both sensitivities should be taken into account in the interpretation of the results of toxicological and ecotoxicological tests

Prva analiza teških metala u perima sivih čaplji Ardea cinerea iz hrvatskih kolonija

This study represents the first analysis of heavy metals: lead and cadmium, as well as two metalloids: arsenic and selenium, in the feathers of Grey Herons breeding in Croatia. The Grey Heron was chosen as a model apex predator of the wetland ecosystem due to high trophic level, feeding habits, long lifespan and abundance. Sampling was conducted during the 2019 breeding season in April and May from six Grey Herons colonies in Croatia (Mrsunjski lug, Kopački rit, Piljenice, Kravarsko, Čepin, Slovinci), whose environment is heavily influenced by rivers Danube, Drava and Sava tributaries. The primary and secondary flight feathers were collected under the colonies as shed feathers or from dead birds. The feathers are a suitable non-invasive method for metal and metalloid analysis, and can reflect internal concentrations, depending on the metal. In the Slovinci colony, we measured the highest lead (Pb) and cadmium (Cd) concentrations (2194.53 ± 416.20 μg kg-1 dw and 79.30 ± 9.37 μg kg-1 dw, respectively). The highest arsenic (As) and selenium (Se) concentrations were found in the Čepin colony (359.70 ± 301.46 μg kg-1 dw and 3375.61 ± 2502.80 μg kg-1 dw, respectively). All the measured levels of heavy metals and metalloids are below concentrations that can cause adverse health effects in birds.Antropogenim aktivnostima povećava se razina metala i polumetala u okolišu rezultirajući njihovim zagađenjem. Mjerenje teških metala i polumetala neinvazivnim metodama dobar je pokazatelj stanja okoliša. U radu su predstavljeni rezultati prve analize teških metala: olova i kadmija te dva metaloida: arsena i selena u perju sivih čaplji Ardea cinerea koje se gnijezde u Hrvatskoj. Uzorkovanje pera sivih čaplji provedeno je tijekom sezone gniježđenja, u travnju i svibnju 2019. iz šest hrvatskih kolonija (Mrsunjski lug, Kopački rit, Piljenice, Kravarsko, Čepin i Slovinci). Primarna i sekundarna letna pera odraslih ptica prikupljena su ispod kolonija. Najveće koncentracije olova (Pb) zabilježene su u perju iz Slovinaca (2194,53 ± 416,20 μg kg-1 suhe tvari) i Kopačkog rita (1955,85 ± 1373,07 μg kg-1 suhe tvari). U Čepinu su izmjerene najviše koncentracije arsena (As; 359,70 ± 301,46 μg kg-1 suhe tvari) i selena (Se; 3375,61 ± 2502,80 μg kg-1 suhe tvari), a najviša razina kadmija (Cd) izmjerena je u koloniji Slovinci (79,30 ± 9,37 μg kg-1 suhe tvari). Sve izmjerene razine metala i polumetala su ispod koncentracija koje se smatraju toksičnima za ptice. Preliminarni rezultati ovog istraživanja ukazuju na relativno nisku zagađenost okoliša kontinentalne Hrvatske, no potrebna su dodatna istraživanja i uspostava kontinuiranog praćenja metala i polumetala u okolišu te njihov utjecaj na vršne predator

ANTIFUNGAL EFFECT OF ESSENTIAL OILS FROM THE NATIVE CROATIAN FLORA ON THE SUPPRESSION OF THE Botrytis cinerea FUNGUS IN GRAPEVINES

U istraživanju je ispitivano djelovanje eteričnih ulja domicilnoga bilja Hrvatske na supresiju rasta micelija Botrytis cinerea izoliranoga s vinove loze. U pokusu su korištena ulja sljedećih biljaka: metvice paprene (Mentha x piperita), kadulje (Salvia officinalis), ružmarina (Rosmarinus officinalis), lavandina (Lavandula hybrida), mravinaca (Origanum compactum), timijana (Thymus vulgaris), gospine trave (Hiperici oleum), stolisnika (Achillea millefolium) i smilja (Helichrysum italicum). Rezultati in vitro volatilnogq testa i makrodilucijskoga testa pokazali su da ulja timijana i paprene metvice mogu imati pozitivan utjecaj na inhibiciju rasta gljive Botrytis cinerea pri svim ispitivanim koncentracijama te da fungistatski učinak traje i preko 96 h. Primjena ulja stolisnika i gospine trave u makrodilucijskoj metodi dala je bolje rezultate u odnosu na volatilnu metodu. Eterično ulje smilja stimuliralo je porast micelija B. cinerea u obje metode. S obzirom na smanjenje porasta micelija B. cinerea u uvjetima in vitro, eterična ulja, kao biofungicidi, predstavljaju moguću zamjenu za sintetske pripravke u suzbijanju razvoja sive plijesni na vinovoj lozi, no potrebna su dodatna istraživanja kako u uvjetima in vitro, tako i u uvjetima in vivo.The aim of this research was to determine the effect of nine essential oils from the Croatian native flora (Mentha x piperita, Salvia officinalis, Rosmarinus officinalis, Lavandula hybrida, Origanum compactum, Thymus vulgaris, Hiperici oleum, Achillea millefolium and Helichrysum italicum) on the Botrytis cinerea mycelial growth suppression. The research results demonstrated that the oils of Thymus vulgaris and Mentha x pipereta in both investigated methods (the volatile and the macrodillution one) have exerted a positive influence on the suppression of mycelial growth. Also, the aforementioned oils had a fungistatic effect in all investigated concentrations long after 96 hours. The essential oils of Achillea millefolium and Hiperici oleum in the volatile method have not manifested a suppression effect, while the effect of suppression of a mycelial growth was recorded in the macrodillution method. The essential oil of Helichrysum italicum stimulated the growth of B. cinerea mycelium in both investigated methods. Considering a reduction of the mycelium growth, the essential oils, as a biocontrol agent, could be a positive substitution for the traditional pesticides in grapevine gray mold control. Additional investigation with regard to the essential oils in the Botrytis control are needed both the in vitro and the in vivo conditions

Commercial preparations of pesticides exert higher toxicity and cause changes at subcellular level in earthworm Eisenia andrei

Background: The environmental impact of pesticides has been an increasingly discussed issue over the last decades. Constant usage of pesticides presents a burden for soil and causes a decrease in its health, including the negative effects on earthworms which are indicators for soil quality. The objective of this research was the assessment of the effects of two insecticides and two herbicides on the earthworm Eisenia andrei. Namely, the following active ingredients and respective commercial preparations were investigated: esfenvalerate (Sumialfa), thiacloprid (Calypso), dimethenamid-p (Frontier) and prosulfocarb (Filon). Lethal concentrations (48 h) of both active ingredient and commercial preparations were determined using the filter paper contact test. Results: The results showed that Calypso and Frontier were significantly more toxic than the active ingredient. Therefore, all further measurements were performed after exposure of earthworms to the commercial preparations of the pesticides. Specifically, several enzymatic biomarkers and multixenobiotic resistance activity were assessed. Additionally, a fluorescence-based assay for the determination of oxidative stress was established. Significant changes were detected for catalase, carboxylesterase and multixenobiotic activities after 48-h exposures. Also, a significant change in oxidative stress parameters could be observed for both Calypso and Frontier. Conclusions: The obtained results show that commercial preparations can be more toxic than the active ingredients, and the formulations being distributed in the environment can affect earthworms on a molecular level already after short exposures. This emphasizes the importance of a more integrated eco-toxicological assessment of commercial pesticide preparations not to underestimate their effects on the environment

Antifungal Activity of Earthworm Coelomic Fluid Obtained from Eisenia andrei, Dendrobaena veneta and Allolobophora chlorotica on Six Species of Phytopathogenic Fungi

The functioning of soil ecosystems greatly depends on the interactions occurring between soil biota communities. It is well known that earthworms are an important soil component that substantially affects its function, including their meaningful impact on the development of different phytopathogenic soil fungi. Phytopathogenic fungi are responsible for crop disease and cause great economic damage. It has previously been established that earthworms’ coelomic fluid can suppress the growth of phytopathogenic fungi, but the exact molecular mechanism is unknown. The present study aimed at broadening the proof of this observed phenomenon by investigating the effects of the coelomic fluid extract of three different earthworm species (Eisenia andrei, Dendrobaena veneta and Allolobophora chlorotica) on the growth of six different phytopathogenic fungi species (Berkeleyomyces basicola, Fusarium culmorum, Globisporangium irregulare, Rhizoctonia solani, Macrophomina phaseolina, and Sclerotinia sclerotiorum). Coelomic fluid extract was obtained by electrostimulation or usage of extraction buffer (only in case of A. chlorotica) and prepared in three different concentrations by diluting the obtained coelomic fluid with physiological saline. The coelomic fluid extract of the three investigated earthworm species had an inhibitory effect on the growth of all six phytopathogenic fungi species. The greatest inhibitory effect was achieved with the E. andrei coelomic fluid extract reducing the growth of R. solani fungi. The findings of this research confirm the antifungal activity of coelomic fluid obtained from earthworm species belonging to different ecological categories and may be of potential use in crop protection against phytopathogenic fungi

Application of non-destructive methods: biomarker assays in blood of white stork (Ciconia ciconia) nestlings

White stork (Ciconia ciconia) nestlings can provide quantitative information on the quality of the surrounding environment by indicating the presence of pollutants, as they depend on locally foraged food. This study represents the first comparison of biomarkers in two fractions of white stork nestling blood: plasma and S9 (the post-mitochondrial fraction). The aim of this study was to evaluate acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as to establish a novel fluorescence-based method for glutathione (GSH) and reactive oxygen species (ROS) detection in plasma and S9. Considering the enzymatic biomarkers, lower variability in plasma was detected only for AChE, as CES, GST, and GR had lower variability in S9. Enzyme activity was higher in plasma for AChE, CES, and GST, while GR had higher activity in S9. Regarding the fluorescence-based method, lower variability was detected in plasma for GSH and ROS, although higher GSH detection was reported in S9, and higher ROS was detected in plasma. The present study indicated valuable differences by successfully establishing protocols for biomarker measurement in plasma and S9 based on variability, enzyme activity, and fluorescence. For a better understanding of the environmental effects on nestlings’ physiological condition, biomarkers can be measured in plasma and S9