Search

Background Zebrafish have been increasingly used for monitoring and assessing the effects of different contaminants in the aquatic environments. In the present study, zebrafish embryos and larvae were used to study the effects of the insecticide diazinon and the herbicide diuron in regard to occurrence of oxidative stress-related cellular responses, multixenobiotic resistance (MXR)-related efflux transporter activity—which represents the first line of defense against xenobiotics in many aquatic organisms—and responses of different molecular and biochemical biomarkers. Results The recently established non-invasive quantitative plate assay, which uses the fluorescent probes CM-H2DCFDA and CellTracker™ Green CMFDA and measures the fluorescence in whole zebrafish larvae, was applied to assess changes in reactive oxidative species (ROS) and glutathione (GSH) after exposure to the investigated pesticides. The results showed a significant increase of GSH after 1h exposure of zebrafish larvae to both diazinon and diuron. Regarding the ROS induction, no significant increases in fluorescence could be detected after 2h exposure to the investigated pesticides. Applying a newly adapted assay for MXR activity, it was determined that diuron caused no change after a 24-h exposure, but caused a significant induction of MXR activity after a 48-h exposure (indicated by a decreased amount of accumulated rhodamine B). On the other hand, diazinon caused an inhibition of MXR activity after both 24h and 48h exposure (indicated by an increased amount of accumulated rhodamine B). Regarding the biomarkers, different set-ups and exposure periods were applied and both molecular (gene expression) and biochemical (enzymatic activities) responses were assessed. Diazinon caused an inhibition of carboxylesterase (CES) and acetylcholinesterase (AChE) activity in zebrafish larvae, diuron inhibited AChE activity in in vitro testing, and both pesticides significantly affected gene expression and activities of some of the cytochrome P450 (CYP) family enzymes. Conclusions The obtained results show various effects of the investigated pesticides and will help to elucidate how aquatic animals cope with pesticides present in their environment. Additionally, the recently developed fluorescence-based assay and the newly adapted MXR activity assay proved to be useful tools for ecotoxicological risk assessment to further investigate pesticide effects.

With the rapid expansion of modern society in both industrial and technological aspect, it is becoming important to assess the risk of compounds which are released daily in our environment in enormous quantities. For this reason, a broad spectrum of ecotoxicological assays with various model organisms has been developed. In this study, toxicity of the Sava River has been assessed by using different model organisms: freshwater algae Chlorella vulgaris, clover Trifolium repens, common wheat Triticum aestivum and zebrafish Danio rerio embryos. Model organisms have been exposed to filtered samples of the Sava River from five different locations (Jesenice, Jankomir, Hrušćica, Rugvica, and Lukavec). Pollution profiles of samples were assessed by chemical analysis. Exposure of Chlorella vulgaris to samples caused growth inhibition and decreased chlorophyll a concentration. Root and/or sprout elongation of Trifolium repens and Triticum aestivum was observed to have lowest rates after exposure to same samples in both model organisms. Exposing the zebrafish embryos to samples resulted in minor mortality rate and sublethal effects. Activity of ABC transporters was inhibited in all samples. The use of these ecotoxicological bioassays, combined with chemical analysis, has resulted in comprehensive toxicity assessment of water from five different sites in Sava River, offering basis for monitoring of lentic and lotic waters. Uzevši u obzir brzo širenje modernog društva u industrijskom i tehnološkom aspektu, postaje nužno procijeniti rizik tvari koje se svakodnevno ispuštaju u naš okoliš u velikim količinama. U tu svrhu, razvijen je široki spektar ekotoksikoloških testova na različitim modelnim organizmima. Unutar ovog rada, istraživana je toksičnost rijeke Save upotrebom različitih modelnih organizama: slatkovodne alge Chlorella vulgaris, djeteline Trifolium repens, pšenice Triticum aestivum i embrija ribe zebrice Danio rerio. Modelni organizmi izlagani su filtriranim uzorcima rijeke Save s pet lokacija (Jesenice, Jankomir, Hrušćica, Rugvica i Lukavec). Prisutnost toksikanata na ovim postajama utvrđena je kemijskom analizom vode. Izloženost alge Chlorella vulgaris uzorcima uzrokovala je inhibiciju njihovog rasta i smanjenu koncentraciju klorofila a. Produljenje korijenja i/ili izdanka kod vrsta Trifolium repens i Triticum aestivum imalo je najnižu stopu nakon izloženosti istim uzorcima kod oba modelna organizma. Izlaganje embrija zebrica uzorcima rezultiralo je niskom stopom mortaliteta i subletalnih efekata. Aktivnost ABC transportera kod zebrica inhibirana je prilikom izlaganja svim ispitivanim uzorcima. Ovakva upotreba ekotoksikoloških testova u kombinaciji s kemijskom analizom, omogućila je sveobuhvatnu procjenu toksičnosti uzoraka rijeke Save s pet različitih postaja, ali i pružila temelj za praćenje stanja tekućica i stajaćica.

With the rapid expansion of modern society in both industrial and technological aspect, it is becoming rather important to assess the risk of compounds which are released daily in our environment in enormous quantities. For this reason, a broad spectrum of ecotoxicological assays with various model organisms has been developed, in order to assess toxic effects that these compounds might cause. In this study, toxicity of the Sava River has been assessed by using different model organisms with different sensitivities to the various kinds of pollutants and their mixtures: freshwater algae Chlorella vulgaris, clover Trifolium repens, common wheat Triticum aestivum and the zebrafish Danio rerio embryos. Model organisms have been exposed to filtered samples of the Sava River from five different locations (Jesenice, Jankomir, Hrušćica, Rugvica, and Lukavec), with different pollution profiles, which was assessed by chemical analysis of 571 organic pollutants. The use of ecotoxicological bioassays performed on different model organisms, combined with chemical analysis, has provided an accurate toxicity assessment of water from the Sava River, allowing us to define and divide the sampling sites accordingly.

Multixenobiotic resistance is a phenomenon in which ATP-binding cassette (ABC) family proteins transfer harmful compounds out of cells. Daphnia magna and Lumbriculus variegatus are model species in aquatic ecotoxicology, but the presence and activity of ABC proteins have not been well described in these species. The aim of this work was to study the presence, activity, and inhibition of ABC transport proteins in D. magna and L. variegatus. The presence of abcb1 and abcc transcripts in 8-9-day-old D. magna was investigated by qRT-PCR. The activity of MXR in D. magna and L. variegatus was explored by influx of the fluorescent ABC protein substrates rhodamine B and calcein-AM, with and without the model inhibitors verapamil (unspecific ABC inhibitor), reversin 205 (ABCB1 inhibitor) and MK571 (ABCC inhibitor). Juvenile D. magna possessed all examined abcb and abcc transcripts, but only reversin 205 inhibited MXR activity. The MXR activity in L. variegatus was inhibited by MK571, and to a lesser extent by verapamil, whereas reversin 205 seemed to stimulate the transport activity. Whereas calcein-AM worked better as an MXR substrate in D. magna, rhodamine B was a better substrate for L. variegatus MXR activity measurements. This is the first report on MXR activity in the order Lumbriculida, subclass Oligochaeta, and class Clitellata.