Časovna odvisnost odziva modelnega nevretenčarskega organizma (kopenski rak enakonožec, Porcellio scaber, Isopoda, Crustacea) na nanodelce TiO [spodaj] 2 v hrani : doktorska disertacija

Menard, Anja (avtor), Drobne, Damjana (mentor)

V doktorski disertaciji smo preučevali strupene učinke nanodelcev TiO2 na modelni nevretenčarski organizem (kopenski rak enakonožec, Porcellio scaber, Isopoda, Crustacea). Nanodelci TiO2 se proizvajajo v velikem obsegu in imajo široko uporabnost v številnih industrijskih in potrošniških proizvodih, posledica česar je njihovo sproščanje v okolje, kjer lahko zaradi svoje majhnosti in specifičnih lastnosti povzročijo strupene učinke na različne organizme v okolju. Nekatere predhodne študije so pokazale, da imajo nanodelci TiO2 strupene učinke na različne organizme v okolju. Glavni namen doktorske disertacije je bil opisati časovno odvisnost odziva modelnega nevretenčarskega organizma P. scaber na nanodelce TiO2 v hrani. Za preučevanje strupenih učinkov nanodelcev TiO2 na modelni organizem P. scaber smo uporabili ekotoksikološki laboratorijski test določanja strupenosti kemikalij s kopenskim rakom, ki omogoča merjenje odzivov na različnih nivojih biološke organizacije. Poleg fizioloških označevalcev (smrtnost, sprememba mase in stopnja prehranjevanja), ki kažejo odzive na nivoju organizma, smo spremljali tudi citološki (destabilizacija celičnih membran) in histološka označevalca (debelina epitelija prebavnih žlez in količina lipidnih kapelj v B celicah prebavnih žlez). Na celičnem nivoju smo določali destabilizacijo celičnih membran prebavnih žlez modelnega organizma z uporabo prilagojene metode diferencialnega barvanja s flourescentnima barviloma (akridin oranžem in etidijevim bromidom) in flourescentno mikroskopijo. Z opazovanjem histoloških sprememb v prebavnih žlezah kopenskega raka P. scaber pa smo preučevali učinke zaužitih nanodelci TiO2 na debelino epitelija (ocenjene na podlagi oblike in velikost B celic prebavnih žlez) in količino lipidnih kapelj v B celicah prebavnih žlez. Na podlagi skupne obravnave debeline epitelija prebavnih žlez in količine lipidnih kapelj v B celicah prebavnih žlez pa smo ocenili vpliv zaužitih nanodelcev TiO2 na fiziološko stanje živali. V doktorski nalogi smo predvideli, da potencialna strupenost zaužitih nanodelcev TiO2 za modelni organizem P. scaber ni odvisna le od odmerka, ampak tudi od trajanja izpostavitve ter da se s trajanjem izpostavitve učinek stopnjuje, vendar je le ta odvisen od merjenega označevalca. Ugotovili smo, da različne koncentracije nanodelcev TiO2 (1000, 2000 in 5000 μg/g suhe mase lista) pri različnih izpostavitvenih časih (3, 7, 14 in 28 dni) nimajo očitnega vpliva na preživetje in spremembo mase modelnega organizma P. scaber. Tudi sprememba stopnje prehranjevanja je bila neodvisna od časa izpostavljenosti ali koncentracije zaužitih nanodelcev TiO2, opazili pa smo stohastični tip odziva. Prav tako učinek nanodelcev TiO2 na destabilizacijo celičnih membran celic prebavnih žlez ni bil odvisen od koncentracije niti časa izpostavitve. Merjeni fiziološki označevalci strupenosti in citološki označevalec so pokazali, da se s podaljševanjem časa izpostavitve (28 dni) modelnega organizma P. scaber in povišanjem koncentracije nanodelcev TiO2 v hrani (5000 μg/g suhe mase lista) učinek ne stopnjuje. V naši raziskavi s fiziološkimi označevalci (preživetje, sprememba mase in stopnja prehranjevanja) in citološkim označevalcem (destabilizacija celične membrane) nismo opazili bistvenih strupenih učinkov nanodelcev TiO2 na modelni organizem P. scaber po 28 dneh prehranjevanja s hrano, kateri smo dodali različne koncentracije nanodelcev TiO2 (1000, 2000 in 5000 μg/g suhe mase lista). Izpostavitev modelnega organizma nanodelcem TiO2 pa je povzročila histološke spremembe v B celicah prebavnih žlez. Z višanjem koncentracije nanodelcev TiO2 v hrani se je povečal delež živali s tanjšim epitelijem prebavnih žlez in delež živali z zmanjšano količino lipidnih kapelj v B celicah prebavnih žlez. Stanjšanje debeline epitelija in zmanjšanje količine lipidnih kapelj v B celicah prebavnih žlez smo opazili že pri najnižji testirani koncentraciji (1000 μg/g suhe mase lista). Nanodelci TiO2 so povzročili tudi spremembo fiziološkega stanja živali pri koncentraciji 1000 μg/g suhe mase lista. Z višanjem koncentracije nanodelcev TiO2 v hrani se je povečeval delež živali pod stresom. Histološke spremembe v B celicah prebavnih žlez in sprememba fiziološkega stanja živali so posledica odziva modelnega organizma na stresne razmere, ki so nastale zaradi prehranjevanja modelnega organizma z nanodelci TiO2. Ugotovili smo, da je za interpretacijo strupenih učinkov nanodelcev TiO2 potrebno uporabiti več različnih označevalcev, ki omogočajo merjenje odzivov na različnih nivojih biološke organizacije. Rezultati našega raziskovalnega dela so pokazali, da je pri ugotavljanju potencialnih strupenih učinkov nanodelcev TiO2 za kopenskega raka P. scaber ključnega pomena izbira pravega označevalca. Na podlagi skupne obravnave fizioloških označevalcev in histoloških označevalcev smo ugotovili, da nanodelci TiO2 v hrani nimajo strupenih učinkov na modelni organizem, povzročijo pa stanje stresa, ki je povezano s koncentracijo nanodelcev TiO2 v hrani. Z rezultati našega raziskovalnega dela smo pokazali, da je potrebno ekotoksikološki laboratorijski test določanja strupenosti kemikalij s kopenskim rakom nadgraditi z merjenjem ustreznih histoloških označevalcev, ki so tudi mera fiziološkega stanja živali, preden se zaključi, da nanodelci TiO2 nimajo učinka na modelni organizem, v našem primeru P. scaber. V doktorski disertaciji smo na podlagi rezultatov 28 dnevnega laboratorijskega testa strupenosti nanodelcev TiO2 za modelni organizem P. scaber in podatkov v prosto dostopni literaturi pripravili oceno tveganja za kopensko okolje. Za oceno tveganja smo izračunali razmerje med predvideno koncentracijo v okolju (PEC) in ocenjeno varno koncentracijo (PNEC). Pri pripravi ocene tveganja smo uporabili konzervativne predpostavke. Rezultati ocene tveganja za okolje so pokazali, da je razmerje med PEC in PNEC (RCR) < 1, kar nakazuje, da nanodelci TiO2 uporabljeni v naši raziskavi ne predstavljajo potencialnega tveganja za organizme v tleh. Ugotovili smo, da trenutno dostopni podatki niso zadostni, da bi lahko izdelali zanesljivo oceno tveganja. Naša ocena tveganja vsebuje številne predpostavke, ki vodijo v številne negotovosti. Ocena tveganja se lahko spremeni, ko bo na voljo več podatkov o dejanskih izpustih in koncentracijah nanodelcev TiO2 v okolju, podatkov o strupenosti nanodelcev TiO2 za kopenske organizme ter podatkov o usodi in obnašanju nanodelcev v okolju.

• Jezik: Slovenski jezik
• Ključne besede: nanodelci, okolje, organizmi, onesnaževanje, raziskave, disertacije
• Vrsta gradiva: Doktorska disertacija
• Tipologija: 2.08 - Doktorska disertacija
• Organizacija: FFA - Fakulteta za farmacijo
• Kraj izida: Ljubljana
• Založnik: [A.M. Srpčič]
• Leto izida: 2016
• Št. strani: IV, 90 str.
• PID: 20.500.12556/RUL-143760
• UDK: 504.5:620.3 (043.3)
• COBISS.SI-ID: 283526656
• Datum objave v RUL: 11.01.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Time dependent response of model invertebrate organism (terrestrial isopod, Porcelio scaber, Isopoda, Crustacea) to TiO [sub] 2 nanoparticles in food

Izvleček

In this doctoral thesis, the adverse effects of TiO2 nanoparticles to the model invertebrate organism (terrestrial isopods, Porcellio scaber, Isopoda, Crustacea) were studied. TiO2 nanoparticles are produced on a large scale and have broad applicability in many industrial and consumer products, resulting in their release into the environment where due to their small size and specific properties could cause adverse effects on various organisms. Some previous studies have shown that TiO2 nanoparticles could have adverse effects on the variety of organisms in the environment. The main purpose of the doctoral thesis was to describe the time dependence of the response of the model invertebrate organism P. scaber to the TiO2 nanoparticles in the food. To study the adverse effects of TiO2 nanoparticles on the model organism P. scaber the ecotoxicological laboratory test for assessing the effects of chemicals on terrestrial isopods, which enables measurement of responses at different levels of biological organization, was used. In addition to the physiological biomarkers (mortality, weight change and feeding rate) which show the responses at the organism's level, cytotoxicological and histological biomarkers (epithelium thickness of digestive glands and amount of lipid droplets in the B cells of the digestive gland) were monitored. At the cellular level, we have determined destabilization of cell membranes of digestive glands of model organism by using modified method of differential staining with fluorescent dyes (acridine orange and ethidium bromide) and fluorescent microscopy. By observing the histological changes in the digestive gland of P. scaber, the effects of ingested TiO2 nanoparticles on the epithelium thickness (assessed based on the shape and size of B cells in digestive glands) and amount of lipid droplets in the B cells of the digestive gland were assessed. Based on epithelium thickness of the digestive glands and amount of lipid droplets in the B cells of the digestive glands, the impact of the ingested TiO2 nanoparticles on the physiological condition of the animal was assessed. In the doctoral thesis it was assumed that the potential toxicity of ingested TiO2 nanoparticle for the model organism P. scaber is not only dose-dependent, but also time-dependent and that the duration of exposure intensifies the effect, but this only depends on the measured biomarker. We have found that different concentrations of TiO2 nanoparticles (1000, 2000 in 5000 μg/g leaf dry weight) at different exposure times (3, 7, 14 and 28 days) have no effect on survival and weight change of model organism P. scaber. Feeding rate was neither concentration- nor exposure duration dependent but the stochastic type of response was observed. Also the effect of TiO2 nanoparticle on the destabilization of the cell membranes of cells of digestive glands was neither concentration- nor exposure duration dependent but occurs sporadically. Measured physiological biomarkers and cytotoxic biomarkers have shown that by prolonged exposure (28 days) of the model organism P. scaber to food dosed with TiO2 nanoparticles and high concentration of nanoparticles TiO2 in the food (5000 μg/g leaf dry weight) does not intensify the response. In our study, no significant adverse effects of TiO2 nanoparticles on the model organism P. scaber measured with physiological biomarkers (survival, weight change and feeding rate) and cytotoxicological biomarker (cell membrane destabilisation) were observed after 28 days of dietary exposure to different concentrations of TiO2 nanoparticles (1000, 2000 in 5000 μg/g leaf dry weight). The exposure of model organism to TiO2 nanoparticles have caused histological changes in the B cells of the digestive glands. With increasing concentration of TiO2 nanoparticles in the food the proportion of animals with a thinner epithelium of digestive glands and the proportion of animals with a reduced amount of lipid droplets in the B cells of digestive glands have increased. Thinning of the thickness of epithelium and reduction of the amount of lipid droplets in the B cells of digestive glands were observed already at the lowest tested concentration (1000 μg/g leaf dry weight). TiO2 nanoparticleshave caused the change in the physiological condition of the animals at concentration 1000 μg/g leaf dry weight. With increasing concentration of TiO2 nanoparticles in the food the proportion of animals under the stress have increased. Histological changes in the B cells of the digestive gland and change in the physiological condition of the animals are result of the response of model organism on the stressful conditions that have arose from dietary exposure to TiO2 nanoparticles. We have found that for the interpretation of the adverse effects of TiO2 nanoparticles it is necessary to use different biomarkers that allow the measurement of responses at the different levels of biological organization. The results of our research have shown that for identifying of potential adverse effects of TiO2 nanoparticles for the terrestrial isopod P. scaber is crucial to choose the suitable biomarker. Looking at the physiological biomarkers and histological biomarkers together we found that TiO2 nanoparticles in the food have not adverse effects on the model organism but they cause the stressful state which is related to the concentration of TiO2 nanoparticles in the food. Theresult obtained in this work has shown that it is necessary to upgrade the ecotoxicological laboratory test for assessing the effects of chemicals on terrestrial isopods with measuring the suitable histological biomarkers which are also measure of the physiological condition of the animal before could be concluded that TiO2 nanoparticles have no effect on the model organism P. scaber. We have performed the risk assessment of nanoparticles TiO2 for terrestrial environment based on the result of 28 days laboratory toxicity test with TiO2 nanoparticles for the model organism P. scaber and available data in the literature. For the assessment of risk the ratio between the predicted environmental concentration (PEC) and the predicted no effect concentration (PNEC) was calculated. The conservative assumptions were used. The results of the environmental risk assessment have shown the relationship between the PEC and PNEC (RCR) < 1 which indicates that TiO2 nanoparticles used in our study do not pose a potential risk to soil organisms. We have found that currently available data are not sufficient to make a reliable assessment of the risk. Performed risk assessment for the terrestrial environment is based on a number of assumptions that may lead into the number of uncertainties. The risk assessment may change when more data will be available on actual emissions and concentrations of TiO2 nanoparticles in the environment, data on the toxicity of TiO2 nanoparticles for terrestrial organisms and information on the fate and behavior of nanoparticles in the environment.

Projekti

• Financer: Drugi - Drug financer ali več financerjev
• Program financ.: European Community's Seventh Framework Programme
• Številka projekta: 263147
• Naslov: NanoValid - Development of reference methods for hazard identification, risk assessment and LCA of engineered nanomaterials