Chemometric characterization of heavy metals in the soil and shoots of pioneer species growing near the polluted water bodies in the close vicinity of mining-metalurgical complex in bor : aspects of phytoextraction and biomonitoring

[J. Petrović]

90 listova

Tehnološko inženjerstvo - Hemija, hemijska tehnologija i hemijsko inženjersto / Technological engineering - Chemistry, chemical technology, and chemical engineering

Predmet i cilj ove doktorske disertacije je procena autentičnih biljnih fitoekstrakcionih i biomonitoring potencijala u odnosu na teške metale, u odabranim pionirskim biljnim vrstama koje rastu pored zagađenih voda u neposrednoj blizini rudarsko-metalurškog kompleksa u Boru i to korišćenjem hemometrijskog pristupa i izračunavanjem rata bioakumulacije za svaki metal(oid) u nadzemnim biljnim delovima (bioakumulacioni faktori izraženi kroz takozvane odnose mobilnosti, MR). Hemometrijski pristup realizovan je najsavremenijim analitičkim metodama za detekciju i kvantifikaciju metal(oid)a, kao što je masena spektrometrija sa indukovano spregnutom plazmom uz prethodno mikrotalasno rastvaranje pripremljenih uzoraka, koje su dalje bile kombinovane sa statističkim metodama kao što su: Pearson-ova korelaciona studija, hijerarhijska klaster analiza (HCA) i jednosmerna analiza varijanse (One-Way ANOVA). Posmatrani metal(oid)i bili su: Cu, Zn, Pb, As i Cd, a kao test-biljke za navedenu procenu, iskorišćene su dve zeljaste višegodišnje biljne vrste: mlečika suncogled (Euphorbia helioscopia L.) i obična kopriva (Urtica dioica L.), jer su jedino one bile pronađene na svih pet lokacija od interesa (osim koprive na jednoj od njih). Preciznije rečeno, kod ovih biljaka uzorkovani su i dalje analizirani njihovi nadzemni delovi (izdanci), kao i odgovarajući uzorci zemljišta/sedimenta. Dodatno, izvršena je i analiza nekih važnih zemljišnih parametara kao što su: pH, električni konduktivitet, EC, organska materija, OM i ukupni N i P, kao i procena njihovog uticaja na procese usvajanja i akumulacije ispitivanih metal(oid)a u sakupljenim biljnim izdancima. Rezultati ove disertacije i primenjeni hemometrijski pristup, kao i izračunavanje odgovarajućeg bioakumulacionog faktora za svaki metal(oid) u obe biljne vrste sa svake lokacije, obezbedili su brojne korisne informacije o fitoekstrakcionim i fitomonitoring potencijalima ispitivanih pionirskih vrsta, gde su najvažnije sledeće: 1) Prvo, pronađena je i analizirana biljka, tj. mlečika suncogled (Euphorbia helioscopia L.), za koju ranije nisu postojali literaturni podaci u odnosu na njene praktične fitoremedijacione i fitomonitoring potencijale. 2) Kada su u pitanju ispitivani uzorci zemljišta/sedimenta, rezultati statističke metode One-way ANOVA, pružili su dokaz da su detektovane koncentracije svakog pojedinog zemljišnog metal(oid)a značajno varirale, osim u slučaju Cd, gde su koncentracije varirale samo minimalno. Rezultati ove disertacije su takođe ukazali i na izuzetno visoke koncentracije Cu u svim uzorcima prikupljenim pored zagađenih voda u Boru i njegovoj najbližoj okolini i najvažnije - ove koncentracije bile su mnogo veće od propisanih graničnih (LV)- i remedijacionih (RV)- vrednosti, a takođe su bile u opsegu fitotoksičnih (zemljišnih) koncentracija. Fitotoksične koncentracije su bile dostignute i u slučaju zemljišnog As na dve lokacije u urbano/industrijskoj (UI) zoni. I u ovom slučaju, odgovarajući LV i RV, su bili prevaziđeni i to u velikoj meri. LV i RV za Cd su takođe bili prevaziđeni na istim UI lokacijama. Na jednoj od njih, LV i RV za Zn su takođe bili prevaziđeni, kao i za Pb na drugoj. Sve ostale koncentracije (u zemljištu/ sedimentu) nisu bile na nivoima koji bi se mogli smatrati povišenim, ili alarmantnim. 3) Ovi nalazi su dalje sugerisali da, generalno, najveći rizik za sve žive organizme, a posebno za prisutnu vegetaciju, može doći od zemljišnog Cu (na svim istraživanim mestima), a zatim i As (na mestima iz UI zone). Ipak, uprkos ovoj činjenici, obe ispitivane biljne vrste su normalno rasle na svojim prirodnim staništima, bez ispoljavanja bilo kakvih vidljivih simptoma toksičnosti Cu, ili As, što ih dalje kvalifikuje kao perspektivne kandidate za obnavljanje vegetacije na ugroženim mestima. Štaviše, obe biljne vrste bile su u stanju da tolerišu ekstremno visoke koncentracije Cu u svojim izdancima. Praktično, u svim izdancima, ovaj element je bio najobilniji i sve detektovane koncentracije bile su na nivou fitotoksičnosti. Fitotoksične koncentracije su takođe bile otkrivene za Pb (u većini slučajeva) i za As (u nekoliko slučajeva). Sve ostale koncentracije metal(oid)a u izdancima, bile su na normalnim nivoima, a redosled obilnosti je bio: Cu > Zn > Pb > As > Cd (za svaku biljnu vrstu, na svakoj lokaciji). One-way ANOVA je dodatno pokazala veliki broj statistički značajnih razlika između detektovanih koncentracija pojedinih metal(oid)a u obe biljke. Najmanje statistički značajnih razlika (kod obe biljke) nađeno je u slučaju Cd i naročito As. 4) U najvećem broju slučajeva, najveća koncentracija svakog pojedinog metal(oid)a u ispitivanim izdancima, bila je zabeležena u slučaju mlečike suncogled (na svakoj lokaciji), što je ukazalo da generalno, ova biljna vrsta može razviti efikasnije mehanizme za usvajanje i akumulaciju metal(oid)a (preko lista, ili putem korena) od koprive, a posebno u pogledu Cu. Međutim, u osnovi, sposobnosti obe ispitivane biljke za usvajanje preko korena, tj. iz zemljišta/sedimenta nisu bile na nekim značajnijim nivoima, jer su vrednosti većine izračunatih MR bile mnogo niže od 1. Najefektniji rezultati uočeni su u slučaju Cu kod mlečike suncogled sa dve UI lokacije, gde su detektovane koncentracije u izdancima bile iznad nivoa odgovarajućih koncentracija u zemljištu (MR > 1). One su takođe bile iznad nivoa koji je karakterističan za biljke hiperakumulatore, što je posebno interesantno za fitoremedijaciju, praktično, fitoekstrakciju. Kako su obe UI lokacije bile karakteristične i po niskim vrednostima pH zemljišta, jasno je da je, u Cu ekstrakciji, mlečika suncogled bila najefikasnija u okolnostima kiselog zemljišta, što je dodatno bilo potvrđeno i rezultatima izvedene Pearson-ove korelacione studije, dok je kopriva bila najefikasnija na mestima sa najvećim vrednostima EC (situacija sa ostalim metal(oid)ima nije bila tako uniformna i jednostavna). Takođe, bilo je jasno da su koncentracije skoro svih ispitivanih metal(oid)a u izdancima bile u negativnim korelacijama sa pH zemljišta, a u pozitivnim korelacijama sa sadržajem OM, dok je situacija sa EC zemljišta, kao i sa zemljišnim P i N bila komplikovanija. Interesantno - zemljišni N bio je u pozitivnim korelacijama sa detektovanim koncentracijama svih metal(oid)a u izdancima koprive i u negativnim korelacijama sa koncentracijama metal(oid)a u izdancima mlečike suncogled. Međutim, poslednje pomenute korelacije nisu bile na statistički značajnim, tj. pouzdanim nivoima. Rezultati iste statističke metode takođe su potvrdili da koncentracije metala u izdancima nisu uvek bile u pozitivnim korelacijama sa odgovarajućim koncentracijama u zemljištu (osim u slučaju As u mlečiki suncogled, gde je uočena jaka pozitivna korelacija sa zemljišnim As), ali su u isto vreme bile u značajnim pozitivnim korelacijama sa nekim drugim zamljišnim metalima: As-Pb, As-Cd i Cd-Pb kod mlečike suncogled i Zn-Cd kod koprive. Ovo sve ukazuje da, u većini slučajeva, koncentracije metal(oid)a u izdancima nisu bile u nekoj značajnijoj meri zavisne od odgovarajućih zemljišnih koncentracija, kao i da su neki metal(oid)i bili u kompeticiji pri usvajanju preko korena (korelacije sa negativnim predznakom), a neki ne (korelacije sa pozitivnim predznakom). Ovo u konačnom sugeriše da su obe biljne vrste usvajale metal(oid)e pre svega u skladu sa svojim individualnim potrebama i takođe, sve ovo ide u prilog opšte poznate činjenice da biljke usvajaju metale ne samo preko korena, već i preko lista. 5) Isti nalazi dalje indikuju da su obe biljne vrste, u slučajevima izuzetno visokih (praktično - rizičnih) zemljišnih koncentracija, za većinu metal(oid)a, usvojile strategiju njihovog ograničenog usvajanja (preko korena). U suštini i pored svih razlika u usvajanju i akumulaciji metala između dve različite ispitivane biljne vrste (koje su bile uočene kroz primenjene analize), na bazi veoma niskih izračunatih vrednosti MR ( 1), može se reći da su se, u odnosu na većinu metal(oid)a, obe biljne vrste, ponašale kao tolerantne, ali nehiperakumulatorske vrste, a moguće čak i kao ekskluderi. 6) Dobijeni rezultati, uključujući i rezultate HCA, korišćenjem Ward-ove metode, koji se odnose na klasifikaciju lokacija po zagađenosti, potvrdili su da izdanci obe ispitivane biljke ne mogu tačno da odraze postojeću situaciju u zemljištu, što ih dalje ne može preporučiti kao pouzdano sredstvo u fitomonitoringu zagađenja zemljišta. 7) Međutim, u slučaju zagađenja atmosferskog porekla, obe vrste mogu biti od koristi, jer su rezultati HCA sa primenjenom metodom povezivanja srednjih vrednosti između grupa, pokazali da obe biljne vrste imaju veoma dobar potencijal da odraze poreklo metal(oid)a u svojim izdancima (tj., predominantno iz - zemljišta/sedimenta, ili pak – iz atmosfere), pa su tako pružile i jednu adekvatnu ilustraciju opšteg stepena zagađenosti u konkretnoj oblasti, u konkretno vreme (uzorkovanja), što znači da one mogu poslužiti kao precizno sredstvo u praćenju zagađenja jednog sezonskog i pre svega atmosferskog karaktera, praktično tokom kompletnog vegetacionog perioda i u osnovi, za procenu generalnog kvaliteta životne sredine. Ova poslednja metoda je takođe pružila uvid i u moguće izvore metal(oid)a u ispitivanoj oblasti, tako da je kombinacija ove poslednje pomenute vrste HCA i izračunavanja Pearson-ovih koeficijenata, uspešno ukazala: - da se poreklo Cu u uzorcima zemljišta/sedimenta, u značajnom stepenu razlikuje u odnosu na ostale ispitivane metal(oid)e, kao i da se najmanje može pripisati zagađenju koje dolazi iz atmosfere, a pre svega iz topionice kao glavnog izvora ove vrste zagađenja u regionu Bora; poreklo Cu u zemljištu/sedimentu je predominantno prirodno, tj. geološko, ali svakako manjim delom potiče i od povremenih izlivanja zagađenih voda iz neposredne blizine; ostali metal(oid)i su takođe različitog porekla – i geološkog i antropogenog, ali pre svega atmosferskog, pa tek zatim i poreklom od izlivanja zagađenih voda, - da je poreklo metal(oid)a u izdancima mlečike suncogled bilo najviše u vezi sa otpadnim gasovima emitovanim iz topionice, pri čemu su prednjačili metal(oid)i kao što su: Cu, As i Cd, što praktično znači da je mlečika usvajala metal(oid)e pre svega iz atmosfere (tj. preko listova), a manjim delom iz podloge (osim u slučajevima usvajanja Cu iz kiselog zemljišta) i - da su izdanci koprive, iz atmosfere usvajali pre svega Zn i As (najvećim delom), dok su metali Cd i Pb verovatno poticali iz oba izvora: iz tla i iz atmosfere (i to prilično ujednačeno); konačno, najveći deo Cu u koprivi sa izabranih mesta u Borskom regionu bio je poreklom iz podloge na kojoj su biljke rasle.

The objective and goal of this doctoral dissertation is the evaluation of authenthic plant phytoextraction and biomonitoring potentials with regard to heavy metals in the selected pionir plant species, which are growing near polluted water bodies in the close vicinity of the mining/metallurgical complex in Bor, using a chemometric approach and the calculation of bioaccumulation rates for each metal(oid) in the aboveground plant parts (bioaccumulation factors expressed through the so called mobility ratios, MRs). Chemometric approach was realized by the most current analytical methods for metal(oid)s detection and kvantification such as inductively coupled plasma-mass spectrometry with previous microwave digestion of the samples, which further were combined with the statistical methods such as: Pearson’s correlation study, hierarchical cluster analysis (HCA), and one-way analysis of variance (One-Way ANOVA). The observed metal(oid)s were: Cu, Zn, Pb, As, and Cd, while the two herbaceous perennial plant species: sun spurge (Euphorbia helioscopia L.), and common nettle (Urtica dioica L.) were used as the test- plants for the mentioned evaluation, because only they were found at all five locations of interest (except the common nettle at one of them). Precisely speaking, from these plants were collected and then analyzed their aboveground parts (shoots), as well as the corresponding samples of soil/sediment. In addition, the analysis of some important soil parameters such as: pH, electrical conductivity, EC, organic matter, OM, and the total N, and P, was done, as well as the estimation of their influence on the processes of the uptake and accumulation of the investigated metal(oid)s in the collected plant shoots. The results of this dissertation and the applied chemometric approach, as well as the calculation of related bioaccumulation factot for each metal(oid) in both plant species from each location, provided numerous informative data on the phytoextraction and biomonitoring potentials of the investigated pioneer species, where the most important are the following: 1) Firstly, it was found and analyzed the plant, namely, the sun spurge (Euphorbia helioscopia L.), for which, there was no any literature data regarding their practical phytoextraction and phytomonitoring potentials. 2) As for the investigated samples of soil/sediment, the results of the statistical method One-way ANOVA, offered evidence that the detected concentrations of each individual soil metal(oid) varied significantly, except in the case of Cd, where the concentrations varied only minimally. The results of this dissertation also pointed to the extremely high concentrations of Cu in all samples collected near polluted water boddies in Bor and its close vicinity, and most importantly – these concentrations were much higher than the defined limit (LV)- and remediation (RV)-values, and also, they were in the range of phytotoxic (soil) concentrations. Phytotoxic concentrations were reached in the case of soil As at the two locations in urban/industrial (UI) zone, too. In this case, the related LV, and RV were exceeded in a high quantity. LV, and RV for Cd were also surpassed at the same UI locations. At one of them, LV, and RV for Zn were surpassed too, as well as for Pb at the other. All other concentrations (in soil/sediment) were not at the levels that could be considered elevated or alarming. 3) These findings are further suggested that, generally, the greatest risk for all living organisms, and especially for the present vegetation, may come from the soil Cu (at all investigated sites), and then from the soil As (at the sites from UI zone). However, inspite of this fact, the growth of both investigated plant species was normal at their natural habitats, without the expression of any of visible toxicity symptoms of Cu or As, which further qualifies them as prospective candidates for revegetation of endangeres sites. What is more, both plant species were capable to tolerate extremely high Cu concentrations in their shoots. Practically, in all shoots, this element was the most abundant and all detected concentrations were at the level of phytotoxicity. Phytotoxic concentrations were also found for Pb (in most cases), and for As (in several cases). All other concentrations of metal(oid)s in the shoots were at normal levels; the order of abundance was: Cu > Zn > Pb > As > Cd (for each plant species, at each location). Additionally, one-way ANOVA showed a great number of statistically significant differences between the detected concentrations of individual metal(oid)s in both plants. The minimum of statistically significant differences (for both plants) was found in the case of Cd, and especially As. 4) In most cases, the highest concentration of each individual metal(oid) in the investigated shoots was recorded in the case of sun spurge (at each location), which pointed that, in general, this plant species can develop more efektive mechanisms for the uptake and accumulation of metal(oid)s (via leaf or via root) than nettle, especially with regard to Cu. However, basically, the capabilities of both investigated plants regarding the uptake via root, i.e. from the soil/sediment, were not at some significant levels; namely, the values of most calculated MRs were much lower than 1. The most effective results were noticed in the case of Cu in sun spurge from the two UI locations, where the detected shoot concentrations were above the corresponding soil concentrations (MRs > 1). They were also above the level charachteristic for plant hyperaccumulators, which is especially interesting for phytoremediation, practically, phytoextraction. Given that both UI locations were charachterised with their low values of soil pH, it is clear that in Cu extraction, sun spurge was the most efficient in the circumstances of acidic soils, which was additionally confirmed also by the results of the performed Pearson correlation study; the nettle was the most efficient at the sites with the highest EC values (the situation with other metal(oid)s was not so uniform and simple). It was also clear that the concentrations of almost all investigated metal(oid)s in the shoots were in negative correlations with soil pH, but in positive correlations with OM content, whereas the situation with soil EC, as well as wit soil P, and N was more complicated. Interestingly – soil N was in positive correlations with the detected concentrations of all metal(oid)s in nettle shoots and in negative correlations with the metal(oid)s concentrations in the shoots of sun spurge. However, the recently mentioned correlations were not at the statistically significant, i.e. confident levels. The results of the same statistical method also confirmed that the concentrations of metal(oid)s in the shoots were not always in positive correlations with the corresponding soil concentrations (except in the case of As in sun spurge, where a strong positive correlation with soil As was noticed), but simultaneously, they were in significant positive correlations with some other soil metal(oid)s: As- Pb, As-Cd, and Cd-Pb in the case of sun spurge, and Zn-Cd in the case of nettle. This all points that, in most cases, the concentrations of metal(oid)s in shoots were not dependent, in some significant extent, on the corresponding soil concentrations, as well as that some metal(oid)s were in competition during the root uptake (correlations with negative sign), whereas some of them were not (correlations with positive sign). In final, this all suggests that both plant species assimilated metal(oid)s predominately in accordance with their individual needs, and also, this all is in favour of a well known fact that plants assimilate metal(oid)s not only via root but also via leaf. 5) The same findings indicate further that, in the cases of very high (practically - dangerous) soil concentrations, both plant species adopted a strategy of limited root uptake, for most metal(oid)s. Mainly, regardless all differences in metal(oid)s uptake and accumulation between the two different plant species (that were perceived through the applied analyses), on the basis of very low values of the calculated MRs ( 1), it can be said, that, regarding most metals, both plant species acted as tolerant but nonaccumulator species, possible even as excluders. 6) The obtained results, including the results of HCA, using the Ward's method, which are related to the classification of location according to their contamination, confirmed that the shoots of both investigated plants can not reflect accurately, the existing situation in the soil, which further, can not recommend them as a confident tool in soil phytomonitoring. 7) However, in the case of contamination of atmospheric origin, both species can be of use, because the results of HCA with the applied method of average linkage between groups, showed that both plant species have a very good potential to reflect the origin of metal(oid)s in their shoots (i.e., predominately from the soil/sediment, or from the atmosphe). They offered an adequate illustration of a general extent of contamination in the concrete area, in the actual time (of sampling), which means that they can serve as an accurate tool in the screening of contamination of a seasonal and the atmospheric character, practically, during the complete vegetation period, and basically, for the estimation of a general environmental quality. Also, the last method offered an insight into the possible sources of metal(oid)s in the examined area, so that, the combination of the last kind of HCA and the calculation of Pearson's coefficients is successfully pointed that: - the origin of Cu in the soil/sediment samples differ, in a significant extent, from the other investigated metal(oid)s, as well as that it can be less ascribed to the contamination of atmospheric origin, and above all – to the smelter plant as the main source of this kind of contamination in the region of Bor; the origin of Cu in the soil/sediment is predominately natural, i.e., geogenic, but in a less part it comes from the occasional overflows of polluted waters in the close vicinity; the rest of metal(oid)s are also of different origin – geogenic and anthropogenic, but primarily, they come from the atmosphere and then from the occasional overflows of polluted waters, - the origin of metal(oid)s in the shoots of sun spurge is mostly linked to tho waste gases emmited from the smelter, where Cu, As, and Cd, were leading metal(oid)s, which means that the sun spurge assimilated metal(oid)s predominately from the atmosphere (via leaves), and in a less extent, from the ground (except in the cases of Cu assimilation from the acidic soil) and - the shoots of the common nettle assimilated Zn, and As mostly from the atmosphere, while Cd i Pb originated probably from both sources, i.e., from the ground and from the atmosphere (preety evenly); finally, the greatest part of Cu in the nettle from the selected sites in the Bor region, was from the ground where the plants were growing.

hemometrija, fitoremedijacija, fitoekstrakcija, biomonitoring, mlečika suncogled (Euphorbia helioscopia L.), obična kopriva (Urtica dioica L.), teški metal(oid)i, zemljište/sediment, biološka akumulacija

chemometry, phytoremediation, phytoextraction, biomonitoring, sun spurge (Euphorbia helioscopia L.), common nettle (Urtica dioica L.), heavy metal(oid)s, soil/sediment, biological accumulation

66.012.1:504.5(497.11)(043.3)

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