Ann. Limnol. - Int. J. Lim. 51 (2015) 139–146 Ó EDP Sciences, 2015 DOI: 10.1051/limn/2015010 Available online at: www.limnology-journal.org Diversity of algae in a thallium and other heavy metals-polluted environment Bartosz J. Płachno1, Konrad Wołowski2*, Joanna Augustynowicz3 and Magdalena Łukaszek2 1 2 3 Department of Plant Cytology and Embryology, Jagiellonian University in Kraków, Gronostajowa 9 St., 30-387 Kraków, Poland Department of Phycology, W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46 St., PL-31-512 Kraków, Poland Faculty of Biotechnology and Horticulture, Institute of Plant Biology and Biotechnology, University of Agriculture in Kraków, Al. 29 Listopada 54, 31-425 Kraków, Poland Received 5 September 2014; Accepted 3 March 2015 Abstract – Thallium (Tl) compounds are extremely toxic to living organisms, including algae, but there is a dearth of basic information regarding the mechanisms of action of Tl in the environment and its effects on algae in natural conditions. This study examined algal diversity in an environment highly polluted by Tl. Graniczna Woda stream is contaminated by Tl and other heavy metal compounds (Cd, Pb and Zn). There we found 66 algae taxa representing five phyla, among which euglenophytes prevailed. We found that euglenophytes, including Phacus species, can survive and can show high species diversity in the presence of high Tl concentrations. The fact that these small organisms covered only by a pellicle are able to thrive in such inhospitable habitat, shows a great plasticity of these organisms. It is unclear whether the algae of Graniczna Woda stream have a broad tolerance to harmful conditions or rather represent new varieties/clones that evolved in metal-contaminated waters and are adapted to this environment. Key words: Euglenophytes / Cd / Zn / Pb / thallium / water pollution Introduction Thallium (Tl), a metallic element placed in group 13 (formerly 3A) of the periodic table on account of its high density ( > 11.6 g.cmx3), is classified as a heavy metal. Its name, from Greek, is connected with the green spectral line that identified this element (Fleischer, 1997). Tl in the environment exists at two oxidation states: the more stable Tl(I) (e.g., Tl2O) and the less stable Tl(III) (e.g., Tl(OH)3). The natural background level of Tl in the Earth’s crust is put at 0.85 mg.kgx1, and its minerals, associated with K or S, are quite rare but widespread. Its mean concentration in natural water systems is 10 ng.dmx3 (Kabata-Penidias and Mukherjee, 2007). Under the quality standards of both the United States Environmental Protection Agency (EPA) and the Polish Ministry of the Environment, the maximum permitted concentration of Tl in surface waters is 2 mg.dmx3. Anthropogenic sources of Tl in the environment are related mainly to coal combustion and ferrous or nonferrous smelting (Peter and Viraraghavan, 2005). Commercial use of Tl is associated with the manufacture of refractive glass and formerly for pesticide production, *Corresponding author: k.wolowski@botany.pl and it is used as a radioisotope for scintigraphy (Kabata-Pendias and Mickherjee, 2007). Both Tl(I) and Tl(III) compounds are readily soluble and therefore bioavailable. Tl compounds are extremely toxic to living organisms. Its toxicity to mammals is higher than that of Hg, Cd and Pb (Peter and Viraraghavan, 2005; Babula et al., 2008). Up to 1984, however, Tl was not considered an environmental pollutant. In Poland, research on the effects of Tl on biota started in the late 1990s, focused on plants and small mammals occurring in the vicinity of Olkusz, a highly industrialized area in southern Poland (e.g., Dmowski et al., 1998; Wierzbicka et al., 2004). As with other heavy metals, the effect of Tl on organisms can be explained by its binding to –SH groups of cysteine residues in proteins, leading to changes in the activity of a broad range of enzymes. The most pronounced toxic effect of the Tl ion is related to the similarity of Tl(I) to the potassium ion, due to their similar chemical structure and properties. Monovalent Tl disrupts Kcontrolled activity of enzymes and membrane processes such as the mitochondrial respiratory chain, and also stabilization of ribosomes (Léonard and Gerber, 1997; Arzate and Santamaria, 1998; Peter and Viraraghavan, 2005). Article published by EDP Sciences 140 B. J. Płachno et al.: Ann. Limnol. - Int. J. Lim. 51 (2015) 139–146 Fig. 1. Oxbow of Graniczna Woda stream in the former De˛by Boruszowickie Reserve. Unlike for the heavy metals Cd, Pb, Hg, Ni and Zn, the literature contains little data on the effects of Tl on plants. Only three plant species have been classified as Tl hyperaccumulators, that is, plants able to accumulate > 100 mg.kgx1 d.w. in natural conditions: Iberis intermedia Guersent, Biscutella laevigata L. (both from the Brassicaceae family) and Silene latifolia Poir. (Caryophyllaceae) (van der Ent et al., 2013). These are all terrestrial species originating from southern France. Studies showed that Tl can accumulate especially in plants of the Brassicaceae family (Leblanc et al., 1999; Al-Najar et al., 2005). There is far less information about aquatic vegetation in Tl-contaminated environments, although these organisms, immersed as they are, are far more exposed to harmful substances. In Synechocystis, Avery et al. (1991) demonstrated competition between Tl(I) and K ions, and Lustigman et al. (2000) showed Tl(I) toxicity to the cyanobacterium Anacystis nidulans (Richter) Drouet & Daily and the chlorophyte Chlamydomonas reinhardtii P.A. Dang. Ralph and Twiss (2002) reported differential toxicity of Tl to the unicellular chlorophyte Chlorella, which depended on the oxidation state of the metal. They observed the same degree of Chlorella growth inhibition under treatment with Tl (III) at a dose of 2 r 10x13 [mol.dmx3] (4.1 r 10x2 ng.dmx3) and with Tl(I) at a dose of 10x8 [mol.dmx3] (2.0 mg.dmx3). They noted that Tl(III) toxicity was orders of magnitude greater than Tl(I) to this phytoplankton but that the bioavailability of Tl(III) was significantly limited, and pointed to the lack of fundamental information regarding Tl in the environment. In experiments on Lemna minor L., Tl(I) induced generation of reactive oxygen species, resulting in damage to DNA and cell proteins (Babić et al., 2009). The intrinsic toxicity of Tl and the mechanism of its transport through the cell membrane were investigated by Turner and Furniss (2012) in the marine alga Ulva lactuca L. Upper Silesia (southern Poland) is a highly contaminated mining and industrial region. Some watercourses in this area, including Graniczna Woda stream, are polluted by heavy metal compounds from chemical plants and mines. Graniczna Woda stream was polluted by chemical plants in the town of Tarnowskie Góry and by zinc smelters in the town of Miasteczko Śla˛skie (Reczyńska-Dutka, 1986). In our latest work (Augustynowicz et al., 2014), we conducted the Microtox1 toxicity test on the Graniczna Woda strem and showed that studied water exhibited second class of acute toxicity. In the above-mentioned work, we also found high Tl pollution of the water in Graniczna Woda stream with almost complete absence of higher aquatic plants in its streambed. In the present study, we examined there the diversity of algal flora. In an unpolluted location in this area we had recorded a rich algal flora in an earlier study (Wołowski et al., 2013a). Material and methods Study site Material was obtained from Graniczna Woda stream and its oxbow (Fig. 1) in the former De˛by Boruszowickie Reserve near Tarnowskie Góry (Upper Silesia, Poland: ca. 50x30kN/18x49kE). The stream bed is silty. The study B. J. Płachno et al.: Ann. Limnol. - Int. J. Lim. 51 (2015) 139–146 141 Table 1. Algae taxa occurrence in De˛by Boruszowickie water bodies during sampling with comparison to the species occurrence in Poland (rare – noted up to three times from Poland, often – noted four or more times from Poland). Taxa October 2008 Cyanophyta Cyanophyceae Oscillatoria sp. Vaucher ex Gomont + Phormidium sp. Kützing ex Gomont + Planktothrix agardhii (Gomont) Anagnostidis and Komárek Heterokontophyta Bacillariophyceae Achnanthes sp. Bory de Saint-Vincent Cyclotella sp. (Kützing) Brébisson Eunotia bilunaris (Ehrenberg) Schaarschmidt Eunotia exigua (Brébisson ex Kützing) Rabenhorst Eunotia sp. Ehrenberg + Gomphonema parvulum (Kützing) Kützing Navicula sp. Bory de Saint-Vincent Nitzschia obtusa W.Smith + Nitzschia palea (Kützing) W.Smith + Pinnularia cf. ferrophila K. Krammer Pinnularia nodosa (Ehrenberg) W.Smith Pinnularia viridis (Nitzsch) Ehrenberg + Heterokontophyta Xanthophyceae Characiopsis subulata var. ensiformis (Hermann) Lemmermann + Tribonema sp. Derbès & Solier + Euglenophyta Euglenophyceae Euglena agilis H.J. Carter Euglena archaeoviridis B. Zakrys and P.L. Walne Euglena archaeoplastidiata M. Chadefaud + Euglena hemichromata Skuja Euglena mutabilis F. Schmitz + Euglena sp. Ehrenberg Lepocinclis spirogyroides Marin & Melkonian + Euglena viridis (O.F. Müller) Ehrenberg Lepocinclis fusca (Klebs) Kosmala and Zakrys + Lepocinclis acus (O.F.Müller) Marin & Melkonian Lepocinclis oxyuris (Schmarda) Marin and Melkonian Lepocinclis ovum (Ehrenberg) Lemmermann Monomorphina pyrum (Ehrenberg) Mereschkowsky Petalomonas mediocanellata Stein Phacus acuminatus Stokes + Phacus angustus Drezepolski + Phacus caudatus Hübner + Phacus curvicauda Svirenko + Phacus ichthydion Pochmann Phacus indicus Skvortzov Phacus inflexus (I. Kiselev) Pochmann + Phacus longicauda var. tortus Lemmermann Phacus orbicularis Hübner + Phacus obolus Pochmann Phacus parvulus Klebs Phacus pleuronectes (O.F. Müller) Nitzsch + Phacus pusillus Lemmermann Phacus unguis Pochmann Trachelomonas bacillifera Playfair Trachelomonas cervicula Stokes Trachelomonas hispida (Perty) F.Stein + Trachelomonas oblonga Lemmermann Trachelomonas perforata Awerinzew Trachelomonas volvocinopsis Swirenko May 2009 July 2010 June 2011 October 2011 Often Often Often + + + + + + + + + + Reported from Poland + + + + + + + + Often Often Often Often Often Often Often Often Often Rare Often Often Often + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Often Rare Rare Often Often Often Often Often Often Often Often Often Often Rare Often Rare Often Often Rare Rare Rare Often Often Rare Often Often Rare Rare Often Often Often Often Rare Often 142 B. J. Płachno et al.: Ann. Limnol. - Int. J. Lim. 51 (2015) 139–146 Table 1. (Contd.) Taxa October 2008 Chlorophyta Chlorophyceae Chlamydomonas sp. Ehrenberg Desmodesmus armatus (R.Chodat) E.Hegewald + Desmodesmus sp. (R.Chodat) S.S.An, T.Friedl and E.Hegewald + Oedogonium sp. Link ex Hirn + Scenedesmus ecornis (Ehrenberg) Chodat + Scenedesmus cf. armatus (R.Chodat) R.Chodat + Scenedesmus intermedius Chodat + Scenedesmus sempervirens Chodat + Scenedesmus sp. Meyen + Stigeoclonium tenue (C.Agardh) Kützing Ulothrix sp. Kützing Chlorophyta Trebouxiophyceae Microthamnion kuetzingianum Nägeli ex Kützing Chlorophyta Ulvophyceae Ulothrix sp. Kützing Chlorophyta Zygnematophyceae Closterium moniliferum Ehrenberg ex Ralfs + Closterium ehrenbergii Meneghini ex Ralfs + stream is in the Stoła River basin, which has the annual discharge of 1.46 m3.sx1 (Program of the Environmental Protection for the Tarnowskie Góry community). Part of the oxbow borders the peat bog. The fieldwork was done during the 2008, 2009, 2010 and 2011 vegetation seasons, together with studies on the occurrence of carnivorous Utricularia in this area. Samples for taxonomic research were taken once a year (except in 2011 when sampling was done twice) using a plankton net (0.25 mm mesh) and a big pipette (phytobenthos). The samples were placed in 35 ml plastic containers and refrigerated. Fresh samples or samples fixed with 4% formaldehyde were observed with a Nikon ECLIPSE 600 light microscope with Nomarski phase contrast. The collected water samples were transported to the W. Szafer Institute of Botany (Polish Academy of Sciences) in Cracow, where they were analyzed as previously described (Wołowski et al., 2011). Chemical analysis of water samples Inductively coupled plasma mass spectrometry (ICPMS) (ELAN 6100, Perkin Elmer) (PN-EN ISO 99631:2001) as well as titration methods (PN-ISO 9297:1994; PN-EN ISO 17294-1:2007) were applied to measure the chemical composition of samples. The spectrometer was calibrated to the ICP multi-element standard (Merck). Results Water chemistry analyses showed the following average amounts of elements (mg.dmx3): inorganic C 78.17, N 7.41, P 0.45, S 151.03, K 36.07, Fe 0.86, May 2009 July 2010 June 2011 + + October 2011 + + + + + + + + Often Often Often Often Often Often Often Rare Often Often Often Often + + Reported from Poland + Often Often Often Mg 8.71, Mn 0.26, Ca 123.10. The amounts of heavy metals and metalloids such as Cr, Ni, Cu, Zn, Ag, Hg, As and Se did not exceed the relevant norms. For Cd (0.06 mg.dmx3) the upper limit was exceeded by a factor of 16 (Polish standard) or 40 (US EPA standard), for Pb (0.05 mg.dmx3) by a factor of 7 (US EPA standard) and for Zn (1.06 mg.dmx3) by a factor of 10 (US EPA standard). The concentration of Tl (0.24 mg.dmx3) exceeded the surface-water limits most spectacularly: by a factor of 120 under both Polish and US EPA standards (Rozporza˛dzenie Ministra Środowiska z dn. 9 listopada 2011 r.; US EPA Water Quality Standards, 2013). In the group of biogenic substances, the upper limits were x3 (Polish standard exceeded for NOx 3 at 7.15 mg.dm 3x x3 j 5 mg.dm ) and for PO4 at 0.45 mg.dmx3 (Polish standard j 0.31 mg.dmx3). Other water parameters were as follows: pH 7.0–7.5, electrical conductivity 1.04 mS.cmx1 and redox potential (Eh) 258 mV. We identified 66 algal taxa representing five phyla (Table 1). The euglenophytes of Graniczna Woda stream showed great diversity, including 9 species of Euglena, 5 of Trachelomonas, 15 of Phacus, 4 of Lepocinclis, and one each of Monomorphina and Petalomonas (see Fig. 2). Among the other algal groups were 3 taxa of Cyanophyceae, 2 of Xanthophyceae, 14 of Bacillariophyceae and 15 of green algae (Chlorophyceae). The euglenophytes dominated among the observed algae in every sampling year, and they also were the group with the highest diversity, numbering 37 taxa. Some of them are taxa rarely noted from Poland: Euglena archaeoviridis B. Zakrys & P.L. Walne, Euglena archaeoplastidiata M. Chadefaud, Petalomonas mediocanellata Stein, Phacus angustus Drezepolski, Phacus ichthydion B. J. Płachno et al.: Ann. Limnol. - Int. J. Lim. 51 (2015) 139–146 143 Fig. 2. Euglenophytes documented in samples from Graniczna Woda stream: (A) Phacus indicus, (B) Phacus acuminatus, (C) Phacus alatus, (D) Phacus curvicauda, (E) Phacus obolus, (F) Phacus parvulus, (G) Phacus caudatus, (H) Phacus unguis, (I) Phacus longicauda var. tortus, (J) Euglena mutabilis, (K) Euglena hemichromata, (L) Euglena archaeoviridis, (M) Trachelomonas bacillifera, (N) Monomorphina pyrum. Scale bar = 10 mm. Pochmann, Phacus indicus Skvortzow, Phacus inflexus (Kiselev) Pochmann, Phacus obolus Pochmann, Phacus pusillus Lemmermann, Phacus unguis Pochmann and Trachelomonas perforata Awerinzew. The other determined algal taxa are often reported from contaminated waters. In the area we examined, the water chemistry analyses clearly indicated contamination with biogenic compounds and heavy metals (Tl, Cd, Zn and Pb). This is the first time so many Phacus taxa (15) have been recorded in such a contaminated habitat; Phacus caudatus Hübner, Phacus curvicauda Svirenko and Phacus parvulus Klebs were recorded repeatedly throughout the study. Among the euglenas, Euglena mutabilis F. Schmitz, Euglena viridis (O.F. Müller) Ehrenberg, E. archaeoplastidiata Chadefaud and Euglena agilis H.J. Carter were constant in the study area. E. agilis occurred en masse in the pallmeloid stage at sampling time in summer 2011. Trachelomonas hispida (Perty) F. Stein was noted in almost every studied sample. Diatoms were equally represented, and Nitzschia palea (Kützing) W. Smith and Pinnularia viridis (Nitzsch) Ehrenberg were noted in every sample. Among the 15 Chlorophyceae taxa the one most commonly noted was Desmodesmus sp. We observed that the shape and arrangement of the chloroplasts in E. viridis and E. mutabilis varied and did not always fit the classical description, and that the dimensions of euglenophyte specimens were at their lower limits. 144 B. J. Płachno et al.: Ann. Limnol. - Int. J. Lim. 51 (2015) 139–146 Discussion Previously we studied algal flora in the JeleniakMikuliny Reserve, situated in the same large forest complex as De˛by Boruszowickie. The area of the Jeleniak-Mikuliny reserve consists of two shallow, overgrown water ponds lying in the lowland between two sand dunes. In this reserve, we found 96 algal taxa [Cyanophyceae (4) Bacillariophyceae (20), Chrysophyceae (1), Raphidophyceae (1), Xanthophyceae (1), Cryptophyceae (2), Dinophyceae (1), Euglenophyceae (24), Chlorophyceae (19), Zygnematophyceae (27)] and 11 morphotypes of chrysophyte stomatocysts (Wołowski et al., 2013a). The algal flora of polluted Graniczna Woda stream was poorer, with 66 species. It is known that heavy metal pollution alters algal diversity and also community structure. Different species may be more or less tolerant to pollution and some may dominate in a polluted environment (Say and Whitton, 1980; Whitton et al., 1981; Podda et al., 2013; Trzcińska and PawlikSkowrońska, 2013). Some cyanobacteria of the genera Oscillatoria, Phormidium, Plectonema and Schizothrix are often abundant in alkaline waters polluted by heavy metal compounds (Say and Whitton, 1980; Whitton et al., 1981). Thus we might expect such taxa to be very abundant in the alkaline, metal-polluted water of Graniczna Woda stream, but we observed Oscillatoria sp. and Phormidium sp. during only one season. The low occurrence of filamentous cyanobacteria may also be related to a lack of N limitation (as the mean N: mean P was 16.4:1 which is very close to the Redfield ratio of 16:1) (see Reynolds, 1984), or to the presence of other organic compounds that were present in the studied water. The high diversity of euglenoids in Graniczna Woda stream indicates high tolerance of Tl by these algae. Years of research on euglenophytes have shown that they are remarkably tolerant to various kinds of pollution with heavy metals such as Fe, Zn, Cu, Cd, Mn, Pb, Ni and Al (Albergoni et al., 1980; Tam et al., 1981; Fasulo et al., 1983; Walne and Kivic, 1990). They have also been found in waters polluted with diesel oil (Dennington et al., 1975), phenol (Pawlitz and Werner, 1978) and herbicides and insecticides (Poorman, 1973; Butler, 1977), and can survive in highly radioactive water (Lackley, 1968). Euglenophytes are also found living under very high salinity, for example, in Great Salt Lake (Jones, 1944). Several features help euglenophytes to survive in an unfavorable environment, such as fast reproduction (division), formation of cysts (Hindák et al., 2000) and mixotrophy. Euglenophytes thrive very well in eutrophic water and as a consequence they are often used as bioindicators of water contamination (Starmach, 1983; Sládeček and Sládečková, 1996; Wołowski, 1998, 2011, Wołowski and Hindák, 2005). They usually inhabit a-mesosaprobic and polysaprobic waters. The contamination of the studied stream with organic phosphates and nitrates explains the abundant occurrence of euglenophytes. It is known that high concentrations of heavy metals limit a site’s availability to different groups of organisms but in this case they apparently had no major effect on the algal taxa that occurred in De˛by Boruszowickie. It is generally known that acidophilic E. mutabilis (Lane and Burris, 1981) and Euglena gracilis (Cook, 1968) are able to grow in highly polluted habitats. E. mutabilis colonizes highly acidified waters, tolerates pH of ca. 1, and can be dominant among the eukaryotes in habitats such as the metal-contaminated ponds of the Smoking Hills region of the Canadian Arctic (Tam et al., 1981; Havas and Hutchinson, 1983) and acidic post-mining ponds contaminated with heavy metals (Wołowski et al., 2008, 2013b). In the investigated area of De˛by Boruszowickie, E. mutabilis was present but did not develop en masse. Probably its growth was limited by the high pH (7–7.5) of the water. Our results confirm the higher tolerance spectrum of E. mutabilis. Presumably the very low pH of post-mining ponds also limits the growth of euglenophytes, such as Phacus species, which prefer slightly acidic or neutral water. We found no literature data about the occurrence of these Phacus species in waters contaminated with heavy metals, including Tl. Ph. caudatus and Ph. curvicauda are common, well-known species that occur in waters polluted with organic compounds. There are somewhat fewer records published for Ph. parvulus, which occurred in Graniczna Woda stream throughout the studied period. That this small organism covered only by a pellicle can thrive in such an inhospitable habitat is an intriguing finding. Future studies should determine whether the algae of Graniczna Woda stream accumulate heavy metals or possess physiological adaptations endowing them with a mechanism to effectively exclude harmful heavy metals. Acknowledgements. We would like to thank Michael Jacobs for improving the English version of the manuscript. For this research, K.W. received funding from the Polish Ministry of Science and Higher Education/National Science Centre (grant N N304 220135), K.W. and M.Ł. received funding from the statutory fund of the Institute of Botany, Polish Academy of Sciences, and J.A. received funding from the National Science Centre (grant DEC-011/03/B/NZ9/00952). B.J. 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