Soil and Biota of Serpentine: A World View 2009 Northeastern Naturalist 16(Special Issue 5):297–308 Plant Colonization on a Contaminated Serpentine Site Stefano Marsili1, Enrica Roccotiello1, Cristina Carbone2, Pietro Marescotti2, Laura Cornara1, and Mauro G. Mariotti1,* Abstract - This study evaluated relationships between the serpentine soil from a waste-rock dump of the abandoned Libiola sulphide mine (NW Italy) and its pioneer vegetation. We identified the tolerance of various species to environmental conditions and evaluated physical or chemical factors that influenced the first plants to colonize this stressful environment. Thirteen sampling sites were identified in the rock dump from characterization of surface or near-surface oxidation zone and vegetation type. Sampling sites were analyzed for slope, pH, mineralogy, soil chemistry, floristic composition, and the percent coverage of each species. In all the plots, species richness and vegetation cover were extremely low. The flora showed an acidophilous character. Introduction The soils of mine tailings are often comparable to other pioneer soils because they harbor metal-tolerant plants that colonize from the surrounding plant communities (Marrs and Bradshaw 1993, Rajakaruna 2004). Plants that grow on mine tailings show adaptations to stressful edaphic situations such as the lack of nutrients and ionic toxicity associated with heavy metals (Shu et al. 2005). Plant communities established on metalliferous waste provide an example of primary plant succession in which the major limiting factors are edaphic. With a more complete understanding of the natural colonization of plants on mine waste, it should be possible to achieve restoration of visually acceptable, biodiverse, and self-sustaining ecosystems quickly and cheaply by accelerating the natural succession process (Bradshaw 1992, 1997; Dobson et al. 1997). Combining backfilling and revegetation can reduce acid loads from current mining operations or abandoned mine sites. Covering pyritic refuse or other acid-producing materials on a site with good soil material and then establishing vegetation can have a major impact on reducing acid concentrations in leachate (Tordoff et al. 2000). Such remediation efforts often also decrease the flow of water from these sites by encouraging infiltration into the soil and evapotranspiration by plants (Skousen et al. 1998). Existing vegetation inventory and evaluation of the habitat requirements are listed in the international guidelines for surface-mining reclamation programs and projects (Darmer and Dietrich 1973). 1DIP. TE. RIS. Polo Botanico Hanbury, University of Genova, Corso Dogali 1M, I-16136, Genova, Italy. 2DIP. TE. RIS. University of Genova, Corso Europa 26, I-16132 Genova, Italy. *Corresponding author - m.mariotti@unige.it. 298 Northeastern Naturalist Vol. 16, Special Issue 5 Although a number of studies related to plant communities development on metalliferous soils have been reported, their main emphasis has been on the original vegetation assemblages on undisturbed soils. The excessive concentrations of heavy metals in these soils are the result of natural mineralization caused by the presence of undisturbed ore bodies near the surface (Baker and Proctor 1990, Brooks and Malaisse 1985, Wu 1990). Most investigations on vegetation of man-made mine wastes emphasize the selection of metal-tolerant species, and few are related to initial stages of plant succession on these wastelands (Chambers and Sidle 1991, Ernst 1988, Gibson 1982, Raskin and Ensley 2000). In Italy, the study of the initial stages of plant succession on these wastelands has been directly addressed by Brooks et al. (1998), along with studies of the relationships between ultramafic soils and plant growth in relation to revegetation (Chiarucci 2004; Chiarucci et al. 1998, 1999, 2001). The objectives of this study were to evaluate the pioneer vegetation of the main waste-rock dump of the Libiola mining area and to verify the relationships between plant communities and the chemical parameters of the soils. Both objectives provide background knowledge for planning an appropriate revegetation strategy of the metalliferous sites. Field-site Description The Libiola mine is located 8 km from Sestri Levante (eastern Liguria, Italy; Fig.1). Between 1864 and 1962, it was one of the most important Italian Figure 1. Location of Libiola mine (inset) and map of the waste-rock dump (light grey). Area A, black dots: sampling sites with vegetation in the low-slope basal part of the dump. Area B, black squares: sampling sites with vegetation in the upper part. Area C, white dots: sampling sites without vegetation in the vertical cut on the upper part. 2009 S. Marsili, E. Roccotiello, C. Carbone, P. Marescotti, L. Cornara, and M.G. Mariotti 299 centers for Fe-Cu-sulphide exploitation (Marescotti and Carbone 2003). The sulphide ores occur toward the top of a pillow basalt sequence, which lies upon serpentinites and minor rodingitized gabbros. The sulphides are in pyrite-rich and chalcopyrite-rich massive lenses, stockwork-like epigenetic veins, and disseminated mineralizations (Garuti and Zaccarini 2005). The study site was in the main waste-rock dump of the mining area. It was located near a small river and was about 100 m in height. The upper slopes of the dump were steep (about 45–50°); down-slope the gradient decreased to about 20–25°, which in turn graded to 5° adjacent to the river. The deposited material showed high heterogeneity, with alternating fine and coarse layers and variation in lithology on the 1–10 m scale. The waste-rock fragments are mainly represented by non-economic mineralizations, serpentinites, and basalts. Serpentinites were scattered throughout the entire dump and reached considerable concentrations (≥50%). The natural vegetation around the mine area was characterized by xerophilous communities with bushy acidophilus stages dominated by Buxus sempervirens L. (Box) and/or Erica arborea L. (Arboreus Heather), with other Mediterranean shrubs such as Juniperus oxycedrus L. subsp. oxycedrus, (Red Juniper), Arbutus unedo L., (Strawberry Tree), Calicotome spinosa (L.) Link., (Thorny Broom). Areas of old forest with Pinus pinaster Aiton (Maritime Pine) were also present. Small areas of more-developed communities were present, such as forests of Quercus ilex L. (Holm Oak) and Q. pubescens Willd. (Downy Oak) or mixed thermophilous woods. The shrub stages are characterized by pseudomaquis formed by Box and/ or Genista desoleana Valsecchi (Salzmann’s Broom), Euphorbia spinosa L. subsp. ligustica (Fiori) Pignatti (Thorny Euphorbia), Helichrysum italicum (Roth.) G. Don (Italian Immortelle), Minuartia laricifolia (L.) Schinz. & Thell subsp. ophiolitica Pignatti (Ophiolitic Minuartia), Thymus sp. pl. (thyme), and Satureja montana L. (Winter Savory). The described natural vegetation represented typical serpentine vegetation for NW Italy (Mariotti 1994, Vagge 1997). Methods Sampling, field, and laboratory analysis Thirteen sampling sites were subjectively selected in the main wasterock dump of Libiola mine based on surface or near-surface oxidation zones and the vegetation type found in the study area. Six sites were located in the low-slope basal part of the dump (area A; Fig. 1, black dots) where the progressive accumulation of debris created a heterogeneous landscape of waste-rock materials mainly consisting of serpentinites and minor basalts. The only exception was represented by site 4, which was strongly enriched in sulphides, as this location was previously used for dumping mined material after preliminary processing. Three sites were sampled in the upper part of the dump (area B; Fig. 1, black squares) on a flat terrace near to an open pit excavated close to the contact between serpentinites and basalts. Four sites 300 Northeastern Naturalist Vol. 16, Special Issue 5 were located in a vertical cut, along an erosion channel, in the northeastern part of the dump (area C; Fig. 1, white dots) where undisturbed waste-rock materials devoid of vegetation cover outcropped. For each sampling site, we have recorded slope, pH, mineralogy, and soil chemistry (Cd, Co, Cr, Cu, Ni, Zn, V, K, and P). At the 9 sampling sites (6 from area A and 3 from area B; Fig. 1) exhibiting the first steps of plant colonization, we also recorded the floristic composition and percent cover of each species. The pH of soils was measured in situ using a portable pH meter (WTW PH330i) equipped with a glass electrode. The mineralogy of the samples has been determined by a combination of several techniques, including optical (binocular, transmitted- and reflected-light) microscopy, X-ray powder diffraction (XRPD), scanning electron microscopy (SEM) with microanalysis (EDS), and grain-size analyses. Soil chemistry has been determined by fluorescence x-ray (XRF) and ICP-AES analyses. All analyses were performed using the analytical conditions reported in Marescotti et al. (2008). Statistical analysis A total of 17 species were collected from 13 sampling sites. Two matrices were considered in analyzing the data: (1) a matrix of sampling plots x species abundances and (2) a matrix of sampling plots x environmental factors (physical-chemical variables of soils). To detect and exclude possible outliers from the analysis, an exploratory multivariate analysis was carried out using PC-ORD (McCune and Mefford 1999). We then used principal component analysis (PCA) as ordination technique. Analyses were performed with PC-ORD version 4.25 (McCune and Mefford 1999). Results Mineralogy and chemistry Soil samples were generally incoherent or weakly cemented by iron-oxide and -oxyhydroxides and varied from gravel-dominated to sandy-gravel sediments, with a uniform particle size distribution in the range of 2–64 mm. Samples from area A (Table 1) showed the highest content of serpentinites and basalts. They presented a low concentration of primary sulphides that were mostly replaced by secondary Fe-oxides (hematite) and -oxyhydroxides (goethite). Sample 4 was very different, containing 400 g kg-1 of primary sulphides and 450 g kg-1 of secondary Fe-oxides and -oxyhydroxides deriving from sulphide oxidation. Serpentinites and basalts were almost in the same proportions and represented the remaining 150 g kg-1 of the constituents. Samples from area A showed the highest content of MgO, SiO2, Al203, and minor and trace elements, such as Cr and Ni (Table 1) related to mafic and ultramafic minerals (serpentines, spinels, pyroxenes, and olivines). Sample 4 showed the highest sulphur and Cu contents due to the presence of high amount of pyrite and chalcopyrite. Samples from area B (Table 1) had the highest content of secondary minerals, similar serpentinite and basalt 2009 S. Marsili, E. Roccotiello, C. Carbone, P. Marescotti, L. Cornara, and M.G. Mariotti 301 Table 1. Mineralogy and chemistry of the dumped materials. Mineralogical values represent minimum and maximum concentrations of the main recognized minerals, expressed as g kg-1. Minerals have been abbreviated according to Kretz (1983). Ccp = chalcopyrite, Chl = chlorite, Fe-ox = Fe-oxides and -oxyhydroxides (mainly goethite and hematite), Mag = magnetite, Pl = plagioclase, Py = pyrite, and Srp = serpentine group minerals. Trace elements and macronutrients are expressed as mg kg-1. Macro- Trace element nutrients concentrations (mg kg-1) (mg kg-1) Area Site Mineralogy (min–max, g kg-1) Cr Co V Cu Zn Ni Cd K P A 1 Srp (300–400), Pl (50–100), Mag (10–20), Chl (10–30), Fe-ox (300–400), Py-Ccp (10–20) 1117 74 239 1910 197 967 12 867 579 A 2 Srp (400–450), Pl (50–100), Mag (10–20), Chl (10–30), Fe-ox (300–400), Py-Ccp (10–20) 1259 96 223 3272 196 1214 13 471 577 A 3 Srp (600–750), Pl (50–100), Mag (10–30), Chl (10–50), Fe-ox (100–150), Py-Ccp (10–20) 2524 170 180 1240 192 3579 9 1910 376 A 4 Srp (20–50), Pl (20–50), Fe-ox (300–600), Py-Ccp (50–400) 253 78 200 13,347 74 93 1 166 44 A 7 Srp (500–600), Pl (50–100), Mag (10–50), Chl (10–50), Fe-ox (100–150), Py-Ccp (10–20) 2005 150 126 902 88 3207 6 760 1198 A 9 Srp (600–700), Pl (50–100), Mag (10–50), Chl (10–50), Fe-ox (50–100), Py-Ccp (10–20) 498 94 288 989 78 436 6 1331 1005 B 5 Srp (350–450), Pl (50–100), Mag (10–20), Chl (10–30), Fe-ox (350–400), Py-Ccp (10–20) 473 62 403 1258 282 175 15 1556 301 B 6 Srp (300–400), Pl (10–50), Mag (10–20), Chl (10–20), Fe-ox (500–550), Py-Ccp (20–30) 549 46 371 4180 516 356 12 1203 969 B 8 Srp (250–350), Pl (50–100), Mag (10–20), Chl (100–150), Fe-ox (350–400), Py-Ccp (5–10) 541 408 541 1615 317 252 16 647 680 C 10 Srp (300–600), Pl (50–150), Mag (10–30), Chl (10–20), Fe-ox (200–300), Py-Ccp (10–20) 972 66 121 4251 161 1063 2 1660 3706 C 11 Srp (300–400), Pl (50–100), Mag (50–100), Chl (100–200), Fe-ox (50–100), Py-Ccp (10–20) 1304 70 83 2527 139 1480 1 747 1962 C 12 Srp (350–450), Pl (50–100), Mag (50–150), Chl (150–200), Fe-ox (100–150), Py-Ccp (10–20) 1108 77 107 3755 144 1207 1 1079 2834 C 13 Srp (350–450), Pl (10–50), Mag (50–150), Chl (150–200), Fe-ox (150–200), Py-Ccp (10–20) 825 79 139 4479 154 928 1 1245 3052 302 Northeastern Naturalist Vol. 16, Special Issue 5 contents, and relatively low sulphide concentrations. Chemical composition varied from site to site according to the variable proportions of mafic and ultramafic rocks, sulphide mineralizations, and secondary minerals. Samples from area C (Table 1) were characterized by a generally high content of serpentinite and basalt, with a high concentration of secondary Fe-oxides and -oxyhydroxides that acted as cement-forming superficial crusts (hardpan layer) and filled interstices. Sulphides were generally minor components. In this area, the high contents of Ni, Cr, V, and Co were related to serpentinite and basalt, whereas the highest Cu and Zn concentrations were assigned to the Fe-secondary minerals (which effectively scavenged most of these elements from contaminated solutions; Cornell and Schwertmnann 1996 and references therein). Finally, major nutrient (P and K) concentrations ranged widely across the sampling sites (Table 1). The soils were acidic, with pH values varying from 3.8 in area A to 4.0 in areas B and C. Flora Few species (17) were able to colonize the waste-rock dump. Deschampsia flexuosa (L.) Trin. (Wavy Hairgrass) and Ophiolitic Minuartia covered about 80% of the examined area. In addition, the contribution of Festuca ovina (Fescue Grass), Maritime Pine, and Sesamoides interrupta (Boreau) G. López (Pygmean Weld) was important, with coverage up to 40% (Table 2). Plant-soil relationships The first axis of the PCA ordination (Fig. 2) reveals that the transition from stages without vegetation (Area C) to the first stage of vegetation Table 2. Sampling areas and sites with plant species, their coverage (%), and the slope (degree). A B C 1 2 3 4 7 9 5 6 8 10 11 12 13 Slope degrees 20 20 80 10 20 30 0 0 50 80 80 80 80 Total Plant Coverage (%) 9 31 4 2 7 45 40 42 2 0 0 0 0 Species Deschampsia flexuosa 8 30 4 1 7 12 2 3 1 0 0 0 0 Pinus pinaster 0 1 0 1 0 1 1 1 0 0 0 0 0 Minuartia laricifolia ophiolitica 1 0 0 0 0 30 2 4 0 0 0 0 0 Sesamoides interrupta 0 0 0 0 0 1 0 1 1 0 0 0 0 Galium lucidum 0 0 0 0 0 1 2 2 0 0 0 0 0 Festuca ovina 0 0 0 0 0 0 28 25 0 0 0 0 0 Echium vulgare 0 0 0 0 0 0 1 0 0 0 0 0 0 Dianthus sylvestris 0 0 0 0 0 0 1 0 0 0 0 0 0 Jasione montana 0 0 0 0 0 0 0 1 0 0 0 0 0 Euphorbia spinosa ligustica 0 0 0 0 0 0 0 1 0 0 0 0 0 Helichrysum italicum 0 0 0 0 0 0 1 0 0 0 0 0 0 Reichardia picroides 0 0 0 0 0 0 1 0 0 0 0 0 0 Poa pratensis 0 0 0 0 0 0 1 0 0 0 0 0 0 Silene paradoxa 0 0 0 0 0 0 0 1 0 0 0 0 0 Dittrichia viscosa 0 0 0 0 0 0 0 1 0 0 0 0 0 Juniperus oxycedrus 0 0 0 0 0 0 0 1 0 0 0 0 0 Scrophularia canina 0 0 0 0 0 0 0 1 0 0 0 0 0 2009 S. Marsili, E. Roccotiello, C. Carbone, P. Marescotti, L. Cornara, and M.G. Mariotti 303 Figure 2. PCA analysis. A: axis 1 (50.7% variation explained) vs axis 2 (19.9% variation explained). B: axis 1 (50.7% variation explained) vs axis 3 (14.6% variation explained). Species are listed in table 2, and numbers indicate sampling sites. 304 Northeastern Naturalist Vol. 16, Special Issue 5 cover in areas A and B (increasing coverage rate, axis 1, r = 0.885) was influenced by a decrease of the land slope (r = -0.736), a decrease in the amount of Cr (r = -0.448) and Ni (r = -0.441), and increasing amounts of Zn (r = 0.647), V (r = 0.618), and to a lesser extent, Cd (r = 0.562). The species that exhibited a concomitant increase in coverage, were Ophiolitic Minuartia (r = 0.841) and Fescue Grass (r = 0.880). Wavy Hairgrass showed a negative correlation with P (r = -0.750) and a positive correlation with Fe-oxides and -oxyhydroxides (r = 0.730) and magnetite (r = 0.548) on axis 3. These species are the most common in the sampling sites. Discussion The surveyed vegetation represents an early successional plant community characterized by an extremely poor flora, probably related to the high concentration of trace elements that represent a limiting and selecting factor for plant colonization (Antonovics et al. 1971). The waste materials examined showed high concentrations of trace elements of environmental concern, notably exceeding the Italian legal limits for Cr, Co, V, Cu, Zn, Ni, and Cd (Legislative Decree 2006, Ministerial Decree 1999). Moreover, low nutrient levels were recorded, which could affect the abundance and diversity of plant communities, as previously observed by Bradshaw and Chadwick (1980). The pH showed values below 5, which are indicative of unavailability of some elements, such as Ca and P (Torres et al. 1993), and can also signal the presence of toxic amounts of Zn, Mn, Al, and/or Ni. Many of the potentially toxic elements are incorporated and/or adsorbed by Fe-oxyhydroxides (goethite) and -oxides (hematite) that represent the main stable mineral phases of these soils (Marescotti et al. 2008). It is well known that succession on mine waste is often slow, and the poor species richness and the limited colonization can persist even for a hundred years (Kalin and van Everdingen 1988, Kimmerer 1981). The flora of the first stages of colonization are characterized by species that have not been anthropogenically introduced, such as the dominant Wavy Hairgrass that is present in the surrounding natural vegetation of the area. This species is acid tolerant, as described by Bradshaw (1997), and formed plant communities of first colonizers with Fescue Grass, Ophiolitic Minuartia, and Pygmean Weld. These species are typical pioneer plants characteristic of ultramafic vegetation of Liguria and Italian North Apennines. The same plant communities are also present in rock and scree habitats of the surrounding areas and probably represented the first step of natural recolonization of soils containing high concentrations of heavy metals (Bradshaw 1997). It is well known that abandoned mine sites support metal-tolerant populations of common grasses that could be used to revegetate other mine dumps (Bradshaw 1952). Several authors described the successional pathway of abandoned mine vegetation (Jaffré et al. 1994, Malaisse and Brooks 1982, Malaisse and Grégoire 1978). Some of them evaluated the possibility of employing native plants to revegetate mine sites (Jaffré and Pellettier 1992, Jaffré and Rigaut 1991, Jaffré et al. 1994, 2009 S. Marsili, E. Roccotiello, C. Carbone, P. Marescotti, L. Cornara, and M.G. Mariotti 305 Pellettier and Esterle 1995, Robinson et al. 1997). Their results support the idea of a good recolonization potential by our native flora. We found the second step of plant colonization characterized by few species with high coverage at sites where Cr and Ni decrease and Zn, V, and Cd increase. In particular, species that better respond to these physical and chemical changes are also more abundant in the landfill and are therefore more tolerant for elements such as Zn and V, confirming that trace element concentrations exercised strong selective pressure on natural plant communities in metalliferous soils (Nicolls et al. 1965). In contrast to other observations for mine soils (Nicolls et al. 1965), an increase of the vegetation cover did not seem to be directly correlated to an increase of macronutrients in the soil. For example, Wavy Hairgrass even showed a negative correlation with phosphorus. A slope greater than 45° is a critical factor limiting the first plant colonizers (Bochet and García-Fayos 2004). The more developed soils do not have steep slopes and probably represent a first mature soil, also exhibiting higher species diversity and representing the third stage. This is certainly a successional stage mostly unaffected by the landfill and the precursor to subsequent stages like the garrigue vegetation and bushes that represent the natural vegetation of the surrounding areas. In conclusion, this work has enabled us to chemically and floristically characterize plant pioneer stages in this mine landfill, which are certainly the most critical points for a future redevelopment of the area. The considerations on the parameters that influence the first colonization by plants allow us to plan a regeneration of the area in order to develop future land restoration of degraded serpentine sites that minimize interventions and costs. 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