Plant and Soil

, Volume 418, Issue 1–2, pp 205–218 | Cite as

Is annual or perennial harvesting more efficient in Ni phytoextraction?

  • George C. Adamidis
  • Maria Aloupi
  • Petros Mastoras
  • Maria-Ioanna Papadaki
  • Panayiotis G. Dimitrakopoulos
Regular Article

Abstract

Aims

The use of perennial metal hyperaccumulators in phytoextraction provides an excellent gateway toward the removal of heavy metals from polluted sites, and the opportunity for the phytomining of valuable metals. In order to further advance our understanding of the effect of cropping systems on metal phytoextraction, it is important to investigate the effect of harvesting time. This study focuses on the variation in biomass production, Ni concentration and Ni mass across the different phenological stages, populations and organs of Alyssum lesbiacum, in order to evaluate when Ni phytoextraction is at a maximum.

Methods

We sampled 60 single-phenological stage plots in three A. lesbiacum populations and we determined biomass production and Ni concentration at the plant organ level.

Results

Based on spontaneous A. lesbiacum vegetation, we were able to record remarkably high values of Ni phytoextraction. Biomass production and Ni concentration were found to be maximal on the third and fourth year of the A. lesbiacum life cycle respectively, while maximum phytoextraction capacity was reached in the third year.

Conclusions

Our results: (1) demonstrate the significant variation in Ni phytoextraction across different phenological stages, populations and organs of A. lesbiacum, (2) imply that its phytoextraction potential is mostly influenced by biomass production and (3) suggest that perennial harvests could be an interesting alternative to consider in the future.

Keywords

Alyssum lesbiacum Intra-specific variation Nickel hyperaccumulation Phenology Phytomining Phytoremediation 

Introduction

The increasing release of heavy metals to the environment due to human activities such as mining and smelting operations, industry, agriculture and domestic use, raise concern for food safety and human health worldwide (Moon et al. 2000; Kachenko and Singh 2006; Zhang et al. 2015; Bednářová et al. 2016) and present challenges for rehabilitation (Bian et al. 2012). Remediation of polluted soils using conventional technologies (e.g. excavation, chemical stabilization, capping) is considered costly (Berti and Cunningham 2000) and thus after 1990 a number of cost-effective and eco-friendly techniques have emerged. These techniques, collectively known as “phytoremediation” ("the use of plants and associated soil microbes to reduce the concentrations or toxic effects of contaminants in the environment"; Greipsson 2011), include phytoextraction, phytofiltration, phytostabilization, phytovolatilization and phytodegradation (Alkorta et al. 2004). Among these, phytoextraction, that is "the uptake of soil or water metals by plant roots and their translocation to and accumulation in aboveground biomass" (Chaney 1983) is considered as the most efficient phytoremediation technique for the removal of certain trace metals (e.g. Ni, Zn, Cd) from soils, sediments or water with low to moderate contamination levels as well as from naturally metal enriched soils (Cluis 2004; Cherian and Oliveira 2005; Milic et al. 2012; Li et al. 2003). Phytomining (or its variant, agromining) is a subset of phytoextraction aiming to mine valuable metals from polluted or mineralized soils using plants in order to recover marketable quantities of metals from bio-ores (plant biomass) (Brooks et al. 1998). Recently, Robinson et al. (2015) presented serious limitations of phytoextraction, by investigating the reasons for the lack of commercial use of phytoextraction technologies and for the current inefficiency of phytomining. However, the authors of this study declared that they had no intention to discourage research related to phytoextraction technologies and that future research, providing practical calculations, is needed in this area.

Since the 90’s numerous phytoremediation (e.g. Rafati et al. 2011; Belouchrani et al. 2016; reviewed by Mahar et al. 2016) and phytomining experiments (e.g. Durand et al. 2016; Bani et al. 2015a; Chaney et al. 2007; reviewed by Nkrumah et al. 2016) have been undertaken and our understanding of the effect of cropping systems on the phytoextraction potential of different species has advanced. The potential of a plant species to phytoextract metals is mainly determined by its above-ground tissue metal concentration and biomass (Li et al. 2010). However, hyperaccumulation and hypertolerance are considered more important traits than the high biomass yield (Chaney et al. 1997) and thus most of the aforementioned phytoextraction experiments use metal hyperaccumulating species (sensu Reeves and Baker 2000). In addition, most of these experiments utilize perennial hyperaccumulating species (e.g. Chardot et al. 2005; Robinson et al. 1997a, 1997b, 1999) which can also maintain the substrate-stabilizing function of the established crops (van der Ent et al. 2015). The amount of the phytoextracted trace metals from the soil by a metal hyperaccumulating plant species is a function of above-ground tissue metal concentration multiplied by the produced biomass (Macek et al. 2008). Therefore, in order for a metal hyperaccumulator to be considered as a suitable candidate for application as “crop” in phytoextraction operations, its total biomass production has to be sufficiently high and the high-metal tissue should be a high proportion of the total biomass (Zhang et al. 2014). However, when referring to perennial metal hyperacumulating species, phytoextraction potential is not only a question of selecting the most appropriate plant species but also the optimal harvesting time. In other words, we can hypothesize that when using a perennial metal hyperaccumulator on phytoextraction operations, annual harvests may not provide higher yields compared to perennial harvests (i.e. harvesting once every few years). In addition, considering the cost of harvesting, we can hypothesize that annual harvests may not provide the highest profit in order to make phytomining commercially viable (Li et al. 2003). In order to investigate the previous hypotheses, data on the biomass production and metal storage on different organs across the different life stages of perennial metal hyperaccumulating species are needed.

Alyssum lesbiacum (Candargy) Rech. f. is a monocarpic perennial species (with a four year life cycle) and a well-established Ni hyperaccumulator (Brooks et al. 1979; Reeves et al. 1997; Kazakou et al. 2010) endemic to the serpentine soils of the island of Lesbos, Greece (Strid and Tan 2002). It is not only a “hypernickelophore” (sensu Chaney et al. 2007) but also one of about 15 species belonging to the genus Alyssum that have Ni concentrations above c. 2%. Alyssum lesbiacum holds the 7th highest recorded Ni concentration (23,650 mg kg−1; c. 2.4%) amongst the Alyssum species that meet the definition of Ni hyperaccumulators, while Alyssum murale, on the same rank, holds the 14th place (Kazakou et al. 2010). A. lesbiacum is considered as a ‘micro edaphic’ endemic Ni-hyperaccumulator, since Ni hyperaccumulation varies considerably amongst different natural populations, depending on soil Ni concentration (Kazakou et al. 2010). Furthermore, the same populations have been shown to present significant variation in Ni tolerance, accumulation and translocation patterns, under hydroponic conditions (Adamidis et al. 2014a). Finally, it has been shown to maintain a high metal phytoextraction efficiency even in soils marginally contaminated with polycyclic aromatic hydrocarbons (PAHs) (Singer et al. 2007).

In the present study, we aim to evaluate on site the variation in Ni phytoextraction capacity and in metal storage in different plant organs across the four different life stages and three different populations of A. lesbiacum. The four year life cycle of A. lesbiacum provides the opportunity to investigate differentiation between phenological stages, which would provide useful information on the effect of cropping systems on the phytoextraction potential of perennial metal hyperaccumulators. In addition, the significant intra-specific variation in leaf Ni concentrations along with the significant genetic differentiation between A. lesbiacum populations (Adamidis et al. 2014b) is particularly useful for studying the effect of inter-population variability and making suggestions at the population level (Adamidis et al. 2014a) for potential use in phytoremediation and/or phytomining technologies. Specifically, the following four questions are addressed: For which phenological stage, population and organ of A. lesbiacum is (1) biomass production and (2) Ni concentration maximized? (3) Is biomass production or Ni hyperaccumulation the most important factor in terms of determining the potential of A. lesbiacum to phytoextract metals? (4) For which phenological stage and population of A. lesbiacum is the phytoextraction capacity maximized?

Materials and methods

Study species and material collection

This study was conducted in the central and southeastern part of the island of Lesbos (Greece), at three serpentine sites (Ampeliko, Olympos and Loutra), encompassing three large populations of A. lesbiacum (AM, OL and LO populations respectively). At all three sites A. lesbiacum is the dominant species (its relative contribution to the total biomass of dominant species is 47–99% at the three sites; Adamidis et al. 2016). These sites are open grasslands with no history of agricultural use; a more detailed description of the sites is given in Adamidis et al. (2014c) and in Kazakou et al. (2010). A. lesbiacum has a life cycle of four years a distinct and easily identifiable phenological stage for each one of the four years as follows (for typical stands): in the 1st year a high number of small individuals with no branches (or one branch at the most), in the 2nd year fewer but larger individuals with more than one branches, in the 3rd year even fewer and larger individuals with numerous branches and in the 4th year fewer and flowering individuals (G.C. Adamidis and P.G. Dimitrakopoulos, unpublished data). In each of the three populations all different A. lesbiacum phenological stages (hereafter “years”) coexist while there are relatively large patches covered by single-stage individuals (e.g. single-stage 3 (hereafter year 3) patches: patches covered exclusively by three-year-old individuals).

Within each site, large patches covered by single-stage A. lesbiacum individuals were identified and within each patch five 1 m × 1 m plots were randomly selected (N = 3 sites × 4 years × 5 plots = 60 plots). All selected plots presented similar A. lesbiacum cover (~75%). Herbaceous species found in all the selected plots (presenting minor contribution to the plots’ biomass) included Plantago lagopus L., Aegilops biuncialis Vis. and Crepis commutata L. (Nomenclature for plant species follows Flora Europaea; Tutin et al. 1964–1980). In each of these plots, the aboveground standing biomass was harvested in late spring 2015. The A. lesbiacum biomass samples were separated into different plant organs (stems, leaves and flowers), oven-dried at 80 °C for 48 h, and weighed.

Ni concentration and phytoextraction

The previously mentioned harvested plant organs were gently cleaned with a soft brush in order to remove adhering soil dust. The samples were pre-frozen at −20 °C and then freeze-dried for 48 h in a LabconcoFreeZone 4.5 laboratory apparatus, at −40 °C collector temperature under <5 mBar vacuum. This was followed by pulverization of the freeze-dried samples in a laboratory mixer-mill. The pulverized samples were digested with conc. HNO3 in a Mars Xpress system (CEM), according to the USEPA’s method 3051A (2007). Ni determination was performed by Flame Atomic Absorption Spectrometry in a Perkin–Elmer5100ZL spectrometer. Ni concentrations in different organs were calculated on a dry weight basis.

In order to calculate the phytoextracted Ni mass (mg/m2) in each plot, the total A. lesbiacum biomass per different organ was multiplied by the corresponding Ni concentration (e.g. the total leaf biomass of two-year-old individuals on a specific plot of Ampeliko site, was multiplied by the average leaf Ni concentration of the same year, plot and site) and then the phytoextracted Ni values of the different organs were summed.

Data analysis

We tested for the effects of Year, Organ and Population as independent variables on Biomass production, Ni concentration and Phytoextracted Ni mass, using generalized linear models (GLMs). In all three cases we used a Gamma error with a log link. The initial models included all interaction terms, and then models were simplified by removing all non-significant interactions and main effects. The main effects involved in significant interactions were retained in the models in order to satisfy the principle of marginality. The best fitting models were obtained by minimizing the Akaike Information Criterion (AIC). Initial trials also included tests of generalized linear mixed effects models (GLMM’s) using Plot as a random factor. However, the best-fitting models found were mathematically described by the following GLM formula:

$$ \begin{array}{l}\mathrm{Response} \sim \mathrm{Gamma}\ \left({\mu}_i,\tau \right)\kern1em \\ {} E\left({\mathrm{Response}}_i\right)={\mu}_i\ \mathrm{and}\ \mathrm{var}\ \left({\mathrm{Response}}_i\right)=\frac{\mu_i^2}{\tau}\kern1em \\ {} \log \left({\mu}_i\right)={\beta}_1+{\beta}_2\times \mathrm{Year}+{\beta}_3\times \mathrm{Organ}+{\beta}_4\times \mathrm{Population}+{\beta}_5\times \left(\mathrm{Year}\times \mathrm{Organ}\right)+{\beta}_6\times \left(\mathrm{Year}\times \mathrm{Population}\right)+{\beta}_7\times \left(\mathrm{Organ}\times \mathrm{Population}\right)\kern1em \end{array} $$

Where Responsei is the value of one of the three response variables used in this study (Biomass production, Ni concentration and Phytoextracted Ni mass) for the ploti, β1 is the intercept and β2, β3, β4, β5, β6 and β7 the slopes of the regression parameters. The response variables are gamma distributed with mean μ and parameter τ.

To investigate the significance of the main effects and interactions we performed a chi-squared likelihood ratio test. Finally, Bonferroni multiple comparisons were used to test for pairwise differences between organs, years, populations and their interactions. All statistical analyses were carried out using the R v.3.3.1 environment (R Development Core Team 2016).

Results

Biomass production

The ranges for all biomass production values across the different phenological stages (years), organs and populations of A. lesbiacum are presented in Table 1a. The main effects of Year and Organ on A. lesbiacum biomass production were significant (P < 0.001) while there was no significant effect of Population (P = 0.169). As the pairwise comparisons revealed, the Year 3 plants presented significantly higher values of biomass production (P < 0.001) while Year 4 plants had the lowest biomass values (P < 0.001). No difference in the biomass production was detected between Years 1 and 2 (P > 0.05). The different organs of A. lesbiacum showed significant variation in biomass production values, with stems presenting the highest values, followed by leaves (P < 0.001). Flower biomass was significantly lower than of either leaf (P = 0.03) or stem (P < 0.001). In addition, all three interactions included in the model were found to significantly affect biomass production at the 0.05 level (Year x Organ: χ2 = 1.57, df = 3, P < 0.001; Year x Population: χ2 = 3.36, df = 6, P < 0.001; Organ x Population: χ2 = 1.42, df = 4, P < 0.001). In all four years of the A. lesbiacum life cycle leaves had lower values of biomass than stems (P < 0.01; Fig. 1a). In Year 4, when the plants flower, the flower biomass production was significantly higher than leaf biomass (P < 0.001; Fig. 1a), but lower than the biomass of stems (P < 0.001; Fig. 1a). However, the three populations did not follow the same pattern of biomass production across the four years of the life cycle. In particular, for the AM population, the highest biomass production was recorded in Year 3 although it did not differ significantly from the Year 1 (P = 0.456; Fig. 1b), whereas the lowest values were found in Year 4 (P < 0.003 in all cases; Fig. 1b). For the LO population, the biomass production did not differ significantly across the first three years of the life cycle (P > 0.55 in all cases; Fig. 1b), while Year 4 presented significantly lower values when compared with Years 2 and 3 (P = 0.002 and P < 0.001 respectively; Fig. 1b). Similarly for the OL population, A. lesbiacum biomass production was similar in the first three years (P > 0.172 in all cases; Fig. 1b), while in the 4th year it was significantly lower than in Years 2 and 3 (P = 0.004 and P < 0.001 respectively; Fig. 1b). The highest biomass production was found in Year 3 but without any significant differentiation between the three different populations (P > 0.74 in all cases). As for the variation in biomass production of the examined plant organs between the three sampling sites, no significant differentiation was revealed for flower (P > 0.132 in all cases), leaf (P > 0.9 in all cases) and stem (P > 0.24 in all cases) biomass production among the three populations (Fig. 1c). The stems had significantly higher biomass compared to the other two organs in all populations (P < 0.001 in all cases; Fig. 1c). Higher leaf than flower biomass was found only in AM whereas there was no significant differentiation between leaf and flower biomass production was revealed at LO and OL, (P > 0.05).
Table 1

The minimum, maximum and mean (S.E. in parentheses) of biomass production, Ni concentration and phytoextracted Ni mass across the different phenological stages (years), organs and populations of A. lesbiacum

Year

Organ

Ampeliko

Loutra

Olympos

min/max

mean (SE)

min/max

mean (SE)

min/max

mean (SE)

(a) Biomass production (g/m2)

1

Leaf

79–169

126(15)

85–170

118(16)

42–118

80(16)

Stem

251–739

448(87)

123–277

172(28)

98–251

181(29)

2

Leaf

31–126

75(19)

81–208

143(24)

106–221

154(20)

Stem

101–464

261(71)

137–516

245(69)

140–415

268(45)

3

Leaf

129–412

250(62)

131–217

192(16)

115–225

178(20)

Stem

306–1546

817(258)

305–506

385(36)

240–451

353(34)

4

Leaf

14–38

20(4)

1–13

6(3)

16–30

23(3)

Stem

121–552

224(82)

112–1007

384(210)

144–305

230(35)

Flower

11–54

23(8)

38–183

85(34)

54–98

71(9)

(b) Ni concentration (mg/kg)

1

Leaf

7478–10121

8918 (436)

7399–11156

8984 (704)

8915–11367

9944 (512)

Stem

1475–3453

2383 (369)

2592–4758

4054 (381)

3094–4959

3942 (305)

2

Leaf

8330–11154

9363 (520)

6014–15462

11209 (1521)

6193–10916

8937 (810)

Stem

2363–3811

2804 (258)

2858–4201

3528 (238)

3527–6612

4728 (559)

3

Leaf

10157–13578

11914 (708)

7371–11233

9194 (624)

7048–12920

8865 (1047)

Stem

3344–5293

4363 (420)

3087–4322

3570 (228)

3052–5873

4403 (456)

4

Leaf

19224–22240

20665 (484)

12267–17163

14569 (1146)

10854–14300

12,099 (609)

Stem

3862–6898

5328 (534)

1023–2161

1507 (238)

2110–5715

4348 (690)

Flower

9123–10609

9890 (318)

6488–7520

7064 (213)

5700–8932

7317 (681)

(c) Phytoextracted Ni mass (mg/m2)

1

Leaf

798–1588

1123 (146)

754–1296

1032(90)

463–1121

771(133)

Stem

528–2033

1066 (276)

324–1163

710(142)

358–1158

719(134)

2

Leaf

310–1242

692 (185)

909–2830

1557(332)

748–2102

1406(261)

Stem

272–1268

704 (185)

430–2168

914(321)

846–1928

1234(220)

3

Leaf

1456–5284

2978 (755)

1243–2189

1756(165)

1141–1931

1522(132)

Stem

1451–7838

3875(1467)

942–1955

1378(164)

1023–1885

1520(146)

4

Leaf

286–836

422(104)

18–161

80(30)

174–344

270(31)

Stem

531–3340

1260(529)

157–1408

574(294)

489–1555

997(231)

Flower

113 - 536

228(79)

240–1373

615(260)

354 - 873

535(120)

AM Ampeliko, LO Loutra, OL Olympos

Fig. 1

Biomass production of different organs across years a and of different years b and organs c across populations. Different lower case letters indicate significant differences (at P < 0.05) among years, populations and/or organs, by Bonferroni multiple comparison test. All results are means of original data ± SE

Ni concentration

The ranges for all Ni concentration values across the different phenological stages (years), organs and populations of A. lesbiacum are presented in Table 1b. The main effects of Year and Organ on Ni concentration were significant (P < 0.001) while no significant main effect of Population (P = 0.903) was revealed. In particular, Ni concentration was significantly higher in Year 4 as compared to Year 1 (P < 0.001), Year 2 (P < 0.001) and Year 3 (P = 0.024). Ni concentration in the organs of A. lesbiacum followed the order leaves > flowers > stems (P < 0.001 in all cases). The three interactions included in the model were found to be significant at the 0.05 level (Year x Organ (χ2 = 1.57, df = 3, P < 0.001), Year x Population (χ2 = 3.36, df = 6, P < 0.001) and Organ x Population (χ2 = 1.42, df = 4, P < 0.001). In all four years of the life cycle, Ni was higher in leaves than in stems (P < 0.001 in all cases; Fig. 2a), whereas the flower Ni concentration in Year 4, was higher than the stem concentration (P < 0.001; Fig. 2a), but lower than the leaf concentration (P < 0.001; Fig. 2a). No significant differentiation in Ni concentration was detected across different populations in the first three years (P < 0.001 in all cases; Fig. 2b). However, higher values were found in AM compared to LO (P < 0.001) and OL (P = 0.005) in the Year 4. In the AM population, the values of Ni concentration were similar in the first two years (P > 0.05; Fig. 2b), while the highest values were presented in Year 4 (P < 0.008 in all cases; Fig. 2b). In the LO and OL populations, no significant differentiation was revealed in Ni concentrations across different years (P > 0.05 in all cases; Fig. 2b). No significant differentiation was revealed in leaf, flower and stem Ni concentration among the different populations (P > 0.05 in all cases; Fig. 2c). Moreover, Ni was lower in the stems in all populations (P < 0.027 in all cases; Fig. 2c) whereas Ni concentration in the leaves was either similar to (at AM and OL; P > 0.05) or higher than (at LO; P = 0.008) concentration in the flowers (Fig. 2c).
Fig. 2

Ni concentration for different organs across years a and on different years b and organs c across populations. Different lower case letters indicate significant differences (at P < 0.05) among years, populations and/or organs, by Bonferroni multiple comparison test. All results are means of original data ± SE

Phytoextracted Ni mass

The ranges for all phytoextracted Ni mass values across the different phenological stages (years), organs and populations of A. lesbiacum are presented in Table 1c. The main effects of Year and Organ on Ni mass (Ni mg/m2) were significant (P < 0.001), while no significant main effect of Population (P = 0.188) was found. As the pairwise comparisons revealed, the 3rd year presented the highest values of phytoextracted Ni (P < 0.001). Years 1 and 2 presented similar values of phytoextracted Ni (P > 0.05) while year 4 presented the lowest values of Ni mass (P < 0.001 in all cases). The values of phytoextracted Ni from the stems of A. lesbiacum were significantly higher when compared with both the leaves (P = 0.043) and the flowers (P < 0.001). The flowers presented the lowest values of phytoextracted Ni (P < 0.001 in all cases). In addition, significant Year x Organ (χ2 = 11.76, df = 3, P < 0.001), Year x Population (χ2 = 9.64, df = 6, P < 0.001) and Organ x Population (χ2 = 6.92, df = 4, P < 0.001) interactions were revealed, indicating that differences among populations, organs and years affected the response of phytoextracted Ni mass. For the first three years of A. lesbiacum life cycle the phytoextracted Ni mass was similar in leaves and stems (P > 0.05 in all cases; Fig. 3a). However, in Year 4, the stems presented significantly higher values of phytoextracted Ni when compared to the leaves (P < 0.001; Fig. 3a) while values of phytoextracted Ni for flowers did not differ significantly when compared with either stems (P = 0.056; Fig. 3a) or leaves (P = 0.23; Fig. 3a). For the AM population, phytoextracted Ni was markedly higher in Year 3 when compared with the other three years (P < 0.018 in all cases; Fig. 3b). A less clear time dependence of the phytoextracted Ni mass was observed at LO and OL (Fig. 3b). No significant differentiation was revealed for leaf, flower and stem Ni phytoextraction among the different populations (P > 0.05 in all cases; Fig. 3c). For the AM population, values of leaf and stem Ni phytoextraction were similar (P > 0.05), while the flowers presented significantly lower phytoextracted Ni values (P < 0.001 in all cases; Fig. 3c). For the LO and OL populations, no significant differentiation was revealed on the values of phytoextracted Ni among the different organs (P > 0.05 in all cases; Fig. 3c).
Fig. 3

Phytoextracted Ni mass for different organs across years a and on different years b and organs c across populations. Different lower case letters indicate significant differences (at P < 0.05) among years, populations and/or organs, by Bonferroni multiple comparison test. All results are means of original data ± SE

Discussion

Differences in biomass production among phenological stages, populations and organs

The closest to our experimental design is the work of Bani et al. (2015a) and thus comparisons between these two studies are presented throughout the discussion. However, it should be taken into account that the plots in Bani et al. (2015a) were much larger (36m2) and were not standardized for their plant density. Our results revealed that, on average, the highest biomass production was achieved in the 3rd year of A. lesbiacum life cycle. Although, the separate values of stem and leaf biomass did not significantly differ across the first three years, an increasing pattern between overall biomass production and year of harvest was revealed for the first three years. A similar positive relationship was observed between biomass yield and harvesting time in a field study of spontaneous Alyssum murale vegetation (Bani et al. 2015a). The average biomass production significantly dropped in the 4th year of A. lesbiacum life cycle with regard to previous years. This is primarily due to the significantly lower leaf biomass (Fig. 1a), which is the result of the increased leaf senescence and shedding during the flowering stage (reported also for A. murale; Bani et al. 2015a).

Leaf biomass was found to be significantly lower than stem biomass in all years but also relative to flower biomass in the 4th year of the life cycle (Fig. 1a). Similar resource allocation patterns have been reported for A. murale, with the stems having the greatest biomass, followed by the flowers and with leaves having the lowest biomass production (Bani et al. 2015a). Although this is not an unexpected result, it demonstrates the importance of the stems and flowers during phytoextraction process, despite the fact that the leaves are considered the main storage organ for heavy metals in metal hyperaccumulating species (Krämer 2010; Coinchelin et al. 2012). However, an analysis of the organs of 14 hyperaccumulators from the genera Alyssum, Leptoplax and Bornmuellera showed that, on average, stems presented the highest biomass values, followed by leaves whilst flowers and seeds accounted for less than 20% of the plant biomass proportion (Zhang et al. 2014). Although, total biomass production did not significantly differ across populations, the significant interactions between Population and both Year and Organ that have been detected indicate the presence of inter-population variation in the temporal dynamics of resource allocation. Li et al. (2003) has reported a significant differentiation in biomass production across different ecotypes of Alyssum species, but without investigating patterns at the plant organ level. The highest values of biomass production were recorded in the 3rd year of the AM population of A. lesbiacum (Fig. 1b), though without significantly differing from the values recorded for the AM population in year 1 and for the OL and LO populations in years 2 and 3 (Fig. 1b). This result is not in congruence with Bani et al. (2015a) reporting that the highest biomass yield for A. murale was during the mid-flowering stage. However, this is an expected incongruity, considering that A. murale is a biennial plant and the inter-annual differences are expected to be more marked. The flower biomass at AM was significantly lower when compared with leaf biomass, while for the other two populations biomass of flowers and leaves were similar (Fig. 1c). At the same time, the AM population presented the highest stem biomass, but without significantly differing from stem biomass of OL and LO populations. This result implies that AM population may invest higher percentage of resources to structural compounds (stem growth) than to reproduction (flower formation) with regard to the other two populations. This may demonstrate a possible differentiation in resource allocation across the different populations of A. lesbiacum that face trade-offs between growth and reproduction in harsh serpentine environments. Considering that A. lesbiacum is considered a ‘micro-edaphic’ endemic Ni-hyperaccumulating species (Kazakou et al. 2010) and its populations present significant genetic differentiation (Adamidis et al. 2014b), this result may be in congruence with the alternative strategies for reproductive success and survival reported for different species ecotypes across different soil conditions (Adamidis et al. 2014d). Finally, with respect to our first question, the biomass production was found to be maximized in the third year of A. lesbiacum life cycle, with stems being the most productive organ and AM population presenting the highest recorded values of biomass production.

Differences in Ni hyperaccumulation among phenological stages, populations and organs

The highest Ni concentration was measured in the 4th year of A. lesbiacum life cycle. Considering that the stem Ni concentration was similar through the study period this finding is due to the significantly higher leaf Ni concentration during the flowering stage of A. lesbiacum (4th year) when compared to the first three years (Fig. 2a). This result is in accordance with Bani et al. (2015a) who also recorded the highest Ni concentration during the mid-flowering stage of A. murale. The highest values of leaf Ni recorded in the 4th year of A. lesbiacum life cycle may be the result of newly accumulated Ni from the soil and translocated to the leaves. However, a hypothesis worth future investigation is whether there is some kind of metal re-translocation from the senescenced leaves to other organs (e.g. flowers) during the leaf senescence, similar to the nutrient resorption mechanism (Aerts 1996), which would enable metal hyperaccumulating species to re-use previously stored metals. Such a process could be feasible during the flower formation stage, when metal hyperaccumulating plants allocate metals to flowers (Bani et al. 2015a, b) and seeds (Adamidis et al. 2014a), if the expense of resource acquisition (i.e. metal accumulation and translocation to reproductive organs) is higher than resource conservation (i.e. metal re-translocation). In this context, Deng et al. (2016) recently demonstrated the Ni translocation from older to younger leaves in the Ni-hyperaccumulator Noccaea caerulescens and showed that phloem re-distribution of Ni plays an important role for Ni accumulation in younger leaves. When taking account all different years and organs, no significant differentiation in Ni concentration emerges between different populations. However, the highest values of Ni concentration were recorded in the 4th year of the AM population (Fig. 2b). The AM population was previously found to present the highest shoot and leaf Ni concentration values under both hydroponic and field conditions (Adamidis et al. 2014a; Kazakou et al. 2010 respectively) when compared to the other existing A. lesbiacum populations.

All Ni concentrations recorded in the different plant organs in this study meet the definition of metal hyperaccumulation (Ni concentrations above 1000 mg kg−1; Reeves and Baker 2000). In particular, the lowest Ni concentration (1023 mg kg−1) recorded in this study was measured in the stems of the 4th year of LO population while the highest Ni concentration was recorded on the leaves of the 4th year of AM population and had a value of 22.240 mg kg−1. Stems had significantly lower Ni concentration than leaves and flowers in all the populations (Fig. 2c). This result is in agreement with the suggestion that stems act mostly as transportation routes for Ni and “store” limited amounts of Ni (Zhang et al. 2014). Finally, with respect to our second question, the AM population in the fourth year of A. lesbiacum life cycle had the highest values of hyperaccumulated Ni, with leaves being the most efficient organ in terms of Ni hyperaccumulation.

Differences in Ni phytoextraction among phenological stages, populations and organs

As mentioned before, Ni mass is a matter of both biomass production and Ni concentration. So far, the highest biomass production has been recorded in the stems of the 3rd year while the highest Ni concentration in the leaves of the 4th year of A. lesbiacum life cycle. Although the AM population presented the highest values of both biomass production and Ni concentration, on average no significant differentiation was revealed among the different populations. Therefore, the consideration of biomass production and Ni hyperaccumulation alone cannot guide to solid conclusions about Ni phytoextraction. However, the calculation of Ni mass provides information about the combined effect of biomass production and Ni concentration.

The highest values of phytoextracted Ni mass were recorded in the 3rd year of A. lesbiacum life cycle. This result is due to the significantly higher values of biomass production at this stage of A. lesbiacum life cycle (Fig. 3a). A rather unexpected result was that the 4th year of A. lesbiacum life cycle had the lowest Ni mass, even though the highest values of Ni concentration were recorded in this year. This result implies that when using metal hyperaccumulating species in phytoextraction technologies, high biomass production may be more important than the hyperaccumulation capacity of a species and/or an ecotype, although this might be dependent on physiological and phenological traits of the species of interest. Another surprising result was that stems accounted for significantly higher phytoextracted Ni than both leaves and flowers despite the fact that Ni enrichment occurs mainly in the leaves of hyperaccumulators (Broadhurst et al. 2004; Zhang et al. 2014) and that the stems store significantly lower amounts of Ni (Fig. 2a and c). However, it appears that due to their high biomass production, the stems play a key role in Ni phytoextraction (Fig. 1a and c). Hence, with respect to our third question, the potential of A. lesbiacum populations to phytoextract metals seems to be more influenced by biomass production than by Ni hyperaccumulation capacity. The highest phytoextracted Ni was calculated in the 3rd year of the AM population, though not statistically differing from the other two populations in the same year (Fig. 3b). This result is rather expected considering that, the AM population of A. lesbiacum had both the highest biomass production (Fig. 1b) and Ni concentration (Fig. 2b). Previous studies have also demonstrated that this population was the most efficient in terms of Ni hyperaccumulation (Kazakou et al. 2010; Adamidis et al. 2014a) and stem biomass production (Adamidis et al. 2014a) compared with the other A. lesbiacum populations. In addition, there is some evidence that the AM population may invest more resources on stem growth than on flower formation (see discussion about biomass production) and thus it is the only population in which flower Ni phytoextraction is significantly lower than leaf and stem Ni phytoextraction. So, with respect to our fourth question, it appears that the values of phytoextracted Ni are maximized in the third year of A. lesbiacum life cycle, especially in the AM population.

Implications for Ni phytoextraction technologies

In this study, using spontaneous A. lesbiacum vegetation of different phenological stages, we were able to record remarkably high values of biomass production. The obtained A. lesbiacum biomass (cumulative biomass of the different organs per plot) on the 1st year of AM population had an average value of 5.7 t ha−1. This obtained biomass is 1.8-fold higher that the A. murale biomass obtained on the 1st year (3.2 t ha−1) from unfertilized plots and it is comparable to the A. murale biomass from fertilized plots (6.3 t ha−1) (Bani et al. 2015a). The highest value of obtained A. lesbiacum biomass in our study was recorded in the 3rd year of AM population and had an average value of 10.7 t ha−1 which is 3.3-fold higher than the A. murale obtained biomass (3.2 t ha−1) from the unfertilized plots on the 1st year. In other words, without even taking into account the cost of harvesting, in terms of biomass production it is surely more profitable to harvest spontaneous vegetation from AM population of A. lesbiacum every three years, than spontaneous vegetation of A. murale every year. The leaf Ni concentration recorded on this study had a mean value of 11.2 ± 0.6 g kg−1, which is similar to previous reports from the same Α. lesbiacum populations (Kazakou et al. 2010). However, stems accounting for the greater part of the harvested biomass proportion had a mean Ni concentration of 3.8 ± 0.3 g kg−1. The stem Ni concentrations recorded in this study are similar to the stem Ni concentrations recorded for A. murale, with mean value of 3.54 ± 0.6 g kg−1 (Bani et al. 2015a).

The AM population of A. lesbiacum in the first year phytoextracted 21.9 kg Ni ha−1 (Fig. 3b). This is a remarkably high value of phytoextracted Ni, considering that this study investigated spontaneous A. lesbiacum vegetation without any addition of fertilizers or other agronomic management practices. Spontaneous A. murale vegetation was previously reported to phytoextract 1.7 kg Ni ha−1 in plots with no addition of fertilizers and 22.6 kg Ni ha−1 in fertilized plots (Bani et al. 2015a). In other words, annual harvests of the AM population of A. lesbiacum phytoextract 12.9-fold higher amounts of Ni than a similar A. murale population, while they are able to phytoextract similar amounts of Ni mass when compared with a fertilized A. murale population. Moreover, a mean phytoextraction capacity as high as 68,6 kg Ni ha−1 (Fig. 3b) was calculated for the AM population in the third year, which accounts for more than threefold higher values of phytoextracted Ni compared to the first year of A. lesbiacum life cycle. Taking all these into consideration, it appears that it is more profitable to harvest spontaneous vegetation from the AM population of A. lesbiacum every three years than having annual harvests. The profit of perennial harvesting becomes even more considerable if someone takes into account the extra cost of annual harvesting. Although the cost of harvesting may differ across regions and years (e.g. in Albania and for 2014 the cost of production was 390$ ha−1 yr.−1) it is evident that harvesting once every three years would be more cost-efficient than harvesting every year. In addition, perennial yields ensure that the substrate-stabilizing function of the established A. lesbiacum vegetation is maintained uninterruptedly. Finally, the option to harvest once every few years in phytomining technologies offers flexibility in decision-making by providing the opportunity to avoid years with low price of metals on world markets.

In our study, we were able to record remarkably high values of phytoextracted Ni using spontaneous A. lesbiacum vegetation. However, agronomic practices such as fertilization, would most probably increase biomass production of metal hyperaccumulating crops without decreasing Ni concentration and thus multiply the amounts of phytoextracted Ni (Robinson et al. 1997a, b). It has been reported that fertilization was able to double the biomass production of A. murale crops while at the same time the amounts of phytoextracted Ni increased by up to 15-fold (Bani et al. 2007). Several studies have reported considerable increases in metal phytoextraction with the use of different agronomic practices (e.g. plant density, sown vs natural crops, fertilization and weed control; Li et al. 2003; Bani et al. 2015a, b). Although this study provided very encouraging new insights related to the phytoextraction capacity of A. lesbiacum, it is almost certain that the use of agronomic practices would result in significantly higher yields. Future research focusing on the effect of different agronomic practices on sown crops of A. lesbiacum are needed for a better understanding of the economic feasibility of the potential use of this Ni hyperaccumulator in phytoextraction technologies (phytoremediation and/or phytomining).

Notes

Acknowledgements

We would like to thank Professor Triantaphyllos Akriotis for editorial assistance, and the Editor van der Ent and three anonymous reviewers for their constructive comments on an earlier version of the manuscript.

References

  1. Adamidis GC, Aloupi M, Kazakou E, Dimitrakopulos PG (2014a) Intra-specific variation in Ni tolerance, accumulation and translocation patterns in the Ni-hyperaccumulator Alyssum lesbiacum. Chemosphere 95:496–502CrossRefPubMedGoogle Scholar
  2. Adamidis GC, Dimitrakopoulos PG, Manolis A, Papageorgiou AC (2014b) Genetic diversity and population structure of the serpentine endemic Ni hyperaccumulator Alyssum lesbiacum. Plant Syst Evol 300:2051–2060CrossRefGoogle Scholar
  3. Adamidis GC, Kazakou E, Baker AJM, Reeves RD, Dimitrakopoulos PG (2014c) The effect of harsh abiotic conditions on the diversity of serpentine plant communities on lesbos, an eastern Mediterranean island. Plant Ecol Divers 7:433–444CrossRefGoogle Scholar
  4. Adamidis GC, Kazakou E, Fyllas NM, Dimitrakopoulos PG (2014d) Species adaptive strategies and leaf economic relationships across serpentine and non-serpentine habitats on lesbos, eastern Mediterranean. PLoS One 9:e96034. doi:10.1371/journal.pone.0096034 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Adamidis GC, Kazakou E, Aloupi M, Dimitrakopoulos PG (2016) Is it worth hyperaccumulating Ni on non-serpentine soils? Decomposition dynamics of mixed-species litters containing hyperaccumulated Ni across serpentine and non-serpentine environments. Ann Bot 117(7):1241–1248CrossRefPubMedPubMedCentralGoogle Scholar
  6. Aerts R (1996) Nutrient resorption from senescing leaves of perennials: are there general patterns? J Ecol 84:597–608CrossRefGoogle Scholar
  7. Alkorta I, Hernández-Allica J, Becerril J, Amezaga I, Albizu I, Garbisu C (2004) Recent findings on the phytoremediation of soils contaminated with environmentally toxic heavy metals and metalloids such as zinc, cadmium, lead, and arsenic. Rev Environ Sci Biotechnol 3:71–90CrossRefGoogle Scholar
  8. Bani A, Echevarria G, Sulçe S, Morel J, Mullai A (2007) In-situ phytoextraction of Ni by a native population of Alyssum murale on an ultramafic site (Albania). Plant Soil 293:79–89CrossRefGoogle Scholar
  9. Bani A, Echevarria G, Sulçe S, Morel JL (2015a) Improving the agronomy of Alyssum murale for extensive phytomining: a five-year field study. Int J Phytoremediat 17:117–127CrossRefGoogle Scholar
  10. Bani A, Echevarria G, Zhang X, Benizri E, Laubie B, Morel JL et al (2015b) The effect of plant density in nickel-phytomining field experiments with Alyssum murale in Albania. Aust J Bot 63:72–77. doi:10.1071/bt14285 Google Scholar
  11. Bednářová Z, Kalina J, Hájek O, Sáňka M, Komprdová K (2016) Spatial distribution and risk assessment of metals in agricultural soils. Geoderma 284:113–121CrossRefGoogle Scholar
  12. Belouchrani AS, Mameri N, Abdi N, Grib H, Lounici H, Drouiche N (2016) Phytoremediation of soil contaminated with Zn using canola(Brassica napus L). Ecol Eng 95:43–49CrossRefGoogle Scholar
  13. Berti WWR, Cunningham SD (2000) Phytostabilization of metals. In: Raskin I, Ensley BD (eds) Phytoremediation of toxic metals – using plants to clean up the environment. John Wiley & Sons, Inc, New York, pp 71–88Google Scholar
  14. Bian Z, Miao X, Lei S, Chen S-E, Wang W, Struthers S (2012) The challenges of reusing mining and mineral-processing wastes. Science 337(6095):702–703CrossRefPubMedGoogle Scholar
  15. Broadhurst CL, Chaney RL, Angle JS, Erbe EF, Maugel TK (2004) Nickel localization and response to increasing Ni soil levels in leaves of the Ni hyperaccumulator Alyssum murale. Plant Soil 265:225–242CrossRefGoogle Scholar
  16. Brooks RR, Morrison RS, Reeves RD, Dudley TR, Akmans Y (1979) Hyperaccumulation of nickel by Alyssum Linnaeus (Cruciferae). P Roy Soc Lond B 203:387–403CrossRefGoogle Scholar
  17. Brooks RR, Chambers MF, Nicks LJ, Robinson BH (1998) Phytomining Trends Plant Sci 1: 359–362Google Scholar
  18. Chaney RL (1983) Plant uptake of inorganic waste constituents. In: Parr JFEA (ed) Land treatment of hazardous wastes. Noyes Data Corp, Park Ridge, pp 50–76Google Scholar
  19. Chaney RL, Malik M, Li YM, Brown SL, Brewer EP, Angle JS, Baker AJM (1997) Phytoremediation of soil metals. Curr Opin Biotechnol 8:279–284CrossRefPubMedGoogle Scholar
  20. Chaney RL, Angle JS, Broadhurst CL, Peters CA, Tappero RV, Sparks DL (2007) Improved understanding of hyperaccumulation yields commercial phytoextraction and phytomining technologies. J Environ Qual 36:1429–1433CrossRefPubMedGoogle Scholar
  21. Chardot V, Massoura ST, Echevarria G, Reeves RD, Morel JL (2005) Phytoextraction potential of the nickel hyperaccumulators Leptoplax emarginata and Bornmuellera tymphaea. Int J Phytoremediat 7:323–335CrossRefGoogle Scholar
  22. Cherian S, Oliveira MM (2005) Transgenic plants in phytoremediation: recent advances and new possibilities. Environ Sci Technol 39:9377–9390CrossRefPubMedGoogle Scholar
  23. Cluis C (2004) Junk-greedy greens: phytoremediation as a new option for soil decontamination. Bio Teach J 2:61–67Google Scholar
  24. Coinchelin D, Bartoli F, Robin C, Echevarria G (2012) Ecophysiology of nickel phytoaccumulation: a simplified biophysical approach. J Exp Bot 63:5815–5827CrossRefPubMedGoogle Scholar
  25. Deng T, Tang Y, van der Ent A, Sterckeman T, Echevarria G, Morel JL, Qiu RL (2016) Nickel translocation via the phloem in the hyperaccumulator Noccaea caerulescens (Brassicaceae). Plant Soil 404:35–45CrossRefGoogle Scholar
  26. Durand A, Piutti S, Rue M, Morel JL, Echevarria G, Benizri E (2016) Improving nickel phytoextraction by co-cropping hyperaccumulator plants inoculated by plant growth promoting rhizobacteria. Plant Soil 399(1–2):179–192CrossRefGoogle Scholar
  27. van der Ent A, Baker AJM, Reeves RD, Chaney RL (2015) Agromining: farming for metals in the future? Environ Sci Technol 49:4773–4780CrossRefPubMedGoogle Scholar
  28. Greipsson S (2011) Phytoremediation Nat Educ Knowl 3(10), 7Google Scholar
  29. Kachenko AG, Singh B (2006) Heavy metals contamination in vegetables grown in urban and metal smelter contaminated sites in Australia. Water Air Soil Pollut 169(3):101–123CrossRefGoogle Scholar
  30. Kazakou E, Adamidis GC, Baker AJM, Reeves RD, Godino M, Dimitrakopoulos PG (2010) Species adaptation in serpentine soils in Lesbos Island (Greece): metal hyperaccumulation and tolerance. Plant Soil 332:369–385CrossRefGoogle Scholar
  31. Krämer U (2010) Metal hyperaccumulation in plants. Annu Rev Plant Biol 61:517–534CrossRefPubMedGoogle Scholar
  32. Li YM, Chaney RL, Brewer E, Roseberg RJ, Angle JS, Baker AJM, Reeves RD, Nelkin J (2003) Development of a technology for commercial phytoextraction of nickel: economic and technical considerations. Plant Soil 249:107–115CrossRefGoogle Scholar
  33. Li JT, Liao B, Lan CY, Ye ZH, Baker AJM, Shu WS (2010) Cadmium tolerance and accumulation in cultivars of a high-biomass tropical tree (Averrhoa carambola) and its potential for phytoextraction. J Environ Qual 39:1262–1268CrossRefPubMedGoogle Scholar
  34. Macek T, Kotrba P, Svatos A, Novakova M, Demnerova K, Mackova M (2008) Novel roles for genetically modified plants in environmental protection. Trends Biotechnol 26:146–152CrossRefPubMedGoogle Scholar
  35. Mahar A, Wang P, Ali A, Awasthi MK, Lahori AH, Wang Q, Li R, Zhang Z (2016) Challenges and opportunities in the phytoremediation of heavy metals contaminated soils: a review. Ecotoxicol Environ Saf 126:111–121CrossRefPubMedGoogle Scholar
  36. Milic D, Lukovic J, Ninkov J, Zeremski-Skoric T, Zoric L, Vasin J, Milic S (2012) Heavy metal content in halophytic plants from inland and maritime saline areas. Cent Eur J Biol 7:307–317Google Scholar
  37. Moon JW, Moon HS, Woon NC, Hahn JS, Won JS (2000) Evaluation of heavy metal contamination and implication of multiple sources from Hunchun basin, northeastern China. Environ Geol 39(9):1039–1052CrossRefGoogle Scholar
  38. Nkrumah PN, Baker AJM, Chaney RL, Erskine PD, Echevarria G, Morel JL, van der Ent A (2016) Current status and challenges in developing nickel phytomining: an agronomic perspective. Plant Soil 406(1–2):55–69CrossRefGoogle Scholar
  39. R Core Team (2016) R: a language and environment for statistical computing. Foundation for Statistical Computing, ViennaGoogle Scholar
  40. Rafati M, Khorasani N, Moattar F, Shirvany A, Moraghebi F, Hosseinzadeh S (2011) Phytoremediation potential of Populus alba and Morus alba for cadmium, chromuim and nickel absorption from polluted soil. Int J Environ Res 5:961–970Google Scholar
  41. Reeves RD, Baker AJM, Kelepertsis A (1997) The distribution and biogeochemistry of some serpentine plants of Greece. In: Jaffre T, Reeves RD, Becquer T (eds) Ecologie des milieux sur roches ultramafiques et sur sols metalliferes. ORSTOM, Noumea, pp 205–207Google Scholar
  42. Reeves RD, Baker AJM (2000) Metal-accumulating plants. In: Raskin I, Ensley BD (eds) Phytoremediation of toxicmetals. Wiley, New York, pp 193–229Google Scholar
  43. Robinson BH, Brooks RR, Howes AW, Kirkman JH, Gregg PEH (1997a) The potential of the high-biomass nickel hyperaccumulator Berkheya coddii for phytoremediation and phytomining. J Geochem Explor 60:115–126CrossRefGoogle Scholar
  44. Robinson BH, Chiarucci A, Brooks RR, Petit D, Kirkman JH, Gregg PEH, De Dominicis V (1997b) The nickel hyperaccumulator plant Alyssum bertolonii as a potential agent for phytoremediation and phytomining of nickel. J Geochem Explor 59:75–86CrossRefGoogle Scholar
  45. Robinson BH, Brooks RR, Clothier BE (1999) Soil amendments affecting nickel and cobalt uptake by Berkheya coddii: potential use for phytomining and phytoremediation. Ann Bot-London 84:689–694CrossRefGoogle Scholar
  46. Robinson BH, Anderson CWN, Dickinson NM (2015) Phytoextraction: where’s the action? J Geochem Explor 151:34–40CrossRefGoogle Scholar
  47. Singer AC, Bell T, Heywood CA, Smith JAC, Thompson IP (2007) Phytoremediation of mixed-contaminated soil using the hyperaccumulator plant Alyssum lesbiacum: evidence of histidine as a measure of phytoextractable nickel. Environ Pollut 147:74–82CrossRefPubMedGoogle Scholar
  48. Strid A, Tan K (2002) Flora Hellenica, vol 2. A.R.G. Gantner Verlag KG, RuggellGoogle Scholar
  49. Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walters SM, Webb DA (1964-1980) Flora Europaea. Cambridge University Press, CambridgeGoogle Scholar
  50. US Environmental Protection Agency (2007) Method 3051A, microwave assisted acid digestion of sediments, Sludges, soils, and oils, revision 1. Test Methods for Evaluating Solid Waste, USEPA Washington DCGoogle Scholar
  51. Zhang X, Houzelot V, Bani A, Morel JL, Echevarria G, Simonnot M-O (2014) Selection and combustion of nickel-hyperaccumulators for the phytomining process. Int J Phytoremed 16:1058–1072CrossRefGoogle Scholar
  52. Zhang X, Zhong T, Liu L, Ouyang X (2015) Impact of soil heavy metal pollution on food safety in China. PLoS One 10(8):e0135182CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2017

Authors and Affiliations

  • George C. Adamidis
    • 1
  • Maria Aloupi
    • 2
  • Petros Mastoras
    • 1
  • Maria-Ioanna Papadaki
    • 1
  • Panayiotis G. Dimitrakopoulos
    • 1
  1. 1.Biodiversity Conservation Laboratory, Department of EnvironmentUniversity of the AegeanMytileneGreece
  2. 2.Water and Air Quality Laboratory, Department of EnvironmentUniversity of the AegeanMytileneGreece

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