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Pteris melanocaulon Fée is an As hyperaccumulator

Rene Juna R. Claveria Ateneo de Manila University, [email protected]

Teresita R. Perez Ateneo de Manila University, [email protected]

Mary Jean B. Apuan Xavier University

Dennis A. Apuan Mindanao University of Science and Technology

Rubee Ellaine C. Perez University of the Philippines Los Banos

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Recommended Citation Claveria, R. J. R., Perez, T. R., Apuan, M. J. B., Apuan, D. A., & Perez, R. E. C. (2019). Pteris melanocaulon Fée is an As hyperaccumulator. Chemosphere, 236, 124380.

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Short Communication Pteris melanocaulon Fee is an As hyperaccumulator

* Rene Juna R. Claveria a, , Teresita R. Perez a, b, Mary Jean B. Apuan c, Dennis A. Apuan d, Rubee Ellaine C. Perez e a Department of Environmental Science, Ateneo de Manila University, Quezon City, Philippines b Institute of Biology, University of the Philippines, Diliman, Quezon City, Philippines c Xavier University, Cagayan de Oro City, Philippines d Department of Environmental Science and Technology, Mindanao University of Science and Technology, Cagayan de Oro City, Philippines e National Institute of Molecular Biology and Biotechnology, University of the Philippines at Los Banos,~ Laguna, Philippines highlights

P. melanocaulon thrives very well in some CueAu mines in the Philippines. P. melanocaulon is a Cu phytostabilizer and a newly discovered As hyperaccumulator. P. melanocaulon has similar As TF values as P. vittata and P. calomelanos. article info abstract

Article history: Pteris melanocaulon is noted to thrive very well in selected CueAu mines in the Philippines such as the Received 26 April 2019 Carmen Cu mine in Cebu and the Manila Mining CueAu mine in Surigao. Soil and plant samples were Received in revised form collected from field surveys as well as potted experiments. The computed bioaccumulation factor (BF) 3 July 2019 and translocation factor (TF) values from the analyses of As contents of the soil and the belowground and Accepted 15 July 2019 above ground components of the plant, apparently were comparable to known As hyperaccumulators Available online 20 July 2019 Pteris vittata and Pityrogramma calomelanos. It is inferred with probability that the factors that influence Handling Editor: T Cutright the bioavailability of As as well as the uptake mechanisms for P. melanocaulon would be similar to the 2 known As hyperaccumulators. Previous works have noted P. melanocaulon as a very good Cu accumulator Keywords: with very high Cu values in the belowground components and this study further identified it to be an Arsenic excellent As hyperaccumulator with high As values in the aboveground components. Hyperaccumulator © 2019 Elsevier Ltd. All rights reserved. Ferns Bioaccumulation Translocation

1. Introduction is > 1.00 indicating preferential partitioning of metals to the shoot (Baker and Whiting, 2002). The process by which metals from the Hyperaccumulators are conventionally defined as species ground are taken up by plants is called phytoextraction (Anderson capable of accumulating metals at levels 100-fold greater than et al., 1999). The success of phytoextraction depends on the those typically measured in non-accumulator plants (Baker, 2008; bioavailability of metals in the soil and the capacity of plants to Lasat, 2002). The ultimate confirmation of hyperaccumulator sta- accumulate the metals (Japenga et al., 2007). There are a number of tus is based on the following: (a) the plant should be tolerant to ferns identified as As hyperaccumulators and has As accumulated extremely high concentrations of bioavailable metal while values ranging from 1000 to >2000 mg kg 1. These are Pity- remaining healthy, (b) the metal accumulation is in its shoot or leaf rogramma calomelanos, Pteris vittata, Pteris cretica, Pteris longifolia, tissues in exceptional concentrations relative to other species; and Pteris umbrosia, Pteris biaurita L, Pteris quadriaurita Retz and Pteris (c) the shoot/root or leaf/root quotient for metal concentration ryukyuensis Tagawa to name a few (Rathinasabapathi et al., 2006; Srivastava et al., 2006). The occurrences of Pteris melanocaulon are rather limited to some CueAu mining areas in the provinces of Cebu and Surigao. * Corresponding author. E-mail address: [email protected] (R.J.R. Claveria). Clusters of P. melanocaulon thrive very well along open slopes https://doi.org/10.1016/j.chemosphere.2019.124380 0045-6535/© 2019 Elsevier Ltd. All rights reserved. 2 R.J.R. Claveria et al. / Chemosphere 236 (2019) 124380 particularly in open pit mines. Along the open slopes are soils enriched with Cu and other elements such as As and other base metals being products of weathering of the exposed CueAu ores. A previous study on the P. melanocaulon growing in an open pit mine in Surigao indicated the efficient capability of the plant in accu- mulating Cu into the belowground (BG) components particularly the root system than translocating the Cu into the aboveground (AG) components such as the stem and leaves thus classifying it to be a phytostabilizer (De la Torre et al., 2014). There are a number of fern species that were also identified as Cu phytostabilizers with their roots containing higher amounts of Cu than the shoots. Ex- amples of these are Dennstaedtia davallioides and Hypolepis muelleri both of the Family Dennstaedtiaceae, Nephrolepis cordifolia of the Family Nephrolepidaceae (Kachenko et al., 2006), Pityrogramma calomelanos of the Family Pteridaceae (Dahilan and Dalagan, 2017) and Athyrium yokoscense of the Family Athyriaceae (Nishizono et al., 1987). Being an indigenous fern and grows well in metal enriched soils, there was a need to assess further the extraction efficiency of P. melanocaulon not for Cu but for other elements such as As. The objective of the study is to determine whether P. melanocaulon being an efficient Cu accumulator is also an As hyperaccumulator.

2. Materials and methods Fig. 1. a) Geographic location of the different mining study sites in the provinces of Benguet, Cebu and Surigao. b), c), d) and e) the sampling sites at Carmen Cu mine in Field surveys were done in selected CueAu mines in the prov- Cebu. f), g), h), i) and j) sampling sites at Manila Mining CueAu mine in Surigao. inces of Benguet, Cebu and Surigao (Fig. 1). In Benguet, only Photographs of the open pit mines as well as P. melanocaulon and associated P. calomelanos. The ferns thrive very well in relatively Cu and As enriched soils. The P. vittata and P. calomelanos were observed to grow together while Philippine map was modified after De la Torre et al. (2014). in Cebu and Surigao, P. melanocaulon was noted to thrive very well with the 2 other ferns. Samples of P. melanocaulon as well as P. vittata and P. calomelanos were gathered taking into consider- potential of P. melanocaulon with those of P. vittata and ation the roots, stem and leaves of the plants as well as the soil from P. calomelanos. which the plants have grown. Potted experiments on The results (data) of analyses were statistically processed using P. melanocaulon were also done following appropriate propagation Analysis of Variance (ANOVA) to determine significant differences protocols (e.g. Apuan et al., 2016). In the different setups, the or- of values among plant species. Significant differences were identi- dinary uncontaminated soils were conditioned with varying con- fied with r values < 0.05. With notable significant differences, the centrations of sodium arsenate solution (0, 500, 1000 and data were presented in boxplots to appreciate the differences and 2000 mg kg 1). After 5 growth weeks’ growth, no toxicity symp- distribution. The absolute values were transformed to logarithmic toms were observed from which representative samples of the values in order to appreciate the distribution of high and low plant and soil were gathered for analysis. The samples collected values. from the field surveys and potted experiments were subjected to appropriate sample preparation protocols (Kalra, 1989; Wei and Chen, 2006). The plant samples were discriminated as roots, to 3. Results and discussions represent the BG components and as stem and leaves to represent the AG components. Representative samples (of the soil and plant 3.1. Field survey components) were sent to chemical laboratories for As analyses. The analytical method in determining As content is ashing-acid In the Carmen Cu mine in Cebu and in the Manila Mining CueAu digestion/hydride vapor generation Atomic Absorption Spectro- mine in Surigao, P. melanocaulon thrived relatively well along open- photometry (Association of Official Analytical Chemist Analysis pit benches and walls and abandoned tailings ponds overlain by (AOAC) AOAC International, 2012). soils derived from the weathering of mineralized host rocks. The From the results of the As analysis, the Bioaccumulation Factor soils contain As values ranging from a low 2.23 mg kg 1 to a high (BF) and the Translocation Factor (TF) were calculated using the 100 mg kg 1 with an average of about 29.36 ± 26.75 mg kg 1. Other ratio between plant and soil (BF ¼ Plant/Soil) and the ratio between indigenous ferns such as P. vittata and P. calomelanos were noted to the AG and BG components (TF ¼ AG/BG) (Claveria et al., 2019; Wei grow and flourish with P. melanocaulon in the different mine sites. and Chen, 2006). For plants to show capability of extracting As, they The As values in the BG components of P. melanocaulon ranged should manifest computed TF values > 1.00 indicating that the AG from 1.10 to 20.20 mg kg 1 with an average of 8.70 ± 5.01 mg kg 1 values are higher than the BG values (Reichman, 2002). and the AG values ranged from 1.30 to 149.20 mg kg 1 with an In appreciating the hyperaccumulation potential of average of 34.60 ± 40.54 mg kg 1 (Table 1). Similarly the As values P. melanocaulon, a comparative study was made with known As in the BG and AG components of P. vittata ranged from 4.09 to hyperaccumulator ferns such as P. vittata and P. calomelanos found 521.67 mg kg 1 (244.26 ± 231.21) and 80.10e722.33 mg kg 1 to co-exist with P. melanocaulon and also thrive very well in the (416.36 ± 251.59) respectively. For P. calomelanos, the As values in Carmen Cu mine in Cebu and in the Manila Mining CueAu mine in the BG and Ag components ranged from 5.16 to 548.33 mg kg 1 Surigao. In order to have a general view of the As extraction (143.68 ± 154.50) and 51.40e1386.67 mg kg 1 (374.73 ± 363.15) capability of P. vittata and P. calomelanos, samples from some respectively. The As values of the BG and AG components of CueAu mines in Benguet were included. The purpose of this P. melanocaulon in comparison with those of P. vittata and comparative study is to confirm similarities on the extraction P. calomelanos, indicated significant differences (BG R.J.R. Claveria et al. / Chemosphere 236 (2019) 124380 3

F(2,39) ¼ 6.7826, r ¼ 0.0029; AG F(2,38) ¼ 7.1052, r ¼ 0.0024)) with 3.2. Potted experiments the As values of P. melanocaulon notably lower than those of P. vittata and P. calomelanos (Fig. 2a and b). To further appreciate the inferences made from the field sur- The implications of the BG and AG values are further appreci- veys, potted experiments were done using variable inputted ated with the determination of the BF and the TF values. The As BF amounts of As solution. A single type of soil was used in all of the values of P. melanocaulon ranged from 0.69 to 6.76 with an average experiments. The initial data on the biogeochemical composition of of about 2.62 ± 1.75 and the As TF values ranged from 0.21 to 21.97 the soil provided the following: texture (sandy loam), pH (6.8), with an average of about 5.05 ± 6.06. The computed average As BF cation exchange capacity (CEC) (35.4 meq/100 mg of soil), N value of P. melanocaulon (2.62 ± 1.75) in comparison with the other (310 mg kg 1), P (47.8 mg kg 1), K (872 mg kg 1), Cu (34.2 mg kg 1) ferns indicated significant differences (BF F(2,35) ¼ 5.7911, and As (3.2 mg kg 1). After 6 weeks of experiment, soil and plant r ¼ 0.0067) with the As BF values of P. melanocaulon notably lower samples from each set-up were analyzed for As content. The plant than the average As BF value of P. vittata (68.829 ± 110.077) and that samples analyzed were discriminated as BG components (e.g. of P. calomelanos (4.546 ± 5.189) (Fig. 2c). The higher As BF values of roots) and AG components (e.g. stem and leaves). The results of both P. vittata and P. calomelanos are consistent to previous studies analyses from the potted experiments are presented in Table 2. which actually classified both ferns as excellent As accumulators The results of the experimental As treatment for P. melanocaulon (Ma et al., 2001; Chen et al., 2002). It is noted from this study that provided insights on the capability of the fern in absorbing As into the average As BF value of P. melanocaulon is > 1.0 thus classifying it its anatomical parts at different As concentrations. The increased to be a good As accumulator. The computed average As TF values of increments of As concentration into the different set-ups appar- P. melanocaulon (5.052 ± 6.055) in comparison with the computed ently indicated proportional increases of As content in the soil. The averages of As TF values of P. vittata (4.798 ± 6.223) and increase in As treatments in the different set-ups also indicated P. calomelanos (4.910 ± 4.807) manifested no significant differences corresponding increases of As content in the BG and AG compo- of values (TF F(2,38) ¼ 0.0056, r ¼ 0.9944)). It was apparent that the nents of the plant. Interestingly the As accumulations by the AG computed TF values of P. melanocaulon are similar to values components in the different set-ups were significantly higher than exhibited by P. vittata and P. calomelanos. TF values that are the BG components as well as the soil. With increasing As treat- apparently > 1.00 manifest ferns to be efficient As hyper- ment, the computed As BF values apparently tend to decrease accumulators and these are consistent to studies on P. vittata and manifesting an exclusion mechanism of the plant to any increases P. calomelanos (Chen et al., 2002). Based from the data results of the of As making it tolerant to possible phytotoxicity (Fig. 2e). In all of field surveys, the As TF values of P. melanocaulon were noted to be the computed BF ratios, the values ranging from 2.828 to 4.434 are distinctly within the range of the As TF values for P. vittata and > 1.00 indicating that P. melanocaulon is an accumulator of As, with P. calomelanos thus it is inferred that P. melanocaulon is an As the plant having higher amounts of As than that of the soil. This is hyperaccumulator. consistent with the observations made in the field surveys. With increasing As treatment, the computed TF values apparently did not

Table 1 Results of analyses of samples taken during the field surveys. Most of the P. melanocaulon samples were taken from the Carmen Cu mine in Cebu and in the Manila Mining CueAu mine in Surigao. P. vittata and P. calomelanos were taken from 3 study sites which were in Benguet, Cebu and Surigao. The soil and the belowground (BG) and the aboveground (AG) components of the plants were analyzed for As (values in mg kg 1). With the determined values, the bioaccumulation factor (BF) and translocation factor (TF) were computed.

Sample site Arsenic

P. vittata P. calomelanos P. melanocaulon

Soil BG AG BF TF Soil BG AG BF TF Soil BG AG BF TF

Benguet 53.70 175.00 3.26 13.70 15.50 106.00 8.87 6.84 4.75 504.30 642.00 241.33 1.27 33.83 12.67 113.00 3.71 8.92 4.75 504.30 531.00 217.96 1.05 60.67 64.67 377.33 7.29 5.84 25.80 4.09 80.10 3.26 19.61 307.00 208.33 583.00 2.58 2.80 188.00 521.67 633.33 6.14 1.21 59.67 36.00 301.67 5.66 8.38 645.00 246.33 722.33 1.50 2.93 138.73 210.17 1.51 113.33 94.33 327.00 3.72 3.47 253.33 95.67 226.00 1.27 2.36 3158.50 215.33 467.33 0.22 2.17 1243.33 386.67 460.33 0.68 1.19 548.33 1386.67 2.53 2072.67 473.33 1227.33 0.82 2.59 240.00 112.67 562.00 2.81 4.99 48.00 76.00 267.00 7.15 3.51 Cebu 4.01 5.16 86.90 22.96 16.84 4.88 1.83 5.96 1.60 3.26 2.23 1.10 14.00 6.76 12.73 Surigao 46.60 58.40 159.10 4.67 2.72 67.70 41.60 147.20 2.79 3.54 16.90 20.20 11.70 1.89 0.58 64.67 61.33 388.00 6.95 6.33 49.00 81.90 51.40 2.72 0.63 22.80 13.20 78.00 4.00 5.91 209.33 464.67 2.22 18.90 7.60 45.90 2.83 6.04 81.90 51.40 0.63 16.80 8.63 15.70 1.45 1.82 25.00 33.67 43.67 3.09 1.30 100.00 6.79 149.20 1.56 21.97 64.67 14.00 244.67 4.00 17.48 16.67 11.00 40.00 3.06 3.64 16.67 9.80 10.00 1.19 1.02 16.67 4.90 10.00 0.89 2.04 11.00 9.70 41.00 4.61 4.23 11.00 12.00 27.00 3.55 2.25 11.00 6.30 1.30 0.69 0.21 4 R.J.R. Claveria et al. / Chemosphere 236 (2019) 124380

Fig. 2. a) and b) are boxplots of the As contents in the BG and AG components respectively of P. melanocaulon in comparison to P. vittata and P. calomelanos for samples taken in the field surveys. ANOVA indicated significant differences of values for both components (As BG values: F(2,39) ¼ 6.7826, r ¼ 0.0029; As AG values: F(2,38) ¼ 7.1052, r ¼ 0.0024). c) and d) are boxplots of the computed As BF and AS TF values of the 3 ferns respectively. ANOVA indicated significant differences in the As BF values (F(2,35) ¼ 5.7911, r ¼ 0.0067) while no significant differences in the As TF values (F(2,38) ¼ 0.0056, r ¼ 0.9944). e) and f) are boxplots of the computed As BF and As TF values of P. melanocaulon taken from the potted experiments. The values were logarithmically transformed. exhibit any decrease similar to the BF values indicating the capa- that with higher amounts of As in the soils, sufficient and tolerable bility of P. melanocaulon to efficiently absorb and store As into its AG increases in the amount of As absorbed by the plants are expected components (Fig. 2f). The computed TF values which ranged from and are either taken up by the plants into their AG components or 4.463 to 5.818 are > 1.00 indicating that P. melanocaulon is an are immobilized in the plant roots (Bohn et al., 1979; Ma et al., extractor of As with AG values greater than that of the BG com- 2001; Wang et al., 2002; Kertulis et al., 2005; Nouri et al., 2009; ponents. This is also consistent to observations made during the Claveria et al., 2019). The As accumulation by P. melanocaulon in field surveys. Considering the significance of the computed BF and the potted experiments also indicated a similar trend of increasing TF values, it is interpreted that P. melanocaulon is an efficient As As accumulation by the plant from 601.44 to 1984.72 mg kg 1with accumulator and hyperaccumulator. the increase of As treatment of the soil from 500 to 2000 mg kg 1. The pH of soils is another factor that could influence As uptake 3.3. As hyperaccumulation affecting particularly the solubility of As and thus the bioavailability of As. The soils in the Manila Mining CueAu mine in Surigao are With P. melanocaulon classified as an As hyperaccumulator, it slightly acidic and the low pH of the soil may have provided the was interesting to identify the possible factors that could influence acidic conditions for As to be in solution and bioavailable for plant the uptake of As into the anatomical parts of the plant. One factor absorption (John and Leventhal, 1995). Another factor could be the could be the amount of As in the soil from which the amounts of heavy metal (e.g. Cu) contents in the soil which may also have an accumulated As in the plants could be in proportion with (John and effect on As accumulation by the plants. It was noted that with low Leventhal, 1995; Cao et al., 2004; Claveria et al., 2019). This meant concentrations of Cu in the soil, the uptake of As by plants is

Table 2 Results of analysis of the potted experiments. The data provided are average values (with standard deviation) for 3 trials. The samples analyzed were soil and the belowground (BG) and above ground (AG) components of the plants (values in mg kg 1). Computations were made for the bioaccumulation and translocation factors (BF and TF).

Plant species Arsenic

P. melanocaulon Soil BG AG BF TF 0 9.706 ± 3.382 11.106 ± 0.832 17.503 ± 5.761 2.947 ± 1.036 1.576 ± 0.4 500 135.626 ± 22.026 92.151 ± 7.913 509.296 ± 95.199 4.434 ± 1.323 5.526 ± 1.462 1000 303.056 ± 34.39 164.535 ± 16.227 957.413 ± 170.684 3.702 ± 0.833 5.818 ± 0.68 2000 701.626 ± 106.353 363.26 ± 73.966 1621.46 ± 364.94 2.828 ± 0.354 4.463 ± 0.299 R.J.R. Claveria et al. / Chemosphere 236 (2019) 124380 5 positively affected (Fayiga and Saha, 2016). The apparent accumu- understanding metal hyperaccumulation. New Phytol. 155, 1e4. ’ lation of Cu into the roots may actually increase the uptake of As to Bohn, H.L., McNeal, B.L., O Connor, G.A., 1979. Soil Chemistry. John Wiley and Sons, Inc, NY. the aboveground components of the plant. Kader et al. (2018) Cao, X., Ma, L.Q., Tu, C., 2004. Antioxidative responses to arsenic-hyperaccumulator indicated that the As uptake of plants is significantly increased Chinese brake fern (Pteris vittata L.). Environ. Pollut. 128, 317e325. with the minimal presence of Cu and similarly the Cu accumulation Chen, T.C., Wei, C., Huang, A., Huang, Q., Lu, Q., Fan, Z., 2002. Arsenic hyper- fi fl accumulator Pteris vittata L. and its arsenic accumulation. Chin. Sci. Bull. 47, is not signi cantly in uenced by As. With the minimal presence of 902e905. Cu in the above ground components, it is of likelihood that As Claveria, R.J.R., Perez, T.R., Perez, R.E., Algo, J.L., Robles, P.Q., 2019. The identification would accumulate. of indigenous Cu and as metallophytes in the Lepanto Cu-Au Mine, Luzon, Philippines. Environ. Monit. Assess. 191, 185. Dahilan, J.K.A., Dalagan, .J.Q., 2017. Bioavailability and accumulation assessment of 4. Conclusions and recommendations copper in Pityrogramma calomelanos. Philipp. J. Sci. 146, 331e338. September. De la Torre, J.B., Claveria, R.J.R., Perez, R.E.C., Perez, T.R., Doronila, A.I., 2014. Copper uptake by Pteris melanocaulon Fee from a Copper-Gold mine in Surigao del P. melanocaulon is presented in this study in a rather different Norte, Philippines. Int. J. Phytoremediation 18 (5), 434e441. classification where it is an excellent accumulator of Cu particularly Fayiga, A.O., Saha, U.K., 2016. Arsenic hyperaccumulating fern: implications for by the BG components and is also an excellent accumulator of As by remediation of arsenic contaminated soils. Geoderma 284, 132e143. the AG components. In comparison with known As hyper- Japenga, J., Koopmans, G., Song, J., Romkens, P., 2007. A feasibility test to estimate the duration of phytorextraction of heavy metals from polluted soils. Int. J. accumulators such as P. vittata and P. calomelanos, the computed As Phytoremediation 9 (2), 115e132. TF values manifested no significant differences in values between John, D.A., Leventhal, J.S., 1995. Bioavailability of metals, ch. 2. In: du, Bray (Ed.), the 3 fern species. In addition to the initial work of De la Torre et al. Preliminary Compilation of Descriptive Geoenvironmental Mineral Deposit Models. US Department of the Interior. US Geological Survey Open-File Report (2014) on the Cu accumulation of P. melanocaulon, this paper has 95e831, Denver, Colorado. presented data and has provided interpretation on the As accu- Kachenko, A.G., Singh, B., Bhatia, N.P., 2006. Heavy metal tolerance in common fern e mulation potential of the fern. This study confirms the identifica- species. Aust. J. Bot. 55 (1), 63 73. Kader, M., Lamb, D.T., Wang, L., Megharaj, M., Naidu, R., 2018. Copper interaction on tion of P. melanocaulon as an As hyperaccumulator. It is highly arsenic bioavailability and phytotoxicity in soil. Ecotoxicol. Environ. Saf. 148, recommended that further research studies be done in determining 738e746. the As uptake mechanisms of P. melanocaulon as well as the factors Kalra, Y.P. (Ed.), 1989. Handbook of Reference Methods for Plant Analysis. CRC Press fl Taylor and Francis Group, New York. that in uence the phytoavailability of As. Kertulis, G.M., Ma, L.Q., Macdonald, G.E., Chen, R., Winefordner, J.D., Cai, Y., 2005. Arsenic speciation and transport in Pteris vittata L. and effects on phosphorus in Acknowledgements the xylem sap. Environ. Exp. Bot. 54, 239e247. Lasat, M.M., 2002. Phytoextraction of toxic metals: a review of biological mecha- nisms. J. Environ. Qual. 31, 109e120. The authors would like to thank the following: DOST-PCIEERD Ma, L.Q., Komar, K.M., Tu, C., Zhang, W., Cai, Y., Kennelley, E.D., 2001. A fern that for the extended financial support for the research and mining hyperaccumulates arsenic. Nature 409, 579. companies such as Lepanto, Carmen Copper, Manila Mining and Nishizono, H., Suzuki, S., Ishii, F., 1987. 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