当前世界环境

Introduction

Natural environment, particularly the aquatic ecosystem is being disturbed by anthropogenic activities with rise in industrialization and urbanization. The decline in water quality of fresh water systems threatens its sustainability and has become one of the major environmental issues. The current scenario of Indian freshwater resources has been studied by many workers and appealed for their management as environmental problems. The increased concentrations of heavy metals in receiving water, especially lakes and rivers worsened the situation and threatened the biological components through bioaccumulation along the food chain and ultimately affect human being as well^[1-5].

Zn which is an essential micro nutrient associated with metabolic activities in organisms can turn toxic at higher concentrations. Its concentration in various Indian aquatic ecosystems has reached alarming levels as reported by a number of scientific studies^[4,6-9]. The main sources of Zn in fresh water ecosystems includes effluents from electroplating industries, smelting and refining, mining, paper industries, domestic sewage and agricultural run-off^[10]. The immersion of idols and tazias is also one of the major reasons for increase level of Zn in lakes and rivers all over India^[11-13]. Zn is one of the major constituents of paints for decorating both idols and tazias, and it has been reported that the number of idol immersion is being increased each year, which could increase load of heavy metals^[14].

Although Zn is a micro nutrient, it becomes hazardous at high levels^[15]. The growing concern in environmental aspects as well as the narrow window between its essentiality and toxicity^[16]has generated interest in studying its effects on aquatic plants which play an important role in ecosystem functioning. In addition, a regular testing of Zn pollution in fresh water is required in the Indian context. Thus, the present study was carried out with the aim of exploring the potential of an aquatic macrophyte,Ipomoea aquaticaForsk. to be used in bio monitoring and phytotoxicity studies. This plant was selected on the basis of its geographical distribution, availability and adaptability. It is widely distributed not only in India, but in the entire South, South East, and East Asia^[17]and well adapted to wide range of habitats. It is also reported to be grown in other parts of the world like Africa, Australia and United States of America (Austin, 2007)^[18]. Further, it is perennial and mostly grows in moist soil, inundated floodplains, ditches, ponds, canals and sluggish rivers^[19]and easy to cultivate due to its ability to proliferate by fragmentation, and produce adventitious roots and lateral branches that bear flowers and leaves, from its nodes^[20].

A number of studies have been conducted on Zn toxicity using aquatic plants. Growth inhibition was observed inEichhornia crassipes^[21],Lemna gibba^[22],Phragmites australis^[23],Iris psuedacarus^[24],Spirodela polyrhiza^[25]andSalvinia natans^[26]. Another study reported the inhibition of growth induced by Zn stress in three aquatic macrophytes viz.Lemna minor,Elodea canadensisandLeptodictyum riparium, whereL. ripariumturned out to be the most resistant species with 50% growth inhibition at 100 µM Zn^[27]. In contrast to this, studies on Zn toxicity toA. aquaticais scarce. This study was, therefore, taken up to understand and assess the toxic effects of Zn on this aquatic macrophyte.

Materials and Methods

Ipomoea aquaticaForsk. was collected from uncontaminated ponds of Irongmara area in Cachar district, Assam, India, and washed with tap water. Stock cultures were grown following standard method^[28]. The plants were grown in hydroponic tubs till new branches developed. These new branches were cut and planted in pots containing soil flooded with 50 % Hoagland nutrient media. The pH of nutrient media was adjusted at the range of 5.8-6.2. Healthy and fully grown shoots of similar shoot height were cut from the same mother plant, washed with tap water and acclimatized in 50 % Hoagland nutrient media for one week at 25-27°C, 12 h light with an intensity of 100-120 µmol^-2s^-1and 12 h dark periods. These acclimatized plants were exposed to half strength Hoagland nutrient media added with graded concentrations of Zn as ZnSO₄. 7H₂O (actual Zn concentrations: 0.23 mg L^-1, 2.27 mg L^-1, 4.09 mg L^-1, 7.26 mg L^-1, 12.71 mg L^-1and 22.7 mg L^-1) for 15 days. Plants grown in 50 % Hoagland media without added Zn served as control. Water loss due to evaporation or transpiration was compensated by renewal of solutions every week. At the termination of the experiment on 15^thday all control and Zn-treated plants were removed, washed with distilled water, and softly blotted to remove excess water before fresh weight of the plants, their root length and lateral roots, and leaf area were measured. This was followed by drying in hot air oven at 60⁰C至恒重。其他生长参数苏ch as shoot length, new leaves, and number of nodes were measured at 3 day intervals. The percentage of primary roots bearing lateral roots (PRL %), and the ratio of lateral roots (LR) to the number of primary roots bearing lateral roots (PRL) was calculated. All these data were also obtained before the plants were exposed to Zn. Leaf area was measured by using ImageJ (http://imagej.nih.gov/ij/) software. All leaves were neatly clipped at their petioles and properly spread to take the image of the entire leaf area. Toxicity symptoms on leaf and root, such as darkening of roots and appearance of chlorosis were noted during the experiment. For chlorophyll estimation, fresh leaf was homogenized with 80 % acetone, centrifuged, and the absorbance of the supernatant was taken at 662, 645, and 470 nm for chlorophyll a (chl a), chlorophyll b (chl b) and total carotene, respectively, in a spectrophotometer. The concentrations of these pigments were calculated following standard formula^[29]with the extraction solution used as blank.

Statistical significance of differences among the data sets was determined with One-way ANOVA, with multiple comparisons made by Tukey test. All tests were done using SPSS 20 software for Windows.

Results and Discussion

Plants at all concentrations of Zn could survive the total exposure period of 15 days, although some toxicity symptoms were encountered in plants exposed to higher concentrations of Zn. Thus the survival pattern of the plant reflected its tolerance capacity up to Zn concentrations of 22.7 mg L^-1. Among aquatic macrophytes,Typha agustifoliaandColocasia esculentawere found to be the most tolerant plants to heavy metals^[30]. These two plants did not show any significant toxicity symptoms when they were grown in sediment contaminated with Zn concentration of 363 mg kg^-1.Eichhornia crassipeswhen exposed to 20 mg L^-1Zn did not show any morphological symptom of toxicity^[31]. The present study revealed that plants treated at 22.7 mg L^-1showed yellowing of older leaves (Fig. 1), while significant blackening of root tips occurred at 22.7 mg L^-1Zn on the 3^rdday of exposure (Fig. 2).

Figure 1: Zn induced change in appearance of leaf inI. aquatica; A- Non chlorotic leaf (NCL) of control; B- Chlorotic leaf CL) of plant treated at 22.7 mg L-1Zn. Click here to View figure

Figure 2: Zn induced darkening of root inI. aquatica; A- Normal root(NR) of control; B- Affected root (AR) of plant treated at 22.7 mg L-1Zn. Click here to View figure

Blackening of root tips was also observed in plants treated at other concentrations with longer exposure time (Table 1). One way ANOVA showed significant reduction in length of PR at 7.26 - 22.7 mg L^-1Zn (Table 1) which might be correlated to gradual decaying of roots along the length. Zn at 200 µg ml^-1reduced root biomass inIris psuedacorus^[24]. However, symptoms like blackening and decaying of roots were not reported, although plants exposed to other heavy metals like Cd showed browning of root tips^[32]. Dark root tips inVicia faba^[33]and cell death in root tissues ofTalinum triangulare^[34]were also reported due to oxidative stress induced by Pb accumulation. Thus, these symptoms of toxicity in roots can be used as a tool in biomonitoring of Zn pollution in water. The suppression of development of new PR led to a significant reduction in the PRL % as well as lateral roots in plants treated at 12.71 – 22.7 mg L^-1Zn (Table 2). A significant reduction at p < 0.05 in LR: PRL was also observed in plants treated with 7.26 – 22.7 mg L^-1Zn at the end of 15 day exposure as revealed by one way ANOVA and multiple comparisons by Tukey test (Table 2). Zn at concentrations of 0.23 and 2.27 mg L^-1, on the other hand, enhanced growth of roots inI. aquatica.

Table 1: Appearance of darkening of roots inI. aquaticaon exposure to graded concentrations of Zn (n=5)

Zn concentration (mg L-1)	Number of plants with darkened roots
	Day 3	Day 6	Day 9	Day 12	Day 15
0.23	0	0	0	1	1
2.27	0	0	0	1	1
4.09	0	1	2	2	2
7.26	0	1	2	3	3
12.71	1	2	2	4	5
22.7	3	5	5	5	5

Table 2: Effects of Zn on root length, number of PR, PRL % and LR:PRL ofI. aquaticaat the end of 15 days.

Zn concentration (mg L-1)	Increase in length of PR (cm)	No. of new PR	PRL %	LR:PRL
Control	23.77±2.43ab	15.67±2.67a	53.26±14.77abc	5.81±1.33a
0.23	39.23±9.68a	16.33±1.33a	68.88±6.20ab	5.00±0.25a
2.27	32±5.04ab	19.33±2.40ab	72.14±10.90a	3.49±0.60ab
4.09	20.7±2.92b	24±1.15b	40.09±11.25abc	2.92±0.79ab
7.26	3.33±4.08c	9.67±1.45c	38.15±5.08bc	0.15±0.88bc
12.71	-1.97±4.22c	5±1.73cd	32.81±8.88c	-1.20±1.69c
22.7	-4.73±1.17c	1.67±1.20d	-20.21±11.40d	-1.92±1.05c

LR – Lateral roots; PR – Primary roots; PRL % - percentage of primary roots bearing lateral roots; Values are given as mean±SE; Values with different superscript letters in the column indicate significant differences at p < 0.05; ‘-’Decrease in each parameter with respect to the corresponding initial value

Despite the toxic symptoms exhibited byI. aquatica, growth in terms of fresh weight and dry weight was not affected at Zn concentrations of 12.71-22.7 mg L^-1, while 0.23-7.264.09 mg L^-1Zn enhanced growth (Fig. 3 & 4). On the other hand, one way ANOVA with multiple comparisons by Tukey tests showed that Zn concentrations of 7.26 – 22.7 mg L^-1had significant effect on number of new nodes (Fig. 4). There was also decline in increase in shoot length with significant effect at 22.7 mg L^-1Zn as revealed by one way ANOVA at p < 0.05. Zn being a micronutrient probably enhanced photosynthesis and protein metabolism, thus increasing the growth of plants at low level of Zn^[35]. Growth ofSesuvium portulacastrumwas enhanced by Zn concentrations up to 300 mg kg^-1[36]. On the other hand, Zn at 2mM concentration could decrease shoot height substantially inPhragmites australis^[23]. In another study, growth ofHydrilla verticillatawas slightly affected by 0.1 and 1 mg L^-1ZnO nano particle treatment for 3 weeks while 1000 mg L^-1significantly reduced growth of the plant^[37]. Thus, there is a wide variation in response to heavy metals among plants^[38,39].

Figure 3: Effects of Zn on fresh and dry weight ofI. aquaticaat the end of 15 days exposure;Values are given as mean±SE; Values with different superscript letters indicate significant differences at p < 0.05 Click here to View figure

Figure 4: Effects of Zn on increase in shoot length and number of nodes inI.aquaticaat the end of 15 days exposure; Values are given as mean±SE; Values with different superscript letters indicate significant differences at p < 0.05 Click here to View figure

The results of the present study revealed that Zn at low concentrations induced new leaf development as well as increased leaf area inI. aquatica, while at higher concentrations it affected leaf growth (Table 3). One way ANOVA showed significant differences in number of new leaf as well as leaf area among control and 22.7 mg L^-1Zn treated plants at p < 0.05. Similar results were also reported inSesuvium portulacastrumgrown in soil amended with 100 – 600 mg kg^-1Zn^[36]. High concentrations of Zn also enhanced shedding of older leaf with significantly higher number of leaf fall at 12.71 and 22.7 mg L^-1Zn from that in control. High Zn concentration resulted growth retardation and induces leaf senescence by disturbing key metabolic processes such as photosynthetic activity, pigment content and antioxidant systems^{[40, 41]}.

表3:锌的影响n leaf fall, new leaf and leaf area ofI. aquaticaat the end of 15 days

Zn concentration (mg L-1)	No. of leaf fallen	No. of new leaf	Total leaf area (cm2)
Control	1.33±0.33ab	3±0abd	29.30±4.3a
0.23	0.33±0.33a	2±0ad	33.46±3.65a
2.27	1.67±0.33ab	2.67±0.33abc	35.47±1.36a
4.09	1.67±0.67ab	3.67±0.33bc	32.37±7.03a
7.26	3±0.58bc	2.67±0.88cd	25.14±4.15a
12.71	3.67±0.33c	2.33±0.33d	17.72±2.99a
22.7	4.33±0.88c	0±0e	15.65±1.51b

Values are given as mean±SE; Values with different superscript letters in the column indicate significant differences at p < 0.05

The present study revealed that low Zn concentrations (0.23 and 2.27 mg L^-1) enhanced the content of chl a in leaf ofI. aquaticawhile it was significantly affected at 7.26 – 22.7 mg L^-1Zn concentrations on 5^thday of exposure as shown by one way ANOVA at p < 0.05. A dose and time – dependant reduction of chl a content was observed in subsequent days of exposure (Fig. 5). Similar results were also observed in case of chl b and total carotene content (Fig. 6 & 7). Chlorophyll and total carotene content can be considered as effective biomarkers since significant decline was obtained at 7.26 mg L^-1Zn after 5^thday of exposure. In addition, symptoms like leaf yellowing was not observed at this concentration till the end of the experiment. Significant reduction in chlorophyll content with leaf yellowing was also observed inPhragmites australistreated with 1000 mg L^-1ZnO nanoparticles^[37], although this dose is much higher than the dose of Zn which showed significant effect in the present study. Chl a, Chl b and carotenoid content inSalvinia natanswas also significantly reduced by Zn treatment at the level of 10 mg L^-1[26].

Figure 5: Effect of Zn on content of chl a in leaf ofI. aquaticaon day 5, 10 and 15 of exposure; Values are given as mean±SE; Values with different superscript letters indicate significant differences at p < 0.05 Click here to View figure

Figure 6: Effect of Zn on content of chl b in leaf ofI. aquaticaon day 5, 10 and 15 of exposure; Values are given as mean±SE; Values with differentsuperscript letters indicate significant differences at p < 0.05 Click here to View figure

Figure 7: Effect of Zn on content of total carotene in leaf ofI. aquaticaon day 5, 10 and 15 of exposure; Values are given as mean±SE; Values with different superscript letters indicate significant differences at p < 0.05 Click here to View figure

Thus, the results of this study revealed thatI. aquaticacan successfully be employed in toxicity studies in aquatic ecosystems polluted with Zn at a threshold limit of about 12 mg L^-1by using root browning, reduction of growth in root and shoot and pigment content as tools for biomonitoring. This plant being resistant to Zn in terms of survival capacity coupled with its adaptive nature to wide range of habitats, has the prospect of being used in further studies like phytoremediation of Zn polluted areas.

Acknowledgment

LBC would like to thank the University Grant Commission, New Delhi, India for financial assistance in the form of UGC-BSR Fellowship during the course of the study.

参考文献

1. Kumar, J.I.N., Soni, H. & Kumar, R.N., Biomonitoring of selected freshwater macrophytes to assess lake trace element contamination: a case study Nal Sarovar Bird Sanctuary, Gujarat, India.Journal of Limnology, 65(1): 9-16 (2006).
   CrossRef
2. Kaur, S. & Mehra, P., Assessment of heavy metals in summer & winter seasons in River Yamuna Segment flowing through Delhi, India.Journal of Environment and Ecology, 3(1): 149-165 (2012).
   CrossRef
3. Das, S., Mitra, A., Zaman, S., Pramanick, P., Chaudhuri, T.R. & Raha, A.T., Zinc, copper, lead and cadmium levels in edible finfishes from lower gangetic delta,American Journal of Bio-pharmacology Biochemistry and Life Sciences, 3(1): 8-19 (2014).
4. Gupta, S.K., Chabukdhara, M., Kumar, P., Singh, J. & Bux, F., Evaluation of ecological risk of metal contamination in river Gomti, India: A biomonitoring approach,Ecotoxicology and Environmental Safety, 110: 49-55 (2014).
   CrossRef
5. Bagul, V.R., Shinde, D.N., Chavan, R.P. & Patil, C.L., Causes and impacts of water pollution on rivers in Maharashtra- A review,Research Journal of Chemical and Environmental Sciences, 3(6): 1-4 (2015).
   CrossRef
6. 女王,一个Ramaiah, M, Harikrishna Khan, I. & Veena, K., Heavy metal pollution and chemical profile of Cauvery River water.E-Journal of Chemistry, 6(1): 47-52 (2009).
7. Subramanian, R., Nesakumari, C.S.A. & Thirunavukkarasu, N., Status of water quality and heavy metal pollution from Coovum River, Tamilnadu, India.Universal Journal of Environmental Research & Technology, 3(4): 483-489 (2013).
8. Dhanakumar, S., Solaraj, G. & Mohanraj, R., Heavy metal partitioning in sediments and bioaccumulation in commercial fish species of three major reservoirs of river Cauvery delta region, India.Ecotoxicology and Environmental Safety, 113: 145-151 (2015).
9. Anas, A., Jasmin, C., Sheeva, V.A., Gireeskumar, T.R. & Nair, S., Heavy metals pollution influence in the community structure of Cyanobacteria in nutrient rich Tropical Estuary.Oceanography, 3(1): 1000137, doi:10.4172/2332-2632.1000137 (2015).
10. http://www.atsdr.cdc.gov/substances/toxsubstance.asp?toxid=54/ (accessed on 29/02/2016).
11. Reddy, M.V., Babu, K.S., Balaram, V. & Satyanarayan, M., Assessment of the effects of municipal sewage, immersed idols and boating on heavy metal and other elemental pollution of surface water of the eutrophic Hussainsagar Lake (Hyderabad, India).Environmental Monitoring and Assessment, 184: 1991-2000 (2012).
12. Kaur, R., Effect of idol immersion on marine and fresh water-bodies.Advances in Applied Science Research, 3(4): 1905-1909 (2012).
13. Malik, D., Singh, S., Thakur, J., Singh, R.K., Kaur, A. & Nijhawan, S., Heavy pollution of Yamuna river: An Introspection.International Journal of Current Microbiology and Applied Sciences, 3(10): 856-863 (2014).
14. Aniruddhe, M. A case study of idol immersion in the context of Urban Lake Management. http://parisaraganapati.net/archives/83 (accessed on 24/02/2016).
15. Nagajyoti, P.C., Lee, K.D. & Sreekanth, T.V.M., Heavy metals, occurrence and toxicity for plants: a review.Environmental Chemistry Letters, 8: 199-216 (2010).
    CrossRef
16. Dibofori-Orji, A.N. & Edori, O.S., Analysis of some heavy metals (Pb, Cd, Cr, Fe, Zn) in processed cassava flour (garri) sold along the road side of a busy highway,Archives of Applied Science Research, 7(2): 15-19 (2015).
17. Khumanleima Chanu, H. & Gupta, A., Necrosis as an adaptive response to copper toxicity inIpomoea aquaticaand its possible application in phytoremediation.Acta Physiologiae Plantarum, 36, 3275-3281(2014).
    CrossRef
18. Austin, D.F., Water spinach (Ipomoea aquatica, Convolvulaceae) a food gone wild.Ethnobotany Research & Applications, 5, 123-146 (2007).
19. Prasad, K.N., Shivamurthy, G.R. & Aradhya, S.M.,Ipomoea aquatica, An underutilized green leafy vegetable: A review.International Journal of Botany, 4(1):123-129 (2008).
    CrossRef
20. Edi, H.H., & Ho, B.W.C.,Ipomoea aquaticaas a vegetable crop in Hong Kong.Econmic Botany, 23 (1): 32-36 (1969).
21. Hasan, S.H., Talat, M. & Rai, S., Sorption of cadmium and zinc from aqueous solutions by water hyacinth (Eichhornia crassipes).Bioresource Technology, 98: 918-928 (2007).
22. Megateli, S., Semsari, S. & Couderchet, M., Toxicity and removal of heavy metals (cadmium, copper and zinc) byLemna gibba.Ecotoxicology and Environmental Safety, 72(6): 1774-1780 (2009).
23. Caldelas, C., Dong, S., Araus, J.L. & Weis, D.J., Zinc isotope fractionation inPhragmites australisin response to toxic levels of zinc.Journal of Experimental Botany, 62(6): 2169-2178 (2011).
24. Caldelas, C., Araus, J.L., Febrero, A & Bort, J., Accumulation and toxic effects of chromium and zinc inIris psuedacorusActa Physiologiae Plantarum, 34: 1217-1228 (2012).
25. Hu, C., Liu, Y., Li, X. & Li, M., Biochemical responses of duckweed (Spirodela polyrhiza) to zinc oxide nanoparticles.Archives of Environmental Contamination and Toxicology, 64: 643-651 (2013).
26. Hu, C., Liu, X., Li, X. & Zhao, Y., Evaluation of growth and biochemical indicators ofSalvinia natansexposed to zinc oxide nanoparticles and zinc accumulation in plants.Environmental Science and Pollution Research, 21: 732-739 (2014).
    CrossRef
27. Basile, A., Sorbo, S., Conte, B., Cobianchi, R. C., Trinchella, F., Capasso, C. & Carginale, V., Toxicity, accumulation, and removal of heavy metals by three aquatic macrophytes.International Journal of Phytoremediation, 14(4): 374-387 (2012).
28. Göthberg, A., Greger, M., Holm, K. & Bengtsson, B-E., Influence of nutrient levels on uptake and effects of mercury, cadmium, lead in Water spinach.Journal of Environmental Quality, 33:1247-1255 (2004).
29. Lichtenthaler, H.K. & Wellburn, A.R., Determination of total carotenoids and chlorophylls a and b of leaf in different solvents.Biochemical Society Transactions, 11, 591-592 (1983).
30. Chayapan, P., Krautrachue, M., Meetam, M. & Pokethitiyook, P., Effects of amenmends on growth and uptake of Cd and Zn by wetland plants,Typha augustifoliaandColocasia esculentafrom contaminated sediments.International Journal of Phytoremediation, 17: 900-906 (2015).
31. Mishra, V.K. & Tripathi, B.D., Accumulation of chromium and zinc from aqueous solutions using water hyacinth (Eichhornia crassipes).Journal of Hazardous Materials, 164(2-3): 1059-1063 (2009).
32. Jamal, Q., Durani, P., Khan, K., Munir, S., Hussain, S., Munir, K. & Anees, M., Heavy metal accumulation and their toxic effects: Review.Journal of Bio-Molecular Sciences, 1(1-2): 27-36 (2013).
33. Shahid, M., Pinelli, E., Pourrut, B., Silvestre, J. & Dumat, C., Lead-induced genotoxicity toVicia fabaroots in relation with metal cell uptake and initial speciation.Ecotoxicology and Environmental Safety, 74(1): 78-84 (2011).
34. Kumar, A., Prasad, M. N. V., Achary, V. M. M. & Panda, B. B., Elucidation of lead- induced oxidative stress inTalinum triangulareroots by analysis of antioxidant responses and DNA damage at cellular level.Environmental Science and Pollution Research, 20: 4551–4561(2013).
35. Srivastava, S., Mishra, S., Dwivedi, S., Tripathi, R.D., Tandon, P.K. & Gupta, D.K., Evaluation of zinc accumulation potential ofHydrilla verticillata.Biologia Plantarum, 53(4): 789-792 (2009).
    CrossRef
36. Kalaikandhan, R., Vijayarengan, P., Sivasankar, R. & Mathivanan, S., The effect of copper and zinc on the morphological parameters ofSesuvium portulacastrumInternational Journal of Current Research and Academic Review, 2(3): 105-120 (2014).
37. Song, U. & Lee, S., Phytotoxicity and accumulation of zinc oxide nanoparticles on the aquatic plantsHydrilla verticillataandPhragmites australis: leaf-type-dependant responses.Environmental Science and Pollution Research, DOI 10.1007/s11356-015-5982-5 (2016).
    CrossRef
38. Yang, J., Tam, N.F-Y. & Ye, Z., Root porosity, radial oxygen loss and iron plaque on roots of wetland plants in relation to zinc tolerance and accumulation.Plant Soil, 374: 815-828 (2014).
39. 林,yf。&艾亚茨,M.G.M分子机制of zinc and cadmium stress response in plants.Cellular and Molecular Life Sciences, 69: 3187-3206 (2012).
    CrossRef
40. Nagajyoti, P.C., Lee, K.D. & Sreekanth, T.V.M., Heavy metals, occurrence and toxicity for plants: a review.Environmental Chemistry Letters, 8: 199-216 (2010).
41. Gomes, M.P., Duarte, D.M., Carneiro, M.M.L.C., Barreto, L.C., Carvalho, M., Saores, A.M., Guilherme, L.R.G. & Garcia, Q.S., Zinc tolerance modulation inMyracrodruom urundeuvaplants.Plant Physiology and Biochemistry, 67: 1-6 (2013).
    CrossRef