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Ratio of Plant Numbers to Total Mass of Contaminant Determines the Phytotoxicity Effects HARMIN SULISTIYANING TITAH1,4, SITI ROZAIMAH SHEIKH ABDULLAH2, MUSHRIFAH IDRIS3, NURINA ANUAR2, HASSAN BASRI1 & MUHAMMAD MUKHLISIN1 1

Department of Civil and Structural Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, MALAYSIA 2 Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, MALAYSIA 3 Tasik Chini Research Centre, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, MALAYSIA 4 Department of Environmental Engineering, Faculty of Civil Engineering and Planning, Institut Teknologi Sepuluh Nopember (ITS), 60111, Keputih, Sukolilo, Surabaya, INDONESIA

[email protected], [email protected], [email protected] Abstract: -A phytotoxicity bioassay is applied to select plant species for phytoremediation. It needs to be conducted before plant species can be chosen for phytoremediation. Among the factors that affect the toxic effects on phytotoxicity in soil were source of contaminant, nature of soil type and plant species. Besides that, the ratio of number plants to total mass of contaminant was expected to give effects on the phytotoxicity. A study was conducted to determine the affect of that ratio against arsenate [As(V)] toxic effects on L. octovalvis. Based on the results,L. octovalvis could survive when the ratio was more than 11 on preliminary phytotoxicity study using sand matrix and no addition nutrient during test for 28 days of exposure. Meanwhile, L. octovalvis could survive with the ratio was more than 19 on prolonged phytotoxicity test at the same condition for 35 days of exposure. On a pilot scale study at still the same condition showed L. octovalvis could survive with only 5% of percentages of toxic effect when use ratio of plant to total mass was ≥ 34 for 42 days of exposure. The percentages of toxic effect reached 86 % and 92% when the ratio was only 4 and 2, respectively. The possibilities of toxic effects to plants increased if the ratio of number of plants to the total mass of contaminant was lower. It can be concluded that ratio of number of plants to the total mass of contaminant could be one factor that determined the phytotoxicity effects but not the contaminant concentration.

Key-Words: - phytotoxicity, phytoremediation, number of plants, total mass, contaminant (Cd), copper (Cu), zinc (Zn), nickel (Ni), and mercury (Hg), includes industrial operations such as mining, smelting, metal forging, combustion of fossil fuels, and sewage sludge application in agronomic practices [2]. Moreover, agricultural activities like application of agrochemicals and

1 Introduction Intense industrial activity in the 20th century has been particularly deleterious to environment, resulting in a large number and variety of contaminated sites [1]. The primary source of metal pollution such as lead (Pb), arsenic (As), cadmium

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long-term usage of sewage sludge in agricultural practices also adds a significant amount of metals to the soil [3, 4]. Excess heavy metal accumulation in soils is toxic to humans and other animals. According to Glick [5], ATSDR (Agency for Toxic Substances and Disease) have listed the chemicals that were often found in contaminated areas including dangerous and toxic chemicals. According to the list released by ATSDR, there were 275 hazardous materials that had an impact on human health. According to this list, As was a chemical in the first rank of dangerous and toxic, then Pb and Hg were second and third. Nowadays, there are many different types of technologies to remediate contaminated areas. The technology using plants is conducted to remediate contaminated soil and groundwater, including heavy metals remediation. The concept is one of the effective approaches in this period and is considered as a green technology. Phytoremediation uses plants and related microorganisms to perform the degradation of pollutants in the soil to be non-toxic [6, 7, 8, 9, 10, 11]. Phytotoxicity is a term used to describe the toxic effect of a compound on plant growth. Such damage may be caused by a wide variety of compounds, including heavy metals. Toxicity result when the plant is incapable of sequestering or excluding excess concentrations of heavy metals. Plants have evolved mechanisms to deliberately exclude, excrete or biologically transform these metals into less toxic forms [12, 13]. Sheppard [14] reported that the source of As and the nature of soil type were the key factors for the phytotoxic effects of As in soils. Besides that, the species of plant could affect the results of heavy metal phytotoxicity [15]. A phytotoxicity bioassay was used to select plant species for phytoremediation that were able to reduce heavy metal in contaminated site. The bioassay can potentially reduce the number or pot or greenhouse studies that need to be conducted before plant species can be chosen for phytoremediation [16]. The aim of the study was to determine the relationship of the ratio of plant numbers of Ludwigia octovalvis to the total mass of As(V) against As(V) phytotoxicity effects. As(V) was an inorganic form As that most found in contaminated area [17]. Besides that, L. octovalvis has been described as a plant that can survive on a contaminated site in Malacca, Malaysia [18]. The data will be used in further investigation in As phytoremediation using L. octovalvis.

2.1 Plant Propagation The seeds of L. octovalvis were grown in the greenhouse using garden soil with ratio of top soil: organic: sand (3: 2: 1) until the next generation plants had produced seeds in large quantities. Only plants from this generation were used to run the massive phytotoxicity test. The seeds were planted in plastic crates (37 x 27 x 10 cm). After three weeks, individual seedlings were selected from the nursery and transferred to polybags, two plants per polybag. All of the plants used in the phytotoxicity experiment were eight weeks old at the beginning of the testing.

2.2 Preparation of Phytotoxicity Test All study was conducted in a greenhouse. The experimental plants were planted in sand matrix to obtain the pure toxic effects of contaminants As(V) because the amount of iron oxide, aluminum oxide and clay minerals in the sand is very small [19, 20] so that the impact of the adsorption of As(V) by iron oxide, aluminum oxide can be reduced and concentrations of bioavailable As in the sand increase [14]. The sand was first sieved using a sieve (4.75 mm). As(V) solution was prepared from sodium arsenate dibasic heptahydrate (AsHNa2O4.7H20) (FlukaChemika, Switzerland) salts. As(V) solution was mixed thoroughly into the sand to achieve homogeneity using manually method and then allowed to equilibrate for seven days in plastic pot/crates under controlled greenhouse conditions before planting. No additional nutrient solution was added throughout this study. During the study, plants were watered alternate days since the sand had a low moisture-holding capacity. Moisture was monitored using a moisturemeter (Decagon ECH2O, USA), while pH and temperature of the spiked sand were monitored using a pH-meter (Cyberscan pH 300, Singapore). 2.2.1 Range Finding Test Plastic pots with 25 cm in diameter contained 3 kg of sieved sand spiked with As(V) at six different concentrations of As(V) : 0 mg kg-1 as control, 4, 20, 40, 60 and 80 mg kg-1[21]. The range finding test was carried out for 28 days. Each pot was planted with two L. octovalvis plants. 2.2.1 Prolonged Phytotoxicity Test A prolonged phytotoxicity test was conducted for 91 days. Each plastic crate (41 x 31x 11 cm) used in the experiment contained 12 kg of sieved sand and 9 L. octovalvis plants. Six different concentrations of As(V) were prepared: 0 mg kg-1 as control and 5,

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10, 21, 39 and 65 mg kg-1, in three replicates using a completely randomized experimental design.

was known as senescence, indicating one of the plant detoxification mechanisms. According to Dahmani-Muller et al. [25], heavy metals translocation root, stems, leaves, and finally the falling leaves was considered as detoxification process to assist the removal of As in plant. The ratio of number of plant to As(V) total mass (g) on the range finding test is summarized in Table 2. The ratio showed the ability of the plants to survive under As(V) exposure using an experimental sand matrix. The ratio of As(V) concentration of 4, 20, 40, 60 and 80 mg kg-1 were 166, 33, 16 and 11, respectively. The percentages of lethal toxic effect was 0% at 4, 20 and 40 mg kg-1 As(V), meanwhile it reached 100% at 60 and 80 mg kg-1 As(V). It showed L. octovalvis could survive if the ratio more than 11 for 28 days of exposure. On the prolonged phytotoxicity test, L. octovalvis could still survive at As(V) concentration of 5, 10, 21 and 39 mg kg -1 until at Day-35 with growing branches and new leaves. Flower and fruit were able to grow at control and As(V) concentration of 5, 10 and 21 mg kg-1. Table 3 shows the images of prolonged phytotoxicity test. All plants were dried in control and As(V) concentrations of 5, 10, 21 and 39 mg kg-1after 91 days of exposure. The situation occurred due to no additional nutrient solution was added throughout this study. While L. octovalvis plant were wilted in As(V) concentration of 65 mg kg-1on the first day. For this concentration, all plants died at Day-14 of exposure until the end of exposure. This condition is similar to the range finding test in which L. octovalvis could not survive at As(V) concentration of 60 mg kg-1. Table 2 shows the ratio of plant numbers to As(V) total mass (g) on the prolonged phytotoxicity test. The ratio for As(V) concentration of 5, 10, 21, 39 and 65 mg kg-1 were 150, 75, 35, 19 and 11, respectively. The percentages of toxic effects for 35 days of exposure were 0% at As(V) concentration of 5, 10, 21 mg kg-1. Meanwhile, the percentages of toxic effects was 22% at As(V) concentration of 39 mg kg-1. It indicated that L. octovalvis could survive when the ratio was more than19 for 35 days of exposure. Total 50 plants of L. octovalvis which were planted in each pilot scale reed bed for the first treatment. Table 4 shows the photos in the first pilot scale reed bed exposure. The ratio of the number of plants used to the total mass (g) of As in one reed bed were 21, 4 and 2, respectively in As(V) concentrations of 5, 22 and 39 mg kg-1(Table 2). After 28 days of exposure, the percentages of the phytotoxicity effects were 30, 86 and 92% under the

2.2.1 Application in Pilot Scale Reed Bed The pilot reed beds were constructed of fiberglass tanks, the walls of which were 0.5 cm in thickness and black in color with dimensions of 92 x 92 x 60 cm. A layer of medium gravel (Ф in 2 cm) was placed at the bottom of the reed bed, and another layer of fine gravel (Ф in 1 cm) was placed at the top. The thicknesses of both the medium and fine gravel layers were 10 cm. Each reed bed was completed with a pipe at the bottom for sampling the leachate. The following four different concentrations of As(V) were prepared: 0 mg kg-1as the control, 5, 22, and 39 mg kg-1. The As concentration were selected based on a range finding test as reported by Titah et al. [21].There were two treatments conducted in pilot scale reed beds. A total of 465 kg of As spiked sand was placed into each reed bed and planted with 50 of L. octovalvis plants so that the ratio was low in the initial treatment. Meanwhile in the second treatment to increase the ratio, a total of 131 kg of As-spiked sand was placed into each reed bed and planted with 100 of L. octovalvis plants.

3 Results and Discussion Based on the plant observation during the range finding phytotoxicity (Table 1), L. octovalvis could survive up to 40 mg kg-1 As(V). Plants were wilting after 3 days of exposure in 40, 60 and 80 mg kg-1 As(V), but plants could still survive and became healthy in 40 mg kg-1 after 7 days of As(V) exposure. Meanwhile L. octovalvis were wilted and dried at 60 and 80 mg kg-1 As(V) after 3 days of exposure until the end of exposure (Day-28).It indicates thatsurvival rate of plants was low. According to Rattanawat et al. [22], the low survival rate indicated the phytotoxicity. According to Kabata-Pendias and Pendias [23] and Quaghebeur and Rengel [24], the symptoms of As toxicity to plants are variously described as leaf wilting, inhibition of root growth and plant death. Several leaves in control fell after 21 days could be due to decreasing nutrient since no additional nutrient was added. The falling leaves at As(V) concentrations of 4 and 20 mg kg-1 also significantly occurred after 21 days due to the additional effect of As(V) toxicity. Meanwhile the falling leaves at As(V) concentrations of 40 mg kg-1 started at 7 days after exposure. Wilting, especially of the leaves, was observed as an initial symptom of phytotoxicity resulting from As(V) exposure. The falling of leaves

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Table 1 Images of L. octovalvis plants in range finding test Day of exposure Day-0

Ratio of plant numbers to As(V) total mass 33 16

Control

166

All plants healthy

All plants healthy

All plants healthy

All plants healthy

All plants healthy

All plants healthy

11

8

All plants healthy

All plants healthy

All plants healthy

All plants healthy

All plants died

All plants died

Day-28

Table 2 Affect of ratio of plant numbers to As total mass in phytotocixity effects against L. Octovalvis As(V) in spiked sand

Test

Study

Range finding test (28 days)

Concentration of As(V) (mg kg-1)

0

4

20

40

60

80

Σ plants : total mass (g As)

2:0

2 : 0.012

2 : 0.06

2 : 0.12

2 : 0.18

2 : 0.24

166

33

16

11

8

0%

0%

100%

100%

*)

Ratio

Prolonged phytotoxicity test (91 days)

First pilot reed bed treatment (42 days)

Second pilot reed bed treatment (42 days)

Percentages of toxic effects at 28 days Concentration of As(V) (mg kg-1)

0% 0

5

10

21

39

65

Σ plants : total mass (g As) (g As)**) Ratio

9:0

9 : 0.06

9 : 0.12

9 : 0. 252

9 : 0.468

9 : 0.78


0%

150

75

35

19

11

0%

0%

0%

22%

100%

5

22

39

Σ plants : total mass (g As) (g As)****) Ratio

50 : 2.33

50 : 10.23

50 : 18.14

21

4

2


30%

86%

92%

5

22

39

Σ plants : total mass (g As) (g As)***) Ratio

100 : 0.655

100 : 2.899

100 : 5.116

150

34

19

Percentages of toxic effects at 42 days

1%

5%

24%

Table 3 Pictures of L. octovalvis plants in prolonged phytotoxicity test Day of exposure Day-7


Control

150

All plants healthy

All plants healthy

All plants healthy

All plants healthy

All plants healthy

All plants healthy

19

11

All plants healthy

All plants healthy

All plants wilted

All plants healthy

Two plants wilted

All plants died

Day-28

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As(V) concentrations of 5, 22, and 39 mg kg-1 as compared with the number of healthy plants. It indicates that plant had low survival rate in lower the ratio of number plants to total mass of As(V).

Fewer plants with a higher As(V) total mass will give a smaller ratio, indicating that plants could not survive.

Table 4 Pictures of L. octovalvis plants in pilot reed beds first treatment Day of exposure Day-0

Control


2

50 plants in good condition





15 plants wilted

43 plants wilted

46 plants wilted

Day-28

and wilted, occurred in the As spiked sand. The falling leaves occurred in control and all As concentrations. The percentages of the phytotoxicity effect at 42 days of exposure were 1, 5 and 24%, respectively under the As(V) concentrations of 5, 22, and 39 mg kg-1 as compared with the number of healthy plants.

The second treatment in pilot reed beds was performed using an increased ratio of the plant numbers to the total mass of As (V) in the sand matrix. Table 5 shows the images in the second treatment using pilot reed bed. A total 100 L. octovalvis were planted in each pilot reed bed. All plants were healthy after 42 days under the control conditions, whereas phytotoxicity symptoms, such as the wilting leaves and the plants becoming dried

Table 5 Second treatment images of L. octovalvis plants in pilot reed beds Day of exposure Day-0

150





1 plant wilted

5 plants wilted

24 plants wilted

Day-42

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Pollutants, Sustainable Agriculture Reviews 1, 2009, pp. 319-350. Springer Science and Business Media B.V. [3] Giller K.E., McGrath S.P. & Hirsch P.R. Absence of nitrogen fixation in clover grown on soil subject to long-term contamination with heavy metals is due to survival of only ineffective Rhizobium. Soil Biology Biochemistry, Vol. 21, 1989, pp. 841-848. [4] McGrath S.P., Chaudri A.M., Giller K.E. Longterm effects of metals in sewage sludge on soils, microorganisms and plants. J. Ind. Microbiol., Vol. 14, 1995, pp. 94-104. [5] Glick, B.R. Research review paper: using soil bacteria to facilitate phytoremediation. Biotechnology advanced, Vol. 28, 2010, pp. 367-374. [6] Cho-Ruk, K., Kurukote, J., Supprung, P., & Vetayasuporn, S. Perennial plants in the phytoremediation of lead-contaminated soils. Biotechnology, Vol. 5(1), 2006, pp. 1-4. [7] Ghosh, M., & Singh, S.P. A Review on phytoremediation of heavy metals dan utilization of its byproduct. Applied Ecology and Environmental Research, Vol.3(1), 2005, pp. 1-18. [8] Henry, J.R. An overview of the phytoremediation of lead dan mercury. National Network of Environmental Management Studies (NNEMS) Report. Washington, D.C. 2000, pp. 3-9. [9] ITRC. Technical dan regulatory guidance document, phytotechnology. Interstate Technology Regulatory Council USA, 2001, http://www.itrcweb.org/documents/Phyto-2.pdf [21 April 2009]. [10] Pilon-Smith, E. Pytoremediation. Annual Review. Plant Biology, Vol. 56, 2005, pp. 1539. [11] Sao, V., Nakbanpote, W., & Triravetyan, P. Cadmium accumulation by Axonopus compressus (Sw.) P. Beauv dan Cyperus rotundas Linn growing in cadmium solution dan cadmium-zinc contaminated soil. Journal Science Technology, Vol. 29(3), 2007, pp. 881892. [12] Prasad, M.N.V. Cadmium toxicity and tolerance in vascular plant. Environmental and Experimental Botany, Vol. 35, 1995, pp.525545. [13] Sneller, F.E.C., Van Heerwaarden, L.M., Kraaijeveld-Smith, F.J.L, Ten Bookum, W.M., Koevoets, P.L.M., Schat, H, Verkleij, J.A.C. Toxicology of arsenate in Silene vulgaris, accumulation and degradation of arsenate-

The ratio of the number of plants to the total mass (g) of As was used in one reed bed for As(V) concentration of 5, 22 and 39 mg kg-1 were 150, 34 and 19, respectively (Table 2). It showed that L. octovalvis could survive with 5% of toxic effect percentages when the ratio was ≥ 34 for 42 days of exposure. Therefore, the ratio of plant number to contaminant mass, and not the contaminant concentration, in the sand that determines the phytotoxicity.

4 Conclusion Based on a preliminary phytotoxicity study, prolonged phytotoxicity test, and pilot scale reed bed using sand matrix and no addition nutrient during test, the limit of ratio of number of plants to As(V) total mass can be determined to avoid the As(V) toxic effect to L. octovalvis. The ratio was more than 11 for 28 days of exposure, and more than 19 for 35 days of exposure. It was more better to use the ratio more than 34 for 42 days of exposure to reduce the As(V) toxic effects to L. octovalvis plants at the phytotoxicity test using sand matrix and no addition nutrient. The possibilities of toxic effects to plants increase if the ratio of number of plants to total mass of contaminant was lower.It can be concluded that ratio of number of plants to total mass of contaminant could be one factor that determines the phytotoxicity effect by a contaminant under similar conditions during a test.

Acknowledgment The authors would like to thank Tasik Chini Research, Universiti Kebangsaan Malaysia (UKM) and Ministry of Higher Education, Malaysia of ERGS/1/2011/TK/UKM/02/24 for funding this research, and the Ministry of National Education of the Republic of Indonesia for funding the first author’s study. References: [1] Roy, S., Labelle, S., Mehta, P., Mihoc, A., Fortin, N., Masson, C., Leblanc, R., Cha’teauneuf, G., Sura, C., Gallipeau, C., Olsen, C., Delisle, S., Labreue, M. & Greer, C.W. Phytoremediation of heavy metal and PAH-contaminated brownfield. Journal of Plant and Soil, Vol. 272, 2005, pp. 277-290. [2] Khan, M.S., Zaidi, A., Wani, P.A. & Oves, M. Role of plant growth promoting rhizobacteria in the remediation of metal contaminated soils: A review. Lichtfouse, E. (Edt.). Organic Farming, Pest Control and Remediation of Soil

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[20] Smith, E., Naidu, R.,&Alston. A.M. Arsenic In The Soil Environment: A Review. Academic Press, 1998. http://www.arsenic.lk/content/Effects/Environ mental_effects/Soil02/Soil02.pdf[12 July 2011]. [21] Titah, H.S., Abdullah, S.R.S, Anuar, N., Idris, M., Basri, H. & Mukhlisin, M. Arsenic range finding phytotoxicity test against Ludwigia octovalvis as first step in phytoremediation. Research Journal of Environmental Toxicology, Vol. 6(4), 2012. pp.151-159. [22] Rattanawat, C., Rujira, S., Narupot, P., Maleeya, K. & Prayad, P. Effect of soil amendments on growth and metal uptake by Ocimum gratissimum grown in Cd/Zncontaminated soil. Water Air Soil Pollution, Vol. 214, 2011, pp. 383-392. [23] Kabata, A.P. &Pendias, H. Trace elements in soils and plants. CRC Press, Inc. Boca Raton, Florida, 2001. [24] Quaghebeur, M. & Rengel, Z. Arsenic speciation governs arsenic uptake and transport in terrestrial plant. Microchimica Acta, Vol. 151, 2005, pp. 141-152. [25] Dahmani-Muller, H., van Oort, F., Gelie, B. & Balabane, M. Strategies of heavy metal uptake by three plant species growing near a metal smelter. Environmental Pollution, Vol. 109, 2000, pp. 231-238.

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