324 Chiang Mai J. Sci. 2014; 41(2)

Chiang Mai J. Sci. 2014; 41(2) : 324-333 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

Responses of Water Spinach (Ipomoea aquatica Forssk.) on Growth, Morphology, Uptake Rate and Nutrients Allocation under High Ammonium Concentration Sutthathorn Chairuangsri [a,b], Niwooti Whangchai [c] and Arunothai Jampeetong* [a,b] [a] Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. [b] Environmental Science Program, Faculty of Science, Chiang Mai University, Chiang Mai 50202, Thailand. [c] Faculty of Fisheries Technology and Aquatic Resources, Maejo University, Chiang Mai, 50290, Thailand. *Author for correspondence; e-mail: [email protected]

Received: 29 October 2012 Accepted: 17 June 2013

ABSTRACT + + The effects of external NH4 concentration on growth, morphology, NH4 uptake and mineral allocation in Ipomoea aquatica were investigated under greenhouse conditions. Similar sized plants were grown on full strength Smart and Barko growth medium [1] with + different levels of NH4 -N (0.5, 1, 5, 10 and 15 mM) for four weeks. Relative growth rate + was high in plants fed with NH4 at concentrations of 0.5, 1 and 5 mM, but at higher concentrations the plants were stunted with few and short roots, old leaves were lost and growth of new ones was suppressed. Submerged stems and roots were damaged especially + + in plants supplied with 15 mM NH4 . The NH4 uptake rate tended to decrease with increasing + NH4 supply. This may be due to damaged roots and stems which decreased efficiency of nutrient uptake. However, we found only small changes in mineral concentration in the tissue of both leaves and roots. We suggest that I. aquatica can be used for water treatment but the + NH4 concentration must be less than 5 mM to prevent root and stem damage that cause minerals to be released from the plants and returned to the water treatment system.

+ + Keywords: Ipomoea aquatica, NH4 uptake, NH4 toxicity, water spinach, water treatment

1. INTRODUCTION Growing human populations, agricultural to wide spread eutrophication and water activities and industrialization cause purification technologies that remove degradation of the environment. Water bodies pollutants are costly. Therefore, eco-friendly are the main targets for disposing pollutants and cheaper ecological methods are interesting both directly and indirectly. High nutrient alternatives for treating water. Many species loadings from inland to lakes and rivers lead of aquatic macrophytes have been used in Chiang Mai J. Sci. 2014; 41(2) 325

constructed wetlands (CWs) to remove large depends on species and their environments. amounts of nutrients and heavy metals from Water Spinach (Ipomoea aquatica Forssk.) is a wastewater [2-6]. Inorganic nitrogen in free-floating macrophyte often found in a polluted water is commonly discharged from wide variety of environments. This plant has households, agricultural fertilizers and animal been used in water treatment system for waste from farms [7]. Generally, inorganic removal of nutrients and also some heavy nitrogen is an important nutrient for enhancing metals [18-21]. Moreover, the plant is edible, growth of aquatic macrophytes, which mostly so after harvesting it can be used to feed are fast-growing. Previous studies have shown animals because of its high total N and crude that most aquatic macrophytes prefer protein content [22]. A study by Jampeetong + - ammonium (NH4 ) over nitrate (NO3 ) et al. [23] showed that I. aquatica grew well in + [8-12]. However, excess nitrogen can cause growth medium with NH4 and the plants + water pollution, particularly in lotic systems. had a high NH4 uptake rate compared to - Recently, use of CW technologies is NO3 . However, ability of the plants to grow + increasing because they are cheap and under high NH4 concentrations is unknown. energy-efficient. The performance of CWs It is evident that wastewater containing high is usually based on its design, water retention concentrations of ammonium constitute a time, dimension and substrate used [4, 13]. problem for plants used in CWs (unpublished Plant selection for use in constructed data). Therefore, this study is aimed at wetlands is equally important, but few plants assessing the growth and morphological + have been evaluated [14]. Among aquatic responses, NH4 uptake and nutrient macrophyte growth forms, submerged allocation in the plant tissue of Ipomoea aquatica + macrophytes are sensitive to light intensity supplied with different NH4 concentrations. in the water column. In the case of high The results of this study will be useful for loading of nitrogen and phosphorus, the selecting aquatic macrophytes for use in + plants may suffer from low light intensity treatments of high NH4 strength wastewater caused by algae blooms [15-16]. The wetland in tropical CWs. species used in the CWs commonly are emergent macrophytes. Many studies have 2. MATERIAL AND METHODS been carried out on fast growing wetland 2.1 Experimental Set Up and Growth species such as Phragmites spp. in temperate Study regions [2, 10] but so far, there have been only Ipomoea aquatica was collected from a few studies done on tropical free-floating natural ponds in Chiang Mai, Thailand. macrophytes. Furthermore, many previous The plants were cultivated in the greenhouse studies have been done on evaluation of the of the Department of Biology, Faculty of efficiency of CW systems [5, 17], but there is Science, Chiang Mai University at a little published data on the efficiency of temperature range of 32-35°C during the day macrophytes in terms of growth, uptake and 22-25°C at night. The light regime was capacity and nutrient allocation in the plant approximately 80% of full sun and the tissue. light:dark cycle was 14:10 h. The growth The potential to grow and uptake medium was a full-strength standard nutrients are important characteristics used nitrogen-free Smart and Barko nutrient in the selection of aquatic macrophytes for solution [1], to which micronutrients and -1 CWs. The growth and uptake capacity 100 μmol L of KH2PO4 were added. The 326 Chiang Mai J. Sci. 2014; 41(2)

pH of the growth medium was adjusted of water samples were withdrawn each to 7.0. hour and 4 mL of distilled water was added For the experiment, similar sized into the water sample before measurement + + plants (35-40 cm shoot length) from the of NH4 concentrations. The NH4 stock cultures were placed in 5-L containers concentration in all samples was analyzed (n=8) in the greenhouse. The experimental using a modified salicylate method + treatments consisted of five levels of NH4 - (Quikchem Method no. 10-107-06-3-B; N: 0.5 mM, 1 mM, 5 mM, 10 mM and 15 Lachat Instruments, Milwaukee, WI, USA). + mM prepared from (NH4)2SO4. The NH4 After the experiment (6 hours), all plants were concentration was increased in steps over a cleaned and separated into shoots and roots, + period of four days to the final treatment and then freeze-dried. The NH4 uptake rate + levels. During the experiment, the growth was calculated from NH4 depletion curve medium was changed every second day, with linear regression analyses and related to and epiphytic algae were removed gently by volume and root dry weight (DW). hand. All treatments were arranged in a + randomized complete block design. After four 2.4 Inorganic NH4 in The Plant Tissue weeks, the plants were harvested and cleaned. The freeze-dried leaves and roots were Then, the plants were freeze-dried to constant cut into small pieces. Five milligrams of dry weight. The relative growth rate (per day) dried plant material was extracted with for each treatment was calculated using the 15 mL of distilled water at 98°C in a water + formula: RGR = lnW2-lnW1/ (t2-t1), where W1 bath for exactly 20 minutes. Then, the NH4 and W2 are the initial and final dry weights concentration in the extracts was analysed

(gram), and t1 and t2 are initial and final time by a modified salicylate method (Quikchem (days). Method no. 10-107-06-3-B; Lachat Instruments, Milwaukee, WI, USA). The 2.2 Plant Morphology absorbance of the extracts was measured at After four weeks, root number were 690 nm using a UV-VIS spectrophotometer counted, root length and leaf area of the (Lambda 25 version 2.85.04, USA). plants were measured individually, then averaged for each treatment. Then, the plants 2.5 Chlorophyll Contents were separated into shoots and roots before The contents of chlorophyll a (Chl a), being freeze-dried. chlorophyll b (Chl b), total chlorophylls (total Chl a+b) and carotenoids were 2.3 Ammonium Uptake determined according to Lichtenthaler + NH4 uptake rates of I. aquatica were method [24]. The freeze-dried leaves were determined after four weeks of growth in cut into fragments. The plant materials the treatments under the same conditions as (approximately 8 mg) were extracted with 8 the growth study. Four replicates of similar mL of 96% ethanol in the dark and at room sized plants from each treatment were temperature for 24 hours. Then, the + pre-incubated in a container with a NH4 absorbance of the extracts was measured at free growth medium for 18 h. After pre- 648.6 and 664.2 nm using a UV-VIS incubation, the plants were placed in 240 mL spectrophotometer (Lambda 25 version + medium with 500 μM NH4 . One milliliters 2.85.04, USA). Chiang Mai J. Sci. 2014; 41(2) 327

2.6 Mineral Elements performed by the Tukey HSD’s test at a 5% The concentration of total N, significance level. phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) in both leaves and 3. RESULTS roots was determined from subsamples 3.1 Growth and Morphology (150-180 mg) of finely ground freeze-dried The growth of Ipomoea aquatica was + plant material. The samples were digested by affected by external NH4 supply and it was

7 mL acid solution (K2SO4 100 g, selenium 1 significantly different between treatments g in concentrate H2SO4 1 L) at a temperature (Figure 1a). The plants grew well in the + range of 100-330°C. Total N was analyzed medium with NH4 concentration of 0.5, by method followed Hanlon et al. [25] and 1 and 5 mM, but their growth was suppressed the concentrations of P, K, Ca and Mg were when they were supplied with 10 and 15 mM + analyzed according to Chapman and Pratt [26]. NH4 . Root length and root number of the + plants grown on 10 and 15 mM NH4 2.7 Statistics decreased (Figure 1b,c), especially at 15 mM + All statistics were carried out using NH4 , and some dead plants and rotted the software Statgraphics Plus ver. 4.1 roots and stems were encountered. + (Manugistics, Inc., MD, USA). the Cochran’s Furthermore, high NH4 concentrations had C-test was used to test for normal distribution negative effects on leaves of Ipomoea, and homogeneity of variance. Data was log- producing small leaves and the loss of some transformed to ensure homogeneity of old leaves when they grew on 10 and 15 mM + variance, if necessary. The data was tested by NH4 and it was significantly different one-way analysis of variance (ANOVA). from the treatments of 0.5, 1 and 5 mM + Differences between treatments were NH4 (Figure 1d).

Figure 1. The relative growth rate, RGR (a), Root length (b), root number (c) and leaf area + (d) (mean±SE) of Ipomoea aquatica grown at different NH4 -N concentrations (0.5, 1, 5, 10, 15 mM). Different letters above columns indicate significant differences between treatments. 328 Chiang Mai J. Sci. 2014; 41(2)

+ 3.2 Chlorophyll Contents on NH4 concentration higher than 5 mM. Overall, Chl a, Chl b, total Chl a+b and The carotenoids decreased slightly in + + carotenoids were affected by NH4 plants grown at high NH4 concentrations concentrations and significant differences were (10, 15 mM) and there was a significant + found between treatments (Table 1). The Chl difference from the plants grown on NH4 a and Chl b were low in the plants grown concentration of 1 and 5 mM. Table 1. Contents of chlorophylls and carotenoids of Ipomoea aquatica (mean ± SE) grown at + different NH4 -N concentrations. Different letters superscripts between columns indicate significant differences between treatments. + NH 4 concentrations 0.5 mM 1 mM 5 mM 10 mM 15 mM F-ratio 1. Chl a (mg g-1 dw) 12.6±2.2b 13.2±1.9b 11.1±1.7ab 9.7±2.1a 9.2±2.0a 5.98** 2. Chl b (mg g-1 dw) 5.4±1.1b 5.4±0.8b 4.5±0.6ab 4.0±0.7a 3.9±0.9a 5.62** 3. total Chl a+b 18.0±3.3b 18.5±2.6b 15.6±2.3ab 13.7±2.8a 13.1±2.8a 5.94** 4. Ca/ Cb 2.4±0.1 2.4±0.1 2.5±0.1 2.4±0.2 2.3±0.2 3.57 5. Carotenoids 1.9±0.3ab 2.1±0.3b 2.0±0.3b 1.6±0.4a 1.6±0.3a 1.2* (mg g-1 dw) * (P<0.05), ** (P<0.01)

+ + 3.3 NH4 Uptake The NH4 concentration in the plant + The NH4 uptake rate of I. aquatica was tissue of both leaves and roots was lowest in + approximately 50% decreased in plants grown the plants supplied with 10 mM NH4 + on NH4 concentration of 5, 10 and 15 mM and it was significantly different from + and it was significantly different from the other treatments. At 15 mM NH4 , the + plants grown on 0.5 and 1 mM (Figure 2a) concentration of NH4 increased reaching the same levels as in the plants grown on + + + 3.4 NH4 Hot Water Extractable NH4 at 1 and 5 mM NH4 (Figure 2b).

+ + -1 -1 + Figure 2. NH4 uptake rate (μmol NH4 g root dw h ) (a) and NH4 concentration in leaves (dark column) and roots (grey column) (μmol g-1 plant dw) (mean±SE) of Ipomoea + aquatica grown at different NH4 -N concentrations (0.5, 1, 5, 10, 15 mM). Different letters above columns indicate significant differences between treatments. Chiang Mai J. Sci. 2014; 41(2) 329

3.5 Minerals anions, concentrations of P in both Minerals concentrations were slightly leaves and roots were decreased. The P + affected by external NH4 supply (Figure concentrations in the roots of the plants 3a-j). Generally, total N concentration in leaves supplied with 1 and 5 mM decreased by was higher than in roots, and the total 18% and 36%, respectively and were found N concentrations in the leaves was to be significantly different from the treatment + significantly increased when the plants were supplied with 0.5 mM NH4 . Other mineral + supplied with a high NH4 concentration cations (K, Ca, Mg) were slightly affected + especially at 10 and 15 mM. For mineral by NH4 concentrations.

Figure 3. Concentrations of N (a, f), P (b, g), K (c, h), Ca (d, i) and Mg (e, j) in leaves and + roots (mean±SE) of Ipomoea aquatica grown at different NH4 -N concentrations (0.5, 1, 5, 10, 15 mM). Different letters above columns indicate significant differences between treatments. (n.d. not determined). 330 Chiang Mai J. Sci. 2014; 41(2)

4. ISCUSSION AND CONCLUSION + D The NH4 uptake of I. aquatica was + Growth of Ipomoea aquatica was affected by external NH4 concentration. + affected by external NH4 concentration. The present study shows that uptake rates As demonstrated in the present study, I. were significantly decreased in the plants + + aquatica grew well in a wide range of NH4 supplied with NH4 at concentrations of 5, concentrations from 0.5-5 mM which 10 and 15 mM. Generally, the root is an correspond to levels commonly found in important part of plants for nutrient + hypereutrophic water and wastewater from uptake. Under high NH4 concentrations, households or agriculture areas [27-28]. many species, including I. aquatica, showed + At higher NH4 concentration, the growth of damage to roots [29, 31-32]. The plants had the plants was suppressed and negative short roots and low numbers of roots effects on their morphology were found resulting in a low root surface area for including short roots, small leaves and nutrient absorption. Thus, it is possible that + chlorosis. Furthermore, old roots and stems I. aquatica grown on NH4 at very high were damaged particularly in the plants concentrations may suffer from nutrient + supplied with NH4 concentration at 10 mM deficiency caused by root damage, not by + and 15 mM. Similar results were found in NH4 uptake [29]. + other free-floating macrophytes, for example Many free-floating species were NH4 Salvinia natans [29]. I. aquatica has been used in tolerant, with their shoots exposed to light water treatments and it has been intensively as an advantage. This is in contrast to studied in East and Southeast Asia because it submerged species in which the plants may is edible and people can use it for food and suffer from low light intensity in eutrophic animal feed after harvest. I. aquatica has high water [33]. However, shoots of I. aquatica + contents of crude protein and vitamin A and were affected by the high external NH4 C [22]. Hence, using this species in constructed supply, leaves of the plants decreased in leaf wetlands under the influence from household surface area particularly in the plants grown +; and farm lands could be a good way of on 15 mM NH4 these also had loss of old reducing inorganic nitrogen and other leaves, and growth of new leaves was nutrients from polluted water. The ability of suppressed resulting in very small young this species to exist and remove excess N leaves. The results revealed that I. aquatica + from wastewater depends on the type of can grow on wastewater with NH4 as high + wastewater. At high concentrations of as 5 mM and the NH4 toxicity found in this wastewater from farmlands such as piggery species was small. Therefore we suggest + farms in which NH4 concentration may reach that I. aquatica can be used in highly polluted + + 20 mM, NH4 would stress the efficient N water treating systems in cases where NH4 + removal by this species even though NH4 concentration in the polluted water is less than + was the preferred inorganic N form for 5 mM in order to prevent NH4 toxicity that many aquatic macrophytes [9-12, 30]. To leads to the plant damage and nutrients + protect the plants from the NH4 toxicity returned to water treatment systems via and maintain their high N uptake rate, I. submerged stems and rotted roots. + aquatica should be used in post-treatment Generally, plants fed with NH4 had + + systems where the NH4 level does not low concentrations of mineral cations K , exceed 5 mM. Ca2+, Mg2+ in their tissue [29, 32]. Many Chiang Mai J. Sci. 2014; 41(2) 331

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