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Comparative analysis of growth and physio-biochemical responses of Hydrilla verticillata to different sediments in freshwater microcosms
Ecological Engineering 36 (2010) 1285–1289
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Comparative analysis of growth and physio-biochemical responses of Hydrilla verticillata to different sediments in freshwater microcosms Haichan Yu a,b , Chun Ye b,∗ , Xiangfu Song c , Jie Liu b a
College of Life Sciences, Beijing Normal University, Beijing 100875, China Research Center of Lake Ecological Environment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China c Shanghai Academy of Agricultural Sciences, Shanghai 201106, China b
a r t i c l e
i n f o
Article history: Received 14 March 2009 Received in revised form 13 April 2010 Accepted 5 June 2010
Keywords: Sediment Hydrilla verticillata Growth Physio-biochemical response
a b s t r a c t The long-term effect of Hydrilla verticillata to different dredged sediments was investigated in laboratory aquatic microcosms. Two kinds of lake sediments, i.e., fertile sludge and brown clay, which were acquired by deep or slight dredging and differed in their nutrients levels and particle sizes, were used as growth substrate. One year after H. verticillata seedlings were transplanted into the microcosms, growth and physio-biochemical characters of the plants were estimated. In our study, sludge sediment eventually promoted the increase of the biomass, shoot length, bifurcation, nitrogen, phosphorus, soluble protein content of H. verticillata, but not root number and root mass ratio. Additionally, the soluble sugar content was significantly lower in summer (August) in the sludge sediment. Sediment nutrients also increased photosynthetic pigment content, while POD and SOD activities were not affected. The fertile sludge and brown clay formed by different dredging are quite different, but both sediments could meet the requirement of H. verticillata during early growth stage. The fertile sediment after moderate dredge-up fit the growth of H. verticillata. The brown clay formed after excessive dredging showed a lack of nutrients and therefore led to the weak growth of the aquatic plants. It is concluded that moderate dredging was suitable for H. verticillata growth and for absorbing nitrogen and phosphorus from eutrophic fresh water. The experiment also suggested that submersed aquatic plant restoration in freshwater can be promoted by human assistance. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The eutrophication of shallow lakes has increased in recent years, with the major phenomenon of overgrown algae, low water clarity, oxygen deficiency, bad smell and polluted sediment slurry deposit. As we know, cutting down the nutrients in lakes is the basic measure against eutrophication; however, it is still difficult to improve the lake environment in a shorter period even when the external pollution sources have been controlled efficiently. A feasible option is eliminating the internal pollution source by dredging the polluted sediments and recovering the aquatic vegetation by improving their habitat conditions (Zhang et al., 2010). Sediment slurry is the main inner nutrient source and habitat for most of the submersed aquatic plants in shallow lakes, and its nutrient content influences the growth of these plants. Sediment from different dredging depths will show very different properties and influence the restoration of submersed aquatic plants.
∗ Corresponding author. E-mail address: [email protected] (C. Ye). 0925-8574/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2010.06.004
The overabundance of sediment nutrients (especially nitrogen and phosphorus) and organic matter are two important indicators of shallow lake eutrophication, and can affect aquatic plant growth directly or indirectly (Ni, 2001a,b; Matheson et al., 2005; Newbolt et al., 2008). According to previous studies, fertile sediment can provide plentiful nutrients, and benefit growth, bourgeoning, propagation of aquatic plants and increase in their bifurcation and biomass (Li and Cui, 2000; Zhang et al., 2008), but sediment with excessive fertilizer tends to produce stress effects on aquatic plants (Phillips et al., 1978; Wijk et al., 1992; Smolders and Roelofs, 1993). An excess of nutrients can be deleterious to plants, e.g., excess ammonia enrichment will trigger the promotion of oxidative stress in aquatic macrophytes (Medici et al., 2004; Nimptsch and Pflugmacher, 2007; Ni, 2001a,b; Brun et al., 2002; Cao and Ni, 2004; Cao et al., 2004, 2007; Wang et al., 2008) since most sediments are anoxic, with NH4 + being the dominant nitrogen form (Wetzel, 1983). The Ministry of Science and Technology of the People’s Republic of China (MOST of China) established several projects on water pollution control and water remediation of Chinese lakes in recent years. Sediment dredging is a commonly used method in those
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projects to control and clean up pollutants and is helpful for large-scaled ecological restoration in lakes. However, the nutrition, ventilation and other conditions might be quite different after dredging, which will affect the restoration of macrophytes and nutrient recyclings. Hydrilla verticillata (H. verticillata) was selected for study because of its strong capability for growth in water. The objectives of this study were to (1) simulate the weak light in the eutrophic lake and clarify the response of morphology and physio-biochemistry of H. verticillata on two types of sediments from different dredging treatments; (2) analyze the influences of different dredging depths on submersed aquatic plant restoration, (3) guide the dredging project for submersed plant restoration. 2. Materials and methods 2.1. Experiment plants H. verticillata, a member of the Hydrocharitaceae family, is a perennial submersed aquatic plant, widely used in lake ecological restoration because of its extensive spread in fresh lakes of China, strong adaptability, fast growth and reproduction and its function of improving water quality. The experimental H. verticillata was collected from Wuli Bay, Taihu Lake, China, an eutrophic lake at the outskirts of the city with no heavy metal pollution in its sediment. 2.2. Microcosm preparation Microcosms were prepared in culture containers in September 2004. A 12 cm depth of sediment was laid on the bottom of each culture container (inner size: 73 cm × 53 cm × 59 cm). The visible decayed plant matter and inorganic matter were removed from the sediments, which were then mixed thoroughly. The H. verticillata seedlings were selected by specific standards: Plant length 16.2 ± 1.62 cm, root length 13.8 ± 4.44 cm, bifurcation 2.3 ± 1.08, and wet biomass 1.42 g. The seedlings were washed several times to clean up the original sediments around their roots and then planted in the culture containers. Forty individuals were planted in each container, and the replicate container number of each type of sediment was 8. The water depth in the culture container was kept at 33 cm. The containers were placed in the greenhouse covered with a sun-shading net (transmittance was about 10%). Dissolved oxygen (DO) in water was keep around 5.0 mg/L. The experimental period was over one year, from September to October of the next year. The sediment and overlying water were also collected from Xiwuli Lake (located in Wuxi City, Jiangsu Province) of Lake Taihu. The average nitrogen and phosphorus of overlying water were 2.70–5.01 mg/L, and 0.056–0.104 mg/L, respectively. Two sediments were used, namely, brown clay (named as BC) and fertile sludge (named as FS), which were collected from the sediment after deep and slight dredging in the original fishery area of Xiwuli Lake, respectively. Of the two sediments, fertile sludge was enriched of organic matter (OM), nitrogen (TN) and phosphorus (TP). The brown clay sediment had low levels of OM, TN, and TP, equal to about 12%, 23% and 60% of those in fertile sludge, respectively (Table 1; Ye et al., 2009). However, the fertile sludge had lower strength, stability and smaller particle size (Table 2; Ye et al., 2009). Table 1 Content of organic matter (OM), total nitrogen (TN), and total phosphorus (TP) of the sediments at the beginning of the experiment. Sediment type
OM (%)
TN (%)
TP (%)
Brown clay Fertile sludge
0.272 2.294
0.034 0.147
0.048 0.080
Table 2 Size distribution of particle sizes of sediments used in the experiment. Sediment type
Brown clay Fertile sludge
Size distribution of particle sizes (%) <0.01 mm
0.01–0.05 mm
>0.05 mm
16 18
15 20
69 62
2.3. Analytic methods H. verticillata seedlings in four replicated containers of each sediment were sampled at two stages: the next year’s summer (15 August) and autumn (15 October). The samples were washed by distilled water, and then dried with absorbent papers. The shoot length, root length and number, and bifurcation were measured or counted as soon as possible. The shoots and roots of the plants were then separated and dried in oven at 105 ◦ C for 1 h, and then at a constant temperature of 80 ◦ C until the weight of the samples reached constant. The dry weights of the whole plant, shoot and root were then measured. Dry shoot materials were used to measure total nitrogen and phosphorus content. Total nitrogen and phosphorus contents were determined following standard sulfuric acid–hydrogen peroxide digest–Nesster’s reagent colorimetric method and sulfuric acid–hydrogen peroxide digest–molybdenum blue colorimetry, respectively. Fresh leaves on the top 5 cm of the fresh branches were prepared for physiological and biochemical parameters determination. Chlorophyl and carotinoid contents were analyzed according to modified Arnon’s method; soluble protein content was measured following Coomassie Brillant Blue G-250 Dye Binding method, soluble sugar following anthrone colorimetric method, superoxide dismutase (SOD) activity following nitroblue tetrazolium (NBT) method and peroxidase (POD) activity following Guaiacol method. For analyzing plant biomass, bifurcation, shoot height, root length and root number, 20 individuals were used to calculate the means; for all the other analysis including overlying water and sediment, four replicates were used. Other parameters, such as total nitrogen, phosphorus, dissolved oxygen in the overlying water and the illuminance inner or outer of the greenhouse, were determined twice a week. Total nitrogen and total phosphorus in the overlying water were measured using a colorimetric method adapted to auto-analyzer (Skalar San Plus system, Skalar, Inc., The Netherlands). Dissolved oxygen was determined by U-22XD Water Quality Monitoring System (Horiba, Ltd., Kyoto, Japan). Illuminance was measured by ZDS-10 luminometer (Jiadingxuelian Co., Shanghai, China). 2.4. Statistical analysis Data were analyzed using t-test and non-parametric test for comparison of means (P < 0.01 or 0.05) with statistical software SPSS10.0.7. 3. Results 3.1. Plant biomass and morphological changes Table 3 shows the influence of sediment type on the characteristics of H. verticillata biomass and morphologic. As illustrated in Table 3, the single plant biomass of H. verticillata growing on FS was higher than that growing on BC (P < 0.05), and the single plant biomass of H. verticillata for FS was 1.57 ± 0.29 g, which was four times the BC in August 2005. From August to October, the biomass of BC did not show significant increase (t = −0.325, P > 0.05), while
H. Yu et al. / Ecological Engineering 36 (2010) 1285–1289 Table 3 Effects of different sediments on phenotypic characteristics of H. verticillata (means ± SD). Date
Parameter
Brown clay
August
Biomass (g/plant) Shoot height (cm) Bifurcation Root length (cm) Root number Root weight ratio, R/T
0.38 50.3 3.30 19.9 10.93 0.112
± ± ± ± ± ±
0.22 14.10 0.64 3.60 3.70 0.035
1.57 73.1 2.01 18.9 4.36 0.017
± ± ± ± ± ±
0.29** 20.28* 0.88* 4.36 2.18* 0.006**
Biomass (g/plant) Shoot height (cm) Bifurcation Root length (cm) Root number Root weight ratio, R/T
0.41 41.8 2.97 15.4 8.15 0.044
± ± ± ± ± ±
0.20 7.92 0.85 4.77 2.49 0.015
3.89 77.9 4.43 22.2 5.29 0.019
± ± ± ± ± ±
1.15* 26.17* 0.42* 4.92* 2.06** 0.008*
October
* **
Fertile sludge
P < 0.05 vs brown clay group. P < 0.01 vs brown clay group.
that of FS increased significantly (t = −4.247, P < 0.05). Consequently, the increased rates of plant biomass show a significant difference for different sediments, the biomass of FS was about 9.5 times of BC. The variation of shoot length has a trend similar to the biomass. The shoot length of H. verticillata of FS was higher than BC (P < 0.05) during the two sampling times. The shoot length of FS was about 40–50 cm and over 70 cm. However, according to statistic results, the shoot length on two kinds of sediments did not show significant increase from August to October (BC: t = 1.462, P > 0.05; FS: t = −0.349, P > 0.05). The root number of BC and FS produced 8 per plant and only 4–6, respectively. A similar trend was observed for root mass ratio (R/T), which was 1.7% (August) and 1.9% (October) for FS; and 11.2% (August) and 4.4% (October) for BC. No significant difference in root numbers was found between August and October (BC: t = 0.926, P > 0.05; FS: t = −0.594, P > 0.05). Furthermore, R/T value for BC showed significant decline from August to October (t = 3.205, P < 0.05); however, R/T value for FS had no statistical difference during two sampling times (t = −0.340, P > 0.05). For root length, no statistical difference was found on two sediments in August (P > 0.05), about 19 cm; but in October the root length for FS was 6.8 cm, which was longer than BC. Statistical differences of bifurcation from August to October were observed. In August, the bifurcation for BC was 1.29 higher than that of FS. But in October, the bifurcation for FS increased twice compared to that in August As a result, the bifurcation was higher for FS than BC in October. 3.2. Antioxidase activity The data of POD and SOD activities are shown in Table 4. According to statistical test, no difference was demonstrated between the two experimental designs. Though POD for BC increased from 16.2 ± 2.8 U/g min to 28.7 ± 2.8 U/g min, others changed little.
Table 4 Effects of different sediments on POD and SOD activities of H. verticillata (means ± SD). Date
Parameter
Brown clay
Fertile sludge
August
POD (U/g min) SOD (U/g)
16.2 ± 2.8 57.0 ± 7.6
19.2 ± 6.3 55.6 ± 11.1
October
POD (U/g min) SOD (U/g)
28.7 ± 2.8 89.5 ± 31.4
24.1 ± 4.7 89.3 ± 18.7
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Table 5 Effects of different sediments on pigment contents of H. verticillata (means ± SD). Date
Parameter
Brown clay
August
Chlorophyl a (mg/g) Chlorophyl b (mg/g) Chlorophyl (a + b) (mg/g) Carotenoid (mg/g)
1.270 0.448 1.718 0.462
± ± ± ±
0.410 0.147 0.556 0.092
1.319 0.444 1.763 0.405
± ± ± ±
0.366 0.156 0.522 0.114
October
Chlorophyl a (mg/g) Chlorophyl b (mg/g) Chlorophyl (a + b) (mg/g) Carotenoid (mg/g)
0.701 0.248 0.949 0.222
± ± ± ±
0.278 0.086 0.364 0.076
1.644 0.563 2.207 0.434
± ± ± ±
0.158** 0.052** 0.209** 0.036*
* **
Fertile sludge
P < 0.05 vs brown clay group. P < 0.01 vs brown clay group.
Table 6 Effects of different sediments on contents of some biochemical items of H. verticillata (means ± SD). Date
Parameter
Brown clay
August
Soluble sugar (%) Soluble protein (mg/g) N (%) P (%)
0.198 3.019 1.432 0.243
± ± ± ±
0.035 0.424 0.378 0.095
0.131 5.807 2.802 0.239
± ± ± ±
0.042* 1.160* 0.497* 0.093
October
Soluble sugar (%) Soluble protein (mg/g) N (%) P (%)
0.259 4.774 1.775 0.060
± ± ± ±
0.031 1.255 0.313 0.015
0.307 8.935 2.614 0.180
± ± ± ±
0.147 0.840* 0.691* 0.012**
* **
Fertile sledge
P < 0.05 vs brown clay group. P < 0.01 vs brown clay group.
3.3. Photosynthetic pigments In August, chlorophyl a, chlorophyl b, carotenoid and chlorophyl (a + b) between different sediments were not significantly different (Table 5). Chlorophyl contents in August from BC and FS were 1.718 ± 0.556 mg/L and 1.763 ± 0.522 mg/L, respectively, while carotenoid contents were 0.462 ± 0.092 mg/L and 0.405 ± 0.114 mg/L, respectively. In October, these four parameters displayed the same trend, the rank order was FS > BC. 3.4. Tissue contents of total nitrogen, phosphorus, soluble sugar and protein In August, the nitrogen, soluble sugar and soluble protein content showed significant difference between the two sediments, while tissue phosphorus content showed no difference (Table 6). The content of soluble sugar for BC was higher than FS, while the contents of soluble protein and nitrogen were just the opposite. In October, this trend changed, and there was not noticeable difference in soluble sugar between different sediments, while the soluble protein content for FS was still higher than BC. Furthermore, the content of nitrogen and phosphorus for FS was much higher than BC. Comparing the data of October to that of August, the content of both soluble sugar and soluble protein increased, besides the nitrogen contents (P = 0.256 for BC; P = 0.740 for FS), while phosphorus content for BC declined markedly (t = 3.589, P = 0.037). 4. Discussion The exchange of nutrients between the sediment and overlying water was always existing, and thus sediment conditions will inevitably influence sediment–water interface and overlying water conditions, such as nutrient, physical–chemical, light and other conditions, and finally affects plant growth. The freshwater microcosms used in this study simulated lake environments, with the same light, water, oxygen and other conditions suitable for the
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growth of submersed macrophytes among each microcosm except for sediments. The results of this study helps us concentrating just one factor, i.e., sediment condition/composition (such as particle size and nutrient content, etc.), because all the different responses of H. verticillata can be finally attributed to different sediment conditions. H. verticillata can acclimatize itself to different sediment conditions via self-adjust, e.g., via physiological and morphological changes, and our results were consistent with others (Spencer and Ksander, 2005; Xie et al., 2005; Chu et al., 2006; Dooris and Martin, 2007). As demonstrated in our study, H. verticillata has a higher adaptability to the soil and they can grow on different sediments, but they differed greatly in their morphology, biomass, physiological and biochemical parameters. Plants can achieve the trade-off between internal aeration and nutrient acquisition by adjusting the structure of the root system and the pattern of biomass allocation (Xie et al., 2007; Jiang et al., 2008). Biomass production increased with fertilization and changes either in allocative ratios or in tuber production patterns were shown in response to nutrient availability for H. verticillata (Mony et al., 2007). Consistent with previous reports, high nutrient availability, e.g., organic matters and nitrogen, etc., increased the biomass, shoot length, bifurcation and shoot nitrogen content of H. verticillata, but decreased the root weight ratio due to the promotion of plant photosynthesis and metabolism (Cronin and Lodge, 2003; Wang and Yu, 2007). Nitrogen and phosphorus content will also affect the morphology of plant root system, e.g., the number of rice roots and its root formation capability enhanced along with the increase of nitrogen content of the plants, but excessive nitrogen supplement results in the decrease of the root number, root length and root mass ratio. Furthermore, there are also reports demonstrating that root formation rate of the rice declined along with the increase of nitrogen supplement when plants were at early growth stage (Wang et al., 1997). According to the above results, the abundant nitrogen supplement during early growth stage might be one of the reasons explaining the smaller root number and equal root length on fertile sludge in summer. Plants will enlarge the interface between their root system and the soil to adapt to the situation of phosphorus deficiency, and then show the elongation of lateral roots and root hair and the increase of their number. At the same time, more photosynthesis products are transferred to the root system, which leads to the accelerated relative growth rate of the roots and the increase of root-top ratio (R/T), and consequently promote the uptake and usage of the root to the phosphorus (Liu et al., 1999). Thus, the root morphology in this experiment indicated the effect and interaction of all the nutrient factors. Nutrient content will also affect the antioxidase system of the plant, e.g., within definite extension, an increase of nitrogen supplement will lead to an increase of antioxidase activity (Cao and Ni, 2004; Cao et al., 2004). Except phosphorus deficiency and nitrogen supplement, deoxidizing sediment will also lead to he growing stress. The fact that the activities of SOD and POD were not significantly different between different sediments in our study is because the condition in our study did not create enough stress to the plants during the whole plant life cycle. Nitrogen is one of the constituents of plant photosynthetic pigment and it plays important role in plant chlorophyl content. Phosphorus also affects the photosynthesis procedure and an increase of phosphorus supplement will result in significant increase of plant leaf area and biomass. In August when plants were flourishing, different sediments did not influence the photosynthetic pigment content significantly. The reason leading to such result is that the nutrients, especially nitrogen, contained in two kinds of sediments are both adequate; when the synthesis and transfer of chlorophyl reach a balance, excessive nitrogen supple-
ment is nonsense and an increase of chlorophyl content was not found. Accordingly, nitrogen content of 0.034% in brown clay under our experimental condition can meet the demand of chlorophyl synthesis and transfer. The adequate nitrogen supplement will help maintain a high chlorophyl content and lead to late maturation, while low nitrogen treatment will make the plant die earlier. Due to the above reasons, photosynthetic pigment content from fertile sludge was higher in autumn (October). At that time, the capability of photosynthesis of H. verticillata from fertile sludge sediment was kept relatively stronger. Total soluble sugar, including glucose, sucrose, fructose, etc., acts as osmoregulation substance when stress especially osmotic stress happened. In August when plants were flourishing and substance metabolism was active, soluble sugar in plant was relatively low, and it was higher in plants on brown clay than on fertile sledge sediment. In October, the sediment nutrients were largely consumed during plant growth stages, which reduces the fertile level of the sediments, along with the senescence of plants, the soluble sugar content increased and the difference of the content from these two kinds of sediments became insignificant. Within a definite range, nitrogen will promote the increase of tissue nitrogen content and nitrogen assimilation rates, and along with the enhancing of the photosynthesis. Plants on fertile sludge sediments promoted nitrogen metabolism; therefore, the soluble protein content of H. verticillata on fertile sediment was higher than that from brown clay. Gerloff and Krombholz (1966) found that the minimum limits of tissue nitrogen and phosphorus content for the growth of hydrophytes are 1.3% and 0.13%, respectively. The nitrogen content and phosphorus content of the H. verticillata in August had met that requirement and no significant difference was found between the two sediments; Nevertheless, the tissue phosphorus content declined sharply and was merely 0.06% in October, which shows a symptom of phosphorus deficiency. The symptom in October indicated that the phosphorus content of brown clay was quite low and it could not meet the requirement for the growth of H. verticillata. Phosphorus deficiency will lead to stunt cell division and make it difficult for new cell to produce, and it also influences the elongation of the cell; so the morphology of the plant was found to be stunted growth, dwarfism and lower bifurcation ratio, which were observed in our experiment. The fertile sediment increased shoot and root length, bifurcation, biomass, ratio of shoot biomass of H. verticillata, and helped plants to accumulate soluble protein and nitrogen. It was also advantageous for plants to maintain high chlorophyl content, and then to keep a higher photosynthesis capability in the autumn. Our results demonstrated that the recovery of the submersed macrophytes in eutrophica shallow lakes could be promoted by human assistance, e.g., moderate dredging and plant or even seed bank transplantation. The research results provide academic and technical support for the ecological restoration of the lakes.
Acknowledgements The research described in this paper was supported by the grants from national water pollution control and management technology major projects of China (2009ZX07101-009) and national natural science foundation of China (40971277). We gratefully appreciate Prof. Gaomin Jiang, Dr. Linfeng Li, and Dr. Yinghao Li of Institute of Botany, the Chinese Academy of Sciences for their warm help in experiment execution and their valuable suggestion on this paper.
H. Yu et al. / Ecological Engineering 36 (2010) 1285–1289
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Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Comparative analysis of growth and physio-biochemical responses of Hydrilla verticillata to different sediments in freshwater microcosms Haichan Yu a,b , Chun Ye b,∗ , Xiangfu Song c , Jie Liu b a
College of Life Sciences, Beijing Normal University, Beijing 100875, China Research Center of Lake Ecological Environment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China c Shanghai Academy of Agricultural Sciences, Shanghai 201106, China b
a r t i c l e
i n f o
Article history: Received 14 March 2009 Received in revised form 13 April 2010 Accepted 5 June 2010
Keywords: Sediment Hydrilla verticillata Growth Physio-biochemical response
a b s t r a c t The long-term effect of Hydrilla verticillata to different dredged sediments was investigated in laboratory aquatic microcosms. Two kinds of lake sediments, i.e., fertile sludge and brown clay, which were acquired by deep or slight dredging and differed in their nutrients levels and particle sizes, were used as growth substrate. One year after H. verticillata seedlings were transplanted into the microcosms, growth and physio-biochemical characters of the plants were estimated. In our study, sludge sediment eventually promoted the increase of the biomass, shoot length, bifurcation, nitrogen, phosphorus, soluble protein content of H. verticillata, but not root number and root mass ratio. Additionally, the soluble sugar content was significantly lower in summer (August) in the sludge sediment. Sediment nutrients also increased photosynthetic pigment content, while POD and SOD activities were not affected. The fertile sludge and brown clay formed by different dredging are quite different, but both sediments could meet the requirement of H. verticillata during early growth stage. The fertile sediment after moderate dredge-up fit the growth of H. verticillata. The brown clay formed after excessive dredging showed a lack of nutrients and therefore led to the weak growth of the aquatic plants. It is concluded that moderate dredging was suitable for H. verticillata growth and for absorbing nitrogen and phosphorus from eutrophic fresh water. The experiment also suggested that submersed aquatic plant restoration in freshwater can be promoted by human assistance. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The eutrophication of shallow lakes has increased in recent years, with the major phenomenon of overgrown algae, low water clarity, oxygen deficiency, bad smell and polluted sediment slurry deposit. As we know, cutting down the nutrients in lakes is the basic measure against eutrophication; however, it is still difficult to improve the lake environment in a shorter period even when the external pollution sources have been controlled efficiently. A feasible option is eliminating the internal pollution source by dredging the polluted sediments and recovering the aquatic vegetation by improving their habitat conditions (Zhang et al., 2010). Sediment slurry is the main inner nutrient source and habitat for most of the submersed aquatic plants in shallow lakes, and its nutrient content influences the growth of these plants. Sediment from different dredging depths will show very different properties and influence the restoration of submersed aquatic plants.
∗ Corresponding author. E-mail address: [email protected] (C. Ye). 0925-8574/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2010.06.004
The overabundance of sediment nutrients (especially nitrogen and phosphorus) and organic matter are two important indicators of shallow lake eutrophication, and can affect aquatic plant growth directly or indirectly (Ni, 2001a,b; Matheson et al., 2005; Newbolt et al., 2008). According to previous studies, fertile sediment can provide plentiful nutrients, and benefit growth, bourgeoning, propagation of aquatic plants and increase in their bifurcation and biomass (Li and Cui, 2000; Zhang et al., 2008), but sediment with excessive fertilizer tends to produce stress effects on aquatic plants (Phillips et al., 1978; Wijk et al., 1992; Smolders and Roelofs, 1993). An excess of nutrients can be deleterious to plants, e.g., excess ammonia enrichment will trigger the promotion of oxidative stress in aquatic macrophytes (Medici et al., 2004; Nimptsch and Pflugmacher, 2007; Ni, 2001a,b; Brun et al., 2002; Cao and Ni, 2004; Cao et al., 2004, 2007; Wang et al., 2008) since most sediments are anoxic, with NH4 + being the dominant nitrogen form (Wetzel, 1983). The Ministry of Science and Technology of the People’s Republic of China (MOST of China) established several projects on water pollution control and water remediation of Chinese lakes in recent years. Sediment dredging is a commonly used method in those
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projects to control and clean up pollutants and is helpful for large-scaled ecological restoration in lakes. However, the nutrition, ventilation and other conditions might be quite different after dredging, which will affect the restoration of macrophytes and nutrient recyclings. Hydrilla verticillata (H. verticillata) was selected for study because of its strong capability for growth in water. The objectives of this study were to (1) simulate the weak light in the eutrophic lake and clarify the response of morphology and physio-biochemistry of H. verticillata on two types of sediments from different dredging treatments; (2) analyze the influences of different dredging depths on submersed aquatic plant restoration, (3) guide the dredging project for submersed plant restoration. 2. Materials and methods 2.1. Experiment plants H. verticillata, a member of the Hydrocharitaceae family, is a perennial submersed aquatic plant, widely used in lake ecological restoration because of its extensive spread in fresh lakes of China, strong adaptability, fast growth and reproduction and its function of improving water quality. The experimental H. verticillata was collected from Wuli Bay, Taihu Lake, China, an eutrophic lake at the outskirts of the city with no heavy metal pollution in its sediment. 2.2. Microcosm preparation Microcosms were prepared in culture containers in September 2004. A 12 cm depth of sediment was laid on the bottom of each culture container (inner size: 73 cm × 53 cm × 59 cm). The visible decayed plant matter and inorganic matter were removed from the sediments, which were then mixed thoroughly. The H. verticillata seedlings were selected by specific standards: Plant length 16.2 ± 1.62 cm, root length 13.8 ± 4.44 cm, bifurcation 2.3 ± 1.08, and wet biomass 1.42 g. The seedlings were washed several times to clean up the original sediments around their roots and then planted in the culture containers. Forty individuals were planted in each container, and the replicate container number of each type of sediment was 8. The water depth in the culture container was kept at 33 cm. The containers were placed in the greenhouse covered with a sun-shading net (transmittance was about 10%). Dissolved oxygen (DO) in water was keep around 5.0 mg/L. The experimental period was over one year, from September to October of the next year. The sediment and overlying water were also collected from Xiwuli Lake (located in Wuxi City, Jiangsu Province) of Lake Taihu. The average nitrogen and phosphorus of overlying water were 2.70–5.01 mg/L, and 0.056–0.104 mg/L, respectively. Two sediments were used, namely, brown clay (named as BC) and fertile sludge (named as FS), which were collected from the sediment after deep and slight dredging in the original fishery area of Xiwuli Lake, respectively. Of the two sediments, fertile sludge was enriched of organic matter (OM), nitrogen (TN) and phosphorus (TP). The brown clay sediment had low levels of OM, TN, and TP, equal to about 12%, 23% and 60% of those in fertile sludge, respectively (Table 1; Ye et al., 2009). However, the fertile sludge had lower strength, stability and smaller particle size (Table 2; Ye et al., 2009). Table 1 Content of organic matter (OM), total nitrogen (TN), and total phosphorus (TP) of the sediments at the beginning of the experiment. Sediment type
OM (%)
TN (%)
TP (%)
Brown clay Fertile sludge
0.272 2.294
0.034 0.147
0.048 0.080
Table 2 Size distribution of particle sizes of sediments used in the experiment. Sediment type
Brown clay Fertile sludge
Size distribution of particle sizes (%) <0.01 mm
0.01–0.05 mm
>0.05 mm
16 18
15 20
69 62
2.3. Analytic methods H. verticillata seedlings in four replicated containers of each sediment were sampled at two stages: the next year’s summer (15 August) and autumn (15 October). The samples were washed by distilled water, and then dried with absorbent papers. The shoot length, root length and number, and bifurcation were measured or counted as soon as possible. The shoots and roots of the plants were then separated and dried in oven at 105 ◦ C for 1 h, and then at a constant temperature of 80 ◦ C until the weight of the samples reached constant. The dry weights of the whole plant, shoot and root were then measured. Dry shoot materials were used to measure total nitrogen and phosphorus content. Total nitrogen and phosphorus contents were determined following standard sulfuric acid–hydrogen peroxide digest–Nesster’s reagent colorimetric method and sulfuric acid–hydrogen peroxide digest–molybdenum blue colorimetry, respectively. Fresh leaves on the top 5 cm of the fresh branches were prepared for physiological and biochemical parameters determination. Chlorophyl and carotinoid contents were analyzed according to modified Arnon’s method; soluble protein content was measured following Coomassie Brillant Blue G-250 Dye Binding method, soluble sugar following anthrone colorimetric method, superoxide dismutase (SOD) activity following nitroblue tetrazolium (NBT) method and peroxidase (POD) activity following Guaiacol method. For analyzing plant biomass, bifurcation, shoot height, root length and root number, 20 individuals were used to calculate the means; for all the other analysis including overlying water and sediment, four replicates were used. Other parameters, such as total nitrogen, phosphorus, dissolved oxygen in the overlying water and the illuminance inner or outer of the greenhouse, were determined twice a week. Total nitrogen and total phosphorus in the overlying water were measured using a colorimetric method adapted to auto-analyzer (Skalar San Plus system, Skalar, Inc., The Netherlands). Dissolved oxygen was determined by U-22XD Water Quality Monitoring System (Horiba, Ltd., Kyoto, Japan). Illuminance was measured by ZDS-10 luminometer (Jiadingxuelian Co., Shanghai, China). 2.4. Statistical analysis Data were analyzed using t-test and non-parametric test for comparison of means (P < 0.01 or 0.05) with statistical software SPSS10.0.7. 3. Results 3.1. Plant biomass and morphological changes Table 3 shows the influence of sediment type on the characteristics of H. verticillata biomass and morphologic. As illustrated in Table 3, the single plant biomass of H. verticillata growing on FS was higher than that growing on BC (P < 0.05), and the single plant biomass of H. verticillata for FS was 1.57 ± 0.29 g, which was four times the BC in August 2005. From August to October, the biomass of BC did not show significant increase (t = −0.325, P > 0.05), while
H. Yu et al. / Ecological Engineering 36 (2010) 1285–1289 Table 3 Effects of different sediments on phenotypic characteristics of H. verticillata (means ± SD). Date
Parameter
Brown clay
August
Biomass (g/plant) Shoot height (cm) Bifurcation Root length (cm) Root number Root weight ratio, R/T
0.38 50.3 3.30 19.9 10.93 0.112
± ± ± ± ± ±
0.22 14.10 0.64 3.60 3.70 0.035
1.57 73.1 2.01 18.9 4.36 0.017
± ± ± ± ± ±
0.29** 20.28* 0.88* 4.36 2.18* 0.006**
Biomass (g/plant) Shoot height (cm) Bifurcation Root length (cm) Root number Root weight ratio, R/T
0.41 41.8 2.97 15.4 8.15 0.044
± ± ± ± ± ±
0.20 7.92 0.85 4.77 2.49 0.015
3.89 77.9 4.43 22.2 5.29 0.019
± ± ± ± ± ±
1.15* 26.17* 0.42* 4.92* 2.06** 0.008*
October
* **
Fertile sludge
P < 0.05 vs brown clay group. P < 0.01 vs brown clay group.
that of FS increased significantly (t = −4.247, P < 0.05). Consequently, the increased rates of plant biomass show a significant difference for different sediments, the biomass of FS was about 9.5 times of BC. The variation of shoot length has a trend similar to the biomass. The shoot length of H. verticillata of FS was higher than BC (P < 0.05) during the two sampling times. The shoot length of FS was about 40–50 cm and over 70 cm. However, according to statistic results, the shoot length on two kinds of sediments did not show significant increase from August to October (BC: t = 1.462, P > 0.05; FS: t = −0.349, P > 0.05). The root number of BC and FS produced 8 per plant and only 4–6, respectively. A similar trend was observed for root mass ratio (R/T), which was 1.7% (August) and 1.9% (October) for FS; and 11.2% (August) and 4.4% (October) for BC. No significant difference in root numbers was found between August and October (BC: t = 0.926, P > 0.05; FS: t = −0.594, P > 0.05). Furthermore, R/T value for BC showed significant decline from August to October (t = 3.205, P < 0.05); however, R/T value for FS had no statistical difference during two sampling times (t = −0.340, P > 0.05). For root length, no statistical difference was found on two sediments in August (P > 0.05), about 19 cm; but in October the root length for FS was 6.8 cm, which was longer than BC. Statistical differences of bifurcation from August to October were observed. In August, the bifurcation for BC was 1.29 higher than that of FS. But in October, the bifurcation for FS increased twice compared to that in August As a result, the bifurcation was higher for FS than BC in October. 3.2. Antioxidase activity The data of POD and SOD activities are shown in Table 4. According to statistical test, no difference was demonstrated between the two experimental designs. Though POD for BC increased from 16.2 ± 2.8 U/g min to 28.7 ± 2.8 U/g min, others changed little.
Table 4 Effects of different sediments on POD and SOD activities of H. verticillata (means ± SD). Date
Parameter
Brown clay
Fertile sludge
August
POD (U/g min) SOD (U/g)
16.2 ± 2.8 57.0 ± 7.6
19.2 ± 6.3 55.6 ± 11.1
October
POD (U/g min) SOD (U/g)
28.7 ± 2.8 89.5 ± 31.4
24.1 ± 4.7 89.3 ± 18.7
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Table 5 Effects of different sediments on pigment contents of H. verticillata (means ± SD). Date
Parameter
Brown clay
August
Chlorophyl a (mg/g) Chlorophyl b (mg/g) Chlorophyl (a + b) (mg/g) Carotenoid (mg/g)
1.270 0.448 1.718 0.462
± ± ± ±
0.410 0.147 0.556 0.092
1.319 0.444 1.763 0.405
± ± ± ±
0.366 0.156 0.522 0.114
October
Chlorophyl a (mg/g) Chlorophyl b (mg/g) Chlorophyl (a + b) (mg/g) Carotenoid (mg/g)
0.701 0.248 0.949 0.222
± ± ± ±
0.278 0.086 0.364 0.076
1.644 0.563 2.207 0.434
± ± ± ±
0.158** 0.052** 0.209** 0.036*
* **
Fertile sludge
P < 0.05 vs brown clay group. P < 0.01 vs brown clay group.
Table 6 Effects of different sediments on contents of some biochemical items of H. verticillata (means ± SD). Date
Parameter
Brown clay
August
Soluble sugar (%) Soluble protein (mg/g) N (%) P (%)
0.198 3.019 1.432 0.243
± ± ± ±
0.035 0.424 0.378 0.095
0.131 5.807 2.802 0.239
± ± ± ±
0.042* 1.160* 0.497* 0.093
October
Soluble sugar (%) Soluble protein (mg/g) N (%) P (%)
0.259 4.774 1.775 0.060
± ± ± ±
0.031 1.255 0.313 0.015
0.307 8.935 2.614 0.180
± ± ± ±
0.147 0.840* 0.691* 0.012**
* **
Fertile sledge
P < 0.05 vs brown clay group. P < 0.01 vs brown clay group.
3.3. Photosynthetic pigments In August, chlorophyl a, chlorophyl b, carotenoid and chlorophyl (a + b) between different sediments were not significantly different (Table 5). Chlorophyl contents in August from BC and FS were 1.718 ± 0.556 mg/L and 1.763 ± 0.522 mg/L, respectively, while carotenoid contents were 0.462 ± 0.092 mg/L and 0.405 ± 0.114 mg/L, respectively. In October, these four parameters displayed the same trend, the rank order was FS > BC. 3.4. Tissue contents of total nitrogen, phosphorus, soluble sugar and protein In August, the nitrogen, soluble sugar and soluble protein content showed significant difference between the two sediments, while tissue phosphorus content showed no difference (Table 6). The content of soluble sugar for BC was higher than FS, while the contents of soluble protein and nitrogen were just the opposite. In October, this trend changed, and there was not noticeable difference in soluble sugar between different sediments, while the soluble protein content for FS was still higher than BC. Furthermore, the content of nitrogen and phosphorus for FS was much higher than BC. Comparing the data of October to that of August, the content of both soluble sugar and soluble protein increased, besides the nitrogen contents (P = 0.256 for BC; P = 0.740 for FS), while phosphorus content for BC declined markedly (t = 3.589, P = 0.037). 4. Discussion The exchange of nutrients between the sediment and overlying water was always existing, and thus sediment conditions will inevitably influence sediment–water interface and overlying water conditions, such as nutrient, physical–chemical, light and other conditions, and finally affects plant growth. The freshwater microcosms used in this study simulated lake environments, with the same light, water, oxygen and other conditions suitable for the
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growth of submersed macrophytes among each microcosm except for sediments. The results of this study helps us concentrating just one factor, i.e., sediment condition/composition (such as particle size and nutrient content, etc.), because all the different responses of H. verticillata can be finally attributed to different sediment conditions. H. verticillata can acclimatize itself to different sediment conditions via self-adjust, e.g., via physiological and morphological changes, and our results were consistent with others (Spencer and Ksander, 2005; Xie et al., 2005; Chu et al., 2006; Dooris and Martin, 2007). As demonstrated in our study, H. verticillata has a higher adaptability to the soil and they can grow on different sediments, but they differed greatly in their morphology, biomass, physiological and biochemical parameters. Plants can achieve the trade-off between internal aeration and nutrient acquisition by adjusting the structure of the root system and the pattern of biomass allocation (Xie et al., 2007; Jiang et al., 2008). Biomass production increased with fertilization and changes either in allocative ratios or in tuber production patterns were shown in response to nutrient availability for H. verticillata (Mony et al., 2007). Consistent with previous reports, high nutrient availability, e.g., organic matters and nitrogen, etc., increased the biomass, shoot length, bifurcation and shoot nitrogen content of H. verticillata, but decreased the root weight ratio due to the promotion of plant photosynthesis and metabolism (Cronin and Lodge, 2003; Wang and Yu, 2007). Nitrogen and phosphorus content will also affect the morphology of plant root system, e.g., the number of rice roots and its root formation capability enhanced along with the increase of nitrogen content of the plants, but excessive nitrogen supplement results in the decrease of the root number, root length and root mass ratio. Furthermore, there are also reports demonstrating that root formation rate of the rice declined along with the increase of nitrogen supplement when plants were at early growth stage (Wang et al., 1997). According to the above results, the abundant nitrogen supplement during early growth stage might be one of the reasons explaining the smaller root number and equal root length on fertile sludge in summer. Plants will enlarge the interface between their root system and the soil to adapt to the situation of phosphorus deficiency, and then show the elongation of lateral roots and root hair and the increase of their number. At the same time, more photosynthesis products are transferred to the root system, which leads to the accelerated relative growth rate of the roots and the increase of root-top ratio (R/T), and consequently promote the uptake and usage of the root to the phosphorus (Liu et al., 1999). Thus, the root morphology in this experiment indicated the effect and interaction of all the nutrient factors. Nutrient content will also affect the antioxidase system of the plant, e.g., within definite extension, an increase of nitrogen supplement will lead to an increase of antioxidase activity (Cao and Ni, 2004; Cao et al., 2004). Except phosphorus deficiency and nitrogen supplement, deoxidizing sediment will also lead to he growing stress. The fact that the activities of SOD and POD were not significantly different between different sediments in our study is because the condition in our study did not create enough stress to the plants during the whole plant life cycle. Nitrogen is one of the constituents of plant photosynthetic pigment and it plays important role in plant chlorophyl content. Phosphorus also affects the photosynthesis procedure and an increase of phosphorus supplement will result in significant increase of plant leaf area and biomass. In August when plants were flourishing, different sediments did not influence the photosynthetic pigment content significantly. The reason leading to such result is that the nutrients, especially nitrogen, contained in two kinds of sediments are both adequate; when the synthesis and transfer of chlorophyl reach a balance, excessive nitrogen supple-
ment is nonsense and an increase of chlorophyl content was not found. Accordingly, nitrogen content of 0.034% in brown clay under our experimental condition can meet the demand of chlorophyl synthesis and transfer. The adequate nitrogen supplement will help maintain a high chlorophyl content and lead to late maturation, while low nitrogen treatment will make the plant die earlier. Due to the above reasons, photosynthetic pigment content from fertile sludge was higher in autumn (October). At that time, the capability of photosynthesis of H. verticillata from fertile sludge sediment was kept relatively stronger. Total soluble sugar, including glucose, sucrose, fructose, etc., acts as osmoregulation substance when stress especially osmotic stress happened. In August when plants were flourishing and substance metabolism was active, soluble sugar in plant was relatively low, and it was higher in plants on brown clay than on fertile sledge sediment. In October, the sediment nutrients were largely consumed during plant growth stages, which reduces the fertile level of the sediments, along with the senescence of plants, the soluble sugar content increased and the difference of the content from these two kinds of sediments became insignificant. Within a definite range, nitrogen will promote the increase of tissue nitrogen content and nitrogen assimilation rates, and along with the enhancing of the photosynthesis. Plants on fertile sludge sediments promoted nitrogen metabolism; therefore, the soluble protein content of H. verticillata on fertile sediment was higher than that from brown clay. Gerloff and Krombholz (1966) found that the minimum limits of tissue nitrogen and phosphorus content for the growth of hydrophytes are 1.3% and 0.13%, respectively. The nitrogen content and phosphorus content of the H. verticillata in August had met that requirement and no significant difference was found between the two sediments; Nevertheless, the tissue phosphorus content declined sharply and was merely 0.06% in October, which shows a symptom of phosphorus deficiency. The symptom in October indicated that the phosphorus content of brown clay was quite low and it could not meet the requirement for the growth of H. verticillata. Phosphorus deficiency will lead to stunt cell division and make it difficult for new cell to produce, and it also influences the elongation of the cell; so the morphology of the plant was found to be stunted growth, dwarfism and lower bifurcation ratio, which were observed in our experiment. The fertile sediment increased shoot and root length, bifurcation, biomass, ratio of shoot biomass of H. verticillata, and helped plants to accumulate soluble protein and nitrogen. It was also advantageous for plants to maintain high chlorophyl content, and then to keep a higher photosynthesis capability in the autumn. Our results demonstrated that the recovery of the submersed macrophytes in eutrophica shallow lakes could be promoted by human assistance, e.g., moderate dredging and plant or even seed bank transplantation. The research results provide academic and technical support for the ecological restoration of the lakes.
Acknowledgements The research described in this paper was supported by the grants from national water pollution control and management technology major projects of China (2009ZX07101-009) and national natural science foundation of China (40971277). We gratefully appreciate Prof. Gaomin Jiang, Dr. Linfeng Li, and Dr. Yinghao Li of Institute of Botany, the Chinese Academy of Sciences for their warm help in experiment execution and their valuable suggestion on this paper.
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