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Cadmium-induced colony disintegration of duckweed (Lemna paucicostata Hegelm.) and as biomarker of phytotoxicity
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 59 (2004) 174–179
Cadmium-induced colony disintegration of duckweed (Lemna paucicostata Hegelm.) and as biomarker of phytotoxicity T.Y. Li and Z.T. Xiong* Department of Environmental Science, Wuhan University, Wuhan, Hubei 430072, China Received 15 April 2003; received in revised form 3 October 2003; accepted 12 November 2003
Abstract The toxic effect of cadmium on Lemna paucicostata was investigated with hydroponic culture in a culture facility. Cadmium treatment (0.4–6.4 mmol L1 Cd) induced L. paucicostata to release daughter fronds from the mother frond before maturity, resulting in colony disintegration. The 8-h and 24-h EC50 values for colony disintegration in L. paucicostata plants were 0.12 and 0.11 mg L1, respectively. The maximum permissible concentrations (MPCs) were 0.012 and 0.011 mg L1 accordingly (MPC=10% EC50). These values were lower than the values of most of these biomarkers in duckweed reported in the literature, suggesting that colony disintegration in L. paucicostata may serve as a sensitive biomarker for the phytotoxicity test. Nutrient concentrations (1/2, 1/10, 1/20, 1/40, and 0-fold concentrations of Hoagland’s solution) and Cd salt form (CdCl2 or CdSO4) did not have a significant effect on colony disintegration. In addition, resistance to Cd stress differed significantly among clones of the plants. Approximately 2% of colonies in the wild population of L. paucicostata were tolerant of cadmium. These results indicate that colony disintegration of L. paucicostata could be used as a sensitive, cost-effective, and valuable biomarker to assess the acute phytotoxicity of cadmium and other heavy metals. r 2003 Elsevier Inc. All rights reserved. Keywords: Colony disintegration; Biomarker; Bioassay; Cadmium; Lemna paucicostata Hegelm
1. Introduction Heavy metals, such as cadmium, chromium, mercury, and lead, are common environmental pollutants, particularly in areas where there is anthropogenic contamination. They can cause serious problems for all organisms, and their bioaccumulation through the food chain can pose risks to human beings (Sanita` di Toppi and Gabbrielli, 1999; Scheifler et al., 2002). Therefore, there has been ever-increasing concern over the potency of heavy metals as environmental pollutants over the last several decades. Many researchers have investigated heavy metal effects on higher plants (Wang, 1986; Wang and Williams, 1990; Huebert et al., 1993; Sajwan and Ornes, 1994; Muller et al., 2001; Yin et al., 2002). Several standard methods for measuring the phytotoxicity of heavy metals have been published (ASTM, 1993; USEPA, 1996; APHA AWWA WEF, 2000; OECD, 2002). Duckweed plants, Lemna minor, Lemna gibba, and Lemna perpusilla, have frequently been used to test *Corresponding author. E-mail address: [email protected] (Z.T. Xiong). 0147-6513/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2003.11.007
the phytotoxicity of pollutants because of their small size, easy culture, and rapid reproductive rate (doubling time, 1–4 days or shorter) (Lewis, 1995). In previous studies with duckweed, plant responses used as endpoints included growth inhibition (expressed as number of fronds, dry or fresh weight of fronds, area of fronds, and colony number), chlorophyll content, and enzyme activity. The toxicity parameters included ED50, EC50, NOEC, and LOEC (Wang, 1986; Cowgill et al., 1991; Zhang and Jin, 1997; APHA AWWA and WEF, 2000; Muller et al., 2001; OECD, 2002). The methods for testing phytotoxicity are currently standardized (USEPA, 1996; APHA AWWA WEF, 2000; OECD, 2002). Selected strains of Lemna species are exposed to toxicants under axenic culture conditions for 4–7 days or longer. However, this test methodology is open to criticism (Wang, 1990), and the need for improvement has been suggested. The method of phytotoxicity assay using duckweed has not been commonly used in China though some studies have been done (Zhang and Jin, 1997; Yin et al., 2002). Cadmium is a widespread, nonessential heavy metal pollutant in the environment resulting from agricultural,
ARTICLE IN PRESS T.Y. Li, Z.T. Xiong / Ecotoxicology and Environmental Safety 59 (2004) 174–179
mining, and industrial activities as well as automobile emissions (Foy et al., 1978; Xiong, 1998). Aquatic organisms may accumulate Cd directly from contaminated water or through the food chain (Hart and Scaife, 1977). Cadmium is considered an extremely significant pollutant due to its high solubility in water, which results in wide distribution in aquatic ecosystems, and its strong toxicity to organisms (Lockwood, 1976). The toxic effects of Cd on biological systems have been reported in numerous studies (Mukherjee et al., 1984; Sharma et al., 1985; Sanita` di Toppi and Gabbrielli, 1999; Xiong and Peng, 2001). A number of investigations reported genotoxicity and ecotoxicity of cadmium to animals and other organisms (Webb, 1979; Nriagu, 1980; Degreave, 1981; Bhattacharya and Chaudhuri, 1995). In addition, indirect effects of the metal on physical environment factors, such as pH and dissolved oxygen concentrations in the water, resulting from toxic effects of the metal on photosynthesis in the freshwater ecosystem, were also reported (Ravera, 1984). Several studies have demonstrated the interaction of cadmium with essential elements like Fe, P, and Mn (Das et al., 1997). Lemna paucicostata, a species similar to L. minor in many aspects (Wang, 1990), is a duckweed species more common than other species of this genus in China. Some ecotoxicological studies were conducted using this species (Nasu and Kugimoto, 1981; Nasu et al., 1983). L. paucicostata is widely distributed and hence convenient for use as a test species in phytotoxicity research, especially in China. Like other Lemna species, it rapidly reproduces by asexual propagates (fronds) (Lemon and Posluszny, 2000; Lemon et al., 2001). Generally, daughter fronds are released from the mother frond after maturity. New fronds remain attached to a mother frond so that the mother and daughter fronds constitute a ‘‘family,’’ or a colony. A colony is an individual with one or more fronds. The objectives of this study are to investigate the response of L. paucicostata to cadmium, using daughter frond release as an endpoint, and to identify a duckweed plant capable of assessing the phytotoxicity of heavy metals such as cadmium. The results may increase our understanding of the effects and mechanisms of elevated environmental cadmium in duckweed. It is also possible to identify frond release as a sensitive endpoint for monitoring Cd pollution in the water.
2. Materials and methods
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laboratory in a plastic bucket with 10 L of pond water and 5 L of tap water, containing sediment collected from the same pond. The plants were cultured in a greenhouse. Every 3 days tap water was added to the bucket to maintain the water level. Stock plants were washed with distilled water and transferred into 1/10 Hoagland’s solution, and cultured in a growth chamber (2573 C, 4000 lx) for 7 days. Then, the stock plants were placed in a small tray containing tap water. The plants for toxicity tests were selected from this tray and transferred to a second tray containing only distilled water. All of the plants for toxicity tests appeared healthy, each with three fronds of approximately the same size. 2.2. Toxicity tests Toxicity tests were conducted in a growth chamber with pot experiment. Two hundred and fifty milliliters of 1/20 Hoagland’s nutrient solution was added to each plastic pot. Cd treatment concentrations were determined in pre-experimental tests. Based on the results of the pre-experiments, six treatment concentrations were used in this study: 0, 0.4, 0.8, 1.6, 3.2 and 6.4 mmol Cd L1 (as CdCl2 or CdSO4), each treatment with four replicates. The terminal pH of the solution was 6.7. Twenty colonies (60 fronds) of duckweed were transferred into each pot. The pots were covered with glass. The incubation time was 48 h, unless otherwise specified. Illumination was provided with cool-white fluorescent light with an intensity of 6500 lx. The experiments were conducted at room temperature, 15–25 C in spring and 25–35 C in summer. The Influence of nutrient concentration on cadmium toxicity was also determined by using redistilled water, 1/40, 1/10, or 1/2 Hoagland’s solution, and surrogated 1/20 Hoagland’s solution as culture medium. The cumulative number of colonies that were induced to release fronds during Cd exposure and the alteration in appearance of the fronds were recorded hourly for the first 8 h. Observations were also recorded at 24 and 48 h. EC50 was calculated as the cadmium concentration at which 50% of colonies were induced to disintegrate. The maximum permissible concentration (MPC) was calculated with the equation MPC=10% EC50 (Wang, 1986). At the end of each experiment, there were colonies that could not be induced to disintegrate by cadmium treatment. These colonies were tolerant ones that showed less sensitivity to cadmium exposure. They were also counted cumulatively.
2.1. Plant material 2.3. Statistics The test species, L. paucicostata, was collected from a local pond adjacent to Wuhan University. According to Wang (1986), the collected plants were maintained in the
A one-way ANOVA was used to compare mean differences among treatments (at the 0.01 significance
ARTICLE IN PRESS T.Y. Li, Z.T. Xiong / Ecotoxicology and Environmental Safety 59 (2004) 174–179
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level). EC50 (8 h) and EC50 (24 h) were estimated using graphical interpolation.
3.4. Effect of nutrient concentration As was shown in Table 3, the concentration of Hoagland’s solution did not have significant influence
3. Results
Disintegrated colony number
3.1. Release of daughter fronds 1
After 6 h exposure to 0.8 mmol L Cd, the colony disintegration number of L. paucicostata was significantly higher than that of the control (Table 1). At 3.2 mmol L1 and higher, significant effects were detected after 3 h exposure. After 24 h, most colonies had disintegrated. These results indicate that the response to cadmium had a time lag, and the lag decreased as the concentration increased. The number of nondisintegrated colonies was negatively correlated with cadmium concentration. Colony disintegration occurred largely within 24 h. The plants exhibited chlorosis after 24 h Cd exposure. 3.2. EC50 and MPC of colony disintegration at 8 and 24 h The cumulative number of disintegrated colonies varied with cadmium concentration at 8 and 24 h (Fig. 1), demonstrating a dose–response relationship. The EC50 values of colony disintegration at 8 and 24 h were 1.09 mmol L1 (approximately 0.123 mg L1) and 0.94 mmol L1 (approximately 0.106 mg L1), respectively. The MPCs were 0.012 and 0.011 mg L1 accordingly. 3.3. Effect of Cd salt form There was no significant difference between the toxicity of CdCl2 and CdSO4 (P40:01) (Table 2). Therefore, colony disintegration of duckweed in response to Cd2+ ion was independent of the salt form.
25
8h
20
24h
15 10 5 0 0 0.4 0.8 1.6 3.2 6.4 Cadmium-added concentration (mol/L)
Fig. 1. Colony disintegration induced by cadmium exposure at 8 and 24 h.
Table 2 Effect of Cd salt form on colony disintegration of L. paucicostata for 24 h cadmium exposure in a representative experiment Exposure time (h)
Cadmium concentrations (m mol L1) and salt form added 0.4
0 1 2 3 4 5 6 7 8 24
0.8
1.6
CdSO4
CdCl2
CdSO4
CdCl2
CdSO4
CdCl2
0 0 0 0 0 0 1 1 2 2
0 0 0 0 0 0 0 2 2 3
0 0 0 0 2 4 5 7 9 9
0 0 0 0 3 6 6 6 8 10
0 0 0 4 6 9 12 14 14 15
0 0 0 3 7 8 12 13 13 14
Note: A total of 20 colonies were transferred originally in each treatment.
Table 1 Colony disintegration of L. paucicostata exposed to cadmium for various time Time (h) 0 1 2 3 4 5 6 7 8 24 48
Cadmium-added concentration (mmol L1) Control
0.4
0.8
1.6
3.2
6.4
0 0 0 0 0 0 0 0 0 0.2570.50 0.2570.50
0 0 0 0 0 0 0 0 0.7570.50 1.0070.82 1.0070.82
0 0 0 0 2.2571.71 2.7571.26 5.2571.71 6.0071.15 8.0070.00 8.7570.50 8.7570.50
0 0 0 0 4.2571.29 5.5071.29 9.0070.82 11.7571.71 13.5070.58 14.0070.82 14.0070.82
0 0 0 5.5071.29 7.7570.96 10.2571.71 13.2570.96 14.0070.82 16.2570.50 18.5070.58 18.5070.58
0 0 2.0071.82 6.0070.00 8.2570.96 12.5071.29 15.0070.82 17.5071.29 18.7570.96 19.0070.58 19.0070.58
Note: The results are means7SD (n ¼ 4). A total of 20 colonies were transferred originally in each treatment. Significantly different from the control at the 0.01 level.
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Table 3 Effect of nutrient concentration on colony disintegration of L. paucicostata for 24 h cadmium exposure Medium
1/2 H 1/10 H 1/20 H Redistilled water
Control
0 0 0 0
Cadmium-added concentrations (mmol L1) 0.4
0.8
1.6
3.2
6.4
1.0070.82 1.0070.82 0.7570.50 1.0070.82
8.0070.00 8.7570.50 8.0070.00 8.0070.00
13.5070.58 13.5070.58 13.5070.58 13.5070.58
18.5070.58 18.5070.58 18.5070.58 18.5070.58
18.7570.96 19.0070.58 18.7570.96 19.0070.58
Note: Results are means7SD (n ¼ 4). A total of 20 colonies were transferred originally in each treatment.
on colony disintegration. There were no significant differences in colony disintegration between the 0, 1/40, 1/20, 1/10, and 1/2 concentrations of Hoagland’s solution in the culture medium. 3.5. Cd-tolerant colony The percentage of colonies tolerant to cadmium in the population of L. paucicostata was approximately 2% (Table 4). This proportion of colonies did not manifest disintegration at 6.4 mmol Cd L1 in this study.
4. Discussion The present results clearly indicate that cadmium could induce L. paucicostata to release daughter fronds prematurely from the mother frond, resulting in colony disintegration. It is a phenomenon that has not been reported so far in the literature. The frond abscission induced in this study occurred within 5–8 h at lower Cd treatments, and after 3 h at higher Cd concentrations (Table 1). Colony disintegration was almost complete in 24 h, and little effect was observed when the colonies were further exposed to Cd. These findings correspond to the stress ethylene production as reported in other plants under cadmium stress. For example, Rodecap et al. (1981) reported that in Phaselus vulgaris plants exposed to Cd for 5–10 h, ethylene production reached peak concentrations, and then gradually declined to control levels within 1 day. Cadmium stimulates ethylene biosynthesis via the MSAE (methionine, Sadenosylmethionine-1-aminocyclopropane-1-carboxylic acid, ethylene) pathway (Adams and Yang, 1979), directly enhancing (during the first 8 h) and then inhibiting the in vivo activity of ACC synthase (Fuhrer, 1982a). It is well known that ethylene is an endogenous regulator of plant growth, development, abscission of fruits and leaves, and senescence (Abeles, 1973). Ethylene production by plants under normal conditions is very low; it increases when living tissues are subjected to a variety of stress conditions (Lieberman, 1979). Thus it is reasonable to infer that the Cd-induced colony disintegration observed in this study could be attributable to stress ethylene production.
Table 4 The number of colonies of L. paucicostata unaffected by cadmium after 24 h exposure in four experiments Experiment
First Second Third Fourth
Cadmium-added concentrations (mmol L1) Control
0.4
0.8
1.6
3.2
6.4
20 20 20 20
20 20 19 19
13 12 13 13
6 6 7 6
3 2 2 3
3 1 2 2
Note. A total of 20 colonies were transferred originally in each treatment.
Frond abscission might play a role in plant survival in the heavy metal-contaminated environment. In higher plants, several mechanisms of resistance to heavy metal stress have been suggested, such as heavy metal immobilization (Nishizono et al., 1989), exclusion (Rivetta et al., 1997), compartmentalization (Abrahamson et al., 1992), synthesis of stress proteins (Czarnecka et al., 1988) and phytochelatins (Grill et al., 1985), and production of stress ethylene (Sanita` di Toppi and Gabbrielli, 1999; Fuhrer, 1982a, b). The frond abscission observed in this study might be of benefit to plant survival in the Cd-contaminated environment. Perhaps it prevents cadmium transportation from the mother frond to daughter fronds. Release of daughter fronds from the Cd-stressed mother frond would increase the chance of survival of the daughter fronds. Approximately 2% of colonies were tolerant to cadmium (Table 4). The reason for this might lie in the genetic biodiversity of the population used in this study. Here, we used the wild-type L. paucicostata as the test sample. It is reasonable that in the natural population of L. paucicostata, some clones may be sensitive to cadmium while others may be tolerant. This could explain why some investigations used duckweed for bioassay of a pollutant in the environment, whereas other investigations used the same species to remove the pollutants (Wang, 1990). Studies with different purposes can employ stocks different in heavy metal tolerance. Duckweed toxicity tests, generally using growth and multiplication as the test endpoint, required sufficient plant nutrients for optimum growth conditions (Wang,
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1990). Nasu and Kugimoto (1981) found that the pH, concentration and composition of the nutrients in the medium, and the temperature at which cultures were maintained affected the response of L. paucicostata to heavy metals. The growth inhibition of Lemna plants was proportional to the amount of metal absorption. In our toxicity test, however, the end point, frond abscission of L. paucicostata, was not affected by nutrient concentration and Cd salt form (Tables 2 and 3). The reason is unclear, and further research is needed. Responses of plants to exposure to pollutants have been recommended for environmental assessment (Burkard et al., 1995; Chan, 1995). The 8- and 24-h EC50 values of colony disintegration obtained in this study were 0.123 and 0.106 mg L1, respectively (see Fig. 1). The corresponding MPCs were 0.012 and 0.011 mg L1. These MPC are close to the highest permissible Cd level for surface water in China, 0.01 mg L1(China EPA, 1992). Also, these values are lower than those observed for L. minor, for which the EC50 (4-day) and MPC were 0.2, 0.02 mg L1, respectively (Wang, 1986). These results indicate that if we use colony disintegration of L. paucicostata as a biomarker to assess the acute phytotoxicity of cadmium, the results would be better than using other biomarkers. Furthermore, colony counting is simple, convenient, and cost-effective. L. paucicostata is more common than other species of Lemna in freshwater bodies in China. In this respect, this species will be more valuable in the assessment of the contamination by cadmium and possibly other trace metals. This species may also be employed for pollution assessment in other countries where it is naturally distributed.
5. Conclusion Mother fronds of L. paucicostata can release daughter fronds before maturity in response to cadmium exposure, resulting in colony disintegration. Daughter frond release may involve ethylene production stimulated by cadmium stress. A small proportion of colonies are tolerant to cadmium in the wild population of L. paucicostata. The reason for this might lie in the genetic biodiversity of the population. The 8- and 24-h EC50 values for colony disintegration in L. paucicostata plants were significantly lower than those observed in L. minor. The MPCs were close to the highest permissible Cd level for surface water in China. From these results, it is suggested that the prematurity colony disintegration of L. paucicostata first reported in this study is a sensitive and cost-effective biomarker that can be used to assess the contamination by cadmium and other heavy metals in China and other countries where it is naturally distributed.
Acknowledgments The authors thank Ms. Lan Huang and Dr. Jianhong Yang for their help. We also thank Miss Jun Qu, Mr. Liangsheng Shi, and Yihua Liu, the members of the team for the third ecological survey in Shennongjia Natural Conservation, Wuhan University. We especially thank Dr. Michael Lewis, USEPA, who not only gave us good advice, but also checked throughout the manuscript and improved the English.
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Ecotoxicology and Environmental Safety 59 (2004) 174–179
Cadmium-induced colony disintegration of duckweed (Lemna paucicostata Hegelm.) and as biomarker of phytotoxicity T.Y. Li and Z.T. Xiong* Department of Environmental Science, Wuhan University, Wuhan, Hubei 430072, China Received 15 April 2003; received in revised form 3 October 2003; accepted 12 November 2003
Abstract The toxic effect of cadmium on Lemna paucicostata was investigated with hydroponic culture in a culture facility. Cadmium treatment (0.4–6.4 mmol L1 Cd) induced L. paucicostata to release daughter fronds from the mother frond before maturity, resulting in colony disintegration. The 8-h and 24-h EC50 values for colony disintegration in L. paucicostata plants were 0.12 and 0.11 mg L1, respectively. The maximum permissible concentrations (MPCs) were 0.012 and 0.011 mg L1 accordingly (MPC=10% EC50). These values were lower than the values of most of these biomarkers in duckweed reported in the literature, suggesting that colony disintegration in L. paucicostata may serve as a sensitive biomarker for the phytotoxicity test. Nutrient concentrations (1/2, 1/10, 1/20, 1/40, and 0-fold concentrations of Hoagland’s solution) and Cd salt form (CdCl2 or CdSO4) did not have a significant effect on colony disintegration. In addition, resistance to Cd stress differed significantly among clones of the plants. Approximately 2% of colonies in the wild population of L. paucicostata were tolerant of cadmium. These results indicate that colony disintegration of L. paucicostata could be used as a sensitive, cost-effective, and valuable biomarker to assess the acute phytotoxicity of cadmium and other heavy metals. r 2003 Elsevier Inc. All rights reserved. Keywords: Colony disintegration; Biomarker; Bioassay; Cadmium; Lemna paucicostata Hegelm
1. Introduction Heavy metals, such as cadmium, chromium, mercury, and lead, are common environmental pollutants, particularly in areas where there is anthropogenic contamination. They can cause serious problems for all organisms, and their bioaccumulation through the food chain can pose risks to human beings (Sanita` di Toppi and Gabbrielli, 1999; Scheifler et al., 2002). Therefore, there has been ever-increasing concern over the potency of heavy metals as environmental pollutants over the last several decades. Many researchers have investigated heavy metal effects on higher plants (Wang, 1986; Wang and Williams, 1990; Huebert et al., 1993; Sajwan and Ornes, 1994; Muller et al., 2001; Yin et al., 2002). Several standard methods for measuring the phytotoxicity of heavy metals have been published (ASTM, 1993; USEPA, 1996; APHA AWWA WEF, 2000; OECD, 2002). Duckweed plants, Lemna minor, Lemna gibba, and Lemna perpusilla, have frequently been used to test *Corresponding author. E-mail address: [email protected] (Z.T. Xiong). 0147-6513/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2003.11.007
the phytotoxicity of pollutants because of their small size, easy culture, and rapid reproductive rate (doubling time, 1–4 days or shorter) (Lewis, 1995). In previous studies with duckweed, plant responses used as endpoints included growth inhibition (expressed as number of fronds, dry or fresh weight of fronds, area of fronds, and colony number), chlorophyll content, and enzyme activity. The toxicity parameters included ED50, EC50, NOEC, and LOEC (Wang, 1986; Cowgill et al., 1991; Zhang and Jin, 1997; APHA AWWA and WEF, 2000; Muller et al., 2001; OECD, 2002). The methods for testing phytotoxicity are currently standardized (USEPA, 1996; APHA AWWA WEF, 2000; OECD, 2002). Selected strains of Lemna species are exposed to toxicants under axenic culture conditions for 4–7 days or longer. However, this test methodology is open to criticism (Wang, 1990), and the need for improvement has been suggested. The method of phytotoxicity assay using duckweed has not been commonly used in China though some studies have been done (Zhang and Jin, 1997; Yin et al., 2002). Cadmium is a widespread, nonessential heavy metal pollutant in the environment resulting from agricultural,
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mining, and industrial activities as well as automobile emissions (Foy et al., 1978; Xiong, 1998). Aquatic organisms may accumulate Cd directly from contaminated water or through the food chain (Hart and Scaife, 1977). Cadmium is considered an extremely significant pollutant due to its high solubility in water, which results in wide distribution in aquatic ecosystems, and its strong toxicity to organisms (Lockwood, 1976). The toxic effects of Cd on biological systems have been reported in numerous studies (Mukherjee et al., 1984; Sharma et al., 1985; Sanita` di Toppi and Gabbrielli, 1999; Xiong and Peng, 2001). A number of investigations reported genotoxicity and ecotoxicity of cadmium to animals and other organisms (Webb, 1979; Nriagu, 1980; Degreave, 1981; Bhattacharya and Chaudhuri, 1995). In addition, indirect effects of the metal on physical environment factors, such as pH and dissolved oxygen concentrations in the water, resulting from toxic effects of the metal on photosynthesis in the freshwater ecosystem, were also reported (Ravera, 1984). Several studies have demonstrated the interaction of cadmium with essential elements like Fe, P, and Mn (Das et al., 1997). Lemna paucicostata, a species similar to L. minor in many aspects (Wang, 1990), is a duckweed species more common than other species of this genus in China. Some ecotoxicological studies were conducted using this species (Nasu and Kugimoto, 1981; Nasu et al., 1983). L. paucicostata is widely distributed and hence convenient for use as a test species in phytotoxicity research, especially in China. Like other Lemna species, it rapidly reproduces by asexual propagates (fronds) (Lemon and Posluszny, 2000; Lemon et al., 2001). Generally, daughter fronds are released from the mother frond after maturity. New fronds remain attached to a mother frond so that the mother and daughter fronds constitute a ‘‘family,’’ or a colony. A colony is an individual with one or more fronds. The objectives of this study are to investigate the response of L. paucicostata to cadmium, using daughter frond release as an endpoint, and to identify a duckweed plant capable of assessing the phytotoxicity of heavy metals such as cadmium. The results may increase our understanding of the effects and mechanisms of elevated environmental cadmium in duckweed. It is also possible to identify frond release as a sensitive endpoint for monitoring Cd pollution in the water.
2. Materials and methods
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laboratory in a plastic bucket with 10 L of pond water and 5 L of tap water, containing sediment collected from the same pond. The plants were cultured in a greenhouse. Every 3 days tap water was added to the bucket to maintain the water level. Stock plants were washed with distilled water and transferred into 1/10 Hoagland’s solution, and cultured in a growth chamber (2573 C, 4000 lx) for 7 days. Then, the stock plants were placed in a small tray containing tap water. The plants for toxicity tests were selected from this tray and transferred to a second tray containing only distilled water. All of the plants for toxicity tests appeared healthy, each with three fronds of approximately the same size. 2.2. Toxicity tests Toxicity tests were conducted in a growth chamber with pot experiment. Two hundred and fifty milliliters of 1/20 Hoagland’s nutrient solution was added to each plastic pot. Cd treatment concentrations were determined in pre-experimental tests. Based on the results of the pre-experiments, six treatment concentrations were used in this study: 0, 0.4, 0.8, 1.6, 3.2 and 6.4 mmol Cd L1 (as CdCl2 or CdSO4), each treatment with four replicates. The terminal pH of the solution was 6.7. Twenty colonies (60 fronds) of duckweed were transferred into each pot. The pots were covered with glass. The incubation time was 48 h, unless otherwise specified. Illumination was provided with cool-white fluorescent light with an intensity of 6500 lx. The experiments were conducted at room temperature, 15–25 C in spring and 25–35 C in summer. The Influence of nutrient concentration on cadmium toxicity was also determined by using redistilled water, 1/40, 1/10, or 1/2 Hoagland’s solution, and surrogated 1/20 Hoagland’s solution as culture medium. The cumulative number of colonies that were induced to release fronds during Cd exposure and the alteration in appearance of the fronds were recorded hourly for the first 8 h. Observations were also recorded at 24 and 48 h. EC50 was calculated as the cadmium concentration at which 50% of colonies were induced to disintegrate. The maximum permissible concentration (MPC) was calculated with the equation MPC=10% EC50 (Wang, 1986). At the end of each experiment, there were colonies that could not be induced to disintegrate by cadmium treatment. These colonies were tolerant ones that showed less sensitivity to cadmium exposure. They were also counted cumulatively.
2.1. Plant material 2.3. Statistics The test species, L. paucicostata, was collected from a local pond adjacent to Wuhan University. According to Wang (1986), the collected plants were maintained in the
A one-way ANOVA was used to compare mean differences among treatments (at the 0.01 significance
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level). EC50 (8 h) and EC50 (24 h) were estimated using graphical interpolation.
3.4. Effect of nutrient concentration As was shown in Table 3, the concentration of Hoagland’s solution did not have significant influence
3. Results
Disintegrated colony number
3.1. Release of daughter fronds 1
After 6 h exposure to 0.8 mmol L Cd, the colony disintegration number of L. paucicostata was significantly higher than that of the control (Table 1). At 3.2 mmol L1 and higher, significant effects were detected after 3 h exposure. After 24 h, most colonies had disintegrated. These results indicate that the response to cadmium had a time lag, and the lag decreased as the concentration increased. The number of nondisintegrated colonies was negatively correlated with cadmium concentration. Colony disintegration occurred largely within 24 h. The plants exhibited chlorosis after 24 h Cd exposure. 3.2. EC50 and MPC of colony disintegration at 8 and 24 h The cumulative number of disintegrated colonies varied with cadmium concentration at 8 and 24 h (Fig. 1), demonstrating a dose–response relationship. The EC50 values of colony disintegration at 8 and 24 h were 1.09 mmol L1 (approximately 0.123 mg L1) and 0.94 mmol L1 (approximately 0.106 mg L1), respectively. The MPCs were 0.012 and 0.011 mg L1 accordingly. 3.3. Effect of Cd salt form There was no significant difference between the toxicity of CdCl2 and CdSO4 (P40:01) (Table 2). Therefore, colony disintegration of duckweed in response to Cd2+ ion was independent of the salt form.
25
8h
20
24h
15 10 5 0 0 0.4 0.8 1.6 3.2 6.4 Cadmium-added concentration (mol/L)
Fig. 1. Colony disintegration induced by cadmium exposure at 8 and 24 h.
Table 2 Effect of Cd salt form on colony disintegration of L. paucicostata for 24 h cadmium exposure in a representative experiment Exposure time (h)
Cadmium concentrations (m mol L1) and salt form added 0.4
0 1 2 3 4 5 6 7 8 24
0.8
1.6
CdSO4
CdCl2
CdSO4
CdCl2
CdSO4
CdCl2
0 0 0 0 0 0 1 1 2 2
0 0 0 0 0 0 0 2 2 3
0 0 0 0 2 4 5 7 9 9
0 0 0 0 3 6 6 6 8 10
0 0 0 4 6 9 12 14 14 15
0 0 0 3 7 8 12 13 13 14
Note: A total of 20 colonies were transferred originally in each treatment.
Table 1 Colony disintegration of L. paucicostata exposed to cadmium for various time Time (h) 0 1 2 3 4 5 6 7 8 24 48
Cadmium-added concentration (mmol L1) Control
0.4
0.8
1.6
3.2
6.4
0 0 0 0 0 0 0 0 0 0.2570.50 0.2570.50
0 0 0 0 0 0 0 0 0.7570.50 1.0070.82 1.0070.82
0 0 0 0 2.2571.71 2.7571.26 5.2571.71 6.0071.15 8.0070.00 8.7570.50 8.7570.50
0 0 0 0 4.2571.29 5.5071.29 9.0070.82 11.7571.71 13.5070.58 14.0070.82 14.0070.82
0 0 0 5.5071.29 7.7570.96 10.2571.71 13.2570.96 14.0070.82 16.2570.50 18.5070.58 18.5070.58
0 0 2.0071.82 6.0070.00 8.2570.96 12.5071.29 15.0070.82 17.5071.29 18.7570.96 19.0070.58 19.0070.58
Note: The results are means7SD (n ¼ 4). A total of 20 colonies were transferred originally in each treatment. Significantly different from the control at the 0.01 level.
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Table 3 Effect of nutrient concentration on colony disintegration of L. paucicostata for 24 h cadmium exposure Medium
1/2 H 1/10 H 1/20 H Redistilled water
Control
0 0 0 0
Cadmium-added concentrations (mmol L1) 0.4
0.8
1.6
3.2
6.4
1.0070.82 1.0070.82 0.7570.50 1.0070.82
8.0070.00 8.7570.50 8.0070.00 8.0070.00
13.5070.58 13.5070.58 13.5070.58 13.5070.58
18.5070.58 18.5070.58 18.5070.58 18.5070.58
18.7570.96 19.0070.58 18.7570.96 19.0070.58
Note: Results are means7SD (n ¼ 4). A total of 20 colonies were transferred originally in each treatment.
on colony disintegration. There were no significant differences in colony disintegration between the 0, 1/40, 1/20, 1/10, and 1/2 concentrations of Hoagland’s solution in the culture medium. 3.5. Cd-tolerant colony The percentage of colonies tolerant to cadmium in the population of L. paucicostata was approximately 2% (Table 4). This proportion of colonies did not manifest disintegration at 6.4 mmol Cd L1 in this study.
4. Discussion The present results clearly indicate that cadmium could induce L. paucicostata to release daughter fronds prematurely from the mother frond, resulting in colony disintegration. It is a phenomenon that has not been reported so far in the literature. The frond abscission induced in this study occurred within 5–8 h at lower Cd treatments, and after 3 h at higher Cd concentrations (Table 1). Colony disintegration was almost complete in 24 h, and little effect was observed when the colonies were further exposed to Cd. These findings correspond to the stress ethylene production as reported in other plants under cadmium stress. For example, Rodecap et al. (1981) reported that in Phaselus vulgaris plants exposed to Cd for 5–10 h, ethylene production reached peak concentrations, and then gradually declined to control levels within 1 day. Cadmium stimulates ethylene biosynthesis via the MSAE (methionine, Sadenosylmethionine-1-aminocyclopropane-1-carboxylic acid, ethylene) pathway (Adams and Yang, 1979), directly enhancing (during the first 8 h) and then inhibiting the in vivo activity of ACC synthase (Fuhrer, 1982a). It is well known that ethylene is an endogenous regulator of plant growth, development, abscission of fruits and leaves, and senescence (Abeles, 1973). Ethylene production by plants under normal conditions is very low; it increases when living tissues are subjected to a variety of stress conditions (Lieberman, 1979). Thus it is reasonable to infer that the Cd-induced colony disintegration observed in this study could be attributable to stress ethylene production.
Table 4 The number of colonies of L. paucicostata unaffected by cadmium after 24 h exposure in four experiments Experiment
First Second Third Fourth
Cadmium-added concentrations (mmol L1) Control
0.4
0.8
1.6
3.2
6.4
20 20 20 20
20 20 19 19
13 12 13 13
6 6 7 6
3 2 2 3
3 1 2 2
Note. A total of 20 colonies were transferred originally in each treatment.
Frond abscission might play a role in plant survival in the heavy metal-contaminated environment. In higher plants, several mechanisms of resistance to heavy metal stress have been suggested, such as heavy metal immobilization (Nishizono et al., 1989), exclusion (Rivetta et al., 1997), compartmentalization (Abrahamson et al., 1992), synthesis of stress proteins (Czarnecka et al., 1988) and phytochelatins (Grill et al., 1985), and production of stress ethylene (Sanita` di Toppi and Gabbrielli, 1999; Fuhrer, 1982a, b). The frond abscission observed in this study might be of benefit to plant survival in the Cd-contaminated environment. Perhaps it prevents cadmium transportation from the mother frond to daughter fronds. Release of daughter fronds from the Cd-stressed mother frond would increase the chance of survival of the daughter fronds. Approximately 2% of colonies were tolerant to cadmium (Table 4). The reason for this might lie in the genetic biodiversity of the population used in this study. Here, we used the wild-type L. paucicostata as the test sample. It is reasonable that in the natural population of L. paucicostata, some clones may be sensitive to cadmium while others may be tolerant. This could explain why some investigations used duckweed for bioassay of a pollutant in the environment, whereas other investigations used the same species to remove the pollutants (Wang, 1990). Studies with different purposes can employ stocks different in heavy metal tolerance. Duckweed toxicity tests, generally using growth and multiplication as the test endpoint, required sufficient plant nutrients for optimum growth conditions (Wang,
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1990). Nasu and Kugimoto (1981) found that the pH, concentration and composition of the nutrients in the medium, and the temperature at which cultures were maintained affected the response of L. paucicostata to heavy metals. The growth inhibition of Lemna plants was proportional to the amount of metal absorption. In our toxicity test, however, the end point, frond abscission of L. paucicostata, was not affected by nutrient concentration and Cd salt form (Tables 2 and 3). The reason is unclear, and further research is needed. Responses of plants to exposure to pollutants have been recommended for environmental assessment (Burkard et al., 1995; Chan, 1995). The 8- and 24-h EC50 values of colony disintegration obtained in this study were 0.123 and 0.106 mg L1, respectively (see Fig. 1). The corresponding MPCs were 0.012 and 0.011 mg L1. These MPC are close to the highest permissible Cd level for surface water in China, 0.01 mg L1(China EPA, 1992). Also, these values are lower than those observed for L. minor, for which the EC50 (4-day) and MPC were 0.2, 0.02 mg L1, respectively (Wang, 1986). These results indicate that if we use colony disintegration of L. paucicostata as a biomarker to assess the acute phytotoxicity of cadmium, the results would be better than using other biomarkers. Furthermore, colony counting is simple, convenient, and cost-effective. L. paucicostata is more common than other species of Lemna in freshwater bodies in China. In this respect, this species will be more valuable in the assessment of the contamination by cadmium and possibly other trace metals. This species may also be employed for pollution assessment in other countries where it is naturally distributed.
5. Conclusion Mother fronds of L. paucicostata can release daughter fronds before maturity in response to cadmium exposure, resulting in colony disintegration. Daughter frond release may involve ethylene production stimulated by cadmium stress. A small proportion of colonies are tolerant to cadmium in the wild population of L. paucicostata. The reason for this might lie in the genetic biodiversity of the population. The 8- and 24-h EC50 values for colony disintegration in L. paucicostata plants were significantly lower than those observed in L. minor. The MPCs were close to the highest permissible Cd level for surface water in China. From these results, it is suggested that the prematurity colony disintegration of L. paucicostata first reported in this study is a sensitive and cost-effective biomarker that can be used to assess the contamination by cadmium and other heavy metals in China and other countries where it is naturally distributed.
Acknowledgments The authors thank Ms. Lan Huang and Dr. Jianhong Yang for their help. We also thank Miss Jun Qu, Mr. Liangsheng Shi, and Yihua Liu, the members of the team for the third ecological survey in Shennongjia Natural Conservation, Wuhan University. We especially thank Dr. Michael Lewis, USEPA, who not only gave us good advice, but also checked throughout the manuscript and improved the English.
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