Physiological and biochemical perturbations of some submerged macrophytes, including Ceratophyllum demersum, Vallisneria spiralis and Potamogeton maackianus, were investigated following 25-day exposures to different concentrations of dibutyl phthalate. Plants were assayed for malon-diadehyde, soluble carbohydrate, chlorophyll, protein and glutathione. Results showed that contents of malon-diadehyde in submerged macrophytes decreased irregularly with increase in dibutyl phthalate. Contents of soluble carbohydrate in the Vallisneria spiralis had negative correlations with the concentrations of dibutyl phthalate and contents of soluble carbohydrate in the Potamogeton maackianus had positive correlation with increase in dibutyl phthalate. The contents of chlorophyll in Vallisneria spiralis changed irregularly and those of Ceratophyllum demersum and Potamogeton maackianus were higher than the control. The contents of protein and glutathione in these submerged macrophytes were less than the control. Contents of glutathione in Vallisneria spiralis had significantly negative correlations with increasing concentrations of dibutyl phthalate. Content of protein and glutathione might be considered as sensitive indexes of early damage to submerged macrophytes by dibutyl phthalate.
Phthalic acid esters (PAE) are widely used industrial chemicals serving as additives in polyvinyl chloride, polyvinyl acetate, cellulosic and polyurethane resins (Staples et al., 1997). There were approximately 14 kinds of PAE for commercial use; 6 of them have been ranked by the U.S. Environmental Protection Agency as pollutants, and are blacklisted for use in China. Furthermore, half of PAEs are environmental hormone pollutants (Gan, 2002).
These esters can enter the environment through losses during manufacturing processes and by leaching from final products, because they are not chemically bonded to the polymeric matrix (Hermann et al., 2002). The phthalates analyzed in the present study possess low water solubility (0.04–0.4 mg l−1). They have been found in all types of environmental and many biological samples (Hermann et al., 2002). Acute toxicity of PAE to plants is not obvious but they are known to be teratogenic and mutagenic to animals in high concentration.
It has been generally accepted that PAE are global pollutants and their environmental effects are important. Now these chemicals have attracted many workers in this field (Wolverton and McDonald, 1975; Cai, 1997; Chen, 1998; Zeng et al., 2000). The PAEs most widely found in the environment include dibutyl phthalate (DBP), di-ethyl phthalate (DEP), butyl benzyl phthalate (BBP), DEHP and di-octyl phthalate (DOP), If these, DEHP and DBP are the most abundant (Hariklia et al., 2003). Most researches had attached importance to analysing, monitoring and biodegradation of PAE,
The effects of PAEs on plants are poorly researched. Therefore, in this paper, we focused on effects of DBP pollution on the physiological indices of Ceratophyllum demersum, Vallisneria spiralis and Potamogeton maackianus to find which indicated of early damage to submerged macrophytes by DBP. Contents of malon-diadehyde, soluble carbohydrate, chlorophyll, glutathione and protein were measured in plants. Through the experiment, we also wanted to get some useful information to understand the degradation mechanism of hydrophytes in PAE polluted aquatic system.
Materials and methods
Materials and experimentation
Dibutyl phthalate was obtained from the Shanghai Jiuyi Chemical Reagent Co., Ltd. Submerged macrophytes were obtained from the Dongshan area of the city of Suzhou. They were grown in clean lake water in aquaria made to simulate a shallow lake. They were 100 cm long, 50 cm wide and 80 cm deep. Ten cm sand covered the bottoms of the aquaria. The sand was dried after biocidal treatment. Twenty healthy and uniform plants of the three macrophytes were cultivated in each aquarium.
The concentrations of DBP solution were 0.03, 0.05, 0.07, 0.12 and 0.15 mg l−1. Three replicates were used for each concentration. Three aquaria with no DBP added were set up as controls. The plants were cultivated in natural conditions. And the cultivation period was 25 d in natural daylight and laboratory temperatures. At 25 d, the plants were washed with distilled water, weighed and ground with extraction agents which were 10% trichloroacetic acid (m/v) for MDA and soluble carbohydrate, 80% acetone (v/v) for chlorophyll, distilled water for protein and 0.3 mol l−1 mercuric acetate, 30% sodium acetate (m/m), 1 mol l−1 hydrochloric acid for glutathione (SPC, 1999). Aliquots of these materials were centrifuged. Subsequently, for MDA and soluble carbohydrate analysis the materials were treated with 2-thiobarbituric acid and for protein analysis with coomassie brilliant blue G250. The materials for glutathione analysis were titrated with 1 mmol l−1 potassium iodate (Chen, 2002). Contents of MDA and soluble carbohydrate in the blades were tested by bi-component spectrophoto-metric method, contents of chlorophyll by spectrop- hotometry.
Data was assessed by SPSS 12.0 statistical software. Single factor effects were analysed by binary variable correlation analysis. Comparison among groups was checked by 2-tail t-method of inspection. Differences were significant if P ≤ 0.05.
Results and discussion
Effect on malon-diadehyde content
Malon-diadehyde is a lipid peroxidation product of the biomembrane system. Content of MDA always shows the extent of lipid peroxidation and indirectly indicates the extent of cell damage. Contents of MDA in C. demersum blades increased up to 0.05 mg l−1 DBP and then decreased rapidly in concentrations up to 0.12 mg l−1. Therefore, lipid peroxidation in different DBP concentrations was not apparent. It was perhaps because permeability of water and gas in environment was in good working order and tissue cell could breathe well. Consequently, lipid peroxidation was not obvious and contents of MDA did not increase but decrease. As is shown in Figure 1, with the concentration of DBP increasing, contents of MDA in V. spiralis blades decreased generally suggesting that the normal physiological process was interfered with. The MDA contents of P. maackianus showed that these concentrations of DBP also affected the normal growth of this macrophyte. There are similar results with V. spiralis.
With SPSS, there were no significance differences but contents of MDA in C. demersum and P. maackianus had negative correlations with the concentrations of DBP. The influence of DBP to MDA of C. demersum was not apparent and the concentration of DBP to the other two submerged macrophytes was perhaps lower than 0.03 mg l− 1.
Effect on soluble carbohydrate
The changes of soluble carbohydrate in V. spiralis blades were similar with changes of MDA (Figure 2). Contents of soluble carbohydrate in C. demersum blades changed alternatively. The reasons for these changes were perhaps that photosynthesis was good, and that protein and insoluble carbohydrate were decomposed and transported well (Horsfall, 1953). The aerogenic rate of C. demersum photosynthesis in DBP solution was also tested specifically tested. Results showed photosynthesis was not hindered but accelerated for the most part.
With the concentration of DBP solution increasing, contents of soluble carbohydrate in P. maackianus blades decreased. There was a low value in the concentration of 0.03 mg l− 1. But contents of soluble carbohydrate increased rapidly and there was a maximum value in 0.12 mg l− 1. While there were no significant differences, contents of soluble carbohydrate in the V. spiralis had negative correlations with the concentrations of DBP and contents of soluble carbohydrate in P. maackianus had positive correlation with the concentrations of DBP.
Effects on chlorophyll
The changes of contents of chlorophyll in the C. demersum blades were similar with those of contents of soluble carbohydrate (Figure 3). Contents of soluble carbohydrate in the V. spiralis had negative correlations with contents of chlorophyll, showing to a certain extent correlation between photosynthesis and contents of chlorophyl and soluble carbohydrate (Liu, 2002). Contents of chlorophyll in V. spiralis and P. maackianus were higher than in the control. They had positive correlation with the concentrations of DBP solution. The correlation of P. maackianus was better. However, there were no significance differences. Results showed that contents of chlorophyl could not reflect the DBP menace to submerged macrophytes.
Effects on protein
Under the stress of DBP, protein contents of these submerged macrophytes were less than the control (Figure 4), and had negative correlations with the concentrations of DBP solution. The possible reason was that DBP may have inhibited absorption of nutritive materials for synthesis of protein and DNA. The hindering of protein synthesis led to the drop of contents of protein (Zhou et al., 2003). Decrease of protein contents in C. demersum was more obvious than other two submerged macrophytes. As well, this macrophyte also died earlier than the others. Therefore, results showed that there were correlations between decrease of protein content and plant death. The protein contents could indicate the harm of DBP exposure to submerged macrophytes.
Effects on glutathione content
Glutathione is an important component of the antioxidation defense system. Changes in concentration of glutathione may indicate oxidation problems (Kocsy et al., 2001). As shown in Figure 5, with increasing DBP concentration, glutathione contents in stressed plants were 1ess than those in the control plants. Glutathione contents in V. spiralis had significantly negative correlations with DBP concentration. Thus, DBP could impair or destroy the antioxidation defense system in submerged macrophytes. Therefore, content of glutathione might be considered as a sensitive index of early damage to submerged macrophytes by DBP.
Physiological and biochemical perturbations of some submerged macrophytes were investigated. Most of contents of MDA, chlorophyl, soluble carbohydrate and protein showed no significant changes after 25-day exposure to DBP. But contents of protein and glutathione in these submerged macrophytes were less than the control. And glutathione contents in V. spiralis had negative correlations with the concentrations of DBP solution significantly. So among these parameters, contents of glutathione can be used as a more sensitive index of early damage to submerged macrophytes by DBP. But there should be further studies on detail degradation mechanism of these plants so that the research results will be applied in the recovery of submerged macrophytes.
This work was funded by National Basic Research Program (2002CB412307) and Social Development Project of Science and Technology Office, Jiangsu Province.