Water Air Soil Pollut (2014) 225:1865 DOI 10.1007/s11270-014-1865-5
Antagonist Effects of Sodium Chloride on the Biological Responses of an Aquatic Plant (Ceratophyllum demersum L.) Exposed to Hexavalent Chromium Fatih Duman & Fatih D. Koca & Serkan Sahan
Received: 25 June 2013 / Accepted: 6 January 2014 / Published online: 25 January 2014 # Springer Science+Business Media Dordrecht 2014
Abstract In this study, the concentration-dependent joint action of chromium (Cr) and salt (NaCl), two important environmental stressors, was examined in aquatic plants. Ceratophyllum demersum L. plants were exposed to Cr (0–10 mM) for 5 days in the presence and absence of NaCl (0–500 mM). The effect of Cr, Na, and Cl accumulations on certain biological parameters (water content, ion leakage, relative growth rate, photosynthetic pigments, and protein and proline contents) was determined. Furthermore, the interactive effects of NaCl and Cr were evaluated using a mathematical model developed on the basis of the theory of probabilities. The highest Cr accumulation (0.42 mmol g−1) was found in plants treated with 10 mM Cr+ 125 mM NaCl. Treatment with 125 mM NaCl resulted in an increase in Cr accumulation compared with that in the control. However, 250 and 500 mM NaCl concentrations decreased Cr accumulation. Proline and water contents were not affected by increasing Cr concentration. However, NaCl did have a significant effect on any of the studied parameters. Furthermore, the interactive effects of Cr and NaCl on all studied parameters except
F. Duman (*) : F. D. Koca Department of Biology, Faculty of Sciences, Erciyes University, 38039 Kayseri, Turkey e-mail:
[email protected] S. Sahan Department of Chemistry, Faculty of Sciences, Erciyes University, 38039 Kayseri, Turkey
for proline and water contents were determined. Except for photosynthetic pigments and proline content, effect of NaCl was higher than Cr on all studied parameters. The interactive effects were mostly antagonistic or additive. However, the mode of action for ion leakage was synergistic or additive. These results suggest that the coexistence of NaCl and Cr in aquatic ecosystems does not pose an additional ecological risk for aquatic plants. Keywords Salt stress . Chromium . Interactive effects . Biological response . Ceratophyllum demersum
1 Introduction A short but accurate definition of pollution is the ‘occurrence of something in the wrong location at a high amount’ (Phillips and Rainbow 1994). Due to the uncertainty of source and exact type of pollutants, water pollution is considered to be rather problematic. Because of the high levels of evapotranspiration and irrigation as well as the discharge of salt (NaCl)-polluted water, salinity has become an important pollutant that affects many freshwater resources (Jampeetong and Brix 2009). Furthermore, drained surface water from urban roads transports a large amount of NaCl and heavy metals to freshwater ecosystems. Salinity affects plant growth, ion toxicity, and nutrient imbalances (Roache et al. 2006). High NaCl concentrations in aquatic environments impose osmotic ionic stress on plants and allow high concentrations of Na+ and Cl− to enter cells. Eventually, many enzymatic processes
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become non-functional (Chang et al. 2012). Chromium (Cr) is a toxic metal that is present in raw wastewater originating from iron and steel factories, tanneries, electroplating, and sewage sludge (Maine et al. 2004; Shanker et al. 2005). Given its high redox potential and ability to penetrate cell membranes, Cr(VI) compounds are considered to be extremely toxic carcinogens (Kotas and Stasicka 2000). High quantities of NaCls in water might affect ionic strength and thus the biosorption of metals decreases (Aksu and Balibek 2007). Some industries are known to release wastewater that contains high quantities of NaCl and Cr (Stoneham 1985). Determination of the effect of interactions of NaCl and Cr(VI) is important to identify aquatic plants that can be used to purify water polluted with these stressors. Salinity is known to affect heavy metal accumulation, physical and biochemical properties, and other such properties of freshwater species (Leblebici et al. 2011). Munda and Hudnik (1988) investigated a seaweed (Fucus vesiculosus) and concluded that its zinc accumulation capacity decreased at higher salinity. In a laboratory study on two different seaweeds (Ascophyllum nodosum and F. vesiculosus), copper toxicity was found to be increased under reduced salinity (Connan and Stengel 2011). In a similar laboratory study conducted by Yilmaz (2007), nickel accumulation of duckweed (Lemna gibba) was found to decrease with salinity. Some studies also investigated the NaCl–metal interaction in terrestrial plants. Yermiyahu et al. (2008) found that when salinity and boron stressors occur simultaneously, salinity might reduce or increase the toxic effect of boron on plants. Smith et al. (2010) found a significant interaction between salinity and boron that affected the biological responses of broccoli, such as yield, biomass distribution, and water use. In the literature, there are reports on metal–metal interactions. Previous studies have shown that, in polluted aquatic ecosystems, macrophytes play a significant role in filtering pollutants (Williams 2002; Shah and Nongkynrih 2007). Macrophytes are known to have the ability to accumulate pollutants at concentrations several thousand times higher than those in the surrounding water, provided the appropriate chemical form of the pollutant is available in the water (St-Cyr et al. 1994). Ceratophyllum demersum L. (coontail) is known to be a phytoremediator of polluted waters and, in a previous study conducted by Garg and Chandra (1990), this plant was evaluated as a Cr accumulator. This aquatic plant is also preferred for phytoremediation studies of polluted
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water because of features such as being a submerged, floating, rootless macrophyte with a rapid growth rate. Many studies have shown the effects of individual stress factors such as salinity, heavy metals, and pesticides (Yilmaz 2007; Kungolos et al. 2009). Although all types of stressors coexist in nature, their interactions are not sufficiently taken into consideration. Although there have been many studies investigating the effects of Cr(VI) (Augustynowicz et al. 2010; Paiva et al. 2009) and NaCl (Chang et al. 2012; Jampeetong and Brix 2009) on aquatic plants, little information is available about the ecotoxicological risk of their combination in the aquatic environment. In the present study, we used C. demersum as a model plant and investigated the independent and interactive effects of NaCl and Cr(VI) on the biological responses of this aquatic plant. Interestingly, the results of this study show an interaction between the two most important pollutants in exerting their effects on biological responses of aquatic p l an t s a nd m i g h t be us ef ul f or co nd uc t i n g phytoremediation, plant physiology, and toxicology studies.
2 Materials and Methods C. demersum samples were collected from a freshwater pond (Soysalli Spring, Kayseri, Turkey). Collected samples were brought to a laboratory and acclimated in a large aquarium for 5 days under laboratory conditions (115 μmol m−2 ·s−1 light with 14 h photoperiod at 25± 2 °C) in 10 % Hoagland’s solution. Exposure experiments were designed as two different series. In the first series, plants were exposed to nutrient solutions (10 % Hoagland’s solution) with initial Cr(VI) (K2Cr2O7) concentrations of 0, 1, 5, and 10 mM without added NaCl (NaCl) in 400-mL conical flasks under the abovementioned laboratory conditions for 5 days. In the second series, plant samples were exposed to nutrient solutions containing Cr(VI) (0, 1, 5, and 10 mM) and NaCl (0, 125, 250, and 500 mM) for a total of 16 treatments. The pH of the solutions was initially adjusted to 7.5 using a 0.1 M KOH or HCl solution, and no pH adjustment was made during the experiments. The experiments were performed in triplicate, and each replicate contained approximately 5-g plant samples. Doubledistilled water was added daily to each flask to compensate for the water lost. After harvesting, plants were washed with deionized water, blotted on paper
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towels for 2 min, and used for the studying various parameters. The relative growth rate (RGR) was calculated according to Hunt’s equation (Tanhan et al. 2007) as follows: RGR ¼ ½lnðW 2 Þ−lnðW 1 Þ=ðt 2 −t 1 Þ
ð1Þ
where W1 and W2 are the initial and final fresh weights (grams), respectively, and t2 - t1 is the length of the experimental period. The relative water content (RWC) was determined by recording the fresh weight of plant parts and then floating them on deionised water for 5 h. The turgid plant parts were rapidly blotted dry before the determination of turgid weight. The samples were oven dried at 65 °C for 48 h and weighed. The RWC was calculated using the equation of Smart and Birgham (1974): RWCð%Þ ¼ ½ðfresh weight−dry weightÞ= ð2Þ ðturgid weight−fresh weightÞ 100 The Cr, sodium (Na), and chlorine (Cl) contents were determined by drying the plant samples at 70 °C. Each dried sample was digested with 10 mL pure HNO3 by using a CEM-MARS 5 (CEM Corporation Mathews, NC, USA) microwave digestion system. After digestion, the volume of each sample was adjusted to 50 mL by using double-deionized water. The Cr concentrations in all samples were determined using inductively coupled plasma mass spectroscopy (Agilent, 7500a). An aquatic plant (BCR-670) was used as a reference material, and all analytical procedures were also performed on the reference material. Na analysis of solutions was conducted using a Jenway model flame photometer. The Cl content of the solutions was determined using the titrimetric precipitation method. All chemicals were of analytical grade. The ion leakage was determined by rinsing 500-mg plant samples with ultra-pure water to remove any adhering NaCls and placed in flasks containing 100 mL ultra-pure water for 24 h. The conductivity of solutions (microsiemens) was measured using a WTW model conductometer. The level of lipid peroxidation was measured in terms of malondialdehyde (MDA; extinction coefficient, 155 mM−1 cm−1), a product of lipid peroxidation in the plant samples (Heath and Packer 1968). Photosynthetic pigment analyses were conducted
according to the acetone extraction method. Details of the photosynthetic pigment assay can be found elsewhere (Duman et al. 2010). The protein content was determined using the method of Lowry et al. (1951) by using bovine serum albumin as the standard protein. The amount of proline was determined according to the procedure of the modified method of Bates (1973). A 0.25-g sample from each plant was homogenised in 3 % aqueous sulphosalicylic acid, and the proline content was estimated using a ninhydrin reagent. Absorbance of the upper phase was recorded at 520 nm against a toluene blank. Results are presented as the mean±standard error of the mean. Normality was determined using the Kolmogorov–Smirnov statistic, and logarithmic transformations were performed when heterogeneity occurred. Two-way analysis of variance (ANOVA) was used to determine the effects of Cr and NaCl and the interaction of these two parameters on biological responses of C. demersum. Post hoc pairwise comparisons were performed using Tukey’s honestly significant difference test. Further, the effect size of the association of each factor to the ANOVA model was determined by calculating the partial Eta-squared value (η2). The level of significance was p≤0.05. Correlations between all studied parameters were also determined using with Pearson’s correlation coefficient. The theoretically expected effect of the binary mixtures on C. demersum was evaluated using a simple mathematical model (Kungolos et al. 2009; Vellinger et al. 2012) as shown in the following equation: E M ¼ E S þ E Cr −E S ECr =100;
ð3Þ
where EM represents the mixture’s expected effect, Es is the value of the parameter in a certain concentration of NaCl, and ECr is the value of the parameter in a certain concentration of Cr. EM values were calculated for all studied parameters. After a normal distribution test of observed (E0) and calculated values (EM) was conducted, both values were compared using Student’s t test. If the difference between (E0) and (EM) was not significant (p≥0.05), the mode of interaction was considered additive. If (E0) was significantly higher than (EM), the interaction was regarded as synergistic. By contrast, if (E0) was significantly lower than (EM), the interaction was regarded as antagonistic. Statistical tests were performed using SPSS version 15.0.
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3 Results 3.1 Growth of C. demersum Figure 1a shows the effects of Cr and NaCl on the RGR of C. demersum according to exposure concentrations. The lowest RGR value (−1.57 % day−1) was observed in plants exposed to 10 mM Cr+500 mM NaCl. The RGR was strongly affected by Cr concentration (η2 =0.806, p
