Antagonist Effects of Sodium Chloride on the Biological Responses of an Aquatic Plant (Ceratophyllum demersum L.) Exposed to Hexavalent Chromium

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1 Water Air Soil Pollut (2014) 225:1865 DOI /s 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, Kayseri, Turkey fduman@erciyes.edu.tr S. Sahan Department of Chemistry, Faculty of Sciences, Erciyes University, 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

2 1865, Page 2 of 12 Water Air Soil Pollut (2014) 225:1865 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) founda 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 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; Kungolosetal. 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 plants and might be useful for conducting phytoremediation, plant physiology, and toxicology studies. 2MaterialsandMethods 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) (K 2 Cr 2 O 7 )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

3 Water Air Soil Pollut (2014) 225:1865 Page 3 of 12, 1865 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 ÞŠ= ðt 2 t 1 Þ ð1þ where W 1 and W 2 are the initial and final fresh weights (grams), respectively, and t 2 -t 1 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): RWC ð% Þ ¼ ½ðfresh weight dry weightþ= ðturgid weight fresh weightþš100 ð2þ 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 HNO 3 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 (η 2 ). The level of significance was p 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 E Cr =100; ð3þ where E M represents the mixture s expected effect, E s is the value of the parameter in a certain concentration of NaCl, and E Cr is the value of the parameter in a certain concentration of Cr. E M values were calculated for all studied parameters. After a normal distribution test of observed (E 0 ) and calculated values (E M ) was conducted, both values were compared using Student s t test. If the difference between (E 0 )and(e M ) was not significant (p 0.05), the mode of interaction was considered additive. If (E 0 ) was significantly higher than (E M ), the interaction was regarded as synergistic. By contrast, if (E 0 ) was significantly lower than (E M ), the interaction was regarded as antagonistic. Statistical tests were performed using SPSS version 15.0.

4 1865, Page 4 of 12 Water Air Soil Pollut (2014) 225:1865 3Results 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 (η 2 =0.806, p<0.01) and by the presence of NaCl in the solution (η 2 =0.857, p<0.01; Table 1). An increase in Cr and NaCl concentrations in the solution resulted in a decrease in the RGR values compared with those of the control. Further, Cr and NaCl had a strong interactive effect on RGR (η 2 =0.571, p<0.01). The interaction of NaCl and Cr might be characterised as antagonistic to the growth of C. demersum (Table 2). As it is seen at Table 3, the most important relationship between RGR and all other studied parameters was with carotenoid (R=0.646, p<0.01). 3.2 Relative Water Content Two-way ANOVA indicated that Cr had no significant effect on water content (η 2 =0.169, p>0.05; Table 1). However, there was a significant effect of NaCl on this parameter (η 2 =0.541, p<0.01). At NaCl concentrations of 250 and 500 mm, water content was found to be significantly lower than that in the control. In comparison to that in the control, an approximately 2 % increase was observed with the application of 1 mm Cr (Fig. 1b). The maximum rate of decrease (3 %) was recorded for an exposure of 10 mm Cr+500 mm NaCl. For all tested concentrations, the mode of action was antagonistic (Table 2). The highest correlation coefficient value was observed between RGR and RWC (R=0.646, p<0.01) 3.3 Cr accumulation Exposure to Cr(VI) and/or NaCl caused an increase in Cr accumulation concentration (η 2 =0.986, p<0.01). Furthermore, NaCl strongly affected Cr accumulation (η 2 =0.635, p<0.01). In this study, Cr and NaCl had a strong interactive effect on Cr accumulation (η 2 =0.526, p<0.01). According to a post hoc analysis, application of 125 mm NaCl significantly increased Cr accumulation compared with that of the controls. Furthermore, there was a significant synergistic interaction observed on Cr accumulation in the plants treated with 10 mm Cr+125 mm NaCl (Table 2). The highest Cr accumulation (0.42 mmol g 1 ) was observed in plants exposed to 10 mm Cr+125 mm NaCl (Fig. 1c). Post hoc comparisons showed that 500 mm NaCl application decreased Cr accumulation compared with that in the control (p<0.05). Similarly, for 1 mm Cr+250 mm NaCl and 1 mm Cr+500 mm NaCl applications, the mode of action was antagonistic (Table 2). There was no significant correlation between Cr accumulation and Na and Cl accumulation in plants. 3.4 Na and Cl Accumulation The concentration of both Na and Cl in plants increased in the presence of NaCl in the solution (η 2 =0.979, p<0.01; η 2 =0.978, p<0.01, respectively). Cr concentration in solution significantly affected both Na (η 2 = 0.556, p<0.01) and Cl (η 2 =0.517, p<0.01) uptake. Further, Cr and NaCl had a strong synergistic interactive effect on Na (η 2 =0.763, p<0.01) and Cl (η 2 =0.741, p<0.01) uptake. Na uptake increased in 10 mm Cr solution compared with that in the controls, whereas Cl uptake decreased in plants treated with 5 mm Cr solution compared with that in the controls. The mode of action was found to be synergistic for 10 mm Cr+ 500 mm NaCl (Table 2). However, for all other combinations, the mode of action was antagonistic or additive. The highest Na accumulation (4.79 mmol g 1 )was observed at 10 mm Cr+500 mm NaCl. However, the highest Cl accumulation (1.25 mmol g 1 ) was observed at 1 mm Cr+500 mm NaCl (Fig. 1d, e). Both Na and Cl showed a strong relation with electric conductivity (EC). 3.5 Ion Leakage Exposure to both Cr (η 2 =0.925, p<0.01) and NaCl (η 2 = 0.994, p<0.01) significantly affected the measured EC. Exposure to Cr and/or NaCl increased EC values. The highest EC value (113 mmhos cm 1 g 1 fw) was observed in plants exposed to 10 mm Cr+500 mm NaCl (Fig. 1f). Furthermore, a strong interaction (η 2 =0.633, p<0.01) between Cr and NaCl on EC values was observed by two-way ANOVA. The mode of action was synergistic or additive for the tested concentrations (Table 2). 3.6 Lipid Peroxidation Both Cr (η 2 =0.934, p<0.01) and NaCl (η 2 =0.960, p<0.01) had a significant effect on lipid peroxidation.

5 Water Air Soil Pollut (2014) 225:1865 Page 5 of 12, 1865 A B C D E F Fig. 1 Effects of Cr(VI) alone and in combination with NaCl on RGR (a), RWC (b), Cr accumulation (c), Na accumulation (d), Cl accumulation (e)andec(f)ofc. demersum. Values are expressed as means. Vertical bars indicate the standard error of three separate experiments

6 1865, Page 6 of 12 Water Air Soil Pollut (2014) 225:1865 Table 1 Two-way ANOVA summary for studied parameters (partial eta-squared (η 2 ) shows effect size of factors and interactions contribution to the overall effects; degrees of freedom (df)) df F P Effect size (η 2 ) RGR Salt < Cr < Salt Cr < RWC Salt < Cr Salt x Cr Cr accumulation Salt < Cr < Salt x Cr < Na accumulation Salt < Cr 3 20 < Salt x Cr < Cl accumulation Salt < Cr < Salt x Cr < EC Salt < Cr < Salt x Cr < MDA Salt < Cr < Salt x Cr < Total Chlorophyll Salt Cr < Salt x Cr < Carotenoid Salt < Cr < Table 1 (continued) There was a significant interactive effect of Cr and NaCl on lipid peroxidation (η 2 =0.838, p<0.01; Table 1). The highest MDA levels (31.59 μmol g 1 fw) were observed in plants exposed to 10 mm Cr+500 mm NaCl (Fig. 2a). Post hoc comparisons suggested that MDA values increased with increasing Cr and NaCl concentrations. For most of the tested concentrations, there was an antagonistic effect between NaCl and Cr (Table 2). 3.7 Photosynthetic Activity df F P Effect size (η 2 ) Salt x Cr < Protein Salt < Cr < Salt x Cr < Proline Salt Cr Salt x Cr The total chlorophyll and carotenoid contents were significantly affected by the presence of both NaCl and Cr in the solution, with a significant interactive effect (η 2 = 0.579, p<0.01; η 2 =0.640, p<0.01, respectively). The total chlorophyll and carotenoid contents significantly decreased in the presence of 250 and 500 mm NaCl. The total chlorophyll content decreased in the presence of 5 and 10 mm Cr, while the carotenoid content decreased depending on the concentration of Cr. The interaction of NaCl and Cr might thus be characterised as antagonistic towards both total chlorophyll and carotenoid contents (Table 2). The highest correlation between carotenoid and other parameters was between carotenoid and total chlorophyll (R=0.671, p<0.01). 3.8 Protein and Proline Contents Exposure to both Cr (η 2 =0.530,p<0.01)andNaCl(η 2 = 0.864, p<0.01) significantly affected the protein content (Table 1). A strong synergistic interaction (η 2 =0.562, p<0.01) was observed between Cr and NaCl on protein

7 Water Air Soil Pollut (2014) 225:1865 Page 7 of 12, 1865 Table 2 Interactive effects of Cr and salt on studied parameters for all tested combinations Concentration combinations RGR RWC Cr accumulation Na accumulation Cl accumulation EC NaCl (mm) Cr (mm) MoA p value MoA p value MoA p Value MoA p value MoA p value MoA Anta <0.001 Anta <0.001 Add Anta <0.001 Anta <0.001 Add Anta Anta <0.001 Add Anta <0.001 Anta Syn Anta <0.001 Anta <0.001 Syn 0.04 Anta <0.001 Anta <0.001 Syn Anta <0.001 Anta <0.001 Anta Anta Add Add Anta Anta <0.001 Add Add Anta Syn Anta 0.04 Anta <0.001 Add Add Add Syn Anta <0.001 Anta <0.001 Anta Anta 0.02 Syn Add Add Anta <0.001 Add Anta 0.03 Anta Syn Add Anta <0.001 Add Syn Add Syn Concentration combinations MDA Total chlorophyll Carotenoid Protein Proline NaCl (mm) p value MoA p value MoA p value MoA p value MoA p Value 125 <0.001 Anta <0.001 Anta <0.001 Anta Add <0.001 Anta <0.001 Anta <0.001 Anta Add <0.001 Anta <0.001 Anta <0.001 Anta Add <0.001 Anta <0.001 Anta <0.001 Anta <0.001 Add <0.001 Anta <0.001 Anta <0.001 Anta <0.001 Add Anta Anta <0.001 Anta Add Add 0.06 Anta <0.001 Anta Add Add 0.06 Anta Anta <0.001 Add Anta Anta <0.001 Anta Add p values show the significant difference between expected observed values according to Student s t test (p<0.05) MoA mode of action, Anta antagonistic, Add additive, Syn synergistic

8 1865, Page 8 of 12 Water Air Soil Pollut (2014) 225:1865 Table 3 Correlation between studied parameters Cr Cl Na EC MDA RGR Total chlorophyll Prolin RWC Protein Carotenoid Cr 1 Cl Na ** 1 EC ** 0.907** 1 MDA 0.457** 0.584** 0.791** 0.828** 1 RGR 0.486** 0.548** 0.638** 0.758** 0.654** 1 Total 0.390** ** 0.367** 0.428** 0.514** 1 chlorophyll Proline 0.263* 0.366** 0.395** 0.390** 0.414** 0.259* RWC ** 0.595** 0.607** 0.582** 0.626** 0.512** Protein Carotenoid 0.605** * 0.390** 0.390** 0.646** 0.671** ** *0.05; **0.01 significance level of correlation content. The protein content increased with increasing NaCl concentration and decreased in the presence of 5 mm Cr compared with that in the control. The mode of action between NaCl and Cr on protein content was antagonistic. A two-way ANOVA indicated that Cr had no significant effect on the proline content (η 2 =0.102, p>0.05), whereas NaCl had (η 2 =0.15, p <0.05). Furthermore, there was no significant interactive effect of Cr and NaCl on the proline content. Proline content increased in the presence of 500 mm NaCl. For proline, the interaction of NaCl and Cr was characterised as additive. 4 Discussion Our results suggest that 125 mm NaCl adversely affects the growth of C. demersum. Jampeetong and Brix (2009) also indicated that the growth of Salvinia natans was reduced after treatment with 50 mm NaCl compared with that of the control. The reason for the slower growth might be the inhibition of K + uptake, membrane dysfunction, and reactive oxygen species generation in the cells (Jampeetong and Brix 2009). Higher Cr concentrations in the medium also decreased plant growth. Vallisneria spiralis plants have been shown to have lower biomass when exposed to Cr (Vajpayee et al. 2000). A significant interactive effect of NaCl and Cr on the growth of C. demersum was also found in this study. Generally, the mode of interaction was antagonistic, but it became additive at the highest concentrations of NaCl and Cr. Similar results were observed by Vellinger et al. (2012) for Gammarus pulex. Dönmez and Aksu (2002) showed that Cr uptake from saline waters by dried Chlorella species decreases because of increasing ionic strength. Furthermore, competition between Cl and Cr 2 O 7 2 for binding sites and decrease of cell membrane permeability because of the inhibitor effect of NaCl might be the reason for the reduced Cr accumulation. Another factor that affects bioaccumulation is the molecular size of the competitors, which determines whether they can pass through the pores of cell walls and cell membranes. Unlike large molecules, small ones are known to pass easily through cell membranes. Density also affects the transition ratio of matter. Passing through membranes is considerably easier for dense matters than diluted ones. For example, when 125 mm NaCl is dissolved in water, 125 mm Na + and Cl are formed; hence, the concentration of Cl - becomes higher than that of Cr(VI). This suggests that the competition is in favour of Cl. An electrostatic interaction is known to occur between Na + and Cr 2 O 7 2. However, at the ph level (7.5) used in our study, NaCr 2 O 7 dissociates easily. NaCl is primarily known to have an osmotic effect on plant growth. Osmotic stress is induced by lowering of the water potential causing reduced turgor and cellular water loss. In this study, high NaCl concentrations (250 and 500 mm) in the growth medium negatively affected plant water content. Under NaCl stress, C. demersum

9 Water Air Soil Pollut (2014) 225:1865 Page 9 of 12, 1865 A B C D E Fig. 2 Effects of Cr(VI) alone and in combination with NaCl on MDA (a), total chlorophyll (b), carotenoid (c), protein (d) and proline content (e) ofc. demersum. Values are expressed as means. Vertical bars indicate the standard error of three separate experiments

10 1865, Page 10 of 12 Water Air Soil Pollut (2014) 225:1865 reacted by significantly decreasing the water potential. Little information is available on the exact effect of Cr on the water relations of higher plants (Shanker et al. 2005). Excess Cr has been shown to decrease the water potential in cauliflower (Chatterjee and Chatterjee 2000). Our study showed that the water content was not significantly affected by the presence of Cr in the growth medium. Similartothatreportedbypreviousstudies(Duman et al. 2009; Mishra and Tripathi 2009; Augustynowicz et al. 2010), uptake of Cr was found to be concentration dependent in our study. In this study, a significant effect of NaCl on Cr accumulation was found. In the literature, reports on interactions between NaCl and trace elements on the element concentration in plants are contradictory. For example, Yilmaz (2007) reported that salinity decreased nickel accumulation in duckweed (L. gibba), whereas it increased boron concentration in cucumber (Alpaslan and Gunes 2001). Furthermore, Fritioff et al. (2005) found that lead concentrations in aquatic plants were not affected by salinity. Alpaslan and Gunes (2001) found that boron increases because of a decreased transpiration rate in plants. However, there are no reports in the literature on the effect of NaCl on Cr uptake by aquatic plants. Cr accumulation increased in the presence of 125 mm NaCl. These results suggest that the addition of 125 mm NaCl might be effective at increasing Cr accumulation and can be used for the phytoextraction of Cr. Higher NaCl concentrations (250 and 500 mm) reduced Cr accumulation. These findings are consistent with those reported by Yilmaz (2007). An increased concentration of Na and Cl is expected in plants with an increase in NaCl concentration (Eraslan et al. 2007; Ismail2003). The Na + /K + ratio is crucial for the NaCl tolerance of plants. When the external concentration of Na + is substantially higher than that of K +, plant cells cannot maintain Na + /K + homeostasis. In this study, 10 mm Cr was found to increase the Na content. Thus, the Na + /K + ratio might be thought to have changed. When K 2 Cr 2 O 7 is dissolved in water, 2K + and Cr 2 O 7 2 are produced, Cr 2 O 7 2 competes with the Cl present in the media. Consequently, the uptake of Cl by plants decreases. Therefore, 5 and 10 mm of Cr(VI) or Cr 2 O 7 2 might decrease the Cl content of plants. Cell membrane stability has been widely used as a criterion to determine abiotic stress tolerance (Devi and Prasad 1998; McCannandSolomon2000). In this study, ion leakage of C. demersum was assessed to study the extent of membrane damage. Both NaCl and Cr stresses were found to increase ion leakage. Similar to our findings, Masood et al. (2006) showed that NaCl stress increased ion leakage at NaCl concentrations of 30 and 40 mm in two varieties of Azolla, an aquatic fern. Cr exposure is known to increase membrane damage in aquatic plants (Sinha et al. 2005). NaCl stress was found to have a stronger effect on ion leakage than Cr stress in our study. Interestingly, unlike the other studied parameters, the interaction between NaCl and Cr was synergistic or additive. The interactive effect of chemicals on the studied parameters might thus be different. This finding needs to be taken into consideration for ecotoxicological studies. MDA is a cytotoxic product of lipid peroxidation and an indicator of free radical production and consequent tissue damage (Ohkawa et al. 1979). An increase in the MDA level due to increasing pollutant concentration and a longer exposure period indicates oxidative stress. Chang et al. (2012) showed that high concentration of NaCl (200 mm) inhibited plant growth and increased lipid peroxidation. Our results showed that both NaCl and Cr stress increased lipid peroxidation. However, similar to the effect on ion leakage, NaCl stress had a larger effect than Cr stress on lipid peroxidation. Consequently, the synergistic effect of NaCl and Cr indicated that NaCl stress increases the deleterious effect of Cr on membrane stability. In addition, NaCl and Cr were found to have an antagonistic or additive influence on MDA content. The most important visible response of plants against stressors is chlorosis. Chlorosis is related to the degradation of photosynthetic pigments and reduction in photosynthetic activity. Similar to our findings, Jampeetong andbrix(2009) found that, when an aquatic plant (Salvinia natans) was exposed to increasing NaCl concentrations, photosynthetic activity significantly decreased. Previous studies also showed that Cr exposure reduces photosynthetic activity (Ganesh et al. 2008; Augustynowicz et al. 2010). Decreased photosynthetic activity might be attributed to various factors such as peroxidative breakdown of pigments and an increase in the rate of chlorophyllase activity (Ozturk et al. 2010). Our findings are in agreement with those of the previous studies. In addition, Cr had a stronger effect than NaCl on photosynthetic pigment content. Furthermore, for all tested combinations, NaCl and Cr had an antagonistic interaction in their effects on both total chlorophyll and carotenoid contents.

11 Water Air Soil Pollut (2014) 225:1865 Page 11 of 12, 1865 Changes in protein levels in plant cells are an indicator of changing environmental conditions. Low concentrations of both NaCl and Cr increased the protein content compared with that of the controls; however, at higher concentrations, protein content decreased. The increase might be due to the production of stress proteins that are required to cope with the stress conditions. As mentioned above, protein content increased at higher concentrations of NaCl and Cr. Pinheiro et al. (2002) stated that protein content decreases with increasing lipid peroxidation. Our findings are in agreement with this conclusion. In this study, there were significant synergistic effects of NaCl and Cr on the protein content of C. demersum. For all tested combinations, the NaCl Cr interaction was antagonistic. When a plant is exposed to salinity stress, the first response is an adjustment of cell turgor pressure and then the production of some osmotically active compounds such as proline, provided the stress continues (Nejrup and Pedersen 2008). Proline is known to play a significant role as an enzyme protectant (Shah and Dubey 1998). According to our findings, Cr had no significant effects on proline levels, whereas salt had. Rout et al. (1998) also found that salt stress increased proline levels in three different aquatic macrophytes. 5 Conclusions The following conclusions can be drawn from this study: 1. The mode of action might change depending on the concentrations of binary exposure. For example, while 125 mm NaCl exposure increased Cr accumulation, high NaCl concentrations might decrease Cr accumulation. The interaction of chemicals needs to be investigated across a larger range of concentrations to better understand the toxicological effects of combinations of pollutants. 2. Changes in Cr concentrations did not significantly affect proline and water contents. However, NaCl significantly affected all the studied parameters. 3. There were significant interactive effects of Cr and NaCl on all the studied parameters except for proline and water contents. Although Cr had a greater effect on photosynthetic pigments and proline than NaCl, NaCl had a larger effect on other parameters. 4. Except for effects on EC and proline content, interactions between Cr and NaCl can be characterised as antagonistic. 5. These results suggested that C. demersum might be useful for the bioremoval of Cr from moderately NaCly water. References Aksu, Z., & Balibek, E. (2007). Chromium(VI) biosorption by dried Rhizopus arrhizus: effect of salt (NaCl) concentration on equilibrium and kinetic parameters. Journal of Hazardous Materials, 145, Alpaslan, M., & Gunes, A. (2001). Interactive effects of boron and salinity stress on the growth, membrane permeability and mineral composition of tomato and cucumber plants. Plant and Soil, 236, Augustynowicz, J., Grosicki, M., Hanus-Fajerska, E., Lekka, M., Waloszek, A., & Kołoczek, H. (2010). Chromium(VI) bioremediation by aquatic macrophyte Callitriche cophocarpa Sendtn. Chemosphere, 79, Bates, L. S. (1973). Rapid determination of free proline for waterstress studies. Plant and Soil, 39, Chang, I. H., Cheng, K. T., Huang, P. C., Yen, Y. L., Lee-Ju, C., & Tai-Sheng, C. (2012). Oxidative stress in greater duckweed (Spirodela polyrhiza) caused by long-term NaCl exposure. Acta Physiologiae Plantarum, 34, Chatterjee, J., & Chatterjee, C. (2000). Phytotoxicity of cobalt, chromium and copper in cauliflower. Environmental Pollution, 109, Connan, S., & Stengel, D. B. (2011). Impacts of ambient salinity and copper on brown algae: 1. Interactive effects on photosynthesis, growth, and copper accumulation. Aquatic Toxicology, 104, Devi, S. R., & Prasad, M. N. V. (1998). Copper toxicity in ceratophyllum demersum L. (Coontail), a free floating macrophyte: response of antioxidant enzymes andantioxidants. Plant Science, 138, Dönmez, G., & Aksu, Z. (2002). Removal of chromium(vi) from saline wastewaters by Dunaliella species. Process Biochemistry, 38, Duman, F., Leblebici, Z., & Aksoy, A. (2009). Growth and bioaccumulation characteristics of watercress (Nasturtium officinale R. Br.) exposed to cadmium, cobalt and chromium. Chemical Speciation and Bioavailability, 21, Duman, F., Ozturk, F., & Aydın, Z. (2010). Biological responses of duckweed (Lemna minor L.) exposed to the inorganic arsenic species As(III) and As(V): effects of concentration and duration of exposure. Ecotoxicology, 19, Eraslan, F., Inal, A., Savasturk, O., & Güneş, A. (2007). Changes in antioxidative system and membrane damage of lettuce in response to salinity and boron toxicity. Science Hortic- Amsterdam, 114, Fritioff, A., Kautsky, L., & Greger, M. (2005). Influence of temperature and salinity on heavy metal uptake by submersed plants. Environmental Pollution, 133,

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