Effects of copper and cadmium on physiology and antifouling defense of the marine macroalga Ulva reticulata

The green macroalga Ulva reticulata Forsskal 1775 was collected from the Obhur Creek (21°42′33.52″N; 39°5′45.71″E), the central Red Sea, Saudi Arabia. Salinity, temperature and pH of the creek water at the time of sampling were 39 PSU, 25.5°C and 8.3, respectively. These parameters were measured in situ using a portable multiparameter water quality monitor (Hanna). Many previous studies described hydrographic and environmental properties of the Obhur Creek waters (Alsaafani et al. 2017; Salama et al. 2018). The collected algal samples were brought to the laboratory in a bucket with seawater. In the laboratory, they were initially rinsed with filtered (Millipore, 0.47 µm) seawater to eliminate the epifauna and debris. Ulva reticulata was identified based on morphological characters according to the available identification keys (Dhargalkar & Kavlekar 2004; Brodie et al. 2007). Samples were kept in glass tanks containing filtered seawater. The tanks were kept at 25°C under 80 µmol E m⁻² s⁻¹ illumination (14 h light/10 h dark cycle) with gentle aeration. Salinity and pH of seawater in the tank during the experiment were 38 PSU and 8.3, respectively. Salinity and pH of water were measured using a refractometer and pH meter (Hanna), respectively. Samples were acclimatized under laboratory conditions for 5 days prior to experiments.

Two heavy metals, copper (Cu) in the form of copper sulfate (CuSO₄) and cadmium (Cd) as cadmium chloride (CdCl₂), were used as toxicants to study their effects on the alga U. reticulata. To prepare a stock solution, 5 g of a metal compound was dissolved in 100 ml distilled water in dark bottles. Fresh stock solution was prepared for each day of the experiment.

The acclimatized macroalgal samples (100 g) were transferred to small glass tanks (5 l) with filtered seawater. Three different toxicant concentrations, 1 mg l⁻¹, 3 mg l⁻¹ and 5 mg l⁻¹, were used to study the effects. The toxicant was added from the stock solution prepared for each metal compound. The experiment was conducted for 7 days in replicates (n = 3, three independent experiments were conducted for each metal). Macroalgae samples kept in the tank without the addition of any of the metal solutions were used as a reference. Seawater in the tanks was changed on each day of the experiment and a fresh metal solution was added. Samples (about 20 g) were collected from each tank after 2 and 7 days of exposure to metals. One portion of the collected samples was processed immediately for pigment analysis (Chlorophyll-a and carotenoids). The other portion of an algal sample was dried under shade for one week, powdered and used for the analysis of other parameters. Ethanolic extract of dried algal powder was prepared with 2.5 g of dried algae samples with 10 ml of ethanol. The mixture was kept in a shaker for 3 days and centrifuged at 3000 rpm for 15 min at 4°C. The resulting crude extract was filtered and used for the experiments.

Macroalgal samples (500 mg of fresh algal tissue from each sample) were macerated with 10 ml of acetone in a pestle and mortar under dark conditions. The homogenate was centrifuged at 3000 rpm for 15 min at 4°C. The supernatant was collected in test tubes, which were covered with aluminum foil and the absorbance at 470 nm, 647 nm and 664 nm was measured immediately in a UV-Vis spectrophotometer. The content of chlorophyll-a (Chl-a) and carotenoid was calculated using the formulae provided in the related literature (Jeffrey & Humphrey 1975; Torres et al. 2014).

The total phenolic content in algal samples was measured by the method described by Singleton & Rossi (1965) with some modifications. Distilled water (1.58 ml) and the Folin–Ciocalteu reagent (100 µl) were added to the macroalgal extract (20 µl). The mixture was then allowed to settle for 5 min at room temperature. Sodium carbonate solution (300 µl) was then added and agitated carefully for 10 min. The mixture was kept for 2 h under dark conditions at 20°C. The optical density of the mixture was measured at 765 nm using a spectrophotometer. The total polyphenolic content in the analyzed samples was determined from the absorbance of the standard (gallic acid) and the values obtained were presented as mg gallic acid equivalents g⁻¹ of algal dry weight.

The antioxidant capacity of algal samples was determined by the method described by Prieto et al. (1999). In brief, sulfuric acid (0.6 M), sodium phosphate (28 mM) and ammonium molybdate (4 mM) were mixed to prepare a total antioxidant capacity (TAC) reagent. The TAC (3 ml) was added to 50 µl of algal extracts and kept at 95°C for 90 min in a water bath. After removal from the water bath, the TAC and algal extract mixture was cooled for 10 min at room temperature. The optical density (OD) was then measured at 695 nm in a spectrophotometer using ethanol as blank. The OD of ascorbic acid was used as a standard to calculate the total antioxidant capacity, and the values obtained were presented as equivalents of ascorbic acid (µg ml⁻¹).

This experiment was conducted to understand the variation in antibacterial activity of U. reticulata samples after being treated with heavy metals. Two biofilm-forming bacteria strains, Pseudomonas shioyasakiensis (NCBI: KY224086) and Vibrio harveyi (NCBI: KY266820), maintained in our laboratory, were used as target organisms for a growth inhibition assay. These bacteria strains were isolated from the artificial material submerged in the Obhur Creek (Balqadi et al. 2018). An overnight grown bacteria culture (marine broth, Difco) was used for the experiment. The bacterial culture (3 ml) was collected into test tubes and 30 µl algal extracts were added. Two types of controls, one with 30 µl of ethanol and another without solvent or algal extracts (only bacteria culture) were maintained. The OD of the culture was measured at 570 nm in a spectrophotometer immediately after the addition of the extracts. The test tubes were cotton plugged and kept in an incubator at 30°C for 24 h. After that, the final OD was measured and the percentage of bacteria growth inhibition by the extracts was calculated using the following formula: Growth Rate(%)=Final OD value - Initial OD valueInitial OD value×100 Growth\;Rate\left( \% \right) = {{Final\;OD\;value\, {\text -}\, Initial\;OD\;value} \over {Initial\;OD\;value}} \times 100

The content of heavy copper and cadmium in algal samples treated with different concentrations of solutions of respective metals was analyzed by the method described in Topcuoglu et al. (2003). The dried algal samples (5 g) were placed in Teflon vessels and digested by adding H₂SO₄ (5 ml). The samples were then heated on a hot plate at 70–80°C for 15 min. The sample was then allowed to cool at room temperature and 2 ml of concentrated HNO₃ was added slowly. This mixture was again heated for 30 min and allowed to cool. After cooling, 15 ml of H₂O₂ was added and heated for 2 h at 150°C. Finally, the solution was diluted to 100 ml with 2% concentrated HNO₃ in a volumetric flask. Blank samples were prepared using the same protocol without algal samples. The content of copper and cadmium in the samples was analyzed (minimum detection limit: 0.001 µg g⁻¹) by inductively coupled plasma mass spectrometry (ICP-MS). The obtained values were presented as µg g⁻¹ dry weight of an algal sample.

Data were analyzed for differences in different physiological parameters and bacterial growth inhibitory activity between control and metal-treated algal samples using three-way ANOVA. Concentration, treatment duration and metals were used as factors in ANOVA. Tukey’s post-hoc test was used when ANOVA results showed significant differences between algal samples treated with different concentrations of metals. Two-way ANOVA was also used to examine the differences in bioaccumulation of copper and cadmium in algal samples. Treatment concentration and exposure duration were used as factors in two-way ANOVA. Further, simple correlation analysis was performed to determine the relationship between bacterial growth, bioaccumulation of metals and different physiological parameters. All statistical analyses were carried out using STATISTICA and p <0.05 was considered significant.

Chl-a content in a control algal sample was 200.31 µg g⁻¹ after 2 days and 133.41 µg g⁻¹ after 7 days (Fig. 1). Samples treated with copper and cadmium showed a decrease in Chl-a after 2 and 7 days of exposure. A concentration-dependent decrease was observed in samples treated with both copper and cadmium (not significant, three-way ANOVA; Table 1). The Chl-a content in samples treated with 1 mg l⁻¹ of Cu was 165.8 µg g⁻¹ after 2 days and 143.72 µg g⁻¹ after 7 days. Similarly, the Chl-a content in algal samples treated with 3 mg l⁻¹ of Cu showed a decrease (159.88 µg g⁻¹) after 2 days and 7 days (99.75 µg g⁻¹). The lowest Chl-a content (79.93 µg g⁻¹) was determined in U. reticulata samples treated with 5 mg l⁻¹ of copper for 7 days (Fig. 1).

Figure 1

The carotenoid content in the control algal sample was 88.05 µg g⁻¹ after 2 days and 62.04 µg g⁻¹ after 7 days (Fig. 1). Algal samples treated with copper and cadmium varied in carotenoid content. While a gradual decrease in carotenoid content with treatment concentration was observed in samples treated with copper for 2 and 7 days. A slight increase in carotenoid levels was observed in samples treated with 1 mg l⁻¹ and 3 mg l⁻¹ of cadmium for 7 days. The lowest carotenoid content (27.17 µg g⁻¹) was measured in algal samples treated with 5 mg l⁻¹ of cadmium for 7 days (Fig. 1). Three-way ANOVA results indicated that the differences in carotenoid content due to copper and cadmium treatments were not significant (Table 1).

The control algal sample showed a total phenolic content of 3.02 mg GAE g⁻¹ after 2 days and 1.86 mg GAE g⁻¹ after 7 days. Samples treated with 1 mg l⁻¹ of copper showed a decrease in phenolic content after 2 days (2.204 mg GAE g⁻¹) and 7 days (1.103 mg GAE g⁻¹). Samples treated with 3 mg l⁻¹ and 5 mg l⁻¹ of copper also showed a decrease in phenolic content after 2 and 7 days of treatment (Fig. 2). A very low phenolic content of 0.778 mg GAE g⁻¹ was observed after 7 days of a copper treatment at a dose of 3 mg l⁻¹. Contrary to the copper treatment, samples treated with cadmium showed an increase in total phenolic content after 2 days of exposure (Fig. 2). In algal samples exposed to cadmium for 7 days, a decrease in total phenolic content was observed in samples treated with 3 mg l⁻¹ and 5 mg l⁻¹ (1.48 and 0.98 mg GAE g⁻¹, respectively). Further, ANOVA results showed significant differences in the total phenolic content in algal samples depending on the treatment duration (Table 1). Changes in the phenol content in algal samples also showed significant differences between Cu and Cd treatments (Table 1).

Figure 2

U. reticulata samples (control) revealed the antioxidant capacity of 330.87 and 262.64 µg ml⁻¹ after 2 and 7 days under laboratory conditions. However, samples treated with copper and cadmium showed higher levels (significant variation between the metals; Table 1) compared to the control after both 2 and 7 days of exposure (Fig. 3). The antioxidant capacity was remarkably high in samples observed after 2 days (706.5 µg ml⁻¹ for copper-treated samples; 1696.65 µg ml⁻¹ for cadmium-treated samples). In general, a concentration-dependent increase in antioxidant capacity was observed in samples treated for 2 days with both copper and cadmium. The highest antioxidant capacity of 1696.65 µg ml⁻¹ was observed in the extract of algal samples treated with 5 mg l⁻¹ of cadmium for 2 days. Three-way ANOVA results indicated a significant variation in antioxidant capacity as a function of metal concentration and exposure duration (Table 1). Further, significant variations were observed for all interactions between the factors used in ANOVA. While the antioxidant capacity for all concentrations of copper treated samples differed significantly from the control, an algal sample treated with 1 mg l⁻¹ of cadmium showed no significant difference from the control (Table 2).

Figure 3

The extract obtained from control algal samples showed a strong growth inhibitory effect on the bacterium P. shioyasakiensis. However, algal samples treated with different concentrations of copper and cadmium for 2 days exhibited a relatively weak growth inhibitory effect on P. shioyasakiensis (Fig. 4). Further, extracts obtained from algae treated with 1 mg l⁻¹ of copper and cadmium for 2 days showed very low growth inhibitory activity. Algal samples treated with copper and cadmium for 7 days also showed lower growth inhibitory activity (except the extract obtained from samples treated with 5 mg l⁻¹ of cadmium). Algal extracts obtained from samples treated with copper and cadmium exhibited a weak growth inhibitory effect on the bacterium V. harveyi (Fig. 4). A concentration-dependent reduction in bacterial growth inhibitory activity was observed for extracts obtained from samples treated with cadmium for 2 days. The growth inhibitory activity of algal samples treated with copper and cadmium was very low except for the extract obtained from samples treated with copper at 5 mg l⁻¹ for 2 days.

Figure 4

The ANOVA results indicated a significant variation in bacterial growth inhibitory activity of algal extracts depending on the concentration of metals and days of exposure against P. shioyasakiensis (Table 1). On the other hand, a significant difference was observed between metal concentration and growth percentage of V. harveyi (Table 1). Tukey’s post-hoc test revealed significant variation in bacterial growth inhibitory activity between algal samples treated with different concentrations of metals and control samples (Table 2). The growth rate of P. shioyasakiensis also showed a significant negative correlation with the bioaccumulation of copper in algal samples (Table 3), but V. harveyi showed a significant positive correlation. A positive correlation was determined between the antioxidant activity and the growth rate of P. shioyasakiensis (Table 3).

The content of copper and cadmium in algal samples treated with different concentrations of copper sulfate and cadmium chloride is presented on Figure 5. In control samples, the copper content was 0.057 and 0.049 µg g⁻¹ after 2 and 7 days under laboratory conditions. However, copper accumulation in the algal tissue was observed in samples treated with copper sulfate with a maximum of 17.71 µg g⁻¹ (samples treated with 5 mg l⁻¹ of copper for 7 days). Similarly, cadmium accumulation was observed in algal samples treated with 5 mg l⁻¹ of cadmium for 7 days, with a maximum of 0.72 µg g⁻¹. The cadmium content in control algal samples was 0.012 and 0.003 µg g⁻¹ after 2 and 7 days. In general, a significant increase in metal content was observed in algal samples with increasing exposure duration and treatment concentrations (Table 4). The bioaccumulation level of copper in algal samples indicated a significant negative correlation with Chl-a and the total phenolic content in algal samples (Table 5). However, cadmium concentration in algal samples showed no significant correlation with physiological parameters.

Figure 5