Macroalgal communities play a key role in coastal ecosystem functioning by acting as a primary producer and providing refuge for a number of organisms (Wang et al. 2014; Schiel & Foster 2015). Marine pollution is considered to be one of the factors contributing to the decline of macroalgal populations in coastal ecosystems (Martins et al. 2012; Scherner et al. 2013). Among the pollutants, heavy metals have a significant impact on macroalgal communities. Due to the sensitivity of macroalgae to anthropogenic pressure and environmental factors, these systems are used as a valuable indicator to assess the health of coastal ecosystems (Mannino & Micheli 2020). The effects of metals on algae have been previously studied due to the ability of these organisms to uptake metal ions and accumulate them in their tissues (Flouty & Estephane 2012). Further, heavy metal accumulation in macroalgae has both direct and indirect effects on organisms at higher trophic levels (Contreras-Porcia et al. 2017).
Once accumulated, metals can cause physiological damage depending on the sensitivity of the organisms (Polo et al. 2014; Costa et al. 2016). Higher concentration of metals has severe effects on the growth and metabolism of many algae species (Ismail & Ismail 2017). In brief, exposure of algae to higher concentration of heavy metals resulted in an increase in lipoperoxides and a decrease in antioxidant enzymes, reduction in reactive oxygen species (ROS) production, protein content, free amino acids and carbohydrates (Contreras et al. 2009; Huang et al. 2010; Yadav 2010; Foyer & Noctor 2011). Moreover, heavy metals can also reduce the content of photosynthetic pigments, the growth and cause cell damage (Xia et al. 2004; Saleh 2015; Zhu et al. 2017).
In addition to physiological effects, heavy metals affect the chemical defense properties of marine macroalgal communities (Lurling 2012; Warneke & Long 2015). Macroalgae are characterized by a strong chemical defense mechanism against herbivores and colonizing marine organisms (Paul et al. 2001; Sudatti et al. 2018). Epibiosis or biofouling on surfaces can have detrimental effects on marine macroalgae, mainly reduction in growth and reproduction, biomass loss, depletion of nutrients and tissue damage (Jormalainen & Honkanen, 2008; Da Gama et al. 2014). To reduce fouling, most macroalgae are equipped with antifouling defense mechanisms against both micro- and macrofoulers (da Gama et al. 2008). The antifouling defense is mainly achieved through the production of secondary metabolites. Many bioactive metabolites play an important role in macroalgal defense against fouling organisms and herbivores (Paul et al. 2006; Pereira & Da Gama 2008). In addition, pollutants can interfere with the production and composition of bioactive metabolites in marine algae (Pinto et al. 2011; Gressler et al. 2011). Any change in the biosynthesis of secondary metabolites can reduce the defense properties of macroalgae against fouling organisms and herbivores. Further, algae with fouling organisms on their surface are more attractive to herbivores (Da Gama et al. 2008).
While the physiological effects of metal pollution on macroalgal communities have been studied in detail (Huang et al. 2010; Jiang et al. 2013; Saleh 2015; Costa et al. 2016), the effects of metals on the antifouling defense of marine macroalgae have received little attention. Most of the previous studies have focused on the effects on antioxidant enzymes and total phenolic content (Toth & Pavia 2000; Tzure-Meng et al. 2009; Costa et al. 2016). Therefore, in this study, the effects of two selected metals, copper and cadmium, on macroalgae physiology and antifouling defense were assessed using
The green macroalga
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
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-
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-
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
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
Chl-
Three-way ANOVA results showing the variations in antifouling defense and physiological parameters of the macroalga
Factors | df | Chl- |
Carotenoids | Phenol | Total antioxidant capacity | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
F | F | F | F | F | F | ||||||||
Concentration | 3 | 18.68 | 0.000 | 15.15 | 0.000 | 1.57 | 0.213 | 1.02 | 0.395 | 1.40 | 0.258 | 89.65 | 0.000 |
Days | 1 | 40.29 | 0.000 | 0.91 | 0.345 | 1.61 | 0.213 | 3.48 | 0.070 | 18.08 | 0.000 | 133.51 | 0.000 |
Metals | 1 | 0.15 | 0.695 | 1.98 | 0.168 | 0.00 | 0.934 | 0.48 | 0.490 | 9.10 | 0.004 | 16.22 | 0.000 |
Concentration × Days | 3 | 8.08 | 0.000 | 1.42 | 0.253 | 0.44 | 0.723 | 1.22 | 0.317 | 2.07 | 0.123 | 40.36 | 0.000 |
Concentration × Metals | 3 | 0.52 | 0.666 | 0.77 | 0.517 | 0.08 | 0.970 | 0.66 | 0.582 | 1.99 | 0.134 | 34.77 | 0.000 |
Days × Metals | 1 | 7.98 | 0.008 | 2.28 | 0.140 | 1.38 | 0.248 | 0.08 | 0.776 | 5.68 | 0.023 | 120.35 | 0.000 |
Concentration × Days × Metals | 3 | 4.98 | 0.006 | 3.03 | 0.043 | 0.20 | 0.889 | 0.04 | 0.984 | 1.25 | 0.306 | 29.64 | 0.000 |
Error | 32 | ||||||||||||
Total | 47 |
Changes in chlorophyll-
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).
Effects of heavy metal treatment on total phenol content (mean ± SE, n = 3) in the macroalga
Post-hoc Tukey HSD test results for the effects of different concentrations of copper and cadmium on the macroalga
Factor 1 | Factor 2 | Total antioxidant activity | Bacterial growth |
Bacterial growth |
|||
---|---|---|---|---|---|---|---|
Cu-treated samples | Cd-treated samples | Cu-treated algal extracts | Cd-treated algal extracts | Cu-treated algal extracts | Cd-treated algal extracts | ||
Control | 1 mg l−1 | 0.049 | 0.966 | 0.028 | 0.001 | 0.001 | 0.000 |
3 mg l−1 | 0.000 | 0.000 | 0.020 | 0.007 | 0.001 | 0.005 | |
5 mg l−1 | 0.000 | 0.000 | 0.000 | 0.001 | 0.075 | 0.001 | |
1 mg l−1 | 3 mg l−1 | 0.013 | 0.000 | 1.000 | 0.999 | 1.000 | 0.981 |
5 mg l−1 | 0.668 | 0.000 | 0.669 | 1.000 | 0.811 | 0.999 | |
3 mg l−1 | 5 mg l−1 | 0.473 | 0.000 | 0.755 | 0.999 | 0.789 | 0.999 |
Changes in total antioxidant capacity (mean ± SE, n = 3) in algal samples treated with copper and cadmium for 2 and 7 days. a) Total antioxidant capacity of algal samples treated with copper. b) Total antioxidant capacity of algal samples treated with cadmium.
The extract obtained from control algal samples showed a strong growth inhibitory effect on the bacterium
Effects of heavy metal treatment on antifouling defense of the marine macroalga
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
Correlation between bacterial growth and physiological parameters of the macroalga
Copper-treated samples | Cadmium-treated samples | ||||
---|---|---|---|---|---|
Antioxidant capacity | 0.39 | 0.31 | Antioxidant capacity | 0.44* | 0.39 |
Total phenol | 0.20 | −0.37 | Total phenol | 0.38 | 0.29 |
Chl- |
0.16 | −0.13 | Chl- |
−0.05 | −0.19 |
Carotenoid | 0.05 | −0.08 | Carotenoid | 0.05 | 0.02 |
Cu accumulation | −0.43* | 0.43* | Cd accumulation | −0.008 | 0.047 |
= significant
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-
Bioaccumulation of copper (a) and cadmium (b) in the macroalga
Two-way ANOVA of bioaccumulation of copper and cadmium in the macroalga
Factors | df | Cu accumulation | Cd accumulation | ||
---|---|---|---|---|---|
F | F | ||||
Concentration | 3 | 58.427 | 0.000 | 35.030 | 0.000 |
Days | 1 | 229.51 | 0.000 | 68.458 | 0.000 |
Concentration × Days | 3 | 59.790 | 0.000 | 37.720 | 0.000 |
Error | 16 | ||||
Total | 23 |
Correlation between metal accumulation and physiological parameters of the macroalga
Metal accumulation | Chl- |
Carotenoid | Total phenol | Antioxidant capacity |
---|---|---|---|---|
Copper | −0.444* | −0.264 | −0.481* | 0.208 |
Cadmium | −0.178 | −0.277 | −0.356 | 0.158 |
= significant
Heavy metal pollution is one of the major anthropogenic stressors affecting the marine environment throughout the world (Tzafriri-Milo et al. 2019). After entering the coastal waters through various sources, heavy metals accumulate in marine organisms (Saez et al. 2012; Lozano-Bilbao et al. 2019). This study showed that the macroalga
The results indicated a decrease in chlorophyll-
Most algae species adapt easily to metal pollution through various physiological mechanisms (Contreras-Porcia et al. 2017). The resistance mechanisms against heavy metal toxicity are mainly involved in the production of polyphenols, which act as chelating agents (Toth & Pavia 2000). In general, phenolics prevent the catalytic functions of metals by acting as metal chelators (Wu & Hansen 2008). The results of the present study indicate that the total phenol content was reduced in algal samples treated with copper, while it showed higher values (after 2 days) in samples treated with cadmium. Further, algal samples treated with copper and cadmium showed an increase in antioxidant capacity. As antioxidant activity is directly related to the total phenolic content (Zhang et al. 2007), the increase in total antioxidant capacity in this study may be a resistance strategy of
In this study, antifouling mechanisms of
Macroalgae exhibit constitutive (permanent) and induced (temporary) chemical defenses as protective mechanisms against attacks of herbivores and fouling organisms (Cronin & Hay 1996). The induced chemical defense involves increased production of secondary metabolites due to grazing or other competitors. Therefore, the induced defense trait may be more sensitive to abiotic and biotic factors. In a previous study, Warneke & Long (2015) reported that copper contamination reduced the inducible defense in the marine alga
Of the two metals used in this study, copper is considered an essential trace element for physiological functions of macroalgae (Leal et al. 2018). However, higher concentrations of copper in seawater are reported to have toxic effects on macroalgal communities and other aquatic organisms (Kramer & Clemens 2006; Gouveia et al. 2013). The significant negative correlation between copper accumulation in algal samples and Chl-
In this study, statistically significant differences were observed between the effects of two metals on total antioxidant capacity and total phenolic content of algal samples, with higher phenolic content found in samples treated with cadmium. It has been reported that phenols are secondary metabolites of algae responsible for the antioxidant activity (Ganesan et al. 2008; Pereira et al. 2017). Antioxidant compounds protect algae from the production of reactive oxygen species (ROS) by acting as free radical scavengers (Kokilam & Vasuki 2014). In a previous study, the correlation between stress tolerance and higher levels of antioxidants were observed in brown algae from the genus
In conclusion, the results of the present study indicate that exposure of