Anthropogenic releases of heavy metals into the marine environment have become increasingly frequent in recent decades, causing public concern for the health of nearshore ecosystems. In particular, coastal ecosystems located near large metropolitan areas experience constant exposure to industrial effluents, urban and residential wastes, and recreational pollution, all of which add heavy metals to the ocean (Manahan 1991, Deheyn and Latz 2006, Kimbrough et al. 2008). In addition, excessive use of fertilizers and both organic and inorganic chemicals in urban environments also introduce heavy metals into the ocean, especially after rain events, when storm Durwater runoff carries them to the sea (e.g., Kimbrough et al. 2008). Along the coastal environments of southern California, USA, heavy metal contamination primarily results from non-point sources, and it thereby becomes untraceable regarding its point of origin (Flegal and Sanudo-Wilhelmy 1993, Lenihan et al. 2003). For instance, San Diego Bay, CA is major source of Cu and Zn contamination, due to its high number of recreational and commercial boating marinas, shipyards, and US Naval facilities spreading throughout the bay (San Diego Port District 2009). The use of copper in anti-fouling paints on the hulls of boats (Blossom 2002), and zinc sacrificial anode in the marina’s iron and steel structures (Bird et al. 1996) increase these metals’ concentrations within the bay waters. As a consequence, tidal exchange can then transport this contaminated water out of the bay and into the adjacent Point Loma kelp forest, where the water’s residence time can range from a few of days to a week (Jackson and Winant 1983), during which the metals can become available for uptake by the kelp forest organisms. Specifically, bay water can introduce Cu concentrations of 27 to 78 nmol L-1 km-1 and Zn concentrations of 120 to 200 nmol L-1 km-1 into the Point Loma kelp forest (Volpe and Esser 2002). In addition, the water entering the coastal environment from the adjacent, large metropolitan areas due to storm water runoff can contain heavy metal concentrations exceeding 30 ppb Cu and 100 ppb Zn (Schiff et al. 2001). Together, these can subject the Point Loma kelp forest to both chronic and acute heavy metals exposures that can last up to a week, affecting the ecosystem’s health.
The term “heavy metal,” as used in ecotoxicological studies, encompasses elements that industry commonly uses, that impact aerobic and anaerobic processes, and that researchers generally consider toxic when concentrated (Smith and Scott 1981, Duffus 2002). These metals can generally be categorized depending on how they affect organisms; “essential” metals are those needed, in trace amounts, for many physiological processes, and “non-essential” metals are those that are potentially toxic even at relatively low concentrations (Krzesłowska 2011). This study focused on copper (Cu) and zinc (Zn) and their uptake by the giant kelp,
Researchers have studied heavy metal bioaccumulation in plants and algae from terrestrial (e.g., Fu et al. 2008), tropic (e.g., Amado Filho et al. 1997), and temperate (e.g., Conti and Cecchetti 2003, Gaudry et al. 2007) marine environments. In terrestrial environments, bioaccumulation from contaminated soils may cause plant tissue heavy metal concentrations to exceed soil concentrations, making such plants toxic to consume (Food and Nutrition Board 2001). Likewise, in the marine environment, bioaccumulation from metal contaminated sites can cause marine algal tissue concentrations to exceed such concentrations from non-contaminated sites (e.g., Al-Homaidan 2007, Gaudry et al. 2007). This generally occurs in a two-step process: an initial, rapid, passive uptake, followed by a slower active uptake (Bates et al. 1982). During passive uptake, the cell surface absorbs metal ions within a few seconds to minutes. During active uptake, the cell membrane transports metal ions across and into the cytoplasm, via a metabolism-dependent route whereby heavy metals bind to intercellular compounds and exhibit intracellular precipitation (Kadukova and Vir?ikova 2004). In some cases, this can occur through passive diffusion, since metals increase the cell membrane’s permeability (Gadd 1988, Mehta and Gaur 2005). These metals can then bind either to the cell membrane, one of its constituents, or intercellular molecules, such as metallothioneins, cytoplasmic ligands, and phytochelatins. For example, brown algae cell walls primarily comprise cellulose, for structural support, alginic acid (10-40% DW), in the intercellular matrix, and fucoidins (i.e., fucans, 5-20% DW sulfated polysaccharides) in the extracellular mucilage (Graham and Wilcox 2000, Mehta and Gaur 2005). Of these components, alginic acid and fucans have an affinity for binding metals, as they are abundant in carboxyl (-COOH) groups (Bryan 1971, Krzesłowska 2011). In addition, other functional groups within the cell wall, such as hydroxyl (-OH), amino (-NH2) and sulfhydryl (-SH) increase the metals’ abilities to bind, since they produce negative charges. In water, such metals usually appear in their cationic forms, which allows them to either absorb into, or bind to, the cell wall. Consequently, as metal concentrations increase in the organism’s tissues, they have a greater toxicity potential (Rainbow 2002). For example, the bioaccumulation of metals in macroalgae can prevent the normal compound transport though the cell wall (Manahan 1991), inhibit growth (Amado Filho et al. 1997), prevent settlement (Bryan 1971), and, ultimately, result in mortality (Anderson et al. 1990, Huovinen et al. 2010).
In addition to their impacts on organism growth and survival, heavy metals can adversely affect human health as they build up in the marine organisms’ tissues, through human consumption and utilization of contaminated species. Consequently, numerous US government agencies, such as National Oceanic and Atmospheric Administration (NOAA), US Geological Survey (USGS), and the Evironmental Protection Agency (EPA), have established programs to monitor trends in contaminates, such as heavy metals, within the coastal environment. Many of these monitoring programs rely on bioindicator species that sequester metals from the water column into their tissues, thus reflecting the degree of pollution over time (Martin and Coughtrey 1982). While many of these programs can identify longer-term (months to years) variations in metal concentrations, these programs often sample too infrequently to address short-term (days to weeks) variations. If exposures to elevated metals over krthese short time periods result in elevated heavy metal tissue concentrations in marine organisms, this can profoundly affect commercial and recreational fisheries. This may be especially important to the kelp forest ecosystems in southern California, where runoff from storms can result in Cu and Zn concentrations of 30-60 ppb Cu and 100-150 ppb Zn to enter the forests (Schiff et al. 2001), and where the residence times suggest such elevated concentrations can persist for several days to a week (Jackson and Winant 1983). Thus, understanding how marine algae take up heavy metals from the water column when exposed to these concentrations over a period of a few days may allow researchers to better understand their full impact on coastal ecosystems.
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Collection site and study species
We collected meristematic tissues of the giant kelp,
Prior to running our experiments, we cleaned all glassware, tanks, and dissection materials using the following protocols: we first washed 10 L tanks with 7× cleaning solution for laboratory use, rinsed them with fresh water, and air dried them. Then we allowed the tanks to sit, filled with 2% HCl, for three days, rinsed them with double distilled water, and then air dried and covered them with plastic wrap, to prevent subsequent contamination. In addition, we soaked all glassware and dissection materials in 2% 7× clean solution for two days, rinsed them with fresh water, and placed them in a 1-2% HCl acid bath for two days. The materials were then rinsed with double distilled water, air dried, and wrapped in plastic wrap.
To prepare the
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Background levels of copper and zinc
To characterize natural variabilities in Cu and Zn background levels in
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Macrocystis pyrifera bioaccumulation
All laboratory aquaria experiments were performed at San Diego State University’s Coastal Waters Laboratory San Diego, CA. We placed an array of 24 ten liter clean plastic aquaria (see above), 12 for assessing Cu uptake and 12, for Zn uptake, in a closed, temperature-controlled cold room and held them at 12°C. To circulate the water and aerate the tanks, we utilized four aquarium air pumps. Full-spectrum, 64-W fluorescent bulbs irradiated the room, producing 19-22 micromoles of photons above each tank.
Once the aquaria were set up, we prepared experimental treatments of elevated Zn and Cu from stock solutions. Specifically, we chose three treatments consisting of elevated Cu (30 ppb), elevated Zn (100 ppb), and both elevated Cu and Zn (30 ppb + 100 ppb, respectively), as they represent the expected levels following contamination events that storm water runoff might produce (Schiff et al. 2001). For Cu, we prepared a 1,000 ppm stock solution of copper sulfate (CuSO4), and for Zn we prepared a 600 ppb solution of zinc sulfate (ZnSO4) by dissolving analytical grade CuSO4 and ZnSO4 each into 1 L of double distilled water. Next, we weighed the salts for their respective elements, dissolved them in 1 mL of HCl, and diluted each result to 1 L with double-distilled water. Samples of the stock solutions were then sent to Enviromatrix Analytical Inc., San Diego, CA to validate their Cu and Zn concentrations prior to use. This revealed that the CuSO4 stock solution contained 1,140 ppm Cu, and the ZnSO4 stock solution contained 594 ppm Zn. Following this, we added 0.21 mL CuSO4 stock solution to seawater to create the 30 ppb Cu treatment, 1.35 mL ZnSO4 stock solution to seawater to create the 100 ppb Zn treatment, and used a combination of these two to make the 30 ppb Cu + 100 ppb Zn treatment. All solutions were added to 8 L of seawater collected from the SCRIPPS pier, La Jolla, CA (hereafter “clean” sea water), using a calibrated Pasteur pipette. In addition, we established a fourth, control treatment of clean seawater with no added metals.
After establishing the experimental aquaria, we examined
To determine whether
All data were analyzed using SYSTAT version 12 (Systat Inc., Chicago, IL, USA). To provide a value for each tank (replicate), we averaged the values of the two
Experimental metal concentrations used for the accumulation of Cu and Zn by Macrocystis pyrifera
Bartlett’s tests, and for normality, via graphical inspection of the residuals. We transformed and retested any data not meeting the required tests’ assumptions, to correct any problems. This occurred only once, for Zn accumulation data. Consequently, we applied a square root transformation to these data, which corrected the problem.
Following the meristems’ three-day exposures to elevated metal(s) concentrations and the assumptions testing, we compared
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Background levels of copper and zinc
The concentrations of Cu and Zn in naturally-occurring
concentrations ranged from 0 to 29.8 ppb (mean = 17.7 ppb, Fig. 1). Interestingly, the values of these two metals appeared to co-vary with each other, possibly reflecting contamination events in the water.
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Macrocystis pyrifera bioaccumulation of Cu and Zn
After three days of exposure to elevated metal concentrations in seawater, metal concentrations in the
Cu and Zn uptake by
Anthropogenic actives along the coast of southern California, USA have produced higher heavy metals contamination in its coastal environments (Manahan 1991, Schiff et al. 2001, Deheyn and Latz 2006). Specific to this study, the numerous marinas, shipyards, and commercial activities in San Diego Bay, along with storm water runoff from the surrounding San Diego urban areas, have introduced elevated levels of Cu and Zn into the coastal waters (San Diego Port District 2009). While natural concentrations of these metals in the coastal zone of southern California are typically below 3 ppb Cu and 7 ppb Zn, they can temporarily increase to more than 30 ppb Cu and 100 ppb Zn following rain events, when storm water runoff carries pollutants from the surrounding urban areas into the bay and coastal waters (Schiff et al. 2001). This can cause the kelp forest organisms to accumulate these metals in their tissues, with possible negative impacts on this ecosystem’s health. Our study indicates the giant kelp,
Natural variation in
While these highest levels are well above those one would expect to occur naturally in the Point Loma kelp forest, they do suggest that, under extreme contamination events, such as might occur during sewage spills, industrial accidents, rain events, or re-suspension of metals from contaminated sediments, accumulation of these metals can produce
The different processes by which metals bind within the thalli of algae can allow some marine algae to develop tolerance to increased metal concentrations. Levitt (1980) suggests plant tolerance occurs when plants can neutralize metals inside their cells by removing them from the protoplast and / or neutralizing their toxic effects. Studies have shown brown algae, such as
Both macroalgae and marine invertebrates are well-documented as bio-indicator species for marine pollution (Bryan 1971, Phillips 1976, Ratte 1999, Rainbow 2007, Kimbrough et al. 2008). A bio-indicator species is one that can accumulate and integrate concentrations of several metals in seawater over relatively long intervals (Conti and Cecchetti 2003), reflecting pollution levels in their surrounding environment. Macroalgae generally have higher metal concentrations in their tissues than the surrounding seawater does (Mehta and Gaur 2005), but researchers still commonly use them as bio-indicators. Some commonly-used macroalgae are species of green (e.g.,