Metal Accumulation From Dietary Exposure in the Sea ...

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Mar 9, 2012 - Department of Biology, Valdosta State University,. Valdosta, GA 31698, USA e-mail: [email protected]. S. Ryan Á P. McLoughlin.
Metal Accumulation From Dietary Exposure in the Sea Urchin, Strongylocentrotus droebachiensis Gretchen K. Bielmyer, Tayler A. Jarvis, Benjamin T. Harper, Brittany Butler, Lawrence Rice, Siobhan Ryan & Peter McLoughlin Archives of Environmental Contamination and Toxicology ISSN 0090-4341 Volume 63 Number 1 Arch Environ Contam Toxicol (2012) 63:86-94 DOI 10.1007/s00244-012-9755-6

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Author's personal copy Arch Environ Contam Toxicol (2012) 63:86–94 DOI 10.1007/s00244-012-9755-6

Metal Accumulation From Dietary Exposure in the Sea Urchin, Strongylocentrotus droebachiensis Gretchen K. Bielmyer • Tayler A. Jarvis • Benjamin T. Harper • Brittany Butler • Lawrence Rice • Siobhan Ryan • Peter McLoughlin

Received: 6 November 2011 / Accepted: 13 February 2012 / Published online: 9 March 2012 Ó Springer Science+Business Media, LLC 2012

Abstract Metal contamination is a common problem in aquatic environments and may result in metal bioaccumulation and toxicity in aquatic biota. Recent studies have reported the significance of dietary metal accumulation in aquatic food chains, particularly in species of lower trophic levels. This research investigated the accumulation and effects of dietary metals in a macroinvertebrate. The seaweed species Ulva lactuca and Enteromorpha prolifera were concurrently exposed to five metals (copper, nickel, lead, cadmium, and zinc) and then individually fed to the green sea urchin Strongylocentrotus droebachiensis for a period of 2 weeks. Body mass, test length, total length, and coelomic fluid ion concentration and osmolality were measured. The sea urchins were also dissected and their organs (esophagus, stomach, intestine, gonads, and rectum) digested and analyzed for metals. The results demonstrated that metal accumulation and distribution varied between seaweed species and among metals. In general, there were greater concentrations of metals within the sea urchins fed E. prolifera compared with those fed U. lactuca. All of the metals accumulated within at least one organ of S. droebachiensis, with Cu being most significant. These results indicate that E. prolifera may accumulate metals in a more bioavailable form than within U. lactuca, which could impact the grazer. In this study, no significant differences in body length, growth, or coelomic fluid ion concentration and osmolality were detected between the control and G. K. Bielmyer (&)  T. A. Jarvis  B. T. Harper  B. Butler  L. Rice Department of Biology, Valdosta State University, Valdosta, GA 31698, USA e-mail: [email protected] S. Ryan  P. McLoughlin Waterford Institute of Technology, Waterford, Ireland

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metal-exposed sea urchins after the 2-week testing period. This research presents new data concerning metal accumulation in a marine herbivore after dietary metal exposure.

Many marine ecosystems are heavily influenced by metals, which predominantly enter the environment from sewage, storm water runoff, industrial effluents, metal-plating activities, and mining processes (Mance 1987; El-Rayis et al. 1997; Soualili et al. 2008). Due to their common uses and significant anthropogenic inputs, metals are often found concurrently in aquatic environments. Although essential metals, such as copper (Cu), zinc (Zn), and nickel (Ni), are needed by marine organisms in trace amounts, both essential and nonessential metals may accumulate and/or potentially cause toxicity at increased concentrations (Hornberger et al. 2000; Matthiessen and Law 2002; Cebrian et al. 2003; Bielmyer et al. 2005, 2006). To date, research examining metal accumulation and toxicity in aquatic organisms has largely focused on waterborne metal exposure. However, in the last decade the importance of the dietary metal-exposure route has been demonstrated, particularly in invertebrates (Meyer et al. 2002; Bielmyer and Grosell 2010). Significant reproductive impairment and decreased fecundity has been observed in zooplankton fed metal-laden phytoplankton diets previously exposed to metal concentrations near current water-quality criteria (Bielmyer 2000; Hook and Fisher 2001a, b, 2002; De Shamphalaere et al. 2004; Bielmyer et al. 2006). Bielmyer et al. (2006) reported a significant decrease in copepod fecundity after 7 days of dietary exposure to algae containing 120 lg Ag/g, 14.4 lg Zn/g, 32 lg Cu/g, or 58 lg Ni/g, equating to the following waterborne concentrations in the algal exposure media: 5.5 lg Ag/L, 0.62 lg Zn/L,

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1.5 lg Cu/L, and 7.6 lg Ni/L, respectively. More studies are needed to elucidate the distribution of metals and subsequent responses in invertebrates. Metal accumulation has been documented in predatory gastropods, scallops, and polychaetes as a consequence of dietary metal exposure (Wang and Ke 2002; Selck and Forbes 2004; Pan and Wang 2008). Significant metal accumulation has also been reported in field-collected sea urchins that had been primarily feeding on kelp at a metalcontaminated site (Ahn et al. 2009). In addition, Ahn et al. (2009) indicated that metal-transfer pathways to the sea urchin by way of dietary metal exposure was likely different than through waterborne exposure. When exposed through the water, metals may contact the internal organs of the sea urchin more directly; however, if exposed through the diet, metals theoretically must first traverse the intestinal membrane. Metal uptake at the intestine has been shown to be highly regulated (Ahearn 2010). Relatively low waterborne Cu exposure to the adult sea urchin Diadema antillarum has been shown to cause significant mortality, spine closure at low concentrations, spine loss, significantly increased coelomic fluid CO2 concentration, decreased coelomic fluid pH, and significantly decreased coelomic fluid osmolality (Bielmyer et al. 2005). Furthermore, significant developmental abnormalities have been reported in embryos of D. antillarum as well as other sea urchin species after waterborne exposure to relatively low concentrations of Cu, Zn, Ni, silver (Ag), and selenium (Se) (Kobayashi 1980; Radenac et al 2000; Ferna´ndez and Beiras 2001; Bielmyer et al. 2005; Scha¨fer et al. 2009). These herbivores seem to be sensitive to metal exposure and are important components of many food webs. Examining metal-accumulation patterns and elucidating toxic effects in adult sea urchins exposed to dietary metals may be useful in determining metal bioavailability in contaminated waters and provide necessary needed data concerning the dietary metal-exposure pathway in invertebrates (Augier et al. 1989; Ablanedo et al. 1990; Phillips 1990; Flammang et al. 1997). The green sea urchin Strongylocentrotus droebachiensis is one of the most widely distributed of the echinoderms (Stephens 1972). In many rocky subtidal communities, this sea urchin is the dominant grazer and a keystone organism. Sea urchins have been used for decades as bioindicators for polluted marine systems (Kobayashi 1971; Phillips 1990; Flammang et al. 1997). Seaweeds, particularly Chlorophyta, are important food sources for sea urchins and also serve as biomonitors of marine pollution in coastal areas around the world (Ho 1990; Haritonidis and Malea 1995, 1999; Brown et al. 1999; Villares et al. 2002). Ulva lactuca and Enteromorpha prolifera are species of Chlorophyta that occupy a variety of coastal habitats, such as inner bays and estuaries, and are

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therefore commonly exposed to polluted environments (Ho 1990; Rijstenbil et al. 2006; Murphy et al. 2009a). The species’ high capacity to accumulate and tolerate metals made them ideal for use as experimental diets in this study (Ho 1990; Murphy et al. 2009a). U. lactuca and E. prolifera were concurrently exposed to five different metals: Cu, Ni, Zn, cadmium (Cd), and lead (Pb). The metal-laden seaweeds (U. lactuca or E. prolifera) were then used as diets for S. droebachiensis. The objectives of this study were to quantify metal accumulation in the seaweed and sea urchins and measure growth, coelomic fluid ion concentration and osmolality in S. droebachiensis after the 2-week dietary metal exposure.

Materials and Methods Testing Organisms The sea urchin S. droebachiensis was obtained from the Aquatic Resources Division Marine Biological Laboratory in Woods Hole, MA. The urchins were shipped to the Aquatic Laboratory at Valdosta State University. Before the experiment, S. drobachiensis were held at 15°C in tanks containing carbon filters (Aqua Clear, Mansfield, MA) and acclimated to testing conditions for 2 weeks. There were three urchins per tank, and they were fed daily with 5% of their body weight in U. lactuca, which was also obtained from Woods Hole. Saltwater was prepared by adding artificial sea salt (Instant OceanÒ) to ultra-pure 18 mX Milli-Q water to achieve a salinity of 35 g/L. The solution was mixed for 24 h before use. Experimental Diets The green seaweed U. lactuca and E. prolifera were used as diets for S. droebachiensis. The seaweed was collected from Fethard-on-Sea (Wexford, Southeast Ireland) (pristine site 52°110 53.6800 N, 6°490 34.6400 W) and brought back to the Waterford Institute of Technology (WIT) in Waterford, Ireland. The two seaweed species were individually exposed for 48 h to certified 20 mg/L standards of the following metals in combination: Cu as CuSO4, Zn as ZnSO4, Ni as NiCl2, Cd as CdCl2, and Pb as Pb(CH3 COO)2, and the control seaweed were exposed to pristine seawater. After the exposures, metal concentrations were quantified using microwave assisted acid digestion (modified protocol from Caliceti et al. 2002) and inductively coupled plasma–mass spectroscopy (ICP-MS; Varian 710-OES). BCRÒ reference material no. 60 Lagarosiphon Major and National Institute of Standards and Technology Standard Reference Material Tomato Leaves were used as certified reference materials. Percent metal recovery from

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this process was as follows: 90% Cd and 87% Pb using LM and 91% Cu, 90% Ni, and 91% Zn using TL. The ICP-MS detection limits were as follows: \0.5 lg/L Cu, 0.2 lg/L Zn, \0.08 lg/L Cd, \0.7 lg/L Ni, and \1.5 lg/L Pb. The exposed seaweed was then shipped on ice from WIT to the Aquatic Laboratory at Valdosta State University and preserved at 4°C throughout the experiment. The health of seaweed was verified by microscopic observation. Experimental Design Strongylocentrotus droebachiensis were exposed to metalladen (Pb, Zn, Cu, Ni, and Cd) seaweed or control seaweed of each species (U. lactuca and E. prolifera) for 14 days at a temperature of 14.2°C ± 0.39°C in 12 separate 20-L filtered tanks. Each tank contained a carbon filter to maintain water quality. There were 3 replicate tanks for each treatment and each with 1 urchin/tank. All urchins were measured for weight, total length (including spines), and test (body only) length before being placed in the different treatment tanks. The average weight, total length, and test length were 46.3 ± 7.80 g, 4.5 ± 0.43 cm, and 6.6 ± 0.45 cm, respectively. All urchins were fed 2.5% of their body weight once per day throughout the experiment. Before feeding each day, U. lactuca and E. prolifera remaining in the tanks from the previous day were collected and removed. Seawater in the filtered tanks was renewed at day 7 by pumping out 50% of the water and replacing it with newly made saltwater. In addition, at days 0, 7, and 14 during the experiment, water samples were collected from each tank, filtered, acidified, diluted, and measured for metal content to determine if any leaching occurred from the metal-laden diets. With the exception of Cd, metal concentrations were not significantly different between experimental tanks. Waterborne Cd concentrations were increased in the metal-exposed tanks compared with the controls (Table 1). Water Quality Salinity, dissolved oxygen (DO), and temperature were all measured using a YSI model 85 meter daily during the experiment. Ammonia, nitrite, nitrate, and pH were measured at days 0, 7, and 14 during the experiment using a LaMotte saltwater aquaculture test kit (model AQ-2). The average ± SD of the measured water-quality parameters are listed in Table 1. Sampling At the beginning and end of the exposure period (n = 3 per treatment), the total length, test length, and weight of the urchins were recorded. S. droebachiensis were then

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dissected and tissue samples (esophagus, stomach, intestines, rectum, gonads, and spines) collected. Tissue samples were placed in preweighed tubes and then weighed again to determine the net weight of the samples. Concentrated nitric acid (1 mL) was added to all of the tubes to digest the tissues. The tissue digest was heated in a water bath to 80°C for 24 h. Once fully digested, 5 mL of ultrapure Milli-Q water was added to each sample. All tissue and water samples were analyzed for metal (Pb, Zn, Cu, Ni, and Cd) concentrations by way of atomic absorption spectrophotometry (AAS; Perkin Elmer) with a graphite furnace (\1 lg/L detection limit) or flame as appropriate. Coelemic fluid samples were also collected from all of the urchins, diluted, and K?, Ca2?, and Mg2? ion concentrations measured using AAS. Osmolality of the coelemic fluid was measured with a vapor pressure osmometer (model 5600; Wescor). Data Analysis Data were analyzed for normality and equal variance using a Shapiro–Wilk test and Levene’s test, respectively. Significant differences (p \ 0.05) between treatments were determined by analysis of variance followed by Tukey’s test using the commercial software Sigma Plot 8.0 (San Jose, CA).

Results After 48 h of waterborne exposure to 20 mg/L of Cu, Zn, Ni, Cd, and Pb, significant metal accumulation was observed in both seaweed species (Table 2). Cu accumulation was similar in both species; however, U. lactuca accumulated a nearly two-fold increase in Ni and Zn concentrations (Table 2). When comparing between seaweed species, concentrations of the nonessential metals, i.e., Cd and Pb, were similar; however, U. lactuca accumulated slightly greater concentrations of Pb, whereas E. prolifera accumulated slightly greater concentrations of Cd (Table 2). Strongylocentrotus droebachiensis fed metal-laden seaweed for 2 weeks accumulated significant metal concentrations in several organs. Cu accumulation in metalexposed sea urchins was most significant and showed the clearest trend compared with the controls. There was significant Cu accumulation in esophagus, stomach, and intestines of S. droebachiensis fed metal-contaminated E. prolifera compared with the sea urchins fed control E. prolifera (Fig. 1). In addition, there was a trend of Cu accumulation in every organ of the sea urchins fed both species of metal-contaminated seaweed (Fig. 1). The species of seaweed diet clearly impacted the magnitude of Cu

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Table 1 Water chemistry in the S. droebachiensis exposure media Replicate

Ammonia (mg/L)

Nitrate (mg/L)

Nitrite (mg/L)

pH 7.50 ± 0.0

U. lactuca (control A)

1.50 ± 0.86

0.35 ± 0.56

0.10 ± 0.17

U. lactuca (control B)

0.50 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

U. lactuca (control C)

0.83 ± 0.29

0.40 ± 0.53

0.35 ± 0.26

DO (mg/L)

7.75 ± 0.3

Salinity (ppt)

[Cd] (lg/L)

7.68 ± 0.45

35.4 ± 0.61

1.65 ± 2.34

8.03 ± 0.20

34.5 ± 0.17

1.22 ± 1.73

7.62 ± 0.32

35.4 ± 0.28

2.04 ± 1.81

U. lactuca (metals A)

0.75 ± 0.43

0.20 ± 0.26

0.07 ± 0.12

8.00 ± 0.0

7.72 ± 0.38

35.5 ± 0.57

10.8 ± 11.0

U. lactuca (metals B)

1.17 ± 0.76

0.20 ± 0.26

0.07 ± 0.12

8.00 ± 0.0

7.66 ± 0.44

35.6 ± 0.45

14.3 ± 20.9

U. lactuca (metals C)

1.00 ± 0.87

0.43 ± 0.51

0.15 ± 0.13

8.00 ± 0.0

7.63 ± 0.31

35.6 ± 0.21

7.11 ± 4.61

E. prolifera (control A)

0.53 ± 0.29

0.20 ± 0.26

0.07 ± 0.12

8.00 ± 0.0

7.55 ± 0.54

35.5 ± 0.52

1.45 ± 2.01

E. prolifera (control B)

1.50 ± 0.87

0.40 ± 0.53

0.20 ± 0.13

8.00 ± 0.0

7.66 ± 0.36

35.7 ± 0.36

2.21 ± 2.30

E. prolifera (control C)

0.67 ± 0.29

0.70 ± 0.52

0.43 ± 0.49

7.75 ± 0.3

7.73 ± 0.34

35.8 ± 0.28

5.84 ± 8.05

E. prolifera (metals A)

1.08 ± 0.88

0.10 ± 0.13

0.07 ± 0.12

8.00 ± 0.0

7.47 ± 0.51

35.1 ± 0.47

24.1 ± 38.7

E. prolifera (metals B) E. prolifera (metals C)

0.42 ± 0.14 0.67 ± 0.29

1.45 ± 2.21 0.20 ± 0.26

0.57 ± 0.45 0.17 ± 0.10

7.75 ± 0.3 8.00 ± 0.0

7.72 ± 0.32 7.84 ± 0.43

35.6 ± 0.27 35.7 ± 0.39

13.1 ± 20.9 18.1 ± 10.5

Data are presented as average and SDs of three replicate samples (0, 7, and 14 days) or 14 replicate samples (for DO and salinity) during the course of the exposure Table 2 Average metal concentration (mg/kg wet weight), SD, and 95% CI in U. lactuca and E. Prolifera after waterborne exposure to control and 20 mg/L of Cd, Cu, Pb, Ni, and Zn Metal

Control U. lactuca

Metal-exposed U. lactuca

Average

SD

95% CI

Cd

0.07

0.04

0.04

Cu Pb

0 0

0 0

0 0

Ni

1.28

0.12

0.14

Zn

0

0

0

Metal

Control E. prolifera Average

Average

SD

95% CI

72.03

1.10

1.24

123.7 58.07

12.1 6.10

13.7 6.90

70.85 167.1

3.00 8.83

3.39 12.2

Metal-exposed E. prolifera SD

95% CI

Average 81.59

SD

Cd

0.03

0.01

0.01

Cu

0

0

0

Pb

0

0

0

44.03

5.49

Ni

1.13

0.06

0.06

48.40

6.17

Zn

0

0

0

86.71

17.14

125.5

6.74 10.42

95% CI 7.62 14.4 6.22 6.98 23.8

Calculations were based on triplicate samples

accumulation in the organs of S. droebachiensis, with significant increases observed in the intestines of sea urchins fed metal-contaminated E. prolifera compared with the sea urchins fed metal-contaminated U. lactuca (Fig. 1). Likewise, there was a trend toward increased Ni accumulation in S. droebachiensis fed metal-contaminated E. prolifera in every organ except the gonads (Fig. 2), with significant increases in the stomach and rectum compared with S. droebachiensis fed a control E. prolifera diet (Fig. 2). Contrary to the accumulation patterns of Cu and Ni, Zn accumulated the least in all organs, except the rectum, of S. droebachiensis. There was an apparent increase in the oesophagus (although not significant) and

significant Zn accumulation in the rectum of sea urchins fed metal-contaminated E. prolifera compared with the controls. The levels of Zn in the control S. droebachiensis were similar to the concentrations measured in those exposed to metals even though they were fed a diet with at least an 8000-fold greater Zn concentration (Table 2; Fig. 3). Significant Cd accumulation was observed in the esophagus and stomach of sea urchins fed metal-laden U. lactuca and E. prolifera, respectively, compared with initial controls (Fig. 4), and there was a trend of Cd accumulation in the intestine and rectum of sea urchins fed both species of contaminated-seaweed diets. Likewise,

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Arch Environ Contam Toxicol (2012) 63:86–94 90

ABC

Control

Tissue Zinc (mg/kg wet weight)

Tissue Copper (mg/kg wet weight)

90

U.lactuca control U.lactuca metal E. prolifera control E. prolifera metal

80 70

AB

60 50

A

40 30 20 10

Esophagus Stomach

Intestine

Gonads

60 50 40 30 20 10

40

30

20

10

0

A

Tissue Cadmium (mg/kg wet weight)

Tissue Nickel (mg/kg wet weight)

ABC

U.lactuca control U.lactuca metal E. prolifera control E. prolifera metal

14 12

Intestine

Gonads

Rectum

Fig. 2 Ni accumulation in tissues of S. droebachiensis after 14 days of metal exposure by way of two diets (U. lactuca and E. prolifera). Data are presented as mean ± SEM. A significant difference from the initial control, B significant difference from E. prolifera control, C significant difference from metal-exposed U. lactuca (p B 0.5)

there was a trend of Pb accumulation in the esophagus, stomach, and intestine of sea urchins fed metal-contaminated E. prolifera; however, no significant differences were detected (Fig. 5). Both seaweed species were exposed to the same metal concentrations; however, E. prolifera accumulated less Ni, Pb, and Zn than U. lactuca (Table 2). Despite the lower metal concentrations in E. prolifera, sea urchins fed this seaweed accumulated a greater concentration of metals in almost every organ and for every metal analyzed than sea urchins fed U. lactuca. Significant differences in body length, test length, and weight among urchins in the different treatments were not detected because the sea urchins did not grow significantly

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Intestine

Gonads

Rectum

Control U.lactuca control U.lactuca metal E. prolifera control E. prolifera metal

A

10 8 6 4

A

2 0

Esophagus Stomach

Esophagus Stomach

Fig. 3 Zn accumulation in tissues of S. droebachiensis after 14 days of metal exposure by way of two diets (U. lactuca and E. prolifera). Data are presented as mean ± SEM. A significant difference from the initial control, B significant difference from E. prolifera control, C significant difference from metal-exposed U. lactuca (p B 0.5) 16

50

ABC

Rectum

Fig. 1 Cu accumulation in tissues of S. droebachiensis after 14 days of metal exposure by way of two diets (U. lactuca and E. prolifera). Data are presented as mean ± SEM. A significant difference from the initial control, B significant difference from E. prolifera control, C significant difference from metal-exposed U. lactuca (p B 0.5) Control

U.lactuca control U.lactuca metal E. prolifera control E. prolifera metal

70

0

0

Control

80

Esophagus

Stomach

Intestine

Gonads

Rectum

Fig. 4 Cd accumulation in tissues of S. droebachiensis after 14 days of metal exposure by way of two diets (U. lactuca and E. prolifera). Data are presented as mean ± SEM. A significant difference from the initial control (p B 0.5)

during the experiment. Ion (K?, Ca2?, and Mg2?) concentrations in sea urchin coelomic fluid were not significantly different between treatments (Table 3) nor were there significant differences in ion concentrations between the coelomic fluid of sea urchins and the surrounding seawater. Similarly, coelomic fluid osmolality in sea urchins did not significantly differ between treatments (Table 3).

Discussion Seaweed, particularly U. lactuca, is known to rapidly accumulate metals and therefore has been frequently used in field studies as a bioindicator of metal contamination (Ho 1990; Misheer et al. 2006; Ordun˜a-Rojas and Longoria-Espinoza

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Tissue Lead (mg/kg wet weight)

91

U.lactuca control U.lactuca metal E. prolifera control E. prolifera metal

14 12 10 8 6 4 2 0

Esophagus Stomach

Intestine

Gonads

Rectum

Fig. 5 Pb accumulation in tissues of S. droebachiensis after 14 days of metal exposure by way of two diets (U. lactuca and E. prolifera). Data are presented as mean ± SEM (p B 0.5)

2006; Abdallah and Abdallah 2007; Chaudhuri et al. 2007; Gaudry et al. 2007; Omar 2008). Metal concentrations in seaweed collected from metal-contaminated environments range from 2.1 to 100 lg/g dw Cu, 0.4 to 70.5 lg/g dw Ni, 7.2 to 1015 lg/g dw Zn, 0.1 to 10.0 lg/g dw Cd, and 0.1 to 170 lg/g dw Pb (Dutton et al. 1973; Perdersen 1984; Moore and Ramamoorthy 1984; Guisti 2001; Caliceti et al. 2002; Ordun˜a-Rojas and Longoria-Espinoza 2006). The seaweed species in this study contained similar Zn concentrations as those in heavily polluted environments and approximately 2- to 10-fold greater concentrations of Pb, Cu, Cd, and Ni. The exposure regime used in this experiment (high exposure concentration for a short duration) was useful to rapidly load seaweed with metals so they could be used as sea urchin diets; however, it should be noted that exposure to lower metal concentrations for a longer duration could result in a different internal distribution within the seaweed. Exposure time has been shown to impact the distribution of metals in phytoplankton, and several studies have examined the sorption rates of metals by seaweed (Lau et al. 2003; Omar 2008; Murphy et al. 2009a, b; Areco and Afonso 2010). A previous study reported a fast adsorption rate (approximately 1 to 2 h) for Cu, Zn, Cd, and Pb by the seaweed Gymnogongrus torulosus followed by intraparticle

diffusion (Areco and Afonso 2010). These studies concluded that binding sites on the surface of the cell are generally saturated within a couple of hours depending on different environmental conditions. In this study, it is likely that after 48 h of metal exposure, metals were both adsorbed on the surface and absorbed into the interior of the cell. The magnitude of metal accumulation differed to some extent between seaweed species, possibly due to differences in nutrient requirements and modes of uptake by way of specific transporters. A consistency in both species of seaweed was the preferential bioconcentration of Cu followed by Zn compared with Ni, Cd, and Pb. Storelli et al. (2001) conducted a field study that compared metal accumulation between U. lactuca and E. prolifera, and the results showed patterns of accumulation that were similar to those observed in the present study. Furthermore, Omar (2008) demonstrated that U. lactuca had an increased adsorption affinity for Cu compared with Ni and mangenese when exposed individually and in combination to 10 mg/L. Other studies have also reported an increased binding affinity of Cu for the surface of phytoplankton cells compared with other metals (Knauer et al. 1997). Franklin et al. (2002) reported that Cu exposure actually decreased the uptake of both Zn and Cd in the alga Chlorella sp. after mixed-metals exposure. However, Zn was an effective competitive inhibitor of Cd uptake in the alga Scenedesmus vacuolatus when the concentration of Zn was at least 14-fold greater than the concentration of Cd (Tupperwien et al. 2007). In this study, it is likely that competition between metals occurred for metal-binding sites on the surface of the seaweed. Strongylocentrotus droebachiensis fed metal-laden seaweed for 2 weeks accumulated significant metal concentrations in several organs. For every metal tested, a greater concentration was found in the rectum compared with the other organs, suggesting that the sea urchins were expelling some of the metals taken in by the diet. However, many differences were observed in the pattern of metal accumulation within S. droebachiensis depending on both the metal and the species of seaweed used for food. Significant differences in Cu accumulation were most notable among

Table 3 Strongylocentrotus droebachiensis coelomic fluid ion concentrations and osmolality after 14 days of exposure to control and metalexposed diets Treatment

K? (mg/L)

Ca2? (mg/L)

Mg2? (mg/L)

Osmolality (mM)

U. lactuca control

529.5 ± 75.2

366.4 ± 18.7

1163 ± 67.3

1188 ± 31.11

U. lactuca metals

496.9 ± 61.5

370.9 ± 39.5

1182 ± 67.6

1154 ± 19.80

E. prolifera control

489.1 ± 28.6

381.0 ± 33.2

1268 ± 48.1

1365 ± 29.70

E. prolifera metals

544.2 ± 48.3

370.9 ± 5.3

1240 ± 49.0

1247 ± 46.67

Average ± SDs; n = 3

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the metals tested. A previous study demonstrated spine closure, spine loss, and mortality in adult sea urchins exposed to waterborne Cu concentrations\100 lg/L of Cu (Bielmyer et al. 2005); however, none of those reported behaviours were observed in this study due to dietary Cu uptake. In addition, no changes in coelomic fluid ion concentration and osmolality were observed in S. droebachiensis. This result was somewhat expected due to the mode of metal exposure (by way of diet) as well as the osmoregulatory strategy of this animal (osmoconformer). The osmolality values in S. droebachiensis in this study were comparable with those of D. antillarum in a previous study (Bielmyer et al. 2005). Like Cu, Zn and Ni are essential nutrients to many aquatic organisms. However, Ni and Zn did not accumulate in S. droebachiensis as substantially as did Cu, which might be indicative of an efficient excretion mechanism for excess dietary Zn and Ni. Zn is used in reproductive function, and Zn accumulation in the tissues of sea urchins tends to fluctuate throughout the year and among the different sexes (Watling and Watling 1976; Orren et al. 1980). In addition, Zn is a cofactor in the metzincin super-family of proteins, which have many functions in sea urchin development regulation (Brew et al. 2000; Gallego et al. 2005; Angerer et al. 2006). S. droebachiensis in the wild have been shown to exhibit similar internal Zn concentrations as the urchins used in the present study, even though the diets of sea urchins in the wild had a much lower Zn concentration (Ahn et al. 2009). S. droebachiensis may have a greater Zn requirement and a more efficient homeostatic mechanism for Zn regulation than the other metals tested. Cd is not essential to aquatic organisms, yet it has been shown to substantially accumulate in species of lower trophic levels (Wang et al. 1996; Bargagli et al. 1998; Wang and Ke 2002; Wallace and Luoma 2003; Wallace et al. 2003), which is consistent with the results of this study. Both seaweed and sea urchins accumulated significant amounts of Cd, but the seaweed species accumulated a much greater concentration than was observed in the sea urchins (Figs. 1, 5). It is also important to note that minimal leaching of Cd did occur from the metal-laden seaweed to the sea urchin exposure medium (Table 1), supporting the idea that some of the Cd was only loosely associated with the surface of the phytoplankton. In addition, S. droebachiensis were exposed to minimal waterborne Cd as well as dietary Cd, which could have impacted Cd accumulation in sea urchins. Pb has been shown to accumulate at all levels of the food chain from exposure through water and sediments (Goodyear and McNeill 1999; Campbell et al. 2005). The present study suggests that S. droebachiensis may accumulate Pb from dietary intake; however, more replication and perhaps longer exposure

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duration may be necessary to detect significant differences. In addition, it is possible that the combined dietary exposure of the metals could have resulted in competition for uptake into the sea urchin. Guan and Wang (2004) demonstrated that the assimilation efficiency (AE) of Zn in Daphnia magna was not affected by increasing Zn, Cd, or Se concentrations in their phytoplankton diet. Conversely, the Cd AE was significantly decreased in D. magna fed phytoplankton diets containing increased Zn concentrations (Guan and Wang 2004). In this study, metal accumulation primarily occurred in the digestive system, and metal transport across the integument of the digestive system seems to be greatly regulated, especially given the high dietary metal concentrations used. Competition for uptake across the intestine likely occurred, and it is clear that Cu predominated. The gonads of the sea urchins, which are valued in human consumer markets, did not substantially accumulate metals after the 2-week exposure period; however, after a longer exposure duration, metal accumulation in gonads may be more likely. Differences in metal accumulation in S. droebachiensis resulting from the food sources were apparent in this study. Both seaweed species accumulated similar metal concentrations, but the metal accumulation pattern in sea urchins suggest that metals in U. lactuca may not be as bioavailable as they are in E. prolifera. This may be due to the chemical form of the accumulated metals (Bielmyer and Grosell 2010). In any case, due to their high feeding rates, herbivores may be particularly susceptible to metal bioaccumulation and potentially toxicity through dietary intake (Falconer 2001). However, many species-specific factors may affect whether the accumulated metal causes toxicity (Wang 2001; Wallace et al. 2003; Wallace and Luoma 2003; Selck and Forbes 2004; Luoma and Rainbow 2005; Rainbow 2007; Pan and Wang 2008). More research is needed to examine tissue residues of metals and associated toxic effects in aquatic organisms.

Conclusion In this study, accumulation and effects of a metal-laden (Pb, Zn, Cu, Ni, and Cd) diet in the green sea urchin S. droebachiensis were assessed using two different seaweed species: U. lactuca and E. prolifera. Results indicate that metal accumulation in S. droebachiensis through dietary intake is possible and may occur in the environment at lower metal-exposure levels for longer durations. Of the metals tested, Cu and Zn accumulations were most substantial in the seaweed species, and Cu was most significant in sea urchins fed metal-laden seaweed. S. droebachiensis was able to regulate its internal Zn concentrations to a great

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extent, even after exposure to high dietary Zn concentrations. The diet (i.e., species of seaweed) was another important factor in determining metal accumulation in S. droebachiensis because metals appeared to be more bioavailable in E. prolifera diets. Sea urchins are a food source for many fish, crustaceans, birds, and humans around the world (Pearce et al. 2003; Phillips et al. 2009), and consumption of this species may have implications for consumer health as well as ecosystem dynamics. Acknowledgments The authors thank Bryan Murphy for contributions to this research. Funding was provided by a Valdosta State University Seed Grant.

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