FACTORS CONTROLLING THE BIOACCUMULATION OF MERCURY AND METHYLMERCURY INTO BENTHIC INVERTEBRATES AND FISH

 

Robert P Mason, Kelly M McAloon, Joy J Leaner, Jean-Michel Laporte and Sandrine Andres

Chesapeake Biological Laboratory, University of Maryland, Solomons, MD 20688. mason@cbl.umces.edu

 

ABSTRACT

            The extent to which inorganic mercury (Hg) and methylmercury (MMHg) are released from the solid phase (sediment or food) during digestion appears to be the process limiting the extent of bioaccumulation. Studies with crab gut tissues suggest that membrane transport of both mercury complexes is similar, and that uptake into the gut tissue is rapid followed by slower redistribution to other tissues of the body. Sediment organic content appears to directly effect Hg and MMHg bioavailability to benthic invertebrates, through limitation of the degree of solubilization of Hg and MMHg by intestinal fluid. Thus changes in sediment organic content could influence the extent to which Hg and MMHg are bioaccumulated into benthic food chains.

 

INTRODUCTION

The bioaccumulation of methylmercury (MMHg) into aquatic food chains is an important environmental health concern as levels of MMHg in top predator fish are elevated enough to pose a threat to humans, and fish-eating mammals and birds. Much of the research focus has been on bioaccumulation in the water column food chain (phytoplankton/zooplankton/fish). However, in many shallow ecosystems, benthic organisms provide an important link between MMHg in the sediment and the accumulation of MMHg into piscivorous fish. Furthermore, little is known of the mechanisms controlling the accumulation of MMHg and Hg into benthic organisms. For this reason, we have studied the factors controlling the bioaccumulation of Hg, and specifically MMHg, into benthic invertebrates. Our studies have focused on using traditional methods such as bioaccumulation factors, and correlation statistics of field data, as well as laboratory exposure experiments to ascertain the controlling mechanisms for uptake. In addition, we have adapted the methods of in vivo extraction of sediments with the intestinal fluid of benthic organisms to study Hg bioavailability and bioaccumulation. Here we briefly describe the results of the various studies. Overall, our results point to the importance of sediment organic carbon as the overriding control on the amount of Hg and MMHg bioaccumulated by benthic invertebrates. The implications of our results to management of Hg and MMHg in aquatic sediments will be briefly discussed.

 

METHODS

Sediment was collected from Fishing Bay, MD, an unpolluted site; and sand from the Patuxent River mouth. Experimental treatments of varying organic matter were obtained by diluting Fishing Bay sediment with muffled (5500C for 24 hrs) sand, both sieved to less than 250 mm. These mixtures were then spiked with Hg and MMHg solutions made from certified reference standards and stored in the dark at 4 0C for  4 days. For the in vitro experiments, digestive fluid from the midgut of the polychaete, Arenicola marina, was collected in Maine, USA (Lawrence et al., 1999; Mayer et al. 1996), frozen at -800C and shipped to CBL. Fluid from the sea cucumber, Sclerodactyla briareus was collected by dissection of animals at CBL. Our studies have shown that freezing does not affect the ability of intestinal juice to solubilize MMHg (McAloon et al., 2000). Sediment was incubated with digestive fluid in acid-cleaned Teflon centrifuge tubes.  Each digestive fluid incubation was performed in triplicate. The slurries were vigorously shaken and held at room temperature on an orbital shaker during extraction. Control incubations included digestive fluids without sediment. After incubation, fluids were removed from slurries by centrifugation at 2700g for 0.5 hr at room temperature (Lawrence et al., 1999). The fluids were then transferred to acid cleaned Teflon vials and frozen until subsequent analysis.

For exposure studies, filtered (0.2 mm) ambient seawater was added to a layer of sediment mixture in experimental microcosms. The microcosms were kept in a temperature controlled room (25 oC) under 16 hrs of light, continually aerated and allowed to equilibrate for 24 hrs prior to the addition of animals. Fish feeding experiments employed similar protocols but without sediment. Fish were fed using gel-encapsulated food for a short period and then transferred to beakers with clean water to allow them to eliminate their gut contents and to minimize any uptake from contaminated water. Water was again changed after food elimination, and throughout the remainder of the experiment. Water, food and biota samples were collected as appropriate.

Perfusion experiments were performed with the excised gut of the blue crab. The tissue was suspended in physiological medium of appropriate ionic strength and chemistry. The gut was connected to a precision peristaltic pump so that the exposure solution could be pumped through the gut cavity. Samples were collected of the eluent, and the physiological medium, as well as the tissue at the end of the experiments which were of two hour duration.

Samples were analyzed for total Hg and MMHg using standard techniques that are routinely used in our laboratory (Mason and Lawrence, 1999; Lawrence and Mason, in press) and which are based on tin chloride reduction preconcentration and cold vapor atomic fluorescence detection (CVAFS; Bloom and Fitzgerald, 1988) for Hg. Distillation preconcentration, ethylation derivitization and CVAFS methods are used for MMHg quantification (Bloom, 1988; Horvat et al., 1993).Spike recoveries for water and sediment  measurements were in the range of 75-120% for Hg and 80-110% for MMHg. Detection limits for Hg and MMHg were based on three standard deviations of the blank measurement (0.1 ng g-1 Hg and 0.01 ng g-1 MMHg, wet weight). Inorganic Hg (HgI) was determined by difference (total Hg - MMHg).

 

RESULTS

            We have shown that organic content of sediment is an important parameter governing MMHg bioavailability (Lawrence et al., 1999; Lawrence and Mason, in press; Mason and Lawrence, 1999). Indeed, we have demonstrated that for benthic invertebrates feeding on sediment, the degree to which the MMHg is released to solution during digestion is the rate limiting step controlling bioaccumulation (Lawrence et al., 1999). Also, because we conclude that the process of intestinal solubilization is a competitive ligand exchange process, any factors that increase the strength of MMHg binding to sediment will decrease bioavailability. This is contrary to the common notion that the preferential uptake of MMHg over Hg during digestion is due to the lack of inorganic Hg transport across the gut lining. Our results, discussed below, suggest that the membrane is not a substantial barrier to inorganic Hg compared to MMHg.

We have studied solubilization of Hg and MMHg by the intestinal fluids of two benthic organisms. These  invertebrates have very different abilities to solubilize inorganic Hg and MMHg from sediments, as shown in Fig. 1. Clearly, although there is the same trend of decreasing solubilization with increasing sediment organic content for both fluids, the  lugworm (Arenicola marina) intestinal fluid has a greater ability to extract Hg and MMHg from sediment than that of the sea cucumber (Sclerodactyla briareus). Thus, assuming similar feeding rates, the lugworn would likely assimilate higher levels of MMHg if exposed to the same sediment as the sea cucumber. Field results (e.g. Mason and Lawrence, 1999) also suggest that different benthic organisms have different bioaccumulation from the same sediment and our results suggest that these differences are controlled at the most fundamental level - by processes occurring during digestion.

In further experiments (McAloon et al., 2000), the in vitro solubilization was compared to bioaccumulation after a 10 day exposure of the sea cucumber to sediment Hg and MMHg. Given an estimated feeding rate for the organism, and knowing the organic content and MMHg and Hg concentration of the sediment, it was possible to estimate the amount of solubilization that would have been required, assuming no barrier to transport across the membrane, to obtain the measured concentrations in the organism. The estimated degree of solubilization matched the in vitro measurements overall, for both Hg and MMHg. These results therefore strongly suggest that the rate limiting step to bioaccumulation of Hg and MMHg by benthic invertebrates is the digestion process.

            To confirm that solubilization is the rate limiting step, we performed perfusion experiments with the gut of the blue crab, Callinectes sapidus, to ascertain the controls over membrane transport (Laporte et al., 1999). The results of these studies (Fig. 2) show that inorganic Hg and MMHg are both accumulated rapidly into the gut tissue and that the accumulation did not appear to be strongly dependent on the metal-ligand complex. These results suggest active transport across the membrane. We have performed additional experiments at different temperatures, and in the presence of metabollic inhibitors, and have confirmed that the process is likely active uptake rather than passive diffusion. The similarity in the uptake of inorganic Hg and MMHg suggest that the membrane is not a barrier to inorganic Hg to any greater degree than it is to MMHg. More experiments are clearly required but the results of the solubilization studies and the perfusion experiments suggest that for benthic invertebrates, the process of digestion exerts a major control over the extent of Hg and MMHg bioaccumulation by these organisms.

To study the accumulation pathway further we are studying the accumulation of MMHg from food into fish. Results of two feeding experiments with different foods show that there is not a strong difference in uptake when accumulation is normalized to the food concnetration (Leaner and Mason, 1999). Examination of tissue concentrations shows that MMHg is rapidly taken up into the intestinal tissue but that the transport from this tissue to other parts of the body is slower. Thus, as shown in the perfusion studies, the rate of uptake into gut tissue exceeds the rate of transport across the internal membranes to the blood and other tissues. The results of the fish experiment show that the MMHg is redistributed to other tissues with most of the transfer occurring within 60 hours of  feeding. Perfusion studies and solubilization experiments are being designed to further investigate the controls over digestion and accumulation of MMHg by fish.

            The results of these studies indicate that management of sediments in terms of Hg and MMHg exposure cannot be based simply on either the total Hg or total MMHg concentration but that the chemical characteristics of the sediment need to be taken into account. Additionally, efforts to reduce organic loading to aquatic environment may have a detrimental effect in terms of MMHg bioavailability to the organisms that reside in those sediments. Clearly, these interactions and relationships are not simple, and more research needs to focus on the parameters influencing Hg and MMHg bioavailability from sediments.

REFERENCES

 

1. Bloom, NS (1989) Can. J. Fish. Aquat. Sci. 46:1131-1140.

2. Bloom NS, Fitzgerald WF (1988),  Anal. Chim. Acta 208: 151-161.

3. Horvat M, Bloom NS,  Liang L (1993) Anal. Chim. Acta.282: 153-168.

4. Laporte J-M., Andres S, Mason, RP (1999),  Presentation made at SETAC Meeting,      Philadelphia, USA, November 1999.

5. Lawrence AL, McAloon KM, Mason RP, Mayer, LM (1999) Environ. Sci. Technol. 33: 1871‑1876.

6. Lawrence AL, Mason RP (2000), Environ. Poll. (in press).

7. Leaner, JJ, Mason RP (1999), Presentation made at SETAC Meeting, Philadelphia, USA, November 1999.

8. Mayer LM, Chen Z, Findlay RH, Fang J, Sampson S, Self RFL, Jumars PA, Quetel C, Donard OFX (1996), Environ. Sci. Technol. 30: 2641-2645.  

9. Mason RP, Lawrence AL (1999),  Environ. Toxicol. Chem. , 18:2438-2447.

10. McAloon KM, Lawrence AL, Mason RP, Mayer LM (2000),  Presentation made at the International ASLO Meeting, Copenhagen, Denmark, June 2000.

 


Figure 1. Comparison of percent solubilization from digestive fluid of Arenicola marina (left figure) and Sclerodactyla briareus (right figure)