IX. Uptake by Species Higher in the Food Web
The study of the trophic transfer of metals in marine organisms is a largely unexplored field. Organisms exposed to metals may develop mechanisms for sequestering contaminants in their systems in such a way that the metals do not harm them, but these organisms may still pose a threat to the predator that consumes them. To understand metal cycling through trophic levels, the factors (e.g. tissue metal distributions and concentrations, duration of exposure, growth of prey, etc.) which control the bioavailability of metals to predators must be understood.
Two recent studies address this question using 2 different predators: the grass shrimp and the blue crab. How do alterations in the subcellular Cd distribution in prey relate to changes in Cd absorption by predators? To address this question, Wallace (1992a,b, 1995a,b) used Limnodrilus hoffmeisteri as the prey species and the grass shrimp, Palaemonetes pugio, as the predator in a series of feeding experiments.
Feeding Experiments I: Foundry Cove Oligochaetes
In the first series of experiments, Wallace (1992a) exposed individuals of resistant Limnodrilus hoffmeisteri from Foundry Cove to radiolabelled 109Cd in solution. His intent was to determine subcellular Cd distributions in the prey and then to relate these to Cd levels in the grass shrimp, as well as determine absorption efficiencies for shrimp fed whole worms and specific subcellular fractions.
After exposure to Cd, the worms were rinsed, pooled by replicate, and homogenized in distilled water. The homogenate was then split into two portions with one used for feeding experiments with shrimp and the other for subcellular fractionation. Radioactivity was determined for each portion, then the homogenates were frozen at -20oC until further use. Each homogenate was sequentially centrifuged (as in Chapter VIII) to produce a debris pellet, an intracellular pellet, and a cytosol fraction. Subcellular Cd distributions were determined by radioactivity assays on each pellet fraction. The homogenates used in feeding trials were individually mixed with gelatin solution, divided into 6 µl portions(=gelatin discs), then frozen until use in feeding trials. Individual shrimp were fed 1 gelatin disc (made from either whole worm extract or specific subcellular fractions), rinsed, and assayed for radioactivity; shrimp fecal pellets were also assayed.
Feeding Experiments II: South Cove Oligochaetes
In a second series of experiments, non-resistant (South Cove) worms were used (Wallace 1992b, 1995a). In order to alter the subcellular Cd distribution in Limnodrilus from South Cove, individual worms in vials were exposed to 1 of 3 Cd concentrations in 20 ml of solution: 0.5µg, 47µg, or 140µg Cd/l, for a 1 or 6 week duration. 109Cd was added in negligible amounts (1.7 ng/l) as a tracer. Exposure concentrations were chosen to span a range from background levels (i.e. 0.5 µg/l as found in South Cove) to levels toxic to Limnodrilus (170 µg/l). Two replicates (of 20 - 30 worms each) for each concentration and duration combination were used.
Shrimp were collected and acclimated to experimental temperature (23oC) and salinity (5 ppt). Ten shrimp constituted a replicate and two replicates were run for each Cd concentration and duration combination. Individual shrimp were fed 1 gelatin disc, rinsed, and assayed for radioactivity. They were then allowed to feed on unlabelled squid tissue; the shrimp and their fecal material were assayed for radioactivity for a 48 h period.
Feeding Experiments III: Foundry and South Cove Oligochaetes
In a third series of experiments resistant (Foundry Cove) and non-resistant (South Cove) Limnodrilus were exposed to radiolabelled Cd, centrifuged, and used in feeding trials (Wallace 1995b). The intent was to directly compare subcellular Cd distributions among resistant and non-resistant worms as well as further resolve the debris fraction into metal-rich granule and tissue subfractions and the cytosol fraction into heat-stable (includes MT-like proteins) and heat denatured protein subfractions. If resistant worms utilize the detoxification mechanisms differently than non-resistant worms, this could have important implications for transfer to higher trophic levels. Shrimp were handled, fed various gelatin discs, and assayed for radioactivity as described above in series II experiments.
Table 1. Mass balance equations for estimating 109Cd absorption efficiency (A.E.) for shrimp feeding on radiolabelled oligochaetes. Equations use a subcellular 109Cd distribution obtained from the labelled oligochaetes and 109Cd A.E.'s which were determined experimentally for shrimp feeding on the same subcellular fractions (From Wallace 1992a, series I experiments).
|Subcellular Fraction||% of total worm Cd||Absorption Efficiency||% Cd absorbed by shrimp|
|High molecular weight||37.6||85.2||32.0|
Both Cd concentration and duration of exposure affected subcellular Cd distribution in the prey (series II experiments, Wallace 1995a). A 1:1 relationship was found between the amount and percentage of Cd in oligochaete cytosol and the amount and percentage of Cd absorbed by shrimp. Only metal bound to the soluble fraction of prey was available to the predator. The metals deposited in metal granules were relatively indigestible and were not well absorbed. Thus, factors that influence the subcellular metal distribution in prey will directly affect metal transfer to predators.
There were no significant differences in subcellular fractions of worms exposed for 1 week to 0.5µg or 47µg Cd/l, but more Cd was in the cytosol fraction of worms exposed to 140µg Cd/l for a week than in debris or intracellular fractions (non-resistant worms, series II experiments). The importance of cytosol for binding Cd increased after 6 week exposure to both 0.5µg and 47µg Cd/l concentrations, with proportionately more Cd bound to cytosol than the other fractions. The proportion of Cd in the debris fraction was constant in both 1 and 6 week durations. Worms exposed to 140µg Cd/l for 6 weeks all died, thus no assays or feeding trials could be run.
Oligochaete exposure conditions (concentration and duration) significantly affected Cd absorption by shrimp. Shrimp fed gelatin discs from worms exposed for 1 week at the two lower Cd concentrations had similar absorption efficiencies, while shrimp fed discs from worms exposed to 140µg Cd/l absorbed proportionately more of the ingested Cd. A strong positive correlation (r=0.99) was found between the amount of Cd in cytosol and the amount of Cd transferred to shrimp. The MT-like proteins would be concentrated in the cytosol fraction by centrifugation.
These data come from non-resistant worms exposed to Cd in the laboratory. Natural populations in which genetic resistance has evolved may have different subcellular metal distributions with quite different effects on higher trophic levels. Series III feeding experiments addressed this question directly. Foundry Cove (resistant) worms have most Cd bound in the metal-rich granule fraction whereas South Cove (nonresistant) worms have very little Cd in this fraction. South Cove worms have the highest proportion of Cd in the heat stable cytosol fraction (MT-like pool). Foundry Cove worms have a much lower proportion of Cd bound to MT-like proteins. It may be that MT-like proteins are important in the early response to metal exposure in this worm species, but that binding to metal-rich granules becomes the most important detoxification mechanism in resistant populations. This work is still ongoing; a similar study is also being conducted with blue crabs as the predator.