Ingesting biota

During recent years, two different approaches have evolved to describe metal speciation and bioavailability inter dependencies, i.e. absorption (or assimilation) efficiency (AE) and gut juice extraction.

We know today that bio-uptake of metals can occur both from dissolved and particulate sources, whereby the latter is a function of the assimilation efficiency (AE), of the metal concentration in ingested particles, and of the feeding rate. There seems to be no simple relationship between the binding strength of metals in sediment particles, and the AE.In oxidized sediments, TOC (total organic carbon) is known to reduce metal bioavailability. However, recent research shows that the influence of TOC on metal bioavailability is far from being understood and may strongly vary among metals and organism species. This may be partly due to the fact that sediment TOC studies mostly do not differentiate between humic, algal or bacterial matter, each of which can affect bio-uptake and accumulation of metals in a different way. For instance, humic matter decreases metal bioavailability due to high binding coefficients and high ligand concentrations, while the living organic sediment microflora may in contrast increase bioavailability.

To what extent and in what way gut physiology (e. g. pH, enzymatic activity, surfactant concentration, particle processing time and intensity, abrasional mixing) determines the AE is almost unknown and needs further research. Recent studies point to a strong influence of biological factors, like particle selection, flexible digestion, gut redox status, reproductive state, or gut fluid chemistry, on final metal bioavailability. There is also evidence that the 'age' of metal-sediment associations, beside gut residence times and readsorption of initially desorbed metals in the animal gut, may control AE, and/or minimize metal bioaccumulation. Also antagonistic effects between certain trace metals (as shown for Cd and Zn), or adaptive physiological regulation mechanisms by the organisms themselves for essential metals, may affect AEs in aquatic biota.

New experimental results from gut juice extraction studies confirm that bioaccumulation of metals may not be predictable solely based on their bioavailaibility. Experiments with digestive fluids obtained from deposit and suspension feeders, in combination with chemical extraction, showed that metal bioavailability varies among organism species according to the type of digestive physiology. These studies also elucidate the role of some major metal-binding sediment phases, like sulphides or Fe/Mn oxides, in that they can limit metal solubilization by the digestive fluid of deposit feeders.

Metal speciation and bioavailability studies are traditionally decoupled from toxicity studies due to the problem to relate tissue body burden directly to toxicity. In the same way it is difficult to relate bioavailability to bioaccumulation. During recent years, two different approaches have evolved to study metal speciation and bioavailability interdependencies in organisms ingesting sediments, i. e. the absorption or assimilation efficiency (AE) and gut juice extraction. In general, equilibrium partitioning models assume that sediments are not a direct but indirect source for metal accumulation in biota, and that they may act as a buffer or source/sink for metals in the water column and porewater. In this context, we also know that the relative importance of sediment vs aqueous uptake is highly specific for different groups of organisms.

5.4.8.1 Absorption efficiency (AE)

It is well accepted that biouptake can occur both from the dissolved phase, via respiratory membranes, and from particulates, via the gastrointestinal tract, whereby the latter being a function of the assimilation efficiency (AE), i. e. of the metal concentration of ingested particles and of the feeding rate. According to Griscom et al. (2000), AE-values reflect per definition the bioavailability of a specific metal for a given animal under specific conditions (compare to section 5.1.2). It may offer a tool to integrate and quantify geochemical, sediment and species influences on the uptake of metals from food. AE's can be experimentally determined by "pulse-feeding radioactive metal-labeled particles" to test animals and by measuring the metal fraction retained after gut depuration. Griscom and co-workers (2000) showed that it can strongly affect the metal accumulation in mussels. They evaluated AEs of metals (Ag, Cd, Co, Cr, Se, and Zn) ingested by mussels from various sediments and sediment components, and the influence of geochemical properties which may affect metal bioavailability. In addition, the relationship between dietary metal assimlation and AVS binding was addressed in both anoxic and oxic sediments.

In oxic sediments where AVS does not control metal accumulation in porewater, it is supposed that total organic carbon (TOC) is reducing bioavailability of metals, in contrast to a report (ref. given by Griscom et al. 2000) indicating that bioavailability increases with the living organic sediment microflora. In their study, Griscom et al. observed that the influence of TOC on metal bioavailability was not consistent and varied among metals and test species. In particular, the experiments showed that sediment TOC did influence Cd-AEs, but not AEs for Zn and Cr. This may be partly due to the fact that sediment TOC studies generally do not differentiate between humic, algal or bacterial matter, each of which can affect AEs in a particular way. In this context we know that humic matter decreases metal bioavailability due to high binding coefficients and high ligand concentrations. In their study, Griscom et al. (2000) found AEs from bacteria-coated glass beads being slightly higher than from humic-acid coated beads. In this context, Robertson and Leckie (1999) also argue that it may be too difficult to predict the effect of humic substances on metal behaviour and the impact of ambient solution conditions on these effects at the same time, due to the complex heterogeneous nature (molecular weight, composition, structure) of the humic matrix.

We know that there is no simple relationship between the binding strength of metals for sediment particles and the AE. The work of Griscom et al. showed that sediment-bound trace metals were assimilated to varying degrees by suspension-feeding animals, ranging from 1 % (Cr) to 41 % (Zn). AEs of all trace metals associated with anoxic sediment were 1.5 times higher for the bivalve Mytilus edulis when compared to metals bound to oxic sediment (except for Ag). It was also observed that the exposure time of metals in the sediment correlated with the decrease in AE by M. edulis for Co, Cd and Zn (except for Ag). Results from sequential extraction showed at the same time that the labile fraction of all metals decreased over time ("aging"; see further below), which coincided with a decrease of the AE in the bivalve, due to the observed lower bioavailability. Also interesting that 1.5 times more Cd than Zn was extractable in the labile fraction (water rinse, exchangeable and weak-acid extractable metals), although AE of Zn was twice that of Cd. Somewhat surprising was that sediment organic matter apparently did not show a consistent influence on the AE among the selected metals, probably due to the fact that TOC was not further characterized. No statistically significant relationship was observed between the mean AE

(among particle types) and the mean desorption of metals, as obtained from desorption experiments (with seawater adjusted to pH 5 and 8).

It is accepted that geochemical influences affect porewater concentrations, but these influences on dietary uptake are suppposed to be less extreme. The authors argue that correction factors appropriate among species and metals (like AVS or TOC normalization) are not applicable for dietary uptake. They also point out that the observed AE of metals in bivalves is less or equal to AE-values reported for periphyton or seston (see ref. given therein). Also that metals were less assimilable from anoxic than from oxic sediments in M. balthics, but still bioavailable, in contrast to what was the case for M. edulis. Higher AEs for Cd and Co in M. edulis from anoxic than from oxic sediments were unexpected given the supposed strong binding by sulphide and their slow reoxidation (days). The authors explain that M. edulis has a stronger reducing gut than M. balthica as indicated by preliminary experiments. In how far and what way the gut physiology (e. g. pH, enzymatic activity, surfactant concentration, particle processing time and intensity, abrasional mixing) may contribute to the observed AE differences is almost unknown and needs further research. Indeed, several studies (also cited here) point out to a strong effect of biological factors, like particle selection, flexible digestion, gut redox status, reproductive state, or gut fluid chemistry on the final metal bioavailability (see following section).

In this context, Griscom et al. addressed also the 'age' of the metal-sediment association and could show that it had a pronounced effect on the AE in M. edulis (see section 5.5.6). Similar effects appeared after 35 days also on M. balthica, suggesting additionally species-specific differences. For example, we know that M. balthica has a longer gut residence time, which may lead to stronger absorption, which in turn may diminish or mask the effect of exposure time. But also readsorption in the gut of the initially desorbed metal ions, e. g. in the presence of surfactants, may minimize assimilation in animals. On the other hand, desorption experiments showed that the gut juice of different animal species varies in its ability to extract sediment-bound metals (see also following section). A progressive redistribution of added metals over time into more resistant phases in the sediment was observed by SEP, emphasizing the importance of sediment aging on metal AE. It is speculated that trace metals may even become lodged over time into sedimentary mesopores, a mechanism proposed by Mayer (1994) (cited by Griscom et al. 2000) for the observed loss of labile organic matter in sediments.

In a recent survey on extremely contaminated sediments from a marine coastal bay, also Fan et al. (2002) tried to examine the control of sediment geochemistry on the assimilation (expressed as AE) of Cd, Zn and Cr in marine mussels and clams by combining a sequential extraction method (the 'Tessier' procedure), with the 'radiotracer pulse-chase feeding technique', and the AVS approach. Metal assimilation efficiencies were measured in clams and mussels feeding on contaminated sediments radiolabeled with 109Cd, 65Zn and 51Cr. A clear relationship between metal partitioning and total concentrations was only observed for Cd and less for Zn, in that the most labile phases increased with increasing total concentration. This confirms the empirical observation that metals released by human activities in general bind to more labile forms in the sediment. In contrast, but not surprising, Cr was almost completely (72-91%) bound to the residual phase and its sediment partitioning did not reveal a distinct dependence from total concentration. A significant correlation between the AE in both clams and mussels and the partitioning of metals into easily exchangeable and reducible phases was only established for Cd, inspite of a negative correlation between total sediment Cd and AE. Fan et al. suggest that this may be due to either saturation of Cd ligand binding sites in the gut or due to an antagonistic interaction between Cd and Zn occuring in very high concentrations in the sediment (up to 10.000 ppm!), in that Zn successfully competes with Cd for available gut binding sites. The decrease of Cd concentrations in deposit-feeding invertebrates with increasing Zn concentrations has been reported earlier and is a well known phenomenon (ref. given). The increasing Cd AE with increasing partitioning of this metal in the easily reducible phase may be due to increasing desorption of this metal in the bivalves gut. In contrast to previous studies with artificially prepared sediments, the authors did not find a significant relationship between the AEs observed for Cr and Zn and their partitioning in easily exchangeable and reducible phases. The authors interpret this discrepancy by the influence of other geochemical factors existing in natural sediments, like Fe, TOC or varying total metal concentrations, but also by the greater complexity of Cr speciation (with two redox species!), or by possible adaptive physiological regulation mechanisms for Zn (as an essential element!), which has been observed for many aquatic invertebrates.

In addition, differences in [SEM-AVS] did not significantly affect AEs of Cd, Zn and Cr. As shown by others, sediment-dwelling oligochaetes and insects take up most of their Cd from the overlying water column, which was also here the main source for metals, mainly due to the particular behaviour of these aquatic invertebrates, such as burrowing and irrigation. Lee et al. (2000a und 2000b, see section 5.4.3) (cited in Fan et al. 2002) recently found that AVS-bound metals were assimilated by clams with a similar efficiency as metals bound to oxic sediments, and that metal accumulation in the clam was not related to the SEM/AVS ratio but to metal extractability. The authors speculate about the reason for the observed lack of correlation between AE and AVS, and argue that the very high metal contamination would make it difficult to create [SEM-AVS] < 0. Consequently, speciation influences on metal bioavailability may be also confounded by the degree of contamination. However, due to differences in the partitioning of spiked and natural sediments (see above), extrapolating metal AEs by the use of radiotracer techniques to field conditions should be made with great care.

5.4.8.2 Gut juice extraction

Yan and Wang (2002) used a simple kinetic model and could show that sediment ingestion by the deposit-feeding peanut worm Sipuncula nudus was the dominant source for metal accumulation. More than 96% of Cd, > 89% of Cr and > 85% of Zn bioaccumulation could be predicted from sediment ingestion. However they found low uptake rate constants for S. nudus with regard to these metals. The uptake rate for Zn increased with increasing water concentrations. The corresponding AEs were 6-30% for Cd, 0.5-8% for Cr, and 5-15% for Zn.

The metal fraction assimilated by the worm was generally lower than the fraction extracted by the gut juice indicating that AEs may also depend on the actual amount of metals extracted in the animal gut from the ingested sediment. Metal desorption from radiolabelled sediment was measured by gut juice extraction obtained from Sipuncula and yielded up to 63% of Cd, but only up to 4% of Cr. A significant correlation between AE and gut juice extraction was only observed for Cd, but not for extractable Zn and Cr, indicating at least for the latter metals an uncoupling between desorption and assimilitation in the gut and confirming that the bioaccumulation of metals may not be predictable solely based on their bioavailaibility. Zn AEs from most investigated sediment types remained within a narrow range (6-9 %) despite obvious variations of Zn extraction in the gut juice. The extremely low fraction of Cr extracted by the gut juice suggests a strong association of this metal (which indeed often exists in residual phases) with the ingested sediment. However, one additional explanation for the observed differences between AE and gut juice extractable metals may be that the gut juice was collected by centrifugation, which may have altered the original enzyme activity.

More recently, Fan and Wang (2003) compared different extractants (normal and acidic seawater pH5, seawater with 1 % sodium dodecyl sulfate, and gut digestive fluid from Sipunculus nudus) with operationally defined geochemical phases (exchangeable, reducible and carbonate phases) in contaminated coastal sediments. Extracting Cd, Cr, and Zn by gut juice did not correlate with the concentration of SEM or SEM-AVS, indicating that significant differences may exist between the chemical (AVS) and biological (gut) availability of metals.

To demonstrate that the bioavailability of sediment Cu is not only determined by the particular sediment geochemistry, but also by the digestive physiology of exposed organisms, Chen and Mayer (1999) used a biomimetic approach which involved Cu extraction with digestive fluids obtained from 2 deposit feeders and 1 suspension feeder, in comparison to AVS-extraction, to finally define Cu bioavailability as the amount of Cu solubilized by the gut juices. One major goal was to test if sulphide plays a role in limiting Cu solubilization by the digestive fluid of the used deposit feeders considering that sulphide may act as a strong competing Cu-binding phase and hence prevent Cu toxicity in estuarine sediments with varying SEMCu-AVS values (no information was given if these sediments were oxic or anoxic). The experimental results showed that Cu bioavailability varied among organism species having unequal types of digestive physiology, but increased significantly at SEMCu - AVS > 0 according to the AVS premise. As an example, the amount of copper bioavailable to the digestive fluid of Arenicola marina (deposit feeder) was higher in sediments with SEMCU-AVS > 0. A SEMCU-AVS range of 0.4 and 1.5 ^mol/g (sed) was necessary for the 'holothuroid' gut fluids (of Parastichopus californicus and Cucumaria frondosa) before a significant increase in Cu solubilization was observed suggesting AVS is limiting Cu bioavailability.

Positive SEMCU-AVS values may also point out that other additional binding phases than AVS, like organic matter, may successfully compete with dissolved gut ligands, or that Cu(I) and Cu(II) sulphides are present in the sediment (with a smaller amount of sulphide present in Cu2S, in contrast to CuS) (see also Simpson et al., 2000b). The amount of Cu available for the digestive fluids was less than that measured as SEMCU-AVS, suggesting that only a fraction of the HCl-soluble non-AVS bound Cu was solubilized during the digestive process (see Table 5.11), and again the more conservative nature of the AVS concept (cf. section 5.4.3.3). This fraction decreased in the order Arenicola > Parastichopus > Cucumaria, which is consistent with the decreasing concentration of the strongest Cu binding ligands (i. e. histidine) found among the digestive fluids of the tested organisms. The authors conclude that sedimentary Cu availability is not only a measure of the applied geochemical factors (e. g. AVS), but varies also according to the digestive physiology of exposed organisms.

Table 5.11. Example of Cu released by 1 N HCl, gut fluids inArenicola marina, Parastichopus californicus, Cucumaria frondosa and sea water from harbour (BBH and PLH) and estuarine sediments (BIW), in comparison to total sedimentary Cu and acid-volatile sulphidea (AVS) (from Chen and Mayer, 1999)

Sample

1 N HCl

A. marina

P.californicus

C. fron-dosa

seawater

total Cu

AVS

BBHI

7.01±0.30

1.65±0.43

0.78±0.37

0.19±0.05

0.006±0.001

8.59±0

0.48±0.08

(I,sand)

BBH2 (I)

1.19±0.02

0.18±0.01

0.16±0.01

0.04±0.00

0.001±0.000

1.20±0.01

0.02±0.01

(coarse sand)

BBH3

3.66±0.09

1.34±0.24

0.22±0.13

0.14±0.03

0.006±0.002

6.00±0.14

0.84±0.21

(I,sand)

BBH4

5.50±0.09

1.74±0.64

0.59±0.07

0.07±0.01

0.020±0.002

9.56±0.34

0.19±0.02

(I,sandy silt)

BBH5

0.70±0.00

0.09±0.02

0.00±0.00

0.00±0.00

0.000±0.000

1.79±0.02

0.34±0.05

(I,silt)

BBH6

1.57±0.06

0.44±0.04

0.01±0.01

0.00±0.00

0.001±0.000

3.31±0.16

0.12±0.02

(I,clay)

BBH7

0.03±0.00

0.01±0.00

0.00±0.00

0.00±0.00

0.000±0.000

0.06±0.00

0.39±0.12

(I,coarse sand)

BBH8

0.28±0.01

0.07±0.01

0.02±0.01

0.00±0.00

0.000±0.000

0.28±0.00

0.16±0.04

(S,silty clay)

PLH-F

0.40±0.01

0.05±0.00

0.00±0.00

0.00±0.00

0.000±0.000

0.48±0.01

2.24±0.39

(I,silt)

PLH-B

0.18±0.01

0.03±0.00

0.00±0.00

0.00±0.00

0.000±0.000

0.18±0.00

1.27±0.08

(S,silt)

BIW1 (S,silt)

0.15±0.00

0.02±0.00

0.00±0.00

0.00±0.00

0.000±0.000

0.50±0.01

0.09±0.01

BIW2 (S,silt)

0.14±0.01

0.01±0.00

0.00±0.00

0.00±0.00

0.000±0.000

0.30±0.01

0.07±0.00

BIW3 (S,silt)

0.10±0.00

0.01±0.00

0.00±0.00

0.00±0.00

0.000±0.000

0.30±0.01

0.09±0.03

aAll data as mean ± 1 S.D. (n = 2) with a unit of ^mol/g sediment. I = interidal, S = subtidal aAll data as mean ± 1 S.D. (n = 2) with a unit of ^mol/g sediment. I = interidal, S = subtidal

Incubation of digestive fluids with Cu-binding model phases (geothite and sulphide) confirmed the relative unavailability of sulphide-bound Cu, which means a reduced Cu exposure to subsurface deposit feeders feeding on anoxic sediment in comparison to surface feeders. Less Cu was released from sulphide than from geothite by the digestive fluids demonstrating sulphide as the stronger Cu-binding phase. This is in good agreement with the size of the corresponding Cu-binding constants of the sulphide (logKCuS=36.1) and hydroxyl group (logKCuOH=6.5). It is known that field sediments may contain a mixture of a-FeOOH and sulphides over a wide range of concentrations reflecting varing sedimentary redox conditions, which in addition to organism species may influence the bioavailability of Cu in sediments for a given total concentration (see following section).

Chen and Mayer (1999) state that neither the deposit feeding biology nor the digestive physiology have been sufficiently examined yet by current research approaches designed to assess metal bioavailability, although we know that digestive fluids enhance metal solubilization, and vary among different deposit feeders. In addition, histidine containing proteins and peptids in digestive fluids have been found particularly responsible for mobilising and complex-binding sedimentary Cu. But relatively little is known about their role in controlling metal bioavailabilities, bioaccessibility and uptake in natural biota. Moreover, to what extent released metals are influenced by the ratio of gut ligand concentration and actual sediment metal load, or by the reaction time between sediments and digestive fluids, remains still unknown. The fact that 1 N HCl (SEM) released much more Cu than the digestive fluid of the 3 tested deposit feeders, may be also partly due to the acidic extraction pH causing a harsher attack than the neutral pH in the gut. Also the amount of Pb, Zn, Cd and Ni released by digestive fluids was insignificant compared to Cu, although Pb and Zn reached a significant fraction in the 1 N HCl fraction.

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