Full text of DOI:10.1002/etc.5620210618

Environmental Toxicology and Chemistry, Vol. 21, No. 6, pp. 1243–1248, 2002
2002 SETAC
Printed in the USA
0730-7268/02 $9.00 .00
᭧
ϩ
INHIBITION OF DIGESTIVE ENZYME ACTIVITIES BY COPPER IN THE GUTS OF
VARIOUS MARINE BENTHIC INVERTEBRATES
Z
HEN
C
HEN,† LAWRENCE M. MAYER,*† DONALD P. W ESTON,‡ MICHAEL J. BOCK,† and PETER A. JUMARS
†Darling Marine Center, University of Maine, Walpole, Maine 04573, USA
†
‡Department of Integrative Biology, University of California, Berkeley, California 94720, USA
(
Received 15 June 2001; Accepted 4 December 2001)
Abstract—Digestive systems of deposit and suspension feeders can be exposed to high concentrations of copper (Cu) by ingestion
of contaminated sediments. We assessed a potential impact of this Cu exposure on digestive enzyme activities in a wide range of
benthic organisms by monitoring enzyme activities in their gut fluids during in vitro titrations with dissolved Cu, which mimics
Cu solubilization from sediments. Increasing Cu inhibited digestive protease activities at threshold values, which varied widely
among organisms, from 8
containing higher amino acid concentrations because strong Cu-binding sites on amino acids prevent Cu interaction with the
enzymatically active sites. Threshold Cu concentrations were similar for proteases, esterases, lipases, and - and -glucosidases,
␮M for an echinoderm to 0.4 M for an echiuran. More Cu was required to inhibit proteases in guts
␣
␤
suggesting the same inhibition mechanism. Copper was less effective at inhibiting enzymes at lower pH, suggesting that protons
can compete with Cu ion for binding to enzymatically active sites or that enzyme conformation is less vulnerable to Cu inhibition
at lower pH. These results lead to the counterintuitive conclusion that deposit feeders with low enzyme activity, low amino acid
concentration, and high pH values are most vulnerable to harm from sedimentary Cu by this mechanism, although they solubilize
less sedimentary Cu than their counterparts with high enzyme activity, high amino acid concentrations, and low gut pH. In general,
digestive systems of echinoderms may therefore be more susceptible to Cu contamination than those of polychaetes, with various
other phyla showing intermediate susceptibilities. If threshold Cu values are converted to solid-phase sedimentary Cu concentrations,
the thresholds are at least consistent with Cu loadings that have been observed to lead to biological impacts in the field.
Keywords—Copper
Digestive
Enzyme
Invertebrates
INTRODUCTION
complexes with Cu, preventing its inhibition of protease ac-
tivity by interacting with the enzymatically active sites. Results
from this type of experiment suggested that individual lug-
worms with higher gut amino acid concentrations are less vul-
nerable to Cu toxicity than others with lower amino acid con-
centrations.
Different species of macrobenthos contain different levels
of gut amino acids [14], suggesting differential susceptibility
among species to gut enzyme inhibition by Cu. The objectives
of this paper are therefore to extend the previous work on
lugworms to an interphyletic study by comparing the sensi-
tivities (threshold Cu concentration) of digestive proteases
among various deposit and suspension feeders and to examine
effects of physiological conditions in the digestive system of
different species, such as gut pH and amino acid concentra-
tions, on the threshold Cu concentration. A Cu titration ap-
proach was used to mimic the solubilization of sedimentary
Cu during digestion to determine the threshold Cu concentra-
tions.
Copper is a common contaminant in coastal sediments, re-
sulting from urban and industrial developments and shipping
activities [1,2]. Laboratory and field studies indicated that Cu-
contaminated sediments can cause bioaccumulation and toxic
effects on marine benthic organisms [3–6] and result in mor-
tality, reduction of hatching and growth rates, and alteration
of community structure. Ingestion and digestion of metal-con-
taminated sediments have been demonstrated to be an impor-
tant pathway leading to toxic effects [7,8].
A biomimetic method was developed to assess bioavailable
Cu during digestion by incubation of Cu-contaminated sedi-
ments with gut fluids of deposit feeders [9]. Considerable frac-
tions (10–30%) of sedimentary Cu can be solubilized by gut
fluids, resulting in elevated Cu concentrations of 55 to 4,400
␮
M in gut fluids of the deposit-feeding lugworm Arenicola
marina [9,10]. These results suggest that guts of deposit feed-
ers could be exposed to high concentrations of dissolved Cu
during in vivo digestive processes.
Copper can interfere with enzyme function [11–13], and
the gut is a site of intense digestive enzyme activity [14]. To
assess the threat from digestive Cu exposure, activities of ex-
tracellular proteases in gut fluids were monitored during in
vitro incubation with Cu-contaminated sediments and during
Cu titration experiments [10]. Inhibition of proteases occurred
only after dissolved Cu concentration in gut fluid reached a
threshold value, which was determined by the level of gut-
dissolved amino acids. A subset of gut amino acids form strong
MATERIALS AND METHODS
We used gut fluids of various deposit- and suspension-feed-
ing organisms from previous studies that report on sites, meth-
ods of gut fluid extraction, and initial Cu and amino acid
concentrations [15,16]. As reported previously, initial Cu con-
centrations were determined by graphite furnace atomic ab-
sorption spectroscopy and total amino acid concentrations de-
termined after acid hydrolysis using fluorometric detection of
orthophthaldialdehyde derivatives. One sample of gut fluid
from each species was used for the current study (Table 1).
The pH of each gut fluid was measured with pH electrodes
* To whom correspondence may be addressed
(lmayer@maine.edu).
1243

1244
Environ. Toxicol. Chem. 21, 2002
Z. Chen et al.
Table 1. Species used in this study, their feeding mode, collection site, gut pH, dissolved amino acid concentration (AA; mM), copper threshold
concentration (as
␮
M in gut fluid and as
␮g of bioavailable Cu per gram sediment after applying assumptions described in text), and maximum
enhancement of protease activity during the Cu titration (Pmax/Po)
Copper threshold
AA
pH (mM)
Speciesa
Feeding strategy
Collection location (all USA)
␮
M
␮
g/g
Pmax/Po
Strongylocentrotus droebachiensis (E) Herbivore
Damariscotta Estuary, Maine
Puget Sound, Washington
Puget Sound, Washington
7.92
8.29
7.56
7.43
7.46
7.22
5.71
8.53
8.25
—
8.13
6.61
6.36
6.52
6.61
8.02
7.38
5.61
7.05
7.43
7.40
8.28
7.65
7.71
6.56
5.64
5.76
6.94
6.34
7.51
7.33
6.01
6.32
8.42
16
2
10
12
15
58
168
65
2
40
16
14
32
13
44
44
149
216
342
242
102
215
351
202
394
202
381
424
169
116
436
166
430
156
8
46
76
1.0
5.9
9.7
10.9
20.6
22.3
21.7
27.3
33
33
44
48
62
64
69
85
87
110
299
1.2
1.7
1.3
1.4
1.0
1.0
1.2
1.3
1.9
1.5
1.5
1.8
1.1
1.4
1.1
1.9
1.1
1.1
1.2
1.8
1.3
1.3
1.9
1.5
2.1
6.1
2.6
5.4
22
Molpadia intermedia (E)
Parastichopus californicus (E)
Cucumaria frondosa (E)
Brisaster latifrons (E)
Mya arenaria (M)
Crassostrea virginica (M)
Siphonosoma ingens (S)
Echinarachnius parma (E)
Chiridota laevis (E)
Deposit feeder
Deposit feeder
Suspension feeder Damariscotta Estuary, Maine
Deposit feeder Puget Sound, Washington
Suspension feeder Damariscotta Estuary, Maine
Suspension feeder Damariscotta Estuary, Maine
Deposit feeder
Deposit feeder
Deposit feeder
86
162
176
171
215
257
262
346
379
485
500
544
671
686
868
Bodega Bay, California
Damariscotta Estuary, Maine
Pemaquid Point, Maine
Ensis directus (M)
Chirdota spp. (E)
Suspension feeder Damariscotta Estuary, Maine
Deposit feeder
Herbivore
Deposit feeder
Suspension feeder Damariscotta Estuary, Maine
Deposit feeder
Deposit feeder
Suspension feeder Narragansett Bay, Rhode Island
Deposit feeder
Deposit feeder
Deposit feeder
Omnivore
Seldovia, Alaska
Princeton, California
Seldovia, Alaska
Strongylocentrotus purpuratus (E)
Eupentacta quinquesemita (E)
Modiolus modiolus (M)
Protoglossus graveolens (H)
Saccoglossus bromophenolosus (H)
Modiolus demissus (M)
Arenicola marina (P)
Abarenicola pacifica (P)
Travisia foetida (P)
Nereis virens (P)
Damariscotta Estuary, Maine
Damariscotta Estuary, Maine
Various sites, Maine
2,355
8,744 1,111
9,558 1,215
Puget Sound, Washington
Puget Sound, Washington
Sheepscot Estuary, Maine
Puget Sound, Washington
San Francisco, California
Sheepscot Estuary, Maine
Damariscotta Estuary, Maine
10,844 1,378
13,308 1,691
15,253 1,939
35,287 4,485
55,958 7,112
136,766 17,382
137,866 17,522
148,483 18,871
163,566 20,788
191,105 24,288
282,158 35,860
346,474 44,034
388,110 49,326
Abarenicola vagabunda (P)
Arenicola brasiliensis (P)
Amphitrite johnstoni (P)
Nicolea venustula (P)
Myxicola infundibulum (P)
Urechis caupo (U)
Deposit feeder
Deposit feeder
Deposit feeder
Deposit feeder
Suspension feeder Damariscotta Estuary, Maine
Suspension feeder Various sites, California
Clymenella torquata (P)
Nereis diversicolor (P)
Eupolymnia heterobranchiata (P)
Cirratulus cirratus (P)
Spio setosa (P)
Deposit feeder
Deposit feeder
Deposit feeder
Deposit feeder
Deposit feeder
Deposit feeder
Damariscotta Estuary, Maine
Boothbay Harbor, Maine
Little Tutka Bay, Alaska
Damariscotta Estuary, Maine
Damariscotta Estuary, Maine
Seldovia, Alaska
11
8
66
5.6
21
Echiurus echiurus (U)
a E
ϭ
echinoderm, M
ϭ
mollusk, S
ϭ
sipunculan, H
ϭ
hemichordate, P
ϭ
polychaete, U
ϭ
echiuran.
either immediately after the sampling or on samples stored
frozen for approximately eight months at 80 C. No signifi-
cant difference in pH values was found between fresh and
frozen samples ( 9).
measured by adding alanine-methylcoumarinyl-amide (MCA).
Esterase, lipase, and - and -glucosidase activities were mea-
sured by adding the various monomers (butyrate, palmitate,
glucose, and glucose, respectively) attached through the ap-
Ϫ
Њ
␣
␤
n
ϭ
Enzyme activities at various Cu concentrations were mea-
sured according to Mayer et al. [14] except a 0.01-M MOPS
(3-[N-morpholino]propanesulfonic acid) or MES (2-[N-mor-
pholino]ethanesulfonic acid) buffer system (in 0.01 M NaNO3)
was used in this study because of negligible Cu complexation
by MOPS and MES under these conditions. For the cross-
propriate bond (ester, ester, and ␣- and ␤-glucoside, respec-
tively) to methyl-umbelliferone (MUF). Production of the
MCA and MUF fluorophores was monitored fluorometrically
at excitation/emission wavelengths of 340/445 nm with a
Fluostar microplate reader (BMG, Durham NC, USA). Enzyme
activities are expressed as nmol substrate hydrolyzed per mi-
nute per milliliter of gut fluid.
phyletic comparison, we used a constant pH
ϭ 7. We also
tested the pH dependence of this reaction for digestive fluid
from A. marina. Buffers for various pH were obtained by
adding different amounts of 0.1-M potassium hydroxide so-
Copper titration experiments were performed in black 96-
ϩ
well microplates by adding incremental amounts of a Cu2
Ϫ
8
Ϫ2
solution (10 –10 M, as cupric nitrate) to a series of wells
containing 230 l of gut fluids in buffer. Copper was titrated
lution into either MOPS (pKa
ϭ
6.9, for pH 6.5–7.9) or MES
␮
(pKa 6.1, for pH 5.5–6.5). Both MOPS and MES are struc-
ϭ
up to a Cu:amino acid ratio of about 1:1, which, according to
preliminary experiments, is sufficient to detect inhibition ef-
turally similar, and no significant difference was observed in
enzyme activities assayed in these two buffer systems at pH
6.5, indicating that the variation of enzyme activities measured
in the varying buffers was due solely to pH. These buffers
were tested and found to maintain pH during titration with
acidic copper solutions.
ϩ
fects on enzymes. The Cu2 solutions were freshly made each
day by diluting a 0.1-M Cu(NO3)2 standard solution (Orion,
Beverly, MA, USA) with deionized water. The volume of all
wells was then adjusted to 250 ␮l with deionized water before
measuring enzyme activities with the microplate reader, so that
liquid level of the wells had no effect on the fluorescence
reading. To be consistent with previous studies, the Cu thresh-
old is defined as the concentration of total dissolved Cu in gut
fluid above which enzyme activity sharply drops below its
To measure activities of various enzymes, gut fluids were
diluted 1,000
at each Cu concentration); then 25
substrate solutions were added [14]. Protease activities were
ϫ
with appropriate pH buffers (three replicates
␮
l of 100 mM fluorescent

Inhibition of digestive enzymes by copper
Environ. Toxicol. Chem. 21, 2002
1245
Fig. 2. The copper threshold concentration (␮mol Cu/L gut fluid; solid
line) in Arenicola marina gut fluid generally decreases with increasing
pH, while the initial proteolytic activity (nmol MCA/ml gut fluid/min;
dashed line) increases.
Fig. 1. Schematic of relative enzyme activity (compared to initial gut
fluid without added Cu) during titration by ionic copper solution. The
copper threshold concentration is the point at which enzyme activity
falls below its initial value.
sensitive to Cu. Nevertheless, this pH-dependent behavior im-
plies that animals with lower gut pH may be less vulnerable
to Cu with respect to this enzyme inhibition mechanism.
Guts of many deposit feeders have relatively neutral pH,
probably because of the difficulty of acidifying a large through-
put of sediments [18]. However, pH values as low as 5.5 have
been reported in some small polychaetes (M. Ahrens, personal
communication), and a pH range of 5.6 to 8.5 was found for
the species in this study. If the pH dependence found for A.
marina is characteristic of that to be found for other species,
then the threshold Cu concentrations for in vivo enzyme in-
initial (pretitration) level. While Cu is not introduced to animal
guts in vivo as an ionic cupric nitrate solution, the similar
inhibition of enzyme activity by copper solubilized from con-
taminated sediments that we previously reported [10] gives
confidence that cupric nitrate titrations adequately represent
in vivo processes.
RESULTS AND DISCUSSION
hibition in other species, based on a determination at pH
7, could be incorrect by as much as an order of magnitude.
ϭ
Copper concentration–response curve
The response of protease activity to added Cu was found
to be qualitatively similar to that reported for A. marina [10].
Adding Cu initially produced a rise in protease activity, fol-
lowed a sharp decrease in activity on reaching a threshold
concentration (schematically shown in Fig. 1). Our data using
digestive fluids from A. marina in this study agreed with the
previous report in that the initial rise in enzyme activity was
relatively small: approximately 20%. However, for many poly-
chaetes and echiurans, we found much more dramatic increases
in protease activity, ranging up to 66-fold above the starting
activity (Table 1). Only two species, Brisaster latifrons and
Mya arenaria, showed no such increase in activity during the
titration. This nearly ubiquitous pattern corroborates the pre-
vious single-species observation and suggests a potentially
important role of solubilized gut metals in affecting enzyme
activity in the positive as well as the negative direction. Such
metal enhancement of digestive enzymes has been observed
for pigs [17]. The mechanism of this Cu-enhanced activity is
beyond the scope of this study, but it may involve confor-
mational optimization of proteases or activation of ‘‘apopro-
teases’’ (dormant form of proteases) after Cu binding.
Response of various enzyme types to Cu exposure
Enzyme activities found in this study were similar to those
reported in our previous cross-phyletic study [14]. All the
enzyme types studied—protease, esterase, lipase, and
␣- and
␤
-glucosidase—in digestive fluid from A. marina were inhib-
ited by concentrations of Cu within a factor of two of one
another (Fig. 3). Two important conclusions derive from this
result. First, a variety of digestive processes besides the hy-
drolysis of proteinaceous food may be inhibited by Cu. Second,
the relatively small range of threshold Cu concentrations sug-
gests the same mechanism of inhibition, i.e. Cu inhibition of
enzymes, occurs only after saturation of the Cu-binding ca-
pacity of strong binding sites not involved in the catalytic
function of the enzymes.
Esterase seemed to differ from the other enzyme types in
that it maintained significant activity after considerably ex-
ceeding the Cu threshold concentration. Its activity appeared
to stabilize at approximately 50% of original activity over a
wide range of Cu concentrations. Either esterase enzymes are
less affected by Cu complexation than the other enzymes or
multiple types of esterolytic enzymes exist that vary in their
sensitivity, including at least one that is remarkably insensitive.
We previously found that contaminated sediments from the
field could induce inhibition of digestive proteases of A. ma-
rina [10]. By extension, it follows that sufficient Cu levels can
be solubilized from some contaminated sediments to inhibit
different digestive enzymes from a wide variety of species.
Effects of pH on Cu–response curve
We tested the effect of gut pH on Cu inhibition of digestive
proteases in A. marina by varying buffer pH in the assay (Fig.
2). The threshold Cu concentration peaked at pH 6.0 and then
decreased markedly with increasing pH. This general decrease
in threshold accompanies a rise in protease activity even in
the absence of Cu due to the normal pH dependence of this
enzyme. This decrease in threshold with increasing pH sug-
Threshold Cu concentrations among species
ϩ
ϩ
gests that H can compete with Cu2 for binding sites on the
Threshold concentrations varied by more than four orders
of magnitude among the species studied (Table 1) from a low
proteases or that protease conformation at lower pH was less

1246
Environ. Toxicol. Chem. 21, 2002
Z. Chen et al.
Fig. 4. Copper threshold concentration (
␮
g/g sediment) versus dis-
Fig. 3. Relative enzyme activity in Arenicola marina gut fluid (com-
pared to gut fluid without any Cu added) versus concentration of added
solved amino acid concentration (mM) in various species, grouped
according to major taxonomic group. E echinoderm, H hemi-
sipunculan, and U
ϭ
ϭ
Cu (
␮mol Cu/L gut fluid) for protease, esterase, ␣- and ␤-glucosidase,
chordate, M
echiuran.
ϭ
mollusk, P
ϭ
polychaete, S
ϭ
ϭ
and lipase.
of 8
␮M for an herbivorous echinoderm to a high of almost
Species with high amino acid concentrations in their gut
fluids typically dissolve the greatest amounts of Cu from sed-
iments [9,15,19,20]. The somewhat paradoxical implication is
that those species that dissolve the most Cu might have their
digestive enzymes least affected by it, depending on the rel-
ative amounts of Cu solubilized versus the relative Cu thresh-
olds for inhibition. The solution to this apparent paradox lies
in the conclusion that strong Cu-binding ligands accompany
high dissolved amino acid concentrations but that Cu com-
plexation to these strong ligands does not influence enzyme
activity. Only when these strong ligands are saturated can the
added Cu begin to bind to sites that affect enzyme activity.
The cross-phyletic trends in complexation and enzyme inhi-
bition are thus consistent with the binding–inhibition sequence
observed in the titrations (Fig. 1).
0.4 M for a deposit-feeding echiuran. Digestive proteolytic
activities of polychaetes were the among the most resilient to
Cu additions, but their copper thresholds still ranged more
than two orders of magnitude from 2.4 to 346 mM. Echinoderm
proteolytic activities were least tolerant to Cu contamination
(range
ϭ 8–500 ␮M), suggesting that this group of organisms
could be affected relatively easily. Copper thresholds of other
groups (hemichordates, sipuncula, and mollusks) generally
ranked between polychaetes and echinoderms. This rank order
among species may be disrupted if the factors not taken into
account in our experiments (e.g., pH and particle selection)
were capable of overcoming the
Ͼ4 order of magnitude range.
However, it seems unlikely that these other factors would
change susceptibilities more than one to two orders of mag-
nitude (e.g., Fig. 2), so that the distinction between polychaetes
and echinoderms likely is valid. Within phyla, species differ-
ences in rank might well be affected by these other parameters.
We previously showed that different A. marina individuals
had varying threshold concentrations, with greater concentra-
tions of dissolved amino acids in the digestive fluid leading
to higher threshold concentrations [10]. We therefore examined
the role of amino acids in influencing the cross-phyletic results
reported here (Fig. 4) and found a strong positive relationship
between threshold Cu concentration and dissolved amino acids
Relevance to other contaminant impact data
In the absence of in vivo experiments, our data cannot be
used to assess the importance of digestive enzyme activity
inhibition as a mechanism for toxic action in marine sediments.
Inhibition of digestive enzyme activity has been observed un-
der metal stress (e.g., for cladocerans [21]), though it is not
clear if such stresses are due to direct metal–enzyme inter-
actions as seen here or to more indirect effects on enzyme
secretion.
(Pearson product moment correlation:
p Ͻ 0.01). Clearly, this
relationship explains differences among phyla but not among
species within phyla. Polychaetes and echiurans normally have
the most intense digestive conditions, as evidenced by high
enzyme activities and dissolved amino acid concentrations,
while echinoderms typically have much less intense digestive
conditions [14,16]. Despite the excellent correlation, clearly
much variance remains, so that other factors must influence
Cu inhibition of digestive protease activities. The pH of the
gut fluids explains some of this variance; for example, among
polychaetes alone, a significant inverse relationship was ob-
served between threshold Cu concentration and pH. This in-
verse relationship follows the prediction of our experiment
that varied pH only (Fig. 2). Other factors, such as varying
composition of the amino acid pool or the presence of other
protecting ligands, may also be important in determining the
threshold Cu concentration.
We can assess the consistency of our findings with other
types of studies by estimating the sedimentary Cu concentra-
tions necessary to achieve the copper thresholds found in this
study. This solid-phase threshold level was calculated by mul-
tiplying the dissolved copper threshold values by a typical gut
fluid:sediment ratio of 1 ml gut fluid:0.5 g sediment (Table 1),
common in deposit feeders and previously used in biomimetic
assays [9,20]. These solid-phase concentrations represent the
amount of Cu that can be solubilized from the sediment by
the gut fluid and hence are equivalent to the Cu spike levels
used in this study. Fluid:solid ratios may vary 16-fold among
individuals and among species [22]; thus, errors may be in-
troduced in the threshold calculations due to the assumption
of constant fluid:solid ratio among species. Other factors, such
as gut residence time and particle selection by animals from
bulk sediment, also vary among animals and may significantly

Inhibition of digestive enzymes by copper
Environ. Toxicol. Chem. 21, 2002
1247
affect the amount of Cu that can be solubilized from sediments
by various animals. Thus, our calculations likely do not ac-
curately represent the amounts of Cu necessary to inhibit di-
gestive proteases in animals in the field. Nevertheless, these
calculations have value in showing the potential for differential
impacts among species.
The resultant concentrations (Table 1) range from values
similar to those in uncontaminated sediments to those higher
than typically encountered even in highly polluted environ-
ments [23]. For example, digestive protease activities of rel-
atively sensitive species in this study show inhibition at thresh-
old Cu concentrations between 10 (Parastichopus) and 87
ferential response of benthos to Cu inhibition may be useful
for explaining some patterns of stress of benthic organisms in
Cu-contaminated areas. Varying gut amino acid concentrations
among these organisms appear to be the major factor affecting
the bioavailable Cu threshold, although other factors must also
play a role. This short-term Cu toxicity affected all enzyme
types at a similar bioavailable Cu threshold.
Acknowledgement—We thank L. Schick, L. Self, and A. Knowlton.
This work was supported by the U.S. Environmental Protection Agen-
cy, Office of Naval Research, and National Science Foundation and
represents contribution 369 from the Darling Marine Center.
(
Saccoglossus
)
␮
g/g, which correspond to the 10th and 50th
g/g
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Echinoderms, which are here shown to be especially sen-
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pollution-sensitive group, being less frequent than polychaetes
in contaminated areas [4,24]. Tolerance to Cu contamination
also varies greatly among polychaetes [6]. Nereis diversicolor
[25] can live in sediments with more than 1,000 ppm Cu, while
110 ppm Cu has been found to be highly toxic to A. marina
[26]. These trends are consistent with our data on Arenicola
and Nereis species.
Changes in community structure result from many inter-
actions between organisms and their environment. Digestive
enzyme inhibition could be one of many stresses to benthic
communities in Cu-contaminated areas. Our data indicate that
these organisms can be subjected to stress when Cu concen-
trations are above these thresholds, but it does not imply that
these species will not appear in sediments with higher-than-
threshold Cu concentrations. Benthic invertebrates have mech-
anisms to regulate Cu in tissues, thus allowing them to thrive
in heavily contaminated areas, and perhaps means exist to
regulate Cu in digestive fluids as well.
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CONCLUSIONS
These results lead to the counterintuitive conclusion that
deposit feeder taxa with low digestive intensities (low enzyme
activity and low amino acid concentrations) and high pH are
most vulnerable to sedimentary Cu inhibition by this mech-
anism, although they solubilize less sedimentary Cu than taxa
with high digestive intensities and low gut pH. In general,
echinoderms should therefore be more susceptible to digestive
inhibition from Cu contamination than polychaetes. This dif-
19. Chen Z, Mayer LM. 1998. Mechanisms of Cu solubilization dur-
ing deposit-feeding. Environ Sci Technol 32:770–775.
20. Chen Z, Mayer LM. 1999. Sedimentary metal bioavailability de-

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