Environmental Toxicology and Chemistry, Vol. 23, No. 1, pp. 65–71, 2004
Printed in the USA
0730-7268/04 $12.00 ? .00
IMPORTANCE OF EQUILIBRATION TIME IN THE PARTITIONING AND TOXICITY OF
ZINC IN SPIKED SEDIMENT BIOASSAYS
JUNG-SUK LEE,†‡ BYEONG-GWEON LEE,*‡§ SAMUEL N. LUOMA,‡ and HOON YOO‡§
†NeoEnBiz Company, Seoul, Republic of Korea
‡U.S. Geological Survey, 345 Middlefield Rd, Menlo Park, California 94025
§Department of Oceanography, Chonnam National University, 300 Yongbong-Dong, Kwang-Ju 500-757, Republic of Korea
(Received 25 March 2003; Accepted 24 June 2003)
Abstract—The influences of spiked Zn concentrations (1–40 ?mol/g) and equilibration time (?95 d) on the partitioning of Zn
between pore water (PW) and sediment were evaluated with estuarine sediments containing two levels (5 and 15 ?mol/g) of acid
volatile sulfides (AVS). Their influence on Zn bioavailability was also evaluated by a parallel, 10-d amphipod (Leptocheirus
plumulosus) mortality test at 5, 20, and 85 d of equilibration. During the equilibration, AVS increased (up to twofold) with spiked
Zn concentration ([Zn]), whereas Zn–simultaneously extracted metals ([SEM]; Zn with AVS) remained relatively constant. Con-
centrations of Zn in PW decreased most rapidly during the initial 30 d and by 11- to 23-fold during the whole 95-d equilibration
period. The apparent partitioning coefficient (Kpw, ratio of [Zn] in SEM to PW) increased by 10- to 20-fold with time and decreased
with spiked [Zn] in sediments. The decrease of PW [Zn] could be explained by a combination of changes in AVS and redistribution
of Zn into more insoluble phases as the sediment aged. Amphipod mortality decreased significantly with the equilibration time,
consistent with decrease in dissolved [Zn]. The median lethal concentration (LC50) value (33 ?M) in the second bioassay, conducted
after 20 d of equilibration, was twofold the LC50 in the initial bioassay at 5 d of equilibration, probably because of the change of
dissolved Zn speciation. Sediment bioassay protocols employing a short equilibration time and high spiked metal concentrations
could accentuate partitioning of metals to the dissolved phase and shift the pathway for metal exposure toward the dissolved phase.
Keywords—ZincToxicityEquilibration timeSediment agingAcid volatile sulfides
Aquatic sediments in industrialized areas can contain con-
centrations of metals that are orders of magnitude higher than
background levels . Because metal-contaminated sediments
pose a threat to resident benthic organisms [2–4], a primary
goal of managing contaminated sediments is to protect food
webs from that threat. Sediment bioassays are commonly em-
ployed for evaluating the threshold concentrations at which
specific metals have adverse effects on test organisms. Bio-
assays can also aid in understanding the processes that influ-
ence partitioning and bioavailability of metals in sediments.
However, important uncertainties remain in extrapolating
spiked-sediment bioassay results unambiguously across a
range of conditions in nature . In the present study, we
illustrate that the metal concentrations and equilibration time
used for bioassay can be critical in determining the partitioning
of metals to different binding phases of sediment, subsequent
acute toxicity to sediment-dwelling organisms, and thus, the
reliability of extrapolations from sediment bioassays to nature.
A growing body of work has demonstrated that understand-
ing uptake routes of metals to benthic organisms from con-
taminated sediments is critical to determining metal bioavail-
ability from sediments [6–10]. Some contradictory results are
observed among studies concerning major metal exposure
routes (e.g., pore water [PW] vs dietary metals) [8,11–14].
Most equilibrium partitioning–based studies contend that PW
metal is the dominant bioavailable pool in sediment [12,15,16].
Another body of work has employed laboratory microcosm
studies, field transplantation experiments, or biokinetic models
to demonstrate that dietary uptake from contaminated sedi-
* To whom correspondence should be addressed
ments and food particles is a major route of metal exposure
for various marine invertebrates, especially in moderately con-
taminated conditions [6,7,17–19]. Differences in experimental
protocols make it difficult to compare among these contradic-
tory studies. However, one major comparable difference
among studies, as well as between laboratory studies and field
situations, is how the metal contaminants are introduced to
sediments before bioassay.
Many laboratory studies employ a protocol that exposes
sediments to a spike of dissolved, but surface-reactive, metal
and then equilibrating the slurry for days before the bioassay.
Metal contaminants in nature, however, are often introduced
gradually and over a much longer period; therefore, these con-
taminants have long equilibration times with sediments. In a
reanalysis of previous studies (e.g., [12,16,20,21]), Lee et al.
 suggested that the apparent partition coefficient (ratio of
sediment to PW metal concentration) of Cd, Ni, and Zn tends
to decrease with spiked metal levels and increase with metal–
sediment equilibration time. It is possible that high concen-
trations of dissolved metals introduced to sediment slurries
can initially oversaturate the available binding sites on sedi-
ment surfaces until exchange reactions re-equilibrate parti-
tioning . Although numerous studies [10,23–26] have dem-
onstrated effects of various geochemical parameters (e.g., acid
volatile sulfide [AVS], pH, redox potential, organic content,
salinity, etc.) on metal partitioning in sediments, the effects
of metal–sediment equilibration time on metal partitioning re-
main poorly understood.
If the degree of metal contamination and metal–sediment
equilibration time influences directly the partitioning of metals
between PW and sediment, they could also influence the bio-
availability and toxicity of metals in contaminated sediments.
Environ. Toxicol. Chem. 23, 2004J.-S. Lee et al.
In fact, Sae-Ma et al.  reported that bioaccumulation and
mortality of midge (Chironomus tentans) exposed to Cd-
spiked soils decreased with storage time (up to 120 d) of the
experimental sediments. Although a few studies [27–29] have
demonstrated reduction in bioavailability with metal–sediment
equilibration time, this change in bioavailability has not been
systematically linked to changes in metal geochemistry. Fur-
thermore, the actual cause of reduction in bioavailability has
rarely been explained.
In the present study, the influence of the equilibration time
of Zn introduced to estuarine sediment particles on partitioning
and toxicity was evaluated over 95 d. We tried to relate the
temporal change of geochemical parameters, such as AVS,
weak-acid extractable Zn, and pore-water Zn concentration
(PW [Zn]), to that of mortality of test animals. For simulta-
neous sediment toxicity test, estuarine amphipods (Lepto-
cheirus plumulosus) were exposed for 10 d to Zn-spiked sed-
iments after 5, 20, or 85 d of equilibration. The relative con-
centrations of reactive sulfides called AVS (extracted by 1 N
HCl and typically composed of amorphous iron sulfides) and
simultaneously extracted metals (SEM) with AVS in sediments
[SEM ? AVS] can control dissolved metal concentrations in
PW . So, a range of AVS and Zn-SEM in sediments was
employed to investigate the dynamic interaction of these geo-
chemical parameters with equilibration time.
MATERIALS AND METHODS
The experimental sediment containing approximately 30
?mol/g of AVS was collected from a mud flat near Palo Alto
(CA, USA) . The collected sediment was screened through
1-mm nylon mesh at the site to remove macroinvertebrates.
Mean particle size was 8.1 ? (phi), and mean sand, silt, and
clay contents, as analyzed by the pipetting method , were
0.4, 43.8, and 55.8%, respectively. Loss on ignition for 5 h at
450?C was 7.7% ? 0.7% (mean ? standard deviation [SD], n
After being brought to the laboratory, a portion of the col-
lected sediment was manipulated to achieve two nominal AVS
concentrations (5 and 15 ?mol/g) according to an established
protocol . Briefly, the sediment was mixed with an equiv-
alent volume of deoxygenated saline water (20 practical sa-
linity units [psu]) and divided into two batches. One batch of
the sediment was kept in a closed container and purged with
N2gas periodically. The remaining sediment was oxidized by
bubbling continuously with air for one week. The AVS con-
centration after one week of aeration decreased to approxi-
mately 1 ?mol/g. The oxidized and the remaining anoxic sed-
iments (?30 ?mol/g) were mixed at appropriate ratios to
achieve two nominal AVS levels (5 and 15 ?mol/g).
Following the AVS manipulation, the experimental sedi-
ments (containing ?1.2 ?mol Zn/g) were mixed with an ap-
propriate volume of Zn stock solution to achieve nominal Zn
concentrations of 10, 20, 30, and 40 ?mol/g for the low-AVS
(5 ?mol/g) series and 10, 20, 40, and 50 ?mol/g for the high-
AVS (15 ?mol/g) series sediments. The Zn stock solution was
prepared by dissolving ZnCl2in deoxygenated, deionized wa-
ter. Additionally, the control sediments without Zn addition
were included for each of two AVS seriessediments.Following
vigorous mixing with an electric mixer, the Zn-spiked and
control sediments were kept in closed polyvinylchloride bags
purged with N2gas and stored at 20 ? 1?C during the entire
Aliquots of the stored sediments were removed from each
treatment 4 d before the beginning of the bioassays (at t ? 5,
20, and 85 d). The removed sediments were mixed well, and
300 ml were transferred to four replicate, 1-L glass beakers.
Three beakers were used for bioassay replicates and one for
chemical analysis. The transferred sediments were allowed to
consolidate for 3 d in the beakers. The overlying water was
then decanted, 700 ml of aerated seawater (20 psu) added into
each beaker, and the sediments allowed to equilibrate for an-
other day before bioassay.
A 10-d amphipod sediment toxicity test [32,33] was used
to evaluate the influence of equilibration time on the acute
toxicity of spiked Zn in sediments. The estuarine amphipod
Leptocheirus plumulosus (approximately one month old) was
obtained from laboratory culture and maintained in a salinity
of 20 psu at 20?C. At the beginning of each bioassay (at t ?
5, 20, and 85 d), 20 individual amphipods (length, 0.6–1.0
mm) were transferred to each of the four beakers. Each beaker
contained previously consolidated sediments that were equil-
ibrated with overlying water. The overlying water was contin-
uously aerated, and test chambers containing beakers were
illuminated for 24 h. Water quality (temperature, salinity, dis-
solved oxygen, pH, and total ammonia) was monitored at t ?
0, 5, and 10 d of bioassay and met the recommended criteria
for amphipod toxicity test in all cases . Total ammonia
concentration in the overlying water, sampled at 3 cm above
the sediments, was always less than 100 ?M, which is far
below the acute toxicity concentration for this species .
Following 10-d exposure, live animals were collected from
each of three experimental beakers by sieving sediments
through 0.6-mm mesh. The remaining beaker was used for the
later geochemical analysis.
Separate 96-h, water-only Cd and Zn toxicity tests were
conducted using L. plumulosus in 20-psu seawater at 20?C.
The Cd toxicity test was done as a positive control to compare
sensitivity of the test animals used for each of three bioassays.
Mean 96-h median lethal concentration (LC50) estimates (12
? 0.2 ?M) for the three concurrent Cd reference toxicant tests
conducted in conjunction with the spiked-sediment bioassays
were not significantly different, indicating similar sensitivity
to metals among the batches of test organisms used. The LC50
values from the water-only Zn toxicity test would be compared
later to the LC50 values for PW Zn from each of the three
Sediment samples for chemical analysis (AVS, SEM, and
PW) were collected twice, at the beginning and end of each
10-d bioassay, from the beaker dedicated to chemical analysis.
This sampling scheme resulted in six sediment-sampling
events (at t ? 5, 15, 20, 30, 85, and 95 d). The sediment in
the beaker was homogenized with a plastic spatula after the
overlying water was decanted. Following the homogenization,
approximately 20 ml of sediment were taken using a plastic
syringe and immediately analyzed for AVS and SEM with an
established method (see below). Approximately 40 ml of sed-
iment were transferred to a 50-ml centrifuge tube and spun for
30 min at 2,500 g, and then the supernatant was filtered with
a 0.45-?m syringe filter. The filtrate was acidified immediately
with Ultrex? (Baker, Phillipsberg, NJ, USA) HCl to a pH of
1 to 2 and used later for PW Zn analysis. Two replicatesamples
were taken from each treatment for AVS, SEM, and PW Zn
Partitioning and toxicity of zinc in sediments
Environ. Toxicol. Chem. 23, 200467
Fig. 1. Variation of acid volatile sulfide (AVS) with sediment–Zn equilibration time in low-AVS (A) and high-AVS (B) series and with Zn–
simultaneously extracted metals (SEM) with AVS (C). Weak acid extractable Zn concentration [Zn-SEM] in low-AVS series increased from 1
(?), 10 (?), 15 (?), 24 (?), and 30 (□) ?mol/g and those in high-AVS series from 1 (?), 10 (?), 15 (?), 30 (□), and 37 (1) ?mol/g. The
AVS value at each sampling time represents the mean of two replicate measurements. The difference between two measurements was mostly
less than 20%. Relationship between AVS and Zn-SEM in Zn-contaminated sediments in low- and high-AVS series was from the time-averaged
values; error bars represents one standard deviation of AVS and Zn-SEM (n ? 6).
Fig. 2. Variation of pore-water Zn concentrations with sediment equil-
ibration time in low–acid volatile sulfide (AVS) (A) and high-AVS
(B) series (see Fig. 1 for symbols). SEM ? simultaneously extracted
metals with AVS.
The experimental containers and glassware used for sedi-
ment handling, chemical analysis, and sample storage were
acid washed (1 N HCl), then soaked in N2-purged deionized
water for one week. The sediment samples were handled under
a glove bag filled with N2gas. The AVS analysis was con-
ducted by an N2purge and trap method using an ion-specific
sulfide electrode within a week of sample collection [34,35].
The detailed analytical procedures are described elsewhere
. Metal concentrations in SEM, metal extracts from total
sediment digestion (HF-HClO4-HNO3), and PW samples were
determined with inductively coupled argon plasma-atomic
emission spectroscopy and/or flame-atomic emission spec-
troscopy. Pore-water samples were diluted 10-fold with 0.1 N
ultrapure nitric acid (to mitigate chloride interference) before
analysis. The analytical quantification limit (mean ? 10 SD
of procedure blank ? dilution factor) for PW Zn was 1.5 ?M.
The AVS, Zn-SEM, the molar difference betweenSEMwith
with AVS and AVS in sediments, [SEM ? AVS], and PW
[Zn] data were analyzed by multiway analysis of variance
using the Statistica? (StatSoft, Tulsa, OK, USA) package to
test the effects of equilibration time and of AVS and SEM
levels on the biogeochemical parameters. Dry weight–based
concentrations were used for all sediment data. The [SEM ?
AVS] values were calculated by the difference between Zn-
SEM and AVS [10,12]. The LC50 values for 96-h, water-only
Cd and Zn tests and 10-d sediment tests were calculated using
the trimmed Spearman-Karber method . Limited statistical
analyses were done for PW [Zn] data, because many data were
under the detection limit. Statistical significance was set at p
? 0.05 unless otherwise noted. Mortality data for the amphi-
pod were analyzed by two-way analysis of variance to test the
effects of Zn-SEM and equilibration time for both low- and
high-AVS series treatments.
Geochemistry of sediment
The AVS in all sediments was significantly influenced by
both spiked Zn concentration and equilibration time (p ?
0.001) (Fig. 1). The AVS generally increasedwithequilibration
time in the Zn-spiked sediments but remained relatively con-
stant over time in the control sediments. The concentration of
AVS changed most rapidly during the initial 30 d. The range
of measured AVS at the beginning of equilibration (t ? 5 d)
was 3 to 7 ?mol/g in the low-AVS series and 13 to 19
?mol/g in the high-AVS series, close to their nominal values
(5 and 15 ?mol/g). At the end of the experiment, AVS ranged
from 3 to 16 ?mol/g in the low-AVS series and from 13 to
30 ?mol/g in the high-AVS series. The AVS also increased
with Zn-SEM in both series (Fig. 1C).
The Zn-SEM in sediments was little influenced either by
AVS or by equilibration time (p ? 0.05). Mean Zn-SEM (n
? 12) over the ranges of AVS and equilibration time was 1.2
? 0.1 ?mol/g for control and 9.8 ? 1.3, 15 ? 1, 24 ? 1, 30
? 2, and 37 ? 2 ?mol/g in sediments with nominal Zn level
of 10, 20, 30, 40, and 50 ?mol/g, respectively. Mean recoveries
of Zn-SEM (ratio of SEM to total extractable Zn) were 57 ?
10% (n ? 12) for control sediments and 87 ? 6% (n ? 48)
for all the Zn-spiked sediments.
Pore water [Zn] increased with spiked Zn concentrations,
as expected. Pore water [Zn] decreased significantly with
equilibration time, and its decrease was most apparent during
the initial 30 d of equilibration (Fig. 2). Pore water [Zn] de-
creased more in the high-AVS series (decrease of 23-fold) than
in the low-AVS series (decrease of 11-fold) at the end of the
95-d equilibration period. This was least obvious in the sed-
Environ. Toxicol. Chem. 23, 2004J.-S. Lee et al.
Fig. 3. Variation of [simultaneously extracted metals (SEM) ? acid
volatile sulfide (AVS)] with sediment equilibration time in low-AVS
(A) and high-AVS (B) series. Relationship between pore-water Zn
concentrations and [SEM ? AVS] in low-AVS (C) and high-AVS (D)
series at the different equilibration time is also shown.
Fig. 4. Variation of apparent partitioning coefficient of Zn (Kpw) with
sediment equilibration time (A and B) or with [simultaneously ex-
tracted metals (SEM) ? acid volatile sulfide (AVS)] in low- and high-
AVS series at the different equilibration time (C and D). Some Kpw
in high-AVS series could not be calculated, because the pore-water
Zn concentrations decreased below the detection limit. Decimal no-
tation of exponential form is used for y axis scale.
Fig. 5. Mortalities of amphipod Leptocheirus plumulosus with respect
to [simultaneously extracted metals (SEM) ? acid volatile sulfide
(AVS)] in low-AVS (A) and high-AVS (B) series. Amphipods were
exposed to control and Zn-contaminated sediments for 10 d at the 5
(●), 20 (?), and 85 d (?) of equilibration. Error bars represent one
standard deviation (n ? 3).
iments with lower Zn-SEM (?15 ?mol/g), because some PW
[Zn] were below the detection limit (1.5 ?M).
The patterns of [SEM ? AVS] variation over time reflected
the changes in AVS, because SEM changed little over time
(Fig. 3, A and B). The [SEM ? AVS] declined most during
the first 30 d of equilibration, reflecting the rapid increase of
AVS during this period, and remained relatively stable there-
after. The PW [Zn] increased generally with [SEM ? AVS],
as expected from the AVS normalization approach. The PW
[Zn] to [SEM ? AVS] relationship had changed with equili-
bration time; for a given [SEM ? AVS] value, PW [Zn] de-
creased with equilibration time (Fig. 3, C and D).
The apparent partitioning coefficient (Kpw; L/kg) of Zn,
defined as the ratio of Zn concentrations in SEM to PW, in-
creased by 10- to 20-fold with equilibration time but decreased
with spiked [Zn] (Fig. 4, A and B). The results suggest that
the longer sediment aged or the lower the spiked [Zn], the
more Zn partitioned proportionally to the sediment particles
than to PW. The apparent partitioning coefficient decreased
with [SEM ? AVS] when [SEM ? AVS] was greater than
zero and increased with equilibration time for a given [SEM
? AVS] (Fig. 4, C and D).
The mortality of amphipods exposed to sediments for 10
d at day 5, 20, or 85 of Zn–sediment equilibration were com-
pared to [SEM ? AVS] (Fig. 5) or PW [Zn] (Fig. 6). The
mortality of amphipods increased with [SEM ? AVS] but was
not significantly different from controls when [SEM ? AVS]
was less than zero (Fig. 5). The mortality was significantly
reduced as the equilibration time increased. The reduction of
toxicity was most apparent when comparing bioassay results
conducted after 5-d equilibration to those after 20-d equili-
bration (Fig. 5). Most individuals survived at 85-d equilibra-
tion even when [SEM ? AVS] was much greater than zero.
Mortality of amphipods was explained better by PW [Zn]
than by [SEM ? AVS] (Fig. 6). All the mortality data from
both high- and low-AVS treatments could be pooled into one
relationship with PW [Zn] for a given equilibration time (5 d
or ?20 d). Significant mortality always occurred when PW
[Zn] was greater than 20 ?M. The mortality was significantly
more at an equilibration time of 5 d than at 20 d across the
range of PW [Zn], when significant mortality was observed.
Partitioning and toxicity of zinc in sediments
Environ. Toxicol. Chem. 23, 200469
Fig. 6. The relationship between mortality of the amphipod Lepto-
cheirus plumulosus and pore-water Zn concentrations. Amphipods
were exposed to control and Zn-contaminated sediments at the 5 (●),
20 (#), and 85 (▫) d of equilibration. Vertical lines represent either
96-h median lethal concentration (LC50) of water-only Zn exposure
or the limit of detection (LOD) for pore-water Zn.
From the relationship in Figure 6, the 10-d LC50 of dissolved
Zn in PW at 5 d was estimated as 17 ?M (with a 95% con-
fidence interval of 15–21 ?M) and at 20 d as 33 ?M (with a
95% confidence interval of 27–40 ?M). These values were
higher than the mean 96-h LC50 value of 14 ?M Zn, inde-
pendently determined with the water-only Zn toxicity test for
Previous bioassays involving metal-spiked sediments used
a wide range of metal–sediment equilibration times and con-
centration levels. These differences in experimental protocols
could have a critical influence on metal partitioning and, there-
by, could affect toxicity to organisms exposed to the sediments.
We have systematically demonstrated that both sediment aging
and metal contamination levels are major controlling factors
for metal partitioning between sediment particles and the dis-
solved phase and, thus, collectively influence acute toxicity to
exposed benthic organisms. These results help to explain some
of the discrepancies among the studies regarding routes of
metal uptake from contaminated sediments [8,13].
Effect of equilibration time on metal partitioning
The 20-fold decrease of PW [Zn] during 85 d of equili-
bration could be explained mainly by the redistribution of Zn
from labile/weak-binding phases to more insoluble phases, in-
cluding AVS, and other binding phases, such as metal oxides
and organic matter. Among these binding phases, AVS is rec-
ognized as a dominant binding phase in the anoxic sediments,
and its effect on PW metals is relatively well studied .
The influence of AVS relative to SEM on PW metals has
been well established for several divalent metals, including Zn
[12,15,16,26]. Typically, PW [Zn] is very low when [SEM ?
AVS] is less than zero and increases exponentially with [SEM
? AVS] when [SEM ? AVS] is greater than zero. Inthepresent
study, the decrease of [SEM ? AVS] with time, which resulted
in a reduction of PW [Zn], was largely controlled by the in-
crease of AVS, because SEM was relatively constant over the
equilibration period. The increase of AVS concentration
([AVS]) in Zn-contaminated sediments can be explained by
sequestration of AVS (mostly in amorphous FeS) by Zn. If
AVS could be rapidly converted from amorphous FeS to ZnS
in the Zn-spiked sediments, the slower oxidation rate of ZnS
compared to that of FeS could result in the buildup of AVS
that was observed. For example, Simpson et al. [24,38] dem-
onstrated that FeS was rapidly oxidized in aerated waters
whereas ZnS was kinetically stable for oxidation. Similarly,
other studies also observed an increase of AVS on addition of
divalent metals in sediments [12,39].
An increase of AVS alone, however, does not entirely ex-
plain the observed reduction of PW [Zn] with equilibration
time. For a given [SEM ? AVS], PW [Zn] decreased with
equilibration time when [SEM ? AVS] was greater than 0
(Fig. 3). Furthermore, in anoxic sediment, PW [Zn] continu-
ously decreased with time even when [SEM ? AVS] was less
than zero. These results collectively suggest that some other
processes were responsible for further reduction of PW [Zn].
One process, other than AVS sequestration of PW [Zn], that
could be responsible for PW [Zn] reduction is redistribution
of Zn from labile binding sites to more refractory sites. In
fact, the greater extraction efficiency of SEM in Zn-spiked
sediments compared to unspiked control sediments in the pres-
ent study suggests that Zn in spiked sediments is more labile
than in the control sediments, which probably had a much
longer metal–sediment equilibration time in nature. Confirm-
ing this observation, Griscom et al.  showed in the se-
quential extraction of metals in sediment aged up to 35 d that
the labile fraction of Cd, Co, Se, and Zn decreased whereas
the resistant fractions significantly increased with time. The
labile fraction (i.e., low Kpw) includes the adsorbed and ex-
changeable phases of metals bound on the sediment surface;
the resistant phases (i.e., high Kpw) include the hydrous iron
or manganese oxides, organic matter, and mineralized fractions
of sediments . Other laboratory metal adsorption studies
[41,42] demonstrated that slower partitioning processes (e.g.,
physical and microbial transformation) followed the initialrap-
id adsorption of metals that occurred within a few days.
The level of spiked metal is another important factor af-
fecting PW metal concentrations. An order of magnitude de-
crease occurred in Kpwof Zn as Zn-SEM or [SEM ? AVS]
increased (Fig. 4). One possibility is association of Zn with
binding sites of progressively lower stability as the [Zn-SEM]
increases. Consistent with this, adsorption isotherms predict
nonlinearity at high adsorbate (metal) concentrations because
of the saturation of binding sites on the particle surface[22,43].
Other laboratory studies also showed decreases in Kpwof Cd,
Ni, and Zn with the increase of [SEM ? AVS] in laboratory-
spiked sediments .
Bioavailability (expressed as acute toxicity) of spiked Zn
in sediments was also reduced significantly with Zn–sediment
equilibration time. Other studies [39,44] have also evaluated
the role of temporal variation of AVS and SEM for bioavail-
ability and other biological responses, but to our knowledge,
they have not established the potential reasons for the varia-
tion. In the present study, the reduction in toxicity was best
explained by the concurrent reduction of dissolved [Zn] as
represented by PW [Zn]. In contrast, [SEM ? AVS] was a
less accurate indicator of bioavailability/toxicity, because the
dissolved Zn to [SEM ? AVS] relationship was changed by
the equilibration time. However, the reduction of dissolved
[Zn] as represented by PW could not explain all the reduction
of Zn bioavailability over time. The increase in LC50 values
Environ. Toxicol. Chem. 23, 2004J.-S. Lee et al.
of PW Zn in the sediment toxicity test by twofold during
equilibration time suggests that the toxicity of dissolved Zn
decreased over time. A plausible explanation for this obser-
vation could be that the Zn speciation of the dissolved phase
had been changed over time. For example, a decrease in free
Zn ion activity in PW, which could have occurred because of
changes in concentrations and/or composition of dissolved Zn-
binding ligands (e.g., dissolved organic matter), might be re-
sponsible for reduced toxicity of PW Zn. Another possible
explanation could be related to the variation of dissolved Zn
concentration in overlying water, which was not measured in
the present study.
It should be noted that the cause of mortality was assigned
to dissolved Zn (either overlying water or PW Zn) rather than
to PW Zn only. The dissolved [Zn] in the overlying water was
not determined in the present study. However, it is reasonable
to assume that overlying water [Zn] correlated and behaved
similarly to PW [Zn], because [SEM ? AVS] is a major co-
factor controlling [Zn] in both overlying water and PW. Es-
pecially, the static renewal protocol employed in the present
study could facilitate Zn to be equilibrated between PW and
overlying water. Consistent with this idea, previous studies
[19,21] have demonstrated that metal concentrations in over-
lying water strongly correlate with PW metal concentrations.
Many earlier studies (e.g., ) have suggested that acute
mortality of organisms exposed to metal-contaminated sedi-
ments is controlled by exposure to dissolved metals. Our re-
sults generally support the utility of AVS normalization for
predicting no acute toxicity of animals exposed to metal-con-
taminated sediments when [SEM ? AVS] is less than zero. It
is important to recognize, however, that the strong correlation
between dissolved Zn and acute toxicity does not preclude
chronic effects from other routes of uptake, including exposure
to metals via diet, at lower Zn concentrations. In fact, a grow-
ing body of evidence [9,19,26,40,45] shows that bioaccumu-
lation of metals in a variety of benthic invertebrates occurs
when [SEM ? AVS] is less than zero. Such results are little
affected by the variation of [SEM ? AVS]. Rather, bioaccu-
mulation is related best to SEM concentrations and is best
explained by ingestion of contaminated sediments [9,26,45].
One of the reasons for such results is that benthic invertebrates
ingesting AVS-rich, pure-phase particles or anoxic sediments
are able to assimilate metals with assimilation efficienciessim-
ilar to those for oxic particles [8,40,46]. A second reason,
however, is that all such studies were conducted with less-
than-extreme metal concentrations and realistic partitioning
The toxicological significance of complex, chronic expo-
sure conditions are only beginning to be understood (e.g.,
). However, whether the measure is toxicity or contaminant
uptake, both short equilibration times and high spiked metal
concentrations in sediments will accentuate partitioning of
metals disproportionately to the dissolved phase and increase
the probability of exposure and/or toxicity via dissolved met-
als. If metals are introduced gradually into contaminated sed-
iment, equilibrated over long time scales, and/or have con-
centrations that are not extreme, then partition coefficients will
be high, and chronic exposures are the concern. Resultsderived
from spiked-sediment bioassays do not necessarily provide
accurate/relevant estimates of the risk for sediments in nature.
Acknowledgement—The authors thank two anonymous reviewers for
many thoughtful suggestions. This work was supported in part by the
Toxic Substances Hydrology Program of the U.S. Geological Survey
and by research and development funds granted to B.-G. Lee from
the Korean Ministry of Marine and Fisheries.
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