Acute toxicity of interstitial and particle-bound cadmium to a marine infaunal amphipod

Marine Envirionmental Research 26 (1988) 135-153

Acute Toxicity of Interstitial and Particle-bound Cadmium to a Marine Infaunal Amphipod* Paul F. Kemp:l: University of Georgia Marine Institute, Sapelo Island, GA 31327, USA

& Richard C. Swartz US Environmental Protection Agency, Hatfield Marine Science Center, Newport, OR 97365, USA (Received 4 June 1988; accepted 4 August 1988)

A BSTRA C T The relative acute toxicity of particle-bound and dissolved interstitial cadmium was investigated using a new bioassay procedure. Interstitial concentration of Cd was controlled by means of peristaltic pumps, allowing separate manipulation of interstitial and particle properties. Addition of small quantities of organic-rich fine particles to sandy sediment resulted in greatly differing particle-bound Cd concentrations in sediment with similar interstitial Cd concentrations. Analysis of variance indicated no significant difference in the survival or ability to rebury in sediment of the phoxocephalid amphipod Rhepoxynius abronius (Barnard), when exposed to sediment with different total Cd concentrations but nearly equal interstitial Cd concentrations; in one case LCso data indicated slightly increased mortality in sediment with higher total Cd concentration. At least 70.2-87.9% of mortality could be predicted from past data on mortality based on dissolved Cd concentrations. The acute toxicity of Cd to this infaunal amphipod appears to be due principally to Cd dissolved in interstitial water. Our results indicate that static and flow-through bioassay tests of this organism produce * Contribution 618 of the University of Georgia Marine Institute and Contribution NO24 from EPA Envirionmental Research Laboratory, Narrangansett. Present address: Brookhaven National Laboratory, Oceanographic Sciences Division, Upton, NY 11973, USA. 135 Marine Environ. Res. 0141-1136/88/$03"50 (D 1988 Elsevier Science Publishers Ltd, England. Printed in Great Britain

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Paul F. Kemp, Richard C. Swartz comparable results with regard to mortality and survival, while the flowthrough system provides a greater capacity to manipulate experimental conditions.

INTRODUCTION Many anthropogenic contaminants released to the marine environment accumulate in sediment. Regulatory agencies are presently faced with the difficult task of assessing the ecological significance of such contaminants. A major area of uncertainty is the effect of sediment-contaminant interactions on the toxicity of introduced substances. Binding to sediment particles alters the bioavailability and toxicity of contaminants. Although a contaminant dissolved in interstitial water may affect biota similarly to dissolved contaminants in the water column, the toxicity of the same contaminant when particle-bound is poorly understood. Most studies have inferred the relative toxicity of particle-bound contaminants from indirect evidence (Fowler et al., 1978; Roesijadi et al., 1978; Langston, 1984; Schuytema et al., 1984; Swartz et al., 1985a). A primary difficulty in examining the relative toxicity of interstitial versus particle-bound contaminants has been the inability to manipulate their concentration separately. For example, Swartz et al. (1985a) examined the toxicity of Cd in sediment by manipulating total sediment Cd concentrations. Their experimental design allowed no control of interstitial Cd concentrations, and in order to examine the effect of interstitial Cd required exposing an infaunal marine amphipod to abnormal conditions (water in the absence of sediment). A modification of existing bioassay procedures at this laboratory allows greater manipulation of sediment and interstitial water concentrations and a more direct examination of the relative toxicity of interstitial versus particle-bound contaminants. We devised a flow-through bioassay system which maintains constant interstitial concentrations of a contaminant and allows separate manipulation of sediment properties to control total Cd concentrations. Many potential sediment contaminants are increasingly bound by sediment in proportion to various components of sediment, including inorganic mineral components and organic matter (Davies-Colley et al., 1984). With a flowthrough bioassay system, it is possible to produce sediment treatments which have equal interstitial concentrations of a contaminant, yet greatly different particle-bound concentrations at equilibrium with interstitial dissolved Cd because of slight differences in sediment properties. We have used this system to test directly whether the principal route of toxicity of cadmium to an infaunal marine amphipod, Rhepoxynius abronius, was via particle-bound Cd or Cd dissolved in interstitial water.

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METHODS

Collections and general bioassay procedures The collection site (Kemp et al., 1985) and the static bioassay procedures on which the present bioassay is based (Swartz et al., 1985a, b) have been described previously. The collection site (44°37-6'N, 124°3.0'W) is a moderately high-energy sandy bottom adjacent to the central channel of the Yaquina River estuary on the central Oregon coast, USA. Sediment is wellsorted sand (equivalent spherical diameter = 0.165mm) with an organic content (OC) less than 2% (w/w; all organic contents are weight loss after ashing at 500°C). Adults of the phoxocephalid amphipod Rhepoxynius abronius (Barnard) are used for standard bioassays at this and other laboratories. R. abronius was the most sensitive to Cd of 8 species of crustaceans tested by Hong & Reish (1987). R. abronius is an infaunal, burrowing phoxocephalid amphipod which does not construct permanent tubes or burrows, and typically burrows freely within the upper 2 cm of sediment in the laboratory. Gut content analyses indicate that it feeds on particulate matter in the sediment, including diatoms, other algae, and meiofaunal invertebrates as well as unidentified organic particles (Oliver et al., 1982). Aspects of the life history of R. abronius were discussed by Kemp et al. (1985) and Slattery (1985). Adult R. abronius were collected from a small boat using a small dredge which collects the upper 2 cm sediment layer, and were separated by sieving in water through a 1.00mm screen. A 1 to 2 c m layer of sieved (0.5 m m screen) sediment was placed in 1 liter beakers and 0.45/~m filtered seawater was passed continuously through the sediment using the apparatus described below. Standard test conditions were 15°C and 25 parts per thousand (ppt) salinity. Twenty amphipods were added in random order to each beaker. Individuals which did not bury into the sand within 10min were replaced. After 4 days, the contents of beakers Were sieved through a 0"5 mm screen and living and dead amphipods counted. All (living and apparently dead individuals) were placed on clean sediment from the sampling site and the number which reburied within 1 h was used as a measure of sub-lethal effects of contaminants. Sediment to be prepared for experimental use was collected from the Yaquina Bay site with the dredge and sieved through a 0.5 m m screen to remove large particles and macrofauna. All sediment passing the sieve was allowed to settle and then remixed. Since the sediment was collected from a moderately high-energy environment, the separation and remixing procedure was not expected to materially affect sediment properties. Fine

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Paul F. Kemp, Richard C. Swartz

particles were elutriated from additional sediment and collected in settling barrels. This organic-rich fraction (10-9% OC) was then added to a portion of the sieved sediment to produce sediment with a higher organic content and slightly skewed grain size distribution. Properties of the fine particulate matter other than organic content, especially iron oxide content, affect binding of Cd to manipulated sediment (Davies-Colley et aL, 1984). However, evidence suggests that iron oxide-bound Cd may be relatively unavailable whereas Cd bound to organic matter (at least in the form of bacterial exopolymer) may be relatively available (Harvey & Luoma, 1985a); thus, we measured organic content rather than iron oxide content or other inorganic fractions to represent our manipulation of the capacity of sediment to bind potentially available Cd.

Description of apparatus Multiflex (Cole-Parmer) peristaltic pumps were used to deliver a concentrated stock solution and seawater to mixing chambers and then to deliver the Cd-seawater solution to experimental chambers (Fig. 1). The seawater source was filtered seawater stored in a 220 liter polypropylene container (not shown in Fig. 1). Because flow rates of seawater to the mixing chambers were affected by head pressure, the stored water first entered a small secondary tank in which water level was controlled by means of a float valve. Each peristaltic p u m p was equipped with four p u m p heads, allowing four concentrations of Cd in seawater to be delivered to four separate mixing chambers. The flow from each mixingchamber was divided by a 12port syringe manifold (Wheaton) which was connected by Teflon tubing to 12 replicate experimental chambers. Flow rates from a given manifold to individual chambers were equal to within + 10%, provided that the manifold was level and tubing running from manifold to experimental chambers terminated at equal heights (to avoid siphoning between chambers). Manipulations of the sediment within each Cd treatment were possible (e.g. two sediment types within each Cd concentration, each with six replicates). Mixing chambers consisted of Ehrlenmeyer vacuum (sidearm) flasks. C a d m i u m stock solutions and seawater were delivered by Teflon tubing through perforated stoppers and mixed continuously by means of a magnetic stirrer. The sidearm was used as an overflow outlet and the total volume input was set at slightly greater than the volume removed and delivered to the experimental chambers. Stock solutions ofCdC12 in distilled water were 100 x the desired final concentration (distilled water alone for controls) and were replenished every 3 days. Experimental chambers consisted of polypropylene 1000 ml beakers with

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the bottoms removed and replaced with 110/~m polypropylene screening. The chambers were placed in 1.3 liter polypropylene 'jackets'. C a d m i u m seawater was initially delivered to the overlying water in the chamber, passed through the sediment and screen into the jacket, and exited via an outlet in the jacket which controlled water level. The water above the sediment was continually mixed by gentle aeration. Dosing and equilibration of the sediment was monitored by chemical analysis (method described below) of the incoming Cd-seawater solution from the lines connected to the manifolds, the effluent water in the jackets, and the water overlying the sediment. Preliminary experiments using methylene blue dye showed that flow through mixed sandy sediment is horizontally uniform and that no channels of higher or lower flow form.

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Paul F. Kemp, Richard C. Swartz

Experimental design Following testing of the apparatus to calibrate dosing and flow rates, two major experiments were conducted. Experiment I, based on previous 4-day static bioassays (Swartz et al., 1985a), was a preliminary experiment designed to determine required sediment manipulations and the time required to reach constant particle-bound Cd concentrations. Treatments in Experiment I consisted of a seawater control (mixed with distilled water) and three Cd concentrations in seawater. Mean concentrations in Cd treatments were 1.01, 2.04, and 4.99mg/liters final concentration. Control or Cd-treated seawater was delivered to 2 sediment treatments (unamended and + 0.25% OC) and the water and sediment were allowed to equilibrate for 4 days before adding 20 amphipods to each container. Sediment depth was 2 cm. The flow rate was 2 ml/min/chamber, equivalent to the addition of twice the total interstitial water volume every hour. The bioassay was terminated 4 days after the addition of amphipods. The null hypothesis was that Cd bound to particulate organic matter does not contribute directly (i.e. without first partitioning into interstitial water) to toxicity of sediment to R. abronius, and would be rejected if analysis of variance showed significantly reduced survival and reburial in treatments where OC and total sediment Cd concentration are higher but interstitial Cd concentrations are equal. In previous static bioassays, small (× 125%) organic enrichments resulted in large differences in survival and reburial (Swartz et al., 1985a). It was anticipated that unamended sediment would have an organic content of about 1% and the manipulated sediment 1.25%. Organic contents of sand from the collection site range between 1 and 2% OC. Cadmium concentrations in incoming and effluent seawater were monitored daily in replicate chambers containing amphipods but used solely for chemical sampling. Replicate water samples within a single chamber showed very little variation in concentration; consequently, water samples were not routinely replicated. Cadmium concentrations were also determined in effluent. water samples collected and drawn through syringe-mounted 0.45 ~m filters to remove any Cd which might be bound to very fine particulate matter suspended in the effluent seawater. At the end of the bioassay, the chambers which had been used for water sampling were sacrificed and duplicate 1 cm diameter cores were removed for total sediment Cd concentrations determined for the upper and lower 1 cm of sediment in each core. Total sediment Cd concentrations were also determined in samples collected from chambers set up and sacrificed at the initiation of the bioassay. Filtered and unfiltered seawater samples were stabilized by reducing pH to < 2 with concentrated HC1 (Ultrex) and were analysed for Cd by flame atomic

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absorption. Sediment samples were dried at 105°C overnight and 2 g of the mixed sample were extracted with 10ml o f 4 N HCI for 15 min. The extract was filtered (Whatman No. 40) and diluted to 25 ml with distilled water. The diluted extracts were analysed by flame atomic absorption. Sediment Cd concentrations are given on a dry sediment weight basis (i.e. mg Cd per kg dry sediment). The toxicity of dissolved Cd is associated with the concentration of the free Cd 2 ÷ ion, which is strongly influenced by chlorinity and artificial and natural chelating agents (Sunda et al., 1978). Dissolved organic substances released from the sediment could reduce the toxicity of dissolved Cd by reducing the concentration of the free ion. Therefore, Cd 2 + concentrations in incoming and effluent seawater were monitored daily by the method of Sunda et al. (1978) for measuring Cd 2 ÷ in seawater, using a Cd ion-selective electrode (Orion) calibrated by comparison with the electrode response to known solutions of Cd in distilled water (adjusted to the ionic strength of seawater by the addition of NaNO3). Comparisons of incoming and effluent Cd 2 + concentrations served both as a measure of complexation of Cd by dissolved organic substances, and as a means of rapidly determining whether sediment exposed to dissolved Cd was approaching equilibrium (i.e. little or no difference in incoming and effluent concentrations). The results of Experiment I were used to select a single Cd concentration of 2 mg/liter for Experiment II, which by comparison with previous static bioassays was anticipated to produce approximately a 50% mortality if toxicity was entirely due to Cd dissolved in interstitial water (Swartz et al., 1985a). Cadmium-seawater was delivered to two sediment treatments (1 and 2% OC), where the 1% treatment used elutriated sand and the 2% treatment used elutriated sand recombined with the organic-rich fine fraction (10-9%OC). A third treatment with 3 % O C was unsuccessful due to excessive clogging of the sediment or screen with fine particles, and results from this treatment are not reported. Replication was increased from 4 per treatment combination in Experiment I to 6 in experiment II. Controls received seawater plus distilled water (final salinity was effectively unchanged). Because the added fine sediment tended to reduce water flow through the sediment, sediment thickness was reduced to 1 cm and the flow rate to each chamber was reduced to 0.52 ml/min, sufficient to replace the interstitial water once per hour. The second treatment consisted ofelutriated sediment with sufficient fine sediment added back to produce the target value of 2% OC. The equilibration period was extended to 8 days when Cd 2 + measurements indicated slower equilibration than in Experiment I. Chemical sampling was as in Experiment I except water samples were not filtered since Experiment I results indicated filtration was not necessary. Also, sediment cores were not split into upper and lower fractions, since the

Paul F. Kemp, Richard C. Swartz

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results of Experiment I showed little difference in Cd concentration between the upper and lower 1 cm sediment layers (see below). The overlying water in the experimental chambers was sampled daily in addition to incoming and effluent water. RESULTS Experiment I--Water and sediment chemistry Measured incoming Cd concentrations were essentially constant through the bioassay (95% confidence limits _ 1.8-2-5% of mean concentrations). Cd began penetrating the sediment within 24 h (Fig. 2), at which time Cd was detectable in effluent water but concentrations were substantially lower relative to incoming concentrations. However, the water in the jackets was initially uncontaminated and it took some time to equilibrate jacket water concentrations with effluent water from the beaker. By the start of the bioassay 96h after initiating Cd flow, incoming and effluent Cd concentrations were nearly equal. For unknown reasons, Cd 2+ con-

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centrations tended to vary between days consistently in all treatments and for both experiments (Fig. 3a and 3b); for this reason, only comparisons of Cd 2 + concentrations within a given day are considered here. The lack of any substantial difference between free Cd 2 + in concentrations in the incoming and effluent water in Experiment I indicates that the sediment and interstitial

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water were in near-equilibrium during the bioassay (Fig. 3a), and also that incoming Cd was not significantly complexed by dissolved organic matter. There were only slight and inconsistent differences in the Cd concentration measured in filtered versus unfiltered samples, indicating that a very minor portion of the interstitial Cd was lost from sediment while bound to filterable particulate matter. Manipulation of the Experiment I sediment resulted in an unamended sediment with 1.69+_0-14%OC (mean _+95% confidence limits) and manipulated sediment with 1-92 _+0.07% OC, near the target difference of + 0.25% OC. The difference in organic content was intended to represent a 1.25 x increase from lower to higher OC treatments. However, since the ambient OC of collected sediment was higher than anticipated the actual change was only 1-14 x. Total sediment Cd concentrations increased with increasing incoming Cd concentrations and differed only slightly between the upper 1 cm and lower 1 cm of sediment (Table 1); at the start of the bioassay (day 0) the mean concentration in the upper 1 cm of the cadmium treatments was 12% (st. err. -- 1.48%, n = 6) greater than in the lower 1 cm. At the end of the bioassay there was no significant difference between the upper and lower 1 cm of sediment (upper = 98.4% of lower, st. err. = 6-9%, n = 6). There was no significant change in sediment OC (final = 99% of initial OC) in any sediment treatment and little or no net increase in total sediment Cd concentration between the first and fourth days of the bioassay. Averaging over days and core depths, the total sediment Cd concentration in the + 0 . 2 5 % O C treatment was 27-5, 18"8, and 26.0% higher than in the TABLE 2 Mean (_+95% Confidence Limits) Number of Amphipods which Survived or Reburied in Experiment I, in Two Sediment Types (Unamended= 1-69% Organic Content, + 0'25% = 1"92% Organic Content). Treatments are Controls and Three Total Dissolved Cd Concentrations (given as Mean Measured Concentrations During Bioassay, mg/liter). Four Replicates per Treatment, 20 Amphipods per Replicate. LCso and ECs0 shown with 95% Limits in Parentheses Dissolved Cd concentration

Survival Unamended

0 (controls) 1"01 2'04 4"99 LC5o and ECso

19"25 (0"80) 16"25 (2-72) 14"00 (7-00) 0'00 (--) 2.23 (2.03-2.46)

+ 0"25% OC

19.25 (1-52) 14.75 (5"26) 8-75 (4"18) 1"00 (2-25) 1.79 (1.55-2'04)

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19-25 (0-80) 10-00 (3"67) 3"75 (2.00) 0-00 (--) 1.07 (0.77 1.26}

+ 0"25% OC

19-25 (1"52) 9"75 (5"72) 3"25 (4.75) 0-00 (--) 1-04 (0-76-1.26)

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unamended sediment treatment receiving 1, 2 and 5 mg/liter Cd, respectively. Thus, the increase in total sediment Cd concentration was roughly proportional to the increasing organic content of the sediment regardless of the varying incoming Cd concentration.

Experiment I--biological data Survival and reburial decreased with increasing dissolved and total sediment Cd concentration (Table 2). Sediment organic content (and therefore total sediment Cd concentration) had no significant effect on survival or reburial (ANOVA, p > 0"05). The LCso (survival) calculated from mean incoming Cd concentrations was slightly lower for the + 0"25% OC sediment than for the unamended sediment, while the ECso (reburial) values were nearly identical (Table 2).

Experiment II--water and sediment chemistry The second experiment was designed to increase the difference in total sediment concentration between sediment treatments, making use of the observation in Experiment I that total sediment Cd concentrations resulting from a given interstitial concentration were proportional to the organic content of the sediment. As in Experiment I, penetration of Cd through the sediment occurred within 24 h (Fig. 4). Although the screen or possibly pore space in the 2% OC sediment tended to clog, flow through the sediment was maintained by adjusting the height of the overflow outlet in the jacket. Incoming and effluent dissolved Cd concentrations were nearly equal at 1% OC after 96 h of equilibration (Fig. 4), although concentrations in overlying water tended to be slightly higher than incoming concentrations. It is possible that Cd was concentrated in the aerated overlying water by collection of the aerosol on the watch glass, evaporation and dripping back into the chamber. Effluent concentrations were substantially lower than incoming at 2% OC throughout the equilibration period (Fig. 4), which was extended to 8 days, and continued to be lower than incoming during the 4 day bioassay period. Concentrations of free Cd 2 ÷ ion in incoming and effluent water (Fig. 3b) also indicate that equilibrium between interstitial water and sediment was established or approached in the 1% OC treatment but not in the 2% OC treatment. Projecting the slope of increasing dissolved Cd or Cd 2 ÷ during the course of the bioassay, it is apparent that full equilibration would require a period substantially greater than the 8 days allowed during Experiment II. The desired substantial difference in total sediment Cd concentrations in sediment was achieved in Experiment II (Table 3). Although total sediment

Acute toxicity of cadmium

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TABLE 3 Experiment I1. Total Cd Concentrations (mg/kg dry weight) in Sediment with 1% or 2% Organic Content, Days 0 and 4 after Adding Amphipods (Days 8 and 12 after Cd Addition Started). Values Shown are Means (se of Mean; n = 2). Means for all Controls Were Zero

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18.80 (0.62) 72-05 (3'60)

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Paul F. Kemp, Richard C. Swartz

Experiment ll--biological data Survival and reburial of amphipods in the 1% and 2 % O C control sediments was high and did not differ between treatments (Table 4). Survival and reburial were much lower than in controls and were not significantly different between the 1% and 2% OC Cd treatments (ANOVA, p > 0-05). Few individuals were capable of reburying in either treatment. TABLE 4 Mean (+ 95% Confidence Limits) Survival and Reburial in Experiment II, In Two Sediment Types (1 and 2% Organic Content) for Controls and 2 mg/liter Dissolved Cd in Incoming Water. Six Replicates per Treatment, Twenty Amphipods per Replicate Dissolved Cd concentration

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1% OC

2% OC

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20.00 (0.00) 9-17 (3"89)

19-83 (0.43) 0-17 (0.43)

20.00 (0.00) 1.00 (1.76)

DISCUSSION In Experiment I, sediment organic content was significantly higher in the +0"25% OC treatment, but since the unamended sediment OC was higher than expected, the higher OC was only 114% of that in the lower treatment, a substantially lesser difference than intended. Consequently, the results of Experiment I are preliminary and were primarily used to provide LC5o data based on interstial Cd concentration and to plan Experiment II. As might be expected given the small difference in sediment organic content, the average increase in total sediment Cd content was only 23-1% (+ 4.3 %). The results of analysis of variance indicate no significant effect of organic content and the resultant total sediment Cd concentration, on the survival or reburial of amphipods in controls (no Cd) and at three levels of interstitial Cd concentration. However, this conclusion is equivocal because of the small proportional difference in total Cd concentration between the two treatments, which would be expected to have minor impact on toxicity. The LCso was slightly but significantly lower in the +0-25% OC treatment of Experiment I, suggesting that the somewhat higher total sediment Cd concentration in the +0.25% sediment contributed to the toxicity of Cd, although the effect was not detected by the analysis of variance. In contrast with the LCso results, the reburial ECso values in Experiment I were nearly equal between treatments. The inability to clearly discriminate between the

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impact of interstitial and particle-bound Cd in Experiment I results in part from the small increase in total sediment Cd concentration in manipulated sediment relative to unamended sediment. In Experiment II, total sediment Cd concentrations were 146% (initially) to 283% (at end) higher in the 2% OC sediment than in the 1% OC sediment. The difference in effluent Cd and Cd 2 + concentrations between the 1% OC and 2% OC treatments indicates that amphipods in the two sediment treatments were not exposed to equal interstitial concentrations, a necessary condition for the experimental design. However, it is likely that the interstitial concentrations experienced by amphipods in the two treatments were similar, if not identical. Interstitial concentrations presumably decline as water passes through the sediment. Since R. abronius is free-burrowing and moves throughout the surficial 1-2cm of sediment, the average concentration to which an individual was exposed can be reasonably represented by the average of incoming and effluent concentrations. On this basis, the average exposure was to 2.09 _+ 0-05 mg/liter at 1% OC, and at 2% OC was 1-62 + 0"37 mg/liter, 77"5% of that in the 1% OC treatment. Mean C d 2+ concentration (average of incoming and effluent values) was 0.050 + 0"005 mg/liter at 1% OC, and at 2% OC was 0"039 + 0.011 mg/liter, 78"0% of that in the 1% OC treatment. [Note that this similarity of proportions of mean Cd and Cd 2+ concentrations between treatments suggests that the reduced Cd 2 + concentration at 2% OC can be attributed simply to lower total dissolved Cd concentrations rather than to complexing of Cd 2+ by dissolved organic matter.] This small reduction in mean dissolved Cd concentration (proportionally much less than the difference in total sediment Cd concentration) should produce only slightly higher survival and reburial in the 2% OC treatment, if all toxicity is due to dissolved Cd (Swartz et al., 1985a, Table 2). If it is indeed reasonable to assume that the average exposure of amphipods can be estimated in this manner, the requirement for equal interstitial Cd concentrations can be considered nearly satisfied. Despite the large difference in total sediment Cd concentrations, there was no significant difference between survival or reburial in the 1% and 2% OC treatments. Furthermore, mean survival in both sediment treatments was not significantly different from 10 out of 20 (t-test, p > 0"05), the number of survivors which would be predicted from the interstitial-based LCso (ca. 2 mg/liter) estimated in Experiment I. Survival and reburial in the 2% OC treatment were somewhat higher than at 1% OC (though not significantly), as might be expected since the mean interstitial Cd and C d 2 + concentrations at 2% OC were slightly lower than at 1% OC (see above paragraph). Thus, subject to the assumption of nearly equal interstitial concentrations, the acute toxicity of Cd to Rhepoxynius abronius in this experiment appears to

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be due to Cd dissolved in interstitial water, and not to the greatly disparate amounts of Cd adsorbed to sediment particles in the two treatments. As a worst-case interpretation, the maximum difference between dissolved Cd concentrations experienced by amphipods in the two treatments would occur if amphipods were effectively exposed at the effluent concentrations, such that amphipods in the 2% OC treatment experienced dissolved Cd at 50-60% of the concentration experienced by individuals in the 1% treatment. Survival at each concentration can be predicted from our large previous LCs0 data set for amphipods in Cd-treated interstitial water (Swartz et al., 1985a). Based on effluent concentrations of 2 mg/liter at 1% OC and 1.125 mg/liter at 2 % OC (Fig. 4), approximately 9"3 of 20 amphipods in the 1% OC treatment should survive whereas approximately 12-4 of 20 amphipods in the 2% OC treatment should survive. The actual values were 7"83/20 at 1% OC and 9-17/20 at 2% OC. Even in this worst-case interpretation of the data, there is little or no indication of a substantial direct contribution to mortality by particle-bound cadmium: nearly all mortality in Experiment II is explained by the predicted response to dissolved cadmium, as calculated below.

and

% explained = (initial- predicted final survivors)/(initial) x 100 (initial- actual final survivors)/(initial) (20 - 9-3)/20 × 100= 87.9% at 1% OC (20 - 7.83)/20 (20 - 12.4)/20 × 100 = 70"2% at 2% OC (20 - 9" 17)/20

Thus, only about 12-30% of the observed mortality is not predicted by the previous data on toxicity of dissolved cadmium, and may presumably be due to an additional toxic effect of particle-bound cadmium. The predicted LCso and ECso based on dissolved Cd in this flow-through system (from Experiment I) can be compared to the LCso and ECso in previous static bioassays of sediment, the latter based on Cd concentrations measured in interstitial water extracted at day 0 and day 4 (Swartz et al., 1985a). This comparison is useful in assessing whether the flow-through and static bioassay systems are equivalent in measuring toxicity. In the prior bioassays, the LCso and ECso based on interstitial water concentrations at the start of the bioassay were 3-56mg/liter (95% limits 3.13-4.22 mg/liter) and 2"25 mg/liter (95% limits 1.95-2-65 mg/liter), respectively. The corresponding values based on interstitial concentrations at the end of the bioassay were l'33mg/liter (95% limits 1.10-1.70 mg/liter) and 0-80mg/liter (95% limits 0-64-1.03 mg/liter), respectively. The apparent change in LCso was due to decreasing interstitial Cd concentrations during the bioassay,

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such that a given final mortality corresponded to a higher concentration at the start or lower concentration at the end of the experiment (Swartz et aL, 1985a). Values from either sediment treatment in Experiment I of the present bioassay fall between the initial and final values for the prior bioassay, suggesting that Cd toxicity to R. abronius in static bioassays conducted at this laboratory can also be attributed largely-to dissolved interstitial Cd, and that the static and flow-through bioassays produce comparable results. Other studies using less direct methods have also attributed Cd toxicity to the dissolved fraction; however, the apparent importance of dissolved Cd is influenced by the species used. The toxicity of Cd has been attributed primarily to the dissolved fraction for epifaunal (Sunda et aL, 1978) and planktonic (Schuytema et aL, 1984) species, and also for a facultative suspension/deposit-feeding bivalve while suspension-feeding (Harvey & Luoma, 1985b). Dissolved Cd was much less important when the same bivalve was able to ingest Cd-laden natural sediment (Harvey & Luoma, 1985a). Similarly, much of the uptake of Cd by another deposit-feeding bivalve was attributed to ingestion of sediment (Bryan & Uysal, 1978). These studies suggest that particle-bound Cd may be important to deposit-feeding species. Rheopoxynius abronius is infaunal and has been shown to ingest diatoms and meiofaunal-sized prey (Oliver et aL, 1982) but not inorganic sediment particles; presumably any uptake of particle-bound Cd would be in the form of Cd associated with living food items as well as non-living organic particles. Since R. abronius can be maintained for extended periods under similar conditions in the laboratory, we assume that individuals in the bioassay chambers fed on sediment organic particles more or less normally during the course of the bioassays. The importance of predation or microalgal grazing as a vector for uptake of contaminants is not known. With regard to the uptake of dissolved Cd, the route of uptake was not examined in this study but is unlikely to be due to incorporation of Cd associated with dissolved organic matter. In general, studies have shown repeatedly that crustaceans are inefficient at assimilating dissolved organic matter in comparison to other invertebrate species (e.g. Stephens, 1981). Information on the relative availability of dissolved and particle-bound contaminant fractions to organisms would be of value if it led to an improved ability to assess the potential impact of contaminants in a given sediment. For example, if the dissolved fraction of most sediment contaminants controlled toxicity, sediment toxicity could be assessed from measurements of dissolved concentrations and background knowledge of their toxicity in water (e.g. the E.P.A. Water Quality Criteria). For Cd, species, feeding habit and probably other factors must be considered before information on availability can be applied to practical problems. Similarly,

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the bioavailability of the particle-bound fraction of other metals (e.g. Co, Cr, Ni, Pb, Zn, Bryan & Uysal, 1978; As, Langston, 1984; Zn, Ag, Harvey & Luoma, 1985a; Zn, Co, Harvey & Luoma, 1985b), including radionuclides (e.g. Pu, 241Am, Beasley & Fowler, 1976; 241Am, Vangenechten et al., 1983) is influenced by species and sediment characteristics, and varies between metals. There are also substantial differences in the characteristic availability of organic contaminants and metals (Fowler et al., 1978). The bioassay procedure used in our study provides an improved means of partitioning sources of contaminants to organisms. In the case of the present work with R. abronius, the method demonstrated that most or all of Cd toxicity in sediment was attributable to interstitial dissolved Cd, corroborating the inferences drawn from our previous work with static bioassay methods. Our results with cadmium suggest promise in applying this procedure to other contaminants and to other infaunal species. Manipulations of other sediment components such as iron oxide are also possible with this bioassay system, allowing rigorous testing of hypotheses concerning the relative importance of various inorganic and organic sediment components.

ACKNOWLEDGEMENTS The authors thank C. Holcombe for drafting. Funding was provided by Cooperative agreement CR812792-01 between EPA Region X and Oregon State University.

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Harvey, R. W. & Luoma, S. N. (1985b). Separation of solute and particulate vectors of heavy metal uptake in controlled suspension-feeding experiments with Macoma balthica. Hydrobiol., 121, 97-102. Hong, J.-S. & Reish, D. J. (1987). Acute toxicity of cadmium to eight species of marine amphipod and isopod crustaceans from southern California. Bull. Environ. Contain. Toxicol., 39, 884-8. Kemp, P. F., Cole, F. A. & Swartz, R. C. (1985). Life history and productivity of the phoxocephalid amphipod Rheopoxynius abronius (Barnard). J. Crust. Biol., 5, 449-64. Langston, W. J. (1984). Availability of arsenic to estuarine and marine organisms: A field and laboratory evaluation. Mar. Biol., 80, 143-54. Oliver, J. S., Oakden, J. M. & Slattery, P. N. (1982). Phoxocephalid amphipod crustaceans as predators on larvae and juveniles in marine soft-bottom communities. Mar. Ecol. Prog. Set., 7, 179-84. Roesijadi, G., Anderson, J. W. & Blaylock, J. W. (1978). Uptake of hydrocarbons from marine sediments contaminated with Prudhoe Bay crude oil: Influence of feeding type of test species and availability of polycyclic aromatic hydrocarbons. J. Fish. Res. Board Can., 35, 608-14. Schuytema, G. S., Nelson, P. O., Maleug, K. W., Nebeker, A. V., Krawczyk, D. F., Ratcliff, A. K. & Gakstatter, J. H. (1984). Toxicity of cadmium in water and sediment slurries to Daphnia magna. Environ. Toxic. Chem., 3, 293 308. Slattery, P. N. (1985). Life histories of infaunal amphipods from subtidal sands of Monterey Bay, California. J. Crust. Biol., 5, 635-49. Swartz, R. C., Ditsworth, G. R., Schults, D. W. & Lamberson, J. O. (1985a). Sediment toxicity to a marine infaunal amphipod: Cadmium and its interaction with sewage sludge. Mar. Environ. Res., 18, 133 53. Swartz, R. C., DeBen, W. A., Jones, J. K. P., Lamberson, J. O. & Cole, F. A. (1985b). Phoxocephalid amphipod bioassay for marine sediment toxicity. In Aquatic Toxicology and Hazard Assessment: Seventh Symposium, ed. R. D. Cardwell, R. Purdy & R. C. Bahner, ASTM STP 854. American Society for Testing and Materials, Philadelphia, pp. 284-307. Stephens, G. C. (1981). The trophic role of dissolved organic matter. In Analysis of Marine Ecosystems, ed. A. R. Longhurst, Academic Press, New York, pp. 271-91. Sunda, W. G., Engel, D. W. & Thuotte, R. M. (1978). Effect of chemical speciation on toxicity of cadmium to grass shrimp, Palaemonetes pugio: Importance of free cadmium ion. Environ. Sci. Technol., 12, 409-12. Vangenechten, J. H. D., Aston, S. R. & Fowler, S. W. (1983). Uptake of americium241 from two experimentally labelled deep-sea sediments by three benthic species: A bivalve mollusc, a polychaete and an isopod. Mar. Ecol. Prog. Ser., 13, 219-28.