Long-term storage of sediments: Implications for sediment toxicity testing

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Environmental Pollution, Vol. 89, No. 2, pp. 147-154, 1995 Elsevier Science Limited Printed in Great Britain. 0269-7491(94)00058-1

ELSEVIER

LONG-TERM STORAGE OF SEDIMENTS: IMPLICATIONS FOR SEDIMENT TOXICITY TESTING D a v i d W. Moore, a Thomas M. Dillon a & Elayne W. G a m b l e b ~USAE Waterways Experiment Station, 3909 Halls Ferry Road, Vicksburg, Mississippi 39180, USA bAsCI Corp., 1720 Clay Street, Vicksburg, Mississippi 39181, USA

(Received 10 March 1994; accepted 22 July 1994)

ably within 2 weeks of collection but held no longer than 6 weeks prior to testing (USEPA/USACE, 1991). This guidance is based in part on physicochemical changes known to occur in sediments during storage that can affect availability of nutrients and contaminants (Ho & Lane, 1973; Thompson et al., 1980; Macdonald & McLaughlin, 1982; ASTM, 1993). There are few studies evaluating the effects of storage time on sediment toxicity. Published reports that are available suggest toxicity is variable over time (Malueg et al., 1986; Stemmer et al., 1990; Outhoudt et al., 1991; Dillon et al., 1994). Outhoudt et al. (1991) examined effects of storage time on reproduction and growth in Daphnia magna and Chironomus tentans, respectively. Results of this study indicated highly significant changes in toxicity with storage; however there was no trend over time. In acute toxicity tests with D. magna, Malueg et al. (1986) showed increased toxicity with storage of a copper-spiked, freshwater sediment. In contrast, studies by Stemmer et al. (1990) and Weiderholm and Dave (1989) showed decreased toxicity with storage of sediments spiked with selenium and metalcontaminated freshwater sediments, respectively. In the only study to date with an estuarine sediment, Dillon et al. (1994) found toxicity of suspended sediment mixtures prepared from stored sediment increased with increasing holding time. To assess potential changes in sediment toxicity of field collected sediments with increasing holding time, a series of 21 day juvenile growth assays with the marine polychaete, N. arenaceodentata were conducted. Tests were conducted shortly after sediment collection (i.e. within 30 days) and at points in time beyond the prescribed holding limits up to 2 years after collection.

Abstract Juvenile Nereis (Neanthes) arenaceodentata survival and growth were used to evaluate the effect of storage time on the toxicity of sediments with moderate PAH and metal contamination. Seven San Francisco Bay area sediments and a clean control sediment were stored (4°C) and then periodically evaluated (up to two years after collection). During each test, juvenile worms (2-3 weeks post emergence) were exposed for 21 days. Test endpoints were survival and growth rate (mg dry weight~day). In general survival was high (>75%) and long-term cold storage (740 days) did not significantly alter growth or survival. In half of the sediments a cyclical phenomenon was observed associated with the appearance of ammonia in the overlying water of bioassay beakers. The periodicity of this phenomenon was approximately one year. It was not associated with any geophysical characteristic of the test sediments (i.e. grain size, % TOC, % TKN). Significant mortality (0% survival on day 427) was associated with the largest of these peaks in overlying water ammonia concentration. Results of this study suggest that ammonia in stored sediments is an important, potentially confounding factor in sediment toxicity tests.

Keywords: Dredged material, toxicity, storage, polychaeta, Neanthes, ammonia, survival, growth, San Francisco Bay.

INTRODUCTION Both the Marine Protection, Research and Sanctuaries Act (PL 92-532) and the Federal Water Pollution Control Act (PL 92-500) require an evaluation of potential environmental impact prior to dredging and disposal operations. Sediment samples from the area to be dredged are brought into the laboratory for testing. Laboratory dredged material toxicity evaluations follow a tiered, effects-based approach, beginning with shorter term 'acute' lethality and other screening tests and progressing to longer term 'chronic' sublethal tests. Consequently, logistical constraints often dictate that sediments be stored for some period of time prior to testing. Current Federal recommendations for storage of dredged material suggests that sediments be (i) stored between 2°C and 4°C (not frozen) and (ii) tested prefer-

MATERIALS AND METHODS Test species Nereis (Neanthes) arenaceodentata (Moore) is a benthic

infaunal polychaete widely distributed in shallow marine and estuarine benthic habitats of Europe, North America and the Pacific (see citations in Dillon et al. (1993)). This subsurface deposit feeder constructs one or more mucoid tubes in the upper 2-3 cm of sediment and ingests sediment particles up to 70/~m, with a preference for particles around 12 /xm (Whitlatch, 1980). 147

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N. arenaceodentata has been accepted by the regulatory community as an appropriate test species for evaluating sediment (USEPA/USACE, 1991). In addition, a draft guidance document describing sediment toxicity methods for N. arenaeeodentata has been adopted by the American Society of Testing and Materials (ASTM) (ASTM, in press). A large amount of toxicological information already exists for this species (Reish, 1985; Jenkins & Mason, 1988; Anderson et al., 1990; Johns et al., 1991a,b; Pesch et al., 1991). The life cycle and culture methods of N. arenaeeodentata are well-documented (Reish, 1980; Pesch et al., 1987). Adult worms (6-9 weeks old) establish mating pairs and occupy a common tube. The female deposits her eggs and dies shortly after, while the male remains in the tube to 'incubate' the eggs. He draws a current of seawater over the egg mass via rhythmic undulations, presumably to oxygenate the eggs and remove metabolic wastes. Larval development is direct via a non-planktonic metatrochophore larva and occurs entirely within the parental tube. Emergent juveniles (EJs) exit the tube 3 to 4 weeks after egg deposition. Shortly after emergence, juvenile worms begin to feed and construct tubes of their own. Worms grow steadily, increasing in both size and mass. Eggs first become visible in the coeloms of female worms at about 6 weeks. The majority of worms pair with a mate by 8 to 9 weeks. Egg deposition follows 2 to 6 weeks later to complete the life cycle.

Laboratory cultures

Stock populations of N. arenaceodentata were obtained in March 1988 from Dr D. J. Reish, California State University at Long Beach. Laboratory cultures have been maintained continuously since that time using static-renewal methods adapted from Reish (1980) and Pesch and Schauer (1988). Briefly, EJs are raised to sexual maturity in a 38 liter aquaria containing 2-3 cm of uncontaminated natural marine sediment and 30 liters of 30%0 seawater (Instant Ocean@). Aquaria were placed under trickle flow aeration. Seawater was renewed (80% of volume) every 3 weeks. Water quality (temperature, salinity, dissolved oxygen, and pH) was monitored weekly. Temperature and photoperiod were maintained at 20°C and 12 h light, respectively. Animals were fed a combination of ground Tetramarin flakes (2 mg/worm) and alfalfa (1 mg/worm) twice weekly. Survival (>80%) and reproduction (100-1000

eggs/brood; 50-500 EJs/brood) of worms in culture were consistent with that reported for other laboratory populations of Neanthes (Reish, 1980; Pesch et al., 1987; Harrison & Anderson, 1988). After 8-9 weeks, aquaria were broken down and adult worms paired. Sex is confirmed by the presence of eggs in the coelom and the intra-sexual fighting reaction described by Reish and Alosi (1968). Mated pairs were placed in 600 ml beakers covered with watch glasses and provided trickle flow aeration. Pairs were provided with 10 ml of a Tetramarin-alfalfa slurry containing 5 mg of each constituent for initial foraging and tube building activity. They were not fed thereafter since feeding activity in both sexes was greatly reduced prior to egg deposition and during brood incubation (Pesch & Schauer, 1988; personal observations). Saltwater was carefully renewed weekly in a manner which avoided disturbing worm pairs. Beakers were monitored daily for the presence of eggs and EJs. When discovered EJs were mixed with other broods and returned to the 37 liter aquaria to complete the culture cycle. These culture conditions and feeding rations were used in all experiments described below unless otherwise noted. Sediments Test sediments were collected from seven sites in and around the San Francisco Bay area (Table 1, Fig. 1). All sediments except for the control were composites of several cores taken to a depth of 11.6 m (38 ft) below mean low-water mark. The control sediment was collected via grab sampler from Sequim Bay, WA (Table 1, Fig. 1). This control sediment was used to validate experimental results. All sediments were collected by Battelle Pacific Northwest Laboratory. For a complete description of sampling methods and protocols see Mayhew et al. (1992). Sediment samples were immediately refrigerated (4°C) after collection and then shipped via a refrigerated truck to the USACE Waterways Experiment Station (WES). Upon receipt at the WES sediment samples were wet sieved (<2mm), thoroughly homogenized, and refrigerated (4°C) until analysis and testing could be performed. Sediments were collected 9-10 October 1990, arrived at the WES 16 October 1990 and the first test was initiated on 9 November 1990 (i.e. 30 days after collection). Three composites from each of the eight sediments were analyzed for priority pollutant metals (except an-

Table I. Sediments evaluated in 21-day juvenile growth bioassays with N. arenaceodentata

Category

Sediment

Description

Control

Sequim control (SC)

Fine grain, high organic

Test

Alcatraz environs reference (AER) Alcatraz mound reference (AMR) Bay Farm reference (BFR) Point Reyes reference (PRR) Oakland outer (OO) Oakland inner (OI) Oakland contaminated (OC)

Sandy with some fine grain material. Coarse grain. Fine grain. Sandy. Fine grain. Sandy with some fine grain material, Fine grain.

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Fig. 1. Locations of (a) Sequim Bay Control (SC) sediment and (b) San Francisco Bay area test sediments. timony and thallium), chlorinated pesticides and polychlorinated biphenyls (PCBs), and polyaromatic hydrocarbons (PAHs). Analysis was performed by the Analytical Laboratory Group (ALG) at WES according to procedures outlined in USEPA SW-846 (USEPA, 1986). Sediments were also analyzed for tri-, di-, and monobutyltins by the Naval Command and Control and Ocean Surveillance Center (NRaD) in San Diego, CA using procedures outlined by Stallard et al. (1989). Results of bulk chemical analysis are reported in Moore and Dillon (1993). Total organic carbon (TOC) and Total Kjeldahl nitrogen (TKN) analyses were performed by the ALG using Standard Method 505c (APHA, AWWA, and WPCF, 1990) and procedures outlined in USEPA (1979), respectively. Grain size analysis was performed using the methods of Patrick (1958). In addition, pore water was extracted from each of the sediments using methods described by Ankley et al. (1990). Sediment pore water extracts were subsequently analyzed for total NH 3 and H2S. Samples for ammonia analysis were adjusted to a pH of 2 with 1 N HCI and stored at 4°C for no longer than two weeks. Total ammonia (rag NH3-N/liter) was determined with an Orion, ammonia-specific electrode after adjusting sample pH to 12 with 5 N NaOH. Pore water extracts were analyzed for H2S using a HACH, HS-7 test kit. This kit makes use of the color reaction between lead acetate and hydrogen sulfide. Filter pads impregnated

with lead acetate were exposed to effervescing water samples containing hydrogen sulfide. The ensuing color change in the filter pad is compared to a standardized chart accompanying the kit to yield a semi-quantitative measurement of hydrogen sulfide.

Experimental approach Sediments were evaluated using 21-day juvenile growth assays with the marine polychaete Nereis (Neanthes) arenaceodentata. Sediments were tested starting 30 days after collection and then again at day 65, 113, 194, 286, 334, 427, 469, 621, and 740. Sediments were added to 1 liter beakers to a depth of 2.5 cm. Approximately 600 ml of 30%o salinity seawater was gently added to each beaker, carefully avoiding resuspension of the bedded sediment. Beakers were then equilibrated to test conditions for 24 h prior to addition of test animals. To initiate the test, 2-3-week-old juvenile worms (n = 225) were taken from laboratory culture and randomly distributed among 40 beakers (8 sediments, 5 replicates/sediment, 5 animals/replicate). Except for tests conducted on days 30, 65, 113 a subset of worms (n = 25) was retained for initial estimated individual dry weight determinations (see below). The test was conducted under staticrenewal conditions (weekly renewal 80% overlying water) at a temperature of 20°C and a 12 h photoperiod. Gentle aeration was provided to each beaker. Worms were fed twice weekly a combination of finely

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ground Tetramarin and alfalfa prepared in a seawater slurry using the same ration as described above for the laboratory cultures. Dissolved oxygen, salinity, temperature, and pH were monitored weekly. In addition, a 30 ml sample of overlying water was collected from each beaker, fixed with 50/xl of 1 N HC1, refrigerated and subsequently analyzed for total ammonia. Total ammonia (mg NH3-N/liter) was determined with an Orion, ammonia-specific electrode as described above. After 21 days, worms were removed from all beakers and counted. Estimated individual dry weights were determined by placing all surviving worms within a replicate on a preweighed aluminum pan, drying for 24 h at 60°C, weighing to the nearest 0.01 mg on an electrobalance, subtracting the pan weight and dividing the resultant mass by the number of animals recovered within the replicate. Growth was expressed as a rate for each sediment using the following equation G = (WTt2-WTtO/(t2-tO

(1)

where G is the growth rate (mg/day); WTt2 -- mean estimated individual dry weight of worms at test termination; WTt~ -- mean estimated individual dry weight at test initiation; and t2-t~ -- duration of exposure (21 days). In tests conducted on days 30, 65, and 113 (where initial weights were not collected) WTfi : the mean initial estimated individual dry weight of all subsequent tests. Reference toxicant tests The general viability of juvenile worms taken from cultures and used in these experiments was assessed by conducting reference toxicant tests with the heavy metal cadmium (as CdC12). Tests were conducted as described in Dillon et al. (1993). Nominal exposure

concentrations were analytically confirmed in each test with an Orion specific-ion cadmium electrode. Generally, reference toxicant tests were conducted within 1-2 weeks of each sediment storage test. Data analysis All statistical analyses and data transformations were conducted using SYSTAT, statistical software (Wilkinson, 1988). All data were screened for normality and homogeneity of variance via residual plots and Bartlett's test, respectively. Effects of storage time on sediment toxicity (survival and growth) were evaluated via ANOVA. If the F statistic was significant, mean separation was performed using a Tukey's HSD test. If significant differences were observed and there appeared to be a relationship between toxicity and storage time (i.e. increasing/decreasing toxicity with increasing storage time), regression analysis was performed. All tests for significance were analyzed at a significance level of a = 0.05.

RESULTS Sediment analysis Results of bulk sediment chemistry may be found in Moore and Dillon (1993). In general, concentrations of certain metals (e.g. cadmium, chromium, copper, lead, nickle, and zinc), butyltins, and PAHs were several times higher in OC sediments when compared to the other San Francisco Bay sediments and the Sequim control (Table 2). Concentrations of pesticides and PCBs were at or below detection limits in all of the sediments tested. Grain size analysis indicated that AMR, PRR, AER, and OI sediments were mostly sand (i.e. >50% sand) while BFR, OC, OO, and SC sediments were fine grained (i.e. mostly silt and clay) (Table 3). The lowest

Table 2. Mean (standard error) concentrations (mg/kg dry weight) of major contaminants in San Francisco Bay sediments prior to storage (n = 3)

Sediment SC AER AMR BFR PRR Ol OO OC

Cd 0.90 (0.012) 0.25 (0-021) 0.04 (0.012) 0.23 (0.007) 2.34 (0.021) 0.14 (0.006) 0-27 (0.006) 1-01 (0.006)

Cr

Cu

45-9 (0.829) 81.6 (6.047) 39.1 (4.215) 86-5 (1-192) 61-5 (1.823) 57.3 (0-674) 83-8 (1.050) 229 (4-509)

35.2 (1.793) 37-4 (4.594) 5.20 (0.656) 44.6 (0.321) 6-97 (0.033) 21.5 (0-473) 40.3 (0-291) 133 (2.848)

Pb 26-3 (1-447) 52-3 (17.20) 13.0 (0-218) 41.5 (1.300) 12-2 (0.437) 21.2 (0-426) 38.3 (0-606) 122 (16-83)

Hg ND 1.13 (0.124) ND 0.36 (0-001) ND 0.18 (0.033) 0.29 (0-038) 4-07" (0.034)

Ni

Zn

Total butyltin YPAHs

42.0 82.5 (1.137) (2-207) 69.8 76.6 (2.615) (2.041) 31.7 24-0 (0.767) (0.536) 83.9 113 (0.153) (1.000) 40.7 42.4 (0.451) (0.463) 55-0 51-6 (0.684) (0.635) 81.7 109 ( 0 - 3 8 4 ) (11.62) 142 267 (18.94) (4.359)

0.03 (0.001) 0.03 (0.001) 0-02 (0.001) 0.03 (0.002) 0.02 (0.001) 0.04 (0.003) 0.02 (0.001) 0.453 (0.053)

NS 2.41 (0.534) ND ND ND ND ND 172 (7.384)

Cd = cadmium, Cr = chromium, Cu = copper, Pb -- lead, Hg = mercury, Ni = nickel, and Zn = zinc. Total butyltin includes mono-, di-, and tributyltin YPAHs is a summation of the concentrations of naphthalene, acenaphthene, phenanthrene, acenaphthylene, fluorene, anthracene, fluoranthene, chrysene, benzo(b)fluoranthene, pyrene, benzo(a)athracene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(a,h)anthracene, indeno(1,2,3-c,d)pyrene, and benzo(g,h,i)perylene. ND = not detected. UAnalysis of OC sediment by a second analytical laboratory resulted in mean (standard error) mercury concentrations of 0.005 mg/kg dry weight (_+0-001) n -- 5.

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Effect of storage time on sediment toxicity Table 3. Initial physical-chemical analyses of sediments and sediment pore water (n = 3)

Sediment Sand (%) (SE) SC AER AMR BFR PRR OI OO OC

13 (0.0) 55 (5.0) 68 (2.5) 15 (4.0) 59 (1.4) 54 (1.4) 28 (0.0) 19 (1.4)

Silt (%) (SE) 40 (0.0) 32 (2.5) 25 (0.6) 56 (1.2) 29 (1.4) 33 (0.0) 49 (1.4) 53 (1.4)

Clay (%) (SE) 47 (0.0) 12 (2.5) 8 (2.0) 29 (3.2) 12 (1.4) 13 (1.4) 23 (1.4) 27 (2.8)

Pore water TOC (%) (SE)

TKN (%) (SE)

Salinity%o (SE)

0.7 (0.23) 0.5 (0.10) 0.5 (0.10) 0-4 (0.25) 0-5 (0-10) 0.1 (0.06) 0.8 (0.49) 0.2 (0.12)

3.5 (0.54) 0.4 (0.31) 0.02a (N.A.) 0.5 (0.12) 0.4 (0.62) 0.2 (0.52) 0.4 (0.89) 0.6 (0.92)

32 (0.0) 34 (0-0) 34 (0.0) 33 (0.0) 34 (0.0) 30 (0.0) 28 (0.0) 32 (0.0)

Total NH3-N (mg/1) (SE)

HzS (mg/1)

(SE) 133 (57.7)

13.7 (3.21) 4.7 (0.12) 5.5 (0.12) 17-2 (0.29) 22.0 (0.50) 11.2 (0.29) 28.7 (0.28) 42.2 (0.29)

<0.10

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TOC --- Total Organic Carbon, TKN = Total Kjeldahl Nitrogen, SE = standard error. aTwo of the three replicates were below detection limits. levels of organic carbon were measured in OI sediment (e.g 0.1% TOC) while the highest levels were measured in SC and OO sediments (e.g. 0.7 and 0.8% TOC, respectively) (Table 3). Total Kjeldahl nitrogen (TKN), was markedly higher in SC sediment (3.5%) relative to all other sediments tested (0.010~). 500%) (Table 3). Analysis of sediment pore water extracts (collected upon receipt) also showed marked differences between sediment types. Analysis of pore water for total ammonia resulted in a gradient in concentrations ranging from 5 mg NH3-N/liter in AER sediment pore water to 42 mg NH3-N/liter in OC sediment pore water (Table 3). High levels of hydrogen sulfide were measured in the pore water of SC sediment while it was not detected in any of the other sediments tested (Table 3).

and OI. The highest value (mean = 11.9 mg NH3-N/liter, range = 2 to 20 mg NH3-N/liter) correspond to the single instance of low survival (i.e. OC sediment on day 427). i

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With one exception, worm survival was high (80-100%) in all bioassays (Fig. 2). Substantial mortality (0% survival at day 427) was observed in the OC sediment (Fig. 2). High levels of total ammonia (mean = 11 mg NH3-N/liter; range 2.0-20.0 mg NH3-N/liter) were measured in the overlying water during this time period (Fig. 2). Growth was a more variable endpoint than survival (mean CVs of 50% and 13%, respectively). There were no statistically significant differences in growth with sediment storage time (Fig. 3). With the exception of total ammonia, water quality was acceptable (mean D O -- 6.95 mg/liter, pH -- 8-10, salinity = 30.4%0, temperature = 20. I°C) in all sediment treatments. However, total ammonia in the overlying water was elevated on several occasions. These peaks appeared to be cyclical (days 65, 427, 740) and occurred only in four sediments: SC, PRR, OO, and OC (Fig. 4). High ammonia concentrations were never observed in the other four sediments: AER, AMR, BFR,

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Fig. 2. Effect of sediment storage time on mean per cent survival of juvenile N. arenaceodentata exposed to San Francisco Bay sediments. Error bars = standard error of the mean, n -- 5. SC -- Sequim control; AER = Alcatraz environs reference", AMR = Alcatraz mound reference; BFR = Bay Farm reference; PRR = Point Reyes reference; OI -- Oakland inner; OO -- Oakland outer; OC -- Oakland contaminated.

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Reference toxicant tests Both survival and cadmium concentration were more variable in earlier reference toxicant tests but tended to stabilize after about day 200 (Fig. 5). I

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DISCUSSION In developing guidance for sediment storage there are two categories of sediments that must be considered: (i) sediments which are moderately contaminated and show little to no initial toxicity (i.e. at the time of collection) and (ii) sediments with higher levels of contamination which show significant initial toxicity. F r o m a regulatory perspective, one concern is that management decisions m a y change depending on how long the sediment was stored prior to testing. For sediments which are not initially toxic the question is: 'Has toxicity increased with storage time such that they no longer pass regulatory criteria for a given management alternative?' Conversly, for sediment that was initially toxic the question becomes: ' H a s toxicity decreased with storage time such that the sediment now passes the regulatory criteria?' This study suggests that for sediments with moderate P A H and metal contamination, storage time has little effect on the response of juvenile N. a r e n a c e o dentata.

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The eight sediments evaluated in this study showed no initial toxicity (i.e. day 30) and increasing sediment storage time had little effect on survival and growth of juvenile N. a r e n a c e o d e n t a t a . Survival was significantly reduced (100°/, mortality) in a single sediment (i.e. OC) at a single point in time (i.e. the test initiated on day 427). However, measured levels of total a m m o n i a in the overlying water of the OC sediment treatment during this test were the highest recorded for this study. Previous studies have shown reduced survival and

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Fig. 3. Effect of sediment storage time on growth (mg/day) of juvenile N. arenaceodentata exposed to San Francisco Bay sediments. Error bars = standard error of the mean, n = 5. SC = Sequim control; AER = Alcatraz environs reference; AMR = Alcatraz mound reference; BFR = Bay Farm reference; PRR = Point Reyes reference; OI-Oakland inner; OO -- Oakland outer; OC = Oakland contaminated.

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Effect o f storage time on sediment toxicity

growth in juvenile N. arenaceodentata exposed for 21 days to total ammonia concentrations >20 mg NH3-N/liter (Moore & Dillon, 1992; Dillon et al., 1993), suggesting that ammonia may have been at least partially responsible for the observed toxicity. Results of the reference toxicant tests indicate that the response of juvenile N. arenaceodentata exposed to cadmium chloride was consistent over the duration of the study. One of the more significant observations of this study was the cyclical nature of total ammonia levels in the overlying water in several of the test sediments. Peaks in mean total ammonia in the overlying water of SC, PRR, OO, OC sediment occurred on nearly an annual basis (test initiated on days 65, 427, 740). Results of physicochemical analysis indicated little in common among these four sediments (Moore & Dillon, 1993). The SC, OO, and OC sediments were fine grained (mostly silt and clay) while the PRR sediment was mostly sand (i.e. >50% sand). Per cent total organic carbon ranged from 0.2 in the OC sediment to 0.8 in the OO sediment. Total Kjeldahl nitrogen averaged 0.4-0.6% for the PRR, OO, and OC sediments but was markedly higher in the SC sediment (3-5%). Analysis of interstitial water from these sediments shortly after collection indicated high levels of total ammonia ranging from 14 mg NH3-N/liter in the SC sediment to 42 mg NH3-N/liter in the OC sediment. However, two of the sediments which did not show this cyclical pattern (BFR and OI), also had high initial pore water ammonia levels (17 mg NH3-N/liter and 11 mg NH3-N/liter, respectively). While it was not possible to identify the mechanism responsible for this cyclical phenomenon we speculate that succession in sediment-associated microbial communities during storage may have played a role. Under anaerobic conditions (conditions of storage) nitrification (the oxidation of ammonia and nitrite to nitrate) does not occur. Consequently, ammonia can only be removed from a closed anaerobic system via assimilation by microorganisms (Brock, 1970). Therefore, if there were microbial communities in these stored sediments capable of assimilating ammonia, and they followed a 'boom and bust' pattern of population growth, then one would expect ammonia levels to peak when the population of ammonia assimilating micro-organisms was small and decrease as the population grew. Once sediments are removed from storage and placed in a test beaker the high concentrations of interstitial ammonia would then diffuse to the overlying water until equilibrium is established. Based on the findings of this study it appears that storage time has the potential to significantly affect interstitial ammonia concentrations and possibly sediment toxicity. These results emphasize the importance of monitoring ammonia concentrations during sediment toxicity tests. In addition it suggests a need for research on the dynamics of microbial communities in stored sediments and the corresponding effects on the nitrogen cycle.

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ACKNOWLEDGEMENTS Financial support for this work was provided by the San Francisco District through an Intra-Army Order for Reimbursable Services. Additional funding was provided by Headquarters, USACE through the Environmental Effects of Dredging Program Long-Term Effects of Dredging Operations Research Program, Work Unit No. 374--9 Chronic Sublethal Effects. The authors gratefully acknowledge the support provided by Mr Thomas Chase, Mr Kerry Guy, Ms Sandra Lemlich, Mr Duke Roberts, Mr Thomas Wakeman, and Mr Brian Walls of the San Francisco District.

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