Effects of natural sediment features on survival of the phoxocephalid amphipod, Rhepoxynius abronius

Marine Environmental Research 25 (1988) 99-124

Effects of Natural Sediment Features on Survival of the Phoxocephalid Amphipod, Rhepoxynius abronius

Theodore H. DeWitt Oregon State University, Mark O. Hatfield Marine Science Center,

Newport, OR 97365, USA

George R. Ditsworth & Richard C. Swartz US Environmental Protection Agency, Mark O. Hatfield Marine Science Center, Newport, OR 97365, USA (Received 31 August 1987; revised version received 16 October 1987; accepted 3 February 1988)

ABSTRACT EffEcts of sediment particle size and water content on the survival of the amphipod, Rhepoxynius abronius, were examined by manipulating these natural sediment features within static laboratort, microcosms. Mean amphipod survival infine, uncontaminated, field sediments ( >_80% sih-clay ) can be 15%o lower than survival in native sediment. Storage of sediments at 4~C over 7-14 days did not change sediment toxiciO,, but handling (i.e. elutriation and recombination) of muddy sediments increased toxicity. Sediment particle size and organic content had greater impact on the survival of R. abronius than did sediment water content in modifying amphipod survival, but we could not independently separate the eff'ects of these two sediment variables. A new set of criteria is proposed to interpret toxici O, results from the amphipod bioassay in the light of the mortali O, associated with fine sediment particle size. The efficacy of these criteria to separate mortali O, caused byfine particles and chemical contaminants was tested by analyzing field surw,y data J?om 78 Puget Sound ( WA ) Urban sites. Using our new criteria, the toxicity of these sediments was .found to closely reflect the degree of chemical contamination. We propose that an approach similar to this be undertaken for 99 Marine Environ. Res. 0141-1 ! 36/88/$03"50 ~:) 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain

100

Theodore H. De Witt, George R. Ditsworth, Richard C. Swartz toxicity tests whenever natural environmental factors induce mortality above background levels.

INTRODUCTION The physiology and behavior of many benthic invertebrates are highly dependent on subtle differences in the physical and chemical composition of marine sediments. The particle-size distribution, organic content, and topographic structure of sediments, for example, affect amphipod life history parameters (DeWitt, 1985) and habitat choices (Meadows & Reid, 1966; Sameoto, 1969; Oakden, 1984; DeWitt, 1987). Understanding how benthic organisms respond to different sedimentary environments is important in the interpretation of the responses of benthic populations and communities to environmental stresses, including pollution. The phoxocephalid amphipod, Rhepoxynius abronius, has been used extensively on the west coast of the USA as a bioassay organism to test the toxicity of contaminated sediments (Swartz et al., 1979, 1982, 1985a,b, 1986a,b; Oakden et al., 1984a,b; Battelle, 1985; Tetra Tech, 1985; Kemp et al., 1986; Ott, 1986). In its native habitat, R. abronius burrows freely within fine, well-sorted sands (Oakden, 1984; Kemp et al., 1985; Slattery, 1985), and shows strong preference for such sediments over finer and coarser sediments (Oakden, 1984). Although this amphipod has been used frequently to assay the toxicity of a wide variety of marine sediments, little is known about its sensitivity to natural environmental variables associated with these sediments. Some very fine, apparently uncontaminated sediments have been found to be toxic to Rhepoxynius (Battelle, 1985; Tetra Tech, 1985; Ott, 1986). The cause(s) of mortality in these sediments is unknown, but may be associated with the small sediment particle size (especially in the silt-clay size range), high sediment water content, or high sediment organic content that characterize fine sediments. These sediment properties are highly intercorrelated, and it is very difficult to distinguish among them for the cause of mortality using field survey data alone. The purpose of our study was to determine experimentally which of these sediment variables had the greatest impact on the survival of Rhepoxynius abronius. Subsequently, we examined whether mortality due to these natural source(s) could be distinguished from the effects of chemical contaminants. The latter was accomplished by analyzing field survey data using the results of our experiments. A general procedure for interpreting toxicity data for field-collected sediments with various particle-size distributions was formulated from the results of this analysis.

Amphipod sensitivity to natural sediments

101

MATERIALS AND METHODS

Manipulation of sediment properties The effects of variation in the particle-size distributions and water contents of fine sediments collected from Dabob Bay, Puget Sound, Washington, on Rhepoxynius abronius were examined in replicated, static 10-day sediment bioassays (Swartz et al., 1985a). Sediments from these and other sites within Dabob Bay were known to induce mortality in R. abronius although the bay is in a non-urban region of Puget Sound and has negligible concentrations of contaminants (Battelle, 1985; pers. obsv.). We predicted that the toxicity of these sediments would vary with particle size or water content.

Sediment collection and general procedures At each of four stations in Dabob Bay (Fig. 1; station numbers correspond to those of Battelle, 1985), 25-30 liters of sediment were collected as a composite of five 0.06m z van Veen grabs. Sediment, collected on 25-26 March 1986, was stored on ice and transported to our Newport, OR,

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Fig. 1. Sediment toxicity sampling sites in the Pacific Northwest, Puget Sound (WA), and Dabob Bay. From south to north, Reference sediments were collected from two Oregon sites (Yaquina Bay and Netarts Bay) and six Puget Sound sites (Carr Inlet, Case Inlet, Dabob Bay, Sequim Bay, West Beach, and Samish Bay). From south to north, Urban sediments were collected from five Puget Sound sites (Commencement Bay, Sinclair Inlet, Elliott Bay, Everett Harbor, and Bellingham Bay).

102

Theodore H. De Witt, George R. Ditsworth, Richard C. Swartz

laboratory on 26 March where it was held at 4°C prior to manipulation. These manipulations are detailed in the next section. The toxicity of manipulated sediments was assessed using the standard sediment bioassay procedure of Swartz et al. (1985a). Briefly, 20 recently collected Rhepoxynius abronius were maintained for 10 days in a 2-cm deep layer of experimental sediment in a l-liter glass beaker filled to 1 liter with filtered ( < 1/~m) 28%o seawater (salinity adjusted with distilled water) under constant light at 15°C. At the end of 10 days, the amphipods were separated from the substrate by wet sieving through a 0-5-mm screen, decanted into counting dishes, and the number and reburial ability of survivors were noted. Rhepoxynius abronius were collected from Yaquina Bay, OR, 2-3 days prior to starting each experiment and held in flowing seawater in native sediment at ambient bay-mouth temperature and salinity; additional native sediment to be used as a control was collected and held at 4°C. Samples of each manipulated sediment were collected 1 day prior to the addition of amphipods and stored at 4°C until analyzed. Particle-size distributions were determined using the sieve/pipette method (Buchanan, 1984); two to three measurements of % Fines (i.e. % Silt + % Clay) and % Sand were made for all samples. Triplicate estimates of the % H 2 0 content were derived from the per cent of wet weight lost by drying sediment overnight at 90°C. Sediment organic content, also measured in triplicate, was estimated as the per cent weight loss on ignition of dry sediment at 550°C for 1 h. Organic content is reported here as per cent total volatile solids (% TVS) of dry sediment. Oxidation-reduction potentials were measured on all sediments on the 1st, 5th, and 10th days of each bioassay using a 0.7-mm diameter platinum wire Eh probe. Eh readings ranged from 178 to 334 mV indicating aerobic conditions throughout all treatments in each experiment.

Sediment particle-size manipulation We endeavored to synthesize four classes of substrate of various particlesize distributions from each Dabob Bay sediment (i,e. stations [-St.] 2, 14, 15 and 18). The substrate treatments were 'Original' (i.e. unmanipulated sediment), 'Coarse', 'Fine', and 'Recombined' (i.e. Coarse and Fine fractions combined on a dry weight basis in the same proportions found in the Original substrate). A cut-off of approximately 16/~m between the Coarse and Fine substrates was chosen since this was the median size class on a per wt. basis of the sediments from St. 2, 14 and 15. The Recombined substrate was included as a control for possible effects due to handling of sediments. The field sediments were separated into relatively 'Coarse' and 'Fine' fractions by repeated sediment-water mixing and elutriation. Elutriation is

Amphipod sensitivity to natural sediments

103

less destructive of natural sediment aggregates (Johnson, 1974) and more time efficient than wet sieving for the mass of sediment we needed. Furthermore, we wished to avoid any possible contamination of the experimental substrates from abraded metals or metal salts derived from sieve screens. Ten kilograms of the well-mixed sediment from each station were slurried with seawater and quickly poured into a plastic barrel containing 50 liters of seawater. This suspension was mixed thoroughly, backstirred to stop water motion, and then allowed to settle for 10 min. The overlying water was then siphoned into a second barrel at 2.41iters/min, which we predicted would leave coarser particles ( > 16 ~m) in the first barrel and transfer finer sediments to the second barrel. The contents of the second barrel were elutriated in a similar manner, and the fine sediments were collected in a third container. Sediment remaining in the second barrel was combined with that remaining in the first barrel, 50 liters of seawater were added, and the fine particulates were again elutriated. The final contents of the first barrel were defined as the 'Coarse' fraction, and sediment in the second and third containers were combined and defined as the 'Fine' fraction. All sediments were allowed to settle and dewater for 2 days, after which the nearly clear supernatant was siphoned off and discarded. The water contents of the Coarse, Fine, and Recombined substrate treatments from each station were adjusted to that of the Original fraction through centrifugation and mixing. Bioassays of the Original sediments from each Dabob Bay station began 1-2 days after collection. Experiments with the Fine, Coarse and Recombined substrates began 8 days after collection. Each substrate treatment was replicated five times, except for St. 18 sediments for which we had only enough material to prepare three Coarse replicates. No Recombined substrate could be prepared for St. 18.

Sediment water-content manipulation This experiment was implemented to determine whether water content, independent of other sediment properties, affected the survival of Rhepoxynius abronius. Four water-content treatments (80%, 72%, 64% and 57% of wet weight) were created for Recombined sediments from St. 2, 14 and 15 (henceforth referred to as St. 2-R, 14-R and 15-R, respectively) and for unaltered sediment (e.g. Original) from St. 2 (referred to as St. 2-0). The treatments spanned the range of water content for fine sediments we collected in the field (unpubl. data). The water content of the freshly collected sediments from these stations averaged 69.8% (_+ 2.44 SD). Water content was adjusted by a combination of centrifugation (0"3-1 centrifuge tubes at 5000 rpm for 5 min) and addition of filtered, 28%o-salinity seawater. This

104

Theodore H. De Witt, George R. Ditsworth, Richard C. Swartz

experiment began when the amphipods were added to the beakers, 16 days after field collection of sediments. Controls

A control treatment consisting of native sediment (ca. 97% sand, 3% fines) from one Rhepoxynius abronius habitat in Yaquina Bay, OR, was included in all experiments. Prior to use, native sediment was sieved (to remove amphipods) through a 0.5-mm screen into 28%0 salinity seawater. Otherwise, the sediment properties of the native sediment were not manipulated; the control only served to measure the ability of the amphipods collected for each experiment to survive under laboratory conditions. Control survival ranged from 98% to 100% across all experiments. Only 3 of 280 amphipods that were exposed to native sediment died. Some of the field-collected sediments were held up to 2 weeks prior to use and others were extensively altered during this time. Both storage and handling may contribute to sediment toxicity. The effects of sediment storage were examined by comparing changes in amphipod survival in (A) the Recombined particle-size substrates and the 72% water-content substrates for sediments from St. 2, 14 and 15, and (B) St. 2 Original particlesize substrate and St. 2-0 72% water-content substrate. Sediments were stored 7 and 14 days in sets A and B, respectively. The effects of handling on sediment toxicity were examined separately in both experiments. In the particle-size experiment, amphipod survival was compared between Original and Recombined substrates derived from sediments from each Dabob Bay station. In the water-content experiment, treatments were arranged from greatest to least amount of handling (i.e. 72% < 80% < 64% < 57%), assuming that the addition of water (plus mixing) in the 72% and 80% treatments involved less handling than removing water by centrifugation (plus mixing and the addition of some water) in the 64% and 57% treatments.

Analysis of field surveys of sediment toxicity A database of sediment toxicity, geological properties, and chemical contaminants was compiled from field surveys performed in the US Pacific Northwest by our laboratory, Battelle Pacific Northwest Laboratory (Battelle, 1985) and Tetra Tech., Inc. (Tetra Tech., 1985). Surveys were performed in thirteen embayments, inlets and harbors along the Oregon coast and in Puget Sound, WA (Fig. 1). The database was organized into Reference (8 bays) and Urban (5 bays) subsets depending on the proximity of the sampling location to urban or industrial centers. Reference sites were assumed to be less contaminated.

Amphipod sensitivity to natural sediments

105

Only those variables common among the surveys were included in the database. All analytical laboratories were included in an inter-laboratory calibration of the Rhepoxynius abronius sediment-toxicity test (Mearns et al., 1986). Analyses of sediment properties and contaminant chemistry were comparable across field surveys and are described elsewhere (Battelle, 1985; Tetra Tech., 1985). By category, the variables used in this analysis were: (1) sediment toxicity (R. abronius survival after 10 days' exposure to sediment), (2) sediment variables (% Sand, % Silt, % Clay, % Fines, % U20 and % TVS), and (3) chemical contaminants (polycyclic aromatic hydrocarbons [-PAH], polychlorinated biphenyls [PCB], total oil and grease [TOG], Ag, Hg, Pb, As, Cu and Zn). Sediments were assayed from 127 Reference sampling sites and 170 Urban sites. Material from the top 2cm of sediment samples only was retained for analysis. Sediment properties and toxicity were measured in every sample; single measurements were taken for sediment properties, while 1-5 replicate observations of amphipod mortality were made on each sample. Contaminant concentrations were measured at 22 Reference and 109 Urban sites. Only one measurement per sample was made for each chemical variable. Total oil and grease was measured at only 6 Reference and 29 Urban sites. The relationships between amphipod survival and sediment properties within the Reference and Urban databases were examined via product moment correlation. Together with the results of our laboratory experiments, we used the correlations from the Reference database to select the sediment variable that was most strongly associated with Rhepoxynius abronius mortality in the absence of chemical contaminants. This relationship was further analyzed by linear regression. Prediction limits for this regression and previous bioassay results were then used to divide sediments from Urban sites into three toxicity categories: those sites at which amphipod mortality was less than, or equal to, amphipod mortality in native sediments, those sites at which amphipod mortality could be due to sediment properties (such as particle size) alone, and those for which mortality could be due to sediment effects plus an unmeasured factor, presumably contaminants. Discriminant analysis was used to test the concordance in classification of stations by sediment contaminants and these toxicity categories. Nine sediment contaminants were used in this analysis (total organic carbon [TOC], PAH, PCB, Ag, Hg, Pb, As, Cu and Zn). The contaminant data were generally highly skewed among toxicity categories; therefore, each variable was normalized by transformation. For each contaminant, the transformation functions were: sin-l(TOC), Px/-PAH, Loglo(PCB), (x/-~-)- l s (xflH-g) ', Loglo(Pb ), A s - ' , Loglo(Cu ), Loglo(Zn ). These normalizing

106

Theodore H. De Witt, George R. Ditsworth, Richard C. Swartz

transformation functions were selected using the Box-Cox procedure (Sokal & Rohlf, 1981).

RESULTS

Manipulation of sediment properties Particle-size experiment Differences between Coarse and Fine substrates were large only in sediments from St. 15 and 18. The texture of the Coarse and Fine substrates from St. 2 and 14 differed by < 5% Fines, which was slight (although significant) by comparison with the > 50% difference in % Fines between Coarse and Fine substrates from St. 15 and 18 (Tables 1 and 2). Sediment water and TVS were higher in the finer substrates derived from St. 15 (e.g. Fine and Recombined) and 18 (e.g. Fine), but not different among substrate treatments from St. 2 and 14 (Tables 1 and 2). Therefore, we analyzed the amphipod survival data station by station. The particle-size distributions of all experimental substrates were comparable to silty-sand (i.e. St. 18 Original; St. 15 and 18 Coarse) and silt-clay (i.e. the remaining experimental substrates) sediments found in Puget Sound (Battelle, 1985) and Oregon estuaries (G. Ditsworth, unpubl, data). The median grain-size for all Fine and Recombined substrates was <16/~m (Table 1). Rhepoxynius abronius mortality was significantly lower in the Coarse and Original treatments than in the Fine or Recombined treatments for St. 15 sediment (Table 2). Mortality in St. 18 substrates was also lower in the Coarse and Original substrates than in the Fine substrate (no Recombined substrate was available for testing). Mortality was not significantly different between the Coarse and Original substrates derived from either St. 15 or 18, nor was mortality significantly different between the Fine and Recombined substrates from St. 15. For these two stations, lowest mortality was observed in substrates with lowest % Fines, % TVS, and % HzO. Amphipod survival was greatest in the Original substrates and lowest in the Fine substrates derived from sediments from St. 2 and 14 (Table 1). However, survival was not significantly different among the Fine, Coarse, and Recombined treatments for either station (Table 2), and there was little or no difference in particle size, water content, or sediment organic content among these substrates for St. 2 and 14. The cause of decreased survival between the Original and the other substrates for sediments from St. 2 and 14 is unclear, but it may be an artifact of handling the sediment. Combining the sediment data from all stations and substrates, survival was significantly negatively correlated with the fineness of the sediment (%

Amphipod sensitivity to natural sediments

107

TABLE 1 Results of Particle-size Manipulation Experiment: Means and Standard Deviations of Amphipod Survival and Sediment Variables. Graphical Mean and SD for Grain Size are given in q5 units ( ~ - l o g 2 m m ; 6 . 0 = 15.6/~m). N = 5 for Survival (except St. 18 Coarse; N = 3); N >_ 3 for Sediment Variables. The St. 18-Recombined Substrate was not availablc due to Lack of Sediment

Particle-size manipulation substrates Original

St. 2

St. 14

St. 15

St. 18

N u m b e r of survivors Grain size %, Sand % Fines % H20 % TVS N u m b e r of survivors Grain size % Sand % Fines % H20 % TVS Number of survivors Grain size % Sand % Fines % H20 % TVS Number of survivors Grain size % Sand % Fines % H20 % TVS

Coarse

Fine

Reeombined

Mean

SD

Mean

SD

Mean

SD

Mean

SD

12"6 7'5 1.0 99.0 70.1 8"1

3"2 1-4 0.1 0.1 1.1 0"8

104 72 5-2 94.8 70.9 7-8

3"1 1'7 2.6 2-6 0.7 0-2

6"2 7'5 0.4 99.6 71-2 80

1-8 1"5 0.1 0.1 0.9 3"2

8-4 7-5 0-9 99.1 71.3 7"8

4.4 1.4 0.2 02 0.7 0"6

17"0 74 0'6 99.4 68.1 10.1

0"7 1"3 0.2 0.2 2.2 0.6

7-6 7'3 2-0 98.0 71.0 9.3

3"7 1-5 1.1 1.1 0.4 0.2

6"4 7-3 0.2 99.8 70.0 9-6

2"3 1.4 0.1 0.1 0.7 0.2

78 7.4 1-0 99.0 70.2 9.3

3"7 1-4 1.0 1.0 4-3 0.2

14"0 6"4 20.4 79"6 64"6 9"5

1-6 2"6 5"2 5"2 0"6 0'5

17"4 3-2 56'5 42'5 63"8 8"5

1'5 3"6 0"6 0"6 1-6 0"5

7'4 7"4 0"1 99"9 67"7 10'4

4-5 14 0'2 0"2 0"1 0"l

8"6 6-7 11'4 88"6 65"3 9"6

32 2"5 2'7 2.7 1'6 0"4

18"8 3'3 61.9 38-1 28.0 1-8

0"8 1"6 1.1 1.1 0-1 0.1

19"4 3-3 65.2 34.8 33.2 1.9

0-6 1'6 3.3 3.3 0.2 0.1

10"3 7-2 5.9 94.1 67-9 7.7

1"5 1.7 0.2 0-2 2.3 0.1

108

Theodore H. De Witt, George R. Ditsworth, Richard C. Swartz TABLE 2

Station by Station 1-way ANOVAs of the Effect of Particle Size on Amphipod Survival and Sediment Properties. Means Column Presents Results ofa posteriori Multiple Comparisons among Substrate Means (Tukey-Kramer Test: Sokal & Rohlf, 1981) with Substrate Treatments Arrayed in Decreasing Magnitude from Left to Right. Substrates not Statistically Different are Underlined, O = Original, C = Coarse, F = Fine, R = Recombined Substrates Dependent variable

Station

Treatment sum of squares

d[

F

P

Means

Number dead

2 14 15 18

112.40 361.00 328.95 178.26

3,16 3,16 3,16 2,10

3.56 14.67 12-39 102.84

0.038 1 0.0001 0"0002 0.0001

F R C> R C O EC R>O FR> OC F>OC

% Fines

2 14 15 18

47"53 5"43 5501"68 6 986"36

3,9 3,8 3,8 2,8

10.64 3-26 218"62 135"29

0.0026 0-0805 0"0001 0.000 1

F RO>C FORC F>R>O>C F>OC

%TVS

2 14 15 18

0-49 1"16 5.82 68.21

3,11 3,8 3,8 2,6

0'47 3"95 11"31 6090"34

0.7111 0-0534 0"0030 0"0001

_OFCR OF RC FRO>OC F> C O

% H20

2 14 15 18

4.29 13"75 25"87 2825'98

3,11 3,8 3,8 2,6

1'59 0"78 6'53 808-15

0.2472 0"5386 0"0152 0'000 1

RFCO C RFO FR>ROC F>C>O

Fines [ R = - 0 . 8 1 ] , % C l a y [ R = - 0 - 7 8 ] , % Silt [ R = - 0 " 6 8 ] ; p < 0.005 for all), sediment w a t e r c o n t e n t (R = - 0.72, p < 0"002), a n d % T V S (R = - 0.62,

p _< 0-015). Water-content experiment Results o f the w a t e r - c o n t e n t m a n i p u l a t i o n e x p e r i m e n t are s u m m a r i z e d in T a b l e 3. Survival decreased slightly as the sediment w a t e r c o n t e n t increased f r o m 5 7 % to 72%, a n d increased slightly f r o m 7 2 % to 8 0 % water. The n u m b e r o f survivors was n o t significantly affected by experimental variation o f sediment w a t e r content, except between the 8 0 % a n d 7 2 % w a t e r - c o n t e n t t r e a t m e n t s (Table 4). Since the 6 4 % water t r e a t m e n t for St. 2 - 0 was n o t included (due to lack o f sediment), two A N O V A s were run to separately include each o f the missing factors. Results o f the two analyses were a l m o s t identical (Table 4 A a n d B). D u r i n g the 10-day experiment, the sediments in each test b e a k e r settled, c o m p a c t e d , a n d d e w a t e r e d such that the water c o n t e n t o f all substrates was

Amphipod sensitivity to natural sediments

109

TABLE 3 Water-content Experiment: Means and Standard Deviations of A m p h i p o d Survival and Sediment Variables. N = 5 for Survival, N = 3 for H 2 0 and TVS, a n d N = 2 for Fines and Sand. The St. 2-0, 6 4 % H 2 0 Treatment was not R u n due to Lack of Sediment Sediment

Water-content treatments (Initial water content} 57%

N u m b e r of survivors % Sand % Fines Final % H20 % TVS St. 2-O N u m b e r of survivors % Sand "/o Fines Final % H20 % TVS St. 14-R Survival % Sand % Fines Final % H 2 0 % TVS St. 15-R Survival % Sand % Fines Final % H 2 0 % TVS

64%

72%

80%

Mean

SD

Mean

SD

Mean

SD

Mean

SD

138 0.7 99.3 56-7 81

3"3 0.2 0.2 0"1 0"1

14-0 0"8 99.3 59"1 8"0

2"9 0-2 0.2 0"0 0"0

9"5 0.7 99"3 70"0 8'0

3"1 0.2 0-2 0-1 0.2

17"0 0.6 99.4 73'2 8"6

1-4 0.0 0.0 0-1 0-2

15"5 1-0 99'0 54'7 8"0 15-0 0-4 99"6 57.5 9.9 15"0 6-5 935 562 9.3

1'0 0.3 0.3 0.2 0-1 1"4 0.2 0.2 0-1 0.1 2'6 0'7 0.7 0.1 0.1

3'3 0-1 0"1 0"! 0.l 4"0 0-5 0.5 0.4 0.1

12"5 0-5 99.5 69"3 8"5 11'5 0.4 99.6 68-8 10.4 11"3 5"6 94.4 66.2 9.8

3"1 0.5 0'5 0"1 0"I 6.7 0-2 0'2 0.1 0.4 3"5 0"3 0.3 0.4 0-1

11"5 0.8 99.3 73-8 9"0 15"5 0.6 99.4 72.7 10.7 17'3 9"1 90.9 72.5 10.2

5-5 0-1 0-1 0"1 0'2 57 0-2 0"2 0.1 0.2 2.4 3'8 3.8 0.1 02

St. 2-R

12-0 0.4 99.6 62.9 10.0 13.5 5-5 94.5 61-9 9.6

generally lower at the end of the experiment than at the start (Table 3). Nevertheless, the final water content still spanned a range of ca. 15% for each substrate tested (Table 3). Although not measured, the viscosity of the sediments from these water-content treatments ranged from stiff (at 57% water) to nearly flocculent (at 80% water). We were surprised to find significant variation in organic content among water-content-treatment substrates for each station, since each manipulated substrate was derived from a single sediment from a given station. All substrates were very fine-grained ( > 90% Fines), although those from St. 15-R were significantly coarser (mean = 93% Fines) than those from the other stations (all > 9 9 % Fines). There was no significant variation in % Fines among water-content treatments for any station. However, % TVS

110

Theodore H. De Witt, George R. Ditsworth, Richard C. Swartz

TABLE 4 Two-way ANOVAs of RhepoxyniusabroniusSurvival Across Water-content and Sediment Source (Station) Treatments. Because Experimental Design was Unbalanced, each A N O V A has One Treatment Removed. A. St. 2 - 0 was Removed. B. The 64% Water-content Treatment was Removed. Means Column is the Same as in Table 2 with Water-content Treatments Arrayed in Decreasing Magnitude from Left to Right. See Table 3 for Values of Treatment Means Source A. Station 2-0 Removed Water-content

Station Interaction Error Source

d/

MS

F

P

Means

3

68"51

5"06

0"005 l

80% 57% 64% > 57% 64% 72%

2 6 35

4.48 4.00 13"55

0.33 0"30

0'8155 0"935 2

df

MS

F

P

Means

5.37 0.44 1.15

0.009 3 0-725 7 0.3545

80% 57% > 72%

B. 64% Water Treatment Removed Water-content 2 74.88 Station 3 6.14 Interaction 6 16'05 Error 35

increased slightly but significantly as the water content of each substrate was raised (Table 3). This may have been due to loss of dissolved organic matter during centrifugation to create sediments with low water content. Combining data for all substrates used in this experiment, the mean number of survivors was not correlated with the mean of any sediment variable. Correlation was greatest with % Fines (R = -0"29; p = 0.288), intermediate with final water content (R = -0"11; p = 0"707), and lowest for % TVS (R = - 0 - 0 9 ; p = 0.744).

Storage and handling effects There were no differences in amphipod survival in sediments stored 7 days (Recombined substrates versus 72% water treatments for sediments from St. 2, 14 and 15; F~1,2 ) = 0"159, p > 0.05) or 14 days (Original St. 2 substrate versus St. 2 - 0 72% water treatment; t = 0.050,p > 0-05). Survival was higher in all Original substrates than in Recombined substrates (Tables 1 and 2), although the two substrate treatments differed in % Fines at St. 15 (Table 2). The Original and Recombined treatments for St. 2 and 14 differed in two independent ways: Recombined was held 7 days longer than Original prior to being assayed, and Recombined was manufactured from elutriated Fine and Coarse substrates. If storage did not increase mortality, then increased mortality was caused by the elutriation and mixing process. By contrast,

Amphipod sensitivi O, to natural sediments

! 11

increasing or decreasing the water content of four substrates did not significantly change the toxicity of the sediments (Tables 3 and 4A).

Analysis of field survey data Sediment particle size and water content were not significantly different between Reference and Urban sediments, but % TVS was higher on average in Urban sediments (t = 4.48, df = 295, p _< 0.001) (Table 5). As one might expect, contaminant concentrations were greater in Urban sediments, often by an order of magnitude or more. Thus, with respect to all measured sediment variables, Reference and Urban sediments were very similar, with the exceptions of total volatile solids and contaminant concentrations. Survival of Rhepoxynius abronius was negatively correlated with all sediment variables in Reference sediments, and all sediment variables were highly intercorrelated (Table 5). No sediment property stood out as being the best predictor of amphipod survival, so based on our experimental results, we chose sediment particle size (e.g. % Fines) as a predictor of R. abronius survival. This relationship can be summarized by the linear regression of amphipod survival on the % Fines fraction of unpolluted (i.e. Reference sediments: N u m b e r of survivors = 19"13-(0.05 x % Fines)

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which is highly significant (p < 0-001) (Fig. 2). The same relationship is obtained using the arcsin-square root transformations for both variables. Despite this negative trend, R. abronius survival was > 80% in 44% of the tests with very fine Reference sediments (i.e. % Fines > 80%) (Fig. 2), which accounts for the relatively low R 2 value. Ninety-five per cent prediction limits (95%PL) around this regression line may be calculated as follows: 9 5 % P L = P S ++_tto.os.N- 2)* x / M S E * (1/M + 1/N + ((X i - X ) 2 ) / S S x )

(2j

where P S is the predicted number of survivors for a given % Fines value as calculated in eqn (1); N is the number of sample points in the original regression (N = 315 for eqn ( 1)); tto.o 5. N- 2) is the t-statistic value for ~ = 0"05 and N-2 degrees of freedom (t = 2.33 for N = 315); M S E is the Mean Square Error from the regression ( M S E = 8.30 for eqn (1)); M is the sample size for which prediction is being made; X is the % Fines about which prediction is being made; X is the mean % Fines (X = 57"39 for eqn (1)) and S S x is the uncorrected sum-of-squares for % Fines from the regression ( S S x = 401 102.01 for eqn (1)) (Sokal & Rohlf, 1981). Applying these values to eqn (2), the equation for the 95% prediction limits simplifies to: 9 5 % P L = P S + 2"33 • ,,/(0-026 + 8"3/M + (0"00002 • (% Fines - 57"4)2)) {3)

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The high value for S S x causes the hyperbolic prediction limits to closely approximate parallel lines above and below the regression line within the range of 0-100% Fines. Amphipod survival was negatively correlated with most contaminants measured in Reference sediments (Table 5); P A H and T O G were exceptions. In Urban sediments, amphipod survival was poorly correlated with any sediment variable except TVS and TOC. No significant relationship between survival and particle size was found for Urban sediments: N u m b e r of survivors = 15"01 - (0"0001 • % Fines)

R 2 = 0.005 (4)

Amphipod sensitivity to natural sediments

115

(p = 0-897) (Fig. 2). Survival of R. abronius in very fine sediments (i.e. % Fines > 90%) from Urban sites (many of which are not highly contaminated) was not different from Reference sites (t=0.45; p > 0"05). However, in sandy sediments (i.e. % Fines < 40%), amphipod survival was much lower at Urban sites (t = 6"81;p < 0-001). This may reflect the greater relative bioavailability of toxic materials to R. abronius in coarser, less organically enriched sediment (Swartz et al., 1986b).

DISCUSSION

Natural sediment factors affecting survival of Rhepoxynius abronius Rhepoxynius abronius naturally occurs in well-sorted, fine sands, and prefers this substrate over muddier sediments (Oakden, 1984). Survival of R. abronius was most strongly affected by sediment particle size: as the % Fines (i.e. the silt-clay fraction) content of a sediment increased, amphipod survival decreased. This was apparent in sediments naturally varying in particle size and in substrates of artifically manipulated particle size. However, particle size alone cannot account for all of the background mortality in uncontaminated sediments, as evidenced by the high survival of R. abronius in some of the finest-grained Reference sediments (Fig. 21. Although contaminant species concentrations were significantly positively correlated with R. abronius survival in Reference sediments (Table 5), the absolute concentrations of these potential toxicants were within average crustal levels for marine sediments (i.e. metals; Weast, 1981) or similar to background levels previously reported from Puget Sound (Dexter et al., 1981). Thus, we do not believe that these compounds are responsible for R. abronius mortality in unpolluted sediments. Rhepoxynius abronius survival was higher in coarse sediments (e.g. the Coarse substrates from St. 15 and 18, and field collected sediments from the Pacific Northwest) than fine sediments. Alteration of sediment water content did not produce significant changes in survival across the range of sediment moisture examined. Survival was more strongly correlated with % Fines than with % H20. Sediment particle size is probably just a 'super-variable' that is correlated with the actual cause of mortality. Surface-sediment mineral grains are embedded within organic-mineral aggregates (DMA) with particulate organic matter, mucus, and sediment microflora (Johnson, 1974). In sands, most organic matter adheres to the large mineral grains, and there is a distinct particulate texture to the substrate. However, the organic content of muds is typically much higher than sands, and the organic matrix imparts a loosely cohesive, 'cotton-candy' texture to the substrate. Presently, there is a

116

Theodore H. De Witt, George R. Ditsworth, Richard C. Swartz

poor understanding of the micro-scale structure of fine marine sediments and how infaunal organisms perceive sediment particulates and sediment texture. We do not know what specific sediment properties are relevant to the health and wellbeing of benthic animals. This probably precludes us from measuring the appropriate sediment properties, and we are left with using crude characterizations of sediments, such as particle size, water content, organic content, and various shear properties. Nonetheless, our experiments indicate that the cause of death is probably associated with the particles and not the interstitial water. Correlation between amphipod survival and % TVS of Reference and laboratorymanipulated sediments was consistently the lowest of all correlations between survival and sediment variables. Although this does not preclude the importance of the sediment organic content in determining R. abronius survival, at this time it appears that sediment particle size is the best single predictor of amphipod survival in uncontaminated sediments. The mechanism(s) responsible for Rhepoxynius abronius mortality in fine sediments is not apparent. Very fine particles may adhere to the gills and inhibit respiration, or clog the amphipod's feeding appendages. Rhepo.~vnius abronius probably does not starve while in muddy sediments, as it can survive the same duration in azoic foundry sands with high success (Ott, 1986). All sediments remained aerobic throughout the experiments, so anoxia or H2S toxicity were unlikely to have been causes of mortality. Manipulation of the sediments apparently increased the toxicity of very fine sediments, as seen in differences in survival between the Original and Recombined particle-size treatments. Storage of experimental substrates over a 2-week period apparently did not affect Rhepoxynius abronius survival. During elutriation, mixing, or sieving, the organic matrix of fine sediment may become disassociated and the mineral grains freed within interstitial spaces (Ott, 1986). If fine mineral particles increase amphipod mortality, as Ott (1986) suggests, then their disassociation from organicmineral aggregates may increase their deleterious effects. Manipulation of sediments by elutriation may have enhanced such disassociation in our experimental substrates which could account for the handling-associated mortality we observed. It is conceivable that natural conditions, such as scouring, which cause sediment to be transported into the water column may have an effect on sediment particulates similar to that induced by elutriation and recombination in these experiments. We have no data to test these hypotheses. As the handling effects were unexpected and not a primary focus of this project, we have not examined this phenomenon in sufficient detail to state whether or not sediment manipulation will generally increase sediment toxicity. But, we view all forms of sediment manipulation, including elutriation, sieving, and mixing, as possible sources of toxic artifacts.

Amphipod sensitivity to natural sediments

I 17

Sediment compaction, which may hinder burrowing, was not likely to have contributed to Rhepoxynius abronius mortality since very fine sediments typically have high water content and high fluidity (Table 1) and are thus easy to penetrate. Furthermore, artificial compaction of fine sediments in the water-content experiment did not affect survival, even between extreme treatments (Tables 3 and 4). The 57% water-contenttreatment substrates were very stiff (similar to modelling clay) and the amphipods dug short, open burrows into the mud, while the 80% watercontent-treatment substrate was quite fluid and the amphipods easily burrowed into the mud, leaving no burrow opening (R. abronius is typically free-burrowing, leaving no burrow opening to the sediment surface). Since the amphipods had approximately equal success at surviving in substrates of these extremes of compaction it is unlikely that sediment water content directly influences R. abronius mortality. The organic content and particle size of Reference sediments are strongly negatively correlated (Table 5). It is difficult to identify their independent effects on Rhepoxynius abronius survival. Ott (1986) found that mortality of R. abronius was higher for sediments composed of silt-sized particles when the organic content was low. In our experiments, amphipod survival was more strongly correlated with both particle size and % H20 than with % TVS. Among the Reference sediments, % Fines was slightly more highly correlated with Survival than was TVS, although not significantly more so. Of these three bulk sediment properties (particle size, organic content, and water content), sediment particle-size and water content were the best determinants of R. abronius surivival. We suspect that sediment particle size affects survival relatively more than sediment organic content, but the technical difficulties of independently changing sediment particle size and organic content without altering other sediment factors hinder the definitive empirical resolution of this problem.

Regulatory implications Although causal mechanisms are uncertain, our laboratory research and analysis of field survey data strongly indicate that natural sediment properties associated with fine particle-size classes sometimes cause mortality of Rhepoxynius abronius during 10-day bioassays. In the original development of the R. abronius bioassay method, Swartz et al. (1985a) recommended a sediment texture control for sediment with a high silt or clay fraction. The present results support that recommendation and offer a method to calibrate expected survival in relation to the per cent fines (i.e. silts plus clays) of test sediment samples. Equation (3) may be used to determine whether amphipod mortality

118

Theodore H. De Witt, George R. Ditsworth, Richard C. Swartz

observed in a test sediment is significantly different from mortality associated with fine sediments. If the mean number of survivors observed for a test sediment is less than the lower 95% prediction limit calculated for eqn (3), then the observed mortality is probably due to other factors (i.e. contaminants, predators, disease, etc.). For sediments in which mean survival was greater than the lower 95% prediction limit (95%LPL), mortality also could be caused by contaminants, but could also be due to particle-size-associated factors. The lower 95% prediction limit for R. abronius survival in Reference sediment of a particular % Fines fraction is: 9 5 % L P L = P S - 2.33 • x/(0.026 + 8"3/M + (0"00002 • (% Fines - 57.4)2))

where PS, N and M are as defined in eqn (2). The toxicity of test sediment with a given % Fines fraction can be evaluated by comparing the mean survival of R. abronius in the sediment with amphipod survival in native sediments (e.g. >87"5%; Mearns et al., 1986) and Reference sediments (i.e. survival relative to the 9 5 % L P L derived from eqns (3) or (5)). If mean survival in test sediment is _>87"5%, then the sediment should be considered non-toxic (Mearns et al., 1986). If mean survival is > the 9 5 % L P L , mortality would be interpreted as being within the range of survival observed in Reference sediment, although the real source of mortality could still be chemical contaminants. However, if mean survival in test sediment is less than 9 5 % L P L , a contaminant effect would be indicated. The possibility of a contaminant effect could then be examined through additional analyses of sediment chemistry, toxicity, and ecological conditions. We applied this approach to the Urban field-survey database. Of the 170 Urban stations sampled, five replicate measurements of survival were made for sediments from 78 sites. The relationship between mean amphipod survival and the fine content of these test sediments is shown in Fig. 3; the non-toxic boundary (i.e. survival > 87-5%), the regression line for survival due to % Fines (eqn (1)), and the 95% prediction limits (eqn (5) for M = 5) overlay the survival observations. Fifteen Urban stations were thus identified as non-toxic, 51 stations were between the non-toxic limit and the 9 5 % L P L (i.e. intermediate toxicity), and 12 stations were below the 9 5 % L P L . Mortality in the 12 cases below the 9 5 % L P L may have been caused by contaminants. For the 51 sites with mortality between the nontoxic limit and the 9 5 % L P L , mortality also may be due to contamination, but the ranges of mortality observed were not statistically different from mortality caused by particle-size-associated factors alone. We compared the concentrations of sediment contaminants from the sites falling into the three toxicity categories (i.e. non-toxic, intermediate, and

Amphipod sensitivity to natural sediments

119

Puget Sound Urban Sediments

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below the 9 5 % L P L ) to test the hypothesis that sediment contamination increased from the 'non-toxic' to the 'below 9 5 % L P L ' toxicity categories. All mean sediment contaminant concentrations, except Ag, were highest in 'below 95°/oLPL ' sediments, lowest in the 'non-toxic' sediments, and intermediate in the intermediate sediments (Table 6). Multivariate analysis of variance (MANOVA) and predictive discriminant analysis (using G L M & DISCRIM of PC-SAS; SAS Institute Inc., 1985) were used to test the null hypothesis that no differences existed among these toxicity categories in overall sediment contamination. Two stations (one intermediate, one below 9 5 % L P L ) were dropped from the analysis due to missing data for one or more contaminants. From the MANOVA, Pillai's Trace statistic (0.499; p = 0.002) suggests rejection of the null hypothesis and acceptance of the alternative hypothesis that sediment contamination is different among stations classified as non-toxic, intermediate, or below 9 5 % L P L . Predictive discriminant analysis using a quadratic discriminant function found very good concordance in classification of Urban stations by our toxicity criteria and by sediment contaminant data (Table 7), as evidenced by the high per cent agreement between the classification schemes seen along the diagonal of the matrix (Huberty's proportional chance criterion z--6"32; p < 0.001; Huberty, 1984). However, there was significant heterogeneity among the

120

Theodore H. De Witt, George R. Ditsworth, Richard C. Swartz

TABLE 6

Amphipod Survival, Sediment Variables and Contaminant Concentrations for Urban Stations Classified as Non-toxic ( N = 15), Intermediate ( N = 51) and below 95%LPL (N = 12) Based on Toxicity Criteria. Means (+_ SD) of Raw (Untransformed) Data Presented Variable

Toxicity category Non-toxic

Survival (Number) Fines (% Wt) H20 (% Wt) TOC (% Wt) PAH (#g/kg) PCB (pg/kg)" TOG (mg/kg) h Ag (mg/kg) Hg (mg/kg) Pb (mg/kg) As (mg/kg) Cu (mg/kg) Zn (mg/kg~

18.2 51.7 46.l 3.4 4201 99 399 0"26 0"47 46-9 22-6 98 114

(0"5) (29'2) (15'7) (2"0) (4612) (120) (259) (0.10) (0"45) (31"1) (28"1) (90) (52)

Intermediate

Below 95% LPL

15-6 68.4 50-5 4.2 7627 259 647 0'56 1"38 136"6 25'1 138 195

7.4 46.9 53.4 5.2 11 752 276 1 800 0'55 5"04 750"2 1 005 1 260 707

0"6) (20"6) (11.4) (2-6) (7065) (407) (837) (0-62) (4-57) (139"5) (23"1) (124) (166)

(4.6) (29-8) (18.2) (4.4) (14548) (365) (2 180) (0.99) (14.82) (1 763) (2 777) (3251) (955)

" PCB sample sizes: Non-toxic ( N = 15), intermediate (N = 50), < 9 5 % L P L ( N - 111. h TOG sample sizes: Non-toxic ( N - 7), intermediate (N = 32), < 9 5 % L P L (N = 7).

TABLE 7

Discriminant Analysis of the Classification of Urban Stations Based on Amphipod Toxicity Criteria Compared with Classification Based on Overall Sediment Contamination. Classification of Stations Based on Contaminant Data Achieved with Quadratic Discriminant Functions using SAS Procedure DISCRIM. Matrix Elements are the Per Cent of Stations Classified by Toxicity that were Classified into Each Category by Contaminant Data. Per Cent Agreement between Classification Schemes is Found along the Diagonal of the Matrix Class(fled by toxicity criteria

Non-toxic Intermediate Below 95%LPL

ClassO~'ed by contaminant data Non-toxic

Intermediate

< 95%LPL

Total number ~[ stations

93"3 14.0 9"1

6'7 84.0 9"1

0 2-0 81"8

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Amphipod sensitivity to natural sediments

121

within-covariance matrices, despite transformations to normalize predictor variables, thus violating an assumption of both M A N O V A and discriminant analysis. For this reason, Pillai's Trace statistic was used for M A N O V A as it is the most robust of the M A N O V A criteria (Tabachnick & Fidell, 1983; p. 233), and quadratic rather than linear discriminant functions were used to classify Urban stations (Huberty, 1984). A major advantage of the linear regression model for the relationship between amphipod survival and % Fines is the reduction of'false positives' due to the effects of natural sediment properties which may occur in uncontaminated reference sediments. This model could be used in the place of reference sediments having similar particle-size distribution as test sediments. Native-sediment controls and toxicity standards (Ott, 1986) should be included in field surveys to evaluate the health of test animals. The lower prediction limit of this regression line may be used to identify field sediments in which Rhepoxynius abronius mortality is probably caused by chemical contaminants in addition to any particle-size induced mortality. Field sediments in which survival is not different from survival in native R. abronius sediment (e.g. survival > 87"5%; Mearns et al., 1986) would be identified as non-toxic. Sediments in which survival is less than native sediment but greater than the lower prediction limit could still be significantly contaminated, but the observed amphipod mortality could also be due to particle-size-associated factors. Toxicity due to sediment contamination would be indicated when mortality is tess than that predicted by the 95%LPL. Multivariate analysis revealed significant differences in overall sediment contamination among these 15 non-toxic, 51 intermediate, and 12 toxic Urban stations. Toxic stations (i.e. below 95%LPL) generally had the highest concentrations of each sediment contaminant examined. Thus, the pattern of sediment toxicity revealed with Rhepoxynius abronius closely reflects the degree of contamination of the sediment, at least for Puget Sound sediments. This analysis could be routinely applied to field survey data, allowing regulators to identify critically toxic sites. Furthermore, it will no longer be necessary to attempt the difficult task of locating an uncontaminated sediment texture control. Negative controls using native sediment from the amphipod collection site and positive controls using sediment spiked with reference toxicants should be continued to confirm the acceptability of methods and animals used in individual bioassays. We realize that the use of these equations does not provide an exact statistical comparison of amphipod mean survival in test sediments against the predicted limit of acceptable survival. It does, however, provide a reasonable guide to the interpretation of sediment toxicity for regulatory purposes. It also

122

Theodore H. De Witt, George R. Ditsworth, Richard C. Swartz

incorporates most of the historic data concerning marine sediment toxicity in the Pacific Northwest area of the USA. We recommend this type of analysis be applied to any toxicity test when natural environmental factors associated with the materials being tested cause mortality above background levels. Such will often be the case when bioassay organisms are introduced to ecological conditions marginal to their habitat requirements. This has frequently been the case with the Rhepoxynius abronius sediment bioassay: this amphipod naturally occurs in fine, well-sorted marine sands, but has been used to test the toxicity of muds of high silt or clay content. Identification and quantification of the relationship between mortality and natural environmental variables are critical steps, often ignored, in the development of any toxicological assay.

ACKNOWLEDGEMENTS We thank J. W. Chapman, W. R. Davis, G. W. Fellingham, S. Ferraro, R. Fujita, T. C. Ginn, C. Krueger, J. R. Skalski, J. A. Strand, J. Underwood and J. Q. Word for reviewing the manuscript; J. Jones, J. Lamberson, K. Sercu and F. Cole for field and laboratory assistance; and S. Ferraro, S. Overton and M. Winsor for statistical advice. This study was funded by the Puget Sound Estuary Program through EPA Cooperative Agreement No. CX812792-01-0. We thank Catherine Krueger, our Project Officer at EPA Region X, for her assistance. This is contribution No. N040 from the US Environmental Protection Agency Environmental Research Laboratory-Pacific Division, Newport, OR.

REFERENCES Battelle. (1985). Detailed chemical and biological analyses of selected sediments from Puget Sound. Vol. 1. Draft Final Report. EPA Contract DE-AC0676RLO 1830 and Interagency Agreement No. DW89930272-01-1. Pacific Northwest Laboratory, Battelle, Marine Research Laboratory, 439 W. Sequim Bay Rd., Sequim, WA. Buchanan, J. B. (1984). Sediment analysis. In Methods for the Study of Marine Benthos. (2nd edn) ed. N. A. Holme & A. D. Mclntyre, Blackwell Scientific Publications, London, pp. 41-64. DeWitt, T. H. (1985). The behavior and ecology of migration and colonization in the epibenthic, tubicolous amphipod, Microdeutopus gryllotalpa. PhD Dissertation, State University of New York, Stony Brook. DeWitt, T. H. (1987). Microhabitat selection and colonization rates of a benthic amphipod. Marine Ecology--Progress Series, 36, 237-50.

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Dexter, R. N., Anderson, D. E., Quinlan, E. A., Goldstein, L. S., Strickland, R. M., Pavlou, S. P., Clayton, Jr., J. M., Kocan, R. M. & Landolt, M. (1981). A summary of knowledge of Puget Sound related to chemical contaminants. NOAA Technical Memorandum OMPA-13. Huberty, C. J. (1984). Issues in the use and interpretation of discriminant analysis. Psychological Bulletin, 95, 156 71. Johnson, R. G. (1974). Particulate matter at the sediment-water interface in coastal environments. Journal of Marine Research, 32, 313-30. Kemp, P. F., Cole, F. A. & Swartz, R. C. (1985). Life history and productivity of the Phoxocephalid amphipod Rhepoxynius abronius (Barnard). Journal o[" Crustacean Biology, 5, 449-64. Kemp, P. F., Swartz, R. C. & Lamberson, J. O. (1986). Response of the Phoxocephalid amphipod, Rhepoxynius abronius, to a small oil spill in Yaquina Bay, Oregon. Estuaries, 9, 340-7. Meadows, P. S. & Reid, A. (1966). The behavior of Corophium volutator (Crustacea: Amphipoda). Journal of Zoology, London, 150, 387-99. Mearns, A. J., Swartz, R. C., Cummins, J. M., Dinnel, P. A., Plesha, P. & Chapman, P. M. (1986). Inter-laboratory comparison of a sediment toxicity test using the marine amphipod, Rhepoxynius abronius. Marine Environmental Research, 19, 13 37. Oakden, J. M. (1984). Feeding and substrate preference in five species of Phoxocephalid amphipods from central California. Journal of Crustacean Biology, 4, 233-47. Oakden, J. M., Oliver, J. S. & Flegal, A. R. (1984a). Behavioral responses of a phoxocephalid amphipod to organic enrichment and trace metals in sediment. Marine Ecology Progress Series, 14, 253-7. Oakden, J. M., Oliver, J. S. & Flegal, A. R. (1984b). EDTA chelation and zinc antagonism with cadmium in sediment: Effects on the behavior and mortality of two infaunal amphipods. Marine Biology, 84, 125-30. Ott, F. S. (1986). Amphipod sediment bioassays: Experiments with naturallycontaminated and cadmium-spiked sediments to investigate effects on response of methodology, grain size, grain size toxicant interactions, and variations in animal sensitivity over time. PhD Dissertation, University of Washington, Seattle. Sameoto, D. D. (1969). Physiological tolerances and behavioral responses of five species of Haustoriidae to five environmental factors. Journal of the Fisheries Research Board of Canada, 26, 2283-98. Slattery, P. N. (1985). Life histories of infaunal amphipods from subtidal sands of Monterey Bay, California. Journal of Crustacean Biology, 5, 635-49. SAS Institute Inc. (1985). S A S User's Guide: Statistics Version 5 Edition. SAS Institute Inc., Cary, N.C., USA. Sokal, R. R. & Rohlf, F. J. (1981). Biometry. (2nd edn). W. H. Freeman, San Francisco. Swartz, R. C., DeBen, W. A. & Cole, F. A. (1979). A bioassay for the toxicity of sediment to marine macrobenthos. Journal of the Water Pollution Control Federation, 5, 944-50. Swartz, R. C., DeBen, W. A., Sercu, K. A. & Lamberson, J. O. (1982). Sediment toxicity and distribution of amphipods in Commencement Bay, Washington, USA. Marine Pollution Bulletin, 13, 359-64.

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