Marie-Gil Fortin

Catherine M Couillard
email: couillardc@dfo-mpo.gc.ca

Jocelyne Pellerin

Michel Lebeuf

Effects of salinity on sublethal toxicity of atrazine to mummichog (Fundulus heteroclitus) larvae

Keywords: Fundulus heteroclitus, Larval bioassay, Atrazine, Salinity, Condition, Cortisol, Water content

In coastal marshes, fish larvae may be exposed simultaneously to extreme salinities and to atrazine, a widely used herbicide. To assess the effects of salinity on the toxicity of atrazine, newly-hatched mummichog (Fundulus heteroclitus) were exposed to atrazine (0, 5, 50 and 500 µg/L) at three salinities (3, 15 and 35 PSU). Whole body cortisol was measured after 24 h. Body length, condition factor, whole body proteins, lipids, residual masses and water contents were assessed after 96 h. Significants effects were found for both condition factor and water content. Condition factors were lower at salinity 3 and 35 compared to near isoosmotic salinity, 15 PSU. In addition, atrazine decreased condition factor at 500 µg/L. Reduction in condition was likely due to retardation in axial growth since body length, percentages of proteins or lipids were not affected. In the absence of atrazine, salinity had no effect on the prevalence of dehydrated (≤81% water) or hyperhydrated (≥85% water) larvae. In larvae exposed to atrazine, the

Introduction

Atrazine (2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine) is one of the most widely used herbicides in North America for the control of weeds and grasses in corn, sorghum and sugarcane. Runoff from agricultural fields is the main source of atrazine to aquatic ecosystems.

The highest atrazine concentrations in surface water generally occur in pulses following rain events after herbicide application, with spikes lasting up to several days. In North America, atrazine concentrations up to 108 µg/L have been reported in rivers (USEPA, 2002). Estuaries and coastal marshes are vulnerable to atrazine contamination because they receive waters carrying agricultural pesticides from upland sources [START_REF] De Lorenzo | Toxicity of pesticides to aquatic microorganisms: a review[END_REF]. Atrazine has been detected in estuarine and coastal waters of the eastern coast of North America at concentrations as high as 30 µg/L in Chesapeake bay (USEPA, 2002).

Several recent laboratory studies have shown that environmentally realistic concentrations of atrazine cause significant toxic effects to fish. For example, low concentrations of atrazine (1 µg/L) altered olfactory-mediated endocrine function in male Atlantic salmon (Salmo salar) [START_REF] Moore | The impact of two pesticides on olfactory-mediated endocrine function in mature male Atlantic salmon (Salmo salar L.) parr[END_REF]. At 100 µg/L, atrazine altered Na + K + ATPase activity in common carp (Cyprinus carpio) held in fresh water, indicating osmoregulatory disturbances [START_REF] Hanke | Physiological changes in carps induced by pollution[END_REF]. In addition, in vitro studies in fish have shown that atrazine may affect the secretion of cortisol, involved in osmoregulation and stress response [START_REF] Bisson | Cytotoxic and endocrine-disrupting potential of atrazine, diazinon, endosulfan, and mancozeb in adrenocortical steroidogenic cells of rainbow trout exposed in vitro[END_REF].

Studies on the interaction of salinity with atrazine toxicity in fish are scarce. Pre-exposure of Atlantic salmon smolts to atrazine at ≥1 µg/L in fresh water caused mortalities (14-43%) after a sea water challenge with alteration of Na + K + ATPase activity at ≥2 µg/L [START_REF] Waring | The effect of atrazine on Atlantic salmon (Salmo salar) smolts in fresh water and after sea water transfer[END_REF]. Acute lethal toxicity of atrazine on larval sheepshead minnow (Cyprinodon variegatus) increased eight fold with salinity increasing from 5 to 25 PSU [START_REF] Hall | Influence of salinity on the chronic toxicity to a Chesapeake Bay copepod (Eurytemora affinis) and fish (Cyprinodon variegates)[END_REF]. To our knowledge, the impact of salinity on the sublethal toxicity of atrazine to marine fish species has never been studied.

The euryhaline teleost fish Fundulus heteroclitus, or mummichog, has been used as model species to investigate osmoregulatory and toxicological responses (Hall and Anderson, 1995 and references therein;[START_REF] Marshall | Time course of salinity adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus: a multivariable approach[END_REF]. Mummichog live in estuaries and coastal marshes and are broadly distributed along the eastern coast of North America [START_REF] Joseph | Determination of salinity tolerances in mummichog (Fundulus heteroclitus) larvae obtained from hormone-induced spawning[END_REF].

The capacity to osmoregulate is necessary for this species. They repeatedly move from tidal margin shallows, where salinity is low (mimimum reported for mummichog=0.4 PSU) to deeper parts of the estuary during ebb tides, where salinity is high (maximum reported for mummichog=35 PSU) [START_REF] Griffith | Environment and salinity tolerance in the genus Fundulus[END_REF]. In waters differing from isosmotic salinity (ie, ~10-12 PSU for mummichog), compensatory mechanisms (e.g., drinking, urinating, active influx or outflux via ion channels) maintain water and ion concentrations at a constant level [START_REF] Varsamos | Ontogeny of osmoregulation in postembryonic fish: A review[END_REF]. Given that atrazine is used as a pre-emergence herbicide in early spring, when mummichog spawn, larvae could be exposed simultaneously to contaminated water and extreme salinities [START_REF] Joseph | Determination of salinity tolerances in mummichog (Fundulus heteroclitus) larvae obtained from hormone-induced spawning[END_REF][START_REF] Waring | The effect of atrazine on Atlantic salmon (Salmo salar) smolts in fresh water and after sea water transfer[END_REF].

Early developmental stages are considered to be one of the most sensitive stages in the fish life cycle to the toxic effects of chemical contaminants [START_REF] Weis | Pollutants as developmental toxicants in aquatic organisms[END_REF]. Short-term sublethal effects on growth, behaviour or osmotic control may affect the survival of these critical stages and impact recruitment [START_REF] Houde | Fish early life dynamics and recruitment variability[END_REF][START_REF] Houde | Subtleties and episodes in the early life of fishes[END_REF][START_REF] Sclafani | Osmoregulation, nutritional effects and buoyancy of marine larval fish: a bioassay for assessing density changes during the earliest life-history stages[END_REF][START_REF] Alvarez | Environmental levels of atrazine and its degradation products impair survival skills and growth of red drum larvae[END_REF]. For example, loss of osmotic control altering water content may influence larval density and buoyancy. The vertical position of larvae in the water column affects their patterns of drift and their interactions with preys or predators. Thus, a temporary loss of osmotic control in fish larvae may increase their susceptibility to predation or impair their feeding abilities [START_REF] Sclafani | Osmoregulation, nutritional effects and buoyancy of marine larval fish: a bioassay for assessing density changes during the earliest life-history stages[END_REF]. Disruption of normal cortisol secretion in early life stages may also affect their survival by reducing the ability to cope with acute stressful situations and by inducing adverse secondary effects on osmoregulation, growth, development and immune function [START_REF] Benguira | Loss of capacity to elevate plasma cortisol in rainbow trout (Oncorhynchus mykiss) treated with a single injection of o,p'dichlorodiphenyldichloroethane[END_REF][START_REF] Gravel | Disruption of the hypothalamo-pituitaryinterrenal axis in 1+ yellow perch (Perca flavescens) chronically exposed to metals in the environment[END_REF][START_REF] Kennedy | Ion homeostasis and interregnal stress responses in juvenile Pacific herring, Clupea pallasi, exposed to the water-soluble fraction of crude oil[END_REF].

This study investigates the effects of salinity on sublethal atrazine toxicity for mummichog larvae. This information is needed to assess the risk associated with the presence of atrazine in coastal and estuarine fish spawning grounds. Suborganismal and organismal responses that have been linked to population responses are examined. Larval body mass and length, proteins and lipids contents are measured to assess effects on larval condition. Water content is also assessed as an indicator of loss of osmotic control. In addition, cortisol content is determined since this hormone is involved in osmoregulation and stress.

Materials and methods

Water

Filtered (pore size of 0.2 µm) natural seawater (St. Lawrence Estuary, Mont-Joli, QC, Canada, salinities ranging from 20 to 25 PSU) was diluted with Millipore water to obtain salinities 3 and 15 PSU. The test water with the highest salinity (35 PSU) was prepared by diluting hypersaline brine with Millipore water. Hypersaline brine was obtained as remaining liquid of partially frozen filtered natural seawater at -10 to -20°C.

Source of chemicals and preparation of stock solutions

Atrazine (100% purity) and desethylatrazine (99.2% purity) standard solutions used for chemical analysis were supplied by AccuStandards Inc. (New Haven, CT, USA). All gaschromatography-grade solvents used for chemicals analyses were provided by EM Science (Gibbstown, NJ, USA). The atrazine powder (99.9% purity, lot LB16929) used in the toxicity tests was provided by Supelco (Bellefonte, PA, USA). Acetone (99.9% purity, pesticide free) was obtained from EMD Chemicals Inc. (Gibbstown, NJ, USA).

Atrazine was first dissolved in acetone (100 mg atrazine/10 mL acetone). A volume of 100 µL of this solution was added to 1 L of filtered seawater of each salinity to achieve three salinity-adjusted atrazine stock solutions of 1000 µg/L. Therefore, solvent concentration in the exposure media could never exceed 100 µL/L according to recommendations of the American Society for Testing and Materials (ASTM, 2004). Stock solutions were made on the first day of the experiment and kept in the dark at 4°C until the end of the experiment, 3 days later.

Source of mummichog larvae

Adult mummichog were captured at a reference site, Horton's Creek, NB, Canada, and brought alive to the Maurice Lamontagne Institute, Mont-Joli, QC, Canada, where they were kept in a 420-L living stream aquarium with flowing seawater. Filtered ambient seawater (salinity ∼26 PSU) was provided by the Maurice Lamontagne Institute marine laboratory system, located on the south shore of the St. Lawrence Estuary, QC. Previous measurements of PCBs, polychlorinated dibenzodioxins and dibenzofurans in the liver of mummichog captured at Horton's Creek indicated low contamination and supported the selection of this site as a reference [START_REF] Couillard | Organochlorine contaminants in mummichog (Fundulus heteroclitus) living downstream from a bleached kraft pulp mill in the Miramichi Estuary, New Brunswick, Canada[END_REF]. To induce gonadal maturation, the water temperature was raised progressively from 10°C to 20°C, and a photoperiod of 14-h light: 10-h dark was maintained [START_REF] Boyd | Continuous laboratory production of fertile Fundulus heteroclitus (Walbaum) eggs lacking chorionic fibrils[END_REF][START_REF] Couillard | A microscale test to measure petroleum oil toxicity to mummichog embryos[END_REF]. The fish were fed ad libitum twice daily with pelleted cichlid food (Nutrafin®, Rolf C. Hagen Inc., Montreal, QC, Canada) supplemented with dried shrimps.

Approximately 1-6 h after natural fertilization, eggs were collected on spawning substrates. Eggs were incubated outside of water on damp filter paper (moistened with water at salinity 15 PSU) in Petri dish at 22°C. Hatching was induced by immersion in water of salinity 15 PSU on day 11.

Bioassays

A factorial design consisting of three salinities (3, 15 and 35 PSU) and four nominal concentrations of atrazine (0, 5, 50 and 500 µg/L) crossed for a total of twelve treatments.

Salinities were chosen to represent a near isosmotic salinity (15 PSU) and two salinities (3 and 35 PSU) representing the extreme range to which mummichog are exposed in field conditions [START_REF] Griffith | Environment and salinity tolerance in the genus Fundulus[END_REF][START_REF] Varsamos | Ontogeny of osmoregulation in postembryonic fish: A review[END_REF]. A short-term, acute -exposure period (96-h) was selected to represent field exposure conditions. Specific volumes of salinity-adjusted atrazine stock solutions were added to filtered and aerated water (>95% dissolved oxygen) of each salinity to achieve the desired concentrations.

Larvae were exposed in 250-mL glass Mason jars containing 210 mL of test solution.

Newlyhatched larvae were randomly assigned to each jar. Larvae were incubated at 20°C under fluorescent light on a 12:12 light: dark cycle and were not fed during the experiment.

Exposure lasted 96 h (4 replicates of 10 larvae each) with daily renewal of the solutions for assessment of larval condition and water content. For each treatment, solutions were renewed with solutions with the same salinity and the same concentration of atrazine throughout the exposure period. It was not necessary to feed the larvae since they rely on their yolk sac in the first 4 days after hatching. Dissolved oxygen was measured at the beginning of each 24-h exposure period. Temperature, salinity and pH were measured at the beginning and end of each 24-h exposure period.

Cortisol after a 24-h exposure period

Cortisol measurements (3 replicates of 7 larvae each) were made after a 24-h exposure period since cortisol levels generally peak during the first 24 h after an acute salinity challenge in Fundulus heteroclitus and other fish species [START_REF] Marshall | Time course of salinity adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus: a multivariable approach[END_REF][START_REF] Kajimura | Changes in plasma concentrations of immunoreactive ouabain in the tilapia in response to changing salinity: is ouabain a hormone in fish?[END_REF] and are expected to be back to normal levels after 96 h. This transient rise in cortisol triggers the osmoregulatory responses necessary for maintenance of water content and optimal growth. Thus, it is expected that an abnormal cortisol response in the first hours after a salinity challenge could have delayed impact on osmotic control and growth [START_REF] Liebert | Effects of acute stress on osmoregulation, feed intake, IGF-1 and cortisol in yearling steelhead trout (Oncorhynchus mykiss) during seawater adaptation[END_REF].

At the end of the 24-h exposure period, 840 µL of an anaesthetizing solution of clove oil: ethanol 1:10 was added directly to the jars to obtain a final concentration of 40 mg/L clove oil (Sigma, Oakville, ON, Canada, 98% purity) in the exposure media. Clove oil was used since it was found to prevent the cortisol stress response associated with handling in fish [START_REF] Small | Anesthetic efficacy of metomidate and comparison of plasma cortisol responses to tricaine methanesulfonate, quinaldine and clove oil anesthetized channel catfish Ictalurus punctatus[END_REF].

After 3 min immersion in clove oil, larvae were frozen immediately in liquid nitrogen and stored to -80°C until cortisol analysis. In order to minimize stress, length of these larvae was not measured.

Frozen larvae from each replicate were pooled in two groups of two larvae and one group of three larvae, then weighed to the nearest 0.1 mg. They were homogenized in ED1 buffer (R&D Systems, Minneapolis, MN, USA) at a ratio of 150 µL/larva with a polypropylene pellet pestle (Sigma-Aldrich, Oakville, ON, Canada). Steroid displacement reagent (R&D Systems) was added to the homogenates in proportion 1:100 (v/v). Homogenates were centrifuged twice at 4°C (12 500 g, 30 min, and then 10 min) and supernatants kept on ice. Cortisol concentrations were quantified using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) in which cortisol present in samples competes with a fixed amount of alkaline phosphatase-labeled cortisol for sites on a mouse monoclonal antibody. This complex binds a goat anti-mouse polyclonal antibody coated onto the microplate. The ELISA plates were read at 405 nm using a Multiskan® Ascent (Titertek, Huntsville, AL, USA) and readings at 570 nm were subtracted to correct for optical imperfection in the plate. Samples were analyzed in duplicate. Average coefficient of variation (CV) among duplicates was 3.5±3.1%. Cortisol contents in the homogenates were calculated and expressed on a per larva basis (larval cortisol content) or on a wet mass basis (larval cortisol concentration) for purpose of comparison with the literature.

The ELISA method was validated for measuring mummichog cortisol in whole larval homogenates by verifying that serial dilutions of these samples inhibited the binding of alkaline phosphatase-labeled cortisol in the same way as cortisol standards (R&D Systems). The slopes of the relationship between percentage of binding (B/B 0 ) and concentration of standard or dilution factor did not differ between cortisol standard and serial dilutions of larval homogenates (P>0.05). Parallel slopes indicated no apparent interference of larval homogenate components on antibody/cortisol binding parameters.

Larval condition and water content after a 96-h exposure period

For determination of treatment effects on larval condition (length, condition factor, percentages of proteins, lipids and residual masses) and water content, a 96-h static test was conducted with daily renewal of the solutions. At the end of exposure, larvae were anaesthetized in carbonated water (560 mg/L HCO 3 -, 1000 mg/L mineral salts; Saint-Justin Co. Maskinonge, QC, Canada) instead of clove oil that could possibly interfere with lipid determination. Total body length was measured to the nearest 0.1 mm with a binocular microscope equipped with an ocular micrometer. To determine wet mass, larvae were blotted dry, weighed to the nearest 0.1 mg on an analytical balance, then immediately frozen individually in liquid nitrogen and stored at -80°C. Larvae were lyophilized until constant mass was attained. Larvae were kept in a dessicator until dry mass was determined to the nearest 0.001 mg. Wet and dry masses were used to calculate percentages of water. Length and dry masses were used to calculate condition factor (length/ dry mass X 100) as recommended by [START_REF] Safran | Theoretical analysis of the weight-length relationship in fish juveniles[END_REF] for juvenile fish. Lyophilized larvae were individually homogenized with a polypropylene pellet pestle in 150 µL of sodium phosphate buffer (0.2 M, pH=7.0). Two aliquotes (75 µl each) were used for protein and lipid analysis, respectively.

Lipids were extracted following the Bligh and Dyer method [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF].

Extractable lipids were measured using the sulpho-phospho-vanillin method described by [START_REF] Izard | Rapid screening method for quantitation of bacterial cell lipids from whole cells[END_REF]. Briefly, this method consists of oxidizing cellular lipids to small fragments after chemical digestion with hot concentrated sulfuric acid. After the addition of a solution of vanillin and phosphoric acid, a red complex is formed. Samples were analyzed in triplicates at 530 nm with a Multiskan® ascent spectrophometer. Lipid concentration in larval homogenates was determined using olive oil as a standard. Average CV among triplicates was 3.2±6.4%. Lipids in larvae (% dry mass) were calculated.

After centrifugation (10 000 g, 10 min at 4°C) supernatants were analysed for soluble protein content with Bradford reagent. Protein concentration in larval homogenates was determined using bovine serum albumin (BIO-RAD Laboratories, Hercules, CA, USA) as a standard. Samples were analyzed in triplicates at 595 nm using a Multiskan® ascent spectrophotometer. Average CV among triplicates was 6.6±5.0%. Proteins in larvae (% dry mass) were calculated. Residual mass [defined as dry body mass-(lipid+protein content)] was also calculated and expressed as percentage of dry mass [START_REF] Ali | Comparative study of body composition of four fish species in relation to pond depth[END_REF].

Chemical analysis

Atrazine and desethylatrazine were analysed in the following exposure solutions: atrazine nominal concentration of 0 µg/L on day 0 for each test salinity; all atrazine nominal concentrations (0, 5, 50 and 500 µg/L) at the beginning (t-0) and end (t-24) of each 24-h exposure period in salinity 15 PSU; high (500 µg/L) and low (5 µg/L) nominal concentrations at t-0 and t-24 on the last day at salinity 3 PSU and 35 PSU. These measurements were used to assess: potential contamination of the controls; variability associated to the technique of preparation of the solution and/or to the potential degradation of atrazine during 24-hr at salinity 15PSU; the effect of salinity on the potential degradation of atrazine during a 24-hr exposure period. A replicate was prepared specifically for chemical analyses at the beginning (t-0) of each 24-h exposure period. Chemical analyses at the end (t-24) of each 24-h exposure period of the was operated in MS-MS mode. GC-MS analysis was performed by using the following conditions: injector temperature 90°C-310°C (90°C for 0.3 min; 200°C/min to 310°C, held for 44 min), oven temperature 90°C-300°C (90°C for 1.2 min; 30°C/min to 150°C; 1.5°C/min to 190°C, held for 1 min; 8°C/ min to 260°C; 30°C/ min to 300°C, held for 3 min) and ion trap temperature 250°C.

Atrazine and desethylatrazine concentrations were calculated on the basis of their response relative to the one of 13 C 12 PCB-101 in the same sample. Relative response factor was determined on the basis of a four point calibration curve for atrazine and desethylatrazine, while 13 C 12 PCB-101 and TCPMe were kept at constant concentration (100 pg/µL). Atrazine and desethylatrazine concentrations were corrected on the basis of the recovery of the surrogate compound. Limit of quantification was 0.003 ng/L for both atrazine and desethylatrazine.

Analytical precision was 6%.

Statistical analysis

The effects of salinity and of exposure to different concentrations of atrazine on cortisol (24-h exposure) or on body length, condition factors, percentages of proteins, lipids, residual mass and water (96-h exposure) were assessed by two-way ANOVA with interaction conducted on the mean values of the replicates (N=4 jars per treatment). Following significant ANOVA, a Tukey's multiple comparison test was applied. Data met with the requirements for normality (Shapiro-Wilk's test) and homogeneity of variance (Levene's test).

Graphical examination of the linear relationship between water mass and dry mass revealed the presence of severely affected larvae with either very low or very high water content.

Larvae with water percentages ≥2 standard deviations from the mean of control larvae (salinity 15 PSU, atrazine 0 µg/L) were considered to be severely affected (≤81%, dehydrated or ≥85%, hyperhydrated). The prevalence of dehydrated or hyperhydrated larvae were compared between each treatment and control using a two-tailed Fisher's exact test.

The severity of dehydratation of each dehydrated larvae was graded from 2 to 6 based on the deviation of its percentage of water from the mean percentage in control larvae (number of standard deviations from the mean). The severity of dehydratation was compared among treatments with significantly enhanced percentages of severely affected larvae using a Kruskall-Wallis test followed by a Tukey's multiple comparison test.

The prevalence of larvae with condition factor ≥2 standard deviations from control mean was also compared among treatments using a two-tailed Fisher's exact test. Condition factors of dehydrated and hyperhydrated larvae were compared to condition factors of larvae with normal water content by one-way ANOVA within treatments. Statistical analyses were performed with SAS software (SAS Institute, Cary, NC, USA). All differences were tested at a probability (α) of 0.05.

Results

Water quality parameters and chemical analysis

Characteristics of the test water at t-0 for the three different salinities were: salinity=3.1±0.1 PSU, pH=8.0±0.2 and temperature=21.6±0.6°C; salinity=15.1±0.1 PSU, pH=7.8±0.1 and temperature=21.6±0.5°C; salinity=35.1±0.1 PSU, pH=7.9±0.1 and temperature=21.6±0.5°C (mean±standard deviation). At t-24, there were: salinity=3.1±0.1 PSU, pH=7.7±0.2 and temperature=20.6±0.2°C; salinity=15.1±0.1 PSU, pH=7.6±0.2 and temperature=20.6±0.2°C; salinity=35.0±0.2 PSU, pH=7.8±0.1 and temperature=20.6±0.1°C.

Dissolved oxygen remained >95% of saturation throughout the experiment. Water quality conditions (pH, temperature and O 2 ) did not differ among treatments.

Measured atrazine concentrations at t-0 (Table 1) were within 85-105% of the nominal concentrations. The single measurement of atrazine for each treatment at salinities 3 and 35 PSU was within the range observed for the corresponding treatment at salinity 15 PSU. Larvae were exposed to nearly constant atrazine concentrations over the bioassay period. Variations of atrazine concentration within each 24-h period were of the same amplitude as the day to day variation at t-0. In blanks at the three salinities, atrazine was occasionally detected in trace amounts (<0.02 µg/L). Desethylatrazine was not detected, except in trace amounts (0.004 and 0.013 µg/L) at the highest atrazine concentration at salinity 3 and 35 PSU, respectively, on the first day of exposure.

Cortisol

No larvae died during the first 24-h exposure period. Salinity or atrazine concentration did not significantly affect cortisol content in larvae (Table 2). Cortisol content in whole larvae presented a high degree of variability within treatment. Coefficient of variation in control (salinity 15 PSU, and 0 µg/L atrazine) was 13%. The highest coefficient of variation (40%) was observed in salinity 15 PSU, 500 µg/L atrazine. The variability observed could not be reduced by expressing cortisol as concentration (% wet mass) rather than per whole larvae and no significant effect of treatments on cortisol concentrations was detected (ANOVA p>0.05). Cortisol concentration in control was 106±14 ng/g (wet mass).

Larval condition

No larvae died during the 96-h exposure period. Salinity or atrazine had no effect on body length, percentages of proteins, lipids or residual masses (Table 3). Larval dry masses ranged from 0.274 to 0.460 mg. The prevalence of larvae with a condition factor ≥2 standard deviations from control mean did not differ significantly among treatments (Fisher's exact test, P>0.05).

No interaction was detected between atrazine and salinity effects on condition factor (Table 3).

Salinity affected condition factor of larvae, with lower condition factor at salinity 3 PSU and 35 PSU than at salinity 15 PSU. The highest atrazine concentration, 500 µg/L, elicited a significant decrease in condition factor. Condition factors of dehydrated (≤81% water) or hyperhydrated (≥85% water) larvae did not differ from condition factors of larvae with normal percentages of water within treatments (ANOVA, p>0.05).

Water content

No interaction was detected between atrazine and salinity effects on the percentages of water. Mean percentages of water in whole-larvae were affected by salinity; a higher percentage of water was found at salinity 3 PSU than at salinity 15 and 35 PSU. No significant effect of atrazine concentration on mean percentages of water was detected by ANOVA (Table 3).

In the absence of atrazine, salinity had no effect on the prevalence of dehydrated or hyperhydrated larvae (Figure 1). At atrazine concentrations ≥5 µg/L, the prevalence of dehydrated larvae was significantly increased at salinity 15 and 35 PSU (prevalence of dehydrated larvae ranging from 28 to 42% at ≥5 µg/L compared to 7% in controls) (Fisher's exact test, p ≤ 0.05). At salinity 35 PSU, the dehydratation was significantly more severe at the highest (500 µg/L) compared to the lowest (5 µg/L) concentration of atrazine, with water percentages as high as 6 standard deviation from control mean at 500 µg/L (Kruskal WallisW, p ≤ 0.05; Figure 2). At salinity 15 PSU, the severity of dehydration did not differ significantly among atrazine concentrations. The prevalence of hyperhydrated larvae was significantly increased at salinity 3 PSU, 5 µg/L atrazine (prevalence of hyperhydrated larvae of 23% compared to 0% in controls; Fisher's exact test, p ≤ 0.05) whereas it was not affected at higher concentrations of atrazine.

Discussion

This study shows that short-term exposure to environmentally realistic concentrations of atrazine affects osmotic control in mummichog larvae. Without atrazine, no change in the prevalence of severely hyperhydrated or dehydrated larvae was observed in response to changes in salinity. Atrazine affected the prevalence of hyperhydrated and dehydrated larvae without affecting the mean percentages of water, suggesting that some individual larvae were more vulnerable to atrazine toxicity. At higher salinities (15 and 35 PSU) than isosmotic salinity for mummichog (10-12 PSU), larvae faced water loss by passive diffusion [START_REF] Varsamos | Ontogeny of osmoregulation in postembryonic fish: A review[END_REF].

In absence of atrazine, larvae controlled their water content at these salinities. Atrazine induced an osmoregulatory imbalance in some larvae with an increase in the prevalence of dehydrated larvae at 5 -500 µg/L. The most severe effects were observed in larvae exposed to 500 µg/L at 35 PSU. At salinity 3 PSU, larvae faced water invasion by passive diffusion. Prevalence of hyperhydrated larvae increased at 5 µg/L compared to control indicating osmoregulatory disturbances induced by atrazine. In Atlantic salmon smolts, similar atrazine concentrations (≥6.5 µg/L) affected osmoregulation and gill Na + K + ATPase activity which is directly related to the size and number of gill chloride cells [START_REF] Waring | The effect of atrazine on Atlantic salmon (Salmo salar) smolts in fresh water and after sea water transfer[END_REF]. Newly-hatched mummichog larvae have functional chloride cells on their gill filaments that could be affected by atrazine exposure (Kanekoh and Katoh, 2004). Water content was not altered at higher atrazine concentrations. At these concentrations (50, 500 µg/L), water content may be negatively affected by a loss of condition counteracting the positive effect of osmotic imbalance [START_REF] Couture | Biochemical correlates of growth and condition in juvenile Atlantic cod (Gadus morhua) from Newfoundland[END_REF][START_REF] Bureau | Pattern and cost of growth and nutrient deposition in fish and shrimp: Potential implications and applications[END_REF]. Further studies with larger sample size and thus higher precision of the estimate of the prevalence of abnormal larvae are required to better define the concentrationresponse relationship and to determine the no-observed-adverse-effect-concentration.

Loss of osmotic control in fish larvae observed in this study may influence larval buoyancy that is a critical determinant of growth and survival [START_REF] Sclafani | Osmoregulation, nutritional effects and buoyancy of marine larval fish: a bioassay for assessing density changes during the earliest life-history stages[END_REF][START_REF] Moustakas | Combined effects of photoperiod and salinity on growth, survival, and osmoregulatory ability of larval southern flounder Paralichthys lethostigma[END_REF]. To fully evaluate the ecological significance of our findings, the buoyancy of hyperhydrated and dehydrated mummichog larvae should be measured and its impact on the interactions of the larvae with preys or predators should be assessed.

After a 96-h exposure period, salinity had no effect on the reduction in body condition caused by atrazine. Atrazine reduced condition factor of larvae only at the highest concentration tested, 500 µg/L. The lowest atrazine concentration reported to affect fish condition (expressed as mean wet mass and mean length) was 120 µg/L for newly hatched freshwater brook trout (Salvelinus fontinalis) after a 306-day exposure period [START_REF] Macek | Chronic toxicity of atrazine to selected aquatic invertebrates and fishes[END_REF]. Atrazine reduced growth rate after a 9-day exposure period in red drum larvae at 80 µg/L at 27 PSU but this reduction was not significant after a 96-h exposure period [START_REF] Alvarez | Environmental levels of atrazine and its degradation products impair survival skills and growth of red drum larvae[END_REF]. A longer period of observation of the mummichog larvae, following a pulse exposure to atrazine, may be necessary to reveal significant impacts on growth or condition.

The reduction in condition of larvae in response to salinity and/or atrazine could not be attributed to enhance use of body reserves since percentages of proteins, lipids or residual masses were not affected. Prevailing growth mechanisms in larvae are increased in length and axial expansion of muscles and developing organs [START_REF] Couture | Biochemical correlates of growth and condition in juvenile Atlantic cod (Gadus morhua) from Newfoundland[END_REF]. Body length was not affected by the treatments. Reduction in condition probably originated from a reduction in axial growth with a decrease in tissues synthesised, since body composition was not affected. Atrazineexposed red drum larvae were hyperactive at concentration ≥40 µg/L [START_REF] Alvarez | Environmental levels of atrazine and its degradation products impair survival skills and growth of red drum larvae[END_REF]. Thus, hyperactivity could have contributed to reduction in larval condition observed in our study but changes in behaviour were not assessed.

Our results confirm that the near isosmotic salinity of 15 PSU was optimal for mummichog larvae among the three salinities tested. Larvae had slightly higher percentages of water at hyposmotic salinity compared to near isosmotic or hyperosmotic salinities, regardless of atrazine concentration. This indicates incapacity of the larvae to entirely counteract passive water invasion at this low salinity. This result is consistent with the fact that mummichog are better adapted to hyperosmotic than to hyposmotic environments [START_REF] Kaneko | Functional morphology of chloride cells in killifish Fundulus heteroclitus, a euryhaline teleost with seawater preference[END_REF]. It also affected condition of larvae, with lower condition factor at salinity 3 and 35 PSU than at the near isosmotic salinity of 15 PSU. This result is in agreement with the observation that fry body length was highest at the near isosmotic salinity of 20 PSU in mummichog incubated in salinities ranging from 0.5 to 60 PSU from fertilisation to fry stage [START_REF] Tay | Compensatory embryogenic responses in osmoregulative structures of mummichog Fundulus heteroclitus L. incubated at constant temperature and various levels of salinity[END_REF]. Salinity did not affect dissolved concentration or degradation rate of atrazine. Indeed, measured atrazine concentrations for each nominal concentration were similar among salinities and were stable over the 24-h exposure periods. However, salinity may have affected the absorption of atrazine in the larvae and studies with radiolabelled atrazine could be conducted to test this hypothesis [START_REF] Blanchard | Effects of salinity on copper accumulation in the common killifish (Fundulus heteroclitus)[END_REF]. This is the first study that reports cortisol content in mummichog larvae (217±29 pg/larva in controls, salinity 15 PSU, 0 µg/L atrazine). Similar cortisol content (140 pg/larva) has been reported in 1 day post-hatch (dph) silver sea bream (Sparus sarba) larvae (Deane and Woo, 2003). Length and dry masses of 1 dph silver sea bream larvae are in the same range as those of mummichog larvae (Deane et al., 2003). Lower cortisol concentrations have been reported in the larvae of other fish species such as rainbow trout (Onchorynchus mykiss) (0.3 ng/g wet mass 1 dph larvae) or red drum (Sciaenops ocellatus) (undetectable in 0-1 dph larvae) [START_REF] Barry | Ontogeny of the cortisol stress response in larval rainbow trout[END_REF]Pérez-Domingez and Holt, 2006).

Neither atrazine nor salinity had detectable effects on cortisol content. Considering the high variability of cortisol content and the sample size (n=3), the experimental design provided an 80% power to detect a 27% change in cortisol. This power was sufficient to detect a typical cortisol response to an acute salinity challenge with >500% elevation in cortisol concentrations [START_REF] Marshall | Time course of salinity adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus: a multivariable approach[END_REF]Pérez-Dominguez and Holt, 2006). The time course of salinity adaptation has not been described in larval mummichog. It is possible that a cortisol response has occurred shortly after transfer of the larvae in the exposure jars and was no longer detectable after 24 h. In adult mummichog, cortisol levels were elevated 24 h after transfer from freshwater to seawater [START_REF] Marshall | Time course of salinity adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus: a multivariable approach[END_REF]. It is also possible that one day after hatching, mummichog larvae were not able to produce cortisol or that their response was masked by cortisol from maternal origin [START_REF] Stouthart | Stress responsiveness of the pituitary-interrenal axis during early life stages of common carp (Cyprinus carpio)[END_REF]Pérez-Dominguez and Holt, 2006). Measurement of cortisol in blood would have provided a better indication of the response of the larvae than whole-body cortisol but it was not technically possible given the small-size of the mummichog larvae.

Conclusion

Environmentally relevant concentrations (5 µg/L) of atrazine affected the capacity of mummichog larvae to osmoregulate with higher prevalence of dehydrated larvae (at salinity 15 and 35 PSU) or hyperhydrated larvae (at salinity 3 PSU). Severity of dehydration increased with atrazine concentration at salinity 35 PSU. After a 96-h exposure period significant reduction in condition was only observed at 500 µg/L, a concentration rarely encountered in the environment.

Further work is needed to determine the no-observed-adverse-effect-concentration (NOEC) and to assess the effect of observed changes in water content on larval buoyancy, growth and survival of mummichog larvae. The experimental model developed in this study could be used to evaluate the interaction between salinity and other toxic compounds encountered in estuarine and coastal waters.

Figure caption

  96-h assay were performed on pooled water samples of all 4 replicates. Various volumes of exposure solutions were collected, depending on the nominal atrazine concentration, and transferred into a clean glass bottle with 10 ng of the labeled surrogate compound 13 C 12 PCB-101 to assess the extraction efficiency. Extraction was performed 3 times with a volume of dichloromethane corresponding to approximately 25% of the collected solution. Combined extracts were then reduced to about 50 µL and completed with 50 µL of tris(4chlorophenyl)methane (TCPMe, 100 pg/µL) as internal standard. Analysis was conducted on a gas chromatograph (GC) equipped with a DB-5MS capillary column (30 m X 0.25 mm i.d. X 0.25 µm film thickness, J&W Scientific, Folson, CA, USA) coupled to a Varian Saturn 2000 ion trap mass spectrometer (MS) by a transfer line kept at 300°C. The injector was operated in splitless mode. Helium was used as the carrier gas (flow rate, 1.0 mL/min). The ionization was performed by electron impact at 70 eV and the ion trap

Fig. 1 .

 1 Fig. 1. Effect of salinity and atrazine on the prevalence of A) abnormally dehydrated larvae

Fig. 2 .

 2 Fig. 2. Severity of dehydratation in dehydrated larvae in treatments where the prevalence of

  Fig.1

Table 1 .

 1 Nominal and measured atrazine concentrations in exposure solutions at the beginning (t=0) and

	end (t=24) of 24-h exposure periods
	Salinity	Nominal concentrations	Measured concentrations (µg/L)
	(PSU)	(µg/L)	t=0	t=24
	3	0	<0.003	-
		5	4.50	5.49
		500	492	491
	15	0	0.004 (<0.003-0.005)	<0.003 (<0.003)
		5	4.67 (4.38-5.12)	4.25 (2.90-4.81)
		50	45.2 (43.0-47.7)	47.2 (45.1-50.1)
		500	490 (456-579)	456 (368-524)
	35	0	0.018	-
		5	4.74	3.90
		500	476	447
	Values for salinity 15 PSU represent mean (range) of concentrations measured (n=4); for other
	salinities, n=1 (pool of 4 replicates). Limit of quantification (0.003 µg/L) was given to non
	detected values to calculate mean. -Not measured.

Table 3 .

 3 Effects of salinity and atrazine on body length, condition factor, percentages of water, proteins, lipids or residual mass of mummichog larvae after a 96-h exposure period Nominal concentrations of atrazine. Condition factor: (dry mass/length*100). Values are expressed as means (±SE). Sample size was 4 replicates of 10 larvae. Different letters indicate statistically different values (two-ways ANOVA with interaction, P<0.05).

	Salinity	Atrazine	Length	Condition factor	Proteins	Lipids	Residual mass	Water
	(PSU)	(µg/L)	(mm)	(g/cm)	(% dry mass)	(% dry mass)	(% dry mass)	(% wet mass)
	3	0	7.00 (0.05) 5.12 (0.04)	29.8 (0.7)	7.2 (0.8)	63.6 (1.3)	82.6 (0.9)
		5	6.99 (0.05) 5.08 (0.12)	30.3 (1.3)	7.1 (0.7)	62.7 (1.9)	83.4 (0.5)
		50	7.01 (0.06) 5.03 (0.12)	30.6 (0.9)	6.8 (0.8)	62.4 (1.8)	82.5 (0.9)
		500	6.94 (0.03) 4.95 (0.12)	30.8 (1.5)	6.5 (0.9)	62.5 (1.6)	83.0 (0.5)
	15	0	6.99 (0.06) 5.17 (0.08)	29.8 (1.4)	6.9 (0.3)	63.4 (1.3)	82.7 (0.8)
		5	7.00 (0.06) 5.21 (0.13)	31.1 (0.8)	7.3 (0.1)	61.5 (0.9)	81.8 (0.3)
		50	7.06 (0.05) 5.27 (0.10)	30.5 (1.0)	7.1 (0.6)	62.4 (0.9)	81.6 (0.3)
		500	6.98 (0.10) 5.05 (0.11)	29.6 (2.0)	7.3 (0.7)	63.2 (2.3)	81.8 (0.4)
	35	0	6.95 (0.05) 5.16 (0.11)	29.6 (1.3)	7.2 (0.4)	63.4 (1.5)	81.9 (0.7)
		5	6.92 (0.10) 5.10 (0.09)	29.2 (0.9)	6.2 (0.6)	65.1 (0.9)	82.0 (0.5)
		50	7.04 (0.07) 5.13 (0.05)	30.1 (2.6)	6.8 (0.3)	63.1 (2.5)	81.9 (0.9)
		500	7.02 (0.05) 5.01 (0.11)	29.3 (1.4)	6.6 (0.8)	64.2 (1.9)	81.5 (0.6)
	Salinity*atrazine P=0.36	P=0.68	P=0.75	P=0.29	P=0.41	P=0.36
	Salinity effect	P=0.50	P=0.0033	P=0.24	P=0.15	P=0.058	P<0.0001
				3b 15a 35b				3a 15b 35b
	Atrazine effect	P=0.08	P=0.0025	P=0.63	P=0.61	P=0.63	P=0.24
				0a 5a 50a 500b

Acknowledgements

We thank B. Légaré, M.-C. Marquis and H. Talbot for their technical assistance with the bioassays and the fish cultures. We also thank B. Légaré and P. Rioux for their assistance with biochemical measurements and S. Trottier for chemical analyses of test water samples. This project was funded by the Environmental Strategic Science Research Fund and the Pesticide Research Program of the Department of Fisheries and Oceans.


Atsuko Fukunaga
email: atsukofu@hawaii.edu

Julie H Bailey-Brock

Benthic infaunal communities around two artificial reefs in Mamala

Keywords: Bay, Oahu, Hawaii Oahu, Hawaii Artificial reef, Benthic ecology, Polychaete, Multivariate analysis, Mamala Bay, Hawaii distribution

come

Introduction

Non-fishery use of an artificial reef (e.g. ecotourism and environmental mitigation) has been given great attention in recent years [START_REF] Abelson | Artificial reefs vs coral transplantation as restoration tools for mitigating coral reef deterioration: Benefits, concerns, and proposed guidelines[END_REF][START_REF] Ambrose | Mitigating the effects of a coastal power plant on a kelp forest community: Rationale and requirements for an artificial reef[END_REF][START_REF] Brock | Beyond fisheries enhancement: Artificial reefs and ecotourism[END_REF][START_REF] Falace | Algal transplantation as a potential tool for artificial reef management and environmental mitigation[END_REF][START_REF] Fox | Experimental assessment of coral reef rehabilitation following blast fishing[END_REF][START_REF] Reed | Quantitative assessment of different artificial reef designs in mitigating losses to kelp forest fishes[END_REF]. This is partly because, unlike the traditional use in fisheries and aquaculture, non-fishery artificial reefs are unlikely to cause a population decline of reef-associated organisms through aggregation [START_REF] Bohnsack | Are high densities of fishes at artificial reefs the result of habitat limitation or behavioral preference[END_REF][START_REF] Powers | Estimating enhancement of fish production by offshore artificial reefs: uncertainty exhibited by divergent scenarios[END_REF]. The primary concern associated with non-fishery artificial reefs is their effects to the surrounding ecosystems. Artificial reefs constructed by haphazardly dumping scrap materials show not only poor enhancement of organisms but also instability and degradation of the materials over a relatively short time period [START_REF] Brock | An analysis of the efficacy of four artificial reef designs in tropical waters[END_REF]. Materials such as steel, automobile tires and coal ash are of concern because leaching of chemical elements may cause pollution [START_REF] Collins | Acceptable use of waste materials[END_REF].

Deployment of artificial reefs modifies the bottom current and affects sediment grain size [START_REF] Bailey-Brock | Changes in pollution indicators at the Sand Island sewage outfall[END_REF][START_REF] Bailey-Brock | Polychaetes associated with a tropical ocean outfall: synthesis of a biomonitoring program off O'ahu, Hawai'i[END_REF][START_REF] Lee | Temporal changes in the polychaete infaunal community surrounding a Hawaiian mariculture operation[END_REF][START_REF] Swartz | Regional monitoring of benthic fauna in Mamala Bay, O'ahu, Hawai'i[END_REF].

They comprise the most diverse and species rich group in sediment samples from Mamala Bay, with a variety of trophic, motility and reproductive modes that provide insight to the ecological conditions that prevail in the habitat [START_REF] Lee | Temporal changes in the polychaete infaunal community surrounding a Hawaiian mariculture operation[END_REF]. Polychaete community structures were compared with one from a natural reef and existing data from Mamala Bay.

Benthic community structures can be influenced by various environmental factors [START_REF] Fabi | Effects of an artificial reef on the surrounding soft-bottom community (central Adriatic Sea)[END_REF][START_REF] Moura | Effect of depth and reef structure on early macrobenthic communities of the Algarve artificial reefs (southern Portugal)[END_REF], and therefore, be highly variable within the bay. The comparison with existing data was (1) to examine if environmental variables (depth and grain size composition of sediments/sands) affect community structural patterns, and (2) to evaluate polychaete assemblages around the two artificial reefs by comparing them with those representing various natural habitats throughout Mamala Bay. This paper presents the case that the two artificial reefs in Mamala Bay provide beneficial use in ecotourism without causing detrimental impacts to the surrounding environments.

Materials and methods

Study sites

The study was carried out at two artificial reefs and a natural reef in Mamala Bay, off the south shore of Oahu, Hawaii (Fig. 1). The Sea Tiger artificial reef (21º16'47''N, 157º51'35''W) was created in 1999 by sinking a 51 m fishing vessel approximately 1.5 km offshore of Ala Moana Beach at a depth of 38 m. The artificial reef was deployed, after weeks of preparation, as the beginning effort of Voyager Submarines' Lopaka Reef Foundation that was formed to protect and re-establish natural coral reefs. The vessel lies perpendicular to the shore with the bow facing outward to the south. Patchy growth of the seagrass, Halophila decipiense, was observed on the sand around the port and starboard sides of the vessel covering about 50% of the area at the time of sampling. This seagrass growth was assumed to be a relatively new, post-deployment settlement, as it had not been This artificial reef has been used for tourism by Atlantis Submarine and dive charters. Both sunken vessels are used by local fisherman for non-commercial fishing, and YO257 was monitored for spear, net and hook and line fishing for a comparative economic study of commercial fishing and ecotourism use of this site [START_REF] Brock | Beyond fisheries enhancement: Artificial reefs and ecotourism[END_REF]. The main current direction in Mamala Bay is parallel to the shore flowing from east to west, although reversals occur synchronizing with tides [START_REF] Hamilton | Ocean current measurements, Mamala Bay Study Project MB-6[END_REF]. This causes different water flow characteristics at the two artificial reef sites. The vessel at the Sea Tiger artificial reef lies perpendicular to the currents, and the yard oiler at the YO257 artificial reef lies parallel to the currents. The YO257 artificial reef and the 100' hole natural reef typically have stronger currents compared to the Sea Tiger artificial reef because of their proximity to Diamond Head, a headland that affects the current patterns along this coast [START_REF] Hamilton | Ocean current measurements, Mamala Bay Study Project MB-6[END_REF]. Mean predicted currents for a 12 month period at a depth of 35 m at the three study sites are approximately 2 cm sec -1 at Sea Tiger, 6 cm sec -1 at YO257 and 7 cm sec -1 at 100' hole [START_REF] Blumberg | Additional hydrodynamic model results, Mamala Bay Study Project MB-5[END_REF]. Water quality in Mamala Bay has been tested intensively to examine effects of runoff and wastewater outfalls and shown that the discharge areas were not statistically different from reference areas (U.S. [START_REF] Epa | Coastal condition for Alaska, Hawaii, and Island Territories[END_REF]. The study sites are, therefore, unlikely to be under the influence of land discharges. decipiense patches, and the other half outside the patches, at Sea Tiger. Sediments were preserved in 10% buffered formalin for a minimum of 48 hours. Organisms in sand were removed by elutriation with 500 m mesh sieves [START_REF] Sanders | An introduction to the study of deep-sea benthic faunal assemblages along the Gay Head-Bermuda transect[END_REF] and placed in 70% ethanol. Endolithic organisms were removed from carbonate rubbles using the acid dissolution technique [START_REF] Brock | A method for quantitatively assessing the infaunal community in coral rock[END_REF]. All infaunal organisms were identified to the lowest taxon possible.

Infauna data analyses

Infaunal organisms were categorized into major taxonomic groups at phylum to class level. Mean abundances and standard errors of the means (SE) of the taxonomic groups were calculated and dominant taxa at each site were identified. Abundances of these dominant groups were statistically compared among study sites using non-parametric Kruskal-Wallis test, avoiding assumptions of normal distribution and equality of variances, followed by multiple pairwise testing with the family significance level = 0.05. The statistical comparison initially treated samples from the starboard and the port sides of artificial reefs separately. This initial comparison did not show any significant within-site variability, and therefore, samples from the starboard and port sides were lumped together considering each study site (Sea Tiger, YO257 and 100' hole) as one treatment.

Polychaetes were all identified to the species level according to Day (1967a;1967b) and [START_REF] Bailey-Brock | Phylum Annelida[END_REF]. Polychaete species richness and Shannon-Wiener diversity index (log e) were computed for each sample. These indices were statistically compared by oneway ANOVA followed by Tukey multiple comparisons with family error rate = 0.05.

Differences in polychaete species composition among the study sites were examined by multivariate analyses using the statistical software PRIMER 5.2.9 (PRIMER-E 2002).

Bray-Curtis similarity coefficients between samples were computed using nonstandardized, fourth root transformed polychaete species abundance data. A 2-dimentional non-metric multi-dimensional scaling (MDS) was constructed using the Bray-Curtis similarity coefficients. An analysis of similarity (ANOSIM; [START_REF] Clarke | Change in marine community: an approach to statistical analysis and interpretation[END_REF] was performed to examine if the pattern observed in the MDS showed a statistical difference in polychaete species composition among study sites.

The PRIMER similarity percentage routine (SIMPER; [START_REF] Clarke | Change in marine community: an approach to statistical analysis and interpretation[END_REF] was used to compute the average Bray-Curtis similarities within each study site and the average Bray-Curtis dissimilarities between study sites. The similarities and dissimilarities were broken down into contributions from each polychaete species. Similarity and dissimilarity contributions of each polychaete species were averaged among replicates at each study site. A polychaete species with a high average similarity contribution ( i S ) and a low standard deviation ( ( )

i S SD
) at a site indicates that the species is found at a high, consistent abundance throughout the site, and therefore it typifies the site. Similarly, a polychaete species with a high average dissimilarity contribution ( i δ ) and a low standard deviation ( ( ) i SD δ ) between two sites suggests that the species is a reliable discriminator between the two sites.

Polychaete species composition analyses

The polychaete species compositions were compared with existing data from the biannual monitoring in Mamala Bay conducted by the City and County of Honolulu [START_REF] Swartz | Regional monitoring of benthic fauna in Mamala Bay, O'ahu, Hawai'i[END_REF]. The 40 stations for the biannual monitoring are numbered between 31 and 70 and spread throughout the bay (Fig. 1). The biannual monitoring also contains environmental variable data for each station, such as grain size distribution and depth. The infauna sampling core size and elutriation methods used are the same as those employed in this study, and therefore, the data were directly comparable. The data from the biannual monitoring and this study were combined, and Bray-Curtis similarity coefficients were calculated using non-standardized, fourth root transformed data. Cluster analysis was first performed to find groupings of the samples, and a similarity dendrogram was constructed using group-average linking. Station 61 of the biannual monitoring did not contain any polychaetes, and therefore had a similarity of 0% to all sites, so it was excluded from further analyses. Polychaete species that typify each group or discriminate groups were identified by performing SIMPER.

Associations between environmental variables and the biotic grouping observed in the similarity dendrogram were examined using the PRIMER matching of biotic to confidence interval for each study site. Statistical analysis of the percent rubble fraction data was performed using one-way analysis of variance (ANOVA) followed by Tukey multiple comparisons with family error rate = 0.05. The data was log transformed prior to the analysis to meet the ANOVA assumptions of normality and equality of variances.

Results

Infauna analyses

An average of 119 invertebrates (SE = 33.2) were found in the six sediment samples from the port side of the Sea Tiger artificial reef, 143 invertebrates (SE = 25.4) from the starboard side, 49 invertebrates (SE = 5.7) from the port side of the YO257 artificial reef, 45 invertebrates (SE = 6.8) from the starboard side, and 115 invertebrates (SE = 33.7) from the 100' hole natural reef (Table 1). The four taxa, polychaeta, nematoda, crustacea and oligochaeta, explained more than 85% of all infaunal organisms at the Sea Tiger artificial reef, more than 75% at the YO257 artificial reef, and in excess of 90% at the 100' hole natural reef (Table 1, Fig. 2). These four taxa were identified as major components of infaunal communities at the three study sites. The non-parametric Kruskal-Wallis tests showed that abundances of the total infauna (p = 0.001), nematodes (p < 0.0005), oligochaetes (p = 0.002) and polychaetes (p < 0.0005) were significantly different among study sites. The following pairwise testing showed that YO257 had significantly lower abundances of total infauna, nematodes and polychaetes than the other two sites.

Oligochaete abundances were also significantly lower at YO257 than at Sea Tiger. Despite the low infauna abundance at YO257, abundances of crustaceans were not significantly different among the three study sites (p = 0.859). This means a relatively high percent abundance of crustaceans at YO257.

Polychaetes were, overall, the most abundant taxonomic group at all three sites with an exception at the 100' hole natural reef (Fig. 2). This exception at the 100' hole was due to one replicate with an extremely high nematode abundance (235 individuals). Excluding this replicate lowered the mean nematode abundance at the site to 11.8 (SE = 2.1), making polychaetes the most abundant taxonomic group. Polychaete species richness (S) and Shannon-Wiener diversity index (H') were computed (Table 2), and the results of ANOVA showed that S (p < 0.0005) and H' (p = 0.035) were significantly different among the three study sites. The following Tukey multiple comparisons showed that S was significantly lower at the YO257 artificial reef than at the other two sites, and H' was significantly lower at the YO257 artificial reef compared to the Sea Tiger artificial reef.

Multi-dimensional scaling of polychaetes data showed a grouping of polychaete species composition at the Sea Tiger artificial reef, but not at the YO257 artificial reef and the100' hole natural reef (Fig. 3). The result of ANOSIM confirmed this pattern (global test, p = 0.001) with pairwise tests showing that Sea Tiger had a significantly different polychaete species assemblage compared to those at YO257 (p = 0.001) and 100' hole (p = 0.002). Polychaete species assemblage at YO257 and 100' hole did not differ significantly (p = 0.501). When polychaete species compositions at starboard and port sides of the two artificial reefs were analyzed separately, there were no significant differences in polychaete species composition within each artificial reef site (the starboard and the port sides of YO257 and Sea Tiger with p = 0.470 and p = 0.768 respectively). Polychaete species composition at Sea Tiger was affected by the presence of H. decipiense (p = 0.015). A comparison between polychaete assemblages at Sea Tiger without H. decipiense and at YO257 did not show significant differences in species composition (p = 0.461), while other pairwise comparisons showed significant differences in polychaete assemblages between Sea Tiger and the other two sites regardless of the presence/absence of H. decipiense.

The result of SIMPER showed that the average within-site similarity was lowest at the YO257 artificial reef (27.44%), while the Sea Tiger artificial reef and the 100' hole natural reef both had the average similarity around 45% (Table 3). Pionosyllis heterocirrata (Family Syllidae) showed high contributions to within-site similarities at all three study sites (Table 3). Myriochele oculata (Family Oweniidae) and Euchone sp.

(Family Sabellidae) showed high contributions at Sea Tiger (Table 3) and were identified as contributors to the dissimilarities in polychaete species compositions between Sea Tiger and the other two sites (Table 4). Euchone sp. was the most dominant polychaete representing approximately 35% of all polychaetes found inside the seagrass Halophila decipiense patches at Sea Tiger, while it comprised less than 5% of all polychaetes in the replicates taken outside the seagrass patches. The YO257 artificial reef had relatively low values of ( )

i i S SD S
for all top three contributors compared to the other two study sites (Table 3).

The similarity dendrogram constructed using polychaete species data from this study and existing data from Mamala Bay [START_REF] Swartz | Regional monitoring of benthic fauna in Mamala Bay, O'ahu, Hawai'i[END_REF] showed a similar result (Fig. 4). The polychaete species compositions at the three study sites were separated into two main groups and 3 smaller groups; Group ST mostly consisting of samples from the Sea Tiger artificial reef, Group H-Y consisting of samples from the 100' hole natural reef and the YO257 artificial reefs, and groups Y-1, Y-2 and Y-3 mainly consisting of samples from the YO257 artificial reef. Other samples were separated into seven groups at 30% similarity level; Group A through Group G. Top three polychaete species that contributed to within-group similarities of Group H-Y and Group ST mostly matched the three species that typified 100' hole and Sea Tiger respectively. Top two species that typified YO257

showed high contribution to within-group similarity of Group Y-3. P. heterocirrata

showed high contribution to within-group similarities of all groups except Group Y-1 and Group Y-2.

The result of BIOENV showed patterns of dissimilarities in environmental and biotic variables had the highest correlation when depths and percentage of sand fractions

(1 , 2 and 3 ) were considered ( s = 0.450). Depths showed the highest correlation with the biotic pattern ( s = 0.356) as a single environmental variable, and then, percentages of fine sand 3 (mean diameter of 125 m; s = 0.275). MDS of polychaete abundance with superimposed circles representing values of these environmental variables visually showed relationships between polychaete species compositions and the two environmental variables, depth and percentage of 3 fraction (Fig. 5). Although association between depths and percentages of fine sand was somewhat suspected from the plots (higher percentage of fine sand at shallower depth), these two environmental variables did not show a strong correlation (R 2 = 0.024).

Granulometry

The mean percent rubble fraction was highest at the YO257 artificial reef, then the 100' hole natural reef and the Sea Tiger artificial reef (Fig. 6). The result of ANOVA suggested that mean percent rubble fractions were statistically different among study sites (p < 0.0005), and the following Tukey multiple comparisons showed that the port (seaward) side of YO257 had a significantly higher percentage of rubble fraction compared to any other sites. The starboard (shoreward) side of YO257 also had a significantly higher percentage of rubble fraction than the starboard side of Sea Tiger. The rubble fraction from YO257 contained a large amount of gravel. There was no significant difference in the percentages of rubble at Sea Tiger and 100' hole. The analysis of the remaining fractions excluding rubble showed that 100' hole contained, on average, approximately 80% of sediments with grain size below 1 (larger than 0.5 mm), while Sea Tiger and YO257 had approximately 50% (Fig. 7). These results revealed grain size composition of relatively coarse sediments at 100' hole, medium to fine sediments at Sea Tiger, and medium to fine sediments with a large amount of gravel at YO257.

Discussion

Infauna communities around the two artificial reefs, Sea Tiger and YO257, and the natural reef, 100' hole, were mainly dominated by polychaetes. Other dominant taxonomic groups found at the three sites were nematodes, oligochaetes, and crustaceans. Although the YO257 artificial reef had a lower abundance of infaunal organisms, the taxonomic compositions at the three study sites did not differ greatly. Nematodes were equally abundant at the 100' hole natural reef and the Sea Tiger artificial reef, but less abundant YO257. Abundances of nematodes generally have a positive association with levels of organics in their habitat [START_REF] Raffaelli | Pollution monitoring with meiofauna, using the ratio of nematodes to copepods[END_REF][START_REF] Sandulli | Meiobenthic biodiversity in areas of the gulf of Taranto (Italy) exposed to high environmental impact[END_REF], but they are also associated with fine sediments that facilitate burrowing [START_REF] Wenner | Crustaceans and other invertebrates as indicators of beach pollution[END_REF][START_REF] Mccarthy | Benthic communities associated with carbonate rubble and adjacent soft sediments in a shallow coastal area of Oahu, Hawaii[END_REF]. Similar abundances of nematodes at 100' hole and Sea Tiger suggest that the deployment of Sea Tiger had no more effect, in terms of organic levels, on the surrounding environment than a natural patch reef would. The low abundance of nematodes at YO257 may be due to the large amount of gravel in the sediments (Fig. 6).

Polychaete abundance and species richness were significantly lower at the YO257 artificial reef than the other two study sites, while crustacean percent abundance was relatively higher. A previous study comparing a carbonate rubble habitat with adjacent soft sediments in Mamala Bay showed that the rubble habitat supported higher densities, taxonomic richness and benthic biomass of polychaetes and crustaceans [START_REF] Mccarthy | Benthic communities associated with carbonate rubble and adjacent soft sediments in a shallow coastal area of Oahu, Hawaii[END_REF]. YO257 had a higher percent rubble fraction in the sediments compared with the other two sites, but lower polychaete abundance and species richness at this artificial reef contradicted the finding by [START_REF] Mccarthy | Benthic communities associated with carbonate rubble and adjacent soft sediments in a shallow coastal area of Oahu, Hawaii[END_REF]. This may be because the rubble fractions at YO257 mostly contained basalt gravel that was used in the deployment operation, and therefore, they were not calcium carbonate fragments of corals. Coral rubbles increase pore space and present higher structural complexity and habitat for borers, and therefore they support a variety of epifaunal organisms as well as diverse endolithic cryptofauna [START_REF] Mccarthy | Benthic communities associated with carbonate rubble and adjacent soft sediments in a shallow coastal area of Oahu, Hawaii[END_REF]. While the basalt gravel at the YO257 artificial reef did not contain any endolithic boring organisms, such as polychaetes, it still provided a suitable habitat for epifaunal crustaceans, resulting in their relatively high percent abundance at the site.

Consideration of polychaete species composition through multivariate analysis suggested that the Sea Tiger artificial reef and the 100' hole natural reef had different polychaete communities. MDS of polychaete abundance (Fig. 3) and ANOSIM showed a separation of polychaete species composition at Sea Tiger from those at the other two sites.

Although the presence of the seagrass H. decipiense affected the polychaete species composition at Sea Tiger, SIMPER showed that this site still had a relatively high withinsite similarity in the species composition. The similarity dendrogram (Fig. 4) also separated samples from Sea Tiger (Group ST) from those from 100' hole and YO257

(Group H-Y). The high similarity and strong grouping of polychaete assemblage at Sea Tiger was mostly due to the presence of Myriocele oculata in consistently high abundance.

Euchone sp. also showed a high contribution to the within-site similarity at the Sea Tiger artificial reef, but the lower value of ( )

i i S SD S
indicated a high variability in their abundance. This is because Euchone sp. was highly abundant inside the Halophila decipiense patches, but absent or sparse outside the patches. Seagrass vegetation plays an important role in marine soft bottom ecosystems by increasing habitat complexity and affects distribution of infaunal polychaetes [START_REF] Brito | Polychaetes associated to a Cymodocea nodosa medow in the Canary Islands: assemblage structure, temporal variability and vertical distribution compared to other Mediterranean seagrass meadows[END_REF][START_REF] Omena | Polychaete fauna of seagrass beds (Halodule wrightii Ascherson) along the coast of Rio de Janeiro (Southeast Brazil)[END_REF].

Influence of H. decipiense on the polychaete assemblage at Sea Tiger was also evidenced when ANOSIM was performed treating samples inside and outside the seagrass patches separately. Comparisons between polychaete assemblages at Sea Tiger and YO257 resulted in a significant difference when Sea Tiger samples were all combined, but not when only those taken outside the seagrass patches were used. Nevertheless, the similarity dendrogram (Fig. 4) did not show a separation between the samples inside and outside of the patches at Sea Tiger owing the persistent presence of M. oculata.

Co-existence of M. oculata and Euchone sp. at the Sea Tiger artificial reef (especially inside the seagrass patches) is somewhat contradictory. Myriochele oculata is a tubicolous, surface-feeding detritivore and has been characterized as a possible early indicator of organic enrichment in Hawaii [START_REF] Davis | Analysis of sublittoral benthic habitats in the vicinity of the Sand Island deep ocean sewage outfall: community structure and indicator organisms[END_REF]. It was one of the most characteristic polychaetes in sands underneath a fish culture operation in Mamala Bay and likely to be tolerant to attrition from anoxia [START_REF] Lee | Temporal changes in the polychaete infaunal community surrounding a Hawaiian mariculture operation[END_REF]. Euchone sp. is a small tubicolous suspension feeder that is abundant away from the influence of sewage outfalls and the fish cages in Mamala Bay, and is a negative indicator of organic enrichment [START_REF] Bailey-Brock | Changes in pollution indicators at the Sand Island sewage outfall[END_REF][START_REF] Bailey-Brock | Polychaetes associated with a tropical ocean outfall: synthesis of a biomonitoring program off O'ahu, Hawai'i[END_REF][START_REF] Lee | Temporal changes in the polychaete infaunal community surrounding a Hawaiian mariculture operation[END_REF]. These species, therefore, inversely respond to organic enrichment, and the co-existence suggests that factors other than organic levels may control their abundances at the site.

Polychaete species that showed high contribution to within-site similarities and between-sites dissimilarities have been commonly found in Mamala Bay [START_REF] Swartz | Regional monitoring of benthic fauna in Mamala Bay, O'ahu, Hawai'i[END_REF]. Pionosyllis heterocirrata was the most dominant polychaete species at the three study sites as well as almost all stations of the biannual monitoring (Fig. 4). This species is robust and active, and it is possibly the most commonly encountered species in biogenic sands around the island of Oahu (Bailey- [START_REF] Bailey-Brock | Changes in pollution indicators at the Sand Island sewage outfall[END_REF][START_REF] Bailey-Brock | Polychaetes associated with a tropical ocean outfall: synthesis of a biomonitoring program off O'ahu, Hawai'i[END_REF].

Absence of P. heterocirrata in a few samples from the YO257 artificial reef contributed to relatively low within-site similarity in polychaete composition at the site.

The similarity dendrogram (Fig. 4) suggested that Mamala Bay exhibited diverse polychaete communities, and the result of BIOENV showed that environmental variables (depth and grain size) could affect patterns in polychaete species composition. This suggests a difficulty to study effects of artificial reefs by comparing infaunal data from the artificial reefs and a natural reef because different locations have different environmental parameters. While such comparisons have been suggested as an alternative way in case pre-deployment data did not exist [START_REF] Carr | Artificial reefs: the importance of comparisons with natural reefs[END_REF], effects of artificial reefs are better examined with a comparison between pre-and post-deployment data [START_REF] Wilding | Evaluating artificial reef performance: approaches to pre-and post-deployment research[END_REF]. Comparisons between pre-and post-deployment data were not possible for this study due to a lack of pre-deployment data specific to the two artificial reef sites.

Deployments of artificial reefs have the potential to biologically and/or physically change surrounding natural sand bottom communities [START_REF] Davis | Man-made structures on marine sediments: Effects on adjacent benthic communities[END_REF][START_REF] Ambrose | Influence of an artificial reef on the surrounding infaunal community[END_REF]. Biological and physical factors and how these factors affect the surrounding benthic community are likely to differ at each artificial reef. [START_REF] Fabi | Effects of an artificial reef on the surrounding soft-bottom community (central Adriatic Sea)[END_REF] examined biological and physical factors and concluded that the composition and abundance of an infaunal community appeared to be more affected by physical factors,

Conclusion

This research showed the infaunal communities around the two artificial reefs were predominantly represented by polychaetes, nematodes, crustaceans and oligochaetes. The YO257 artificial reef in general had a less abundant total infauna, fewer nematodes and polychaetes compared to the Sea Tiger artificial reef and the 100' hole natural reef possibly due to the gravel spilled during deployment. Similarity and dissimilarity of polychaete species composition were affected by the variation in sediment grain sizes and depths, and polychaete assemblages were more variable at YO257 in comparison to those at Sea Tiger or 100' hole. Nevertheless, comparisons of polychaete data with those from various natural habitats suggested that polychaete assemblages around the two artificial were typical and within the range of natural variation in Mamala Bay. These findings strongly suggest that the beneficial uses of the two artificial reefs would not have negative impact on the surrounding environment beyond the level of natural variation.

Tables Table 1: The mean abundances of total infauna and dominant taxonomic groups in six replicate samples at each sampling site. Each cell shows the mean abundance and the standard error of the mean in parentheses. 4: Top two polychaete species that highly contribute to dissimilarities in polychaete composition between study sites. The species were identified based on SIMPER on Bray-Curtis dissimilarity (standardized and forth root transformed data). Each cell shows the average polychaete species dissimilarities between study sites and the lists of polychaete species with high contribution to the dissimilarities. The percent contribution ( i δ ) and the ratio of the average dissimilarity to the standard deviation of dissimilarities ( ( )

  previously reported by local divers. Hull epifauna includes rock oysters, tube worms, bryozoans, tunicates and sponges. This artificial reef was regularly visited by the Voyager Submarine until its last operation in June 2001. The YO257 artificial reef (21º15'38''N, 157º50'15''W) was created in 1989 by sinking a 53 m yard oiler approximately 2 km offshore of Waikiki Beach at a depth of 35 m. The yard oiler was cleared of toxic materials prior to the sinking, and large holes were cut through the hull to enhance shelter space, circulation and light penetration. It lies parallel to the shore with the bow facing to the west. The hull was filled with a large quantity of gravel to aid in the deployment operation and to stabilize the vessel on the sea floor. Some of the gravel spilled out and is found right around the vessel. Hull epifauna includes bryozoans (also on the gravel) and three species of coral on decks and superstructure. The interior of the hull is covered with sponges and dense growth of the alien anthozoan, Carijoa riisei. C. riisei has covered the steel plates in the last 17 years.

  The 100' hole (21º15'16''N, 157º50'2''W) is a natural patch reef located approximately 1.5 km offshore of Waikiki Beach at a depth of 32 m. It consists of a cluster of volcanic rocks forming ledges, caves and one large open-ended cavern and is a popular dive site for SCUBA divers because of the coral cover and access for spear fishing.Epifauna growing on the reef includes corals, tube worms, tunicates and sponges, being similar to those on the artificial reefs. This raised patch reef is approximately 30 m in length, 10 m wide, 3 m in height and about 70 m in circumference. This natural reef was chosen for the comparative purpose in this study because of its proximity to the two artificial reefs, relatively similar environmental characteristics (e.g. depth and current intensity) to those of the artificial reefs, and its fairly large size as a patch reef in Mamala Bay, although it is still smaller than the artificial reefs.

2. 2

 2 Infauna sampling Divers collected replicate cores of sediment (7.6 cm in diameter, 6 cm in depth) at the port and starboard sides of Sea Tiger in November 2005, at the port and starboard sides of YO257 in February 2005 and at 100' hole in April 2005. Transect lines were placed along the wrecks, a meter away from the hull, and six replicate cores were randomly taken along the transect lines at the four sampling locations; Sea Tiger port, Sea Tiger starboard, YO257 port and YO257 starboard. Similarly, a transect line was laid around 100' hole and six replicate cores were taken. Half of the replicates were taken inside the seagrass H.

  environmental patterns (BIOENV;[START_REF] Clarke | Change in marine community: an approach to statistical analysis and interpretation[END_REF]. Environmental variables used in this analysis were grain size distribution and depth. Dissimilarities in environmental variables between samples were calculated using normalized Euclidean distances. Dissimilarities in polychaete species composition were based on the Bray-Curtis similarity coefficient; [Bray-Curtis dissimilarity] = 1 -[Bray-Curtis similarity]. The match between patterns of dissimilarities in environmental and biotic variables was measured using the Spearman coefficient ( s ) rank correlation method (Clarke and Warwick, 2001), and environmental variables that group sites in a manner consistent with the polychaete species composition pattern were identified. These environmental variables were separately plotted by superimposing the values as different sizes of circles on MDS of polychaete abundances. 2.5 Granulometry Divers collected sediment cores (6.5 cm in diameter, 6 cm in depth) at the port and starboard sides of Sea Tiger in October 2004, at the port and starboard sides of YO257 in October 2005 and at 100' hole in November 2004. Randomization of the samplings was performed in the same way employed in the collection of infauna samples. Five replicates were taken along the transect lines at the five sampling locations; Sea Tiger port, Sea Tiger starboard, YO257 port, YO257 starboard, and 100' hole. Sample processing was carried out using a wet sieve gravimetric procedure (McCarthy, 1996), and sediment fractions were removed to a nested sieve series (2.0 mm, 1.0 mm, 500 m, 250 m, 125 m and 63 m) by gently poring water through the sieves. The finest silt fraction (11 µm) was removed by filtering a set volume of the collected slurry. All fractions were oven dried at 80°C for a minimum of 24 hours before weighing.Grain sizes were presented as phi ( ) sizes, where = -log 2 [mean diameter in mm]. Each sample was characterized by seven grain size fractions; rubble ( -1 ), sands (0 , 1 , 2 , 3 , 4 ) and silt. The percent rubble fraction was calculated by dividing the rubble fraction by the sum of all seven fractions for each sample. The percent rubble fraction and the remaining six fractions were profiled as separate graphs with 95%

  such as bottom current and variations in the sediment grain size distribution, than biological factors, such as predation. On the other hand, Bortone et al. (1998) suggested the potential of artificial reef fish assemblages to alter trophic structures of nearby fauna through increased predation and emphasized importance of the biological factor in management of artificial reefs. Exact physical and biological changes caused by deployment of the two artificial reefs in Mamala Bay cannot be elucidated without pre-deployment data. Nevertheless, further studies should consider biological and physical factors to determine what biological and/or physical factors may structure the infaunal communities around the two artificial reefs in Mamala Bay. Changes in the physical and biological parameters and infaunal community structure along a gradient away from these reefs should be examined, because such studies may lead to an inference of whether the deployment of the two artificial reefs in Mamala Bay actually altered those parameters of the surrounding environments.

FiguresFig. 1 .

 1 Figures

Fig. 2 .

 2 Fig.2. The mean abundances of major taxonomic groups at each study site. The group "others" includes Mollusca, Echinodermata, Nemertea, Chaetognatha, Chordata, Kinorhyncha, Phoronida, Platyhelminthes, Priapulida, and Sipuncula. The error bars show the standard errors of the mean.

Fig. 3 .

 3 Fig.3. MDS of polychaete abundance data from the three study sites using Bray-Curtis similarities calculated on fourth root transformed polychaete species abundance data. The five sampling sites are indicated by different symbols (stress = 0.21). At the Sea Tiger artificial reef, the replicates 1, 2 and 3 were taken outside the seagrass Halophila decipiense patches, and the replicates 4, 5 and 6 inside the patches.

Fig. 4 .

 4 Fig.4. Similarity dendrogram for hierarchical clustering of the 70 sites, using group-average linking of Bray-Curtis similarities calculated on fourth root transformed data. The numbers 31 through 70 are the 40 samples from the biannual monitoring in Mamala Bay. "STP" is the port side of Sea Tiger, "STS" the starboard side of Sea Tiger, "YOP" the port side of YO257, "YOS" the starboard side of YO257, and "HOLE" the 100' hole natural reef. The top three polychaete species that typify each group are shown on the bottom. The percent contribution ( i S ) and the ratio of the average similarity to the standard deviation of similarities ( ( )i i S SD S) for each species are shown in parentheses.

Fig. 5 .

 5 Fig.5. MDS of polychaete abundance data from the 69 sites using Bray-Curtis similarities calculated on fourth root transformed polychaete species abundance data with superimposed circles representing (a) depths and (b) percentages of 3 fraction (mean diameter of 125 m). Larger circles represent (a) deeper depths and (b) higher percentages of 3 fraction. Labels are consistent with the grouping shown in Figure6.

Fig. 6 .

 6 Fig. 6. Mean percentage of the rubble fraction by weight at each study site. The bars are showing 95% confidence interval.

Fig. 7 .

 7 Fig. 7. Mean cumulative percentages (retained) of all fractions by weight excluding rubbles at each study site. The bars are showing 95% confidence interval.

Table 2 :

 2 The mean polychaete species richness (S) and Shannon-Wiener diversity index (H') in six replicate samples at each sampling site. The standard error of the mean is shown in parentheses.

		Sea Tiger Port	Sea Tiger Starboard	YO257 Port	YO257 Starboard	100' Hole
	S	15.33 (1.52)	16.67 (1.60)	8.17 (1.40)	8.33 (1.05)	13.33 (1.02)
	H'	2.128 (0.10)	2.152 (0.07)	1.627 (0.13)	1.885 (0.14)	1.887 (0.22)

Table 3 :

 3 The top three polychaete species that typify each study site based on SIMPER on Bray-Curtis similarity (non-standardized and forth root transformed data). The top row shows the average polychaete species similarity within each site. The bottom row shows lists of polychaete family with high contribution to the similarity within each site.

	The

Table

Acknowledgments

We thank the University of Hawaii Wormlab staff for assistance, D. Pence, University of Hawaii diving safety program for field support, and the University of Hawaii Water Resources Research Center for a research assistantship and use of data for comparative purposes. We are grateful to Drs. C. Birkeland and A. Taylor for their invaluable advice on the statistical analyses, and to Dr. R. Swartz for reviewing a draft of the paper. This research fulfilled part of the MS degree requirements for A. Fukunaga and was funded in part by an Edmondson award through the University of Hawaii, Zoology Department. This is a contributed paper CP-2006-00 of the University of Hawaii, Water Resources Research Center.