A B Filipowicz
email: amyfilipowicz@hotmail.com

J E Weinstein

D M Sanger

Dietary Transfer of Fluoranthene from an Estuarine Oligochaete (Monopylephorus rubroniveus) to Grass Shrimp (Palaemonetes pugio): Influence of Piperonyl Butoxide

Keywords: bioaccumulation, cytochrome P-450, dietary transfer, fluoranthene, Monopylephorus rubroniveus, Palaemonetes pugio, polycyclic aromatic hydrocarbons, piperonyl butoxide

The objective of this study was to determine the potential for dietary transfer of sedimentassociated fluoranthene from tubificid oligochaetes (Monopylephorus rubroniveus) to grass shrimp (Palaemonetes pugio). Grass shrimp, either in the presence or absence of sublethal waterborne concentrations of the metabolic inhibitor, piperonyl butoxide (PBO), were fed fluoranthene-dosed oligochaetes for 5 days. All grass shrimp bioaccumulated fluoranthene; however, bioaccumulation was 3X higher in the presence of PBO. Trophic transfer coefficients (TTCs) were 0.02 and 0.01 in the presence and absence of PBO, respectively. Following the 5-day accumulation period, shrimp in both treatments were allowed to depurate for 3 days. Depuration rates were significantly higher in PBO-exposed shrimp. These results demonstrated that sediment-associated fluoranthene can be transferred through the diet from oligochaetes to grass shrimp, and the presence of PBO enhanced fluoranthene bioaccumulation. However, the comparatively low TTCs suggest that biomagnification of fluoranthene in estuarine food webs is low.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in estuarine ecosystems. Primary sources of PAHs in these systems include the combustion of fossil fuels and direct petroleum inputs [START_REF] Ngabe | Polycyclic aromatic hydrocarbons in storm water runoff from urban and coastal South Carolina[END_REF][START_REF] Ross | Polycyclic aromatic hydrocarbons in the San Francisco Estuary water column: Sources, spatial distributions and temporal trends (1993-2001)[END_REF].

Other sources include creosote oil leachates, historical industrial sites and tire dust [START_REF] Bos | Fluoranthene, a volatile mutagenic compound, present in creosote and coal tar[END_REF][START_REF] Takada | Determination of polycyclic aromatic hydrocarbons in urban street dusts and their source materials by capillary gas chromatography[END_REF]Walker, Dickhut, Chisholm-Brause, Sylva & Reddy, 2005). As a group, these compounds are generally considered to be hydrophobic and lipophilic, with log K ow values ranging from 3.31 for bicyclic naphthalene to 6.51 for hexicyclic benzo [ghi]perylene [START_REF] May | The solubility of polycyclic aromatic hydrocarbons in aqueous systems[END_REF]USEPA, 1982). Consequently, these compounds tend to sorb to suspended particles and sediments where deposit feeding organisms may subsequently bioaccumulate them through ingestion of sediments and/or contact with sediments and pore water.

Recent studies in tidal creek and salt marsh complexes along the South Carolina coast have found comparatively high levels of PAHs in intertidal sediments (mean total PAHs = 3.97 µg/g dry weight, range = 0.12-37.03 µg/g dry weight) within suburbanized, urbanized and industrialized watersheds [START_REF] Sanger | Tidal creek and salt marsh sediments in South Carolina coastal estuaries: I. Distribution of trace metals[END_REF]. Fluoranthene is one of the most common PAHs found in the environment. Sediment concentrations of fluoranthene in South Carolina tidal creeks range from 5.69 µg/g dry weight in creeks that drain urban watersheds (e.g., Vardell Creek) to 0.003µg/g dry weight in creeks that drain forested or reference watersheds (e.g., Sawmill Creek) [START_REF] Sanger | Tidal creek and salt marsh sediments in South Carolina coastal estuaries: I. Distribution of trace metals[END_REF].

Estuarine habitats are highly productive and serve as critical nursery and feeding areas for many resident and transient species [START_REF] Cain | Annual occurrence, abundance, and diversity of fish in a South Carolina intertidal creek[END_REF][START_REF] Weinstein | Shallow marsh habitats as primary nurseries for fishes and shellfish, Cape Fear, North Carolina[END_REF][START_REF] Wenner | Utilization of shallow estuarine habitats in South Carolina, USA, by postlarval and juvenile stages of Penaeus spp. (Decapoda: Penaeidae)[END_REF]. Benthic infaunal organisms, especially oligochaetes, are abundant in tidal creeks and salt marshes, and are believed to play an important role in coastal food webs. Oligochaetes have a high nutritional value and are a major food item in the diet of juvenile fish and crustaceans (Giere & Pfannkucke, 1982). Many oligochaetes also contain lipids which are critical for shrimp reproduction [START_REF] Middleditch | Maturation of Penaeid shrimp: Dietary fatty acids[END_REF]. Grass shrimp are also abundant in estuaries [START_REF] Welsh | The role of grass shrimp (Palaemonetes pugio) in a tidal marsh ecosystem[END_REF] and have been shown to be important in food web dynamics by serving as detritivores, primary consumers and secondary consumers [START_REF] Bell | Field evidence that shrimp predation regulates meiofauna[END_REF][START_REF] Kneib | Predation and disturbance by grass shrimp, Palaemonetes pugio Holthuis, in soft-substratum benthic invertebrate assemblages[END_REF][START_REF] Fleeger | Consumption of microalgae by the grass shrimp Palaemonetes pugio[END_REF]. Although grass shrimp do not feed exclusively on oligochaetes, they do consume both macro-and meiobenthos and seem to feed non-selectively, thereby consuming more of the most dominant taxa [START_REF] Bell | Field evidence that shrimp predation regulates meiofauna[END_REF].

The tubificid oligochaete Monopylephorus rubroniveus is the dominant macroinfaunal organism in the upper reaches of tidal creeks in South Carolina [START_REF] Sanger | Physical, chemical and biological environmental quality of tidal creeks and salt marshes in South Carolina estuaries[END_REF][START_REF] Lerberg | Responses of tidal creek macrobenthic communities to the effects of watershed development[END_REF][START_REF] Gawle | Tidal creek responses to watershed development: A comparison of summer 1994 and winter 2000 data[END_REF][START_REF] Gillett | Ecology of tidal creek oligochaetes: Changes in abundance and secondary production of the numerically dominant species Monopylephorus rubroniveus[END_REF]. Densities of M. rubroniveus can exceed 90,000 individuals/m 2 in some intertidal areas [START_REF] Lerberg | Effects of watershed development on macrobenthic communities in the tidal creeks of the Charleston Harbor Estuary[END_REF]. Previous laboratory toxicological studies involving M. rubroniveus have demonstrated that this species is highly tolerant of fluoranthene, one of the predominant PAH congeners found in the environment. In acute sediment exposures with fluoranthene, the 10-day LC 50 for M. rubroniveus exceeded 3.91 µg/g dry weight [START_REF] Weinstein | Bioaccumulation and toxicity of fluoranthene in the estuarine oligochaete Monopylephorus rubroniveus[END_REF]. In addition, M. rubroniveus has the ability to bioaccumulate large amounts of sediment-associated fluoranthene in its tissues. In the above mentioned study, mean tissue concentrations of fluoranthene were as high as 6,431 µg/g dry weight [START_REF] Weinstein | Bioaccumulation and toxicity of fluoranthene in the estuarine oligochaete Monopylephorus rubroniveus[END_REF]. Using environmentally-relevant sediment concentrations of PAHs (43.2 µg fluoranthene/g dry weight), the laboratory-derived biota-sediment accumulation factor (BSAF) for M. rubroniveus was 8.4 (unpublished data).

The high bioaccumulation potential of fluoranthene by M. rubroniveus suggests the likelihood for dietary transfer of this compound to predators. This potential could be enhanced by factors that affect rates of biochemical metabolism, such as environmental quality, an organism's physiological status, or chemicals like piperonyl butoxide (PBO). PBO is a competitive inhibitor of cytochrome P-450, and a common additive of thousands of pyrethroid pesticide products used in mosquito control (e.g., Scourge ® , Anvil ® ), agriculture (e.g., Pyrenone ® ) and household pest control (e.g., Deep 6 Flea Killer ® , Raid Ant and Roach Killer ® ). Many PAH compounds, including fluoranthene, are metabolized by cytochrome P-450 [START_REF] Stegeman | Molecular responses to environmental contamination: Enzyme and protein systems as indicators of chemical exposure and effect[END_REF][START_REF] Akkanen | Biotransformation and bioconcentration of pyrene in Daphnia magna[END_REF], thus inhibition of this enzyme by PBO could result in increased bioaccumulation and dietary transfer of PAHs.

The objectives of the current study were two-fold. First, we wanted to evaluate the potential for dietary transfer of fluoranthene between the oligocheate (M. rubroniveus) and a model predator species, the grass shrimp (Palaemonetes pugio). Second, we wanted to investigate the influence of PBO on dietary transfer of fluoranthene between these two species.

Methods

Experimental Overview

To achieve the objectives of this study, two different experiments were performed. First, the overall sensitivity of grass shrimp, Palaemonetes pugio, to PBO was evaluated using a 96-hour acute toxicity test. Second, a fluoranthene dietary transfer study was conducted using the oligochaete, Monopylephorus rubroniveus, as the prey item and grass shrimp as the predator. Four treatments were performed. Treatment A consisted of PBO-exposed shrimp fed fluoranthene-dosed oligochaetes. Treatment B consisted of control shrimp fed fluoranthene-dosed oligochaetes. Treatment C consisted of PBO-exposed shrimp fed control oligochaetes. Treatment D consisted of control shrimp fed control oligochaetes (Table 1).

Test Organisms

Grass shrimp for the toxicological test and the dietary transfer experiment were collected with a dip net from Leadenwah Creek on Wadmalaw Island, SC, an unimpacted reference site for PAHs (unpublished data). In the laboratory, shrimp were held for two days in an aerated aquarium and were not fed. Adult shrimp that were free of external parasites or other abnormalities were selected for the experiments.

Oligochaetes for the dietary transfer experiment were obtained from sediment that was collected from the intertidal banks of the upper reach of Okatee Creek, near Bluffton, SC. Sediment was collected in plastic bags, brought back to the laboratory and placed on top of ice in a cooler for four hours. These oligochaetes demonstrate negative taxis to cold and are easily collected from the sediment surface following cold exposure with minimal sieving and sorting required.

Test Compounds

Technical grade PBO (90% purity) was obtained from ACROS Organics and was used for the toxicological test and the dietary transfer experiment. Fluoranthene (98% purity) was obtained from Sigma-Aldrich and was used to spike sediment in the oligochaete culture for the dietary transfer experiment. Neither PBO nor fluoranthene are soluble in water and ethanol and acetone were used as carriers, respectively.

Toxicological Test

Before its use in the dietary transfer experiment, the toxicity of PBO to grass shrimp was assessed in a 96-hour acute toxicity test. Five nominal concentrations of PBO were tested (0, 250, 500, 1000 and 2000 µg/L), in addition to an ethanol carrier control. A constant volume of ethanol (equivalent to 0.003% of total solution volume) was used in all treatments. All treatments were replicated three times. Replicates consisted of 10 grass shrimp in 1000 mL test beakers. The test beakers were aerated during the experiment and the grass shrimp were not fed. Solutions were renewed every other day. Mortality and water quality parameters were assessed every 24 hours.

Oligochaete Dosing

Fluoranthene-dosed and control oligochaetes for the dietary transfer study were produced by establishing two oligochaete cultures: one culture consisted of fluoranthenespiked sediment and the other culture contained control sediment that was not spiked with fluoranthene. Sediment for both cultures was collected from the intertidal banks of the upper reaches of Okatee Creek (near Bluffton, SC). Upon return to the laboratory, sediment was wet sieved through a 500 µm mesh screen, left to settle overnight, drained and frozen to remove any unwanted biota. After 24 hours, sediment was thawed and divided between the two cultures.

For the fluoranthene-spiked culture, ~1.5 kg wet weight sediment was spiked with 0.029 g fluoranthene that was dissolved in 5 mL of acetone. For the control culture, 5 mL acetone was added to ~1.5 kg wet weight sediment. Both fluoranthene-spiked and control sediments were rolled overnight uncovered in a 2 L bottle to allow the acetone to evaporate.

The fluoranthene-spiked and control sediment mixtures were placed in separate glass trays (28 cm x 20 cm x 5 cm, l x w x h) and ~1 L of seawater (25 psu) was added. The sediment and water mixtures were allowed to settle overnight prior to adding the oligochaetes. Oligochaetes were added to the trays two days before the start of the dietary transfer experiment, which allowed the oligochaetes in spiked sediment to bioaccumulate fluoranthene. Previous experiments in our laboratory have demonstrated that M. rubroniveus achieves steady state tissue residues of fluoranthene in spiked sediment in <32 h (unpublished data).

Dietary Transfer Experiment

Dietary transfer of fluoranthene from oligochaetes to grass shrimp was assessed under control conditions and in seawater fortified with PBO. The basic experimental design consisted of a 5-day feeding (bioaccumulation) period, followed by a 3-day nonfeeding (depuration) period. There were four treatments, as previously described (see Table 1).

Grass shrimp (60 individuals) were held separately in 600 mL beakers with 500 mL of seawater (25 psu). Beakers were aerated and covered with screen. PBO-exposed shrimp (treatments A and C) were maintained in seawater fortified with 575 µg PBO/L (nominal). This concentration of PBO represented the approximate LC 10 value determined from the toxicological study. Grass shrimp were introduced to test conditions one day prior to the start of the experiment for acclimation. During the 5-day feeding period, either 20 fluoranthene-dosed oligochaetes (treatments A and B) or 20 control oligochaetes (treatments C and D) were fed to each grass shrimp once per day. During the 3-day depuration period, grass shrimp were not fed. In all treatments, water was changed every other day, and water quality parameters were monitored daily (Table 2).

During the feeding period, three grass shrimp in treatments A and B were sampled for fluoranthene tissue residues each day between four and seven hours after feeding, which allowed time for gut clearance [START_REF] Hoyt | Serological estimation of prey protein gut residence time and quantification of meal size for grass shrimp consuming meiofaunal copepods[END_REF]. In treatments C and D, three grass shrimp were sampled only on days 1 and 5 of the feeding period. During the depuration period, three grass shrimp in treatments A and B were sampled for fluoranthene tissue residues each day. Treatments C and D were not continued into the depuration phase because no fluoranthene uptake was expected, as these shrimp were only fed control oligochaetes.

Analytical Chemistry

The amount of fluoranthene that was transferred to grass shrimp through the diet was quantified using the following procedure. Grass shrimp were rinsed with deionized water, placed on aluminum foil, allowed to air dry at room temperature in the dark for eight hours and weighed. Fluoranthene was extracted from grass shrimp by grinding them in a Ten Broeck tissue homogenizer with 4 mL extraction fluid [cyclohexane:acetone (2:1)] and quantified using reverse phase HPLC. Fifty µL of sample or standard solution was injected directly onto an Alltech 3.9 mm x 15 cm alphaBond C18 column. An isocratic elution was performed with acetonitrile:water (9:1) at 1.0 mL/min. A Hewlett-Packard 1100 Series Fluorescence Detector was used at an excitation wavelength of 360 nm with emission detection set at 460 nm. Peaks were recorded and quantified using Agilent ChemStation software (version A.08.03). The limit of detection for fluoranthene was 0.3 µg/L, or 15.0 pg total mass. The recovery of fluoranthene in spiked grass shrimp tissue samples was 88.8 ± 4.0% (n = 3). All fluoranthene tissue residues are reported on a dry weight basis.

Bioaccumulation of fluoranthene in oligochaetes from the sediment cultures was determined for days 1 through 5 of the dietary transfer experiment. Oligochaete tissue residues were determined in a manner generally similar to that described above for grass shrimp with the following exceptions: 1) three oligochaetes were pooled to form one sample, and 2) extractions were performed with only 1 mL extraction fluid. The recovery of fluoranthene in spiked oligochaete tissue samples was 98.2 ± 5.5% (n = 8).

Fluoranthene concentrations in the sediment cultures were determined by removing ~0.2 g sediment on days 1 and 5. Sediment was placed on aluminum foil and allowed to air dry at room temperature in the dark for eight hours. Once dry, the sediment was homogenized using a mortar and pestle, and then ~50 mg of sediment was subsampled and split into two replicate samples. Each sample was sonicated with 4 mL extraction fluid, centrifuged at 3000 rpm for 20 min in glass centrifuge tubes, and quantified using reverse phase HPLC as described above for tissue residues. The recovery of fluoranthene in spiked sediment samples was 109.0 ± 8.3% (n = 2). Sediment concentrations are reported as the mean of the two replicate samples on a dry weight basis.

Data Analysis

For the PBO acute toxicity test, the 96-hour LC 50 and corresponding 95% confidence intervals were determined using the trimmed Spearman Karber method (1.7% trim) (ToxCalc version 5.0.23). For the dietary transfer experiment, bioaccumulation of fluoranthene in grass shrimp was evaluated with a two-way analysis of variance (ANOVA) and Tukey's honestly significant difference test. The two main effects tested were treatment (only treatments A and B were tested) and day (1 through 5). Grass shrimp depuration rates were evaluated by comparing the linear regression lines of treatments A and B. Results were considered significant at α < 0.05.

Trophic transfer coefficients (TTCs) were calculated in order to assess whether biomagnification of fluoranthene occurred in the presence and absence of PBO. A TTC is the ratio of contaminant residue in the consumer tissue (grass shrimp) to that in the prey tissue (oligochaete). Biomagnification is judged to take place if the TTC is > 1 [START_REF] Suedel | Trophic transfer and biomagnification potential of contaminants in aquatic ecosystems[END_REF]. For this study, TTCs were averaged for each treatment. Water quality data are presented as mean + standard deviation (S.D.) and fluoranthene concentrations are expressed as mean + standard error (S.E.).

Results

Toxicological Test

Grass shrimp mortality in both the control and carrier control treatments after 96hours was 1.6%. Mortality to PBO occurred in a concentration-dependent fashion and ranged from 3.3% at the lowest concentration (250 µg/L) to 100% at the highest concentration (2000 µg/L; Figure 1). The 96-hour LC 50 was 1043 µg/L (95% CI = 915-1189 µg/L). Water quality parameters remained relatively constant throughout the duration of the test (Table 2).

Dietary Transfer Experiment

Fluoranthene concentrations in the spiked sediment culture were 39.0 ± 7.5 µg/g and 58.1 ± 2.5 µg/g dry weight on days 1 and 5 of the dietary transfer experiment, respectively. These concentrations of fluoranthene are similar to the total PAH levels found in tidal creeks with urbanized watersheds [START_REF] Sanger | Tidal creek and salt marsh sediments in South Carolina coastal estuaries: I. Distribution of trace metals[END_REF].

Oligochaetes appeared to achieve steady-state tissue residues of fluoranthene after two days of exposure to the spiked sediment during the acclimation period (Figure 2). During the 5-day feeding period, the mean fluoranthene tissue residue in the oligochaetes was 671.6 ± 32.0 µg/g dry weight. Fluoranthene concentrations in the control culture sediment and the control oligochaetes were below the level of detection on both days 1 and 5.

During the 5-day feeding period, grass shrimp in treatments A and B bioaccumulated fluoranthene in their tissues (Figure 3). Within each treatment, fluoranthene tissue residues did not change over the course of the five feeding days (p = 0.6110). However, tissue residues were significantly different between treatments and were three times higher in treatment A than treatment B (p < 0.0001). Grass shrimp in treatment A (PBO exposed shrimp fed fluoranthene-dosed oligochaetes) had mean fluoranthene tissue residues of 13.9 ± 1.2 µg/g, and grass shrimp in treatment B (control shrimp fed fluoranthene-dosed oligochaetes) had mean fluoranthene tissue residues of 4.9 ± 0.9 µg/g. The TTCs for dietary transfer of fluoranthene from oligochaetes to grass shrimp were 0.02 and 0.01 in treatments A and B, respectively. Grass shrimp in treatments C and D were fed control oligochaetes and had comparatively low levels of fluoranthene in their tissues (Figure 4). Mean fluoranthene residues in grass shrimp in treatment C (PBO exposed shrimp fed control oligochaetes) and D (control shrimp fed control oligochaetes) were 0.07 ± 0.02 µg/g and 0.03 ± 0.01 µg/g, respectively. These low tissue residues of fluoranthene were probably the result of the volatilization of fluoranthene from the fluoranthene-spiked oligochaete cultures, which were maintained in the same laboratory. Throughout the feeding experiment, mortality in grass shrimp was low (1.6%) and water quality parameters remained relatively constant (Table 2).

During the 3-day depuration period, mean fluoranthene tissue residues in grass shrimp from treatments A and B decreased in a time-dependent fashion (Figure 5). Over the three day period, fluoranthene tissue residues in grass shrimp decreased by 80.9% and 72.9% in treatments A and B, respectively. The rate of depuration was significantly faster in treatment A (slope = -3.12 µg fluoranthene/g/day) than in treatment B (slope = -1.79 µg fluoranthene/g/day) (p = 0.0300). However, at the end of the three day depuration period, mean fluoranthene tissue residues from grass shrimp in treatment A were still ~25 times higher than treatment B.

Discussion

The results of the present study demonstrate that PAHs in estuarine sediments can be transferred from benthic infauna to shrimp through dietary transfer. Grass shrimp in this study bioaccumulated fluoranthene from a diet of oligochaete worms that had been cultured in fluoranthene-spiked sediments. These results corroborate those of previous studies in marine systems which have demonstrated that sediment-associated PAHs can be transferred to bottom-feeding organisms through the diet. For example, winter flounder (Pseudopleuronectes americanus) fed polychaetes (Nereis virens) dosed with benzo[a]pyrene accumulated both the parent compound and its metabolites [START_REF] Mcelroy | Trophic transfer of benzo[a]pyrene metabolites between benthic marine organisms[END_REF]. Other studies have demonstrated that waterborne PAHs can also be transferred in the diet. Rotifers (Brachionis plicatilis) fed algae (Isochrysis galbana) cultured in phenanthrene-dosed water bioaccumulated phenanthrene in their tissues [START_REF] Wolfe | Influence of dispersants on the bioavailability and trophic transfer of phenanthrene to algae and rotifers[END_REF]. Likewise, clams (Mercenaria mercenaria) fed diatoms (Thalassiosira pseudomana) cultured in benzo[a]pyrene-spiked water bioaccumulated that PAH in their tissues [START_REF] Dobroski | Accumulation of benzo[a]pyrene in a larval bivalve via trophic transfer[END_REF]. In fact, these authors concluded that dietary transfer and direct uptake from water contributed equally to body burdens of benzo[a]pyrene in clams. This suggests that dietary uptake can, in some cases, be a significant contributor to overall PAH tissue residues in consumers.

The presence of the metabolic inhibitor, PBO, in the water resulted in a three-fold increase in the bioaccumulation of fluoranthene in grass shrimp, demonstrating that PBO enhanced the dietary uptake of fluoranthene. PBO is a general cytochrome P-450 inhibitor and is commonly used as a synergist in synthetic pyrethroid pesticide formulations [START_REF] Osimitz | The safety assessment of piperonyl butoxide[END_REF]. In pyrethroid formulations, PBO impedes the oxidative breakdown ability of cytochrome P-450, which reduces biotransformation and allows the pesticides to accumulate to lethal levels. In addition to pesticides, cytochrome P-450 is also responsible for the metabolism of PAHs in vertebrate and invertebrate species [START_REF] Stegeman | Molecular responses to environmental contamination: Enzyme and protein systems as indicators of chemical exposure and effect[END_REF][START_REF] James | Cytochrome P450 monooxygenases in crustaceans[END_REF][START_REF] James | Review: Cytochromes P450 in crustacea[END_REF]. Biotransformation of PAHs naturally occurs at a slower rate in invertebrates than vertebrates due to lower concentrations of cytochrome P-450 [START_REF] James | Cytochrome P450 monooxygenases in crustaceans[END_REF][START_REF] James | Review: Cytochromes P450 in crustacea[END_REF][START_REF] Lee | Review: Annelid cytochrome P-450[END_REF]. These rates of transformation are further reduced by the presence of metabolic inhibitors like PBO. Water fleas (Daphnia magna) exposed to PBO exhibited a reduction in the biotransformation of pyrene, which was likely mediated through an inhibition of cytochrome P-450 [START_REF] Akkanen | Biotransformation and bioconcentration of pyrene in Daphnia magna[END_REF]. In the current study, increased bioaccumulation of fluoranthene in PBO-exposed grass shrimp was also likely the result of an inhibition of cytochrome P-450 and subsequent reduction in the rate of fluoranthene biotransformation, although this was not directly measured. Nevertheless, the results of the current study support the notion that cytochrome P-450 plays an important role in the metabolism of PAHs in crustaceans.

Exposure to PBO during the accumulation period also influenced fluoranthene depuration kinetics in grass shrimp. Depuration rates were significantly higher in the PBO-exposed grass shrimp than in control grass shrimp, suggesting a rapid recovery of those cytochrome P-450 isozymes that had been inhibited during the accumulation period. Despite the faster rate of depuration, PBO-exposed grass shrimp had significantly higher levels of fluoranthene in their tissues at the end of the 3-day depuration period relative to control shrimp, whose fluoranthene tissue residues were similar to background levels. The continued presence of fluoranthene in the PBO-exposed grass shrimp could have profound physiological implications for dose-and time-dependent responses, such as DNA strand breaks [START_REF] Lee | Exposure of grass shrimp to sediments receiving highway runoff: Effects on reproduction and DNA[END_REF]) and UV-enhanced photosensitization reactions [START_REF] Weinstein | Influence of salinity on the bioaccumulation and photoinduced toxicity of fluoranthene to an estuarine shrimp and oligochaete[END_REF]. Also, the continued presence of fluoranthene in the grass shrimp tissues increases the likelihood that fluoranthene could be transferred to higher trophic levels through the diet. The physiological and ecological implications of lingering fluoranthene residues in PBO-exposed organisms certainly warrant further investigation.

The acute toxicity of PBO has been evaluated in a variety of aquatic species, and these studies have demonstrated that invertebrates are generally more sensitive to PBO than fish [START_REF] Osimitz | An ecological risk assessment of piperonyl butoxide[END_REF]. Previous studies involving invertebrates have demonstrated that PBO is highly toxic, with reported LC 50 values for water fleas (Ceriodaphnia dubia) and amphipods (Hyalella azteca) of 330 µg/L and 530 µg/L, respectively [START_REF] Baily | Development of procedures for identifying pesticide toxicity in ambient waters: Carbofuran, diazinon, chlorpyrifos[END_REF][START_REF] Ankley | Influence of piperonyl butoxide on the toxicity of organophosphate insecticides to three species of freshwater benthic invertebrates[END_REF]. By comparison, studies involving fish have demonstrated that PBO is only moderately toxic to this taxon, with reported LC 50 values ranging from 3,940 µg/L in sheepshead minnow (Cyprinodon variegatus) to 11,200 µg/L in rainbow trout (Salmo gairdneri) [START_REF] Erickson | The effect of piperonyl butoxide on hepatic cytochrome P-450 dependent monooxygenase activities in rainbow trout (Salmo gairdneri)[END_REF]. The results of the current study suggest that grass shrimp (LC 50 = 1,043 µg/L) are somewhat less sensitive to PBO compared to previously tested invertebrate species, but are not as tolerant as previously tested fish species. This finding is corroborated by a study involving freshwater prawns (Palaemon paucidens), which reported an LC 50 value of 3,500 µg/L [START_REF] Kobayashi | Reduction of the toxicity of fenitrothion to prawns by piperonyl butoxide[END_REF].

Despite the fact that fluoranthene bioaccumulation was observed in grass shrimp that were fed fluoranthene-dosed oligochaetes, the calculated TTCs suggest that biomagnification of fluoranthene in the food chain is unlikely. Biomagnification is likely to occur when the TTC values in predator-prey relationships are >1.0 [START_REF] Suedel | Trophic transfer and biomagnification potential of contaminants in aquatic ecosystems[END_REF]. In the current study, TTC values between grass shrimp and oligochaete worms were 0.02 and 0.01 in the presence and absence of PBO, respectively, or 50 to 100-fold less than those values associated with biomagnification. In a previous field study investigating dietary transfer of PAHs between zooplankton and mussels (Mytilus edulis) [START_REF] Broman | An in situ study on the distribution, biotransformation and flux of polycyclic aromatic hydrocarbons (PAHs) in an aquatic food chain (seston-Mytilus edulis L-Somateria mollissima L) from the Baltic: An ecotoxicological perspective[END_REF] reported TTC values of 0.2, 0.3 and 0.6 for phenanthrene, fluoranthene and anthracene, respectively. These TTC values, which are higher than those reported in the current study, are probably a reflection of the limited activity of cytochrome P-450 isozymes and other mixedfunction oxygenases in molluscs compared to crustaceans [START_REF] Livingstone | Cytochrome P-450 and oxidative metabolism in invertebrates[END_REF]. Nevertheless, these results collectively suggest that the potential for biomagnification of PAHs in aquatic food webs is low.

Despite its widespread use in household and agricultural pesticides and mosquito adulticides, few attempts have been made to measure concentrations of PBO in the environment [START_REF] Osimitz | The safety assessment of piperonyl butoxide[END_REF]. Application rates of PBO range from 0.0036 lb/acre and 0.021 lb/acre in mosquitocide formulations (Anvil ® and Scourge ® , respectively) (Bayer Environmental Science, 2002;Clarke Mosquito Control Products, 2005) to 0.037 lb/acre in certain agricultural products [START_REF] Kenega | Factors to be considered in the evaluation of the toxicity of pesticides to birds in their environment[END_REF]. Immediately following mosquito spraying, PBO has been detected in surface water at concentrations ranging from 0.7 to 15,000 ng/L [START_REF] Zulkosky | Acute toxicity of resmethrin, malathion and methoprene to larval and juveline American lobsters and analysis of pesticide levels in surface waters after Scourge ™ , Anvil ™ and Altosid ™ application[END_REF][START_REF] Amweg | Effect of piperonyl butoxide on permethrin toxicity in the amphipod Hyalella azteca[END_REF]. Water samples collected from urban creeks, where household pesticides may have been applied, generally had undetectable levels of PBO [START_REF] Amweg | Effect of piperonyl butoxide on permethrin toxicity in the amphipod Hyalella azteca[END_REF]. At these concentrations, PBO itself is not particularly toxic; however, it has the potential to increase the toxicity of other compounds. Under most natural conditions, PBO is relatively short-lived with half-lives in the water column and sediments of 1.6 days and 24 days, respectively [START_REF] Arnold | The fate and behavior of piperonyl butoxide in the environment[END_REF]. However, under certain conditions, including the absence of light and/or the absence of oxygen, PBO is extremely stable with half-lives in water and sediments that exceeded one year [START_REF] Arnold | The fate and behavior of piperonyl butoxide in the environment[END_REF]. Given that tidal creek-salt marsh systems can occasionally be light and oxygen-limited, future studies investigating the prevalence and persistence of PBO in the estuarine environment is warranted.

Conclusion

This study demonstrates that environmentally-relevant sediment concentrations of fluoranthene can travel through trophic pathways to predatory species in tidal creek and salt marsh complexes of the Southeastern U.S. The oligochaete Monopylephorus rubroniveus is the most abundant macroinfaunal organism in intertidal creeks and has a high toxicological tolerance to fluoranthene, and likely other PAHs. In general, oligochaetes, and other invertebrates, have lower levels of cytochrome P-450 and cannot rapidly metabolize and eliminate PAHs [START_REF] Lee | Review: Annelid cytochrome P-450[END_REF]. As such, oligochaetes can accumulate relatively high levels of contaminants and can pass them on to primary consumers, including crustaceans and demersal fish. The presence of metabolic inhibitors, like PBO, can enhance the dietary transfer of PAHs. However, dietary transfer as a mechanism of PAH bioaccumulation in estuarine food webs is probably limited to these primary consumers, due to their higher metabolic capacities for removing contaminants from body tissues.

Acknowledgements

This research was sponsored by the NOAA Center for Sponsored Coastal Ocean Research/Coastal Ocean Program, through the South Carolina Sea Grant Consortium, pursuant to National Oceanic and Atmospheric Administration Award No. NA960PO113. The authors are grateful to Fred Holland, Chrissy Smith and Keane Phillips for their assistance in this research. This article is contribution number 291 from the Grice Marine Laboratory at the College of Charleston.

Figure Captions Figure 1 .

 Captions1 Figure Captions

Figure 2 .

 2 Figure 2. Mean concentration of fluoranthene (µg FLU/g) in whole tissues of fluoranthene-dosed oligochaetes (Monopylephorus rubroniveus) fed to grass shrimp during the 5-day bioaccumulation period. Error bars represent 1 standard error.

Figure 3 .

 3 Figure 3. Mean concentration of fluoranthene (µg FLU/g) in whole tissues of grass shrimp (Palaemonetes pugio) that were fed fluoranthene-dosed oligochaetes. Grass shrimp in Treatment A were exposed to piperonyl butoxide (PBO) and had significantly higher fluoranthene tissue residues (p > 0.0001). Error bars represent 1 standard error.

Figure 4 .

 4 Figure 4. Mean concentration of fluoranthene (µg FLU/g) in whole tissues of grass shrimp (Palaemonetes pugio) that were fed control oligochaetes. Grass shrimp in Treatment C were exposed to piperonyl butoxide (PBO). Error bars represent 1 standard error.

Figure 5 .

 5 Figure 5. Mean concentration of fluoranthene (µg FLU/g) in whole tissues of grass shrimp (Palaemonetes pugio) during the last day of uptake (U5) and three days of depuration (D1 -D3). Grass shrimp in Treatment A were exposed to piperonyl butoxide (PBO). Error bars represent 1 standard error.

Table 1 :

 1 Experimental design used in dietary transfer experiment involving grass shrimp

				Total
	Treatment	Oligochaete Diet	Water Exposure	Number	Sampled
				Exposed
	A	fluoranthene-dosed	750 µg PBO/L	24	Uptake: Days 1-5 Depuration: Days 6-8
	B	fluoranthene-dosed	control	24	Uptake: Days 1-5 Depuration: Days 6-8
	C	control	750 µg PBO/L	6	Uptake: Days 1 and 5
	D	control	control	6	Uptake: Days 1 and 5

Table 2 :

 2 Summary of water quality parameters from 96-h piperonyl butoxide (PBO) toxicological test and 8-day dietary transfer experiment. Values are reported as means + standard deviations.


Margaret M Krahn

David P Herman

Craig O Matkin

John W Durban

Lance Barrett-Lennard

Douglas G Burrows

Marilyn E Dahlheim

Nancy Black

Richard G Leduc

Paul R Wade

Use of chemical tracers in assessing the diet and foraging regions of eastern North Pacific killer whales

Keywords: FEEDING ECOLOGY, PREDATION, BIOPSY SAMPLING, CHEMICAL POLLUTION, STABLE ISOTOPES, FATTY ACIDS, ORGANOCHLORINES, BROMINATED DIPHENYL ETHERS, ORCINUS

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Assessing the diets and trophic positions of top level marine predators, such as the killer whale (Orcinus orca), is essential in understanding marine food webs. Predation by killer whales has recently been hypothesized to play a role in the population declines of several marine mammal species in Alaskan waters [START_REF] Estes | Killer whale predation on sea otters linking oceanic and nearshore ecosystems[END_REF]National Research Council, 2003;[START_REF] Springer | Sequential megafaunal collapse in the North Pacific Ocean: An ongoing legacy of industrial whaling?[END_REF]. In the North Pacific, two distinct types of killer whales have been termed "residents" and "transients" [START_REF] Bigg | An assessment of killer whale (Ocinus orca) stocks off Vancouver Island, British Columbia[END_REF][START_REF] Ford | Killer whales: The natural history and genealogy of Orcinus orca in British Columbia and Washington State, 2nd Edition[END_REF]. Whales of these types differ in their genetics [START_REF] Hoelzel | Low genetic variation among killer whales (Orcinus orca) in the eastern north Pacific and genetic differentiation between foraging specialists[END_REF], acoustics (Barrett-Lennard, [START_REF] Barrett-Lennard | The mixed blessing of echolocation: differences in sonar use by fish-eating and mammal-eating killer whales[END_REF], morphology [START_REF] Ford | Killer whales: The natural history and genealogy of Orcinus orca in British Columbia and Washington State, 2nd Edition[END_REF] and feeding ecology [START_REF] Ford | Dietary specialization in two sympatric populations of killer whales (Orcinus orca) in coastal British Columbia and adjacent waters[END_REF]. Transients are thought to prey solely on marine mammals and residents are believed to principally consume marine fish, in particular salmon. Thus, transient killer whales are members of a "mammal-eating" ecotype, whereas residents belong to a "fish-eating" ecotype [START_REF] Baird | Occurrence and behaviour of transient killer whales: seasonal and pod-specific variability, foraging behaviour, and prey handling[END_REF][START_REF] Ford | Dietary specialization in two sympatric populations of killer whales (Orcinus orca) in coastal British Columbia and adjacent waters[END_REF][START_REF] Saulitis | Foraging strategies of sympatric killer whale (Orcinus orca) populations in Prince William Sound, Alaska[END_REF]. A third group, the "offshores," has also been proposed [START_REF] Ford | Killer whales: The natural history and genealogy of Orcinus orca in British Columbia and Washington State, 2nd Edition[END_REF] to describe whales most commonly encountered in outer coastal waters between California and Alaska (Dahlheim, unpublished data;Ellis, 2005;[START_REF] Krahn | Status Review of Southern Resident killer whales (Orcinus orca) under the Endangered Species Act[END_REF]. Offshore killer whales have a different mitochondrial DNA (mtDNA) haplotype from those of resident and transient whales [START_REF] Barrett-Lennard | Population structure and mating patterns of killer whales as revealed by DNA analysis[END_REF][START_REF] Hoelzel | Low worldwide genetic diversity in the killer whale (Orcinus orca): implications for demographic history[END_REF]. Although few feeding observations have been reported for the offshores, initial field observations suggest that their diet includes marine fish and perhaps squid (Black, unpublished data;Dahlheim, unpublished data;[START_REF] Ford | Killer whales: The natural history and genealogy of Orcinus orca in British Columbia and Washington State, 2nd Edition[END_REF][START_REF] Jones | A northeast Pacific offshore killer whale (Orcinus orca) feeding on a Pacific halibut (Hippoglossus stenolepis)[END_REF]. In order to evaluate whether any of the killer whale populations have played a role in the marine mammal population declines in Alaska, dietary information for all of the Alaska killer whale groups is needed.

Traditional diet analyses have known biases and limitations when applied to marine predators. For example, in Alaska, the "field season" generally extends from late spring to early fall, so information is limited about feeding habits of killer whales at other times of the year. Stomach content analyses provide data only from relatively recent meals and these analyses are typically biased as a result of differential rates of digestion of hard parts [START_REF] Tollit | Species and size differences in the digestion of otoliths and beaks; implications for estimates of pinniped diet composition[END_REF]Yonezaki, Kiyota, Baba, Koido & Takemura., 2003). Finally, stomach contents are available only from a small number of stranded animals and these whales may not reflect the eating habits of healthy whales. Thus, to provide data that may better reflect the long-term diets of killer whales, chemical analyses-using biopsy samples from presumably healthy animals-have been developed to further elucidate the dietary specializations of the three types of killer whales.

Mammalian predators, such as killer whales, integrate chemical tracers acquired from their prey that reflect both the species consumed and the regions from which the prey were taken. For example, two chemical signalsfatty acid signature analysis of blubber [START_REF] Iverson | Quantitative fatty acid signature analysis: A new method of estimating predator diets[END_REF]) and stable isotope enrichments of 13 C and 15 N in the epidermis [START_REF] Kelly | Stable isotopes of carbon and nitrogen in the study of avian and mammalian trophic ecology[END_REF]-have been used to assess the diet and trophic position of marine mammals. In addition, patterns of persistent organic pollutants (POPs) have been shown to differentiate cetacean stocks [START_REF] Krahn | White whales (Delphinapterus leucas) from three Alaskan stocks: concentrations and patterns of persistent organochlorine contaminants in blubber[END_REF][START_REF] Muir | Persistent organochlorines in beluga whales (Delphinapterus leucas) from the St. Lawrence River Estuary-I. Concentrations and patterns of specific PCBs, chlorinated pesticides and polychlorinated dibenzo-p-dioxins and dibenzofurans[END_REF], presumably due to differences in POPs in their prey. All three techniques were combined by [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF] to qualitatively examine the dietary specializations of eastern North Pacific killer whale populations.

An additional approach-POPs ratios-can be used to assess regional sources of POPs transferred to predators from their prey [START_REF] Calambokidis | Chlorinated hydrocarbon concentrations and their use for describing population discreteness in harbor porpoises from Washington, Oregon and California[END_REF][START_REF] Krahn | White whales (Delphinapterus leucas) from three Alaskan stocks: concentrations and patterns of persistent organochlorine contaminants in blubber[END_REF][START_REF] Muir | Organochlorine contaminants in belugas, Delphinapterus leucas, from Canadian waters[END_REF]. For example, DDTs were used heavily in California before their ban in the 1970s and California marine waters also received long-term, significant discharges of DDTs from a manufacturing plant [START_REF] Eganhouse | Diagenetic fate of organic contaminants on the Palos Verdes Shelf, California[END_REF]. As a result, concentrations of ∑DDTs relative to ∑PCBs (i.e., ∑DDTs/∑PCBs ratio) are typically higher in California marine species than in comparable species from other eastern North Pacific locations-providing a "California signature" [START_REF] Brown | Status, correlations and temporal trends of chemical contaminants in fish and sediments from selected sites on the Pacific coast of the USA[END_REF][START_REF] Calambokidis | Chlorinated hydrocarbon concentrations and their use for describing population discreteness in harbor porpoises from Washington, Oregon and California[END_REF][START_REF] Jarman | Levels of organochlorine compounds, including PCDDS and PCDFS, in the blubber of cetaceans from the west coast of North America[END_REF]. Another characteristic contaminant signature-the "Alaskan signature"-has resulted from the continued use in Asia of pesticides, such as p,p'-DDT and technical hexachlorocyclohexane (HCH), long after their ban in much of the rest of the world (de Wit, [START_REF] De Wit | AMAP Assessment 2002: Persistent Organic Pollutants in the Arctic[END_REF]. Due to transport of contaminants from Asia eastward via air and ocean currents, as well as the eastward migration of pollutantcontaining anadromous fish from Asia to the Aleutian Islands and Bering Sea, pollutants used in Asia are often found in marine biota from Alaska (de Wit et al., 2004). For example, the ratio of p,p'-DDT to ∑DDTs is comparatively high in Alaskan species [START_REF] De Wit | AMAP Assessment 2002: Persistent Organic Pollutants in the Arctic[END_REF], whereas this ratio is lower in California marine biota, indicating an "old" metabolized source [START_REF] Aguilar | Relationship of DDE/tDDT in marine mammals to the chronology of DDT input into the ecosystem[END_REF]. Finally, the flame-retardant polybrominated diphenyl ethers (PBDEs), found in urban runoff and sewage effluents, have emerged as an important class of toxicants because of their recent exponential increase in the environment [START_REF] De Wit | An overview of brominated flame retardants in the environment[END_REF]. Thus, investigation of the ratio of ∑PBDEs relative to ∑PCBs can identify sources of contamination indicative of an urban environment.

The current study was undertaken to advance the findings of the previous study [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF] by providing further insight into diet and movements of the North Pacific killer whales from Alaska. Additional biopsy samples (n = 84) were collected from resident, transient and offshore killer whales and were characterized by comparing POP concentrations, patterns and ratios, stable isotope ratios of carbon and nitrogen and fatty acid profiles by killer whale ecotype and region. Interpretation of the results from the additional samples has further demonstrated the differences among the three killer whale groups in feeding habits and movements. For example, the increased sample size for the resident killer whales revealed a gradient in chemical profiles from west (central Aleutians) to east (Gulf of Alaska), consistent with regional differences in their prey. In addition, offshore killer whales were shown to consume a diet that is clearly distinct from those of sympatric residents and transients. Thus, the current study increases the knowledge about the role of killer whales in the marine food web.

Materials and methods

Killer whales sampled

For this study, killer whale biopsy samples (n = 84) were collected in Alaska during the 2003/2004 sampling years. All samples were obtained from live, presumably healthy whales using remote biopsy sampling techniques [START_REF] Barrett-Lennard | Population structure and mating patterns of killer whales as revealed by DNA analysis[END_REF][START_REF] Hoelzel | Low genetic variation among killer whales (Orcinus orca) in the eastern north Pacific and genetic differentiation between foraging specialists[END_REF][START_REF] Ylitalo | Influence of life-history parameters on organochlorine concentrations in free-ranging killer whales (Orcinus orca) from Prince William Sound[END_REF] and biopsy tips of various lengths (typically 3.0 to 3.5 cm). All biopsy samples were stored at -80 o C until analyzed. Sample sizes were standardized as reported previously [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF]. The blubber acquired from these whales was analyzed for fatty acids and POPs and the epidermis, when available, for stable isotope ratios (Table 1). In several instances, an insufficient quantity of epidermis was available to allow stable isotope analyses to be performed, resulting in data omissions for many samples.

In addition to the samples in

Killer whale group structure

Each killer whale in this study was provisionally classified as resident, transient or offshore based on field observations and in some cases, on long-term studies of well-known populations. These classifications were corroborated by mtDNA haplotype identity [START_REF] Barrett-Lennard | Population structure and mating patterns of killer whales as revealed by DNA analysis[END_REF]. The resident and transient killer whales from Alaska were grouped by geographical collection region in this study and include the Central Aleutian Islands (CAI), Eastern Aleutian Islands (EAI) and Gulf of Alaska (GOA) (Table 1 and Figure 1). The offshore killer whales were combined into a single group. Although offshores have been encountered as far south as Los Angeles [START_REF] Black | Killer whales of California and Western Mexico: A catalog of photo-identified individuals[END_REF][START_REF] Ford | Killer whales: The natural history and genealogy of Orcinus orca in British Columbia and Washington State, 2nd Edition[END_REF], mostly in the winter months, only recently have photo-identification resightings of offshore individuals been recorded between Alaska, Washington State and California, thus suggesting a single population with the same individuals moving throughout the northeast Pacific (Dahlheim, unpublished data).

Fatty acid analyses

Fatty acid concentrations in blubber were determined as reported by Krahn et al. (2004b).

Persistent organic pollutant analyses

Blubber from killer whale biopsies was analyzed for POP concentrations using the procedure of [START_REF] Sloan | Northwest Fisheries Science Center Procedures for Extraction, Cleanup and Gas Chromatography/Mass Spectrometry Analysis of Sediments and Tissues for Organic Contaminants[END_REF]. ∑PCBs is the sum of all 40 PCB congeners analyzed; ∑DDTs is the sum of o,p '-DDD, p,p'-DDD, p,p'-DDE, o,p'-DDE, o,p'-DDT and p,p'-DDT; ∑chlordanes is the sum of oxychlordane, gamma-chlordane, nona-III-chlordane, alpha-chlordane, trans-nonachlor, and cis-nonachlor; the ∑HCHs is the sum of alpha-, beta-, and gamma-HCH isomers and finally ∑PBDEs is the sum of congeners 28, 47, 49, 66, 85, 99, 100, 153, 154, 183. Total lipids in killer whale biopsy samples were measured by a TLC-FID method (Ylitalo, Yanagida, Hufnagle Jr & Krahn, 2005c). All POP concentrations reported in this paper have been lipid-normalized.

POP concentrations in selected putative prey species of killer whales were obtained from a number of sources:

whole bodies of California Chinook salmon (O'Neill, Ylitalo, Krahn, West, Bolton & Brown, 2005;Ylitalo, O'Neill, Brown, West, Bolton & Krahn, 2005a); blubber of gray whales [START_REF] Krahn | Organochlorine contaminant concentrations and lipid profiles in eastern North Pacific gray whales (Eschrichtius robustus)[END_REF]; blubber of California sea lions (necropsy) (Ylitalo, Stein, Hom, Johnson, Tilbury, Hall et al., 2005b)

Analyses for stable isotope ratios of carbon and nitrogen

Stable isotope analyses of epidermis from killer (Alaska), gray (Russia and Alaska) and minke (Aleutian Islands) whales, as well as muscle from northern fur seals (Pribilof Islands) and Steller sea lions (Alaska), were conducted on lipid-extracted tissues as described previously [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF]. The internal laboratory standards were calibrated relative to the following international standards: USGS-24 graphite, NBS-22 oil, IAEA-CH-6 sucrose, IAEA-N-1 ammonium sulfate and IAEA-N-2 ammonium sulfate. A standard reference material (NIST SRM 1946) was processed with every 20 analyses to monitor analytical accuracy.

Stable isotope ratio modeling

Stable isotope modeling was conducted using equations and methods reported previously [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF].

Statistical analyses

All univariate and multivariate analyses were conducted using JMP Statistical Discovery Software (PC profession edition, version 5.01). Prior to all statistical calculations, both untransformed and log-transformed stable isotope and POP concentration data for each regional killer whale group were tested for data normality.

The log-transformed data did not lead to a consistent improvement in data normality compared to the untransformed results. Hence, all statistical comparison tests used to evaluate the statistical significance of differences among the various groups were computed from the raw, untransformed data. Unless indicated otherwise, all univariate comparisons between two group means were significance tested (α = 0.05) using a two sample Student's t-test assuming unequal variances. Significant differences among multiple groups having equal variances were evaluated using a Tukey HSD test (α = 0.05).

Results

Killer whale photoidentification

Of the nine offshore killer whales biopsied in Alaska [this study [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF]], three have been sighted in California and four others that were sampled in the Aleutian Islands have been sighted in SE Alaska, but have not been seen in California. Only two of the Alaska offshore whales have not been matched to another region.

Stable isotope ratios and modeling

The killer whales in this study with sufficient epidermis for stable isotope analysis are indicated in Table 1 and the results are given in Figure 2 [whales from [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF] are also included] and Table 2. In general, the carbon and nitrogen values (Table 2) were not statistically significantly different from those reported previously by [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF]. Among killer whale groups shown in Figure 2, the mean δ 15 N and δ 13 C values for the transients were significantly higher than the means for the residents, whereas the offshores had intermediate values and were not significantly different from the other two groups. For the three regional groups of Alaska residents (Figure 2), a significant increase in mean δ 13 C values was noted from west to east (CAI/R < EAI/R < GOA/R). The mean δ 15 N values for the resident groups showed the same trend, but only CAI/R was statistically different from GOA/R and EAI/R.

Calculated stable isotope values for the diet of EAI/T killer whales were estimated using equations 2 and 3 (see Methods), based on observed predation events and stable isotope values measured for the observed prey species from archived marine mammal prey tissues (epidermis or muscle). EAI/T whales were observed to be preying exclusively on juvenile gray whales in May, but later in the summer their observed diet was estimated to comprise northern fur seals (58%) minke whales (28%) and Steller sea lions (14%) (Matkin & Saulitis, unpublished data). Using these stable isotope values of these four prey species (Table 2) and assuming equal weights for these two observed seasonal diets, the carbon and nitrogen isotope values predicted by Equations 2 and 3 (δ model-C = -16.6 ± 0.4; δ model-N = 18.1 ± 0.4) were very similar to those actually measured for EAI/T whales (δ 13 C = -16.2 ± 0.5, δ 15 N = 18.0 ± 0.8).

Fatty acid profiles

For resident, transient and offshore killer whales, the mean summed fatty acid methyl ester weight percent values in each class of blubber fatty acids were similar to those reported previously by [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF], so fatty acid summary data from these additional 84 killer whale biopsy samples are not reported again here.

However, because of the increased statistical certainty obtained as a result of the substantial increase in the number of biopsy samples analyzed in the current study, a longitudinal variation was observed for the first time in the fatty acid results for the three regional Alaska resident groups, in which the proportion of omega-3 and polyunsaturated fatty acids was observed to increase (p < 0.05) from west to east (CAI/R < EAI/R < GOA/R).

POPs concentrations

Only adult male killer whales were used to study differences in POP concentrations, because reproductive female killer whales transfer a substantial portion of their contaminant burden to their calves. As a result, POP concentrations in females are generally lower than in males and are dependent of the number of times they have given birth [START_REF] Borga | Biological and chemical factors of importance in the bioaccumulation and trophic transfer of persistent organochlorine contaminants in arctic marine food webs[END_REF][START_REF] Ross | High PCB concentrations in free-ranging Pacific killer whales, Orcinus orca: Effects of age, sex and dietary preference[END_REF][START_REF] Ylitalo | Influence of life-history parameters on organochlorine concentrations in free-ranging killer whales (Orcinus orca) from Prince William Sound[END_REF]. Mean concentrations for ∑PCBs, ∑DDTs, ∑chlordanes, ∑HCHs and ∑PBDEs measured in the biopsy blubber of adult male offshores from Alaska, transients from the eastern Aleutian Island and residents from three regions (CAI, EAI and GOA) are shown in Table 3. Two other groups of killer whalesthe West Coast transients from California (Table 3) and the ETP whales from waters near Nicaragua (Krahn et al., in prep)-were used for comparison with the whales from Alaska. Contaminant concentrations in male killer whales generally increase with age [START_REF] Ross | High PCB concentrations in free-ranging Pacific killer whales, Orcinus orca: Effects of age, sex and dietary preference[END_REF], so these concentrations typically have large ranges.

For the killer whales in Table 3, the West Coast (California) transients had significantly higher ∑PCBs, ∑DDTs and ∑PBDEs, whereas the EAI/T whales had significantly higher ∑HCHs, compared to the other whale groups.

Among the Alaska killer whales, ∑DDT concentrations in the offshores were two times higher than those of the Alaska transients and 20 times higher than those of the residents (all values significantly different; Table 3).

Furthermore, ∑PCB concentrations of offshores were similar to those of the Alaska transients and both were significantly higher than those of the residents. In contrast, ∑chlordanes and ∑HCHs were significantly higher in the Alaska transients than in either the offshores or residents. Finally, mean concentrations of ∑PBDEs (flame retardants) in offshore killer whales were significantly higher than those found for the transients and both were significantly higher than ∑PBDEs for the residents. Comparing the three regional groups of Alaska resident killer whales, mean concentrations for both ∑PCBs and ∑DDTs were about two times higher for the CAI/R and EAI/R whales than for GOA/R residents (Table 3), but only the differences between the EAI/R and GOA/R groups were statistically significant. Mean concentrations of ∑chlordanes were significantly higher for the EAI/R than for the GOA/R population, whereas the differences between the EAI/R and CAI/R groups, as well as the GOA/R and CAI/R groups, were not significant. Both the GOA/R and EAI/R whales had higher mean concentrations of ∑HCHs than were found for CAI/R residents but differences were not significant.

Finally, the mean concentration of ∑PBDEs was significantly higher in the GOA/R whales compared to the other two regional groups.

POPs ratios

Only adult male killer whales were used to study differences in POP ratios, because females can transfer individual POPs at differential rates to their offspring [START_REF] Borga | Biological and chemical factors of importance in the bioaccumulation and trophic transfer of persistent organochlorine contaminants in arctic marine food webs[END_REF], resulting in changes in these ratios.

In this study, four different contaminant ratios (∑DDTs/∑PCBs; p,p'-DDT/∑DDTs; ∑chlordanes/∑PCBs; ∑PBDEs/∑PCBs; Figures 3456) were used to evaluate differences among killer whale groups and their prey species and to broadly define regional characteristics of each group. In addition, POPs ratios from two other groups of killer whales-the West Coast transients from California and the ETP whales from waters near Nicaragua-were used for comparison with those from the Alaska whales. Ratios for West Coast (California) transients (n=4) ratios are depicted in Figures 3456and ETP killer whales (n=3) had a mean ∑DDTs/∑PCBs ratio of 57.8 ± 5.2 and a mean p,p'-DDT/∑DDT ratio of 0.061 ± 0.005.

When comparing POPs ratios for all the killer whale groups depicted in Figures 3456, as well as those for ETP whales, the ∑DDTs/∑PCBs ratio was found to be significantly higher for ETP whales (57.8 ± 5.2) than for West Coast (California) transient whales and both ETP and West Coast transient whales, in turn, had ∑DDTs/∑PCBs ratios that were significantly higher than all remaining groups (Figure 3). Mean p,p'-DDT/∑DDT and ∑chlordanes/∑PCBs ratios for West Coast transients were significantly lower than those found for other killer whale groups (Figures 4 and5). Finally, West Coast transients had significantly higher ∑PBDEs/∑PCBs ratios than found for the other whale groups (Figure 6). When comparing POPs ratios among all the Alaska killer whale groups, ∑DDTs/∑PCBs and ∑PBDEs/∑PCBs ratios were significantly higher in the offshores than in the Alaska resident or transient groups. For p,p'-DDT/∑DDT, CAI/R had the highest and the offshores had the lowest ratios; both were significantly different from the other Alaska groups, except offshore and EAI/T whales were not different. For ∑chlordanes/∑PCBs ratios, the EAI/T and GOA/R groups were not significantly different from each other, but both were significantly higher than all other Alaska killer whale groups. When comparing the POPs ratios among the three regional groups of Alaska resident killer whales, the ∑DDTs/∑PCBs ratio was highest in CAI/R and lowest in GOA/R whales, with EAI/R residents falling in between, although only the ratio for GOA/R was statistically different from those for other two groups of Alaska residents. Similarly, two other POPs ratios in the Alaska resident groups showed significant west to east trends-p,p'-DDT/∑DDTs decreased from west to east (CAI/R higher than GOA/R), whereas ∑chlordanes/∑PCBs increased. The GOA/R group had a statistically higher ratio for ∑PBDEs/∑PCBs than found for the other two resident groups.

Discussion

The previous study [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF] demonstrated that chemical profiles for resident and transient killer whales supported results from traditional methods of assessing diet (e.g., field observations and stomach content analysis), but the chemical profiles of offshore whales were seemingly contradictory. Specifically, the fatty acid profiles in the blubber of the offshore whales supported a fish diet, but POP concentrations suggested that these whales might be feeding on prey species that were more contaminated than would expected from a diet of Alaskan fish. The current study adds significant additional data from which to help resolve this apparent conflict and finds that the offshore diet is different from that of the fish-eating resident whales, particularly when contaminant ratios are added to the other chemical profiles.

Mean POP concentrations in the resident and transient killer whales (Table 3) were similar to those previously reported by [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF]. Both in this study and the previous one, ∑chlordanes and ∑HCHs were significantly higher in the transients than in either the offshores or residents. However, in the previous study, male offshores (n = 2) had ∑DDT and ∑PCB levels that were consistently higher than those of the residents and approached, but generally did not exceed, those of the transients [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF]. In contrast, results for the additional offshore whales (n = 4), presumed to be primarily fish-eaters, had concentrations of ∑DDTs and ∑PBDEs that were higher than and ∑PCBs that were similar to those of the Alaska transients, which, in turn, had concentrations of these contaminants higher than those of the residents (Table 3). Thus, the offshores are showing a very different pattern of POP contamination than found for resident and transient whales.

Consequently, if the offshores consume a fish diet similar to that of the sympatric residents, the POP profile differences between the offshores and the residents are difficult to explain.

The ratios of certain POPs (e.g., ∑DDTs/∑PCBs; p,p'-DDT/∑DDTs) in marine sediments and biota can be used to document unusual regional patterns of pollutants [START_REF] Calambokidis | Chlorinated hydrocarbon concentrations and their use for describing population discreteness in harbor porpoises from Washington, Oregon and California[END_REF][START_REF] Krahn | White whales (Delphinapterus leucas) from three Alaskan stocks: concentrations and patterns of persistent organochlorine contaminants in blubber[END_REF][START_REF] Muir | Organochlorine contaminants in belugas, Delphinapterus leucas, from Canadian waters[END_REF]. For example, male ETP killer whales had a mean ∑DDTs/∑PCBs ratio (57.8 ± 5.2) that was significantly higher than found for the other killer whale groups studied (Figure 3), suggesting recent use of the DDT pesticide in the tropical Pacific that resulted in a high relative proportion of ∑DDTs in prey consumed by the killer whales. This finding was supported by a relatively high p,p'-DDT/∑DDT ratio in ETP whales, indicating a recent source of the p,p'-DDT from pesticide use. Surprisingly, although the mean concentration of ∑DDTs in ETP whales (1,200,000 ± 500,000 ng/g lipid) was about 3 times the mean levels found in the Alaska offshore killer whales (i.e., the group of whales biopsied in Alaska with the highest ∑DDTs), the mean concentration of ∑PCBs in ETP whales (20,000 ± 8,300 ng/g lipid) was only about 20% of that in the offshores.

These results are consistent with both the heavy use of DDTs as a pesticide in areas of Central America [START_REF] De Wit | AMAP Assessment 2002: Persistent Organic Pollutants in the Arctic[END_REF] and the more limited use of PCBs in a region in which industrialization has been relatively recent [START_REF] De Wit | AMAP Assessment 2002: Persistent Organic Pollutants in the Arctic[END_REF]. The highest absolute concentrations of ∑DDTs and ∑PCBs were found in West Coast transients sampled off California (Table 3). Both ∑DDT concentrations (Table 3) and ∑DDTs/∑PCBs ratios (Figure 3) for the West Coast transients indicated a "California signature." Furthermore, the low p,p'-DDT/∑DDTs ratio in the West Coast transient whales (Figure 4) was also indicative of foraging in California waters, where the source of DDTs is "old", due to the U.S. ban of DDTs in the 1970s.

For Alaska residents and transients, ∑DDTs/∑PCBs ratios (Figure 3) were much lower than those of the West Coast transients, as would be expected because environmental availability of ∑DDTs is lower in Alaska than in California [START_REF] Brown | Status, correlations and temporal trends of chemical contaminants in fish and sediments from selected sites on the Pacific coast of the USA[END_REF][START_REF] Calambokidis | Chlorinated hydrocarbon concentrations and their use for describing population discreteness in harbor porpoises from Washington, Oregon and California[END_REF][START_REF] De Wit | AMAP Assessment 2002: Persistent Organic Pollutants in the Arctic[END_REF][START_REF] Jarman | Levels of organochlorine compounds, including PCDDS and PCDFS, in the blubber of cetaceans from the west coast of North America[END_REF].

Furthermore, the primary indicator of a recent source of DDT pesticide use (i.e., p,p'-DDT/∑DDTs) was higher in Alaska residents and transients than in the West Coast transients (Figure 4). The input of p,p'-DDT (used in Asia until recently) is consistent with the apparent transport of this pesticide via air or ocean currents to Alaska (the "Alaskan signature") (AMAP, 1998;[START_REF] De Wit | AMAP Assessment 2002: Persistent Organic Pollutants in the Arctic[END_REF]. The West Coast (California) transients had a significantly higher mean ∑PBDEs/∑PCBs ratio than was found for the Alaska residents or transients, consistent with higher input of PBDEs in urban environments [START_REF] De Wit | An overview of brominated flame retardants in the environment[END_REF]. Furthermore, POP ratios for the offshore killer whales were dissimilar to those of the other Alaska groups and consistently fell between those of the West Coast (California) transients and those of the Alaska residents and transients (Figures 6789). In light of these differences between the offshores, residents and transients (i.e., differences in contaminant ratios;

absolute ∑PCB and ∑DDT concentrations; PCB profiles), it is apparent that offshores consume prey species having very different POPs signatures than species consumed by the resident and transient ecotypes.

Among the three regional groups of Alaska resident killer whales, POP ratios demonstrated decreasing west-toeast gradients for mean ratios of both ∑DDTs/∑PCBs and p,p'-DDT/∑DDTs (CAI/R > EAI/R > GOA/R).

These results are consistent with the transport of DDT pesticide (particularly unmetabolized p,p'-DDT) from Asia, resulting in the whales foraging closest to Asia (CAI/R residents) acquiring the highest values. In contrast, GOA/R whales had the highest mean ratios for ∑chlordanes/∑PCBs and ∑PBDEs/∑PCBs, consistent with the use of these chemicals on the mainland of Alaska (de [START_REF] De Wit | AMAP Assessment 2002: Persistent Organic Pollutants in the Arctic[END_REF].

Contaminant ratios in selected representative prey species (Figures 3456) generally showed the same regional distinctions found for killer whales. For example, ∑DDTs/∑PCBs ratios (Figure 3) were highest and p,p'-DDT/∑DDTs ratios (Figure 4) were lowest (indicative of the California signature) in California prey species (Chinook salmon and California sea lions) compared to Alaska prey. In contrast, the ∑chlordanes/∑PCBs ratios (Figure 5) were highest in Alaskan prey (particularly, Steller sea lions and northern fur seals), compared to potential prey from California. Because PBDEs are "emerging" contaminants that have not been measured until recently by many laboratories, data are only available for a relatively small number of putative killer whale prey species (Figure 6). As would be expected for prey species that feed near large urban areas, both California sea lions and California Chinook had much higher ∑PBDEs/∑PCBs ratios than found for Steller sea lion pups from Alaska. The ∑PBDEs/∑PCBs ratio was probably lower for Chinook salmon than for California sea lions, because the salmon spend much of their life cycle far offshore compared to the sea lions that feed regularly in coastal waters with greater urban influence. In addition, differential biomagnification of ∑PBDEs and ∑PCBs by these largely dissimilar taxa could cause the differences in their ratios.

Because each contaminant (or group of summed contaminants) is biomagnified to a different extent, contaminant ratios will also change as contaminants from prey are assimilated by the predator [START_REF] Fisk | Influence of chemical and biological factors on trophic transfer of persistent organic pollutants in the Northwater Polynya marine food web[END_REF][START_REF] Hoekstra | Trophic transfer of persistent orgranochlorine contaminants (OCs) within an Arctic marine food web from the southern Beaufort-Chukchi Seas[END_REF]. However, biomagnification factors (BMFs) appear to be species-specific [START_REF] Fisk | Influence of chemical and biological factors on trophic transfer of persistent organic pollutants in the Northwater Polynya marine food web[END_REF][START_REF] Hoekstra | Trophic transfer of persistent orgranochlorine contaminants (OCs) within an Arctic marine food web from the southern Beaufort-Chukchi Seas[END_REF] and no BMFs have been reported

for killer whales. Consequently, POP ratios cannot be directly and quantitatively compared among killer whales and their likely prey until these BMF values are known. In the interim, qualitative comparisons can be made. For example, West Coast (California) transient killer whales and California sea lions [a major transient prey species (Black, unpublished data)], had qualitatively similar contaminant signatures (specifically, high ∑DDTs/∑PCBs and ∑PBDEs/∑PCBs, as well as low p,p'-DDT/∑DDTs and ∑chlordanes/∑PCBs). Similarly, Steller sea lion pups could be a prey source for Alaska transients, because the same signature (low ∑DDTs/∑PCBs and ∑PBDEs/∑PCBs, as well as high ∑chlordanes/∑PCBs) was identified in both the transients and the sea lions. In addition, other marine mammals from the Gulf of Alaska/Aleutian Islands area had ratios likewise indicative of an Alaskan signature (e.g., northern fur seals, harbor seals; Figures 678) and thus, these species should also be considered as potential prey. Alternatively, two or more marine mammal prey species may have "complimentary" ratios, where a high ratio in one species is balanced by a low ratio in others, allowing the species together to be considered as possible prey as part of a mixed diet. Contaminant ratios are only a single indicator and best used in combination with the other chemical profiles to provide a more comprehensive assessment of the most likely killer whale prey. Currently, additional prey species are being acquired and analyzed for these POPs (as well as for fatty acids and stable isotopes) and the combined results should provide additional data for comparison to the killer whales.

Taking the results from contaminant ratios, as well as the other indicators (stable isotope ratios; chemical contaminant concentrations and patterns) into consideration, insights into the feeding ecology of the Alaska killer whales can be further elucidated. As found in the previous study [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF], all data are consistent with resident killer whales in Alaska consuming a fish diet, based on field observations and comparisons of their chemical signatures to those of other known fish-eating populations, e.g., Southern

Resident killer whales [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF]Krahn et al., 2004b). Interestingly, the regional groups of Alaska residents showed distinct west-to-east differences in carbon and nitrogen isotope ratios (Figure 2). The stable isotope results were consistent with CAI/R whales foraging in deeper, more pelagic waters, whereas GOA/R and EAI/R whales forage on prey found in shallower waters on the continental shelf (Figure 1A). Lower δ 15 N values at the base of the food chain in the Central Aleutian islands [START_REF] Kline | Temporal and spatial variability of 13C/12C and 15N/14N in pelagic biota of Prince William Sound[END_REF][START_REF] Schell | Stable-isotope and electron-microscopic evidence that cyamids (Crustacea: Amphipoda) feed on whale skin[END_REF] may be a result of these oceanographic changes and thus contribute, in part, to the observed lower values in the CAI/R whales relative to the EAI/R and GOA/R groups. Furthermore, although both the EAI/R and GOA/R whales are likely consuming salmon as a substantial part of their diets (Matkin & Saulitis, unpublished data;[START_REF] Saulitis | Foraging strategies of sympatric killer whale (Orcinus orca) populations in Prince William Sound, Alaska[END_REF], the CAI/R residents may also be consuming locally available prey (e.g., pollock, Pacific cod, Pacific halibut), because salmon are presumably less available in the Central Aleutians due to lack of anadromous streams and major migratory pathways. Thus, differences in stable isotope values noted among the regional groups of resident killer whales may be significantly influenced by differences in the signatures at the base of the food chain, as well as by differences in prey specialization.

Because predation by transient (mammal-eating) killer whales may be contributing to the population declines of certain Alaskan marine mammal species, specifically Steller sea lions (National Research Council, 2003;[START_REF] Springer | Sequential megafaunal collapse in the North Pacific Ocean: An ongoing legacy of industrial whaling?[END_REF] and sea otters [START_REF] Estes | Killer whale predation on sea otters linking oceanic and nearshore ecosystems[END_REF], there is great interest in learning more about their diet.

The EAI/T whales were observed to be preying exclusively on juvenile gray whales as they transited the eastern Aleutian Islands in May, but observations later in the summer consisted of predation on northern fur seals (58%) minke whales (28%) and Steller sea lions (14%) (Matkin & Saulitis, unpublished data). These observations have been substantiated through stable isotope ratios measured in the EAI/T whales. Specifically, the carbon and nitrogen stable isotope ratios for the diet of EAI/T killer whales calculated using the additive linear model were very similar to those actually measured for EAI/T transients (see Results). Because the δ 15 N ratio was much higher in Steller sea lion tissues (16.3 ± 1.2) than that predicted for EAI/T prey by the stable isotope dietary model (δ model-N = 14.0), sea lions cannot be the exclusive prey of the EAI/T transients. Instead, their diet must be offset by the intake of substantial quantities of alternative prey species with lower δ 15 N ratios (e.g., gray whales, Dall's porpoise, humpback whales) to be consistent with the actual δ 15 N ratio measured for EAI/T whales. Thus, at least in spring and summer, EAI transient killer whales likely prey on a number of different marine mammal species, based on availability.

The current results for the offshores add to the complexity of determining their prey. Stable isotope ratios of offshores were statistically indistinguishable from those of EAI/R residents, but were significantly lower than those of the transients (Figure 2). Unexpectedly, contaminant ratios for offshores fell between those for the West Coast (California) transients and those for resident and transient whales from Alaska. In addition, mean ∑PCB concentrations in offshores were similar to and mean ∑DDT and ∑PBDE concentrations exceeded those of the Alaska transients, but concentration levels were lower than those found in West Coast (California) transients (Table 3). These results are a strong indication that offshore killer whales feed during certain periods of the year in areas where available prey species have a "California signature." These chemical signature results were then substantiated by photoidentification, following a request to check for matches between the 9 offshore killer whales in this study and offshores photographed in other areas. Three of the offshore killer whales biopsied in Alaska had been photographed in California, indicating that the offshores range widely from Alaska to California. Without exposure to the high levels of ∑DDTs and ∑PBDEs generally present in prey from the numerous highly urbanized areas along the California coast, the offshores probably could not have acquired POP concentrations and ratios indicative of a "California" signature. Furthermore, these results provide evidence that the offshore killer whales may belong to a third killer whale ecotype.

The ∑PCB concentrations found in the male offshores (110,000 ± 22,000 ng/g lipid) were similar to those reported for salmon-eating "Southern Resident" male killer whales [146,300 ± 32,700 ng/g lipid from [START_REF] Ross | High PCB concentrations in free-ranging Pacific killer whales, Orcinus orca: Effects of age, sex and dietary preference[END_REF]]. Thus, it is conceivable that the offshores attained these high levels of POPs solely by consuming highly contaminated salmonids as a large portion of their diet. Alternatively, the specific preferential prey of offshore whales may be very different from those of residents and may comprise longer-lived marine fish species that bioaccumulate high levels of POPs (e.g., rockfish, shark and tuna). Therefore, rather than eating primarily the salmon species thought to be the traditional prey of their resident killer whale counterparts, offshores may alternatively consume substantial quantities of longer-lived marine fish (Black, unpublished data). To better define the prey of offshores, samples of other possible prey species (e.g., mako and thresher shark, yellowfin and albacore tuna, rockfish and squid) are currently being collected for analysis.

Conclusions

In conclusion, the chemical profiles presented here have not yet led to a complete description of the feeding ecology of eastern North Pacific killer whales, but the information provided has advanced our knowledge in several important ways. This study has three significant findings:

1. Offshore killer whales consume a diet that is clearly distinct from those of residents and transients.

Furthermore, the offshores biopsied in Alaska feed at least part of the year in California, presumably on highly contaminated, long-lived, high trophic level marine fish. Thus, these results, together with those presented by [START_REF] Herman | Feeding ecology of eastern North Pacific killer whales from fatty acid, stable isotope, and organochlorine analyses of blubber biopsies[END_REF], provide evidence that the offshores belong to a third killer whale ecotype.

2. Resident killer whales in Alaska exhibited a gradient in carbon and nitrogen stable isotope ratios, as well as in certain POPs ratios, from west (central Aleutians) to east (eastern Aleutians and the Gulf of Alaska) that is likely a result of: (1) the gradient in stable isotopes at the base of the respective food webs across these regions;

(2) a shift from off-shelf (CAI) to continental shelf-based (GOA) prey; and

(3) inclusion of lower trophic level fish prey in the diet of the CAI/R whales relative to the other two regions.

3. The EAI/T spring/summer diet does not appear to be composed exclusively of Steller sea lions, because stable isotope ratios show that their diet must include lower trophic level species to offset the high tropic level of the sea lions. Furthermore, this conclusion is supported by field observation data indicating that gray whales, minke whales, and Northern fur seals comprise a substantial portion of the EAI/T diet during summer months.

6. a Although POP analyses were conducted for all killer whales in this table, only adult males "x" were used for POP concentrations (Table 3) and ratios (Figures 3456). . The ratios ∑PBDEs/∑PCBs ± 1SD in adult male killer whale populations (*females were also used for West Coast transients) and selected putative prey species.

Figure 4 .Figure 5 .

 45 Figure4. The ratios p,p'-DDT/∑DDTs ± 1SD in adult male killer whale populations and selected putative prey species.

  Figure6. The ratios ∑PBDEs/∑PCBs ± 1SD in adult male killer whale populations (*females were also used for West Coast transients) and selected putative prey species.

  Table 1, biopsy samples were analyzed from eastern Tropical Pacific (ETP) male killer whales from waters to the west of Nicaragua [n = 3; animal IDs: 38168, 38170 and 38175; provided by the Southwest Fisheries Science Center (Krahn, Pitman, Burrows, Herman & Pearce, in prep)] and from four "West Coast" (California) male transients (animal IDs: 6/02/00, CA-11, CA-30 and NB040423-1) provided by the Monterey Bay Cetacean Project. Selected POP results from both the ETP whales and the West Coast transients were used for comparison with those from the Alaskan killer whales.

Table 1 . Collection date, age class, ecotype and location for killer whale biopsy samples analyzed for fatty acids (FA), stable isotopes (SI) and persistent organic contaminants (POPs).

 1

		FA SI POPs a Animal Identification	Collection date Age class	Ecotype	Location
	ALASKA OFFSHORES (AK/OFF)
	Males
	1	x		x	EA-03-08	7/10/2003	adult	offshore	Eastern Aleutian Islands
	2	x	x	x	AKW-04-006	7/8/2004	adult	offshore	Kenai Fjords
	3	x	x	x	AKW-04-007	7/8/2004	adult	offshore	Kenai Fjords
	4	x	x	x	9-16-04 ENC 8 #2	9/16/2004	adult	offshore	Southeast Alaska
	Females
	5	x			EA-03-06	7/10/2003	adult	offshore	Eastern Aleutian Islands
	6	x			EA-03-07	7/10/2003	adult	offshore	Eastern Aleutian Islands
	7	x			EA-03-09	7/10/2003	adult	offshore	Eastern Aleutian Islands
	8	x	x		AKW-04-005	7/8/2004	adult	offshore	Kenai Fjords
	9	x	x		9-16-04 ENC 8 #1	9/16/2004	adult	offshore	Southeast Alaska
	CENTRAL ALEUTIAN ISLAND RESIDENTS (CAI/R)
	Males
	10	x	x	x	CP030710-01	7/10/2003	adult	resident	Central Aleutian Islands
	11	x	x	x	CP030710-02	7/10/2003	adult	resident	Central Aleutian Islands
	12	x	x	x	AE040819-01	8/19/2004	adult	resident	Central Aleutian Islands
	EASTERN ALEUTIAN ISLAND RESIDENTS (EAI/R)
	Males
	13	x			EA-03-03	6/29/2003	?	resident	Eastern Aleutian Islands
	14	x	x	x	EA-03-05	7/7/2003	adult	resident	Eastern Aleutian Islands
	15	x	x	x	UNAK-03-001	7/7/2003	adult	resident	Eastern Aleutian Islands
	16	x		x	UNAK-03-002	7/13/2003	adult	resident	Eastern Aleutian Islands
	17	x		x	UNAK-03-003	7/14/2003	adult	resident	Eastern Aleutian Islands
	18	x		x	EA-03-11	8/1/2003	adult	resident	Eastern Aleutian Islands
	19	x		x	UNAK-03-006	8/18/2003	adult	resident	Eastern Aleutian Islands
	20	x		x	UNAK-03-007	8/18/2003	adult	resident	Eastern Aleutian Islands
	21	x			UNAK-03-009	8/22/2003	subadult	resident	Eastern Aleutian Islands
	22	x			EA-04-001	7/6/2004	subadult	resident	Eastern Aleutian Islands
	23	x		x	EA-04-002	7/8/2004	adult	resident	Eastern Aleutian Islands
	24	x		x	EA-04-003	7/10/2004	adult	resident	Eastern Aleutian Islands
	25	x	x		UNAK-04-001	7/13/2004	?	resident	Eastern Aleutian Islands
	26	x	x	x	UNAK-04-003	7/14/2004	adult	resident	Eastern Aleutian Islands
	27	x		x	UNAK-04-004	7/23/2004	adult	resident	Eastern Aleutian Islands
	28	x		x	UNAK-04-005	7/24/2004	adult	resident	Eastern Aleutian Islands
	29	x	x	x	AE040728-02	7/28/2004	adult	resident	Eastern Aleutian Islands
	30	x	x	x	AE040728-03	7/28/2004	adult	resident	Eastern Aleutian Islands
	31	x	x	x	AE040728-04	7/28/2004	adult	resident	Eastern Aleutian Islands
	32	x	x	x	AE040728-05	7/28/2004	adult	resident	Eastern Aleutian Islands
	33	x	x	x	AE040728-06	7/28/2004	adult	resident	Eastern Aleutian Islands
	34	x	x	x	EA-04-004	7/31/2004	adult	resident	Eastern Aleutian Islands
	35	x	x	x	EA-04-005	8/9/2004	adult	resident	Eastern Aleutian Islands
	36	x	x	x	AE040818-02	8/18/2004	adult	resident	Eastern Aleutian Islands
	Females
	37	x			EA-03--04	6/29/2003	adult	resident	Eastern Aleutian Islands
	38	x	x		UNAK-04-002	7/14/2004	adult	resident	Eastern Aleutian Islands

GULF OF ALASKA RESIDENTS (GOA/R) Males

	Sample#
	39	x		x	AKW-03-06	7/21/2003	adult	resident	Prince William Sound/ Kenai Fjords
	40	x		x	AKW-03-08	8/3/2003	adult	resident	Prince William Sound/ Kenai Fjords
	41	x		x	AKW-03-09	8/3/2003	adult	resident	Prince William Sound/ Kenai Fjords
	42	x		x	AKW-03-10	8/4/2003	adult	resident	Prince William Sound/ Kenai Fjords
	43	x	x	x	AKW-03-14	9/5/2003	adult	resident	Prince William Sound/ Kenai Fjords
	44	x	x	x	AKW-04-01	5/22/2004	adult	resident	Prince William Sound
	45	x	x	x	AKW-04-010	8/3/2004	adult	resident	Kenai Fjords
	46	x		x	PWS-04-01	8/4/2004	adult	resident	Prince William Sound
	47	x			PWS-04-02	8/4/2004	subadult	resident	Prince William Sound
	48	x	x	x	PWS-04-03	8/7/2004	adult	resident	Prince William Sound
	49	x	x	x	PWS-04-04	8/7/2004	adult	resident	Prince William Sound
	50	x		x	PWS-04-05	8/7/2004	adult	resident	Prince William Sound
	51	x		x	PWS-04-06	8/10/2004	adult	resident	Prince William Sound
	52	x		x	AKW-04-012	8/14/2004	adult	resident	Kenai Fjords
	53	x			AKW-04-013	8/15/2004	subadult	resident	Kenai Fjords
	54	x		x	PWS-04-07	9/6/2004	adult	resident	Prince William Sound
	55	x			PWS-04-08	9/6/2004	subadult	resident	Prince William Sound
	56	x		x	AKW-03-02	?	adult	resident	Kenai Fjords
	57	x		x	AKW-03-03	?	adult	resident	Kenai Fjords
	58	x		x	AKW-03-04	?	adult	resident	Prince William Sound/ Kenai Fjords
	Females
	59	x	x		AKW-04-02	5/22/2004	adult	resident	Prince William Sound
	60	x			AKW-03-01	?	adult	resident	Kenai Fjords

Table 1 , continued. Collection date, age class, ecotype and location for killer whale biopsy samples analyzed for fatty acids (FA), stable isotopes (SI) and persistent organic contaminants (POPs). FA SI POPs a Animal Identification Collection date Age class Ecotype Location EASTERN ALEUTIAN ISLAND TRANSIENTS (EAI/T) Males

 1

	Sample#
	61	x		x	FP-03-01	5/19/2003	adult	transient	Eastern Aleutian Islands
	62	x		x	FP-03-03	5/20/2003	adult	transient	Eastern Aleutian Islands
	63	x		x	FP-03-06	5/22/2003	adult	transient	Eastern Aleutian Islands
	64	x		x	FP-03-07	5/30/2003	adult	transient	Eastern Aleutian Islands
	65	x	x	x	FP-03-08	5/30/2003	adult	transient	Eastern Aleutian Islands
	66	x	x	x	FP-04-025	5/30/2003	adult	transient	Eastern Aleutian Islands
	67	x	x	x	FP-03-09	5/31/2003	adult	transient	Eastern Aleutian Islands
	68	x		x	EA-03-10	7/30/2003	adult	transient	Eastern Aleutian Islands
	69	x	x	x	FP-04-01	5/4/2004	adult	transient	Eastern Aleutian Islands
	70	x	x	x	FP-04-02	5/4/2004	adult	transient	Eastern Aleutian Islands
	62	x	x	x	FP-04-08	5/5/2004	adult	transient	Eastern Aleutian Islands
	63	x	x	x	FP-04-14	5/8/2004	adult	transient	Eastern Aleutian Islands
	64	x	x		FP-04-15	5/9/2004	sub-adult	transient	Eastern Aleutian Islands
	65	x			UNAK-04-06	7/30/2004	subadult	transient	Eastern Aleutian Islands
	66	x	x	x	AE040804-01	8/4/2004	adult	transient	Eastern Aleutian Islands
	67	x	x	x	AE040815-01	8/15/2004	adult	transient	Eastern Aleutian Islands
	68	x	x	x	AE040817-01	8/17/2004	adult	transient	Eastern Aleutian Islands
	Females
	69	x			FP-03-05	5/22/2003	adult	transient	Eastern Aleutian Islands
	70	x	x		FP-04-03	5/4/2004	adult	transient	Eastern Aleutian Islands
	71	x			FP-04-04	5/4/2004	adult	transient	Eastern Aleutian Islands
	72	x	x		FP-04-05	5/4/2004	adult	transient	Eastern Aleutian Islands
	73	x	x		FP-04-06	5/5/2004	adult	transient	Eastern Aleutian Islands
	74	x	x		FP-04-09	5/5/2004	adult	transient	Eastern Aleutian Islands
	75	x	x		FP-04-11	5/7/2004	adult	transient	Eastern Aleutian Islands
	76	x	x		FP-04-12	5/7/2004	adult	transient	Eastern Aleutian Islands
	77	x	x		FP-04-16	5/13/2004	adult	transient	Eastern Aleutian Islands
	78	x	x		FP-04-17	5/13/2004	adult	transient	Eastern Aleutian Islands
	79	x			FP-04-018	5/19/2004	adult	transient	Eastern Aleutian Islands
	80	x			FP-04-021	5/20/2004	adult	transient	Eastern Aleutian Islands
	81	x	x		FP-04-022	5/21/2004	adult	transient	Eastern Aleutian Islands
	82	x	x		FP-04-023	5/26/2004	adult	transient	Eastern Aleutian Islands
	83	x	x		FP-04-024	5/26/2004	sub-adult	transient	Eastern Aleutian Islands
	84	x	x		AE040807-01	8/7/2004	adult	transient	North Alaska Pennisula

Table 2 . Mean (±1 SD) stable isotope values for adult killer whales a from Alaska by region and ecotype, as well as and prey species of transient killer whales. n δ 13 C δ 15 N Killer whales

 2

	Offshores
	AK/OFF	5	-16.5 ± 0.3	17.0 ± 0.1
	Residents
	CAI/R	3	-18.2 ± 0.5	14.0 ± 0.5
	EAI/R	13	-17.0 ± 1.9	16.4 ± 1.7
	GOA/R	6	-15.4 ± 0.4	17.6 ± 0.5
	Mean (residents) ± SD	22	-16.7 ± 1.7	16.4 ± 1.7
	Transients
	EAI/T	27	-17.1 ± 1.0	17.8 ± 08
	Prey species of transients
	gray whale	7	-16.6 ± 0.7	13.2 ± 0.8
	northern fur seal	4	-19.5 ± 0.8	16.7 ± 0.5
	minke whale	1	-19.2	12.4
	Steller sea lion	5	-17.5 ± 0.5	16.3 ± 1.2

a Whales from Table 1 R=resident; T=transient; OFF=offshore; AK=Alaska; EAI=Eastern Aleutian Islands; CAI=Central Aleutian Islands; GOA=Gulf of Alaska.

Table 3 . Mean (±1 SD) and range of persistent organic pollutants (ng/g lipid) and percent lipid in blubber biopsy samples from adult male offshore, resident and transient killer whales from Alaska a

 3 For mean and standard deviation calculations, <LOQ was set = 0.

	Offshores

a

Yonezaki, S.,Kiyota, M., Baba, N., Koido, T., & Takemura., A. (2003) Size distribution of the hard remains of prey in the digestive tract of northern fur seal (Callorhinus ursinus) and related biases in diet estimation by scat analysis. Mamm. Study, 28, 97-102.

Acknowledgments

We greatly appreciate the technical assistance of D.W. Brown, G.M. Ylitalo, C.A. Sloan, R.H. Boyer, R.W. Pearce and J.L. Bolton in sample and data analysis. We thank G.M. Ylitalo, S. O'Neill, J. West and J. Buzitis for the Chinook salmon samples and K. Beckmen, Alaska Department of Fish and Game for Steller sea lion samples. We thank R.L. Pitman and the Southwest Fisheries Science Center for providing several biopsy samples (Z38169, Z38170, Z38171 and Z38175) and A. Schulman-Janiger for the photo-identifications of the offshore killer whales. We would also like to thank the laboratory staff of the SWFSC Population Identity Program, especially, K. Coultrup, N.M. Hedrick, A.R. Lang and K.M. Robertson for generating a portion of the genetic data used in this manuscript. We are grateful for the funding support of the North Pacific Research Board (NPRB publication #28) and T.K. Rowles from the Marine Mammal Health and Stranding Response Program of NOAA Fisheries. In addition, funding support for field research (Matkin) was provided by the Cooperative Institute For Arctic Research, the Steller Sea Lion Recovery Initiative, the Alaska SeaLife Center and the North Pacific Universities Marine Mammal Research Consortiums. Finally, we thank L. Jones of the NWFSC Marine Mammal Program for both programmatic and financial support. Killer whale biopsy samples were collected under one of the following permits: NGOS NOAA Permit No. 545-1488-03; MMPA Permit Nos. 782-1510 and 782-1719, NMFS Permit Nos. 932-1489-05 and 7774-1714-03.

7.

Gulf of Alaska

Eastern Aleutian Is.

Central Aleutian Is.

Gulf of Alaska

Eastern Aleutian Is.

Central Aleutian Is.

Gulf of Alaska

Eastern Aleutian Is.

Central Aleutian Is.

Gulf of Alaska

Eastern Aleutian Is.

Central Aleutian Is.

Gulf of Alaska

Eastern Aleutian Is.

Central Aleutian Is.

Gulf of Alaska

Eastern Aleutian Is.

Central Aleutian Is.