Appendix A – Cadmium Uptake and Depuration BIOKINETICS OF CADMIUM IN DIFFERENT POPULATIONS OF THE PACIFIC GIGAS Trace Metal Ecotoxicology and Biogeochemistry Laboratory, Hong Kong University of Science and Technology (HKUST) and Integral Consulting Prepared by T.Y.T. Ng, C‐Y Chuang, and Wen‐Xiong Wang

Table of Contents 1.0 INTRODUCTION ...... 1 2.0 METHODS ...... 1 2.1 Cadmium concentration in soft tissues (body burden)...... 1 2.2 Subcellular Cadmium concentration ...... 1 2.3 Metallothionein concentration...... 2 2.4 Clearance rate and Condition index ...... 2 2.5 Assimilation efficiency (AE) ‐ different food types and Different diatom concentrations ...... 2 2.5.1 Assimilation efficiency from phytoplankton ...... 3 2.5.2 Depuration rates...... 3 2.5.3 Assimilation efficiency from different phytoplankton concentrations...... 3 2.5.4 Assimilation efficiency from sediments ...... 3 2.6 Dissolved uptake rate ‐ different Cadmium concentrations and body size ...... 4 2.7 Efflux rate...... 4 2.8 Radioactivity measurement and statistical analysis ...... 4 3.0 RESULTS...... 5 3.1 Subcellular Cd distribution ...... 5 3.2 Metallothionein concentration...... 5 3.3 Clearance rate ...... 5 3.4 Condition Index ...... 5 3.5 Diatom Diet ...... 5 3.6 Sediment Diet...... 6 3.7 Pulse‐feeding depuration ...... 6 3.8 Cd dissolved uptake...... 6 4.0 Discussion ...... 6

1.0 INTRODUCTION

In this study, we investigated the cadmium uptake and efflux kinetics in the oyster Crassostrea gigas collected in two sampling events from five oyster farms along the west coast of the U.S. One of the main goals of this study was to determine if there were any metal physiological differences among the oyster populations and examined the effects of environmental factors, such as metal concentrations, food concentration and food type, in addition to biological factors such as body size on cadmium uptake by the . We also quantified the metallothionein concentration and subcellular cadmium distribution in the oysters so as to provide information on differences of metal detoxification among these populations. 2.0 METHODS

Laboratory experiments of cadmium uptake in U.S. west coast Pacific oysters (C. gigas) were performed at the Hong Kong University of Science and Technology using the radiotracer cadmium‐109 (Cd109). In order to provide seeds of adequate sizes (less than 3‐cm maximum length or width) for laboratory experiments, oyster seed from a single hatchery were collected and placed at five sites to grow at each site’s environmental conditions for three months. This setup ensured the were the correct size and were obtained from a known source. The oysters were shipped overnight to Hong Kong July and October 2005 (original site temperature and salinity ranged from 13°C – 19°C and 24 psu – 29 psu, respectively) and acclimated in the laboratory for 1 week 24 °C at 26 psu prior to the experiments. They were continuously fed with the diatom Thalassiosira pseudonana (clone 3H) during the acclimation period. In order to determine the pathways of cadmium accumulation in oysters, controlled laboratory experiments were designed to ascertain the relative contributions of cadmium from sources such as food (suspended particles vs. phytoplankton) and overlying water (i.e. dissolved phase). The measurements of cadmium influx rates (uptake from dissolved phase), efflux rates (depuration), food ingestion rates, growth rates, assimilation efficiencies (AE) and cadmium partitioning into organs and subcellular fractions were used to predict cadmium concentrations in oysters under different environmental conditions. 2.1 CADMIUM CONCENTRATION IN SOFT TISSUES (BODY BURDEN) For each population, 10 individual oysters were sampled for quantification of cadmium concentration in the soft tissues. The oysters were dissected and the soft tissues were gently blotted in clean paper towels and individually placed into acid‐cleaned glass tubes. The wet weight was determined and the dry weight was obtained by drying the tissue at 80°C to constant weights. Afterward, concentrated Ultrapure HNO3 was added to digest the dry tissues. The digests were subsequently diluted with NANOpure® water and the metal concentrations were analyzed by Inductively Coupled Plasma‐Mass Spectroscopy (ICP‐MS) and Polarized Zeeman Atomic Absorption Spectrophotometer. Random checks were made with a standard reference material (1566b Oyster tissue, US Department of Commerce, Technology Administration, National Institute of Standards and Technology, Gaithersburg, MD, USA) and agreement was within 10 %. The Cd tissue concentrations were expressed as μg/g dry weight and wet weight. 2.2 SUBCELLULAR CADMIUM CONCENTRATION The subcellular fractionation of cadmium was quantified only in the October 2005 experiment. The subcellular concentration of cadmium in oysters was quantified using a modified method of Wallace et al. (2003). Five oysters from each population were dissected and soft tissue wet weights were measured. Afterwards, the soft tissue was homogenized in 2 ml of 20 mM Tris‐base buffer (pH 8.6) with 10 mM antioxidant 2‐Mercaptoethanol and 0.1 mM freshly prepared protease inhibitor, Phenylmethylsufonyl fluoride. The homogenate was centrifuged at 1,500 g (15 min at 4 °C). The supernatant was further centrifuged at 100,000 g for 30 min at 4 °C to separate the cytosol from the organelles (ORG) i.e., intracellular pellets containing nuclear, mitochondrial and microsomal fractions.

Appendix A page 1 Following heat treatment (10 min at 80 °C) and ice‐cooling (15 min), the 100,000 g supernatant was centrifuged again at 5,000 g for 10 min at 4 °C. The final supernatant contained the heat‐stable proteins or metallothionein‐like proteins (MTLPs) and the pellets contained the heat sensitive proteins (HSPs) due to heat denaturation. The 1,500g pellets that included the tissue fragments and cellular debris were dissolved in 1 N sodium hydroxide at 80°C, then centrifuged at 5,000 g for 10 min to separate the cellular debris (CD) and metal‐rich granules (MRG). The metal concentrations in the five fractions – MTLP, HSP, ORG, CD, MRG were determined using the method described above, and were expressed as ng/g wet weight of the whole soft tissue (Figure A‐1). 2.3 METALLOTHIONEIN CONCENTRATION Metallotheionein (MT) concentrations in the oysters was only determined in the October 2005 experiment. The MT concentration in the oysters was determined using a modified silver (Ag) saturation method (Scheuhammer and Cherain 1986, Scheuhammer and Cherian 1991, Leung and Furness 1999). The wet weights of five individuals from each population were determined after dissection by gently blotting the tissue in a paper towel and placing the body mass in a pre‐weighed aluminum cup before weighing. The tissues were then homogenized individually in 2 mL Tris‐base buffer (prepared as above), followed by centrifugation at 20,000 g for 20 minutes at 4 °C. Subsequently, 0.3 mL of the supernatant was mixed with 0.5 mL of 20 μg Ag/mL with 110mAg (7.4 kBq/mL) in 0.5 M glycine buffer. The mixture was incubated at room temperature for 10 minutes to saturate the MT with excessive silver. The silver not bound with MT was then removed by adding 0.1 mL rabbit blood cell haemolysate, heating for 5 minutes at 100°C and centrifuging for 5 minutes at 785 g. The addition of haemolysate, heating and centrifugation were repeated twice. The supernatant obtained was further centrifuged for 20 minutes at 17,100 g. The amount of silver remaining in the final supernatant was quantified by measuring the activity of 110mAg. The MT concentration was calculated as 3.55 times the silver concentration and expressed as μg/g wet weight of the oyster (Scheuhammer and Cherian 1991). Figure A‐2 shows the concentrations of MT for each population. 2.4 CLEARANCE RATE AND CONDITION INDEX Ten oysters from each population were placed individually into polypropylene beakers containing 1.5 L of 0.22 μm filtered seawater. After the oysters opened their valves and pumped water normally (usually within 10 minutes), the diatom Thalassiosira pseudonana (filtered from their growth medium) were added into each beaker at a concentration about 104 cells/ml. In each beaker, the algal cells were kept in suspension with a magnetic stir bar over a stir table. Immediately after adding the algae, a 10 mL aliquot water sample was removed from each beaker and the cell density was counted using a Coulter Counter (Z1, Coulter). Further water samples were taken at 20 minutes and 40 minutes and the cell density was determined. After measuring the clearance rates for each population, the were dissected. The soft tissues and shells were dried at 80 °C overnight to determine the tissue dry weights and shell dry weights, respectively. Figure A‐3 shows the clearance rates for each population. Clearance rates were calculated using the following equation (Widdows et al., 1997)

CR= Vol × [ ln(C1) – ln(C2) ] / t where CR is the clearance rate (L/g ∙ h), C1 is the cell density (cell/mL) at time 1, C2 is the cell density at time 2, t is time of measurement increment (i.e. t2−t1, in hours), and Vol is the volume of water (L). The clearance rate for each individual population was calculated from the mean of the two consecutive measurements. The condition index (CI) of oysters represents an ecophysiological approach to estimate meat quality and yield in cultured bivalve mollusks and is an important indicator of how well the oyster has utilized the internal cavity volume for tissue growth. In addition, CI has been used as a tool to estimate the effect that different environmental factors have on oyster meat quality (Mercado‐Silva 2005). The CI was expressed as 100 % x (tissue dry weight / shell dry weight) (Rainer & Mann 1992). Figure A‐3 shows the condition index for each population. 2.5 ASSIMILATION EFFICIENCY (AE) ‐ DIFFERENT FOOD TYPES AND DIFFERENT DIATOM CONCENTRATIONS

Appendix A page 2 The incorporation of trace metals from food can be the main path of metal uptake into aquatic invertebrates. The following experiments measured assimilation efficiency of Cd from different food types and different diatom concentrations. 2.5.1 Assimilation efficiency from phytoplankton The diatom T. pseudonana (clone 3H) was radiolabeled with gamma‐emitting radioisotopes 109Cd as described in (Wang and Fisher, 1996a). Radioactivity additions were 185 kBq for 109Cd (in 0.1 N HCl). After 4 days, the cells had undergone greater than four divisions and were considered uniformly labeled. These cells were collected on a 3‐ μm polycarbonate membrane and resuspended in 50 mL of water before being added to the feeding beakers. Ten individuals from each population of C. gigas were individually placed in 500 mL of 0.22 μm filtered seawater. Algae were added to each beaker to give a cell density between 4 and 5×104 cells/mL. Further additions were made at 10 minutes intervals to maintain this approximate cell density. After 30 minutes of feeding on radiolabeled diatoms, the oysters were rinsed in seawater and analyzed for their radioactivity. Potential radiotracer uptake from the aqueous phase (due to desorption from the radiolabeled diatoms) was negligible compared with the total ingestion of radiotracers from diatoms within the 30 minutes period (Chong and Wang, 2000). 2.5.2 Depuration rates Five individual oysters with the highest activity counts were selected and placed into separate polypropylene beakers (180 mL seawater) held in a 20 L enclosed recirculating flow‐through aquarium tank containing seawater of 26 psu salinity (Wang et al., 1995). Unlabeled T. pseudonana was fed to the oysters at a ratio of about 1 to 2% of their tissue dry weight per day. In the July 2005 experiment, radioactivity retained in the oysters was measured over a 60‐hours depuration period at intervals from 1.5 to 12 hours, whereas in the other (October 2005) assimilation efficiency experiments, depuration period was shortend and assimilation efficiencies were determined as the percentage of initial radioactivity retained in the oysters after 24 hours of depuration since percentage of cadmium retained in the oysters at 24 hours became constant. Any feces egested by the animals during the depuration period were collected regularly to minimize desorption of radiotracers from fecal matter into the water. 2.5.3 Assimilation efficiency from different phytoplankton concentrations We also tested the effect of food quantity on cadmium AE by C. gigas on two populations. Only one population was selected for the July 2005 experiment – Thorndyke Bay, WA (SQB), and one for the October 2005 experiment ‐ Humboldt Bay, CA (HUM). Four different concentrations of the diatom T. pseudonana were used: 0.2, 0.5, 2 and 5 mg/L, which encompassed the food concentrations below and above the pseudofeces production (Widdows et al., 1979; Wang, 2002). A higher concentration of 10 mg/L was also tested in the October 2005 experiment. In each diatom concentration treatment, there were five individual oysters. The food concentrations were maintained constant within the treatment during both the pulse radioactive feeding and the depuration periods. Other procedures followed the description above. 2.5.4 Assimilation efficiency from sediments The assimilation efficiency (AE) of cadmium by the Pacific oysters fed sediment particles was measured similarly to the procedures described above. Dry oxic sediment (10 mg) from each oyster collection site was radiolabeled with 5 μCi (185 kBq) 109Cd and the pH was equilibrated to 8.0 with 1 N NaOH. Once radiolabeled, the sediment slurry was allowed to rest for 3 days. The sediment was then collected by centrifugation at 2,180 g for 5 minutes twice and resuspended in 1.5 mL of 0.22 μm filtered seawater before the assimilation measurements. The particles were then added to 1 L of 0.22 μm filtered seawater at a particle load of 10 mg/L. Individual oysters were allowed to feed on the radiolabeled particles for 30 minutes, and the sediment was added into each treatment at T=0, 10 and 20 minutes. During which, no radiolabeled feces were produced. Afterward, the oysters were then rinsed and radioassayed.

Appendix A page 3 2.6 DISSOLVED UPTAKE RATE ‐ DIFFERENT CADMIUM CONCENTRATIONS AND BODY SIZE Metal dissolved uptake rate describes the rate at which the oysters take up metals from the dissolved phase. A short exposure time (1 hour) was chosen to measure uptake rate so that • Efflux has minimal influence on tissue concentration • Decrease in metal concentration in ambient experimental water is minimized due to uptake by the oysters • Oyster ventilation activity, caused by the absence of food, is minimized. Stable Cd at 0.5, 2, 10, and 50 μg/L and 109Cd (3.7 kBq/L equivalent to 0.03 μg/L stable Cd) were equilibrated overnight before the uptake experiments. Ten oysters from each population were carefully cleaned and placed individually into 200 mL of 0.22 μm filtered seawater with the addition of stable Cd and 109Cd, for 1 hour. After exposure, the oysters were dissected and the radioactivity of the soft tissues was measured. The tissues were then dried at 80°C overnight to determine the dry weights. The influx rate was calculated as the amount of metal accumulated by the oysters and was standardized as per gram tissue dry weight per hour (ng/g∙h). Dissolved uptake rate constant (L/g∙d) was calculated by dividing the influx rate over the concentration. The rates were subsequently compared among the experimental populations. To evaluate the effect of body size on the dissolved metal uptake, 20 oysters ranging from 0.01 to 6 g dry weight were collected from each population for the July experiment. The oysters were similarly exposed to the radioisotopes and stable Cd (2 g/L) as described above. Since the October oysters had a smaller size range (0.01 ‐ 0.15 g dry wt.), all oysters from populations with variable size ranges that were exposed to different Cd concentrations, were pooled from the October dissolved uptake experiment for data analysis. 2.7 EFFLUX RATE The efflux rate was determined for the July populations only. Ten oysters from each population were maintained in clean seawater and fed with 109Cd radiolabeled (37 kBq/L) diatom T. pseudonana for 2 hours each day. These diatoms had been radiolabeled with 109Cd for 4 days (as described above) and were filtered from the growth medium, rinsed, and resuspended in clean seawater before being fed to the oysters. The seawater was renewed every day after the radioactive feeding period. After 6 days of feeding oysters on radiolabeled food (i.e. 2 h/d), the oysters were radioassayed and then placed into an enclosed recirculating seawater aquarium (as described above) to depurate the 109Cd for a period of 21 days. During the course of depuration, the aquarium seawater was changed every 2 days, and nonradioactive T. pseudonana was fed twice daily at a ration of about 2 % oyster dry weight per day. The radioactivity retained in each individual was counted periodically, and fecal pellets were collected frequently. On the first and the last day of depuration, three individuals from each population were ‐1 dissected to determine the radioactivity in the shell and soft tissue. Efflux rate constant (ke, d ) was determined from the slope of the linear regression between the natural log (ln) % Cd retained in the oysters against the depuration time (d). Since depuration of Cd of all populations was linear over time, ke was calculated from day 0 to 21. 2.8 RADIOACTIVITY MEASUREMENT AND STATISTICAL ANALYSIS 109Cd radioactivity was measured by a Wallac gamma counter. Spillover from the higher energy window to the lower energy window of radioisotopes was corrected and all counts were related to standards for each isotope. The gamma emissions of 109Cd were determined at 88 keV. Counting times in all samples were adjusted so that the propagated counting errors were typically < 5 %. Statistical difference among different treatments was performed by one‐way ANOVA (p < 0.05) and significant differences between populations were analyzed by the Tukey Multiple Comparison Post Hoc tests. Linear

Appendix A page 4 regression analysis was performed for the correlations between dissolved uptake rates and Cd concentration or body size. 3.0 RESULTS

This section summarizes the results of biokinetic experiments performed on oyster seeds. 3.1 SUBCELLULAR CD DISTRIBUTION Subcellular Cd concentration was measured for the October oyster populations only. The oyster tissue was fractioned into soluble (MTLP, HSP and organelles) and insoluble fractions (MRG and CD) for Cd concentration analysis (see Section 4.3 for details). Since the concentration of Cd in the CD was below the detection limit, it was not presented in this study. Generally, concentration of Cd was similar (0.10 – 0.38 g/g wet wt.) in all subcellular fractions with the exception of Oakland Bay (OAK) oysters, which had higher Cd concentrations in the soluble fractions, especially in the MTLP fraction (Figure A‐1).

3.2 METALLOTHIONEIN CONCENTRATION Metallothionein concentration was measured for the October oyster populations only. Metallothionein concentration in the oysters was similar among populations (57.41 – 73.03 g/g wet wt.) (Figure A‐2). 3.3 CLEARANCE RATE The July oyster populations had comparable clearance rates (7.99 – 14.02 L/g∙h), with the exception of the Eld Inlet (ELD) population which had a very low clearance rate of 0.91 L/g∙h (Figure A‐3). The ELD population had a high mortality in the laboratory and the low filtration rate could be associated with stress imposed during transport and acclimation to the new laboratory environment. The temperature and salinity at Eld Inlet in July 2005 averaged from 16°C and 29 psu, respectively, and the new water at the laboratory was at 24 °C at 26 psu prior to the experiments; a difference of 8°C and 3 psu. The October populations survived well in the laboratory. They had variable and higher clearance rates (5.61 – 70.06 L/g∙h) than the July populations. Among the October populations, the Humboldt Bay (HUM) oysters had the highest clearance rate (70.06 L/g∙h) and the triploid oysters from Oakland Bay (OAK T) had the lowest clearance rate (5.61 L/g/h). In addition, when looking at differences in ploidy, OAK T oysters had a lower clearance rate than the Oakland Bay diploid oysters (OAK D). 3.4 CONDITION INDEX Condition index (CI) was similar among the July populations with the exception of Thorndyke Bay (SQB) oysters when compared to oysters from Willapa Bay Central (WBC) and Netarts Bay (NET) (Figure A‐3). Condition index of ELD oysters was similar to the other oyster populations. The CI for ELD was determined from oysters that were sampled immediately after transportation to the laboratory and cannot explain the high mortality for that population during the experimental period. Therefore, when taking into account the large differences in temperature and salinity from its original site as discussed above, it is evident that ELD survived well in the field, but may have been stressed during transport and did not adapt to the laboratory conditions. The CI for the October population was similar to the July population. OAK T had a significantly higher (p < 0.01) CI than any of the other oyster population including OAK D. 3.5 DIATOM DIET There were no significant differences in AEs of Cd from the diatom diet in the July populations (Table A‐1, Figure A‐ 4). All July populations depurated Cd at the same rate for the first and second compartments with an average Cd assimilation efficiency of 44%. Some of the October oysters had higher dietary uptake of Cd from diatom than the

Appendix A page 5 July populations [e.g. 61% for HUM and 64% for Thorndyke Bay, WA (TAY) (Table A‐1, Figure A‐4)]. The October populations can be grouped according to their AEs: TAY ≥ HUM > OAK D = OAK T. Different diatom concentrations affected the assimilation of Cd in the oysters. Both July and October experiments demonstrated that the AE of Cd by the oysters decreased with increasing concentration of diatom cells (Table A‐1). AE of diatom at different food concentrations was only determined for the SQB (July) and HUM (Oct) populations. The AE of Cd at diatom quantity of 0.2 and 0.5 mg/L was similar by the SQB and HUM populations. Then the AE decreased from food quantity of 2 mg/L, although it was not noticeable in the HUM population at 5 mg/L, and most obvious at 10 mg/L. 3.6 SEDIMENT DIET AEs of Cd from a sediment diet was however lower (Table A‐1, Figure A‐4). The SQB population did not feed on sediment. Therefore, no AE was determined for SQB. The highest Cd uptake from the sediment diet was 29.86 % for the ELD population. However, it was 7 % lower than the lowest AE from the diatom diet. Oysters from the Hama Hama (HAM) population had the lowest assimilation efficiency of Cd from the sediment diet among all July populations. 3.7 PULSE‐FEEDING DEPURATION The depuration of Cd by the oysters after a diatom and sediment pulse‐feeding exposure can be divided into two compartments – a fast and a slow compartment as shown in Figure A‐4. The oysters released most of Cd in the first 6 hours (0 – 6 h, fast compartment). Subsequently, the percentage of Cd retained in the oysters was almost constant over the depuration period (> 6 h, slow compartment). Depuration or efflux of Cd after feeding to labeled diatom for 6 days followed a single compartment pattern (Figure A‐5). Rate of loss among populations was significantly different. ELD had the fastest efflux rate and the depuration of Cd in the other populations was 2x slower. 3.8 CD DISSOLVED UPTAKE Dissolved uptake rate constants (Ku) are calculated by dividing the dissolved uptake rate over the concentration of Cd in the oysters exposed to different concentrations of Cd. Dissolved uptake rate of Cd increased with increased Cd concentrations in the water (Figure A‐6). The regression was linear (data were plotted on log scale) for both the July and October populations. There were marked differences in Ku among populations of the July oysters (p < 0.01, Table A‐2), with the exception of NET and SQB populations, which had the highest Ku values. The ELD population had the lowest Ku (Figure A‐6). The October populations had higher Ku values than the July populations (Table A‐2) and the Ku among the October populations were similar except for the TAY population which had the lowest Ku. Body size of most July populations was negatively correlated to the dissolved uptake rate (5 out of 6 populations) (Table A‐3 and Figure A‐7). The relationship was less obvious (significantly different regressions in 2 out of 4 populations) when oysters were pooled from the October populations at different concentrations of Cd (Table A‐3 and Figure A‐8) and they showed positive correlations between body size and dissolved uptake of Cd. In general, relationship between body size and dissolved uptake rate of oysters is population dependent. 4.0 DISCUSSION

Oysters from the OAK population may have better capability of detoxifying Cd than the other oyster populations because they stored more Cd in the MTLP (40 % of total Cd, when Cd in the cellular debris portion was neglected). MT is a common metal detoxification protein in bivalves and it is often induced by metals (Mason and Jenkins, 1995). The Pacific oysters in the chronically Cd contaminated site (Gironade estuary, France) stored 40 – 60 % Cd in the soluble form and 30 – 40 % of this fraction was chelated with the metallothioneins (Boisson et al., 2003). This chelation may reduce the toxicity and bioavailability of Cd to the oysters. The higher association of Cd to the MTLP

Appendix A page 6 may also imply higher bioavailability of Cd to humans through oyster consumption because the soluble fractions ‐ MTLP, HSP and organelles are considered the trophically available fractions (Wallace and Luoma, 2003), whereas the insoluble fractions, i.e., MRG and cellular debris are often not available to the predators (Nott and Nicolaidou, 1989; 1990; 1993; Wallace and Lopez, 1997). Wallace and Luoma demonstrated that metals binding to the trophically available fractions was size dependent in the M. balthica (Wallace and Luoma, 2003), but it was not clearly noticed for C. gigas in this study due to small sample number and similar size of oysters used for the subcellular fractionation. Clearance rate can indicate the physiological condition and affects the metal accumulation in the bivalves. Higher clearance rate means higher water pumping rate and more metals passing through the gills, in addition, it implies more food passing the gut, thus affects the amount of metals assimilated from ingestion. The October populations in general, had a higher clearance rate than the July populations. This biological variation may be caused by the seasonal fluctuation in food availability or biological adaptation to their local food availability. When there is less food in late autumn (October) than summer (July), the oysters may pump faster to attain more food to maintain their basic metabolism. Alternatively, the Oct population may have developed a stronger adductor muscle for filtration to adapt to the local food availability. The food concentration in the field however, was not investigated in this study. Despite of a higher clearance rate, the Oct populations had similar energy reserve (dry to wet wt. ratios) as the Jul populations. It is clearly demonstrated in this study that there was variation in Cd uptake from the dissolved phase among populations of the oysters. In addition, the relationship between the clearance rate and dissolved uptake was population dependent, for instance, SQB had the highest dissolved uptake rate constant within the Jul population, but HUM had the highest clearance rate; whereas the lowest dissolved uptake constant of ELD may relate to their low clearance rate, similarly for the overall higher dissolved uptake constants and clearance rate in the Oct populations than the Jul populations. In general, the oysters had more than 10x higher Cd dissolved uptake rate constant than the other bivalves such as the mussels M. edulis (Wang et al., 1996; Wang and Fisher, 1999b), P. viridis (Chong and Wang, 2001), Septifer virgatus (Wang and Dei, 1999) the R. philippinarium (Chong and Wang, 2001; Ng and Wang, 2004), Potamocorbula amurensis and M. balthica (Lee et al., 1998) and again, the oysters had higher clearance rate (10 – 80 l g‐1 h‐1) than the others (typical clearance rate for the clams and mussels: 1 ‐ 10 l g‐1 h‐1). However, the intra‐specific difference in clearance rate cannot completely account for the difference in dissolved metal uptake rate constants among the bivalves (Chong and Wang, 2001). Smaller oysters had lower Cd dissolved uptake rate than the larger oysters in the Jul populations and this was similarly observed for Cd and Zn uptake in the mussels and clams (Lee et al., 1998; Wang and Dei, 1999). Their influx rate decreased in a power function with increasing body size and it can be explained by the size‐specific metabolic rates (Boyden, 1974; Ringwood, 1989; Newman and Heagler, 1991) or the changes in gill surface area (Wang and Fisher, 1997). This relationship however was not observed for the Oct populations. The relationship may be offset by pooling oysters from different sites. In fact, positive, negative and neutral relationships may be found between tissue metal concentration and bivalve size in natural populations and the relationship is complicated by effects of growth and long‐term changes in metal partitioning among intracellular components (Strong and Luoma, 1981; Wallace et al., 2003). Uptake of metals from diet can be a significant route of metal accumulation in the bivalves. For clams, trophic transfer is more important than the dissolved uptake in the overall Cd and Zn accumulation because they have relatively high Cd and Zn assimilation efficiency and low dissolved uptake rate constant (Chong and Wang, 2001). Assimilation of metals from the phytoplankton by the oysters are generally higher than the mussels and clams e.g., 50‐55 % for Cd in the oysters Crassostrea rivularis, (Ke and Wang, 2001) and 37‐64 % in C. gigas (this study); whereas only 10‐40 % in the mussels M. edulis, P. viridis (Wang et al., 1996; Chong and Wang, 2000) and 22‐55 % in the clam R. philippinarium (Chong and Wang, 2000), therefore it may imply that Cd uptake from the diet is a main exposure pathway for the oysters, however considering a similarly high Cd dissolved uptake rate constant, it is difficult to delineate the importance of dietary and dissolved exposure on the Cd accumulation of the oysters in this study AE of metals generally depends on the gut chemistry and physiology of the bivalves,

Appendix A page 7 e.g., gut pH and passage time (Wang and Fisher, 1996a; Griscom et al., 2002a). AE of Cd from sediment and diatom by the oysters was population dependent with implication that gut physiology of the oysters from different sites may be somewhat different, e.g., concentrations of dissolved organic carbon, enzymes and surfactants in the gut. Generally, AE of metals is lower from the sediment than the living matter like phytoplankton (Wang and Wong, 2003) because surface bound metals on the sediment are more readily to be desorbed and lost during digestion. Environmental factors such as distribution of metals in the cytoplasm of the phytoplankton (Reinfelder and Fisher, 1991), food quality and quantity can also affect the assimilation of metals (Wang and Wong, 2003). Wong and Wang (Wang and Wong, 2003) found that Cd AE was more influenced by the quality (in terms of organic carbon concentration) than quantity of food in P. viridis. The Cd uptake from the phytoplankton by the oysters, was however affected by the diatom concentration. Higher food concentration may reduce the AE of metals because there is relatively shorter gut retention time for high quantity of food, thus it is less efficient to digest and absorb the food. In view of this, oysters living in a higher food concentration environment may have similar accumulation of Cd from the diet as those living in the low food environment as the effect of food quantity on metal accumulation is canceled out by the effect on Cd assimilation. The excretion of metals by the bivalves is generally independent from the environmental conditions (Wang, 2003), similarly found in the oysters that were collected from different sites in this study. Typical efflux rate constants in the bivalves are within 0.01‐0.03 d‐1 and the inter‐species variation in the metal efflux rate is rather small. However, there were reported variations in metal efflux rate among oysters and mussels collected from the coastal and estuarine zones. The coastal oyster S. glomerata had relatively lower Cd and Zn efflux rates, than the estuarine oyster C. rivularis (Ke and Wang, 2001). In addition, the P. viridis collected from the coastal zone had a faster Ag efflux than those collected from the estuarine zone (Ng & Wang 2005), and this can probably be explained by a higher storage of Ag in the insoluble subcellular fractions of the mussels in the estuarine zone (Ng & Wang 2005). The difference in storage of Ag in the mussels may be caused by the different water geochemistry in the two hydrographic zones. The diploid and triploid OAK oysters had similar Cd kinetics and storage of Cd in their subcellular compartments, despite of the genetic variation. Genetically, triploid oysters are made partially sterile so that a change in allocation of energy from reproduction enables them to have a better growth. In addition, the triploid oysters may be more resistant to diseases and associated mortality caused by parasites (Nell, 2002). In this study, the only physiological difference examined between the triploid and diploid OAK oysters were the 2x heavier dry weight of the triploids than the diploids, also the diploids filtered water 3x faster than the triploids This physiological or genetic difference apparently did not affect the Cd accumulation or excretion in the two kinds of oysters. Amiard et al. (Amiard et al., 2005) in fact, hypothesized that the higher energy available for growth of the triploid oysters may have a positive impact on the metal detoxification and excretion since transcription is faster with three copies of each gene. The positive role of heterozygosity on feeding rate and absorption efficiency has also been emphasized (Hawkins et al., 2000). However, the triploid oysters C. gigas were demonstrated to have a lower Ag, Cd and Cu accumulation in their study (Amiard et al., 2005) and it may be due to the physiological changes associated with the more reproductive development of the diploids. On the other hand, Marie et al. (Marie et al., 2006) reported no significant difference in MT gene expression, MT protein synthesis and Cd accumulation in the gills of the diploid and triploid C. gigas. It agreed with our results on MT concentration. Both of our studies suggested that the triploid oysters have the same capacities to respond to Cd as diploids. To summarize, the Cd uptake kinetics in the Pacific oysters varied among populations, but they were similar in Cd detoxification and storage in the cells. Dissolved uptake rate of Cd may have a positive relationship with the clearance rate and negative relationship with the size of oysters. The difference in Cd kinetics may be caused by the variation of physiology and the ambient environment, e.g., gut physiology that contributes to the variation in AE, and metal concentrations, food quantity and quality that contribute to the variations of dissolved uptake rate constant and AE. By understanding the Cd kinetics in different populations of Pacific oysters under variety of environmental conditions helps to provide more information on building the bioenergetic based kinetic model for this commercially important species, thus monitors and protects against the Cd contamination in these oysters.

Appendix A page 8 5.0 REFERENCES

Amiard, J.C., Perrein‐Ettajani, H., Gerard, A., Baud, J.P., Amiard‐Triquet, C., 2005, Influence of ploidy and metal‐ metal interactions on the accumulation of Ag, Cd, and Cu in oysters Crassostrea gigas Thunberg. Arch Environ Contam Toxicol 48, 68‐74. Boisson, F., Goudard, F., Durand, J.P., Barbot, C., Pieri, J., Amiard, J.C., Fowler, S.W., 2003, Comparative radiotracer study of cadmium uptake, storage, detoxification and depuration in the oyster Crassostrea gigas: potential adaptive mechanisms. Mar Ecol Prog Ser 254, 177‐186. Boyden, C.R., 1974, Trace metal content and body size in molluscs. Nature 251, 311‐314. Chong, K., Wang, W.X., 2000, Assimilation of cadmium, chromium, and zinc by the green mussel Perna viridus and the clam Ruditapes philippinarum. Environ Toxicol Chem 19, 1660‐1667. Chong, K., Wang, W.X., 2001, Comparative studies on the biokinetics of Cd, Cr, and Zn in the green mussel and the Manila clam Ruditapes philippinarum. Environ Pollut 115, 107‐121. Griscom, S.B., Fisher, N.S., Aller, R.C., Lee, B.G., 2002a, Effects of gut chemistry in marine bivalves on the assimilation of metals from ingested sediment particles. J Mar Res 60, 101‐120. Griscom, S.B., Fisher, N.S., Luoma, S.N., 2002b, Kinetic modeling of Ag, Cd and Co bioaccumulation in the clam Macoma balthica: quantifying dietary and dissolved sources. Mar Ecol Prog Ser 240, 127‐141. Hawkins, A.J., Magoulas, A., Heral, M., Bougrier, S., Naciri‐Graven, Y., Day, A.J., 2000, Separate effect of triploidy, percentage and genomic diversity upon feeding behaviour, metabolic efficiency and net energy balance in the Crassostrea gigas. Genet Res 76, 273‐284. Ke, C., Wang, W.X., 2001, Bioaccumulation of Cd, Se and Zn in an estuarine oyster (Crassostrea rivularis) and a coastal oyster (Saccostrea glomerata). Aquat Toxicol 56, 33‐51. Lee, B.G., Wallace, W.G., Luoma, S.N., 1998, Uptake and loss kinetics of Cd, Cr and Zn in the bivalves Potamocorbula amurensis and Macoma balthica: effects of size and salinity. Mar Ecol Prog Ser 175, 177‐189. Leung, K.M.Y., Furness, R.W., 1999, Induction of metallothionein in dogwhelk Nucella lapillus during and after exposure to cadmium. Ecotoxicol Environ Safety 43, 156‐164. Marie, V., Gonzalez, P., Baudrimont, M., Boutet, I., Moraga, D., Bourdineaud, J.‐P., Boudou, A., 2006, Metallothionein gene expression and protein levels in triploid and diploid oysters Crassostrea gigas after exposure to cadmium and zinc. Environ Toxicol Chem 25, 412‐418. Mason, A.Z., Jenkins, K.D. (1995) Metal detoxification in aquatic organisms. In: Tessier, A., Turner, D.R. (Eds.) Metal speciation and bioavailability in aquatic systems. John Wiley, Chichester, pp 479‐608. Mercado‐Silva, N. 2005. Condition index of the , Crassostrea virginica (Gmelin, 1791) in Sapelo Island Georgia‐‐effects of site, position on bed and pea crab parasitism. Journal of Research, Volume 24, Issue 1, pp 121‐126. Nell, J.A., 2002, Farming triploid oysters. Aquaculture 210, 69‐88. Newman, M.C., Heagler, M.G. (1991) Allometry of metal bioaccumulation and toxicity. In: Newman, M.C., Mcintosh, A.W. (Eds.) Metal ecotoxicity: concepts and applications. Lewis Publishers, Chelsea, Michigan, pp 91‐ 130. Ng, T.Y.T., Wang, W.X., 2004, Detoxification and effects of Ag, Cd, and Zn pre‐exposure on metal uptake kinetics in the clam Ruditapes philippinarum. Mar Ecol Prog Ser 268, 161‐172. Ng, T.Y.‐T., Wang, W.X., 2005a, Modeling of cadmium bioaccumulation in two populations of the green mussel Perna viridis. Environ Toxicol Chem 24, 2299‐2205.

Appendix A page 9 Ng, T.Y.‐T., Wang, W.X., 2005b, Subcellular controls of silver biokinetics in the green mussel Perna viridis from two hydrographic zones. Mar Ecol Prog Ser 299, 193‐204. Nott, J.A., Nicolaidou, A., 1989, Metals in gastropods ‐ metabolism and bioreduction. Mar Environ Res 28, 201‐205. Nott, J.A., Nicolaidou, A., 1990, Transfer of metal detoxification along marine food chains. J Mar Biol Ass UK 70, 905‐912. Nott, J.A., Nicolaidou, A., 1993, Bioreduction of zinc and manganese along a molluscan food chain. Comp Biochem Physiol A 104A, 235‐238. Rainer, J. S. & R. Mann. 1992. A comparison of methods for calculating condition index in eastern oysters Crassostrea virginica (Gmelin, 1791). J. Shellfish Res. 11(1):55‐58. Reinfelder, J.R., Fisher, N.S., 1991, The assimilation of elements ingested by marine copepods. Science 251, 794‐ 796. Ringwood, A.H., 1989, Accumulation of cadmium by larvae and adults of an Hawaiian bivalve, Isognomon californicum, during chronic exposure. Mar Biol 102, 499‐504. Scheuhammer, A.M., Cherain, M.G., 1986, Quantification of metallothioneins by a silver‐saturation method. Toxicol Appl Pharmacol 82, 417‐425. Scheuhammer, A.M., Cherian, M.G., 1991, Quantification of metallothionein by silver saturation. Methods Enzymol 205, 78‐83. Strong, C.R., Luoma, S.N., 1981, Variations in the correlation of body size with concentrations of Cu and Ag in the bivalve Macoma balthica. Can J Fish Aquat Sci 38, 1059‐1064. Wallace, W.G., Lee, B.G., Luoma, S.N., 2003, Subcellular compartmentalization of Cd and Zn in two bivalves. I. Significance of metal‐sensitive fractions (MSF) and biologically detoxified metal (BDM). Mar Ecol Prog Ser 249, 183‐197. Wallace, W.G., Lopez, G.L., 1997, Bioavailability of biologically sequestered cadmium and the implications of metal detoxification. Mar Ecol Prog Ser 147, 149‐157. Wallace, W.G., Luoma, S.N., 2003, Subcellular compartmentalization of Cd and Zn in two bivalves II: Significance of trophically available metal (TAM). Mar Ecol Prog Ser 249, 183‐197. Wang, W.X., 2002, Interactions of trace metals and different marine food chains. Mar Ecol Prog Ser 243, 295‐309. Wang, W.X. (2003) Metal bioaccumulation in bivalve mollusks: recent progress. In: Villalba, A., Reguera, B., Romalde, J.L., Beiras, R. (Eds.) Molluscan Shellfish Safety. Intergovernmental Oceanographic Commission of UNESCO and Conselleria de Pesca e Asuntos Maritimos da Xunta de Galicia, Santiago de Compostela, Spain, pp 503‐520. Wang, W.X., Dei, R.C.H., 1999, Factors affecting trace element uptake in the black mussel Septifer virgatus. Mar Ecol Prog Ser 186, 161‐172. Wang, W.X., Fisher, N.S., 1996a, Assimilation of trace elements and carbon by the mussel Mytilus edulis: effects of food composition. Limnol Oceanogr 41, 197‐207. Wang, W.X., Fisher, N.S., 1996b, Assimilation of trace elements by the mussel Mytilus edulis: effects of diatom chemical composition. Mar Biol 125, 715‐724. Wang, W.X., Fisher, N.S., 1997, Modelling the influence of body size on trace element accumulation in the mussel Mytilus edulis. Mar Ecol Prog Ser 161, 103‐115. Wang, W.X., Fisher, N.S., 1999a, Delineating metal accumulation pathways for aquatic invertebrates. Sci Total Environ 237/238, 459‐472.

Appendix A page 10 Wang, W.X., Fisher, N.S., 1999b, Effects of calcium and metabolic inhibitors on trace element uptake in two marine bivalves. J Exp Mar Biol Ecol 236, 149‐164. Wang, W.X., Fisher, N.S., Luoma, S.N., 1995, Assimilation of trace elements ingested by the mussel Mytilus edulis: Effects of algal food abundance. Mar Ecol Prog Ser 129, 165‐176. Wang, W.X., Fisher, N.S., Luoma, S.N., 1996, Kinetic determinations of trace elements bioaccumulation in the mussels Mytilus edulis. Mar Ecol Prog Ser 140, 91‐113. Wang, W.X., Wong, S.K., 2003, Combined effects of food quantity and quality on Cd, Cr, and Zn assimilation to the green mussel, Perna viridis. J Exp Mar Biol Ecol 290, 49‐69. Widdows, J., Fieth, P., Worrall, C.M., 1979, Relationships between seston, available food and feeding activity in the common mussel Mytilus edulis. Mar Biol 50, 195‐207. Widdows, J., Nasci, C., Fossato, V.U., 1997, Effects of pollution on the scope for growth of mussels (Mytilus galloprovincialis) from the Venice lagoon, Italy. Mar Environ Res 43, 69‐79.

Appendix A page 11

Table A‐1. Assimilation efficiency (%) of Cd by the oyster Crassostrea gigas feeding on different food types and concentrations. Mean ± standard deviation (n = 5).

Population Food type Diatom Sediment Jul experiment NET 48.06 ± 5.72a 21.52 ± 7.58ab WBC 37.28 ± 5.90a 24.58 ± 9.33a SME 47.41 ± 6.01a 17.73 ± 6.91ab SQB 47.62 ± 5.74a ND HAM 44.60 ± 5.04a 8.59 ± 0.89b ELD 38.48 ± 6.67a 29.86 ± 9.19a

Oct experiment HUM 60.78 ± 6.91a ND OAK D 48.01 ± 5.05b ND OAK T 48.05 ± 4.97b ND TAY 64.24 ± 4.83a ND

Diatom concentration Jul experiment (SQB) 0.2 mg l‐1 38.73 ± 7.42ab 0.5 mg l‐1 40.47 ± 5.81ab 2 mg l‐1 40.64 ± 4.03a 5 mg l‐1 28.39 ± 7.37b

Oct experiment (HUM) 0.2 mg l‐1 55.89 ± 9.53a 0.5 mg l‐1 45.35 ± 6.33ac 2 mg l‐1 41.06 ± 7.13c 5 mg l‐1 44.04 ± 4.30ac 10 mg l‐1 17.90 ± 2.59b Note: Data were not determined (ND) for the assimilation efficiency from sediment by SQB because they did not feed on sediment. Assimilation efficiency of each population sharing the same letter as an index do not differ significantly by the Tukey Multiple Comparison post hoc test (p > 0.05).

Appendix A page 12

Table A‐2. Dissolved uptake rate constants (l g‐1 d‐1) of the oyster Crassostrea gigas. Dissolved uptake rate constants are calculated by dividing the dissolved uptake rate over the concentration of Cd in the oysters exposed to different concentrations of Cd. Mean ± standard deviation (n = 40). Dissolved uptake rate constant of each population sharing the same letter as an index do not differ significantly by the Tukey Multiple Comparision post hoc test (p > 0.05).

Population Dissolved uptake rate constant (L g‐1 d‐1) Jul experiment NET 3.76 ± 1.20a WBC 1.71 ± 0.54b SME 2.90 ± 1.03c SQB 4.32 ± 0.76a HAM 2.30 ± 0.71d ELD 0.68 ± 0.34e

Oct experiment HUM 8.68 ± 2.30a OAK D 9.59 ± 1.90a OAK T 8.42 ± 1.70a TAY 5.97 ± 1.57b

Appendix A page 13 Table A‐3. Regressions of the dissolved uptake rate (ng g‐1 h‐1) and dry weight (g) of the oyster Crassostrea gigas. Dissolved uptake rate of the oysters collected in July were exposed to 2 g L‐1 Cd for different populations (n = 20, 0.01 – 6 g dry wt.), whereas populations of oysters collected in October were pooled and tested for dissolved ‐1 uptake rate at 0.5, 2, 5 and 10 mg L Cd. (n = 40, 0.01 – 0.15 g dry wt.). The regression was tested by linear regression analysis. Asterisk(s) indicate significant regression at p < 0.05 (*) or p < 0.01 (**). r2: regression coefficient.

Population Regression equation r2 Jul experiment NET log y = 0.14 log x + 2.77 0.16 WBC log y = ‐0.27 log x + 1.44 0.49** SME log y = ‐0.15 log x + 1.83 0.27* SQB log y = ‐0.95 log x + 0.92 0.75** HAM log y = ‐0.22 log x + 1.85 0.54** ELD log y = ‐0.28 log x + 1.46 0.63**

Oct experiment 0.5 g l‐1 log y = 4.44x + 2.10 0.22** 2 g l‐1 log y = 3.78x + 2.71 0.49** 10 g l‐1 log y = ‐0.93x + 3.57 0.04 50 g l‐1 log y = 1.01x + 4.15 0.02

Appendix A page 14

0.5 B p < 0.01 B MTLP HSP 0.4 p < 0.01 0.3 A A B AB AB 0.2 A

0.1

0.0 0.5 ORG MRG 0.4

0.3 p = 0.05 p = 0.32

0.2

0.1

0.0

TAY HU HUM TAY O O OAK OAK T M AK D AK T D Population

Figure A‐ 1. Subcellular Cd concentrations in the tissue of the oyster Crassostrea gigas. Data are mean ± standard deviation (n = 5). Significant difference among populations is tested by ANOVA (p < 0.05) and populations with the same letters do not differ significantly as analysed by Tukey Multiple Comparison post hoc tests (p > 0.05). MTLP: metallothionein‐like protein; HSP: heat‐sensitive protein; ORG: organelles; MRG: metal‐rich granules.

Appendix A page 15 100 p = 0.62 80

60

40

20

0

T HU AY O OA M A K D K T

‐1 Figure A‐ 2. Metallothionein (MT) concentration (mg g wet wt.) in the oyster Crassostrea gigas. Data are mean ± standard deviation (n = 5). Significant difference among populations is tested by ANOVA (p < 0.05) and populations with the same letters do not differ significantly as analysed by the Tukey Multiple Comparison post hoc tests (p < 0.05).

Appendix A page 16

) 100 -1

h Jul A Oct -1 80 p < 0.01

60 p < 0.01 C

40 A C A A A 20 A B B

Clearance rate (l g 0 15 p < 0.01 Jul p < 0.01 B Oct A 10 AB A AB A A A AB 5 B Condition index (%) 0 D T ET BC L UM N W SME SQB HAM E H AK TAY OAK D O Population Population

Figure A‐ 3. Clearance rate (L g‐1 h‐1) and condition index (%) of the oyster Crassostrea gigas. Data are mean ± standard deviation (clearance rate: n = 8; condition index: n = 10). Condition index is calculated as 100 % x (tissue dry weight / shell dry weight). Significant difference among populations is tested by ANOVA (p < 0.05) and populations with the same letters do not differ significantly as analysed by the Tukey Multiple Comparison post hoc tests (p > 0.05).

Appendix A page 17

100

AE % (p = 0.06) NET 10 WBC SME SQB HAM ELD Jul - diatom 1 100

10 AE % (p = 0.01) % Cd retained

Jul - sediment 1 100

AE % (p < 0.01)

10 HUM OAK D OAK T TAY Oct - diatom 1 0 204060 Depuration (h)

Figure A‐ 4. Percentage of Cd retained in the oyster Crassostrea gigas after pulse feeding to different food types. Assimilation efficiency (AE, %) is determined as the percentage Cd retained at 24 h. Data are mean ± standard deviation (n = 5). Significant difference of AE among populations is tested by ANOVA (p < 0.05).

Appendix A page 18

100

ke (p < 0.01)

a NET, ke = 0.014 + 0.004 ab WBC, ke = 0.017 + 0.007 a % retained SME, ke = 0.013 + 0.002 a SQB, ke = 0.011 + 0.004 a HAM, ke = 0.013 + 0.003 b ELD, ke = 0.024 + 0.006 10 0 5 10 15 20 25 Depuration (d)

Figure A‐ 5. Depuration of Cd by the oyster Crassostrea gigas (July population) after pulse feeding to the diatom for 6 d. Data are mean ± standard deviation (n = ‐1 5). Efflux rate constant (ke, d ) is calculated from the slope of the linear regression between the ln % Cd retained in the oysters against the depuration time (d). Significant difference of ke among populations is tested by ANOVA (p < 0.05) and ke of the population with the same letters do not differ significantly as analyzed by the Tukey Multiple Comparison post hoc tests (p > 0.05).

Appendix A page 19

105 Jul 4 10 2 r > 0.98**

103 NET 102 WBC SME ) -1 1 SQB

h 10

-1 HAM ELD 100

105 Oct

2 104 r > 0.98** Dissolved uptake rate uptake g (ng Dissolved 103 HUM OAK D 102 OAK T TAY

101 10-1 100 101 102

Cd concentration (μg l-1)

Figure A‐ 6. Dissolved uptake rate (ng g‐1 h‐1) of Cd by the oyster Crassostrea gigas at different Cd concentrations. Data are mean ± standard deviation (n = 10). Lines are plotted when the regression coefficient (r2) is larger than 0.50. Asterisks (**) indicate significant difference at p < 0.01 by the linear regression analysis.

Appendix A page 20

1000

100

10

1 SQB HAM y = 19.293 x-0.4343

Uptake rate (ng Cd/g dwt.h) ELD 0.1 NET R2 = 0.3267 WBC SME 0.01 0.001 0.010 0.100 1.000 10.000 Body size (g dwt.)

Figure A‐7. Correlation between oyster body size and uptake rates from dissolved phase in log/log scale (July Populations).

350 HUM y = - 998.14 x + 190 Oakland D 300 R2 = 0.1368 Oakland T Taylor 250 NET

200

150

100 Uptake rate (ng Cd/g dwt.h) (ng rate Uptake

50

0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 Body size (g dwt)

Figure A‐8. Correlation between oyster body size and uptake rates from dissolved phase in linear scale (October Populations).

Appendix A page 21

Appendix B ‐ Health Risk Characterization for Consumption of Cadmium in West Coast Oysters

HEALTH RISK CHARACTERIZATION FOR CONSUMPTION OF CADMIUM IN WEST COAST OYSTERS

Prepared for Oregon State University Laboratory 2001 Marine Drive Astoria, OR 97103

Prepared by

7900 SE 28th Street, Suite 410 Mercer Island, WA 98040

And

Pacific Shellfish Institute 120 State Avenue NE #142 Olympia, WA 98501

August 2008

Integral Consulting, Inc. 2 Cd Oyster HHRA_Aug2008_is.doc

Health Risk Characterization For Consumption of Cadmium in West Coast Oysters August 2008

TABLE OF CONTENTS

LIST OF FIGURES...... iii LIST OF TABLES ...... iv ACRONYMS AND ABBREVIATIONS...... v 1 INTRODUCTION ...... 1‐1 1.1 RISK ASSESSMENT METHODS ...... 1‐1 1.2 BACKGROUND ON STUDY OF CADMIUM IN OYSTERS...... 1‐2 1.3 OCCURRENCE OF CADMIUM IN THE ENVIRONMENT ...... 1‐3 1.4 ORGANIZATION OF RISK ASSESSMENT REPORT...... 1‐4 2 DATA EVALUATION...... 2‐1 2.1 OYSTER SAMPLING AND ANALYSIS PROGRAM ...... 2‐1 2.2 DATA QUALITY ASSURANCE...... 2‐2 2.3 DATA EVALUATION FOR RISK ASSESSMENT...... 2‐3 2.4 CHARACTERIZATION OF CONCENTRATIONS BY EXPOSURE AREAS...... 2‐3 3 EXPOSURE ASSESSMENT...... 3‐1 3.1 OVERVIEW OF POPULATIONS EVALUATED ...... 3‐1 3.2 CALCULATION OF CADMIUM INTAKE...... 3‐2 3.2.1 Oyster Tissue Concentrations...... 3‐3 3.2.2 Consumption Rates...... 3‐3 3.2.3 Relative Oral Bioavailability ...... 3‐8 3.3 ESTIMATION OF CADMIUM INTAKE VIA OYSTERS ...... 3‐9 3.3.1 Alaska...... 3‐9 3.3.2 California ...... 3‐10 3.3.3 Oregon ...... 3‐10 3.3.4 Washington ...... 3‐10 4 CADMIUM TOXICITY ASSESSMENT ...... 4‐1 4.1 CADMIUM TOXICOKINETICS ...... 4‐2 4.2 NONCANCER HEALTH EFFECTS – HUMAN STUDIES OF ORAL EXPOSURE ....4‐3 4.2.1 Renal Effects...... 4‐3 4.2.2 Other Effects...... 4‐4 4.3 NONCANCER EFFECTS – STUDIES OF ORAL EXPOSURE...... 4‐5 4.3.1 Renal Effects...... 4‐5 4.3.2 Other Effects...... 4‐5

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4.4 ORAL TOXICITY VALUES FOR NONCANCER HEALTH EFFECTS...... 4‐6 5 RISK CHARACTERIZATION...... 5‐1 5.1 NONCANCER HAZARD ASSESSMENT...... 5‐1 5.2 PROBABILISTIC RISK ASSESSMENT RESULTS ...... 5‐1 5.2.1 Alaska...... 5‐2 5.2.2 California ...... 5‐2 5.2.3 Oregon ...... 5‐3 5.2.4 Washington ...... 5‐3 5.3 UNCERTAINTIES...... 5‐3 6 REVIEW OF CURRENT INTAKE GUIDELINES ...... 6‐1 6.1 CODEX ALIMENTARIUS COMMISSION ...... 6‐1 6.2 EUROPEAN COMMUNITIES ...... 6‐2 6.3 UNITED STATES ...... 6‐3 6.4 HONG KONG AND ...... 6‐4 6.5 CANADA...... 6‐4 6.6 DISCUSSION OF REGULATORY LIMITS AND POTENTIAL HEALTH RISKS...... 6‐5 7 CONCLUSIONS...... 7‐1 8 REFERENCES...... 8‐1

Attachment A: Oyster Tissue Descriptive Statistics Attachment B: Risk Calculations Attachment C: Field Sampling Standard Operating Procedures

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LIST OF FIGURES

Figure 2‐1. Sampling Station Locations. Figure 2‐2. Cadmium in Oyster Tissue, Average Concentrations (2004‐2006) ‐ Alaska. Figure 2‐3. Cadmium in Oyster Tissue, Average Concentrations (2004‐2006) ‐ California. Figure 2‐4. Cadmium in Oyster Tissue, Average Concentrations (2004‐2006) ‐ Oregon. Figure 2‐5. Cadmium in Oyster Tissue, Average Concentrations (2004‐2006) ‐ Puget Sound and Washington Coast. Figure 2‐6. Cadmium Tissue Concentrations for Each State. Figure 2‐7. Cadmium Tissue Concentrations for Each Exposure Area within Washington State.

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LIST OF TABLES

Table 2‐1. Sampling Events for Pacific Oyster Tissue Collection. Table 2‐2. Categorization of Sample Stations by Exposure Area. Table 2‐3. Summary Statistics and EPCs for Exposure Areas. Table 3‐1. Suquamish Tribe Oyster Consumption Rates for Adults and Children. Table 3‐2. Asian and Pacific Islander Oyster Consumption Rates for Adults. Table 3‐3. U.S. Population Oyster Consumption Rates for Adults. Table 3‐4. Exposure Factors for Intake Calculation. Table 3‐5. Intake of Cadmium in Oysters for all Exposure Areas. Table 5‐1. Noncancer Hazards for Consumption of Cadmium in Oyster Tissue (Probabilistic Calculation). Table 6‐1. Maximum Levels for Cadmium in Oysters (μg/g) for Various Nations.

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ACRONYMS AND ABBREVIATIONS

API Asian and Pacific Islander ATSDR U.S. Agency for Toxic Substances and Disease Registry BMDL benchmark dose lower limits of a one‐sided 95% confidence interval CAC Codex Alimentarius Commission CSFII Continuing Survey of Food Intakes by Individuals EC European Communities EPA U.S. Environmental Protection Agency EPC exposure point concentration FAO Food and Agriculture Organization FDA U.S. Food and Drug Administration Integral Integral Consulting Inc. JECFA Joint FAO/WHO Expert Committee on Food Additives ML maximum limit NOAEL no‐observed‐adverse‐effect level NTP National Toxicity Program PDFs probability density functions ppm parts per million PRA probabilistic risk assessment PSI Pacific Shellfish Institute PTWI provisional tolerable weekly intake QA/QC quality assurance/quality control RBA relative bioavailability adjustment RfD reference dose SCF Scientific Committee on Food SOPs standard operating procedures TDI tolerable daily intake USDA U.S. Department of Agriculture WDFW Washington State Department of Fish and Wildlife WHO World Health Organization

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1 INTRODUCTION

This report presents the results of a human health risk assessment conducted by Integral Consulting Inc. (Integral) on behalf of the U.S. Department of Agriculture (USDA). The risk assessment is one component of a larger study evaluating cadmium in oysters, conducted by Oregon State University and the Pacific Shellfish Institute (PSI). The study was initiated because cadmium concentrations have been found to sometimes exceed international standards in U.S. West Coast Pacific oysters (Crassostrea gigas). These cadmium concentrations may impact not only human health but also result in decreased interstate and international sales of oysters. Integral conducted this risk assessment to determine potential health risks from consumption of cadmium in Pacific oysters by members of the general population as well as populations with higher rates of oyster consumption (e.g., Native Americans, Asian, and Pacific Islanders).

1.1 RISK ASSESSMENT METHODS

A human health risk assessment is a quantitative evaluation of the risk posed to human health by the actual or potential presence of chemicals in the environment. The risk assessment provides estimates of risks to potentially exposed populations using methods designed to avoid underestimation of risks. The uncertainties inherent in the risk assessment methodology are addressed with protective assumptions regarding chemical exposure and toxicity that, in combination, are likely to overestimate risks.

The human health risk assessment process as defined by the U.S. Environmental Protection Agency (EPA) contains four major steps (USEPA 1989). The first step is the problem formulation, where the type of potential hazard is identified. The second step is the exposure assessment, which evaluates the potential exposure of people to the chemicals being evaluated. The third step is the toxicological evaluation, which evaluates the potential toxicity of the chemicals and identifies doses that may cause toxicity and doses that are not expected to cause health effects. The fourth step is the risk characterization that combines the results of the exposure assessment and the toxicological evaluation to describe potential risks to human health associated with the chemicals of interest.

Estimates of risk may be presented as single fixed values (i.e., point estimates) in which the exposure and toxicity assessment inputs also are single fixed values. This approach to risk assessment is described as deterministic. Risk estimates also can be presented as distributions reflecting the varying degree of risk among individuals in a population (also known as a probabilistic risk assessment [PRA]). With a probabilistic approach, some or all of the exposure and toxicity input parameters are represented as probability distributions, also called probability density functions (PDFs), in the risk calculations. While exposure factors are

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routinely expressed as PDFs, this is not the case for toxicity criteria, for which the use of point values is standard practice.

Over the past 10 years, the PRA has become a common and well‐accepted method for robustly characterizing the range of theoretical health risks. PRA methods are considered a significant improvement over deterministic risk assessment methods, providing a more scientifically rigorous approach (Paustenbach 2002). In the early 1990s, EPA recognized the potential for the typical deterministic human health risk assessment approach to overestimate risks without providing adequate characterization of the degree of conservatism in the assessment, and subsequently amended its risk assessment guidance to allow for PRA (USEPA 1992a, 2001).

For this risk assessment, a probabilistic approach using Monte Carlo analysis with Crystal Ball® software is used. A combination of distributions and point estimates are used, depending on the specific input parameter (see Section 3 for more details).

1.2 BACKGROUND ON STUDY OF CADMIUM IN OYSTERS

Cadmium is a toxic metal found naturally in small amounts in the soil, air, and water. It is of particular concern to human health due to its long biological half‐life and ability to accumulate in soft tissues, primarily the liver and kidneys. Molluscan shellfish such as oysters have a natural proclivity to bind and retain cadmium, resulting in elevated tissue concentrations. The U.S. Food and Drug Administration (FDA), National Marine Fisheries Service, and public health agencies in Washington and Alaska have encountered mean cadmium levels in shellfish ranging from 0.1 to 4 μg/g, wet weight (FDA 1993).

The Codex Alimentarius Commission (CAC), created by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) to develop food standards and guidelines, recently deliberated over establishment of a 1‐μg/g, wet weight, maximum limit (ML) for cadmium in molluscan shellfish. Ultimately, a ML of 2 μg/g, wet weight, for all mollusks was established but oysters and were excluded from the ML requirement (Codex 2006).

Codex‐member countries (including the U.S.) usually adopt Codex standards, and any product exceeding their standards is considered adulterated for both international export as well as domestic interstate sales. Scattered and limited sampling of molluscan shellfish indicated that in certain species and growing areas, cadmium levels may significantly exceed the 1‐μg/g, wet weight, ML that had been proposed by Codex (2006). The reduced screening limit of 2 μg/g wet weight for non‐oyster mollusks, coupled with observed elevated levels of cadmium in shellfish, is a growing public health and economic concern for producers, regulators, and consumers.

The USDA granted funds to Integral, Oregon State University, and PSI to evaluate the spatial and temporal distribution of cadmium in commercially important, West Coast molluscan shellfish and to evaluate the human health risk to consumers of oysters. Other studies

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conducted under this grant evaluate the economic risk to industry if the Codex ML is expanded to include oysters in the future and methods to minimize cadmium residues in shellfish products marketed to consumers. Outreach to the community will provide crucial information on geographic regions with high cadmium concentrations and appropriate culture methods, species, and husbandry practices that may be employed to minimize cadmium residues.

Results of this research may be used by FDA and the Interstate Shellfish Sanitation Conference when evaluating MLs proposed by Codex or other countries. Also, results may be used to aid decisions related to adoption of MLs for cadmium in shellfish tissue under the U. S. National Shellfish Sanitation Program.

1.3 OCCURRENCE OF CADMIUM IN THE ENVIRONMENT

Cadmium, in pure form, is a soft, silver‐white metal. In the environment, cadmium typically occurs as a mineral combined with other elements. Complexes with oxides, sulfides, and carbonates in zinc, lead, and copper ores are most common, while complexes with chlorides and sulfates occur to a lesser extent (U.S. Agency for Toxic Substances and Disease Registry [ATSDR] 1999; National Toxicity Program [NTP] 2005).

Cadmium releases into the environment occur as a result of both natural and human activities. Weathering of cadmium minerals contained in rocks is a significant source of these releases to water in rivers and the ocean. Natural releases to air are contributed by forest fires and volcanoes. Mining activities, burning of fossil fuels and of household wastes, application of fertilizers to crops, and industrial sources also may contribute to cadmium levels in the environment. The type of cadmium compound influences the solubility of cadmium in water. In general, cadmium chlorides and sulfates are more water soluble than other forms.

For the general population, exposures to cadmium may occur with consumption of cadmium in food and drinking water, inhalation of cadmium‐containing particles in ambient air or cigarette smoke, and ingestion of cadmium in soil or dust. The relative contribution of these sources to cadmium intakes by the general population varies considerably depending upon the smoking status of the exposed individual. For instance, in the U.S., food is the primary source of cadmium exposure to nonsmokers, while cadmium in inhaled and ingested particulates from smoke may contribute substantially to intake in tobacco smokers who may be exposed to an estimated 1.7 μg of cadmium per cigarette (NTP 2005).

It is noteworthy that cadmium is taken up into plants from soil and is naturally present in a wide variety of foods in addition to shellfish. Primary food sources of cadmium include grain cereal products, potatoes, and other vegetables. Within the general U.S. population of nonsmoking adults, these food sources are thought to account for the largest contribution of cadmium to total daily intake, which is estimated at approximately 30 μg/day (NTP 2005). FDA

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estimates that 20% or 10 μg/person/day (0.14 μg/kg‐day) of the tolerable daily intake (TDI) of 55 μg/person/day is from cadmium in food (not including shellfish food items; FDA 1993). For people who eat a large quantity of organ meats and shellfish, these food items also may contribute to cadmium intake. Cadmium is found at higher concentrations in shellfish than in other types of fish, and cadmium intake for high shellfish consumers may equal cadmium intake from all other dietary sources (FDA 1993).

1.4 ORGANIZATION OF RISK ASSESSMENT REPORT

The remainder of this report is organized as follows:

• Section 2 is an evaluation of cadmium in oyster tissue data. • Section 3 describes the exposure scenarios and assumptions selected for use in this risk assessment. • Section 4 discusses the toxicity of cadmium and lists the toxicity values associated with this assessment. • Section 5 presents the risk characterization including quantitative estimates of health risks for each scenario evaluated. • Section 6 presents a review of current domestic and international cadmium intake guidelines. • Section 7 presents the study conclusions and recommendations. • Section 8 includes a list of references cited in this report. In addition, Attachment A provides the oyster tissue data and descriptive statistics and Attachment B provides risk calculations. The standard operating procedures (SOPs) for field sampling of oyster tissues are provided in Attachment C.

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2 DATA EVALUATION

PSI and Integral designed a field sampling plan to evaluate cadmium concentrations in Pacific oysters (Crassostrea gigas) throughout U.S. West Coast growing areas and to define geographic locations, culture methods, harvest times, and additional factors that affect cadmium concentrations. The overarching goal of the project is to better understand the factors contributing to cadmium in oysters and determine how cadmium residues in oyster products can be minimized. These findings have been presented (Stupakoff et al. 2007) and a comprehensive field data report that includes seasonal and geographical distribution of cadmium concentrations in oyster tissue, sediment, and seawater of the U.S. West Coast is being published under separate cover (PSI 2008). The following sections focus on tissue chemistry results for the assessment of risk to human health.

2.1 OYSTER SAMPLING AND ANALYSIS PROGRAM

In 2004 a total of 41 oyster sampling stations were selected along the U.S. Pacific West Coast to assess the general distribution of cadmium concentrations in oyster tissue (Christy 2005). Pacific oysters were sampled at four shellfish‐growing areas in California, three areas in Oregon, three areas in Alaska, 28 areas located throughout Washington’s Puget Sound, and three areas in Washington coastal estuaries (Figure 2‐1). Sites were selected based on the presence of bottom‐cultured oysters, geographic representation, and the importance and/or size of the commercial, tribal, or recreational shellfish‐growing area. Site locations were assigned a unique station identification code, and their geographical coordinates were recorded using a global positioning system.

At each station, three composites of 20 oysters each (4 to 6 inches in shell length) were collected and submitted for chemical analyses. Based on the results of the initial 2004 region‐wide sampling event, five sites in the Puget Sound area were selected for seasonal sampling from January 2005 to February 2006 (Eld Inlet, Hamma Hamma, Thorndyke Bay, Samish Bay, and Willapa Bay‐Bay Center; Table 2‐1). Each of the five sites were selected to represent a distinct geographic region (south Puget Sound, Hood Canal, north Hood Canal, north Puget Sound, coastal estuaries) and to represent areas of cadmium concentrations in oyster tissues ranging from low (i.e., <μg/g, wet weight) to high (i.e., >2 μg/g, wet weight). In addition, one to two stations were also selected in California, Oregon, and Alaska for seasonal sampling (Table 2‐1).

Pacific oysters were collected during low tide along a 50‐meter transect positioned parallel to the waterline at the 0 mean low low water tidal elevation. Ten individual oysters were sampled at each 10‐meter increment along the transect line for a total of 60 oysters, according to the field sampling SOPs (see Attachment C). All specimens were intact (i.e., tightly closed, unbroken shell) and of similar size (4‐6 inches).

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Oysters were scrubbed with a plastic bristle brush and rinsed onsite using local seawater to remove sand and debris. Biofouling was removed by hand to the best extent possible. Oysters were rinsed a second time in local seawater prior to being systematically divided into three composites of 20 and placed into sealable plastic bags (Attachment C). Samples were maintained at 4ºC and driven within 24 hours or shipped overnight via FedEx to AmTest Laboratories1 in Redmond, Washington. Chain‐of‐custody forms accompanied shellfish samples from the field to the laboratory.

Tissue samples were processed at AmTest Laboratories within 48‐hours of sample receipt. Oysters were individually scrubbed with a soft bristle brush under cool running tap water. After any remaining debris was removed, oysters were rinsed a second time with deionized water. Each composite of 20 oysters was shucked, weighed, and homogenized (tissue and liquor) in a clean blender fitted with stainless‐steel blades. Composites were transferred into glass jars and held at ‐18ºC until further processing.

Tissue samples were tested for cadmium and zinc using inductively coupled plasma‐atomic emission spectrometry, EPA Method 200.3/6020, or graphite furnace atomic absorption spectrometry, EPA Method 200.9/7131. Tissue cadmium concentrations were reported in μg/g (ppm) based on wet tissue weight.

2.2 DATA QUALITY ASSURANCE

All sampling and analytical procedures were performed in accordance with guidelines and protocols published by the FDA (1993), Puget Sound Water Quality Action Team (Puget Sound Estuary Program 1997), and AmTest Laboratories’ SOPs. Sampling materials and detailed sampling SOPs were provided to each participant in order to maintain consistency between locations (Attachment C). A quality assurance project plan was reviewed by the Washington State Department of Health, Office of Environmental Health Assessment prior to initial sample collection (PSI 2004). Quality assurance/quality control (QA/QC) standards included the use of digestion blanks, duplicates, spiked test portions, appropriate standard reference material, and recovery calculations. One blank, duplicate, and matrix spike was performed for every 10 samples. Less than a 20% difference was allowed between duplicate samples. Greater than 80% recovery was required for both matrix spikes and standard reference materials.

The analytical results from AmTest were validated by Pyron Environmental in Olympia, Washington. Since the laboratory reports from AmTest did not include the performance‐based in‐house control limits for the QC analyses, control criteria specified in the EPA’s Contract Laboratory Program National Functional Guidelines for Inorganic Data Review (USEPA 2004a) were applied to evaluate the data quality. The overall assessment of data usability, based on the

1 AmTest Laboratories, 14603 NE 87th Street, Redmond, WA 98052, is accredited for environmental analyses by the Washington State Department of Ecology.

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information presented in the laboratory reports by AmTest, determined that cadmium data were acceptable for use as qualified in Pyron’s data validation report (Pyron 2006).

2.3 DATA EVALUATION FOR RISK ASSESSMENT

Data were evaluated according to Guidance for Data Usability for Risk Assessment (USEPA 1992b), which provides minimum data requirements to assure that data are appropriate for use in risk assessment. The guidance addresses four main issues pertinent to assessing data quality for risk assessment: data sources, analytical methods and detection limits, data quality indicators, and consistency of data collection methods.

• Data sources—Data were evaluated to determine whether QA/QC samples are available to provide data quality information. As described in Section 2.2, data were deemed acceptable based on EPA’s Contract Laboratory Program National Functional Guidelines for Inorganic Data Review (USEPA 2004a). • Analytical methods and detection limits—Methods were evaluated for appropriateness and sensitivity. It was determined that the detection limit for cadmium of 0.0005 μg/g is adequate for risk‐based evaluation. • Data quality indicators—Laboratory validation reports were reviewed for data quality issues. Cadmium was detected in all samples and no data were assigned qualifiers. Therefore, there was no need to reject or exclude any data from the database. • Consistency of data collection methods—Samples were collected using appropriate methods. All participants of this project used the same sampling equipment and followed SOPs provided by PSI (see Attachment C). An exception to this was samples collected on June and July 2004 at Tomales Bay and Humboldt Bay, California, respectively. Oysters were shucked at the field lab, frozen, and then sent to AmTest for analyses. However, analytical results for these samples fell well within the lower end of cadmium concentrations range for all other samples collected at the same site, indicating that the nonstandard sampling procedures did not contribute to possible sample contamination. A review of field notes for subsequent dates did not reveal issues in sample collection procedures, and procedures appear to have been consistent between field sampling events and sampling teams.

2.4 CHARACTERIZATION OF CONCENTRATIONS BY EXPOSURE AREAS

As described in Section 2.1, oyster tissue samples were collected from both public and private beds in Alaska, California, Oregon, and Washington from June 2004 through January 2006. For the purpose of this risk assessment, sample stations within each state were grouped into

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“exposure areas.” Exposure areas were created to represent areas from which consumers may obtain the majority of oysters for personal consumption. Designation of each exposure area was based on geography (e.g., proximity of sampling stations to one another) and best professional judgment. As shown in Table 2‐2 and Figures 2‐2 through 2‐5, exposure areas have been designated for Alaska, California, Oregon, and Washington. Tissue concentrations from the sampling stations within each exposure area were grouped to form one data set per exposure area. These data are used to estimate potential exposures to oyster consumers who harvest or purchase oysters from each area. Three exposure areas were designated for Alaska (Figure 2‐2) and two exposure areas each were designated for California (Figure 2‐3) and Oregon (Figure 2‐ 4). Seven individual exposure areas were designated in Puget Sound for the state of Washington (Figures 2‐5).

The mean cadmium concentrations in oyster tissue from Washington, Oregon, and California were slightly above 1 μg/g, wet weight, with values ranging from 0.44 to almost 2.7 μg/g, wet weight (Table 2‐3). For the 21 samples collected from Alaska, the mean was 2.3 μg/g, wet weight, and values ranged from 1.6 to 4.0 μg/g, wet weight (Table 2‐3). Histograms for each statewide data set are provided in Attachment A. A statistical comparison of median cadmium concentrations measured in each state indicated that cadmium concentrations measured in California, Oregon, and Washington are not dissimilar but cadmium concentrations in Alaska are significantly higher than those measured in other states 2 (Figure 2‐6).

Additional comparisons between exposure areas within Washington indicated that cadmium concentrations measured in oyster tissue collected in exposure area W4 (Hood Canal) are significantly different, or greater, than cadmium measured in oysters collected from exposure areas W1 (North Puget Sound; p<0.04), W6 (South Puget Sound; p<0.00), and W7 (Coast; p<0.00; Figure 2‐7)3.

Tissue concentrations in exposure areas W2, W3, and W5 (San Juan Islands, the Straits, and Central Puget Sound) are not significantly different from each other. Cadmium concentrations in tissue collected from exposure area W6 (South Puget Sound) are generally lower than other areas, but this difference is significant only when compared to exposure areas W1 (North Puget Sound; p<0.01) and W4 (Hood Canal; p<0.00)4.

The harvest of oysters by the general public in Washington State is open year‐round except as noted on the public beach list of the Washington Department of Fish and Wildlife (WDFW 2008). According to the Mason County news release in December 2007, the oyster season of the Hood Canal area ran from March 1 through December 31, 2007 and recent surveys indicate that the oyster population will support a year‐round season in 2008 (KMAS 2007). Private commercial and tribal shellfish growers also harvest year‐round.

2 Kruskal-Wallis Test H (3, N=270) = 49.8, p<0.00, Exhibit A-1. 3 Kruskal-Wallis Test H (6, N=173) = 66.5, Exhibit A-2. 4 Results of all statistical tests are provided in Appendix A.

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3 EXPOSURE ASSESSMENT

Cadmium concentrations varying from well below the ML of 2 μg/g, wet weight, for other molluscan bivalves to more than double the ML have been found in oysters harvested from the U.S. West Coast beaches and commercial farms. The potential for human exposure to cadmium from oysters is expected to vary based on consumption rates as well as cadmium concentrations in oysters. Consumption rates are also expected to vary with the cultural practices of different populations. The objective of the exposure assessment is to estimate the intake of cadmium via consumption of U.S. West Coast oysters in various populations, including those likely to consume oysters at high rates. For this assessment, some exposure factors are represented as PDFs and others as point values, as described in the following sections.

3.1 OVERVIEW OF POPULATIONS EVALUATED

To estimate the amount of cadmium ingested in oysters, it is necessary to first quantify the rate of oyster consumption. The amount of oysters per meal and number of oyster meals consumed varies considerably between different cultural groups. Oyster consumption patterns will vary for consumers in different regions and countries. Additionally, consumers may either purchase commercially raised oysters or harvest their own oysters locally.

Quantification of a consumption rate is difficult, particularly when rates may vary significantly between individuals and populations. Typically, fish and shellfish consumption rates are determined from interviews with fisherman, also called creel surveys, or from broad‐based population surveys where people are asked to provide consumption information via questionnaires or interviews.

Selection of one consumption rate to estimate oyster intake for all consumers is likely to overestimate consumption for those who rarely eat oysters while underestimating for those who frequently eat oysters. For this reason, consumption rates for two Washington state populations known to consume greater quantities of oysters than a typical U.S. consumer were selected to evaluate a range of oyster intake levels. The Suquamish Tribe and Asian and Pacific Islander (API) community of western Washington State were selected due to the availability of detailed shellfish consumption data for both communities. In addition, a U.S. consumer population was selected to represent a more broadly based oyster consumption rate. Consumption rates for the general U.S. consumer may underestimate actual rates for Pacific Coast residents who typically consume greater quantities of oysters compared to other regions in the U.S. (Office of Environmental Health and Hazard Assessment [OEHHA] 1997).

While the average U.S. consumer may represent the majority of the population, the Suquamish Tribe and API communities are specific to western Washington and may or may not represent

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oyster consumption levels seen in other areas from which oysters were sampled (e.g., Alaska, Oregon, California). Oyster consumption data for high consumer populations in areas outside of western Washington were not available.

WHO (2003) provides mollusk consumption rates for five regional diets—African, European, Far‐Eastern, Middle‐Eastern, and Latin American—which may be used to represent overseas consumers who purchase oysters grown on the West Coast. Because these consumption rates are not specific to oysters, potential oyster intake by overseas consumers will be evaluated qualitatively only.

The target populations introduced above represent a range of oyster consumption levels and also may represent other subpopulations who consume oysters from the West Coast. Other subpopulations not included in this assessment, but who may consume greater quantities of oysters relative to the general U.S. population, include people who work at oyster farms, processing and canning facilities, restaurant workers, and on‐water or near‐water residents with access to oyster beds. Quantitative information on oyster consumption by these populations was not found.

3.2 CALCULATION OF CADMIUM INTAKE

Exposure to cadmium via consumption of oysters is estimated by calculating an average daily dose or intake of cadmium taken into the body, averaged over an exposure period. Daily intake of cadmium via oyster consumption is calculated using Equation 1. It is expected that there will be differences among individuals in the level of exposure due to differences in consumption rates and cadmium concentrations in consumed oysters, resulting in a wide range of average daily intakes among different members of an exposed population.

Intakeoyster = oyster × CRC oyster × × CFRBA Eq. 1

Where:

Intakeoyster = Intake, or average daily dose from ingestion of oysters (mg/kg‐day)

Coyster = Concentration of cadmium in oyster (μg/g)

CRoyster = Oyster consumption rate (g/kg‐day)

RBA = Relative bioavailability adjustment (unitless)

CF = Unit conversion factor (10‐3 mg/μg)

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The variables shown in Equation 1 are called exposure factors. For this probabilistic assessment, distributions were used to represent tissue concentrations and consumption rates. A point estimate was selected for RBA because distributional data were not available. The inputs for individual exposure factors are described in the following sections.

3.2.1 Oyster Tissue Concentrations

In deterministic risk assessment, sample data for each exposure medium are subjected to a statistical evaluation to identify a chemical concentration that will be representative of exposures in each population of interest. These concentrations are referred to as the exposure point concentrations (EPCs). The 95th percentile upper confidence limit of the mean concentration typically is selected to represent the EPC. PRA, on the other hand, incorporates the distributional characteristics of the data to estimate the EPC. In other words, the EPC is represented by a probability distribution.

For probabilistic analysis, distributions were fit to the existing data set for each exposure area using Crystal Ball® software (Version 7.2.1; Decisioneering 2006). For exposure areas with fewer than 15 observations, a lognormal distribution was assumed and defined by the mean and standard deviation. Selected distribution type and defining parameters, as determined using Crystal Ball®, are listed in Table 2‐3.

Table 2‐3 also presents point estimated EPCs calculated using ProUCL software (Version 3.00.02; USEPA 2004b). For data sets with fewer than 15 observations, the maximum concentration was selected as the EPC. This information is provided to allow for deterministic evaluation of risks for comparison with probabilistic results.

3.2.2 Consumption Rates

Identification of consumption rates for oysters is a critical step in this risk assessment. Consumption rates for foods are estimated by a variety of techniques that frequently involve conducting short term surveys in which people are asked to record or recall foods they consume during a period of days. The resulting distributions of consumption rates describe the range of intakes during the short period studied. When attempting to apply these distributions to assessment of long term consumption patterns, it is inevitable that upper percentiles of the short term distribution will markedly overestimate the upper percentiles of long term population intakes, a factor acknowledged by EPA in guidance documents (USEPA 1997).

Consumption rates were obtained for the three populations described in Section 3.1, the Suquamish Tribe, API community, and general U.S. consumer. Both the Suquamish Tribe and API community consumption rate studies attempted to provide estimates of long term average intake rates by asking about frequency and seasonality of consumption. In contrast, the general U.S. consumer consumption estimates are strictly based on a short‐term recall study. The three

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consumption studies, the basis for the consumption rates selected and their limitations are described below.

3.2.2.1 Native American – High Shellfish Consumption Population

As described in Section 3.1, the Suquamish Tribe was selected to represent a high shellfish‐ consuming Native American population. The home of the Suquamish Tribe is the Port Madison Indian Reservation, located in north Kitsap County, Washington, which is in the vicinity of exposure areas W4 (Hood Canal) and W5 (Central Puget Sound) (see Figure 2‐5). Tribal shellfish harvest areas include additional areas of Puget Sound, and members may also harvest and/or purchase some shellfish from other areas. Suquamish tribal members rely on fish and shellfish as a significant part of their diet, primarily from Puget Sound. The Suquamish Tribe’s consumption rates represent the highest quantified consumption rates reported in Washington State up to the year 1999 (Suquamish Tribe 2000).

A fish consumption survey of Suquamish tribal members was conducted in 1998 (Suquamish Tribe 2000). Of the 831 total enrolled tribal members, the survey targeted 284 members living on or near the Port Madison Indian Reservation who also were registered on‐water vessel owners or operators and/or possessed a shellfish license. Ultimately, 92 of 142 eligible adults age 16 and older participated in the study. The survey also included 31 children, ages 6 and younger. To be included in the survey, children had to be living with a tribal member who was enrolled in the study for at least one year.

The survey solicited information on a variety of finfish and shellfish, including oysters. The four parts of the survey included: 1) a 24‐hour dietary recall; 2) finfish identification, portion size, frequency of consumption, preparation method, and harvest location of finfish; 3) shellfish consumption, preparation, harvest location; and 4) changes in consumption over time, cultural information, physical information, and socioeconomic information.

The majority (65%) of adult tribal members reported that they consume oysters and that they typically (97% of the time) eat the oyster whole. A smaller percentage of children (32%) were reported to consume oysters. Tribal members reported that they harvest 84% of the shellfish that they consume primarily from within the Puget Sound (Suquamish Tribe 2000). Shellfish that is not harvested is either purchased at grocery stores or restaurants. Consumption of fish and shellfish is an integral part of tribal ceremonies, gatherings, and community events.

Tribal members were surveyed regarding the preparation and cooking methods they use for shellfish such as clams, oysters, mussels, and crabs. Members reported that they typically (73% of the time) bake, steam, boil, broil, roast, or poach shellfish and that they smoke, can, fry, dry, or eat shellfish raw less frequently. The nectar resulting from shellfish preparation often is consumed. Most tribal members interviewed reported that they drink the nectar or use it in cooking, while less than 20% of the respondents reported throwing the nectar away.

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As reported in Suquamish Tribe (2000), consumption rates were calculated by multiplying the portion size normally eaten by a particular survey participant by the frequency of consumption. The portion size was based on models of raw seafood. This calculation was performed twice for each seafood item, once for when the item is “in season” and again for “during the rest of the year.” The sum of the consumption rate for these two periods yielded the annual consumption rate for a particular seafood item. The annual standardized seafood consumption rate then was calculated according to the following equation:

fish Annual fish consumptio fwg ),(n ( CR ( g/kg − day,fresh weight) = Eq. 2 oyster 365 day)( × body weigh kg)(t

Table 3‐1 provides summary statistics and distribution information for adult and child consumption rates for consumers only. The consumption rate data were found to fit a lognormal distribution (Suquamish Tribe 2000). The mean consumption rate for adult consumers only is 0.164 g/kg‐day. The consumption rate for children is considerably lower, 0.060 mg/kg‐day, than that reported for adults. Although consumption rates for Suquamish children are available, potential risks for child consumers were not estimated because information for children is not available for other studied populations and their consumption rate is much lower than that of adults. Consumption rates are provided as fresh weight so it was not necessary to adjust rates to account for preparation or cooking losses.

The data were not adjusted for potential outliers because it was concluded that elevated consumption rates likely reflect actual consumption rates (Suquamish Tribe 2000). The consumption rates are represented by the adult mean and standard deviation consumption rate, provided in the Suquamish study (Table 3‐1). Use of a PDF to describe Suquamish Tribe consumption rates provides greater information on the magnitude and variability of oyster consumption within the population compared to the use of a single point estimate.

3.2.2.2 Asian and Pacific Islander Groups

Asian and Pacific Islanders are people having origins in the Far East, Southeast Asia, the Indian subcontinent, or the Pacific Islands. Members of the API community are hypothesized to consume greater quantities of seafood as well as different portions of seafood and different species than the general U.S. population (Secheena et al. 1999).

A seafood consumption study was conducted to describe the seafood consumption rates, species, and seafood parts commonly consumed by the API community living in King County, Washington (Secheena et al. 1999). The seafood consumption data were collected to support risk assessments addressing the potential risks associated with seafood consumption in the API community. The study included 202 individuals representing a total of 10 API groups (Cambodian, Chinese, Filipino, Hmong, Japanese, Korean, Laotian, Mien, Samoan, and Vietnamese). All study participants were 18 years of age or older, were first or second

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generation API community members, and were seafood consumers (e.g., nonconsumers were excluded).

A survey questionnaire was used to collect data on seafood consumption, the source of seafood, the frequency and portion size of seafood consumed, demographic information, and respondents’ preferred methods of public outreach. Survey respondents were queried regarding their consumption of finfish and shellfish, including oysters.

Shellfish was the most commonly reported seafood group eaten by the survey respondents, and the majority of shellfish consumers also reported eating oysters. Survey respondents who reported eating oysters said that they eat them whole most often (88%). Respondents reported that they occasionally either removed the oyster’s stomach, siphon, or both before consuming. The most common method of shellfish preparation was reported to be baking, boiling, broiling, roasting, or poaching. Respondents reported that 22% of the time, they consume shellfish that is canned, fried, raw, smoked, or dried. API community members reported that they usually throw away the water in which the shellfish is cooked. When the cooking water is re‐used, respondents reported that they used it for cooking or for drinking.

In contrast to the Suquamish tribal members, API community members reported that the majority of the shellfish that they consume is purchased rather than harvested. Respondents reported that 88% of the shellfish they consume is purchased from grocery stores (67%) or restaurants (21%). Survey respondents reported that when they harvest shellfish, they harvest within King County, which borders Puget Sound.

API seafood consumption rates were determined from questionnaire responses regarding frequency and portion size and were calculated using Equation 2 above, with the exception that rates were developed for shellfish “as prepared.” The weight of the portion size is the weight of the edible shellfish tissue, after cooking and removal from the shell. The mean “as prepared” consumption rate for consumers only is 0.098 g/kg‐day, provided in Table 3‐2. No consumption rates are provided for children.

Consumption rates were converted from “as prepared” to “fresh weight” values to maintain consistency between consumption rate and tissue concentration units. The following equation from USEPA (1997) was used to adjust consumption rates to represent fresh rather than cooked oysters:

Ingestion Rate ( g/kg-day, preparedas ) Ingestion Rate ( g/kg-day, weightfresh ) = Eq. 3 ()−W 100/100

W is the percent change in moisture content between uncooked and cooked oysters. USEPA (1997) reports that the moisture content for raw oysters is 82% and the moisture content for breaded or fried oysters is 65%, resulting in an approximate moisture loss of 17% from food

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preparation and cooking. A 17‐percent moisture loss was assumed for adjustment of API consumption rates, resulting in a mean consumption rate of 0.118 g/kg‐day for consumers only.

3.2.2.3 General U.S. Consumer

A typical U.S. resident may consume oysters purchased in markets or restaurants but also may harvest oysters. Residents living in coastal states where recreational oyster gathering is permitted may consume more oysters than the typical U.S. resident. In Washington, recreational users were estimated to harvest 559,312 oysters in 2001 (Washington State Department of Fish and Wildlife [WDFW] 2005). The majority of oysters were harvested from Twanoh Spit (122,229), Potlach West (92,796), and Dosewallips Spit (54,445) (WDFW 2005). These three popular harvest areas are located in exposure area W4, Hood Canal (see Figure 2‐5). It is possible that these harvest rates translate to oyster consumption levels for local residents that are greater than what would be observed in noncoastal states.

Although consumption rates for the average West Coast resident have not been quantified, USEPA (2002) provides estimates for shellfish consumption for the entire U.S. population based on data from the USDA Continuing Survey of Food Intakes by Individuals (CSFII). CSFII data were collected from residents nationwide, with each survey participant providing two nonconsecutive days of dietary recall information during each one‐year survey period. USEPA (2002) compiled data from four survey years (1994–1996, 1998) into one database. The survey results from 10,459 respondents were projected to the U.S. population size of 59,496,815 by weighting individual results based on sociodemographic and geospatial characteristics of each respondent to achieve an adequate representation of the U.S. population (USEPA 2002).

Detailed data are provided for finfish and shellfish, including means and percentile consumption rates. However, only mean consumption rates are provided for individual seafood species, including oysters. Although the CSFII survey results are intended to represent consumption patterns for the entire U.S., these data may be used to represent a West Coast resident who may not be represented by communities consuming higher quantities of oysters, such as the Suquamish Tribe and API groups.

USEPA (2002) provides mean oyster consumption rates based on the short‐term survey data collected by the USDA. Table 3‐3 provides mean consumption rates for oysters “as prepared” and uncooked oysters for the U.S. population (both those who sometimes consume oysters and those who never consume them, termed nonconsumers). Rates for uncooked oysters are used in this risk evaluation to maintain consistency between consumption rate and tissue concentration units. For individuals 18 years and older, USEPA (2002) provides a mean uncooked oyster per capita consumption rate of 0.174 g/person‐day. Because individual body weight data are not available for survey respondents, an average adult body weight of 70 kg can be assumed to convert the consumption rate to a value of 0.002 g/kg‐day.

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3.2.3 Relative Oral Bioavailability

Absolute oral bioavailability refers to the fraction of an ingested dose of a chemical that is absorbed and reaches the systemic circulation. The bioavailability for a particular chemical may vary depending on the chemical species and its interactions with the matrix in which it is found (Kelley, et al. 2002). Differences in bioavailability between different forms and/or matrices of a chemical are referred to as relative bioavailability. In risk assessment, a relative bioavailability adjustment (RBA) may be used to describe the difference in bioavailability between the chemical in the exposure matrix of interest (e.g., oyster tissue) and the dosing matrix used in the toxicity study on which the reference dose (RfD) is based. Another term, bioaccessibility, is a measure of relative bioavailability and refers to the results of an in vitro test used to estimate bioavailability of a chemical in an environmental matrix, usually soil, within the gastrointestinal tract. A RBA is calculated using Equation 4:

fraction absorbed fraction tissuefrom RBA = Eq. 4 fraction absorbed fraction from dosing medium used in toxicit studyy

For soil, the RBA is usually less than 1.0 because the most bioavailable form of a chemical is most commonly used in toxicity studies, although it is also possible to have an RBA greater than 1.0 if the chemical is more readily absorbed from the environmental medium than from the medium used in the toxicity studies.

The oral toxicity reference values for cadmium are based on a number of chronic studies in humans. A toxicokinetic model was used to estimate the no‐observed‐adverse‐effect level (NOAEL) from cumulative exposures. Traditionally, the EPA has differentiated between exposures to cadmium in food (less available) and water (more available), and provided individual toxicity and risk‐based numbers for each of these forms of exposure. It has been argued that there is no basis for differentiating between these exposures (Diamond, et al. 1997, USEPA 1999); however, that argument may only apply to soluble cadmium forms mixed with laboratory diets and not to cadmium naturally incorporated into plant and animal tissues.

The oral absorption of soluble cadmium in humans and several laboratory animals is generally reported to be very low (1‐8%) (Friberg et al. 1985; USEPA 1999). However, most estimates are based on fecal excretion data and are only approximations because there is evidence of both biliary excretion and the trapping of cadmium in the intestinal wall. It has been suggested that what appeared to be a slightly smaller absorption in laboratory animals than in humans is more related to differences in diet than to differences in physiology (USEPA 1999).

Cadmium absorption is increased by low intakes of iron and calcium, and high levels of zinc may affect cadmium absorption, distribution, or elimination. As with several other metals, younger animals may have greater absorption of cadmium than older animals (Hrudey et al. 1996).

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The bioaccessibility of cadmium in oysters was recently evaluated by Bragigand et al. (2004), using oysters harvested in France and the United Kingdom. Measures of bioaccessibility varied by cadmium concentration and ranged from 44 to 75%. No other bioaccessibility studies of cadmium in other food items were identified. For that reason, it is not known if cadmium in oysters is more or less bioavailable than the cadmium in other foods. Consequently, a point estimate RBA of 1 will be used in the intake calculation (i.e., assuming that the bioavailability of cadmium in oysters is comparable to the bioavailability of cadmium in other foods).

3.3 ESTIMATION OF CADMIUM INTAKE VIA OYSTERS

Intakes for a hypothetical Native American population with high shellfish consumption rates were calculated for each exposure area based on consumption rates for the Suquamish Tribe in Puget Sound. Consumption rates for the API community from the Seattle area were used to generate estimates for high end, non‐Tribal consumers for each exposure area. The range of intakes for the general U.S. resident was based on a nation‐wide study.

A combination of point estimates and PDFs shown in Tables 2‐3 and 3‐4 were used to estimate cadmium intake from individual exposure areas and states using Monte Carlo methodology. PDFs for cadmium intake for each population are provided in Attachment B. Mean, median, and 90th and 95th percentile intake rates are listed in Table 3‐5. The PDFs for oyster consumption by the Suquamish Tribe are defined by the mean and standard deviation provided in the study report (Suquamish Tribe 2000), while distribution for the API community consumption rates was generated from the study raw data (Secheena et al. 1999). The PDF for the general U.S. population was estimated using a mean consumption rate provided by USEPA (2002) and an estimated standard deviation. The standard deviation was a default value selected by Crystal Ball software.

The following section describes cadmium intake rates for each population consuming oysters in Alaska, California, Oregon, and Washington.

3.3.1 Alaska

The median intake rate of cadmium for a Native American population consuming oysters from all Alaska sampling stations is 3.6 x 10‐4 mg/kg‐day, with a 95th percentile cadmium intake rate of 5.9 x 10‐4 mg/kg‐day. The median cadmium intake rate for an API population consuming oysters from all Alaska sampling stations is lower than for a Native American population, 9.4 x 10‐5 mg/kg‐day, but due to the large variability in API consumption rates, the 95th percentile of 1.0 x 10‐3 mg/kg‐day is greater than that for the Native American population. Cadmium intake rates for a typical U.S. consumer population are the lowest of the three populations, with values of 2.0 x 10‐6 mg/kg‐day and 1.7 x 10‐5 mg/kg‐day for the median and 95th percentile, respectively.

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Cadmium intake rates for all populations are lower for exposure areas A1 and A3. Due to higher tissue concentrations in area A2, cadmium intakes also are higher, as shown in Table 3‐6.

3.3.2 California

Intake rates of cadmium consumed in oysters harvested in California are generally lower for all populations than rates of cadmium from oysters harvested in other states. The median cadmium intake rate for a Native American population is greater than those for API and general U.S. populations, although the upper percentiles for an API intake rate are greater than those for Native American and general U.S. populations.

The median Native American population intake rate of cadmium from oysters collected from all sampling stations in California is 1.2 x 10‐4 mg/kg‐day and the 95th percentile cadmium intake rate is 3.8 x 10‐4 mg/kg‐day. The median and 95th percentile cadmium intake rates for an API population are 3.5 x 10‐5 mg/kg‐day and 4.3 x 10‐4 mg/kg‐day, respectively. The cadmium intake rates for the general U.S. population are nearly two orders of magnitude lower than those for the other two populations studied. The median and 95th percentile cadmium intake rates for a typical consumer population are 7.4 x 10‐7 mg/kg‐day and 7.6 x 10‐6 mg/kg‐day, respectively.

Cadmium intake rates for individual exposure areas C1 and C2 are comparable to those for all California sampling stations combined, as shown in Table 3‐6.

3.3.3 Oregon

Tissue concentrations from Oregon are similar to those measured in California and Washington. The intake rates of cadmium in oyster tissue collected from all Oregon sampling stations also are similar to those observed for California and Washington. The median and 95th percentile cadmium intake rates for a Native American population are 1.8 x 10‐4 mg/kg‐day and 3.2 x 10‐4 mg/kg‐day, respectively. The median cadmium intake rate for an API population is 4.7 x 10‐5 mg/kg‐day and the 95th percentile rate is 4.8 x 10‐4 mg/kg‐day. Typical U.S. consumer population median and 95th percentile cadmium intake rates are 1.0 x 10‐6 mg/kg‐day and 8.9 x 10‐6 mg/kg‐day, respectively.

Intake rates for exposure areas O1 and O2 are similar to those for all sampling stations, or both exposure areas, combined, as shown in Table 3‐6.

3.3.4 Washington

Intake rates for all exposure areas, W1 through W7 combined, and exposure areas W1 through W6 combined, were calculated in addition to intake rates for individual exposure areas. The following intake rates were obtained for consumption of oysters from all exposure areas. The median and 95th percentile cadmium consumption rates for a Native American population are

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1.8 x 10‐4 mg/kg‐day and 3.9 x 10‐4 mg/kg‐day, respectively. Median and 95th percentile cadmium intake rates for an API population are 4.9 x 10‐5 mg/kg‐day and 5.1 x 10‐4 mg/kg‐day, respectively. The median intake rate for a typical U.S. consumer population is 1.0 x 10‐6 mg/kg‐ day and the 95th percentile intake rate is 9.1 x 10‐6 mg/kg‐day.

The median cadmium intake rates for a Native American population consuming oysters collected from individual exposure areas range from 1.2 x 10‐4 mg/kg‐day for area W6 to 2.6 x 10‐4 mg/kg‐day for area W4. Median cadmium intake rates estimated for an API population range from 3.2 x 10‐5 mg/kg‐day to 6.4 x 10‐5 mg/kg‐day. The range of cadmium intake rates for a general U.S. consumer population for areas W1 through W7 is 6.7 x 10‐7 mg/kg‐day to 1.4 x 10‐6 mg/kg‐day.

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4 CADMIUM TOXICITY ASSESSMENT

A toxicological assessment is conducted by reviewing relevant toxicity information from governmental health authorities and in peer‐reviewed publications. Toxicity values for carcinogenic and noncarcinogenic health effects have been developed for many chemicals by government agencies, including the EPA, ATSDR, Health Canada, and WHO. These toxicity values are numerical expressions of chemical dose and response, and vary based on factors such as the route of exposure (e.g., oral or inhalation) and duration of exposure. The purpose of this focused toxicity evaluation is to summarize adverse health effects that may be associated with ingestion of cadmium and to identify the doses that may be associated with those effects. The dose‐response assessment then forms the basis for the identification of toxicity values that may be used to predict the risk of adverse health effects from exposure to cadmium.

This assessment is focused on noncancer effects because there is little evidence of an association between oral exposure to cadmium and increased cancer rates in humans. Epidemiological studies of populations in England, Belgium, and Japan with elevated oral cadmium exposures did not show significant increase in cancer rates or mortality from cancer, including prostate, kidney, or urinary tract cancer (ATSDR 1999). Seven studies in rats and mice exposed to various cadmium salts showed no evidence of carcinogenic response, while another study had equivocal findings (USEPA 2007). Oral slope factors, tumorigenic concentrations, or unit risks have not been established for cadmium by USEPA (2007).

The noncancer toxicity value for cadmium represents average daily exposure level at which no adverse health effects are expected to occur with chronic exposures. The toxicity value reflects the underlying assumption that systemic toxicity occurs as a result of processes that have a threshold. The value assumes that a safe level of exposure exists and that toxic effects will not be observed until this level has been exceeded.

The toxicity values for noncancer health effects are generally derived based on laboratory animal studies or epidemiological studies in humans. In such studies, the value is calculated by first identifying the highest concentration or dose that does not cause observable adverse effects (NOAEL) in the study subject. If a NOAEL can not be identified from the study, a lowest‐ observed‐adverse‐effect level may be used. This dose or concentration is then divided by uncertainty factors to calculate a toxicity value, referred to by EPA as a RfD.

The uncertainty factors are applied to account for limitations of the underlying data and are intended to ensure that the toxicity value calculated based on the data will be unlikely to result in adverse health effects in exposed human populations. For example, an uncertainty factor of 10 is used to account for interspecies differences (if animal studies were used as the basis for the calculation), and another factor of 10 is used to address the potential that human subpopulations such as children or the elderly may have increased sensitivity to the chemicalʹs adverse effects. Thus, variations in the strength of the underlying data are reflected in the

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uncertainty factors used to calculate the toxicity values and in the low, medium, or high confidence ratings assigned to those values (USEPA 2006).

Toxicity studies most relevant to oral exposure to cadmium are those that evaluate dietary doses/exposures. The critical toxic effect upon which the oral cadmium RfD is based is renal toxicity. Cadmium has a very long residence time in the body and kidney concentrations slowly build up over a lifetime. Consequently the toxicokinetic behavior of this metal is considered in the RfD derivation. The toxicokinetics of ingested cadmium are discussed below, followed by brief summaries of pertinent human and animal studies and an explanation of the derivation of the oral RfD.

4.1 CADMIUM TOXICOKINETICS

Most ingested cadmium passes through the gastrointestinal tract without being absorbed. Approximately 2.5% of total ingested cadmium in food is absorbed, and approximately 5% of total ingested cadmium in water is absorbed (USEPA 2007). Factors affecting cadmium absorption included metal‐metal (e.g., iron, calcium, chromium, magnesium, zinc) and metal‐ protein interactions (glutathione, sulfhydryl containing enzymes) in the body or in the food or water (ATSDR 1999). Dietary zinc has been shown to decrease the oral absorption of cadmium. Cadmium absorption is known to increase with iron or calcium deficiency and increased fat in the diet (ATSDR 1999).

Following absorption from any route of exposure, cadmium widely distributes throughout the body, with the major portion residing in the liver and kidney. Cadmium that is absorbed into the body is excreted very slowly, primarily through urinary and fecal excretion. EPA assumes that 0.01% of the total body burden of cadmium is eliminated per day (USEPA 2007). Cadmium can pass across the placenta and can be excreted in human milk (ATSDR 1999).

Cadmium is not known to undergo any direct metabolic conversion. The cadmium (+2) ion will readily bind to anionic groups in proteins and other molecules. Cadmium interacts with the protein metallothionein, which is capable of binding as many as seven cadmium ions per molecule. Metallothionein is inducible in most tissues by exposure to cadmium, zinc, and other metals as well as organic compounds and physiologic stressors. Parenteral exposure studies in animals have shown that induction of metallothionein by pretreatment with zinc reduces toxicity of cadmium exposure. Zinc can prevent or reduce testicular and prostatic cancers induced by injection of cadmium, but these interactions have been shown to be dependent on dose, route, and target organ (ATSDR 1999).

Cadmium in the liver becomes bound to metallothionein and is released to the bloodstream. Metallothionein‐bound cadmium can be filtered and reabsorbed in the kidney, a process which may induce synthesis of proximal tubular cell metallothionein. The cadmium is then bound to the metallothionein in the kidney. It is hypothesized that the presence of free cadmium in the

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kidney is associated with renal damage; thus, the rationale that a critical concentration of cadmium in the kidney is required before renal effects will occur (ATSDR 1999).

4.2 NONCANCER HEALTH EFFECTS – HUMAN STUDIES OF ORAL EXPOSURE

The health effects of oral exposure to cadmium in humans have been studied for residents living in cadmium‐impacted areas. Cadmium has a long biological half‐life (20 – 30 years in humans) (WHO 1972). Long‐term exposure to cadmium, therefore, results in a variety of cumulative adverse health effects. Cadmium exposure in studied populations is often estimated by blood or urinary cadmium levels in residents. Urinary cadmium levels primarily reflect total body burden of cadmium, while blood cadmium levels may only reflect recent exposure. Urinary cadmium levels are generally reported as microgram per gram cadmium, adjusted for creatinine in the urine (μg/g Cr). Estimates of environmental exposure in these cases may be confounded by exposure to cadmium from smoking (ATSDR 1999).

4.2.1 Renal Effects

The preponderance of human and animal studies to date indicates that the critical organ in chronic cadmium exposure is the kidney. The earliest sign of kidney damage is an increase in tubular proteinuria, characterized by the urinary excretion of a number of proteins (e.g., β2‐ microglobulin) that would normally be reabsorbed in the proximal tubule. The excretion of such proteins in the urine, therefore, indicates damage to the proximal tubules. Tubular proteinuria may be followed by other signs of kidney dysfunction, such as increased urinary excretion of glucose, amino acids, calcium, phosphorus and uric acid (Health Canada 1994).

Elevated incidences of tubular proteinuria have been found in numerous epidemiologic studies of residents in cadmium‐polluted areas of Japan and Belgium, as well as studies of occupationally exposed workers (ATSDR 1999). Proteinuria has been shown to continue even after exposure to cadmium has ceased, indicating that the adverse effect is not readily reversible. However, kidney failure was not a common cause of death among occupationally exposed workers (Health Canada 1994; ATSDR 1999).

Numerous studies in humans and laboratory animals have demonstrated that proteinuria develops only when cadmium concentrations in the kidney exceed a critical level (ATSDR 1999; Health Canada 1994). This critical value was estimated based upon several studies that measured cadmium levels in the kidney either by neutron activation analysis or autopsy/biopsy and compared them with urinary protein levels or histopathological alterations in the same individuals. Estimates of the critical threshold value in the kidney obtained from the major studies ranged from 180 to 385 μg/g, wet weight. For purposes of quantifying potential human

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toxicity, EPA has established the critical level in the kidney as 200 μg/g, wet weight in the renal cortex.

4.2.2 Other Effects

A recent study of occupationally exposed workers found evidence of pancreatic damage in humans associated with elevated concentrations of urinary cadmium (Lei et al. 2007). Urinary cadmium was used as a biomarker for exposure, and serum insulin and amylase were used as biomarkers for pancreatic effects. Benchmark dose lower limits of a one‐sided 95% confidence interval (BMDLs) for 10% excess risk were 3.7 and 5.3 μg/g Cr (urinary cadmium corrected for creatinine) for serum insulin and amylase, respectively. Comparative BMDLs for urinary β2‐ microglobulin and urinary albumin (biomarkers of renal tubular dysfunction) were 3.8 and 5.8 μg/g Cr, respectively (Lei et al. 2007). The results of this study indicate that cadmium exposure can result in pancreatic dysfunction at levels similar to those associated with renal tubular dysfunction.

Studies of cardiovascular effects in humans after oral exposure to cadmium, which also control for smoking, have typically found no association between body cadmium levels (primarily reflecting dietary exposure) and hypertension. Cardiovascular effects such as lower blood pressure, conduction system disorders, and decreased frequency of cardiac ischemic changes were found among elderly women with historical high dietary exposure to cadmium (ATSDR 1999).

Oral exposure to cadmium can reduce gastrointestinal uptake of iron where dietary intake of iron is low. At least one study has found anemia in humans with chronic dietary exposure to cadmium, but other studies have found no significant relationship between dietary cadmium and anemia (ATSDR 1999).

There is substantial epidemiological evidence that dietary cadmium exposure in humans can result in osteomalacia, osteoporosis, and spontaneous/painful bone fractures under certain conditions. Cadmium‐exposed individuals exhibit both a disturbance of renal metabolism of vitamin D to its biologically useful form and increased urinary excretion of calcium. Thus, it is possible that adverse musculoskeletal effects may be a secondary effect of disruption of vitamin D metabolism and resulting problems with calcium absorption/excretion (ATSDR 1999).

There are a few human studies that have examined the neurological effects of oral cadmium exposure. These studies used hair cadmium as a biomarker of exposure and various behavioral endpoints. The potential impacts of several confounding factors and possibly inadequate measure of exposure make the results of these studies highly uncertain, and therefore of limited usefulness in evaluating neurological effects (ATSDR 1999).

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No studies were identified that evaluated reproductive effects in humans after oral exposure to cadmium (ATSDR 1999). Studies of development effects of oral cadmium exposure in humans are limited and inconclusive (ATSDR 1999).

4.3 NONCANCER EFFECTS – ANIMAL STUDIES OF ORAL EXPOSURE

Animals in the dietary and drinking water studies were mostly exposed to cadmium in the form of cadmium chloride. Some studies also evaluated exposure to cadmium in the form of cadmium sulfate (Health Canada 1994).

4.3.1 Renal Effects

Subchronic and chronic oral exposure to cadmium is associated with proteinuria and histopathologic changes in the kidney in several studies of rats. The critical concentration of 200 μg/g in the renal cortex needed before proteinuria occurs is generally supported by the animal data (ATSDR 1999).

4.3.2 Other Effects

Oral exposure of rats, rabbits, and monkeys over subchronic and chronic exposure periods has been found to increase blood pressure in some studies and not in others. Such mixed results may reflect a secondary effect associated with cadmium‐induced anemia or other systemic effects. Overall, ATSDR concluded that animal evidence for cardiovascular toxicity resulting from oral cadmium exposure indicates a slight, but not critical, effect (ATSDR 1999).

Studies of oral exposure to cadmium in rats, mice and rabbits have indicated that anemia is associated with cadmium in food or drinking water, and that additional iron prevents anemia. Some subchronic exposure studies of rats have shown evidence of anemia at doses ranging from 2 to 14 mg/kg‐day, while others have shown no adverse effects.

Results of chronic exposure studies in monkeys, rats, and mice have mixed results. In cases where clinical signs of anemia were observed, biological indicators (such as number of reticulocytes or erythroid progenitor cells in bone marrow) differed, indicating that anemia may be a result of more than simply reduced gastrointestinal absorption of iron (ATSDR 1999).

Musculoskeletal effects, such as decreased calcium content of bone and increased urinary calcium excretion, occurred in rats orally exposed to cadmium at doses ranging from 2 to 8 mg/kg‐day over subchronic and chronic exposure periods (ATSDR 1999). Adverse effects on bone may be exacerbated by a calcium‐deficient diet, by exposure at a young age during bone growth, by ovariectomy, or by gestation and lactation. Some studies in mice have detected effects in bone prior to development of proteinuria (the critical effect upon which the human toxicity value is based) (ATSDR 1999).

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Subchronic oral exposure has been associated with histopathologic changes in the liver at does of 1.6 to 15 mg/kg‐day and metabolic alterations (i.e., changes in enzyme activities) at doses of 0.05 to 10 mg/kg‐day in laboratory animals. Several subchronic and chronic oral exposure studies, however, did not find liver effects in animals at doses up to 14 mg/kg‐day (in rats) (ATSDR 1999).

Limited studies of endocrine effects in animals after oral exposure to cadmium indicated no adverse effects to the parathyroid glands, adrenal glands, or pituitary glands in rats at the tested doses. Pancreatic atrophy and pancreatitis was observed in rats exposed to dietary cadmium at 2.79 mg/kg‐day for 100 days (ATSDR 1999).

Numerous studies in rats, mice, and monkeys have established the capability of cadmium to affect the immune system, but the results are mixed and the clinical significance of the effects is unclear (ATSDR 1999).

Subchronic and chronic exposures to cadmium have been shown to be associated with neurological effects in laboratory animals, including weakness and muscle atrophy, aggressive behavior, anxiety, and changes in brain biochemistry at doses ranging from 5 to 40 mg/kg/day (ATSDR 1999).

Subchronic exposure to cadmium in drinking water in male rats and mice has been associated with necrosis and seminiferous tubule damage, increased testes weight, decreased sperm count, and motility at doses ranging from 5.8 mg/kg/day to 12.9 mg/kg/day. Subchronic exposure to cadmium in females was associated with decreased percentage of pregnancies and increased duration of estrus cycle at doses ranging from 40 to 61 mg/kg/day (ATSDR 1999). Numerous studies of rats and mice have shown that cadmium can be fetotoxic from oral exposures prior to and during gestation. Development effects include reduced fetal or pup weights and skeletal malformations at doses ranging from 1 to 20 mg/kg/day. The most sensitive indicator of development toxicity of cadmium, however, appears to be neurobehavioral development. Neurobehavioral effects include reduced exploratory locomotor activity and decreased conditioned avoidance behavior at doses ranging from 0.04 to 0.7 mg/kg/day (ATSDR 1999).

4.4 ORAL TOXICITY VALUES FOR NONCANCER HEALTH EFFECTS

The EPA RfD for human oral exposure to cadmium is based on a critical effect of significant proteinuria, an early indicator of renal tubular dysfunction. Numerous studies in humans and laboratory animals have demonstrated that proteinuria develops only when cadmium concentrations in the kidney exceed a critical level. and the oral RfD is based on an assumed critical cadmium concentration in the renal cortex of 200 μg/g, wet weight (WHO 2004, USEPA 2007).

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The EPA RfD values of1 × 10‐3 (food) and 5 × 10‐4 (water) mg/kg‐day for intake of cadmium in food are based on application of a toxicokinetic model that assumes that 0.01% of the total body burden of cadmium is eliminated per day. It also assumes 2.5% absorption of cadmium from food or 5% absorption from water. The toxicokinetic model predicts a NOAEL for food that would result in 200 μg/g, wet weight, cadmium in the human renal cortex. An uncertainty factor of 10 was applied to the NOAEL to account for intra‐human variability (USEPA 2007).

TDI similar to the EPA RfD have been developed by several countries. For example, the Canadian TDI of 8 × 10‐4 mg/kg‐day was derived from a lower limit of permissible intake of cadmium of 0.057 mg/day as recommended by WHO and an assumed adult body weight of 70 kg (Health Canada 2007a,b). The permissible daily intake value recommended by WHO was based on a multicompartmental model for cadmium distribution in the body and reflects an estimate of 0.1% of the population reaching the critical cadmium concentration of 200 μg/g in the renal cortex after 50 years (WHO 1972; Kjellstrom and Nordberg 1978).

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5 RISK CHARACTERIZATION

Quantitative estimates of exposure and toxicity were combined to yield numerical estimates of potential health risk. The results of the risk characterization process are provided, followed by a qualitative evaluation of uncertainties associated with the risk estimates and interpretation. Probabilistic risk distributions for all consumer populations and each exposure area were generated using Monte Carlo analysis. Mean, median, and 95th percentile risk results are presented.

5.1 NONCANCER HAZARD ASSESSMENT

To evaluate noncancer risks, the ratio of the average daily intake to the RfD is calculated. This ratio is referred to as the hazard quotient. If the calculated value of hazard quotient is less than 1, no adverse health effects are expected. If the calculated value of the hazard quotient is greater than 1, then further risk evaluation is needed.

For the ingestion of oysters, the hazard quotient was calculated using the following equation:

Intake HQ = Eq. 5 RfD

Where,

HQ = Hazard quotient associated with exposure to cadmium via ingestion (dimensionless)

Intake = Estimated average daily intake of cadmium via ingestion (mg/kg‐day)

RfD = Reference dose for cadmium (mg/kg‐day)

5.2 PROBABILISTIC RISK ASSESSMENT RESULTS

Intake of cadmium from oyster consumption was based on a combination of both point estimates and PDFs. Probabilistic distributions for intake then were combined with a point estimate of noncancer toxicity to generate risk estimates. Probabilistic risk distributions were generated for all populations consuming oysters in all exposure areas, although the consumption rates generated for the Suquamish Tribe and API populations are based on people living in the Puget Sound and may or may not be representative of populations residing in Alaska, California, Oregon, or other parts of Washington. These populations are high‐end consumers of shellfish and may represent other populations that also consume greater

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quantities of oysters than a typical U.S. consumer. Risk distributions were generated using Monte Carlo analysis. Per USEPA guidance, risk estimates in the range of 90th to 99th percentiles are equivalent to reasonable maximum exposures (USEPA 2001). As described in Section 3, the upper percentile oyster consumption estimates for the general U.S. consumer are based on short term consumption surveys and are likely to markedly overestimate the upper percentiles consumption rates on a long term basis, as is needed to accurately characterize chronic risks. In the event that risk to this population is determined to be of potential concern it will be necessary to modify these assumptions. Results for the PRA analysis are presented in the following section and in Table 5‐1. PDFs for risks are provided in Attachment B.

5.2.1 Alaska

Noncancer risks for consumption of cadmium in oysters harvested in Alaska were estimated for the Suquamish, API, and general U.S. consumer and are below levels of concern. Mean and median risks associated with oysters harvested from all Alaska sampling stations are well below a hazard quotient of 1 for all populations. Risks for consumers at the 95th percentile level range from 0.02 for general U.S. consumers to 1 for API consumers.

For oysters harvested exclusively from exposure areas A1 or A3 (Figure 2‐2), consumer risks at the 95th percentile ranged from 0.02 for the general U.S. consumer to 0.9 for the API population. Consumption of cadmium in oysters from these areas is not likely to present a significant health hazard. Due to slightly higher tissue concentrations observed in exposure area A2, the upper risk range increases to 1 for the API consumer although risks for the Suquamish and typical U.S. consumer are well below levels of concern.

5.2.2 California

Consumption of oysters harvested from sampling stations in California did not result in unacceptable risks to consumers. Mean and median risks are below a hazard quotient of 1 for all populations consuming oysters from all exposure areas. The hazard quotient at the 95th percentile is 0.008 for the typical U.S. consumer and the hazard quotient for both the Suquamish and API populations is 0.4 for consumption of oysters from all sampling stations.

Risks for consumers of oysters harvested from exposure area C1 were slightly higher than for oysters from C2. Noncancer risks at the 95th percentile for area C1 for the typical U.S. consumer, Suquamish, and API populations are 0.009, 0.4, and 0.5, respectively. Noncancer risks at the 95th percentile for area C2 for the typical U.S. consumer, Suquamish, and API populations are slightly lower, 0.005, 0.2, and 0.3, respectively. For the populations evaluated in this risk assessment, no hazard quotients greater than 1 are associated with consumption of cadmium in oysters from areas sampled in California (Figure 2‐3).

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5.2.3 Oregon

Potential noncancer hazards associated with consuming oysters collected from all Oregon sampling stations are similar to those calculated for California. Mean and median hazard quotients are below the threshold of 1 for all populations and exposure areas. Noncancer hazard quotients at the 95th percentile range from 0.009 for the typical U.S. consumer to 0.5 for the API population consuming oysters from all Oregon sampling stations. These hazards are unchanged when considering only those oysters collected from exposure areas O1 and O2 (Figure 2‐4). Consumption of cadmium in oysters from the areas sampled in Oregon is not likely to present a significant health hazard.

5.2.4 Washington

Potential noncancer hazards associated with consumption of cadmium in oysters harvested from all sampling stations in Washington were calculated for all populations. Mean and median hazard quotients for all populations consuming oysters from all exposure areas combined were less than the threshold of 1. Risks at the 95th percentile range from 0.009 for the typical U.S. consumer to 0.5 for the API population.

Risks for consumption of cadmium in oysters are consistent with this range for other exposure areas in Washington (Figure 2‐5). Although risks are slightly higher for area W4, Hood Canal, hazard quotients are still well below the target level of 1. The hazard quotient for consumption of cadmium in oysters collected from exposure area W4 for the Suquamish, API, and typical U.S. populations are 0.4, 0.7, and 0.01, respectively. Consumption of cadmium in oysters from the areas sampled in Washington is not likely to present a significant health hazard.

5.3 UNCERTAINTIES

The grouping of data used to identify exposure areas, such as W1 through W6 for Washington State (Figure 2‐5), add uncertainty to risk assessment by not being sensitive to seasonal variations at each site. For instance, in the Hood Canal exposure area W4, measurements of cadmium in oyster tissue showed little seasonal variability except for the Hama Hama sampling station (data not shown). At Hama Hama, cadmium concentrations in oyster tissue were the lowest in summer time and highest in the fall. In addition, cadmium concentrations at the same site were 1.5 to 2 times higher than all other sampling stations within W4 exposure area (Figure 2‐5).

The sources of oysters consumed by local populations were pooled into exposure assessment areas, as described in Section 2.4. Most of these areas contain sampling stations with one‐time measurements and cannot necessarily be extrapolated as being consistent from year to year, as in other sampling stations within the same area.

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Oysters collected for this study were ½ inch to 2 ½ inches larger than most commercially available individuals. There is a weak correlation between body size and cadmium concentrations (data not shown) for all oysters sampled in Washington State, suggesting that tissue concentrations measured in this study are greater than what would be found in commercially grown oysters.

The primary source of uncertainty in the exposure assessment lies in the selection of consumption studies. For example, it is not clear whether or not the two Washington state‐ based populations evaluated in this assessment (i.e., Suquamish Tribe and API communities) are representative of other oyster‐consuming populations residing in California, Oregon, and Alaska. The use of these two high‐end consumer populations and a broad‐based U.S. population was intended to represent a range in consumption rates but without additional consumption studies, it is not known if the risk estimates over‐ or underestimate actual risks to local populations.

Additional uncertainty is found in quantification of the consumption rates for the selected populations. High fish consumption rates were observed in both the Suquamish Tribe and API fish consumption survey results; however, treatment of these rates differed between the two studies. Consumption rates presented for the Suquamish Tribe include all reported rates, as study investigators determined that even elevated rates reflect real fish consumption and did not represent outliers.

Consumption rates presented for the API community have been adjusted for potential outliers. Outliers were defined as consumption rates that were greater than three standard deviations above the mean seafood consumption for the particular seafood group. Outliers were substituted by the mean + 3SD (the rule of “mean plus three standard deviations”) in the individual seafood categories. Although the raw data were used to develop a PDF for this PRA, the contribution of elevated consumption rates resulted in a large standard deviation and, subsequently, considerable uncertainty in estimating upper percentile values.

The PDF for oyster consumption by the Suquamish Tribe is defined by the mean and standard deviation provided in the study report (Suquamish Tribe 2000), whereas the PDF for the general U.S. population was estimated using a mean consumption rate provided by USEPA (2002) and an estimated standard deviation. The standard deviation was a default value selected by Crystal Ball software. Also, the mean consumption rate for the general U.S. consumer includes both consumers and nonconsumers, which may underestimate intake for consumers. Because the data are based on a short‐term consumption survey, a very high percentage of the population surveyed had not eaten oysters during the survey period. This does not accurately represent the fraction of the population that never consumed oysters. Due to the short –term nature of this survey and the high percentage of people who did not consume oysters, it would not be possible to directly estimate a representative distribution of long‐term average consumption rates for people who do consume oysters from this dataset even if upper and

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lower percentiles had been reported (only mean oyster consumption rates were provided). Consequently, the reliability of upper percentile estimates for this population is highly uncertain.

A significant area of uncertainty in the risk estimates is that this risk evaluation focuses only on risks associated with cadmium intake via oysters. Cadmium exposures from cigarettes and other foods besides oysters were not included in this evaluation but are typically the greatest source of exposure. The current average dietary intake of cadmium by adults in the United States is approximately 0.0004 mg/kg‐day (ATSDR 1999). This figure is doubled for smokers (0.0008 mg/kg‐day; ATSDR 1999), resulting in an intake that approaches the FDA’s TDI value of 0.00078 mg/kg‐day (assuming an adult body weight of 70 kg). In the study herein, the intake of cadmium in oyster tissue by high‐end consumers may equal or exceed cadmium intake from other food sources and may result in an exceedance of the FDA’s TDI.

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6 REVIEW OF CURRENT INTAKE GUIDELINES

The European Union and other nations have established MLs for cadmium in shellfish and/or molluscan bivalves. Examples of MLs for cadmium in oysters are provided in Table 6‐1. These values are based on assumed tissue concentrations and consumption rates for various populations and are intended to represent a fraction of the maximum TDI or provisional tolerable weekly intake (PTWI) of cadmium. The TDI and PTWI are estimates of the amount of a chemical that can be ingested over the course of a lifetime without resulting in significant health risks.

This section provides an overview of established TDI and PTWI values that serve as the basis for “maximum levels” for cadmium in shellfish and a discussion of these values in the context of this risk assessment. In some cases, the MLs for cadmium in shellfish may be over‐ or under‐ protective of human health when considering the two Pacific Northwest populations and general U.S. consumer evaluated in this report.

Table 6-1. Maximum levels for cadmium in oysters (µg/g) for various nations. Nation Maximum Level (µg/g) Venezuela, Slovak Republica 0.1 Austriaa 0.2 Switzerlanda 0.6 EC, New Zealand, Czech Republica 1.0 Codex, Hong Kong, Australia, Japana 2.0 United Statesb 4.0b a Information regarding the derivation of these MLs was not located; values presented here for comparison purposes but are not discussed in this report. b This value is not enforced by the FDA (see Section 6.3).

6.1 CODEX ALIMENTARIUS COMMISSION

CAC is an international committee established by the Joint FAO/WHO Food Standards Programme in the 1960s to protect consumer health and facilitate food trade. CAC relies on scientific advisement by various committees, including the Joint FAO/WHO Expert Committee on Food Additives (JECFA). One component of the JECFA’s service to the CAC is to periodically review PTWI5 values established by the CAC by examining newly available data from peer‐reviewed sources.

5 Provisional Tolerable Weekly Intake: An endpoint used for food contaminants such as heavy metals with cumulative properties. Its value represents permissible human weekly exposure to those contaminants unavoidably associated with the consumption of otherwise wholesome and nutritious foods.

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Cadmium as a food additive has been evaluated by the JECFA at various meetings and a PTWI of 400 to 500 μg of cadmium per person was established during early discussions. At later meetings, expression of the PTWI was changed to a weekly intake of cadmium per unit of body weight, or 7 μg cadmium per kg body weight (i.e., equivalent to 420 μg/g per person per week for a 60 kg person). The JECFA has upheld the PTWI of 7 μg/kg despite noted uncertainties in toxicokinetic models predicting the level of cadmium at which adverse renal effects would be expected. The PTWI has not been changed due to a lack of more precise health risk estimates associated with dietary cadmium exposure.

A call for additional studies of the prevalence of renal tubular dysfunction resulted in completion of new investigations designed to address JECFA concerns about cadmium. Nevertheless, it was determined that information remained inadequate to justify revising the PTWI of 7 μg/kg. The assessment of cadmium will be revisited in the future (date unknown) following completion of a separate project being conducted by the Joint FAO/WHO Project to Update the Principles and Methods for the Risk Assessment of Chemicals in Food. This project will address two issues that are hoped to improve the reassessment process for cadmium: 1) the dose–response assessment of biomarkers of effect and their relationship to disease outcome, and 2) the possible specification of longer tolerable intake periods for contaminants with longer biological half‐lives.

The review and periodic update of the Codex PTWI is important in that changes in the PTWI will result in subsequent changes of allowable MLs of cadmium in foods, including oysters. At the same time the PTWI is reviewed by the JECFA, the Committee also reviews MLs for individual foods. In February 2005, the JECFA considered the percent reduction in total cadmium intake via food if the ML for bivalve mollusks were reduced from 2.0 to 1.0 μg/g, wet weight. It was determined that the reduction in cadmium intake, 1%, would have little to no impact on mean intake of cadmium but would result in approximately 25% of bivalve mollusks being in violation of this ML. This would result in no reductions in human health risk for a majority of populations worldwide but would have potentially significant impacts on trade. Ultimately, it was determined that the ML of 2.0 μg/g, wet weight, for cadmium in bivalve mollusks would not be changed.

Most oysters collected for this USDA study do not contain cadmium at levels exceeding the ML established by Codex (2006). Of 270 samples, 44 samples, or 16%, contain cadmium at concentrations greater than 2 μg/g, wet weight.

6.2 EUROPEAN COMMUNITIES

The Commission of the European Communities (EC) has established a limit for cadmium in foodstuffs of 1.0 mg/kg, wet weight (EC 2001). Intake rates for “foodstuffs,” bioavailability

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assumptions, and other medical or scientific information that were used in setting the ML for cadmium was not located. EC reports the following (Commission Regulation No 466/2001):

Cadmium may accumulate in the human body and may induce kidney dysfunction, skeletal damage and reproductive deficiencies. It cannot be excluded that it acts as a human carcinogen. The Scientific Committee on Food (SCF), in its opinion of 2 June 1995, recommended greater efforts to reduce dietary exposure to cadmium since foodstuffs are the main source of human intake of cadmium. Therefore, maximum levels should be set as low as reasonably achievable.

Based on this statement, the ML for cadmium in shellfish set by the EC appears to be based on what was deemed to be technically achievable in shellfish without the derivation of a specific, risk‐based exposure scenario.

Many oyster samples collected in the study reported herein exceed the EC maximum level of 1.0 mg/kg, wet weight, including all oysters collected in Alaska. Of the 270 samples collected in Alaska, California, Oregon, and Washington, 64% contain cadmium at levels greater than 1.0 mg/kg.

6.3 UNITED STATES

The FDA provides an analysis of cadmium levels in shellfish in their Guidance Document for Cadmium in Shellfish (FDA 1993). FDA provides guidelines for establishing “levels of concern” to assist state and local agencies but does not enforce a specific tolerable limit for cadmium in oysters or bivalve mollusks at this time. FDA’s current position is, “These guidance documents represented current agency thinking in regards to the available science at the time they were issued. They no longer represent the current state of science and are presented here for the historical record only.ʺ Because the guidance documents are used by state health departments, the assumptions used to establish a TDI and ML for cadmium in shellfish are considered relevant to this study.

FDA has established a TDI of 55 μg of cadmium per person per day from all sources, including cigarettes, water, diet, air, and soil (385 μg/g per person per week). This value is used as a starting point in estimating a ML for cadmium in shellfish. By combining the TDI with assumptions for consumption rate and tissue concentration, MLs for cadmium in oysters were estimated.

FDA relied on a 1988 Market Research Corporation of America 14‐day study of approximately 25,000 consumers, 4.8% of whom consumed molluscan bivalves, to determine the frequency of oyster consumption. Consumption frequency was reported for three age groups, ages two years and older, ages 2 to 5 years, and ages 18 to 44 years. Standard portion sizes obtained from USDA’s 3‐day National Food Consumption Survey for 1977‐1978 (Pao et al. 1982) were used to

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estimate mean and 90th percentile consumption rates of 12 g/person‐day and 18 g/person‐day for consumers only, respectively. Adjustment of FDA’s values, assuming an adult body weight of 70 kg, results in mean and 90th percentile consumption rates of 0.17 g/kg‐day and 0.26 g/kg‐ day, respectively.

The resulting MLs calculated by FDA for molluscan bivalves, based on mean and 90th percentile consumption rates, are 5 and 3 μg/g, wet weight, respectively. Only one tissue sample collected for the study herein exceeds the estimated ML of 3 μg/g based on the 90th percentile consumption rate.

6.4 HONG KONG AND AUSTRALIA

Kruzynski (2004) reports that Hong Kong and Australia have established an ML for cadmium in bivalves at 2 mg/kg, wet weight. In reference to these countries, Kruzynski (2004) notes that “the medical basis for these figures is not clearly defined in the scientific literature.” No other reference for the Australia ML was located.

Hong Kong’s Food and Environmental Hygiene Department recently conducted a study of dietary exposure to heavy metals in secondary school students in 2002 (HKSAR 2002), assuming a consumption rate of 50.5 g/day for “seafood other than fish.” However, there is no documentation that this information was then used to determine the maximum permitted concentration of cadmium in shellfish.

Most oysters collected for the study reported herein do not contain cadmium at levels exceeding the ML established for Hong Kong, Australia, and New Zealand. Of 270 samples, 44 samples, or 16%, contain elevated concentrations of cadmium.

6.5 CANADA

Canada has no maximum permitted cadmium concentration for oysters. Instead, Health Canada has developed consumption limit recommendations. The Canadian Food Inspection Agency (CFIA) Fact Sheet states: “Following consumption guidelines for oysters will minimize the intake of cadmium. Health Canada recommends that the consumption of B.C. oysters be limited to 460 grams per month for adults and to 60 grams per month for children” (CFIA 2003)6.

In determining these consumption limits, it appears that Health Canada utilized the FAO/WHO, PTWI, and data on cadmium values for BC oysters determined by CFIA. In addition, Health Canada incorporated a factor for bioavailability. Kruzynski (2004) notes that

6 Assuming a mean shucked oyster weight of 44 g as measured for the study herein, the recommended consumption is equal to approximately 10 oysters per month for adults and 1 oyster per month for children.

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Health Canada assumed cadmium in oysters is 5% bioavailable. A value of 5% was assumed to be the mid‐range of data on known cadmium bioavailability from foodstuffs as published in the WHO International Programme on Chemical Safety document, Environmental Health Criteria 134, Cadmium – Addendum (WHO 2004).

With approximately 30 days in one month and an average adult body weight of 70 kg, the consumption guideline may be translated to a maximum recommended oyster consumption rate of 0.22 g/kg‐day. This consumption rate is two orders of magnitude greater than the typical U.S. consumer mean consumption rate of 0.002 g/kg‐day, but is comparable to the API’s 90th percentile consumption rate of 0.2 mg/kg‐day and is approximately 3.6 times lower than the Suquamish Tribe 95th percentile consumption rate of 0.79 g/kg‐day.

6.6 DISCUSSION OF REGULATORY LIMITS AND POTENTIAL HEALTH RISKS

The protectiveness of regulatory limits on cadmium in bivalve mollusks was evaluated qualitatively for the populations studied in this report, a Native American tribe, API, and general U.S. populations. The MLs of 1.0 and 2.0 μg/g cadmium were compared to point estimates of EPCs for all three populations.

The highest oyster tissue concentrations of cadmium are found in the state of Alaska, with EPCs ranging from 2.5 to 4.0 μg/g. Point noncancer hazard quotient estimates obtained for consumption of Alaska oysters ranged from 0.008 for the typical U.S. consumer to 0.7 for the Suquamish Tribe. This indicates that a high level of oyster consumption will not result in unacceptable risks, even when oyster tissue levels are almost two times greater than the ML of 2.0 μg/g. Consumption of oysters at the level of a Suquamish Tribe member would not result in unacceptable risks up to a tissue concentration of 6 μg/g.

Although cadmium concentrations may exceed the ML of 2.0 μg/g imposed by Codex (2006) and others, noncancer risks do not appear to be a human health concern for a typical consumer. Excluding cadmium intake from other dietary sources, intake via oysters at consumption rates as high as 0.164 g/kg‐day (Native American scenario) does not appear to present health risks.

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7 CONCLUSIONS

Potential health risks associated with consumption of cadmium in Pacific Coast oysters was evaluated using probabilistic methods. Two populations, a Native American tribe and an Asian and Pacific Islander group, included high‐end oyster consumers relative to the third population, a typical U.S. consumer. Consumption of Pacific Coast oysters is not expected to result in cadmium intake exceeding the TDI. However, taking into account cadmium intake via cigarettes and other dietary sources aside from oysters, cadmium intake may approach or exceed the TDI for some populations.

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Christy, A.E. 2005. A survey of cadmium in Washington Pacific oysters (Crassostrea gigas) and its implications to the shellfish industry and human health. M.S. Thesis, The Evergreen State College, Olympia, WA.

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FIGURES

CANADA

Drayton Harbor Birch Bay

AKAK

Orcas Island Westcott Bay Samish Island

Eaglek Bay

Windy Bay Dungeness Bay Discovery Bay Sequim Bay

Thorndyke Bay Port Gamble A Port Gamble B Dabob Bay East Sub base Bangor Agate Pass Dosewallips

Hama Hama Belfair Henderson Bay Twanoh Quartermaster Harbor Grays Harbor - South Grays Harbor Potlach Chester Bay Minter Bay Oakland Bay Squaxin Island Totten Inlet Henderson Inlet Nisqually Reach Eld Inlet Willapa Bay - Bay Center

Pacific Willapa Bay - Long Beach

Wahkiakum

WA MT

Clatsop Sonoma

Yaquina Bay IDID Marin Tomales Bay - North OROR

WY

Tomales Bay

Humboldt Bay - North Bay Tillamook Bay Tillamook NVNV UTUT COCO Netarts Bay

CACA

Cadmium (μg/g) wet weight, Cadmium (μg/g) wet weight, sampled once only Seasonal Sampling 0 - 0.5 0 - 0.5 AZAZ NMNM Catalina Island Ferry Terminal 0.5 - 1.0 0.5 - 1.0

1.0 - 1.5 1.0 - 1.5

1.5 - 2.0 1.5 - 2.0 015030075 Kilometers 2.0 - 3.0 2.0 - 3.0 Average cadmium concentrations in C. gigas from 2004 to 2006

Map Document: (O:\Projects\USDA_Cadmium_Shellfish\mxd\Station_Locations_for_Poster_20080320.mxd) TWC -- 3/19/2008 -- 3:34:42 PM ALASKA

Wrangell-St. Elias NP and NPRE

Eaglek Bay*

Windy Bay CANADA A2 A1

Glacier Bay NP and NPRES

Legend Oyster Tissue Concentration Cadmium (µg/g) wet weight Chester Bay* 0 - 0.5

0.5 - 1.0

1.0 - 1.5 A3

1.5 - 2.0

2.0 - 2.5

2.5 - 3.0 Exposure Areas A1 A2 A3 * Site only sampled on one event

0 2550 100 Kilometers Figure 2-2 USDA Cadmium Shellfish Study FEATURE SOURCES: Cadmium in Oyster Tissue Environmental data obtained from Washington State Department of Ecology Environmental Information Management System (EIM) Average Concentration (2004 - 2006) and SEDQUAL database. Alaska Map Document: (O:\Projects\USDA_Cadmium_Shellfish\mxd\AK_Cadmium.mxd) 3/12/2007 -- 3:17:16 PM Sonoma

Humboldt Bay - North Bay

Marin Tomales Bay - North*

C1

Tomales Bay

Tomales Bay - North*

Tomales Bay

Legend Oyster Tissue Concentration Cadmium (µg/g) wet weight

0 - 0.5

0.5 - 1.0

1.0 - 1.5

1.5 - 2.0 C2

2.0 - 2.5 Catalina Island Ferry Terminal*

Exposure Areas C1 C2 * Site only sampled on one event

0 25 50 100 Kilometers Figure 2-3 USDA Cadmium Shellfish Study FEATURE SOURCES: Cadmium in Oyster Tissue Environmental data obtained from Washington State Department of Ecology Environmental Information Management System (EIM) Average Concentration (2004 - 2006) and SEDQUAL database. California Map Document: (O:\Projects\USDA_Cadmium_Shellfish\mxd\CA_Cadmium.mxd) 3/12/2007 -- 3:27:35 PM Tillamook Bay Washington

O1 Tillamook Netarts Bay

Yamhill

Polk

Marion

Legend Oyster Tissue Concentration Cadmium (µg/g) wet weight

Lincoln 0 - 0.5

0.5 - 1.0 O2 1.0 - 1.5 Yaquina Bay* 1.5 - 2.0

Ben2t.o0 n- 2.5 Exposure Areas O1 Linn O2 * Site only sampled on one event

0 5 10 20 Kilometers Figure 2-4 USDA Cadmium Shellfish Study FEATURE SOURCES: Environmental data obtained from Washington State Department Cadmium in Oyster Tissue of Ecology Environmental Information Management System (EIM) Average Concentration (2004 - 2006) and SEDQUAL database. Oregon Map Document: (O:\Projects\USDA_Cadmium_Shellfish\mxd\OR_Cadmium.mxd) 3/12/2007 -- 3:37:51 PM Drayton Harbor* W7 - Coast Inset

Birch Bay SP*

W1

Grays Harbor - South* Orcas Island* Westcott Bay* W2 Samish Island

Willapa Bay - Bay Center

Willapa Bay - Long Beach*

Dungeness Bay*

W3

Discovery Bay* Sequim Bay*

Port Gamble A* Thorndyke Bay Port Gamble B* Dabob Bay East*

Sub base Bangor* Agate Pass* Dosewallips SP* Legend W4 Oyster Tissue Concentration Cadmium (µg/g) wet weight Hama Hama W5 0 - 0.5 0.5 - 1.0

1.0 - 1.5 Belfair SP* Henderson Bay* 1.5 - 2.0 Twanoh SP* Quartermaster Harbor* 2.0 - 2.5 Potlach SP* Exposure Area North Puget Sound - W1 Minter Bay* San Juan Islands - W2

Oakland Bay* Straits - W3 Squaxin Island* Hood Canal - W4

Central Puget Sound - W5 Henderson Inlet* Totten Inlet* W6 South Puget Sound - W6

Nisqually Reach* Coast - W7 Eld Inlet

* Site only sampled on one event

0 5 10 20 Kilometers Figure 2-5 USDA Cadmium Shellfish Study FEATURE SOURCES: Cadmium in Oyster Tissue Environmental data obtained from Washington State Department of Ecology Environmental Information Management System (EIM) Average Concentration (2004 - 2006) and SEDQUAL database. Puget Sound & Washington Coast

Map Document: (O:\Projects\USDA_Cadmium_Shellfish\mxd\Puget_Sound_Cadmium_v92.mxd) TWC -- 11/20/2007 -- 3:32:26 PM Figure 2-6. Cadmium tissue concentrations for each state.

Boxplot by Group Variable: Result 4.5

4.0

3.5

3.0

2.5

2.0 Result (µg/g)

1.5

1.0

0.5

Median 0.0 25%-75% AK CA OR WA Min-Max State Figure 2-7. Cadmium tissue concentrations for each exposure area within Washington state.

Boxplot by Group: Washington Exposure Areas Variable: Result 2.8

2.6

2.4

2.2

2.0

1.8

1.6

1.4

Result (µg/g) 1.2

1.0

0.8

0.6

0.4 Median 0.2 25%-75% W1 W2 W3 W4 W5 W6 W7 Min-Max Exposure Area

TABLES

Table 2-1. Seasonal sampling events for the Pacific Oyster tissue collection. Sampling Events Sampling Station 2004 2005 2006 Jun Jul Sept Jan April May Jun Jul Aug Oct Nov Dec Jan Feb Washington Eld Inlet DDDDD Hama Hama DDDDD Samish Bay DDDDD Thorndyke Bay DDDDD Willapa Bay DDDDD California Humboldt Bay DDDDD Tomales Bay DDDDD Oregon Tillamook Bay DDDDD Netarts Bay DDDDD Alaska Windy Bay DDDD Table 2-2. Categorization of Sample Stations by Exposure Area

Exposure Area Sample Station (No. of Samples) Alaska A1 Windy Bay (12)

A2 Eaglek Bay, NPWS (3)

A3 Chester Bay (6)

California C1 Humboldt Bay (11), Tomales Bay (16), Tomales Bay - North (4)

C2 Catalina Island Ferry Terminal (3)

Oregon O1 Netarts Bay (24), Netarts Bay South (3), Tillamook Bay (15)

O2 Yaquina Bay (3)

Washington W1 - North Puget Sound Samish Bay (18), Drayton Harbor (3), Birch Bay State Park (3)

W2 - San Juan Islands Westcott Bay (3), Orcas Island (3)

W3 - Straits Sequim Bay (3), Dungeness Bay (3), Discovery Bay (3)

W4 - Hood Canal Thorndyke Bay (21), Sub Base Bangor (3), Port Gamble A (3), Port Gamble B (3), Dosewallips State Park (3), Dabob Bay East (3), Twanoh State Park (3), Hama Hama (18), Belfair State Park (3), Potlach State Park (3)

W5 - Central Puget Sound Agate Pass (3), Quartermaster Harbor (2)

W6 - South Puget Sound Minter Bay (North Carr Inlet) (3), Totten Inlet (3), Squaxin Island (3), Oakland Bay (3), Nisqually Reach (3), Henderson Inlet (3), Henderson Bay (3), Eld Inlet (18)

W7 - Coast Willapa Bay - Bay Center (21), Willapa Bay - Long Beach (3), Grays Harbor - south (3)

W1-6 Puget Sound Samish Bay, Drayton Harbor, Birch Bay State Park, Westcott Bay, Orcas Island, Sequim Bay, Dungeness Bay, Discovery Bay, Thorndyke Bay, Sub Base Bangor, Port Gamble A, Port Gamble B, Dosewallips State Park, Dabob Bay East, Twanoh State Park, Hama Hama, Belfair State Park, Potlach State Park, Agate Pass, Quartermaster Harbor, Minter Bay (North Carr Inlet), Totten Inlet, Squaxin Island, Oakland Bay, Nisqually Reach, Henderson Inlet, Henderson Bay, Eld Inlet Table 2-3. Summary Statistics and EPCs for Exposure Areas Point Standard Distribution Calculated Calculated UCL Estimate Exposure Area N Mean Deviation Min. Max Type1,2 UCL1 Statistic EPC 95% Approximate Alaska (all) 21 2.32 0.50 1.64 3.98 Gamma3 2.51 Gamma UCL 2.51 A1 12 2.24 0.37 1.64 2.70 Lognormal ISS ISS; Maximum 2.70 A2 3 2.69 1.17 1.70 3.98 Lognormal ISS ISS; Maximum 3.98 A3 6 2.29 0.23 2.06 2.58 Lognormal ISS ISS; Maximum 2.58

95% Modified-t California (all) 34 1.10 0.58 0.44 2.28 Nonparametric4 1.27 UCL 1.27 95% Approximate C1 31 1.14 0.59 0.44 2.28 Gamma3 1.33 Gamma UCL 1.33 C2 3 0.75 0.04 0.71 0.78 Lognormal ISS ISS; Maximum 0.78

95% Approximate Oregon (all) 42 1.17 0.34 0.52 2.04 Gamma3 1.26 Gamma UCL 1.26 95% Approximate O1 39 1.18 0.35 0.52 2.04 Gamma3 1.28 Gamma UCL 1.28 O2 3 1.12 0.05 1.14 1.06 Lognormal ISS ISS; Maximum 1.06

95% Approximate Washington (all) 173 1.25 0.51 0.44 2.65 Gamma3 1.31 Gamma UCL 1.31 95% Modified-t W1 24 1.15 0.27 0.71 1.48 Nonparametric 1.24 UCL 1.24 W2 6 1.33 0.41 0.89 1.74 Lognormal 1.67 ISS; Maximum 1.74 W3 9 1.22 0.28 0.90 1.62 Lognormal 1.39 ISS; Maximum 1.62 95% Student's-t W4 63 1.63 0.55 0.62 2.65 Normal 1.75 UCL 1.75 W5 5 1.21 0.35 0.79 1.56 Lognormal 1.54 ISS; Maximum 1.56 95% Approximate W6 39 0.81 0.26 0.44 1.34 Gamma3 0.88 Gamma UCL 0.88 95% Student's-t W7 27 1.07 0.24 0.56 1.47 Normal 1.15 UCL 1.15 95% Approximate W1-6 146 1.28 0.54 0.44 2.65 Gamma3 1.36 Gamma UCL 1.36

Tissue concentration units of µg/g, fresh weight EPC = Exposure point concentration ISS = Insufficient sample size N = sample size UCL = 95% upper confidence limit of the mean 1. Distribution type and UCL determined using ProUCL software. 2. When sample size (N) is less than 15, a gamma distribution assumed for PRA analysis and an EPC equal to the maximum detected concentration assumed for deterministic analysis. 3. Gamma distributions defined by location, scale, shape using Crystal Ball software for the following exposure areas: All Alaska Location=1.5; Scale=0.28; Shape=2.88 C1 Location=0.43; Scale=0.67; Shape=1.044 All Oregon Location=0.2; Scale=0.11; Shape=8.602 O1 Location=0.22; Scale=0.12; Shape=7.622 All Washington Location=0.31; Scale=0.29; Shape=3.198 W5 Location=0.44; Scale=0.29; Shape=1.275 'W1-6 Location=0.31; Scale=0.33; Shape=2.935 4. All California data fit to beta distribution defined by: alpha=0.3; beta=0.649 using Crystal Ball software W1 beta distribution defined by: alpha=0.59; beta=0.434 using Crystal Ball software Table 3-1. Exposure Factors for Intake Calculation

Mean / Point Standard Exposure Factor Units Distribution Min Max Estimate Deviation Reference a Exposure Point Concentration Coyster µg/g, ww Area-specific ------

Consumption Rate - General Consumer CR g/kg-day, ww Lognormal -- -- 0.002 -- USEPA 2002

Consumption Rate - Suquamish Tribe CR g/kg-day, ww Lognormal -- 2.01 0.164 0.004 Suquamish Tribe 2000

Consumption Rate - API CR g/kg-day, ww Lognormal 0.001 2.45 0.118 0.260 Secheena et al. 1999

Relative Bioavailability Adjustment RBA unitless Point Estimate NA NA 1 NA Professional judgment

Unit Conversion Factor CF mg/µg Point Estimate NA NA 10-3 NA -- Notes: NA = not applicable ww = wet weight a Exposure area-specific value, See Table 2-3 Table 3-2. Suquamish Tribe Oyster Consumption Rates for Adults and Children

Number of Standard 75th 90th Consumers Only Consumers Mean Deviation Median Percentile Percentile Maximum Adults (g/kg/day, wet weight)

Oysters 60 0.164 0.004 0.068 0.184 0.567 1.490 Oysters (gatherings) 40 0.061 0.002 0.031 0.088 0.152 0.517 Total Oysters: 60 0.225 0.006 0.099 0.272 0.719 2.007

Children

Oysters 10 0.060 0.035 0.015 0.074 0.336 0.320

Source: Suquamish Tribe (2000), Appendix C Table 3-3. Asian and Pacific Islander Oyster Consumption Rates for Adults

Number of Standard 75th 90th Consumers Only Consumers Min Max Mean Deviation Median Percentile Percentile

Oysters - as consumed (g/kg/day, as consumed) 146 0.001 2.036 0.098 0.250 0.035 0.081 0.196

Oysters - wet weight (g/kg/day, wet weight)a 146 0.001 2.453 0.118 0.301 0.042 0.098 0.236

Source: Secheena et al. (1999). a Assumes 17% moisture loss during cooking (USEPA 1997). Table 3-4. U.S. Population Oyster Consumption Rates for Adults Population Mean Units Oysters as "wet weight" Age 18 and older 0.174 g/person-day Age 18 and older 0.002 g/kg-daya All individuals 0.140 g/person-day All individuals 0.002 g/kg-dayb

Oysters "as prepared" Age 18 and older 0.143 g/person-day Age 18 and older 0.002 g/kg-daya All individuals 0.116 g/person-day All individuals 0.002 g/kg-dayb Source: USEPA (2002). a Body weight assumption of 70 kg obtained from EPA (1989). b Body weight assumption of 64.7 kg obtained from EPA (2002). Table 3-5. Intake of Cadmium in Oysters for all Exposure Areas. Intake (mg/kg-day) Statistics Suquamish API General US Alaska - All Mean 3.8E-04 2.6E-04 4.6E-06 Median 3.6E-04 9.4E-05 2.0E-06 90th percentile 5.2E-04 5.9E-04 1.1E-05 95th percentile 5.9E-04 1.0E-03 1.7E-05 Alaska - A1 Mean 3.7E-04 2.4E-04 4.5E-06 Median 3.5E-04 9.1E-05 2.0E-06 90th percentile 5.0E-04 5.5E-04 1.0E-05 95th percentile 5.5E-04 9.0E-04 1.7E-05 Alaska - A2 Mean 4.4E-04 2.9E-04 5.4E-06 Median 4.0E-04 1.0E-04 2.2E-06 90th percentile 7.2E-04 6.7E-04 1.2E-05 95th percentile 8.4E-04 1.1E-03 2.0E-05 Alaska - A3 Mean 3.8E-04 2.6E-04 4.6E-06 Median 3.7E-04 9.7E-05 2.0E-06 90th percentile 4.9E-04 5.8E-04 1.0E-05 95th percentile 5.3E-04 9.5E-04 1.7E-05 California - All Mean 1.7E-04 1.1E-04 2.0E-06 Median 1.2E-04 3.5E-05 7.4E-07 90th percentile 3.3E-04 2.5E-04 4.5E-06 95th percentile 3.8E-04 4.3E-04 7.6E-06 California - C1 Mean 1.9E-04 1.3E-04 2.2E-06 Median 1.5E-04 4.2E-05 8.5E-07 90th percentile 3.4E-04 2.9E-04 4.9E-06 95th percentile 4.3E-04 5.0E-04 8.6E-06 California - C2 Mean 1.2E-04 8.2E-05 1.6E-06 Median 1.2E-04 3.2E-05 6.8E-07 90th percentile 1.6E-04 1.8E-04 3.5E-06 95th percentile 1.7E-04 3.1E-04 5.4E-06 Oregon - All Mean 1.9E-04 1.3E-04 2.4E-06 Median 1.8E-04 4.7E-05 1.0E-06 90th percentile 2.8E-04 2.9E-04 5.4E-06 95th percentile 3.2E-04 4.8E-04 8.9E-06 Oregon - O1 Mean 1.9E-04 1.3E-04 2.3E-06 Median 1.8E-04 4.8E-05 9.9E-07 90th percentile 2.9E-04 2.9E-04 5.3E-06 95th percentile 3.2E-04 4.6E-04 8.5E-06 Oregon - O2 Mean 1.8E-04 1.2E-04 2.3E-06 Median 1.8E-04 4.7E-05 1.0E-06 90th percentile 2.4E-04 2.7E-04 5.0E-06 95th percentile 2.5E-04 4.5E-04 7.9E-06 Table 3-5. Intake of Cadmium in Oysters for all Exposure Areas. Intake (mg/kg-day) Statistics Suquamish API General US Washington - All Mean 2.1E-04 1.4E-04 2.5E-06 Median 1.8E-04 4.9E-05 1.0E-06 90th percentile 3.4E-04 3.1E-04 5.6E-06 95th percentile 3.9E-04 5.1E-04 9.1E-06 Washington - W1 Mean 1.9E-04 1.3E-04 2.2E-06 Median 1.9E-04 4.6E-05 9.9E-07 90th percentile 2.7E-04 2.9E-04 5.0E-06 95th percentile 3.0E-04 4.8E-04 8.1E-06 Washington - W2 Mean 2.2E-04 1.5E-04 2.6E-06 Median 2.0E-04 5.5E-05 1.1E-06 90th percentile 3.2E-04 3.4E-04 6.0E-06 95th percentile 3.7E-04 5.4E-04 9.7E-06 Washington - W3 Mean 2.0E-04 1.3E-04 2.4E-06 Median 1.9E-04 4.8E-05 1.0E-06 90th percentile 2.8E-04 3.0E-04 5.5E-06 95th percentile 3.1E-04 5.1E-04 8.9E-06 Washington - W4 Mean 2.7E-04 1.7E-04 3.2E-06 Median 2.6E-04 6.4E-05 1.4E-06 90th percentile 4.1E-04 4.0E-04 7.4E-06 95th percentile 4.6E-04 6.8E-04 1.2E-05 Washington - W5 Mean 2.0E-04 1.3E-04 2.4E-06 Median 1.9E-04 4.8E-05 1.0E-06 90th percentile 2.9E-04 2.9E-04 5.4E-06 95th percentile 3.3E-04 5.0E-04 8.6E-06 Washington - W6 Mean 1.3E-04 9.3E-05 1.6E-06 Median 1.2E-04 3.2E-05 6.7E-07 90th percentile 2.1E-04 2.0E-04 3.7E-06 95th percentile 2.5E-04 3.5E-04 5.9E-06 Washington - W7 Mean 1.8E-04 1.1E-04 2.1E-06 Median 1.7E-04 4.4E-05 9.2E-07 90th percentile 2.5E-04 2.6E-04 4.9E-06 95th percentile 2.7E-04 4.2E-04 8.0E-06 Washington - W1-6 Mean 2.1E-04 1.3E-04 2.5E-06 Median 1.9E-04 4.7E-05 1.1E-06 90th percentile 3.5E-04 3.0E-04 5.8E-06 95th percentile 4.2E-04 5.2E-04 9.3E-06 Table 5-1. Noncancer Hazards for Consumption of Cadmium in Oyster Tissue (Probabilistic Calculation). Hazard Quotient Statistics Suquamish API General US Alaska - All Mean 0.4 0.3 0.005 Median 0.4 0.09 0.002 90th percentile 0.5 0.6 0.01 95th percentile 0.5 1 0.02 Alaska - A1 Mean 0.4 0.2 0.005 Median 0.4 0.09 0.002 90th percentile 0.4 0.6 0.01 95th percentile 0.5 0.9 0.02 Alaska - A2 Mean 0.4 0.3 0.005 Median 0.4 0.1 0.002 90th percentile 0.7 0.7 0.01 95th percentile 0.8 1 0.02 Alaska - A3 Mean 0.4 0.3 0.005 Median 0.4 0.1 0.002 90th percentile 0.4 0.6 0.01 95th percentile 0.4 0.9 0.02 California - All Mean 0.2 0.1 0.002 Median 0.1 0.03 0.0007 90th percentile 0.3 0.2 0.005 95th percentile 0.4 0.4 0.008 California - C1 Mean 0.2 0.1 0.002 Median 0.2 0.04 0.0009 90th percentile 0.3 0.3 0.005 95th percentile 0.4 0.5 0.009 California - C2 Mean 0.1 0.08 0.002 Median 0.1 0.03 0.0007 90th percentile 0.2 0.2 0.004 95th percentile 0.2 0.3 0.005 Oregon - All Mean 0.2 0.1 0.002 Median 0.2 0.05 0.001 90th percentile 0.3 0.3 0.005 95th percentile 0.3 0.5 0.009 Oregon - O1 Mean 0.2 0.1 0.002 Median 0.2 0.05 0.001 90th percentile 0.3 0.3 0.005 95th percentile 0.3 0.5 0.008 Oregon - O2 Mean 0.2 0.1 0.002 Median 0.2 0.05 0.001 90th percentile 0.2 0.3 0.005 95th percentile 0.2 0.4 0.008 Table 5-1. Noncancer Hazards for Consumption of Cadmium in Oyster Tissue (Probabilistic Calculation). Hazard Quotient Statistics Suquamish API General US Washington - All Mean 0.2 0.1 0.003 Median 0.2 0.05 0.001 90th percentile 0.3 0.3 0.006 95th percentile 0.4 0.5 0.009 Washington - W1 Mean 0.2 0.1 0.002 Median 0.2 0.05 0.001 90th percentile 0.2 0.3 0.005 95th percentile 0.2 0.5 0.008 Washington - W2 Mean 0.2 0.2 0.003 Median 0.2 0.06 0.001 90th percentile 0.3 0.3 0.006 95th percentile 0.3 0.5 0.01 Washington - W3 Mean 0.2 0.1 0.002 Median 0.2 0.05 0.001 90th percentile 0.3 0.3 0.006 95th percentile 0.3 0.5 0.009 Washington - W4 Mean 0.3 0.2 0.003 Median 0.3 0.06 0.001 90th percentile 0.4 0.4 0.007 95th percentile 0.4 0.7 0.01 Washington - W5 Mean 0.2 0.1 0.002 Median 0.2 0.05 0.001 90th percentile 0.3 0.3 0.005 95th percentile 0.3 0.5 0.009 Washington - W6 Mean 0.1 0.09 0.002 Median 0.1 0.03 0.0007 90th percentile 0.2 0.2 0.004 95th percentile 0.2 0.3 0.006 Washington - W7 Mean 0.2 0.1 0.002 Median 0.2 0.04 0.0009 90th percentile 0.2 0.3 0.005 95th percentile 0.2 0.4 0.008 Washington - W1-6 Mean 0.2 0.1 0.003 Median 0.2 0.05 0.001 90th percentile 0.3 0.3 0.006 95th percentile 0.4 0.5 0.009 Appendix C – Economic Effect to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Economic Effect to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Prepared for the Pacific Shellfish Institute

August 2008

Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Prepared for the Pacific Shellfish Institute

August 2008

Prepared by

th 880 H Street, Suite 210, 1108 11 Street, Suite 305 Anchorage, AK 99501 Bellingham, WA 98225 T: 907.274.5600 T: 360.715.1808 F: 907.274.5601 F: 360.715.3588

W: www.northerneconomics.com E: [email protected]

Preparers

Team Member Project Role Company Katharine Wellman Project Manager Northern Economics, Inc. Kent Kovacs Economist Northern Economics, Inc. William Schenken Staff Consultant Northern Economics, Inc.

Please cite as: Northern Economics, Inc. Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium. Prepared for Pacific Shellfish Institute. August 2008.

Contents

Section Page

Acknowledgements ...... iii Abbreviations ...... v Executive Summary...... ES-1 1 Introduction and Purpose ...... 1 2 Existing Conditions: Oyster Production and Industry Revenues in Puget Sound and the Hood Canal ...... 3 2.1 Shellfish Production in the Hood Canal...... 3 2.1.1 Hood Canal Harvesters...... 6 2.1.2 Hood Canal Shellstock Shippers ...... 6 2.1.3 Hood Canal Shucker-Packers...... 8 2.1.4 Market Conditions...... 8 2.2 Oyster Exports from Seattle...... 10 2.2.1 Exports to Asian Countries ...... 12 3 Survey Results ...... 17 3.1 Background ...... 17 3.2 Domestic Markets...... 18 3.3 International Markets...... 20 4 Current and Potential Economic Effect of Cadmium Threshold for Hood Canal Oyster Companies...... 23 4.1 Cadmium in the Hood Canal...... 23 4.2 International Limits on Cadmium...... 24 4.3 Current Economic Losses from Cadmium Limits...... 25 4.4 Potential Economic Losses from the Cadmium Limit ...... 26 4.4.1 No Mitigation of the Cadmium Problem ...... 26 4.4.2 Mitigation of the Cadmium Problem...... 28 5 Conclusions...... 29 6 References...... 31

Table Page Table ES-1. International Oyster Sales of Survey Respondents ...... ES-5 Table ES-2. Domestic and International Oyster Wholesaler Prices – Company F...... ES-6 Table ES-3. Hong Kong Regulation Influence on Survey Respondents...... ES-6 Table 1. Types of Shellfish Companies in Washington ...... 5 Table 2. Summary Statistics of Harvest Sites for Hood Canal ...... 5 Table 3. Sites of Harvesters of Shellfish in the Hood Canal ...... 6

i Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Table 4. Harvest Sites of Shellstock Shippers in the Hood Canal ...... 7 Table 5. Harvest Sites of the Shucker-Packers in the Hood Canal ...... 8 Table 6. Summary Statistics of Harvest Sites for Survey Respondents ...... 17 Table 7. Harvest Sites of Survey Respondents in the Hood Canal...... 18 Table 8. Domestic Oyster Sales from the Hood Canal for Survey Respondents ...... 19 Table 9. Average Oyster Prices in Washington...... 20 Table 10. International Oyster Sales ...... 21 Table 11. Domestic and International Oyster Wholesale Prices ...... 21 Table 12. Cadmium Intake for Consumption of Half Dozen Large Oysters ...... 24 Table 13. Cadmium Exposure Guidelines...... 24 Table 14. Hong Kong Regulation Influence on Survey Respondents...... 26 Table 15. Economic Losses Due to Hood Canal Companies Selling Only to the Domestic Market ....27

Figure Page Figure ES-1. Mean Cadmium Concentrations in Pacific Oysters in the Hood Canal and Puget Sound 2004-06...... ES-1 Figure ES-2. Farmed Pacific Oysters in the Puget Sound and Hood Canal...... ES-3 Figure ES-3. Exports of Oysters to Asia from Seattle ...... ES-4 Figure 1. Reported Farmed Pacific Oysters in the Puget Sound and Hood Canal (Meat Weight)...... 3 Figure 2. Farmed Pacific Oysters from the Hood Canal ...... 4 Figure 3. Prices of Pacific Oysters from the Puget Sound and the Hood Canal ...... 9 Figure 4. Prices of Pacific Oysters from the Hood Canal ...... 10 Figure 5. Exports of Oysters from Seattle ...... 11 Figure 6. Prices of Oyster Exports from Seattle...... 12 Figure 7. Exports of Oysters to Asia from Seattle ...... 13 Figure 8. Prices of Oyster Exports to Seattle from Asia ...... 14 Figure 9. Export Certificates to Asia by Final Destination...... 15 Figure 10. Mean Cadmium Concentrations in Pacific Oysters in the Hood Canal, 2004-2006 ...... 23

ii

Acknowledgements

The authors of this report wish to acknowledge the assistance of the government officials, market experts, scientists, growers, and wild harvesters we interviewed for this report. Their cooperation made this report possible. We would also like to thank the staff of the Pacific Shellfish Institute and the Washington Department of Fish and Wildlife for their invaluable assistance. This analysis was funded by a grant issued to Oregon State University from the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture, under Agreement No. 2004-51110-02156.

iii Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

iv

Abbreviations

BC British Columbia CFIA Canadian Food Inspection Agency EU European Union HK Hong Kong NASS National Agricultural Statistics Service NEI Northern Economics, Inc. NOAA National Oceanic and Atmospheric Administration PSI Pacific Shellfish Institute PTWI Provisional Tolerable Weekly Intake WDFW Washington Department of Fish and Wildlife

v Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

vi

Executive Summary

Cadmium is a naturally occurring metal in Pacific Northwest shellfish. However, it is considered a food additive by the U.S. Food and Drug Administration (FDA) and other international health organizations such as the Food and Agriculture Organization of the United Nations (FAO), and therefore, maximum levels of cadmium concentrations in foods are set to protect human health. A recent scientific study by the Pacific Shellfish Institute, Integral Consulting Inc., Hong Kong University of Science and Technology, and the Oregon State University Seafood Laboratory found that the cadmium concentrations in the Puget Sound are significantly elevated in portions of Hood Canal. Cadmium concentrations in Pacific Northwest shellfish occasionally exceed the individual standards of importing countries. Beginning in 1999 customs at the Special Administrative Region of Hong Kong have, on occasion, rejected shellfish shipments from the Pacific Northwest for violating Hong Kong’s cadmium maximum limit standard of 2.0 parts per million (ppm). For example, as recently as 2005, customs inspectors in Hong Kong rejected a shipment of Washington oysters from a harvest site in the Hood Canal. This report focuses on the economic effect of lost shellfish markets associated with cadmium concentrations in Hood Canal oysters that exceed national standards in importing countries.

Figure ES-1. Mean Cadmium Concentrations in Pacific Oysters in the Hood Canal and Puget Sound 2004-06.

Source: Pacific Shellfish Institute, 2006

ES-1 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Economic Effect This report estimates that if no action is taken to mitigate the elevated levels of cadmium in Hood Canal oysters, then Hood Canal shellfish companies will experience the following losses: • A total loss of $67,500 annually from lost export revenues if international ports reject one oyster shipment per company per year for exceeding cadmium concentration standards. • An ongoing total annual loss of $9,000 to $75,000, depending on market conditions domestically and internationally, from the redirection of oysters to the domestic market where the price for large oysters is lower. The loss associated with rejected exports could largely be avoided by making Hood Canal shellfish companies aware of the potential losses associated with elevated levels of cadmium in their oysters and developing effective mitigation strategies. If the international market is of great interest to large operation Hood Canal companies, the report suggests the following actions to mitigate ongoing losses: • Shift production to harvest sites outside the Hood Canal. • Ship to international ports without cadmium limits (possibly Taipei and Southeast Asia in the short-run). • Test for cadmium to determine the shipments that need to stay domestic. • Develop a method to depurate or treat the oysters to remove Cd; a long-run, and likely costly, strategy. • Strengthen domestic marketing to improve the prices received for smaller Hood Canal oysters, which can be sold directly into domestic markets.

Market Trends The data indicate that production of farmed oysters in Puget Sound and Hood Canal fluctuated within a stable range between 1995 and 2005 (see Figure ES-2). We do note that these data tend to be somewhat underreported, but there is no evidence to suggest that the underreporting differs across the areas. Hood Canal produces fifteen percent of the Puget Sound oysters and roughly five percent of Washington oysters.

ES-2 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Figure ES-2. Farmed Pacific Oysters in the Puget Sound and Hood Canal

4.5

4

3.5

3

2.5

2

Round Pounds (Millions) Pounds Round 1.5

1

0.5

0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Puget Sound Hood Canal

Source: WA Department of Fish and Wildlife

While regional production of oysters remained stable between 1995 and 2005, the export market for oysters from Washington is growing, with the principal export markets located in Asia. Exports to Asian countries are aided by their proximity to the West Coast of the United States and booming regional economies such as China’s. The Seattle export of oysters to Asia from 1995 to 2007 is shown in Figure ES-3. The data show a dramatic rise in exports to Hong Kong and Taipei, Taiwan since 2000. Taiwan has not rejected any oysters for failing a cadmium restriction threshold, making the port the best short-run substitute for Hood Canal companies shipping internationally.

ES-3 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Figure ES-3. Exports of Oysters to Asia from Seattle

2.5

2

1.5

1 Meat Weight in lbs(Millions)Meat Weight in

0.5

0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

Hong-Kong Taipei Japan Other Asia

Source: NOAA Fisheries, Office of Science and Technology

In spite of the strong export market for oysters, most Hood Canal companies currently do not produce for the export market—a factor which limits economic losses. Table ES-1 shows that of the ten Hood Canal companies surveyed, only two have international sales. Furthermore, only about two percent of sales of those two companies are international. The current direct international presence of Hood Canal companies is minimal. However, four survey respondents expressed interest in international sales in the future at a variety of Asia locations and a large number of companies sell their product to domestic or Canadian wholesalers who could be shipping Hood Canal oysters to international markets. If these oysters are mixed in with oysters from other locations it would reduce the chance of Hood Canal oysters being selected for testing, but increase the risk of non-Hood Canal oysters being rejected if a Hood Canal oyster was selected for testing. This interest in selling to international markets increases the likelihood that eventually more producers will have shipments rejected.

ES-4 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Table ES-1. International Oyster Sales of Survey Respondents1

International International New Sales Country Percent Locations (Number of Destinations Sales Plans to Sell Planned for Company Oysters) of Sales International Internationally Sales A 0 — 0.0 Yes China B 0 — 0.0 No -- C 0 — 0.0 Yes Canada D 0 —- 0.0 No -- E 0 — 0.0 Yes Asia

2 Hong Kong, F 16,407 Canada 2.2 Yes Singapore G 0 — 0.0 No -- H 0 — 0.0 No -- Hong Kong, Japan, Canada, Singapore, I 85,886 1.4 Yes Taiwan Australia, China, Malaysia, Indonesia J 0 — 0.0 Yes Malaysia Source: Pacific Shellfish Institute, 2007

Domestic and international markets express different preferences for sizes of oysters as shown in Table ES-2. The export market favors larger oysters, and pays comparatively more for them than the domestic market. A growing export market is expected to increase the divide between domestic and international prices for large oysters if production of large oysters does not increase to meet demand. Prolonged growth in the export market and a greater divide in domestic and international prices could make the development of a system to minimize cadmium residues in oysters a worthwhile investment if the development and operation of the system costs less than the aggregate revenue increases associated with the ability to export more large oysters. The cost per oyster for a cadmium reduction system would need to be less than the difference between the domestic and international price of oysters, taking into account the longer growing time for large oysters.

1 In order to maintain confidentiality, the report identifies companies in tables and text by letter codes. These codes are randomly assigned identifiers, and the same letter code is applied to the same company every time it appears in the report. 2 A high percentage of the product that is exported to Canada is then re-exported to Hong Kong and other Asian cities.

ES-5 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Table ES-2. Domestic and International Oyster Wholesaler Prices – Company F

Size Washington California Canada Small $3.28/dozen $3.20/dozen $2.50/dozen Medium $4.17/dozen $3.70/dozen $4.50/dozen Large $4.42/dozen $4.00/dozen $5.15/dozen X-Large -- -- $5.45/dozen Jumbos -- -- $6.00/dozen Source: Pacific Shellfish Institute, 2007

Hong Kong Cadmium Threshold – Existing Hood Canal Losses Some Hood Canal companies are experiencing losses because the natural cadmium concentration found in some Hood canal oysters exceeds the Hong Kong cadmium threshold. These existing losses to survey respondents are shown in Table ES-3. One company lost a shipment of oysters, and Company C had difficulty entering the export market through at least one Canadian wholesaler because of the threshold. Company I maintains ongoing tests for cadmium to insure international marketability of their Hood Canal oysters. Unfortunately, half of the respondents indicated no awareness of the Hong Kong cadmium limit, which leaves them vulnerable to losing rejected shipments that could otherwise have been diverted into other markets. We conclude: • A greater awareness of the cadmium threshold will prevent the one-time loss of shipments from the rejection of oysters from international ports. • A Hood Canal company planning to enter the export market should test for cadmium. Findings of high levels of cadmium in the oysters suggests that in the short run, the company should focus on business relationships with wholesalers that ship to Taipei or Southeast Asia.

Table ES-3. Hong Kong Regulation Influence on Survey Respondents

Aware of Hong Kong Cost to Date per Year of Company Regulation New Business Practices New Business Practices A Yes None $0 B No N/A N/A Will not accept solicitations from C Yes ~ $25 Hong Kong D No N/A N/A E No N/A N/A Stopped shipments to Hong F Yes ~ $2,594 Kong. G No N/A N/A H No N/A N/A I Yes Ongoing tests for cadmium ~ $440-$880 J Yes None $0 Source: Pacific Shellfish Institute, 2007

ES-6

1 Introduction and Purpose

Cadmium is a naturally occurring metal in Pacific Northwest shellfish. However, it is considered a food additive by the U.S. Food and Drug Administration (FDA) and other international health organizations such as the Food and Agriculture Organization of the United Nations (FAO), and therefore, maximum levels of cadmium concentrations in foods are set to protect human health. A recent scientific study by the Pacific Shellfish Institute, Integral Consulting Inc., Hong Kong University of Science and Technology, and the Oregon State University Seafood Laboratory found that the cadmium concentrations in the Puget Sound are significantly elevated in portions of the Hood Canal. Cadmium concentrations in Pacific Northwest shellfish occasionally exceed the individual standards of importing countries. Beginning in 1999 customs at the Hong Kong Special Administrative Region (HK) have, on occasion, rejected shellfish shipments from the Pacific Northwest for violating Hong Kong’s cadmium contamination standard of 2.0 parts per million (ppm).3 For example, as recently as 2005 customs inspectors in Hong Kong rejected a shipment of Washington oysters from a harvest site in the Hood Canal. This report focuses on the economic effect of lost shellfish markets associated with cadmium concentrations in Hood Canal oysters that exceed national standards in importing countries. Northern Economics has been tasked to 1) broadly determine the markets and economic revenues of Hood Canal oyster producers, and 2) provide baseline information that will contribute to answering the question of how Hong Kong (and future potential international markets) restrictions on cadmium in oysters could impact local oyster growers. This report is structured as follows: • Section 2 describes the markets and economic revenues of Hood Canal oyster producers. • Section 3 describes state and federal secondary data and the data from the study’s survey of ten Hood Canal oyster producers. • Section 4 describes the economic effect of international cadmium regulations on Hood Canal oyster producers. • Section 5 contains the study’s conclusions.

3 The Codex Alimetarius does not contain a standard for cadmium so individual countries are free to establish their own guidelines (Cheney 2008; Codex Alimetarius Commission 2006).

1 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

2

2 Existing Conditions: Oyster Production and Industry Revenues in Puget Sound and the Hood Canal

This section analyzes the current existing conditions and the importance of the Hood Canal to the oyster export market in Washington.

2.1 Shellfish Production in the Hood Canal Hood Canal accounts for approximately 15 percent of Washington State’s oyster production. The area’s portion of total production has been relatively stable across time, and directional changes in Hood Canal production have generally tracked directional changes in Puget Sound production.4

Figure 1. Reported Farmed Pacific Oysters in the Puget Sound and Hood Canal (Meat Weight)

4.5

4

3.5

3

2.5

2

Round Pounds (Millions) 1.5

1

0.5

0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Puget Sound Hood Canal Source: WA Department of Fish and Wildlife, 2008.

The data show that while total Hood Canal production has remained relatively constant, the distribution of that production between the three different areas within the Hood Canal (North,

4 The report notes that reported production is lower than actual production and that production amounts are consistent underreported.

3 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Central and South) has changed substantially (see Figure 2). However, discussions with industry sources indicate that this change in distribution may be an artifact of the data and could indicate a change in where producers landed their product as opposed to an actual change in production location.

Figure 2. Farmed Pacific Oysters from the Hood Canal

11

10

9

8

7

6

5

4

Round Pounds(Millions) 3

2

1

0 19951996199719981999200020012002200320042005

Washington Puget Sound Hood Canal

Source: WA Department of Fish and Wildlife

There are two sources of data used to gauge production in the Hood Canal: the number of shellfish companies from the Washington State Department of Health Office of Shellfish and Water Protection, and the pounds of oysters landed from the Washington Department of Fish and Wildlife (WDFW). The Office of Shellfish maintains a commercial shellfish licensing and certification program that is responsible for issuing licenses to Washington commercial shellfish operations and certifying the sites from which they harvest. The 2007 list of licensed shellfish companies indicates each site where a company is allowed to harvest shellfish. This list allows for the identification of the companies that operate in the Hood Canal, the type of shellfish company (harvester, shellstock shipper, shucker- packer), and how dependent the companies are on the Hood Canal (the percent of its harvest sites located in the Hood Canal). The type of a shellfish company is useful in identifying the company’s position in the supply chain for oysters, and the position in the supply chain helps to assess whether the product from that company is shipped internationally. There are three major types of commercial shellfish operations: • Harvesters, who harvest shellstock (live, unshucked product) and sell only to other licensed Washington shellfish dealers;

4 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

• Shellstock Shippers, who grow and harvest shellstock, and buy and sell in or outside Washington; and • Shucker-Packers, whose activities may include those of harvesters and shellstock shippers, plus shucking product for packing in jars or similar containers. Harvesters who are not involved in marketing their product and who are not part of a vertically integrated company are less likely to know whether their product is shipped internationally. In addition, the product from harvesters is a wild product that is more variable in size and less consistent in quality than the product grown by shellstock shippers. Smaller product is less likely to survive the trip overseas or be in demand once it gets there. The shucker-packers are often larger shellfish companies with the ability to export to international markets. Also, since demand for the shucked product has weakened in US supermarkets, companies that produce shucked product must now market overseas in order to generate equivalent revenue from similar sales (Heyamoto 2005). Table 1 indicates the percentage of each type of company located in Washington, Puget Sound, Hood Canal, and Hood Canal Site 8.5 The three different types of companies have varying percentages of investment in the four regions. The Hood Canal area has a slightly higher percentage of shellstock shippers and shucker-packers and a lower percentage of harvesters as compared to Puget Sound or the state as a whole.

Table 1. Types of Shellfish Companies in Washington

Type of Company Washington Puget Sound Hood Canal Hood Canal Site 8 Number of Companies 332 288 63 23 Harvesters 31% 33% 22% 22% Shellstock Shippers 62% 62% 67% 70% Shucker-Packers 7% 5% 11% 9% Source: WA Department of Health, Office of Shellfish and Water Protection, 2007

Table 2 identifies the average number of harvest sites per company, and the percentage of those sites that are located in the Hood Canal. Shellfish companies of all types have operations in the Hood Canal, but the shellstock shippers who operate in the canal are the most dependent on the area, with an average of 63 percent of their sites being located within Hood Canal. Diversified shucker-packers tend to be the least dependent on the area, but still have 40 percent of their sites within the canal.

Table 2. Summary Statistics of Harvest Sites for Hood Canal

Average # of Average # of Percent Harvest Companies in Hood Canal Total Harvest Sites in Hood Type of Company Hood Canal Harvest Sites Sites Canal Harvesters 14 3.36 5.71 59% Shellstock Shippers 42 2.52 4.02 63% Shucker-Packers 7 3.29 8.29 40% Source: WA Department of Health, Office of Shellfish and Water Protection, 2007

5 The site 8 of the Hood Canal is the site of origin for a shipment of oysters that is known to have been rejected from Hong Kong for excessive cadmium.

5 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

2.1.1 Hood Canal Harvesters Table 3 compares each harvester’s total number of sites with the number of sites in the Hood Canal as reported by the Office of Shellfish and Water Protection. In addition, the table further identifies harvester interests by specifying which harvesters operate in Site 8 of the Hood Canal. The data show that harvesters operating in Hood Canal tend to be very dependent on the canal as measured by harvest sites. All but three of the fourteen shellfish harvesters operating in the Hood Canal have 50 percent or more of their harvest sites located in Hood Canal. There are six operators that are entirely dependent on Hood Canal for their production; only one company has less than 40 percent of its sites within the Hood Canal. While not shown in the table, about half of the harvesters operating in the Hood Canal have sites located outside the canal.

Table 3. Sites of Harvesters of Shellfish in the Hood Canal6

Number of Hood Total Number of Percent Harvest Hood Canal Company Canal Harvest Sites Harvest Sites Sites in Hood Canal Site 8 H 13 14 93% Yes AT 10 20 50% Yes AU 9 19 47% Yes AV 2 9 22% No AW 2 5 40% No AX 2 2 100% No AY 2 2 50% No AZ 1 2 50% No BA 1 2 50% No BB 1 1 100% No BC 1 1 100% No BD 1 1 100% Yes BE 1 1 100% Yes BF 1 1 100% No

2.1.2 Hood Canal Shellstock Shippers Shellstock shippers tend to operate at fewer sites overall and fewer sites in Hood Canal than shellfish harvesters. Only 2 of the 42 shellstock shippers operating at least one site in Hood Canal operate ten or more sites overall. For thirteen of the operators, their single site in Hood Canal is the only site they operate. Shellstock shippers operating in Hood Canal also tend to have their operations concentrated in Hood Canal; only 7 of the 42 Hood Canal shellstock shippers had more than half of their harvest sites outside of Hood Canal. Thus, many of these companies are highly vulnerable to changes in the health of Hood Canal and the canal’s environmental quality.

6 In order to maintain confidentiality, the report identifies companies in tables and text by letter codes. These codes are randomly assigned identifiers, and the same letter code is applied to the same company every time it appears in the report.

6 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Table 4. Harvest Sites of Shellstock Shippers in the Hood Canal

Number of Hood Total Number of Percent Harvest Sites Hood Canal Company Canal Harvest Sties Harvest Sites in Hood Canal Site 8 C 7 10 70% Yes K 7 9 78% Yes L 7 7 100% Yes M 5 7 71% Yes N 5 5 100% Yes F 5 5 100% Yes G 5 5 100% No O 4 7 57% Yes P 4 6 67% No Q 4 5 80% No R 4 4 100% Yes S 3 6 50% No T 3 5 60% Yes U 3 5 60% No J 3 4 75% Yes V 3 4 75% Yes W 2 12 17% No X 2 8 25% No Y 2 5 40% Yes Z 2 4 50% Yes AA 2 4 50% No D 2 3 67% Yes AB 2 3 67% No AC 2 2 100% No A 1 7 14% No AD 1 4 25% Yes AE 1 4 25% No AF 1 4 25% No AG 1 2 50% No AH 1 1 100% Yes AI 1 1 100% No AJ 1 1 100% No AK 1 1 100% No AL 1 1 100% No AM 1 1 100% No AN 1 1 100% No AO 1 1 100% No AP 1 1 100% No B 1 1 100% No AQ 1 1 100% No AR 1 1 100% No AS 1 1 100% No

7 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

2.1.3 Hood Canal Shucker-Packers Compared to harvesters and shellstock shippers, there are relatively few shucker-packers operating in Hood Canal and these can be divided into two groups: larger companies operating approximately twenty sites each and less dependent on Hood Canal, and smaller companies operating fewer than ten sites and highly dependent on Hood Canal (see Table 5). This distribution means than the smaller companies are likely to be much more vulnerable to changes in cadmium import regulations in foreign countries as well as more vulnerable to environmental changes in Hood Canal that are unrelated to cadmium. While larger companies may also feel some effect from changing regulations or environmental conditions, their diversified nature will give them a greater chance of adapting to these changes.

Table 5. Harvest Sites of the Shucker-Packers in the Hood Canal

Number of Hood Total Number of Percent Harvest Hood Canal Company Canal Harvest Sites Harvest Sites Sites in Hood Canal Site 8 BG 6 21 29% Yes BH 6 6 100% No E 5 5 100% No I 2 20 10% Yes BI 2 4 50% No BJ 1 1 100% No BK 1 1 100% No Source: WA Department of Health, Office of Shellfish and Water Protection, 2007

2.1.4 Market Conditions Between 1995 and 2000, the price for Hood Canal oysters was slightly lower than the average price for Puget Sound oysters, but in 2000 and 2001 Hood Canal oysters began to command a substantial price premium over Puget Sound oysters (see Figure 3). One reason for this change could be a change in target market. For example, targeting the half-shell market vs. the shucked market would result in higher prices per pound.

8 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Figure 3. Prices of Pacific Oysters from the Puget Sound and the Hood Canal7

$4.00

$3.50

$3.00

$2.50

$2.00 Price ($/lb) $1.50

$1.00

$0.50

$0.00 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Washington Puget Sound Hood Canal

Source: WA Department of Fish and Wildlife, 2008

Oysters produced in the northern portion of Hood Canal have traded at a price discount to oysters produced in the central or southern regions (see Figure 4). However, Northern oysters have experienced lower price volatility at the same time.

7 Single oysters are typically sold by the piece and not by the pound—therefore, a unit price would be a more dependable figure. Unfortunately, the WDFW data set doesn't account for the size of oysters.

9 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Figure 4. Prices of Pacific Oysters from the Hood Canal

$6

$5

$4

$3 Price ($/lb)

$2

$1

$0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

North Central South

Source: WA Department of Fish and Wildlife

2.2 Oyster Exports from Seattle Oyster exports from Washington State experienced a period of rapid growth between 1995 and 2005 driven by an expanding Asian market and by rapid economic growth in China (see Figure 5). Changes in air transportation links during this same period have allowed Washington producers and exporters to access ports in Mainland China as opposed to relying on Hong Kong. Total exports to Asia have fallen since 2005. NOAA National Marine Fisheries Service reports on the quantity and prices of oysters leaving the port of Seattle (NOAA 2008). These data represent all species of oysters. However, the bulk of the production is Pacific oysters. Most of the oysters exported from Seattle are from Washington State, but the figures from Seattle include production from other areas as well. The data report the destination of the oyster exports as the next destination (often a major port) rather than final destination, which would be a better indication of where consumption occurs. In addition to federal reporting systems, the Washington State Department of Health Office of Shellfish tracks the final destination to allow tracking of where consumption likely occurs (Westerberg 2008).

10 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Figure 5. Exports of Oysters from Seattle 5

4

3

2 Meat Weight in lbs (Millions)

1

0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

Asia Canada Other Source: NOAA, Fisheries, Office of Science and Technology

The prices of the farmed oysters exported from Seattle between 1995 and 2007 are depicted in Figure 6 (NOAA 2008). In general: • The prices listed for oysters exported to the rest of the world (ROW) are more variable than the prices of oysters exported to Asian countries or Canada. The prices could be more variable if the ROW is acting as an overflow valve for the North American and Asian markets or if the ROW is simply receiving small volumes of specialty exports. • Oysters exported to Canada have received a price premium compared to those heading directly to Asian countries. A possible explanation for the higher price for oysters heading to Canada is that exporters from Canada are better connected to the premium importers in Asia than the exporters from the United States (TC Trading 2008; Nexus Seafood 2008). Exporters located in Vancouver have been dominant in the export markets for other shellfish products to Asian markets. For example, the majority of Washington’s production is exported live through Canada to Asian markets.

11 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Figure 6. Prices of Oyster Exports from Seattle

6

5

4

3 Price ($/lb)

2

1

0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

Asia Canada Other

Source: NOAA, Fisheries, Office of Science and Technology

2.2.1 Exports to Asian Countries As noted above, countries located in Asia are the most important market for Washington oyster exports. Export data show that the growth experienced between 1995 and 2005 was driven by access to Hong Kong, Taipei, and “other ports” and that the reduction in exports between 2005 and 2007 is being driven by lower exports to Taipei and Hong Kong (see Figure 7). Some of the resulting slack is being picked up by exports to other markets in Asia including alternative access points to Mainland China. Japan is a small but consistently important importer of Washington oysters.

12 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Figure 7. Exports of Oysters to Asia from Seattle

2.5

2

1.5

1 Meat Weight in lbs (Millions)

0.5

0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

Hong-Kong Taipei Japan Other Asia

Source: NOAA Fisheries, Office of Science and Technology

Over the past four years prices to the major Asian markets have “harmonized” and over the past three years they’ve exhibited an ordinal pattern with Hong Kong bound oysters generating the highest prices followed by Japan, the ROW, and Taipei. One exception to these patterns is that prices for ROW oysters fell over the past several years while prices for oysters heading to other markets increased (see Figure 8).

13 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Figure 8. Prices of Oyster Exports to Seattle from Asia

8

6

4 Price ($/lb)

2

0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

Hong-Kong Taipei Japan Other Asia

Source: NOAA Fisheries, Office of Science and Technology

Figure 9 illustrates the final destinations of Seattle exports to five different importing areas from export certificates (Westerberg 2008). Please note that prior discussions referred to initial ports of entry. An important transition apparent in the data to note is the emergence of these destinations in mainland China, and recently Other Asia as markets for oysters, while Hong Kong appears to have entered a period of decline. The study also notes the relative absence of Taiwan/Taipei from this figure, which indicates that exports from Washington going through Taipei are being transshipped to another major port rather than being consumed domestically in Taiwan. This fact is important because it means that changes in cadmium regulations in Hong Kong or other Chinese ports could affect shipments to more than just Hong Kong; these changes could also affect shipments to Taipei if Taipei is simply a transshipment point for oysters heading to ports with stricter cadmium regulations.

14 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Figure 9. Export Certificates to Asia by Final Destination

8

6

4

Export Certificates (Thousands) 2

0 2002 2003 2004 2005 2006 2007

Hong Kong Taiwan Other China Japan Other Asia

Source: WA Department of Health, Office of Shellfish and Water Protection

15 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

16

3 Survey Results

The Pacific Shellfish Institute conducted a phone survey from December 2007 to January 2008 of shellfish companies that operate in Hood Canal. Ten out of thirty-one companies responded to the survey. The purpose of the survey was to learn if the shellfish companies in Hood Canal sell product internationally and what affect the limit on cadmium in oysters in Hong Kong has had on their business practices. The survey obtained information from the shellfish companies on: • Domestic and international sales • Price and quantity of sales by location of sale • Knowledge and experience with Hong Kong regulation on cadmium limits in oysters • Company characteristics The survey data also helped determine the accuracy of secondary data from state and federal sources on oyster quantities and prevailing prices. Section 3.1 presents background on the survey respondents; Section 3.2 describes the sales and domestic markets of the survey respondents; and Section 3.3 describes survey respondents’ international marketing plans.

3.1 Background While not a scientific sample, the survey provides insight into the practices and characteristics of shellfish companies with operations in the Hood Canal. Table 6 outlines responses to the PSI survey, what type of company the respondents represent, their average number of harvest sites in the Hood Canal, their average number of total harvest sites, and the average percentage of harvest sites located in the Hood Canal.

Table 6. Summary Statistics of Harvest Sites for Survey Respondents

Average Number Average Number Percent Harvest Companies from of Hood Canal of Total Harvest Sites in Hood Type of Company the Survey Harvest Sites Sites Canal Harvesters 1 13 14 93% Shellstock Shippers 7 3.43 5 67% Shucker-Packers 2 3.50 12.50 28% Source: WA Department of Health, Office of Shellfish and Water Protection, 2007

The survey respondents’ dependence on Hood Canal varies. Table 7 summarizes the survey respondent interests in terms of harvest sites in the Hood Canal, and what percentage of their harvest sites was located in the Hood Canal. The survey respondents are generally larger operations, on average, than other companies in the Hood Canal shown in Section 2.1

17 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Table 7. Harvest Sites of Survey Respondents in the Hood Canal

Number of Total Number Percent Hood Canal of Harvest Harvest Sites Hood Canal Company Type of Company Harvest Sites Sites in Hood Canal Site 8 A Shellstock Shipper 1 7 14% No B Shellstock Shipper 1 1 100% No C Shellstock Shipper 7 10 70% Yes D Shellstock Shipper 2 3 67% Yes E Shucker-Packer 5 5 100% No F Shellstock Shippers 5 5 100% Yes G Shellstock Shipper 5 5 100% No H Harvester 13 14 93% Yes I Shucker-Packer 2 20 10% Yes J Shellstock Shipper 3 4 75% Yes Source: WA Department of Health, Office of Shellfish and Water Protection, 2007

3.2 Domestic Markets Hood Canal oysters are sold both whole (in the round) for “on the half shell” service and in shucked form to several different domestic locations in addition to international locations. Table 8 summarizes survey respondents’ annual sales by number of oysters from the Hood Canal. The table also shows the percentage that was sold whole versus the percentage sold shucked, and finally, the location of domestic sales.8 The data suggest that two shellstock shippers sell oysters to shucker-packers for shucking, but most shellstock shippers sell directly to the whole fresh market. More than half of the companies sell oysters outside Washington, but only two sell outside the West Coast. These companies tend to be larger than companies that focus on the West Coast markets; the lower a company’s oyster sales the more likely that company’s domestic sales will be to the Washington market. As noted previously, smaller companies’ operations tend to be geographically concentrated, making them more vulnerable to environmental changes at specific harvest locations. These companies also appear to have geographically concentrated domestic markets. While this concentration limits their options in terms of selling unexportable oysters into domestic markets, it may also mean they have strong ties with one or two domestic distributors that could help them weather a reduction in exports. On balance, small companies that rely on export relationships are likely to be more vulnerable to the effects of foreign regulatory changes or changes in local environmental conditions than larger more diversified companies.

8 The number of oysters sold by the ten survey respondents indicates that WDFW data on the pounds are too low since the pounds sold just by the ten survey respondents is more than the total pounds sold for all of the Hood Canal according WDFW.

18 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Table 8. Domestic Oyster Sales from the Hood Canal for Survey Respondents

Mean Annual Sales of Hood Canal Percent Percent Domestic Destinations 9 Company Oysters (Number) Shucked Whole Fresh of Sales A 58,686 62% 38% Washington, California B 11,058 0% 100% Washington C 1,860,000 0% 100% Washington, California Washington, Oregon, D 288,000 0% 100% California E 100,000 100% 0% Washington Washington, Oregon, F 3,999,996 0% 100% California, Maine G 187,200 0% 100% Washington H 5,100,000 0% 100% Washington Washington, Oregon, I 1,089,540 42% 58% California, New York, 10 Georgia, Illinois, Texas J 440,000 32% 68% Washington, California Source: Pacific Shellfish Institute, 2007

Oyster prices differ across companies and by customer type, making it difficult to calculate a standardized price based on the survey. Table 9 outlines the average prices faced by companies at the processor, wholesaler, retail, and the consumer level. Although these are average prices, there is noticeable variation in the prices between companies. The farms with the largest operations, Company F and Company I, receive the highest prices on average for their oysters, perhaps reflecting their ability to negotiate or set higher prices within the market place.

9 Conditions in Hood Canal result in variable growth and natural reproduction rates resulting in variation between year-to-year harvest levels. 10 These are only the top seven states (in terms of quantity sold) that receive product from Company I.

19 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Table 9. Average Oyster Prices in Washington

11 Company Processor Wholesaler Retail Consumer

12 A $12/gallon $3.00/dozen -- -- B -- -- $3.50/dozen -- C -- $2.50/dozen -- -- D -- $2.25/dozen -- -- E -- $37/gallon $44/gallon -- F -- $3.55/dozen -- -- G -- $3.00/dozen -- -- H -- $1.75/dozen -- -- I N/A $3.25/dozen N/A N/A J N/A $2.50/dozen $3.60/dozen $5.25/dozen Source: Pacific Shellfish Institute, 2007

3.3 International Markets The survey indicates that only large operation companies ship internationally. Two shellfish companies surveyed currently ship internationally, while others sell to domestic wholesalers who likely sell to international market, while four companies have plans to do so. Table 10 details the different international locations of current sales, the percentage of total sales that are international, and the locations planned for international sales. The percentage of international sales is very low, but six companies have new international locations planned for sales. The new international locations are different across the companies, suggesting that there are many locations in Asia where the demand for oysters is strong. The largest operations have plans to sell or already sell directly to the international market. The medium-sized operations sell to a wholesaler that in turn sells to the international market. The smallest companies rely on the domestic market. Recent data indicate that wholesalers that sell to the international market are not purchasing Hood Canal oysters, but this may change if demand from the international market increases (TC Trading 2008). Several survey respondents mention that finding a reliable buyer for the international market is their greatest challenge to selling internationally. This difficulty is why large companies tend to be most active in the international market—they have the resources to incur some risk in sourcing new markets and the ability to deprive customers who fail to meet previously negotiated terms. Smaller companies may not be able to risk the loss of a single shipment or risk a new buyer failing to meet the terms of an agreement. It is easier for these companies to maintain relationships with local and domestic middlemen.

11 Prices do not include shipping and packaging. 12 Prices reflect sales to California wholesalers

20 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Table 10. International Oyster Sales

International New International Country Percent Locations Sales Destinations of Sales Plans to Sell Planned for Company (Number) Sales International Internationally Sales A 0 -- 0.0% Yes China B 0 -- 0.0% No -- C 0 -- 0.0% Yes Canada D 0 -- 0.0% No -- E 0 -- 0.0% Yes Asia Hong Kong, F 16,407 Canada 2.2% Yes Singapore G 0 -- 0.0% No -- H 0 -- 0.0% No -- Hong Kong, Japan, Canada, Singapore, I 85,886 1.4% Yes Taiwan Australia, China, Malaysia, Indonesia J 0 -- 0.0% Yes Malaysia Source: Pacific Shellfish Institute, 2007

Table 11 depicts prices received by a company domestically from wholesalers in Washington and California, and internationally from the wholesaler in Canada. The study notes several trends apparent in the data: • Prices are higher in Washington than in California for similar product, which could reflect market differences or shipping costs. • Prices are higher for larger oysters in all markets. • Prices received for shipments to Canada are higher than prices received from domestic markets for all sizes classes except “small.”

Table 11. Domestic and International Oyster Wholesale Prices

Size Washington California Canada Small $3.28/dozen $3.20/dozen $2.50/dozen Medium $4.17/dozen $3.70/dozen $4.50/dozen Large $4.42/dozen $4.00/dozen $5.15/dozen X-Large -- -- $5.45/dozen Jumbos -- -- $6.00/dozen Source: Pacific Shellfish Institute, 2007

21 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

22

4 Current and Potential Economic Effect of Cadmium Threshold for Hood Canal Oyster Companies

Information from the previous two sections is used in this section to estimate the economic effect of international cadmium limits on the companies in the Hood Canal. Section 4.1 describes the cadmium concentrations in the Hood Canal. Section 4.2 describes the existing cadmium regulations in various countries and the prospects of limits in others. Section 4.3 describes the economic losses that already occurred in the Hood Canal because of Hong Kong cadmium regulation, and Section 4.4 presents the economic losses to the Hood Canal oyster producers if no mitigating action is taken to prevent rejected shipments and expected economic losses if producers take mitigating actions.

4.1 Cadmium in the Hood Canal A recent scientific study by the Pacific Shellfish Institute, Integral Consulting, Hong Kong University of Science and Technology, and the Oregon State University Seafood Laboratory found that the cadmium concentrations in the Puget Sound are significantly elevated throughout portions of the Hood Canal. Mean cadmium concentrations in Hood Canal Pacific oyster composites are shown in Figure 10. The cadmium concentrations are the highest in the central and north parts of the Hood Canal, but the concentration is close to 2.0 ppm at nearly every part of the Hood Canal except the northern end of the canal. These data mean that most oysters from the Hood Canal are at risk of having cadmium concentrations above 2.0 ppm (Hong Kong’s standard).

Figure 10. Mean Cadmium Concentrations in Pacific Oysters in the Hood Canal, 2004-2006

Source: Pacific Shellfish Institute, 2006

23 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

4.2 International Limits on Cadmium In late 1999 and 2000, several shipments of British Columbia oysters were rejected by the Hong Kong Food and Environmental Hygiene Department for having cadmium concentrations in excess of the limit of 2.0 ppm (Kruzynski 2004). A subsequent survey by the Canadian Food Inspection Agency (CFIA) found that the shipments were not anomalous and a mean cadmium concentration of 2.63 ppm exists for British Columbia oysters over a broad geographic area (Schallie 2001). This finding, in combination with recent international limits on cadmium, alerted Pacific oyster farmers that naturally occurring cadmium concentrations present a problem. The provisional tolerable weekly cadmium intake (PTWI), according to the European Union, for a person of 70 kg (154 lbs) is 490 μg per week (European Union 2004). This PTWI includes the intake from all foods in a week, of which shellfish presumably would represent just a small portion. Table 12 and Table 13 indicate the likely amount of cadmium ingested from the consumption of 300g of meat from a half dozen large oysters from Hood Canal. On average, this amount of oyster meat from Hood Canal would result in the ingestion of 585 μg of cadmium, which is above the PTWI. The international limit on cadmium in oysters is, for the most part, consistent with the PTWI. The limits for Hong Kong, Australia, New Zealand, and the United States are above the PTWI, and the limit for the EU and China is below the PTWI. The cadmium concentration for the Hood Canal oysters barely passes the limit in place for Hong Kong, Australia, and New Zealand, but is well within the limit for the United States.

Table 12. Cadmium Intake for Consumption of Half Dozen Large Oysters13

Region Cadmium Concentration (µg Cd/g) Cadmium Ingested (µg Cd) Lowest Hood Canal Concentration 0.94 282 Highest Hood Canal Concentration 2.33 699 Average Hood Canal Concentration 1.95 585 Source: Pacific Shellfish Institute, 2007.

Table 13. Cadmium Exposure Guidelines

Maximum Allowable Cadmium 14 Region Ingestion Limits (µg Cd) Concentration (µg Cd/g) Hong Kong, Australia, New Zealand 2 600 European Union, China 1 300 United States 4 1,200 European Commission PTWI NA 490 Source: Pacific Shellfish Institute, 2007; Kruzynski, 2004; EU Directorate – General Health and Consumer Protection, 2004.

The fact that the Hong Kong cadmium limit for oysters is consistent with the PTWI suggests that the limit represents a primary concern for food safety and not simply a trade barrier. This information may suggest that similar or more stringent limits on cadmium in oysters will be adopted in other countries including the United States. Already of concern is the 1.0 ppm limit in China set in 2001 (Ministry of

13 Half dozen large oysters preferred by the Hong Kong market are about 300g in total. 14 (safe weekly intake from all sources for a 70 kg person)

24 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Agriculture of China 2001) since the final destination of a growing number of shellfish shipments are Chinese ports (not including Hong Kong), as indicated in Figure 9. However, the Chinese limit may be weakly enforced since no evidence of a rejection by Chinese ports (except Hong Kong) exists at this time.

4.3 Current Economic Losses from Cadmium Limits In October 2005, a shipment of oysters from Company F was rejected by the Hong Kong Food and Environmental Hygiene Department because of a cadmium concentration of 3.9 ppm. The citation stated that the importation of oysters from the harvest site, Hood Canal #8, would be “suspended pending investigation and remedial measures taken by the exporter.” Table 14 indicates the cost to Company F due to the rejection of their product. The following year, Company F’s international shipments, through the Canadian wholesaler TC Trading Inc., all went to Taiwan. There is no indication that the Taiwan prices were more or less than Hong Kong oyster export prices. However, data suggest that Taiwan is merely a transshipment point for the exported oysters and the oysters likely entered the Chinese market through mainland ports. Recently, Company C sent a sample of ten dozen oysters to a Canadian wholesaler, which rejected the sample, based solely on the fact that the oysters came from the Hood Canal Area #8. This suggests that wholesalers are aware of the cadmium problem, and there is already difficulty for Hood Canal growers to sell oysters internationally, at least from the harvest site Hood Canal #8. A company taking action to respond to the cadmium problem is Company I. They test regularly for cadmium to obtain information about levels in their oysters. The cadmium problem is not a major concern for this company because the company harvests throughout the Puget Sound; only about 10 percent of the company’s harvest beds are in the Hood Canal. Thus, the international shipments by Company I can be more readily sent from harvest beds outside the Hood Canal. However, this company is an exception, since nearly 90 percent of the companies operating in the Hood Canal are dependent on Hood Canal harvest beds for their operations and very few, if any, have their own testing operations. There are 23 companies with operations in the harvest bed #8, and all of these companies are dependent on the Hood Canal for operations. These are the companies most at risk from the cadmium problem.

25 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

Table 14. Hong Kong Regulation Influence on Survey Respondents

Aware of Hong Kong Cost to Date of New Business Company Regulation New Business Practices Practices A Yes None $0 B No N/A N/A Will not accept solicitations from C Yes ~ $25 Hong Kong D No N/A N/A E No N/A N/A F Yes Stop shipments to Hong Kong. ~ $2,594 G No N/A N/A H No N/A N/A 15 I Yes Ongoing tests for cadmium ~ $440-$880 J Yes None $0 Source: Pacific Shellfish Institute, 2007

4.4 Potential Economic Losses from the Cadmium Limit Section 4.4.1 indicates the economics losses to the Hood Canal oyster market if there is no action taken to mitigate the cadmium problem. Section 4.4.2 describes the mitigating actions companies could take to address the cadmium problem, and the reduction in the economic losses that might occur for each mitigating action.

4.4.1 No Mitigation of the Cadmium Problem Figure 10 indicates that cadmium levels are high throughout the Hood Canal. However, Table 14 indicates that only half of the survey respondents are aware of the Hong Kong cadmium limits, which is somewhat surprising since the survey respondents are larger operations more likely to ship internationally. One shellfish grower indicated in their survey that the Asia market prefers the large, or jumbo, oysters. Unfortunately, these are the oysters likely to have cadmium concentrations above PTWI limits and they take a long time (four to six years) to mature which means higher opportunity costs associated with the lost ability to grow smaller oysters for local markets. If every Asian country adopts a cadmium limit similar to Hong Kong’s, Hood Canal producers may be limited to less restrictive markets. Table 16 illustrates potential annual revenue loss to Hood Canal shellfish producers if export markets are no longer available. Key assumptions used to estimate revenue loss are shown at the top of the table. The assumptions include the number of shellfish companies, oyster production, percent of grower dependence on the Hood Canal, and the percent of growers with plans to market internationally Before Hood Canal shellfish companies switch to the domestic market, one-time losses of shipments may occur as international countries reject the shipments sent to them because of excessive cadmium. The loss of a shipment occurs if the wholesaler or the shellfish company does not test the

15 Assumes five to ten samples test of oysters for cadmium each year for the last eight years.

26 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium shipment for cadmium before shipping and the receiving country rejects the product. Oysters are a fresh product and rejected shipments can not be rerouted to other locations before the product spoils. The one-time revenue losses outlined in Table 15 correspond to the estimated loss associated with each shellfish company with plans to ship internationally, losing a single shipment of oysters of quantity similar to the shipment lost by Company F. Obviously, multiple rejections would result in higher losses. The loss of revenue to Hood Canal shellfish companies because of an inability to ship internationally has a lower and an upper bound to reflect uncertainty about the prices of the international market. The lower bound assumes that only two percent of oyster production from companies with plans for international shipments is shipped internationally, and the difference in price per dozen between domestic and international is $0.75 based on Table 11. The upper bound assumes a much stronger international market, perhaps due to the declining water quality in Asia, so that eight percent of production is shipped internationally, and the difference in price per dozen of oysters doubles to $1.50.

Table 15. Economic Losses Due to Hood Canal Companies Selling Only to the Domestic Market

Scenario Harvesters Shellstock Shippers Shucker-Packers Total Assumptions Number of Shellfish Companies operating in Hood 14 42 7 63 Canal Oysters Production 724,473 972,348 181,179 1,878,000 (Pounds)16 Percent Companies with most 93% 88% 71% 87% operations in Hood Canal Percent Companies with 10% 57% 100% 48% International Plans Potential Total One-time Revenue Loss from Lost $2,500 $52,500 $12,500 $67,500 Shipments Oysters Redirected to 17 Domestic Market Lower Bound Dozens 1,233 8,951 2,373 12,557 Revenue Losses $925 $6,714 $1,779 $9,418 Upper Bound Dozens 4,933 35,806 9,490 50,229 Revenue Losses $7,400 $53,708 $14,235 $75,344 Source: Pacific Shellfish Institute, 2007

16 The pounds of oysters in the Hood Canal are based on the survey of Hood Canal growers by PSI, the Washington Department of Fish and Wildlife, and the USDA NASS Census of Aquaculture. Assume 11 small oysters per pound (Taylor Shellfish 2008). 17 The lower bound corresponds to the assumptions that i) about 2 percent of oysters produced by growers with primary activities in the Hood Canal and with international plans are shipped internationally and ii) the price difference between domestic and international oysters is $0.75. The upper bound corresponds to 8 percent of oysters being shipped internationally and the price difference is $1.50.

27 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

The total annual aggregate loss to Hood Canal companies from the loss of access to the export market because of cadmium limits is estimated to range from $9,418 to $75,344 or 0.22 to 1.75 percent of annual revenues, assuming complete export market closure with no mitigating action. Revenue losses may be greater for the large operation shellfish companies as the probability of these companies shipping internationally is greater.

4.4.2 Mitigation of the Cadmium Problem Economic losses to shellfish companies in the Hood Canal shown in Table 15 are based on the assumption that the companies take no steps to mitigate the losses that result from the international cadmium regulations. However, there are a number of steps that Hood Canal shellfish companies could take to mitigate losses: • Ship oysters to international locations with non-existent or less strict cadmium regulations. • Test for cadmium before shipping internationally. • Develop a method to reduce or minimize cadmium in oysters where cadmium concentrations are high before shipping internationally. This option would likely be a long- term effort with higher costs and may not be practical. The first and easiest step to avoid the cadmium regulation in Hong Kong is to ship oysters to another market with prices similar to Hong Kong. This strategy is likely to be effective in the short run since the overall Asian market is growing. However, more countries are likely to develop and enforce regulations on cadmium. So far, only Hong Kong has rejected oysters due to high cadmium levels, but mainland China has in place an even more stringent standard, though this appears to not be enforced adequately. An alternative longer-run strategy would be to test product regularly. By testing international shipments for cadmium, producers could avoid revenue losses from rejected shipments. Testing for cadmium would provide information to producers on the areas of Hood Canal that are particularly prone to high concentrations, so that harvest from these areas could be avoided. According to Company I, the cost to test for cadmium is $10-$12 per sample of 20 oysters. This cost is trivial compared to the potential one-time losses from a single rejected shipment, but the aggregate cost of testing may be greater than the one-time losses depending on the frequency of rejections and the number of shipments a year to foreign ports. If every international export country were to implement cadmium restrictions, testing for cadmium would serve to alert producers about product that would be best sold in the domestic market assuming no change in the US standard.

28

5 Conclusions

Hong Kong has begun restricting the importation of oysters with cadmium levels above 2 ppm, and many other Asia and international countries are likely to follow. Hood Canal is one area in the Pacific Northwest where cadmium levels have been found to exceed Hong Kong restriction thresholds.18 Northern Economics has presented in this report: 1) an assessment of markets and revenues of Hood Canal oyster producers, and 2) and analysis of the revenue losses resulting from Hong Kong (and potential future international markets) restrictions on cadmium in oysters. The Washington Office of Shellfish and Water Protection certifies 63 companies to harvest in the Hood Canal. These shellfish companies represent roughly 19 percent of the shellfish companies in Washington, and about 5 percent of annual Washington production of oysters. The oyster industry in the Hood Canal is characterized by the operation of numerous shellfish companies that produce a small amount of product relative to the Washington industry as a whole. More than half of the survey respondents produce oysters that are shipped to states outside Washington and two survey respondents produce oysters that are shipped to states external to the West Coast. The Asian ports of Hong Kong and Taipei continue to receive the bulk of exports; however, the prominence of other Asia ports is growing as it becomes easier to ship products directly to these markets. An explanation for the low percentage of production for the international market is that the higher international prices for oysters are only for large oysters and Hood Canal oysters take a long time (four to six years) to grow to a large size. Nevertheless, four other producers surveyed plan to ship internationally in the future, probably in response to the strong international market in the past few years. Hong Kong cadmium regulations have already resulted in revenue losses and additional costs to Hood Canal producers. One producer producer’s shipment was rejected by an international wholesaler based only on the fact that the oysters came from Hood Canal; and Company I now conducts regular tests for cadmium. Other Asian countries have cadmium limits similar to Hong Kong’s, and in some cases even more stringent limits, though they are apparently not always well enforced. Hood Canal oyster producers exporting internationally face the risks associated with stronger enforcement of exiting regulations and the promulgation of new regulations because the concentration of cadmium in oysters is naturally above the 2.0 ppm limit in most of the Hood Canal. The total potential losses to the Hood Canal oyster producers are estimated to be between $9,000 and $75,000 annually depending on the strength of the international and domestic markets if oysters are diverted to domestic markets. Revenue losses to Hood Canal are expected to be limited in the future for a number of reasons: • Most Hood Canal operations are small and unlikely to ship product internationally. • Only a small percentage of annual oyster production from Hood Canal goes to the international market. • Many of the large operations harvest from sites outside the Hood Canal where cadmium concentrations are less.

18 Elevated Cd levels are also common in oysters harvested from certain locations in British Columbia, California and Alaska.

29 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

The mitigating actions available to producers that ship to the international market include the following: • Ship to international countries with less stringent or non-existent cadmium regulations. • Test for cadmium in oysters before shipping. A greater awareness of the cadmium threshold will prevent the one-time loss of shipments from the rejection of oysters from international ports. • A Hood Canal company planning to enter the export market should test for cadmium. Findings of high levels of cadmium in the oysters suggests that in the short run, the company should focus on business relationships with wholesalers that ship to Taipei or Southeast Asia. • Develop a method for reducing or minimizing cadmium residues in oysters prior to shipping. The cost to test oysters is not high compared to potential losses due to product rejection. However, the cost of frequent testing may be significant. Exploring methods to minimize cadmium residue in oysters is a long-run strategy that may address the problem in case the international market becomes particularly strong.

30

6 References

Cheney, Dan. “Characterization of Cadmium Health Risk, Concentration, and Ways to Minimize Residues in Shellfish.” Proceedings of National Integrated Food Safety Initiative Post Award Management Meeting, Olympia, Washington, 2007. http://www.csrees.usda.gov/nea/food/pdfs/cheney.pdf. Codex Alimentarius Commission. Report to Joint FAO/WHO Food Standards Programme. Twenty- ninth Session. International Conference Centre, Geneva, Switzerland: 3 -7 July 2006. European Union. Directorate – General Health and Consumer Protection. Report on tasks for scientific cooperation. Assessment of the dietary exposure to arsenic, cadmium, lead, and mercury of the population of the EU Member States. Report of experts participating in Task 3.2.11, March 2004. Heyamoto, Lisa. “With old ways and new ideas, Willapa Bay’s oystermen face a shifting future.” The Seattle Time Magazine. May 15, 2005. Kruzynski, George M. 2004. “Cadmium in oyster and scallops: the BC experience.” Toxicology Letters. Vol. 148, 159-169. Ministry of Agriculture of the People’s Republic of China. Standards in Agriculture - Concentration Limits of Hazardous Substances in Aquatic Products. (Translation may not be perfect.), NY 5073-2001, September 2001. Nexus Seafood. Spoke to Duby. Personal Communication with the Northern Economics Inc., May 2008. NOAA Fisheries. Office of Science and Technology. Foreign Trade – Raw Data. Accessed on May 2, 2008. http://www.st.nmfs.noaa.gov/st1/trade/build_a_database/TradeSelectIndex.html Northern Economics, Inc., Confidential Client Report, The World Geoduck Market and the Potential for Aquaculture on Washington State Lands. Prepared for Washington Department of Natural Resources, September 2004. Schallie, K., 2001. Results of the 2000 survey of cadmium in B.C. oysters. In: Kruzynski, G.M., Addison, R., Macdonald, R.W. (Eds.), 2002. Proceedings of a workshop on possible pathways of cadmium into the Pacific oyster Crassostrea gigas as cultured on the coast of British Columbia, Institute of Ocean Sciences, March 6–7, 2001, pp. 31–32. Can. Tech. Rep. Fish. Aquat. Sci. 2405, vi + 65 pp. Stanley, Melvin. WA Department of Fish and Wildlife. Information Technology Services. Personal Communication with Northern Economics, May 2008. Stanley, Melvin. WA Department of Fish and Wildlife. Information Technology Services. Personal Communication with the Pacific Shellfish Institute, 2007. Taylor Shellfish, Inc. Spoke to Austin. Personal Communication with the Northern Economics, May 2008. TC Trading. Spoke to Paul. Personal Communication with the Northern Economics, May 2008. U.S. Department of Agriculture, National Agricultural Statistics Service, Census of Aquaculture (2005), Vol. 3, Special Studies Part 2, October 2006. WA Department of Health. Division of Environmental Health, Office of Shellfish and Water Protection. Washington Licensed Shellfish Companies: Alphabetical Listing, December 2007.

31 Economic Effects to Hood Canal Producers of Reduction in Oyster Exports Due to Cadmium

WA Department of Health. Division of Environmental Health, Office of Shellfish and Water Protection. 2006 Annual Inventory of Commercial and Recreational Shellfish Areas in Washington. June 2007. http://www.doh.wa.gov/ehp/sf/growreports.htm WA Department of Health. Division of Environmental Health, Office of Shellfish and Water Protection. Annual Growing Area Review. June 2007. http://www.doh.wa.gov/ehp/sf/growreports.htm Wezenberg, Holly. WA Department of Health. “Export Certs Issued, by Destination.” Personal Communication with Northern Economics, Inc. May 5, 2008.

32 Appendix D – Data Validation Report

Data Review Report

Characterization of the Cadmium Health Risk, Concentrations and Ways to Minimize Cadmium Residues in Shellfish

Prepared for:

Integral Consulting, Inc. 1205 West Bay Drive NW Olympia, WA 98502

Prepared by:

Pyron Environmental, Inc. 3530 32 nd Way NW Olympia, WA 98502

August 18, 2006

ACRONYMS

%R percent recovery CLP U.S. EPA Contract Laboratory Program COC chain-of-custody EPA U.S. Environmental Protection Agency GFAA graphite furnace atomic absorption spectrometer ICP inductively coupled plasma IDL instrument detection limit mg/kg milligram per kilogram mg/L milligram per liter MS matrix spike MSD matrix spike duplicate PCBs polychlorinated biphenyls QA/QC quality assurance/quality control RPD relative percent difference TOC total organic carbon TS total solids TSS total suspended solids

Page 2 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories SUMMARY

I. Sample names, laboratory assigned sample identity, and the correspondent analyses included in this review are summarized in Attachment A – Sample Index.

II. The analytical parameters validated herein, the analytical methods, and the laboratory performing the analyses are summarized as follows:

Parameter Analytical Method Laboratory PCB Aroclors SW8082 Arsenic (Tissue) SW7060A Cadmium (Water) EPA 213.2 Cadmium & Zinc (Water) EPA 200.7 AMTEST Laboratories Cadmium & Zinc (Tissue/Sediment) SW6010 Redmond, WA 98052 TOC (Sediment) SW9060 Total Suspended Solids EPA 160.2 Total Solids (Sediment) SM2540B Grain Size ASTM 2540B Notes: (1) SM Methods – Standard Methods for the Examination of Water and Wastewater, American Public Health Association, 20 th Edition, 1995. (2) EPA Methods - USEPA Methods for Chemical Analysis of Water and Wastes, EPA –600/4-79-020, March 1983 Revision. (3) SW846 Methods - USEPA Test Methods for Evaluating Solid Waste, Physical/Chemical Methods, SW-846, Third Edition, December 1996.

Page 3 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories III. The quality assurance/quality control (QA/QC) parameters validated are summarized as follows:

Analysis

Total Dissolved TOC Grain PCBs TSS QC Parameter Metals Metals TS Size Holding Time √ √ √ √ √ √

Initial Calibration NR NR NR NR NA NA

Calibration Verification NR NR NR NR NA NA

Blanks √ √ √ √ √ NA Surrogate Spikes √ NA NA NA NA NA

MS/MSD NR 4.5 √ √ √ NA Duplicate Analysis NR √ √ √ √ √ Standard Reference NR NA √ √ √ NA Material Reporting Limits and Target NR 4.6 √ √ √ NA Compound Quantitation

Overall Assessment 3.5 4.7 √ √ √ √ Notes: “ √” denotes that the QA/QC parameter met the criteria for the analytical parameter. A number (e.g., “ 3.5 ”) denotes that the QA/QC parameter did not meet the criteria or required further discussion; the number indicates the report section that included discussion. NA – The QC parameter is not applicable for the analytical parameter. NR – The QC results were not reported and were not evaluated as part of this validation.

IV. Data qualification and assigned data qualifiers are summarized as follows:

Report Sample ID Analyte Data Qualifier Reason Section

USDA0510SME1 Insufficient information USDA0510ELD1 UJ (Nondetect) reported with sample PCB Aroclors 3.5 USDA0510SQB1 J (Detect) analyses for accuracy and USDA0510HAM1 precision evaluation.

The matrix spike %R value USDA0507HUMW1 Dissolved Cadmium J- was below the lower 4.5 control limit.

The reported IDL may not Total and Dissolved All water and seston UJ (Nondetect) be representative of 4.6 Cadmium & Zinc sample matrices.

The reported IDL may not J be representative of Total and Dissolved All water and seston (Detections <10x sample matrices and the 4.6 Cadmium & Zinc IDL) reported value may not be accurate.

Cadmium by EPA 200.7 In favor of the results All water if also analyzed with R 4.7 reported from EPA 213.2 EPA 213.2

Page 4 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories

V. Data qualifiers are defined as follows:

Data Qualifier Definition The analyte was detected above the reported quantitation limit, and the reported concentration J was approximate.

The analyte was detected above the reported quantitation limit, and the reported concentration J- was approximate and bias low.

R The analyte was analyzed for, but the result should be rejected.

The analyte was analyzed for, and the associated quantitation or detection limit was UJ approximate.

Page 5 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories DATA VALIDATION FINDINGS

1. Introduction

This report presents and discusses the findings of data review performed by Pyron Environmental, Inc. on data collected during September 2004 through February 2006 for the referenced project. Laboratory reports submitted for this review consisted of summary of sample results, chain-of-custody (COC) documents, and summary of laboratory quality control analyses ( i.e., method blanks, duplicate, standard reference material, and matrix spike analyses). The review did not include initial and verification calibrations or any instrument printout raw data. QC parameters evaluated herein are outlined in the Summary, Section II at the beginning of this report.

Since the laboratory reports did not include the performance-based in-house control limits for the QC analyses, control criteria specified in the U.S. Environmental Protection Agency’s (EPA) Contract Laboratory Program (CLP) National Functional Guidelines for Inorganic Data Review (EPA 2004) were applied to evaluate the data quality. Findings identified in this review are discussed and presented in the section pertinent to each analysis. Qualified data and assigned data qualifiers are summarized in the Summary at the beginning of this report.

2. Sample Delivery Documentation and Preservation

Samples were received at the laboratory intact and consistent with the accompanying chain-of-custody (COC) documentation as noted by the laboratory.

3. PCB Aroclors (SW 8082)

3.1 Holding Times

PCBs analyses were performed on selected tissue samples only. Frozen tissue samples should be extracted within one year of collection, and extracts analyzed within 40 days of extraction. The extraction and analysis of samples met the requirements.

3.2 Method Blanks

Method blanks were prepared and analyzed as required. No target analytes were detected in the method blanks at or above the quantitation limits.

3.3 Surrogate Spikes

Surrogate spikes were added to all samples as required by the method. All surrogate spike percent recovery (%R) values were within 80 – 120 percent.

Page 6 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories 3.4 Target Compound Identification

PCB Aroclors were not detected at or above the quantitation limits in any of the samples requested for this analysis.

3.5 Overall Assessment of Data Usability

Due to the limited quality assurance/quality control (QA/QC) information included in the laboratory reports, the analytical precision and accuracy could not be evaluated further. PCB Aroclor data are subjected to the QA Level II quality as defined by USEPA, and can only be used as screening results. As a precaution to the data usage, PCB Aroclor results are qualified (UJ).

4. Metals (Arsenic, Cadmium, and Zinc)

4.1 Holding Times

Sediment, tissue, and water samples should be analyzed within 180 days for metals. Samples were analyzed within the required holding time.

4.2 Method Blanks

A method blank was prepared and analyzed with each analytical batch. No target analytes were positively reported in the method blanks at or above the instrument detection limits (IDLs), except for zinc in selected method blanks associated with tissue and sediment samples. Zinc concentrations in all associated samples were greater than 10 times the detection in the method blanks. No actions were required on this basis.

4.3 Duplicate Sample Analysis

Duplicate sample analyses were performed on project samples. The relative percent difference (RPD) values met the control criteria (20% for detections ≥ 5xIDL, ±1xIDL for detections < 5xIDL for water samples, and 35%, ±2xIDL for sediment and tissue samples).

4.4 Standard Reference Material Analysis

Standard reference material sample analyses were performed in lieu of laboratory control samples. The %R values were within 80 – 120% for sediment, tissue, and water samples.

4.5 Matrix Spike (MS)

Matrix spike analyses were performed on selected project samples for sediment, tissue, and water samples. The %R values met the control limits (75 – 125%), except for the following:

Page 7 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories

Control Data Spiked Sample ID Analye %R Limits Affected Sample Qualification

USDA0507HUMW1 Cadmium 70.0 75 – 125% USDA0507HUMW J-

In cases where MSD analyses were also performed, the MS/MSD RPD values were within 20% for water samples and 35% for tissue and sediment samples.

4.6 Analyte Quantitation and Detection Limits

For cadmium and zinc analyses on water samples, the laboratory reported results to the IDLs, which were based on studies performed with laboratory reagent water. Water samples collected for this project are seawater, which poses significant interference with the ICP and GFAA analyses. To mitigate the interference for accurate quantitation, sample treatments are required prior to the instrument analysis. Sample treatments separate the interferants from target analytes (such as chelating and co-precipitation techniques), or reduce the interfering stress on instrument (such as sample dilution).

The laboratory reports reviewed herein did not provide documentation to relay any sample treatments performed on the water samples. The reported IDLs (based on studies with reagent water) did not factor the brackish nature of seawater samples, and therefore, may not be representative of the instrument sensitivity for the specific matrices. As a precaution to the data usage, the cadmium (analyzed with ICP and GFAA) and zinc (analyzed with ICP) IDLs are qualified as estimated (UJ) for all non- detects in water and seston samples. Reported values for cadmium and zinc above the IDLs but less than 10 times the IDLs are qualified as estimated (J).

4.7 Overall Assessment of Data Usability

A number of water samples were initially analyzed with ICP technique (EPA Method 200.7), and later analyzed with GFAA (EPA Method 213.2) for cadmium. In such case, results reported from ICP analyses should be rejected (R) in favor of the results reported from the GFAA analyses.

Based on the information presented in the laboratory reports, arsenic, cadmium, and zinc data are acceptable for use as qualified.

5. Grain Size, TOC, TSS, and Total Solids

5.1 Holding Time

TS should be analyzed within 14 days, TSS within 7 days, TOC within 28 days, and grain-size within six months of collection. All samples were analyzed within the required holding time.

Page 8 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories 5.2 Method Blank

Method blanks were prepared and analyzed as required for TOC, TSS, and TS analyses. Target analytes were not detected at or above the quantitation limits.

5.3 Duplicate Analysis

The duplicate analysis RPD values for TS, TSS, and TOC were within 20% for sediment samples. RPD values for selected fractions of the gain-size analyses on a number of samples were greater than 50% mostly due to the relatively small retention amount of the fractions. Data are not qualified on this basis.

5.4 Standard Reference Analysis

Standard reference material analyses were performed with TOC and TSS analyses. The %R values were within 80 – 120%

5.5 Matrix Spike (MS)

Matrix spike analyses were performed for TOC and DOC on project samples. All %R values were within 75 – 125%.

5.6 Overall Assessment of Data Usability

Based on the information presented in the laboratory reports, grain-size, TOC, TSS, and TS data are acceptable for use.

Approved By: Date:

Mingta Lin

Page 9 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories REFERENCES

USEPA Contract Laboratory Program National Functional Guidelines for Inorganic Data Review, Office of Emergency and Remedial Response, U.S. Environmental Protection Agency, October 2004, EPA 540/R-04/004.

USEPA Test Methods for Evaluating Solid Waste, Physical/Chemical Methods, SW-846, Third Edition, December 1996.

USEPA Methods for Chemical Analysis of Water and Wastes, EPA –600/4-79-020, March 1983 Revision.

Method 1637- Determination of Trace Elements in Ambient Waters by Off-Line Chelation Preconcentration and Stabilized Temperature Graphite Furnace Atomic Absorption, U.S. Environmental Protection Agency,Office of Water, Engineering and Analysis Division, January 1996.

Recommended Protocols for Measuring Conventional Sediment Variables in Puget Sound, Puget Sound Water Quality Authority, March 1986.

Recommended Guidelines for Measuring Organic Compounds in Puget Sound Water, Sediment and Tissue Samples, Puget Sound Water Quality Authority, April 1997.

Standard Methods for the Examination of Water and Wastewater , American Public Health Association, 20 th Edition, 1995.

USEPA Methods for Chemical Analysis of Water and Wastes, EPA –600/4-79-020, March 1983 Revision.

Page 10 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories ATTACHMENT A

Sample Index

Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 04-A013490 USDA0409SQU1 09/23/04 Tiss X X

04-A013491 USDA0409SQU2 09/23/04 Tiss X X

04-A013492 USDA0409SQU3 09/23/04 Tiss X X

04-A013493 USDA0409DIS1 09/23/04 Tiss X X

04-A013494 USDA0409DIS2 09/23/04 Tiss X X

04-A013495 USDA0409DIS3 09/23/04 Tiss X X

04-A013496 USDA0409PGB1 09/23/04 Tiss X X

04-A013497 USDA0409PGB2 09/23/04 Tiss X X

04-A013498 USDA0409PGB3 09/23/04 Tiss X X

04-A013499 USDA0409PGA1 09/22/04 Tiss X X

04-A013500 USDA0409PGA2 09/22/04 Tiss X X

04-A013501 USDA0409PGA3 09/22/04 Tiss X X

04-A013502 USDA0409AGP1 09/22/04 Tiss X X

04-A013503 USDA0409AGP2 09/22/04 Tiss X X

04-A013504 USDA0409AGP3 09/22/04 Tiss X X

04-A013505 USDA0409BRC1 09/23/04 Tiss X X

04-A013506 USDA0409BRC2 09/23/04 Tiss X X

04-A013507 USDA0409BRC3 09/23/04 Tiss X X

04-A013508 USDA0409MNT1 09/23/04 Tiss X X

04-A013509 USDA0409MNT2 09/23/04 Tiss X X

04-A013510 USDA0409MNT3 09/23/04 Tiss X X

04-A013511 USDA0409QMH1 09/23/04 Tiss X X

04-A013512 USDA0409QMH2 09/23/04 Tiss X X

04-A013514 USDA0409HNT1 09/22/04 Tiss X X

04-A013515 USDA0409HNT2 09/22/04 Tiss X X

04-A013516 USDA0409HNT3 09/22/04 Tiss X X

04-A013517 USDA0409NQL1 09/21/04 Tiss X X

04-A013518 USDA0409NQL2 09/21/04 Tiss X X

04-A013519 USDA0409NQL3 09/21/04 Tiss X X

04-A013520 USDA0409HNB1 09/22/04 Tiss X X

04-A013521 USDA0409HNB2 09/22/04 Tiss X X

Page 11 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 04-A013522 USDA0409HNB3 09/22/04 Tiss X X

04-A013523 USDA0409SEQ1 09/23/04 Tiss X X

04-A013524 USDA0409SEQ2 09/23/04 Tiss X X

04-A013525 USDA0409SEQ3 09/23/04 Tiss X X

04-A013526 USDA0409OAK1 09/23/04 Tiss X X

04-A013527 USDA0409OAK2 09/23/04 Tiss X X

04-A013528 USDA0409OAK3 09/23/04 Tiss X X

04-A013529 USDA0409DUN1 09/23/04 Tiss X X

04-A013530 USDA0409DUN2 09/23/04 Tiss X X

04-A013531 USDA0409DUN3 09/23/04 Tiss X X

04-A013532 USDA0409SBB1 09/23/04 Tiss X X

04-A013533 USDA0409SBB2 09/23/04 Tiss X X

04-A013534 USDA0409SBB3 09/23/04 Tiss X X

04-A013535 USDA0409ELD1 09/23/04 Tiss X X

04-A013536 USDA0409ELD2 09/23/04 Tiss X X

04-A013537 USDA0409ELD3 09/23/04 Tiss X X

04-A013538 USDA0409TOT1 09/23/04 Tiss X X

04-A013539 USDA0409TOT2 09/23/04 Tiss X X

04-A013540 USDA0409TOT3 09/23/04 Tiss X X

04-A013541 USDA0409DRH1 09/22/04 Tiss X X

04-A013542 USDA0409DRH2 09/22/04 Tiss X X

04-A013543 USDA0409DRH3 09/22/04 Tiss X X

04-A013554 USDA0409HAM1 09/22/04 Tiss X X

04-A013555 USDA0409HAM2 09/22/04 Tiss X X

04-A013556 USDA0409HAM3 09/22/04 Tiss X X

04-A013557 USDA0409DSW1 09/23/04 Tiss X X

04-A013558 USDA0409DSW2 09/23/04 Tiss X X

04-A013559 USDA0409DSW3 09/23/04 Tiss X X

04-A013560 USDA0409DBE1 09/23/04 Tiss X X

04-A013561 USDA0409DBE2 09/23/04 Tiss X X

04-A013562 USDA0409DBE3 09/23/04 Tiss X X

04-A013563 USDA0409SQB1 09/23/04 Tiss X X

04-A013564 USDA0409SQB2 09/23/04 Tiss X X

04-A013565 USDA0409SQB3 09/23/04 Tiss X X

Page 12 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 04-A013566 USDA0409BEL1 09/23/04 Tiss X X

04-A013567 USDA0409BEL2 09/23/04 Tiss X X

04-A013568 USDA0409BEL3 09/23/04 Tiss X X

04-A013569 USDA0409TWA1 09/23/04 Tiss X X

04-A013570 USDA0409TWA2 09/23/04 Tiss X X

04-A013571 USDA0409TWA3 09/23/04 Tiss X X

04-A013572 USDA0409POT1 09/23/04 Tiss X X

04-A013573 USDA0409POT2 09/23/04 Tiss X X

04-A013574 USDA0409POT3 09/23/04 Tiss X X

04-A013575 USDA0409SME1 09/23/04 Tiss X X

04-A013576 USDA0409SME2 09/23/04 Tiss X X

04-A013577 USDA0409SME3 09/23/04 Tiss X X

04-A013578 USDA0409WES1 09/23/04 Tiss X X

04-A013579 USDA0409WES2 09/23/04 Tiss X X

04-A013580 USDA0409WES3 09/23/04 Tiss X X

04-A013581 USDA0409ORC1 09/23/04 Tiss X X

04-A013582 USDA0409ORC2 09/23/04 Tiss X X

04-A013583 USDA0409ORC3 09/23/04 Tiss X X

04-A013877 USDA0409TIL1 09/27/04 Tiss X X

04-A013878 USDA0409TIL2 09/27/04 Tiss X X

04-A013879 USDA0409TIL3 09/27/04 Tiss X X

04-A013880 USDA0409NET1 09/27/04 Tiss X X

04-A013881 USDA0409NET2 09/27/04 Tiss X X

04-A013882 USDA0409NET3 09/27/04 Tiss X X

04-A013883 USDA0409WBC1 09/27/04 Tiss X X

04-A013884 USDA0409WBC2 09/27/04 Tiss X X

04-A013885 USDA0409WBC3 09/27/04 Tiss X X

04-A013886 USDA0409YAQ1 09/26/04 Tiss X X

04-A013887 USDA0409YAQ2 09/26/04 Tiss X X

04-A013888 USDA0409YAQ3 09/26/04 Tiss X X

04-A014098 USDA0409GHS1 09/28/04 Tiss X X

04-A014099 USDA0409GHS2 09/28/04 Tiss X X

04-A014100 USDA0409GHS3 09/28/04 Tiss X X

04-A014101 USDA0409WBL1 09/28/04 Tiss X X

Page 13 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 04-A014102 USDA0409WBL2 09/28/04 Tiss X X

04-A014103 USDA0409WBL3 09/28/04 Tiss X X

04-A014398 USDA0409ANN1 09/28/04 Tiss X X

04-A014399 USDA0409ANN2 09/28/04 Tiss X X

04-A014400 USDA0409ANN3 09/28/04 Tiss X X

04-A014448 USDA0409STE1 09/30/04 Tiss X X

04-A014449 USDA0409STE2 09/30/04 Tiss X X

04-A014450 USDA0409STE3 09/30/04 Tiss X X

05-A000321 USDA0409CAT1 01/10/05 Tiss X

05-A000322 USDA0409CAT2 01/10/05 Tiss X

05-A000323 USDA0409CAT3 01/10/05 Tiss X

05-A000375 USDA0501ELD1 01/10/05 Tiss X

05-A000377 USDA0501ELD2 01/10/05 Tiss X

05-A000376 USDA0501ELD3 01/10/05 Tiss X

05-A000378 USDA0501ELDS1 01/10/05 Sed X

05-A000379 USDA0501ELDS2 01/10/05 Sed X

05-A000380 USDA0501ELDS3 01/10/05 Sed X

05-A000381 USDA0501ELDW1 01/10/05 Water X

05-A000382 USDA0501ELDW2 01/10/05 Water X

05-A000383 USDA0501ELDW3 01/10/05 Water X

05-A000384 USDA0501HAM1 01/10/05 Tiss X

05-A000385 USDA0501HAM2 01/10/05 Tiss X

05-A000386 USDA0501HAM3 01/10/05 Tiss X

05-A000387 USDA0501HAMS1 01/10/05 Sed X

05-A000388 USDA0501HAMS2 01/10/05 Sed X

05-A000389 USDA0501HAMS3 01/10/05 Sed X

05-A000390 USDA0501HAMW1 01/10/05 Water X

05-A000391 USDA0501HAMW2 01/10/05 Water X

05-A000392 USDA0501HAMW3 01/10/05 Water X

05-A000393 USDA0501SQB1 01/10/05 Tiss X

05-A000394 USDA0501SQB2 01/10/05 Tiss X

05-A000395 USDA0501SQB3 01/10/05 Tiss X

05-A000396 USDA0501SQBS1 01/10/05 Sed X

05-A000397 USDA0501SQBS2 01/10/05 Sed X

Page 14 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 05-A000398 USDA0501SQBS3 01/10/05 Sed X

05-A000399 USDA0501SQBW1 01/10/05 Water X

05-A000400 USDA0501SQBW2 01/10/05 Water X

05-A000401 USDA0501SQBW3 01/10/05 Water X

05-A000430 USDA0501SME1 01/11/05 Tiss X

05-A000431 USDA0501SME2 01/11/05 Tiss X

05-A000432 USDA0501SME3 01/11/05 Tiss X

05-A000433 USDA0501SMES1 01/11/05 Sed X

05-A000434 USDA0501SMES2 01/11/05 Sed X

05-A000435 USDA0501SMES3 01/11/05 Sed X

05-A000436 USDA0501SMEW1 01/11/05 Water X

05-A000437 USDA0501SMEW2 01/11/05 Water X

05-A000438 USDA0501SMEW3 01/11/05 Water X

05-A000442 USDA0501NEB1 01/10/05 Tiss X

05-A000443 USDA0501NEB2 01/10/05 Tiss X

05-A000444 USDA0501NEB3 01/10/05 Tiss X

05-A000445 USDA0501TIL1 01/10/05 Tiss X

05-A000446 USDA0501TIL2 01/10/05 Tiss X

05-A000447 USDA0501TIL3 01/10/05 Tiss X

05-A000448 USDA0501TILS1 01/10/05 Sed X

05-A000449 USDA0501TILS2 01/10/05 Sed X

05-A000450 USDA0501TILS3 01/10/05 Sed X

05-A000451 USDA0501TILW1 01/10/05 Water X

05-A000452 USDA0501TILW2 01/10/05 Water X

05-A000453 USDA0501TILW3 01/10/05 Water X

05-A000480 USDA0501NET1 01/11/05 Tiss X

05-A000481 USDA0501NET2 01/11/05 Tiss X

05-A000482 USDA0501NET3 01/11/05 Tiss X

05-A000483 USDA0501NETS1 01/11/05 Sed X

05-A000484 USDA0501NETS2 01/11/05 Sed X

05-A000485 USDA0501NETS3 01/11/05 Sed X

05-A000486 USDA0501NETW1 01/11/05 Water X

05-A000487 USDA0501NETW2 01/11/05 Water X

05-A000488 USDA0501NETW3 01/11/05 Water X

Page 15 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 05-A000489 USDA0501WBC1 01/11/05 Tiss X

05-A000490 USDA0501WBC2 01/11/05 Tiss X

05-A000491 USDA0501WBC3 01/11/05 Tiss X

05-A000492 USDA0501WBCS1 01/11/05 Sed X

05-A000493 USDA0501WBCS2 01/11/05 Sed X

05-A000494 USDA0501WBCS3 01/11/05 Sed X

05-A000495 USDA0501WBCW1 01/11/05 Water X

05-A000496 USDA0501WBCW2 01/11/05 Water X

05-A000497 USDA0501WBCW3 01/11/05 Water X

05-A000605 USDA0409EAG1 07/15/05 Tiss X

05-A000606 USDA0409EAG2 07/15/05 Tiss X

05-A000607 USDA0409EAG3 07/15/05 Tiss X

05-A003384 USDA0409TBN1 07/15/05 Tiss X

05-A003385 USDA0409TBN2 07/15/05 Tiss X

05-A003386 USDA0409TBN3 07/15/05 Tiss X

05-A003387 USDA0409TBN4 07/15/05 Tiss X

05-A003388 USDA0409HB515 07/08/05 Tiss X

05-A003389 USDA0409HB516 07/08/05 Tiss X

05-A003390 USDA0409TBOC10 06/30/05 Tiss X

05-A004459 USDA0504WBC1 04/25/05 Tiss X

05-A004460 USDA0504WBC2 04/25/05 Tiss X

05-A004461 USDA0504WBC3 04/25/05 Tiss X

05-A004462 USDA0504WBCS1 04/25/05 Sed X X

05-A004463 USDA0504WBCS3 04/25/05 Sed X

05-A004464 USDA0504WBCW1 04/25/05 Water X

05-A004465 USDA0504WBCW2 04/25/05 Water X

05-A004466 USDA0504WBCW3 04/25/05 Water X X

05-A004544 USDA0504SQB1 04/26/05 Tiss X

05-A004545 USDA0504SQB2 04/26/05 Tiss X

05-A004546 USDA0504SQB3 04/26/05 Tiss X

05-A004547 USDA0504NET1 04/26/05 Tiss X

05-A004548 USDA0504NET2 04/26/05 Tiss X

05-A004549 USDA0504NET3 04/26/05 Tiss X

05-A004550 USDA0504ELD1 04/26/05 Tiss X

Page 16 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 05-A004551 USDA0504ELD2 04/26/05 Tiss X

05-A004552 USDA0504ELD3 04/26/05 Tiss X

05-A004553 USDA0504HAM1 04/26/05 Tiss X

05-A004554 USDA0504HAM2 04/26/05 Tiss X

05-A004555 USDA0504HAM3 04/26/05 Tiss X

05-A004556 USDA0504TIL1 04/26/05 Tiss X

05-A004557 USDA0504TIL2 04/26/05 Tiss X

05-A004558 USDA0504TIL3 04/26/05 Tiss X

05-A004559 USDA0504SME1 04/26/05 Tiss X

05-A004560 USDA0504SME2 04/26/05 Tiss X

05-A004561 USDA0504SME3 04/26/05 Tiss X

05-A004562 USDA0504SQBS1 04/25/05 Sed X X

05-A004563 USDA0504NETS1 04/25/05 Sed X X

05-A004564 USDA0504ELDS1 04/25/05 Sed X X

05-A004565 USDA0504HAMS1 04/25/05 Sed X X

05-A004566 USDA0504TILS1 04/25/05 Sed X X

05-A004567 USDA0504SMES1 04/25/05 Sed X X

05-A004568 USDA0504SQBS3 04/25/05 Sed X

05-A004569 USDA0504NETS3 04/25/05 Sed X

05-A004570 USDA0504ELDS3 04/25/05 Sed X

05-A004571 USDA0504HAMS3 04/25/05 Sed X

05-A004572 USDA0504TILS3 04/25/05 Sed X

05-A004573 USDA0504SMES3 04/25/05 Sed X

05-A004574 USDA0504SQBW1 04/25/05 Water X

05-A004575 USDA0504NETW1 04/25/05 Water X

05-A004576 USDA0504ELDW1 04/25/05 Water X

05-A004577 USDA0504HAMW1 04/25/05 Water X

05-A004578 USDA0504TILW1 04/25/05 Water X

05-A004579 USDA0504SMEW1 04/25/05 Water X

05-A004580 USDA0504SQBW2 04/25/05 Water X

05-A004581 USDA0504NETW2 04/25/05 Water X

05-A004582 USDA0504ELDW2 04/25/05 Water X

05-A004583 USDA0504HAMW2 04/25/05 Water X

05-A004584 USDA0504TILW2 04/25/05 Water X

Page 17 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 05-A004585 USDA0504SMEW2 04/25/05 Water X

05-A004586 USDA0504SQBW3 04/25/05 Water X X

05-A004587 USDA0504NETW3 04/25/05 Water X X

05-A004588 USDA0504ELDW3 04/25/05 Water X X

05-A004589 USDA0504HAMW3 04/25/05 Water X X

05-A004590 USDA0504TILW3 04/25/05 Water X X

05-A004591 USDA0504SMEW3 04/25/05 Water X X

05-A004600 USDA0405GOE1 04/27/05 Tiss X

05-A004601 USDA0405GOE2 04/27/05 Tiss X

05-A004602 USDA0405GOE3 04/27/05 Tiss X

05-A004858 USDA0504TOM1 05/04/05 Tiss X

05-A004859 USDA0504TOM2 05/04/05 Tiss X

05-A004860 USDA0504TOM3 05/04/05 Tiss X

05-A004861 USDA0504TOMS1 05/04/05 Sed X X

05-A004862 USDA0504TOMS3 05/04/05 Sed X

05-A004863 USDA0504TOMW1 05/04/05 Water X

05-A004864 USDA0504TOMW2 05/04/05 Water X

05-A004865 USDA0504TOMW3 05/04/05 Water X X

05-A005059 USDA0504ANN1 05/10/05 Tiss X

05-A005060 USDA0504ANN2 05/10/05 Tiss X

05-A005061 USDA0504ANN3 05/10/05 Tiss X

05-A005062 USDA0504ANNS1 05/10/05 Sed X X

05-A005063 USDA0504ANNS3 05/10/05 Sed X

05-A005064 USDA0504ANNW1 05/10/05 Water X

05-A005065 USDA0504ANNW2 05/10/05 Water X

05-A005066 USDA0504ANNW3 05/10/05 Water X X

05-A005226 USDA0504HUM1 05/16/05 Tiss X

05-A005227 USDA0504HUM2 05/16/05 Tiss X

05-A005228 USDA0504HUM3 05/16/05 Tiss X

05-A005229 USDA0504HUMS1 05/16/05 Sed X X

05-A005230 USDA0504HUMS3 05/16/05 Sed X

05-A005231 USDA0504HUMW1 05/16/05 Water X

05-A005232 USDA0504HUMW2 05/16/05 Water X

05-A005233 USDA0504HUMW3 05/16/05 Water X X

Page 18 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 05-A006698 USDA0504WIN1 06/15/05 Tiss X

05-A006699 USDA0504WIN2 06/15/05 Tiss X

05-A006700 USDA0504WIN3 06/15/05 Tiss X

05-A006701 USDA0504WINS1 06/15/05 Sed X X

05-A006702 USDA0504WINS3 06/15/05 Sed X

05-A006703 USDA0504WINW1 06/15/05 Water X

05-A006704 USDA0504WINW2 06/15/05 Water X

05-A006705 USDA0504WINW3 06/15/05 Water X X

05-A007301 USDA0507HAM1 07/05/05 Tiss X

05-A007302 USDA0507HAM2 07/05/05 Tiss X 05-A007303 USDA0507HAM3 07/05/05 Tiss X

05-A007304 USDA0507HAMSD1 07/05/05 Tiss X

05-A007305 USDA0507HAMSD2 07/05/05 Tiss X 05-A007306 USDA0507HAMSD3 07/05/05 Tiss X

05-A007307 USDA0507HAMS1 07/05/05 Sed X X

05-A007308 USDA0507HAMS2 07/05/05 Sed X 05-A007309 USDA0507HAMW1 07/05/05 Water X

05-A007310 USDA0507HAMW2 07/05/05 Water X

05-A007311 USDA0507HAMW3 07/05/05 Water X X

05-A007312 USDA0507SQBS1 07/05/05 Sed X X

05-A007313 USDA0507SQBS2 07/05/05 Sed X 05-A007314 USDA0507SQBW1 07/05/05 Water X

05-A007315 USDA0507SQBW2 07/05/05 Water X

05-A007316 USDA0507SQBW3 07/05/05 Water X X 05-A007317 USDA0507SQB1 07/05/05 Tiss X

05-A007318 USDA0507SQB2 07/05/05 Tiss X

05-A007319 USDA0507SQB3 07/05/05 Tiss X 05-A007320 USDA0507SQBB1 07/05/05 Tiss X

05-A007321 USDA0507SQBB2 07/05/05 Tiss X

05-A007322 USDA0507SQBB3 07/05/05 Tiss X 05-A007338 USDA0507NET1 07/05/05 Tiss X

05-A007339 USDA0507NET2 07/05/05 Tiss X

05-A007340 USDA0507NET3 07/05/05 Tiss X 05-A007341 USDA0507NETSD1 07/05/05 Tiss X

Page 19 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 05-A007342 USDA0507NETSD2 07/05/05 Tiss X

05-A007343 USDA0507NETSD3 07/05/05 Tiss X 05-A007344 USDA0507NETS1 07/05/05 Sed X X

05-A007345 USDA0507NETS2 07/05/05 Sed X

05-A007346 USDA0507NETW1 07/04/05 Water X

05-A007347 USDA0507NETW2 07/04/05 Water X

05-A007348 USDA0507NETW3 07/04/05 Water X X

05-A007349 USDA0507TIL1 07/04/05 Tiss X

05-A007350 USDA0507TIL2 07/04/05 Tiss X

05-A007351 USDA0507TIL3 07/04/05 Tiss X

05-A007352 USDA0507TILS1 07/04/05 Sed X X 05-A007353 USDA0507TILS2 07/04/05 Sed X

05-A007354 USDA0507TILW1 07/04/05 Water X

05-A007355 USDA0507TILW2 07/04/05 Water X

05-A007356 USDA0507TILW3 07/04/05 Water X X

05-A007446 USDA0507WBC1 07/06/05 Tiss X

05-A007447 USDA0507WBC2 07/06/05 Tiss X

05-A007448 USDA0507WBC3 07/06/05 Tiss X

05-A007449 USDA0507WBCSD1 07/06/05 Tiss X

05-A007450 USDA0507WBCSD2 07/06/05 Tiss X

05-A007451 USDA0507WBCSD3 07/06/05 Tiss X

05-A007452 USDA0507WBCS1 07/06/05 Sed X X

05-A007453 USDA0507WBCS2 07/06/05 Sed X

05-A007454 USDA0507WBCW1 07/06/05 Water X

05-A007455 USDA0507WBCW2 07/06/05 Water X

05-A007456 USDA0507WBCW3 07/06/05 Water X X

05-A007461 USDA0507ELD1 07/06/05 Tiss X

05-A007462 USDA0507ELD2 07/06/05 Tiss X

05-A007463 USDA0507ELD3 07/06/05 Tiss X

05-A007464 USDA0507ELDSD1 07/06/05 Tiss X

05-A007465 USDA0507ELDSD2 07/06/05 Tiss X

05-A007466 USDA0507ELDSD3 07/06/05 Tiss X

05-A007467 USDA0507ELDS1 07/06/05 Sed X X

05-A007468 USDA0507ELDS2 07/06/05 Sed X

Page 20 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 05-A007469 USDA0507ELDW1 07/06/05 Water X

05-A007470 USDA0507ELDW2 07/06/05 Water X

05-A007471 USDA0507ELDW3 07/06/05 Water X X

05-A007473 USDA0507SME1 07/16/05 Tiss X

05-A007474 USDA0507SME2 07/16/05 Tiss X

05-A007475 USDA0507SME3 07/16/05 Tiss X

05-A007476 USDA0507SD1 07/16/05 Tiss X

05-A007477 USDA0507SD2 07/16/05 Tiss X

05-A007478 USDA0507SMES1 07/16/05 Sed X X

05-A007479 USDA0507SMES2 07/16/05 Sed X

05-A007480 USDA0507SMEW1 07/16/05 Water X

05-A007481 USDA0507SMEW2 07/16/05 Water X

05-A007482 USDA0507SMEW3 07/16/05 Water X X

05-A007579 USDA0507SMESD1 07/16/05 Sed X X

05-A007580 USDA0507SQBSD1 07/16/05 Tiss X

05-A007581 USDA0507SMESD2 07/16/05 Sed X

05-A007582 USDA0507SQBSD2 07/16/05 Tiss X

05-A007583 USDA0507SQBSD3 07/16/05 Tiss X

05-A008158 USDA0507HUM1 07/26/05 Tiss X

05-A008159 USDA0507HUM2 07/26/05 Tiss X

05-A008160 USDA0507HUM3 07/26/05 Tiss X

05-A008161 USDA0507HUMS1 07/26/05 Sed X X

05-A008162 USDA0507HUMS2 07/26/05 Sed X

05-A008163 USDA0507HUMW1 07/26/05 Water X

05-A008164 USDA0507HUMW2 07/26/05 Water X

05-A008165 USDA0507HUMW3 07/26/05 Water X X

05-A008166 USDA0507TOM1 07/25/05 Tiss X

05-A008167 USDA0507TOM2 07/25/05 Tiss X

05-A008168 USDA0507TOM3 07/25/05 Tiss X

05-A008169 USDA0507TOMS1 07/25/05 Sed X X

05-A008170 USDA0507TOMS2 07/25/05 Sed X

05-A008171 USDA0507TOMW1 07/25/05 Water X

05-A008172 USDA0507TOMW2 07/25/05 Water X

05-A008173 USDA0507TOMW3 07/25/05 Water X X

Page 21 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 05-A008534 USDA0507WIN1 07/21/05 Tiss X

05-A008535 USDA0507WIN2 07/21/05 Tiss X

05-A008536 USDA0507WIN3 07/21/05 Tiss X

05-A008537 USDA0507WINS1 07/21/05 Sed X X

05-A008538 USDA0507WINS2 07/21/05 Sed X

05-A008539 USDA0507WINW1 07/21/05 Water X

05-A008540 USDA0507WINW2 07/21/05 Water X

05-A008541 USDA0507WINW3 07/21/05 Water X X

05-A012058 USDA0510SME1 10/10/05 Tiss X X

05-A012059 USDA0510SME2 10/10/05 Tiss X

05-A012060 USDA0510SME3 10/10/05 Tiss X

05-A012061 USDA0510SMES1 10/10/05 Sed X X

05-A012062 USDA0510SMES2 10/10/05 Sed X

05-A012141 USDA0510Eld1 10/10/05 Tiss X X

05-A012142 USDA0510Eld2 10/10/05 Tiss X

05-A012143 USDA0510Eld3 10/10/05 Tiss X

05-A012144 USDA0510ELDSD1 10/10/05 Tiss X

05-A012145 USDA0510ELDSD2 10/10/05 Tiss X

05-A012146 USDA0510ELDSD3 10/10/05 Tiss X

05-A012147 USDA0510ELDS1 10/10/05 Sed X X

05-A012148 USDA0510ELDS2 10/10/05 Sed X

05-A012149 USDA0510SQB1 10/10/05 Tiss X X

05-A012150 USDA0510SQB2 10/10/05 Tiss X

05-A012151 USDA0510SQB3 10/10/05 Tiss X

05-A012152 USDA0510SQBSD1 10/10/05 Tiss X

05-A012153 USDA0510SQBSD2 10/10/05 Tiss X

05-A012154 USDA0510SQBSD3 10/10/05 Tiss X

05-A012155 USDA0510SQBS1 10/10/05 Sed X X

05-A012156 USDA0510SQBS2 10/10/05 Sed X

05-A012157 USDA0510Ham1 10/11/05 Tiss X X

05-A012158 USDA0510Ham2 10/11/05 Tiss X

05-A012159 USDA0510Ham3 10/11/05 Tiss X

05-A012160 USDA0510HAMSD1 10/11/05 Tiss X

05-A012161 USDA0510HAMSD2 10/11/05 Tiss X

Page 22 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 05-A012162 USDA0510HAMSD3 10/11/05 Tiss X

05-A012163 USDA0510HAMS1 10/11/05 Sed X X

05-A012164 USDA0510HAMS2 10/11/05 Sed X

05-A012214 USDA0510SMESSD1 10/10/06 Tiss X

05-A012691 USDA0510WBC1 10/20/05 Tiss X

05-A012692 USDA0510WBC2 10/20/05 Tiss X

05-A012693 USDA0510WBC3 10/20/05 Tiss X

05-A012694 USDA0510WBCSD1 10/20/05 Tiss X

05-A012695 USDA0510WBCSD2 10/20/05 Tiss X

05-A012696 USDA0510WBCSD3 10/20/05 Tiss X

05-A012697 USDA0510WBCS1 10/20/05 Sed X X

05-A012698 USDA0510WBCS2 10/20/05 Sed X

05-A012748 USDA0510NET1 10/18/05 Tiss X

05-A012749 USDA0510NET2 10/18/05 Tiss X

05-A012750 USDA0510NET3 10/18/05 Tiss X

05-A012751 USDA0510NES1 10/18/05 Tiss X

05-A012752 USDA0510NES2 10/18/05 Tiss X

05-A012753 USDA0510NES3 10/18/05 Tiss X

05-A012754 USDA0510TIL1 10/17/05 Tiss X

05-A012755 USDA0510TIL2 10/17/05 Tiss X

05-A012756 USDA0510TIL3 10/17/05 Tiss X

05-A012757 USDA0510NETS1 10/18/05 Sed X X

05-A012758 USDA0510NETS2 10/18/05 Sed X

05-A012759 USDA0510TILS1 10/17/05 Sed X X

05-A012760 USDA0510TILS2 10/17/05 Sed X

05-A013962 USDA0510HUM1 10/16/05 Tiss X

05-A013963 USDA0510HUM2 10/16/05 Tiss X

05-A013964 USDA0510HUM3 10/16/05 Tiss X

05-A014074 USDA0510Tom1 10/15/05 Tiss X

05-A014075 USDA0510Tom2 10/15/05 Tiss X

05-A014076 USDA0510Tom3 10/15/05 Tiss X

05-A014077 USDA0510TOMS1 10/15/05 Sed X X

05-A014078 USDA0510TOMS2 10/15/05 Sed X

05-A015745 USDA0510WIN1 12/19/05 Tiss X

Page 23 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories Analysis Total Diss. Laboratory Field Sampling Cd Cd TOC Cd in Grain Sample ID Sample ID Date Matrix As Zn Zn PCBs TS TSS Seston Size 05-A015746 USDA0510WIN2 12/19/05 Tiss X

05-A015747 USDA0510WIN3 12/19/05 Tiss X

05-A015748 USDA0510WINS1 12/19/05 Sed X X

05-A015749 USDA0510WINS2 12/19/05 Sed X

06-A001026 USDA0601TOM1 01/26/06 Tiss X

06-A001027 USDA0601TOM2 01/26/06 Tiss X

06-A001028 USDA0601TOM3 01/26/06 Tiss X

06-A001029 USDA0601TOMS1 01/26/06 Sed X X

06-A001030 USDA0601TOMS2 01/26/06 Sed X

06-A001031 USDA0601TOMW1 01/25/06 Water X

06-A001032 USDA0601TOMW2 01/25/06 Water X

06-A001033 USDA0601TOMW3 01/25/06 Water X X

06-A002453 USDA0601HUM1 02/27/06 Tiss X

06-A002454 USDA0601HUM2 02/27/06 Tiss X

06-A002455 USDA0601HUM3 02/27/06 Tiss X

06-A002456 USDA0601HUMS1 02/27/06 Sed X X

06-A002457 USDA0601HUMS2 02/27/06 Sed X

06-A002458 USDA0601HUMW1 02/28/06 Water X

06-A002459 USDA0601HUMW2 02/28/06 Water X

06-A002460 USDA0601HUMW3 02/28/06 Water X X Note: Tiss - Tissue samples. Sed - Sediment. As - Arsenic. Cd - Cadmium. Zn - Zinc. Cd in Seston - Cadmium in filter residual (TSS). PCB - Polychlorinated biphenyl. TOC - Total organic carbon, analyzed for sediment samples only. TSS – Total suspended solids. TS - Total solids, analyzed for sediment samples only. X - The analysis was requested and performed on the sample.

Page 24 of 24 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study AMTEST Laboratories

Data Review Report

Characterization of the Cadmium Health Risk, Concentrations and Ways to Minimize Cadmium Residues in Shellfish

Prepared for:

Integral Consulting, Inc. 1205 West Bay Drive NW Olympia, WA 98502

Prepared by:

Pyron Environmental, Inc. 3530 32 nd Way NW Olympia, WA 98502

August 23, 2006

ACRONYMS

%R percent recovery CLP U.S. EPA Contract Laboratory Program CRM certified reference material COC chain-of-custody EPA U.S. Environmental Protection Agency ICP/MS inductively coupled plasma/mass spectrometetry MDL method detection limit g/kg microgram per kilogram g/L microgram per liter LFB laboratory fortified blank MS matrix spike MSD matrix spike duplicate PQL practical quantitation limit QA/QC quality assurance/quality control RPD relative percent difference SD standard deviation SDG sample delivery group TSS total suspended solids

Page 2 of 11 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study Brooks Rand, LLC. SUMMARY

I. Sample names, laboratory assigned sample identity, and the correspondent analyses included in this review are summarized in Attachment A – Sample Index.

II. The analytical parameters validated herein, the analytical methods, and the laboratory performing the analyses are summarized as follows:

Parameter Analytical Method Laboratory Total Cadmium & Zinc (Water) EPA 1640 Modified

Dissolved Cadmium & Zinc (Water) EPA 1640 Modified Brooks Rand LLC. Seattle, Washington 98107 Cadmium & Zinc (Seston) EPA 1638 Modified

Total Suspended Solids (TSS) EPA 160.2

Note: Seston is defined as the material retained on the filter used for dissolved metal sample preparation.

III. The quality assurance/quality control (QA/QC) parameters validated are summarized as follows:

Analysis

Total & Dissolved Cadmium in Seston TSS QC Parameter Cadmium & Zinc

Holding Time √ √ √

Initial Calibration NR NR NA

Calibration Verification √ √ NA

Blanks 3.3 √ NA

MS/MSD √ √ NA

Duplicate Analysis 3.4 √ NR

Certified Reference Material √ √ NA

Laboratory Fortified Blank 3.7 √ NA

Analyte Quantitation and Reporting Limits 3.8 √ NA

Overall Assessment √ √ √

Notes: “ √” denotes that the QA/QC parameter met the criteria for the analytical parameter. A number (e.g., “ 3.3 ”) denotes that the QA/QC parameter did not meet the criteria or required further discussion; the number indicates the report section that included discussion. NA – The QC parameter is not applicable for the analytical parameter. NR – The QC results were not reported and were not evaluated as part of this validation.

Page 3 of 11 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study Brooks Rand, LLC. IV. Data qualification and assigned data qualifiers are summarized as follows:

Data Report Sample ID Analyte Qualifier Reason Section

USDA0510-SME1 J- Method blank contained the USDA0510-TIL J- Total Zinc analyte at a level >1/10 the 3.3 USDA0510-NET J- sample concentration. USDA0510-TOM J-

USDA0510-SME2 0.50 g/L U Method blank contained the USDA0510-TIL J- Dissolved Zinc analyte at a level >1/10 the 3.3 USDA0510-NET J- sample concentration. USDA0510-WBC J-

Method blank contained the Total Cadmium J- USDA0510-WBC analyte at a level >1/10 the 3.3 Dissolved Cadmium J- sample concentration.

USDA0510-Ham2 J- Filter blank contained the USDA0510-Eld2 J- Dissolved Cadmium analyte at a level >1/10 the 3.3 USDA0510-SME2 J- sample concentration. USDA0510-SQB2 J-

Filter blank contained the USDA0510-WIN Dissolved Zinc J- analyte at a level >1/10 the 3.3 sample concentration.

Duplicate analyses RPD Total Cadmium J USDA0510-TOM value exceeded control 3.4 Total Zinc J criterion.

Total Zinc J- LFB %R value was below the USDA0510-WIN 3.7 Dissolved Zinc J- lower control limit

Dissolved analyte concentration was USDA0510-Eld2 J+ Dissolved Zinc significantly greater than the 3.8 USDA0510-SQB2 J+ total recoverable analyte value.

V. Data qualifiers are defined as follows:

Data Qualifier Definition

The analyte was detected above the reported quantitation limit, and the reported concentration J was approximate.

The analyte was detected above the reported quantitation limit, and the reported concentration J- was approximate and bias low.

The analyte was detected above the reported quantitation limit, and the reported concentration J+ was approximate and bias high.

The analyte was analyzed for, and was considered not detected at the associated quantitation U limit.

Page 4 of 11 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study Brooks Rand, LLC. DATA VALIDATION FINDINGS

1. Introduction

This report presents and discusses the findings of data review performed by Pyron Environmental, Inc. on data collected during October through November 2005 for the referenced project. Laboratory reports submitted for this review consisted of summary of sample results, chain-of-custody (COC) documents, and summary of laboratory quality control analyses ( i.e., calibration verifications, method blanks, duplicate, standard reference material, and matrix spike analyses). The review did not include instrument tuning, initial calibrations, or any instrument printout raw data. QC parameters evaluated herein are outlined in Summary, Section II at the beginning of this report.

The laboratory performance-based or method-specific control limits for the QC analyses were applied to evaluate the data quality. The review procedure in general followed requirements specified in the U.S. Environmental Protection Agency’s (EPA) Contract Laboratory Program (CLP) National Functional Guidelines for Inorganic Data Review (EPA 2004). Findings identified in this review are discussed and presented in the section pertinent to each analysis. Qualified data and assigned data qualifiers are summarized in Summary, Section IV .

2. Sample Delivery Documentation and Preservation

Samples were received at the laboratory intact and consistent with the accompanying chain-of-custody (COC) documentation as noted by the laboratory.

Selected samples under sample delivery groups 05BR1518 and 05BR1742 were received at the temperature of 9°C and 11°C respectivel y, exceeding the recommended criterion of 4±2°C. This had no significant effects on m etals or total suspended solids analysis; no actions were taken.

3. Metals (Cadmium & Zinc)

3.1 Holding Times

Water samples should be analyzed within 180 days for metals. Samples were analyzed within the required holding time.

The filtration of sample USDA0510-WIN (for TSS and seston metals analyses) was performed 3 days after collection, exceeding the recommended 2-day holding time. The effects of the holding time exceedance were determined insignificant; no actions were taken on this basis.

3.2 Initial and Continuing Calibration Verification

The percent recovery (%R) values for all initial and continuing calibration verification analyses were within the laboratory control limits (80 – 120%).

Page 5 of 11 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study Brooks Rand, LLC. 3.3 Blanks (Method Blanks & Filter Blanks)

A method blank was prepared and analyzed with each analytical batch. The laboratory analyzed method blanks multiple times (as an alternative approach specified in the method), and reported the average and standard deviation of the results. Sample results less than 10 times the average concentration in the associated method blank were considered affected and qualified as follows:

Data Method Blank Average Concentration Qualification Laboratory ID Analyte in Blank ( g/L) Affected Sample (g/L)

USDA0510-SME1 J- USDA0510-SME2 0.50 U USDA0510-TIL (Total) J- 05-0840 Zinc 0.29 USDA0510-TIL (Dissolved) J- USDA0510-NET (Total) J- USDA0510-NET (Dissolved) J- USDA0510-TOM (Total) J-

05-0899 Zinc 0.10 USDA0510-WBC (Dissolved) J-

USDA0510-WBC (Total) J- 05-0899 Cadmium 0.011 USDA0510-WBC (Dissolved) J-

A filter blank was prepared and analyzed with dissolved cadmium and zinc. Target analytes were detected in selected filter blanks. Sample results less than 10 times the detection in the associated filter blank were considered affected and qualified below:

Filter Blank Concentration Data Qualification Laboratory ID Analyte in Blank ( g/L) Affected Sample (g/L)

USDA0510-Ham2 J- USDA0510-Eld2 J- 05BR1518-17 Dissolved Cadmium 0.040 USDA0510-SME2 J- USDA0510-SQB2 J-

05BR1923-5 Dissolved Zinc 0.840 USDA0510-WIN J-

3.4 Duplicate Sample

Duplicate sample analyses were performed on project samples. The relative percent difference (RPD) values met the control criteria (25% for detections ≥ 5xPQL, and ±1xPQL for detections < 5xPQL), except for the following:

Sample Duplicate Result Result Data Sample ID Analyte (g/L) (g/L) RPD Qualification

USDA0510-TOM Total Zinc 0.01 0.37 >100% J

USDA0510-TOM Total Cadmium 0.038 0.081 71% J

Page 6 of 11 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study Brooks Rand, LLC.

3.5 Certified Reference Material (CRM)

CRM sample analyses were performed in lieu of laboratory control samples. All %R values were within 75 - 125%.

3.6 Matrix Spike (MS)

Matrix spike analyses were performed on selected project samples. All %R values met the control limits (75 – 125%).

In cases where the sample volume was insufficient for the seston matrix spike analysis, a post-preparation spike analysis was performed instead. All %R values were within 75 –125%.

In cases where MSD analyses were also performed, the MS/MSD RPD values were within 25%, except for total recoverable cadmium and zinc in the MS/MSD performed on sample USDA0510-HUM (28.1% and 35.1%). The RPD values for the duplicate analyses were within the criterion; no further actions were taken on this basis.

3.7 Laboratory Fortified Blanks (LFBs)

LFB analyses were performed in lieu of laboratory blank spike analyses. All %R valued were within the laboratory control limits (75 – 125%), except for the following:

LFB ID Control Data (Batch No.) Analye %R Limits Affected Sample Qualification USDA0510-WIN (Total) J- 05-1136 Zinc 70% 75 – 125% USDA0510-WIN (Dissolved) J-

3.8 Analyte Quantitation and Reporting Limits

In some cases, the dissolved analyte concentrations were significantly (5x) greater than the respective total recoverable levels, likely due to artifacts introduced during field or laboratory procedures. Dissolved analyte data were qualified accordingly and summarized as follows:

Total Recoverable Dissolved Concentration Concentration Sample ID Analyte (g/L) (g/L) Data Qualification USDA0510-Eld2 4.080 33.800 J+ Zinc USDA0510-SQB2 4.660 21.800 J+

3.9 Overall Assessment of Data Usability

Based on the information presented in the laboratory reports, cadmium and zinc data are acceptable for use as qualified.

Page 7 of 11 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study Brooks Rand, LLC.

5. Total Suspended Solids (TSS)

5.1 Holding Time

TSS should be analyzed within 7 days of collection. All samples were analyzed within the required holding time.

5.2 Method Blank

Method blanks were prepared and analyzed as required. TSS were not detected at or above the quantitation limits.

5.3 Overall Assessment of Data Usability

Based on the information presented in the laboratory reports, TSS data are acceptable for use.

Approved By: Date:

Mingta Lin

Page 8 of 11 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study Brooks Rand, LLC. REFERENCES

Method 1638 - Determination of Trace Elements in Ambient Waters by Inductively Coupled Plasma-Mass Spectrometry , U.S. Environmental Protection Agency,Office of Water, Engineering and Analysis Division, January 1996.

Method 1637- Determination of Trace Elements in Ambient Waters by Off-Line Chelation Preconcentration and Stabilized Temperature Graphite Furnace Atomic Absorption, U.S. Environmental Protection Agency,Office of Water, Engineering and Analysis Division, January 1996.

USEPA Methods for Chemical Analysis of Water and Wastes, EPA –600/4-79-020, March 1983 Revision.

Recommended Protocols for Measuring Conventional Sediment Variables in Puget Sound, Puget Sound Water Quality Authority, March 1986.

USEPA Methods for Chemical Analysis of Water and Wastes, EPA –600/4-79-020, March 1983 Revision.

Page 9 of 11 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study Brooks Rand, LLC. ATTACHMENT A

Sample Index

Analysis Total Dissolved Laboratory Field Sampling Cadmium Cadmium Cadmium Sample ID Sample ID Date Matrix Zinc Zinc TSS in Seston 05BR1518-1 USDA0510-Ham1 10/11/05 Water X

05BR1518-2 USDA0510-Ham2 10/11/05 Water X

05BR1518-3 USDA0510-Ham3 10/11/05 Water X

05BR1518-4 USDA0510-Ham4 10/11/05 Water X

05BR1518-5 USDA0510-Eld1 10/10/05 Water X

05BR1518-6 USDA0510-Eld2 10/10/05 Water X

05BR1518-7 USDA0510-Eld3 10/10/05 Water X

05BR1518-8 USDA0510-Eld4 10/10/05 Water X

05BR1518-9 USDA0510-SME1 10/10/05 Water X

05BR1518-10 USDA0510-SME2 10/10/05 Water X

05BR1518-11 USDA0510-SME3 10/10/05 Water X

05BR1518-12 USDA0510-SME4 10/10/05 Water X

05BR1518-13 USDA0510-SQB1 10/10/05 Water X

05BR1518-14 USDA0510-SQB2 10/10/05 Water X

05BR1518-15 USDA0510-SQB3 10/10/05 Water X

05BR1518-16 USDA0510-SQB4 10/10/05 Water X

05BR1518-17 Filter Blank 09/23/04 Water X

05BR1564-1 USDA0510-TIL 10/18/05 Water X

05BR1564-2 USDA0510-TIL 10/18/05 Water X

05BR1564-3 USDA0510-TIL 10/18/05 Water X

05BR1564-4 USDA0510-TIL 10/18/05 Water X

05BR1564-5 USDA0510-NET 10/18/05 Water X

05BR1564-6 USDA0510-NET 10/18/05 Water X

05BR1564-7 USDA0510-NET 10/18/05 Water X

05BR1564-8 USDA0510-NET 10/18/05 Water X

05BR1564-9 Filter Blank 10/20/05 Water X

05BR1587-1 USDA0510-WBC 10/20/05 Water X

05BR1587-2 USDA0510-WBC 10/20/05 Water X

05BR1587-3 USDA0510-WBC 10/20/05 Water X

05BR1587-4 USDA0510-WBC 10/20/05 Water X

05BR1587-5 Filter Blank 10/31/05 Water X

Page 10 of 11 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study Brooks Rand, LLC. Analysis Total Dissolved Laboratory Field Sampling Cadmium Cadmium Cadmium Sample ID Sample ID Date Matrix Zinc Zinc TSS in Seston 05BR1711-1 USDA0510-HUM 11/16/05 Water X

05BR1711-2 USDA0510-HUM 11/16/05 Water X

05BR1711-3 USDA0510-HUM 11/16/05 Water X

05BR1711-4 USDA0510-HUM 11/16/05 Water X

05BR1711-5 Filter Blank 11/16/05 Water X

05BR1742-1 USDA0510-Tom 11/15/05 Water X

05BR1742-2 USDA0510-Tom 11/15/05 Water X

05BR1742-3 USDA0510-Tom 11/15/05 Water X

05BR1742-4 USDA0510-Tom 11/15/05 Water X

05BR1742-5 Filter Blank 11/18/05 Water X

05BR1923-1 USDA0510-WIN 12/19/05 Water X

05BR1923-2 USDA0510-WIN 12/19/05 Water X

05BR1923-3 USDA0510-WIN 12/19/05 Water X

05BR1923-4 USDA0510-WIN 12/19/05 Water X

05BR1923-5 Filter Blank 12/22/05 Water X Note: Filter blank samples were generated in the laboratory with 1-L reagent water taken through the same procedure of filtering performed on samples prepared for dissolved metal analysis. TSS – Total suspended solids. X - The analysis was requested and performed on the sample.

Page 11 of 11 Pyron Environmental, Inc. Data Validation Report USDA Cadmium Study Brooks Rand, LLC.