Comparison of domoic acid concentration in king scallops, Pecten maximus from seabed and suspended culture systems.

ABSTRACT Domoic acid (DA) the toxin responsible for amnesic shellfish poisoning (ASP) has proven problematic for king scallop Pecten maximus fisheries and aquaculture in Ireland. Toxin concentration in hepatopancreas of individual scallops and composite samples of gonad and adductor muscle of scallops suspended from a submerged longline, 2 m beneath the water surface and on a seabed site, 12-15 m beneath the suspended scallops were monitored from February 2003 to February 2004 at an aquaculture site in Clew Bay, Ireland. DA concentration in hepatopancreas of scallops from the seabed and longline peaked in April 2003, individual concentrations reaching 1037.1 [micro]g.g.sup.-1] and 1212.6 [micro]g.[g.sup.-1] respectively. No statistically significant differences were exhibited between DA concentration in hepatopancreas of seabed and longline scallops except on 1 of the 10 sampling occasions. Slow depuration of DA toxin from hepatopancreas occurred from April 2003 to June 2003 and the concentration remained relatively stable from June 2003 to Feb 2004. Interpretation of DA concentrations in gonad were complicated by the lower concentrations recorded and the variable size of the gonad caused by the reproductive cycle over the 12-month study duration. DA concentrations in adductor muscle were below the limit of detection throughout the investigation. In summary DA concentration between scallops held in suspension or maintained on the seabed exhibited minor difference, and routine monitoring samples could be collected annually and held in suspended culture systems rather than using more expensive diver collection for sample procurement.

KEY WORDS: amnesic shellfish poisoning (ASP), domoic acid, Pecten maximus, scallop

INTRODUCTION

Amnesic shellfish poisoning (ASP) was first recorded in Canada in 1987 when over 100 people became ill after consuming mussels Mytilus edulis contaminated with the DA toxin. Early symptoms included nausea, gastroenteritis and vomiting followed within 48 h by neurological symptoms such as confusion, lethargy, disorientation and memory loss that persisted indefinitely; three of those affected in this outbreak died (Quilliam & Wright 1989, Todd 1993). The toxin, a small tricarboxylic amino acid belonging to the kainoid class of compounds, has been reported in 10 species of diatoms of the genus Pseudo-nitzschia (Bates 2000). Filter feeding bivalves such as scallops and mussels consuming these toxic phytoplankton species can accumulate the toxin to high concentrations (Zaman et al. 1997). Human consumption of shellfish contaminated with DA results in ASP.

Detection of DA in cultured mussels from Galicia, Spain in 1994 (Arevalo et al. 1997) and a range of Portuguese shellfish in 1995 (Vale & Sampayo 2001) led to the introduction of amendment 61/97 of EU Directive 91/492/EEC, which established the maximum allowable concentration of DA in whole shellfish or edible parts as 20 [micro]g.[g.sup.-1]. Extensive shellfishery closures have since been recorded in many European countries, perhaps the most significant being those applied to the king scallop fishery off the west coast of Scotland from 1999 onwards, which resulted in considerable financial hardship for scallop fishermen (Gallacher et al. 2001, Smith et al. 2005). Prolonged closures of scallop fisheries in Europe because of elevated DA concentration persisting in some instances for months or years (Arevalo et al. 1998, Fernandez et al. 2000), slow rates of depuration in this species, localization of the majority of toxin in the hepatopancreas and high inter-animal variation between scallops led to the introduction of EU Commission Decision 2002/226/EC. This allowed harvesting of scallops with a whole body DA concentration exceeding 20 [micro]g.[g.sup.-1] if the parts to be marketed, principally the adductor muscle and gonad, contained less than 4.6 [micro]g.[g.sup.-1]. Scallop fishing from grounds that otherwise would have remained closed has been allowed, but this EU decision has necessitated that almost all the catch be processed in approved plants prior to sale. Restrictions on the sale of fresh scallops (in-shell), which have traditionally commanded a premium price compared with processed product, has resulted in a 2-tier market for fishermen, a high value one for fresh product, which can rarely be supplied and a lower value one for processed product.

In Ireland high DA concentrations in scallop recorded in late 1999 and early 2000 resulted in the closure of all scallop production/harvesting over the millennium period. Over the last 5 years, as in much of Europe, this toxin has proven particularly problematic in king scallop Pecten maximus fisheries. Recent DA concentrations of 618.2 [micro]g.[g.sup.-1] reported in April 2005 in mussels Mytilus edulis from the west coast of Ireland represented the first time the toxin had been detected above 50 [micro]g.[g.sup.-1] in this species. Interest in DA concentrations in scallop suspended in the water column compared with scallops on the seabed has been expressed from a number of perspectives. From an aquaculture perspective, scallops can be farmed either by sowing juveniles into seabed areas and harvesting at a later date or by more intensive culture in lantern nets suspended from submerged longlines. Both methods have been attempted in Ireland and in some bays both techniques have been used. Current legislation on the monitoring of DA refers to the number of shellfish to be used as a sample, the parts to be analyzed, the analytical technique to be used and the maximum concentrations. If differences in toxin concentration occurred between scallops produced by the two methods, then the possibility exists that scallops produced by one technique might be sold into a different market sector than the other, for example suspended scallops might have to be processed, whereas sown scallops from the seabed could be sold into the fresh market or vice versa. Given the higher value of fresh in-shell product, it is therefore conceivable that the production technique to be used in an area and the economics of scallop farming might be influenced by the likely toxin concentration in finished product. Such a possibility also raised the question that perhaps suspending scallops in shallower, phytoplankton-rich waters might increase rates of toxin deputation. Although bivalves that are actively feeding on nontoxic algae are likely to exhibit faster depuration (Bricelj & Shumway 1998), when mussels Mytilus galloprovincialis, contaminated with PSP toxin, okadaic acid, were transferred to a toxin-free zone the rate of depuration did not decrease significantly (Blanco et al. 1999). Nevertheless, the possibility that maintaining scallops in suspended culture nets until DA concentrations decreased to levels that allowed their sale into the more lucrative fresh market is worthy of further investigation for those faced with regular shellfish closures. The converse, whereby scallops suspended in waters containing a higher concentration of toxic species might accumulate more toxin than scallops on the seabed, should also be considered as a possible outcome of suspending scallops in the water column. Diver collection of farmed scallop has been proposed as a harvesting technique from intensive seabed plots. Though efficient as a means of scallop collection, such a technique is a slow method of harvesting, necessitating the holding of scallops for a period while a consignment is gathered. Concern has been expressed that during this holding period, scallops may accumulate toxins to levels such that their sale into the more valuable fresh market was no longer an option.

From a regulatory perspective, interest in DA concentrations in suspended versus seabed scallops centered on sample collection. In some inshore aquaculture sites in Ireland, divers are used for the collection of scallops for routine toxin monitoring. Recently introduced safety regulations for professional divers have resulted in an escalation of the costs associated with sample procurement using such methods. In circumstances where no differences existed between seabed and suspended scallops, sample provision for routine monitoring could be performed much more economically using scallops maintained for the purpose in suspended culture nets.

In addition to DA production from a range of Pseudo-nitzschia species, a benthic diatom, Nitzschia navis varingica has recently been reported as a further source of the toxin (Kotaki et al. 2004). The distribution of this species is at present largely unknown. If in a comparative study of DA concentration in seabed and suspended scallops, higher toxin concentrations were recorded in seabed scallops, DA production by benthic species might be suspected as a possible contributory factor.

Given the significance of results regarding DA toxin concentration in seabed and suspended scallops to fishermen, aquaculture producers, regulators and scientists; this comparative study was undertaken over a 12-month period at an aquaculture site on the west coast of Ireland.

MATERIALS AND METHODS

King scallop Pecten maximus were collected by diving from a seabed aquaculture site in Clew Bay, County Mayo off the west coast of Ireland and subdivided into 2 batches--a suspended batch, which was held in lantern nets 2 m below the water surface from a submerged longline and a seabed batch, which was returned to the seabed below the longline at a depth of approximately 12 m to 15 m (Fig. 1). Samples from both batches were provided for DA analysis at approximately monthly intervals from February 2003 to February 2004. Each sample comprised 12 individuals from each batch of mean shell length between 100 mm to 115 mm. On three occasions one or two shells in the longline sample were empty on arrival at the laboratory.

[FIGURE 1 OMITTED]

After shell measurement, scallops were dissected and the weight of total soft tissue and individual weight of hepatopancreas, gonad and adductor muscle recorded. The hepatopancreas of each scallop was analyzed individually to provide data on variability in DA concentration within each sample batch on each date. Composite samples of gonad and composite samples of adductor muscle from each sample group on each date were prepared for analysis, because previous studies suggested that DA concentration in these tissues would be much lower than in hepatopancreas; and hence differences, if present, between seabed and longline scallops would be more difficult to confirm because of the lower concentrations. Subsamples of composite tissues were analyzed in triplicate for DA toxin concentration.

DA was extracted from scallop tissue samples using procedures based on Quilliam et al. (1995) with several modifications. Extraction was performed from approximately 4.0 g of tissue homogenate with 16 mL of 50:50 extraction solvent (methanol:water) in a blender (Ultra Turrax T25, IKA-Works.) for 4 min at high speed. DA was extracted from each hepatopancreas individually. Where hepatopancreas weight exceeded 4.0 g, the organ was homogenized and a 4.0 g subsample used for extraction. After extraction, homogenates were centrifuged at 3,800 rpm for 30 min. A sample of supernatant was filtered using a methanol-compatible 0.45-[micro]m syringe filter and the combined concentration of DA and epi-DA in the filtered extract determined using a Shimadzu HPLC/UV and following equipment upgrade a Shimadzu HPLC/DAD (Mason Technology, Dublin). In those instances where the combined DA concentration exceeded that of the highest standard, the filtered extract was diluted with extraction solvent and the measurement of DA and epi-DA repeated. All solvents used were HPLC grade obtained from Lennox Laboratory Suppliers, Dublin. Mobile phase consisted of 10% acetonitrile and 1% TFA (trifluoroacetic acid) prepared with deionized water. HPLC flow rate was 0.5 mL.[min.sup.-1] and injection volume 20 [micro]L. Calibration was performed externally using six DA calibration standards between 0.2 [micro]g.m[L.sup.-1] to 10 [micro]g.m[L.sup.-1] prepared from certified reference standard obtained from the NRC, Canada. Calibration curves were prepared for each sample batch and were always linear ([R.sup.2] > 0.999).

Statistical analysis of all data was performed using SPSS, Version 12.

RESULTS

Scallops used in this comparative study of DA toxin concentration were of a similar size (Table 1). Based on individual scallop measurements, the mean shell length and shell height for seabed scallops was 108.8 [+ or -] 3.9 mm by 96.4 [+ or -] 3.9 mm compared with 111.6 [+ or -] 5.2 mm by 99.4 [+ or -] 4.6 mm for longline scallops.

Mean DA concentration in the hepatopancreas of individual seabed scallops was 326.4 [+ or -] 169.9 [micro]g.[g.sup.-1] compared with 313.9 [+ or -] 214.6 [micro]g.[g.sup.-1] for longline scallops over the 12-month study at this location.

Mean DA concentrations in hepatopancreas and gonad of seabed and longline scallops on the 10 sampling occasions are provided in Table 1. DA toxin concentration in hepatopancreas of seabed and longline scallops exhibited similar trends over the 12-month duration (Fig. 2). The highest mean concentrations of DA in hepatopancreas were recorded on the second sampling occasion (April 3, 2003), individual DA concentrations reaching 1037.1 [micro]g.[g.sup.-1] in seabed scallops and 1212.6 [micro]g.[g.sup.-1] in longline scallops respectively. Comparison of the mean DA concentrations in hepatopancreas of seabed and longline scallops using a t-test demonstrated no statistically significant difference for 9 of the 10 samples. Only the sample from November 12, 2003 showed a significant difference (n = 23, t = 3.263, sig. = 0.004) in the mean DA concentration between seabed scallops (298.3 [+ or -] 89.4 [micro]g.[g.sup.-1]) and longline scallops (172.4 [+ or -] 95.7 [micro]g.[g.sup.-1]).

[FIGURE 2 OMITTED]

DA concentrations in composite samples of gonad tissue never exceeded the regulatory limit of 20 [micro]g.[g.sup.-1] and were considerably lower than concentrations recorded in hepatopancreas. Overall mean DA concentrations based on triplicate analysis of composite gonad samples were 5.0 [+ or -] 2.1 [micro]g.[g.sup.-1] in seabed scallops compared with 3.8 [+ or -] 1.9 [micro]g.[g.sup.-1] in longline scallops. DA toxin concentration in gonads of seabed and longline scallops exhibited considerable fluctuations and dissimilar trends over the 12-mo study duration (Fig. 2). Because DA toxin concentration of gonads is influenced by both the mass of DA in the intestinal loop within the gonad and the mass of gonad tissue itself, the reproductive condition of scallops from both sample batches was determined in an attempt to understand the fluctuations and trends over the 12-mo study (Fig. 3). Relative gonad height (RGH) in both sample groups showed a similar trend with peak RGH at the end of May. Seabed scallops exhibited higher RGH than longline scallops, although differences between sample groups were not significant (n = 236, t = 1.448, sig. = 0.149). The decline in RGH suggested a single prolonged spawning at this study location, although the use of monthly sampling for determination of the number of spawnings is not recommended (Slater 2005). Because of fluctuations in DA concentration within and between sample groups and varying reproductive condition over the 12-month study, the mass of DA in gonad was determined to examine if it better represented the changes in DA levels in gonad (Fig. 4). For comparative purposes the mass of DA in hepatopancreas was also determined (Fig. 4).

[FIGURE 3 OMITTED]

DA concentrations in composite samples of adductor muscle were below the limit of detection (LOD = 0.1 [micro]g.[g.sup.-1]) in all samples over the 12-month study duration.

DISCUSSION

DA concentrations in hepatopancreas were approximately 1 to 2 orders of magnitude greater than in gonad, hence if differences occurred between toxin concentration in longline and seabed scallops; such differences should be more easily recorded in those tissues having the higher DA concentration. Individual analysis of each hepatopancreas showed high inter-animal variability in DA concentration within each sample. Coefficients of variation (CV) of DA concentration in hepatopancreas of longline scallops ranged from 10.7% to 55.5% whereas that of seabed scallops ranged from 17.8% to 49.5%. The mean CV of DA concentration in hepatopancreas of longline and seabed scallops was 42.3% and 31.0% respectively. Similar high levels of inter-animal variability in DA concentration in king scallops have been reported (Campbell et al. 2001, Blanco et al. 2002, Bogan et al. 2006). Over the 12-month duration of the study similar DA concentrations in hepatopancreas of longline and seabed scallops were reported and a statistical difference between the two sample batches occurred on only one occasion. Similarly no significant differences in PSP toxin concentration were reported in a comparative study between sea scallops suspended 2 m beneath the water surface and those on the seabed at 11 m depth (Haya et al. 2003).

Over the 12-month duration of the study both seabed and longline scallops demonstrated rapid accumulation of DA in hepatopancreas in March 2003, slow depuration during April 2003 to June 2003 followed by relative stability from June 2003 to February 2004. The fact that there was no statistical difference in DA content of hepatopancreas between longline and seabed scallops was somewhat unexpected. Reports of toxic Pseudo-nitzschia cells descending after a bloom from nutrient-poor surface waters into higher-nutrient mixed layers suggested that differential profiles in toxicity might have been anticipated between suspended and seabed scallops (Dortch et al. 1997, Trainer et al. 1998, 2000). A time delay in PSP toxin accumulation in suspended and seabed scallops was suggested as toxic cells in surface waters descended towards the seabed as the toxic bloom subsided (Bricelj & Shumway 1998). In this study no temporal difference in peak DA concentration in hepatopancreas between longline and seabed scallops was recorded although this may have been caused by the long sampling intervals between February 26, 2003 to April 3, 2003 and April 3, 2003 to May 23, 2003.

Blooms of toxic Pseudo-nitzschia cells are often regarded as being widely dispersed throughout the water column, however they have also been reported as thin layers in the water column and following sinking as near-bottom thin layers, which may extend over large areas. Cell densities have been reported within these thin layers exceeding [10.sup.6] toxic cells.[l.sup.-1] (Rines et al. 2002). The absence of a significant difference in DA concentration in hepatopancreas between longline scallops suspended 2 m beneath the water surface and seabed scallops from 12 m to 15 m depth, in all but one of the samples, suggested that the source of DA toxicity was relatively evenly distributed throughout the water column in this location.

After the depuration phase during April 2003 to June 2003, concentrations of DA in the hepatopancreas over the remaining 8 month (June 2003 to February 2004) were relatively stable with minor fluctuations between 170 [micro]g.[g.sup.-1] to 330 [micro]g.[g.sup.-1] suggesting that no further depuration of toxin from hepatopancreas occurred, rates of uptake and depuration of DA were balanced or that the rate of depuration was very slow and not recorded over the 8-month period. Prolonged toxicity over an extended period rather than a continual decline in toxicity may have arisen from the continued development of small pulses of toxic Pseudo-nitzschia cells. Such pulses can result from Pseudo-nitzschia cells that had sunk after nutrient depletion being brought back into surface waters during upwelling events (Trainer et al. 2000). This replenishment of cells into surface waters has been proposed as a mechanism to explain why some blooms appear to persist for up to 3 mo in the field and might also explain why toxicity persisted throughout this study (Bates et al. 1989, Smith et al. 1990). Extended periods of toxicity attributed to very slow rates of depuration have also been reported in sea scallops, Placopecten magellanicus with PSP toxin over a 12-month period (Haya et al. 2003).

Many strains of domoic acid-producing diatoms have been reported, all belonging to the genus Pseudo-nitzschia except the benthic species Amphora coffeaeformis (Agardh) Kutzing (Bates 2000, Shimizu et al. 1989). Recently a benthic diatom, Nitzschia navis-varingica, collected from brackish water in Vietnamese shrimp ponds and identified as a new species has been demonstrated to be capable of DA production (Kotaki et al. 2000, 2004). Distribution of this benthic species is presently unknown although it has been reported in Japan and the Philippines and can be found in the water column (Lundholm & Moestrup 2000, Kotaki et al. 2004). The scallop toxicity results over a 12-month sampling period in this site provided no support for the hypothesis that DA production by benthic diatoms occurred.

DA concentrations in gonad were below the regulatory limit of 20 [micro]g.[g.sup.-1] throughout the study and never exceeded 10 [micro]g.[g.sup.-1] in any composite sample of seabed or longline scallop gonad. Early results during the toxin accumulation phase in the hepatopancreas (March 2003) showed an expected increase in DA concentration in gonad. A corresponding decrease in DA concentration in gonad occurred from March 2003 to June 2003, although this was somewhat slower to commence in seabed scallops. During the period of relative stability in DA concentration in hepatopancreas (June 2003 to February 2004), DA concentrations in gonad fluctuated considerably and differences were exhibited between the two groups of scallops. To understand these fluctuations and between-group differences, the reproductive condition of the scallops using relative gonad height, a measure selected because it accounted for small differences in size between the scallop samples was examined. Because the DA is predominantly contained within the intestinal loop that passes through the gonad, it was expected that as the gonad increased in size DA concentration of gonad would decrease and vice versa. The results from the February 2003 to July 2003 period demonstrated the converse, as RGH increased and then declined after spawning, DA concentration in the gonad followed a similar trend. The results demonstrated that despite the variations in gonad size, DA concentration in the gonad was predominantly determined by DA concentration in the hepatopancreas, which exhibited a similar trend over the February 2003 to July 2003 period. The influence of gonad size on DA concentration in gonad was demonstrated by comparing toxin concentration between the scallop batches, higher RGH of seabed scallops being reflected in lower DA concentration whereas lower RGH of longline scallops was reflected in comparatively higher DA concentration. Fluctuations in DA concentration in gonad from Jul 03 to Feb 04 or variations between the two groups of scallop could not explained by consideration of the RGH independently. In an attempt to explain the variations in DA concentration in gonad and gonad size, changes in the mass of DA in gonad were examined over the study duration. For comparative purposes changes in the mass of DA in hepatopancreas were also examined. Changes in the mass of DA in gonad over the 12-month period exhibited a similar trend to that for both the concentration and mass of DA in hepatopancreas. Because fluctuations in DA concentration of gonad from July 2003 to February 2004 could not be attributed to changes in gonad size, it was concluded that fluctuations during this period, and variations between the two scallop batches must have resulted from high inter-animal variability in DA concentration in gonad and experimental error. DA concentrations in gonad have exhibited high inter-animal variability in several other studies (Arevalo et al. 1998, Campbell et al. 2001). Similar difficulties to those encountered in this study in interpreting changes in DA concentration in gonad have been reported elsewhere (FSA 2001). Although regulatory monitoring of DA concentration in gonad for determining its suitability for entering the food chain is vital, the low concentrations compared with other tissues, variations in gonad size during the reproductive cycle and difficulties encountered interpreting results, suggest that its value in understanding temporal changes is questionable.

ACKNOWLEDGMENTS

YMB was the recipient of Higher Education Authority Strand I postgraduate funding under the Technological Sector Research Programme supported by the Irish National Development Plan 2000-2006. The collection and provision of scallop samples was co-ordinated by Mr. Neil O'Boyle, Clew Bay Forum and funded by the Marine Institute.

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YVONNE M. BOGAN, (1) DAVID KENNEDY, (1) ANNE L. HARKIN, (1) JOHN GILLESPIE, (1) PHILIPP HESS (2) AND JOHN W. SLATER (1).

(1) Letterkenny Institute of Technology, Port Road, Letterkenny, Co. Donegal, Ireland; (2) Marine Institute, Galway Technology Park, Parkmore, Galway, Ireland

* Corresponding author. E-mail: [e-mail]

TABLE 1.

Concentration of DA in hepatopancreas and gonad of seabed and longline
scallops from Feb 03 to Feb 04.

                                   Seabed

                        Shell                 Conc. of DA
                        Length                   in HP
            n            (mm)           ([micro]g x [g.sup.-1])

26-Feb-03   12   112.91 [+ or -] 2.71   272.58 [+ or -] 59.77
03-Apr-03   12   108.54 [+ or -] 4.08   697.96 [+ or -] 156.55
23-May-03   12   109.17 [+ or -] 3.69   457.68 [+ or -] 134.61
03-Jul-03   12   107.67 [+ or -] 2.57   216.50 [+ or -] 107.22
30-Jul-03   12   108.08 [+ or -] 3.34   332.70 [+ or -] 140.64
03-Sep-03   12   109.91 [+ or -] 2.84   304.98 [+ or -] 69.39
25-Sep-03   12   111.91 [+ or -] 2.42   223.68 [+ or -] 65.37
12-Nov-03   12   110.08 [+ or -] 1.62   298.33 [+ or -] 89.42
04-Dec-03   12   104.50 [+ or -] 2.97   238.98 [+ or -] 109.41
11-Feb-04   12   104.36 [+ or -] 2.98   245.24 [+ or -] 43.75

                     Seabed

                  Conc. of DA
                    in gonad
              ([micro]g x [g.sup.-1])

26-Feb-03      2.00 [+ or -] 0.24
03-Apr-03      5.35 [+ or -] 0.64
23-May-03      4.07 [+ or -] 0.46
03-Jul-03      2.99 [+ or -] 0.43
30-Jul-03      6.07 [+ or -] 1.21
03-Sep-03      6.72 [+ or -] 0.21
25-Sep-03      8.45 [+ or -] 1.01
12-Nov-03      3.55 [+ or -] 1.12
04-Dec-03      6.56 [+ or -] 2.65
11-Feb-04      4.02 [+ or -] 0.66

                                Longline

                        Shell                 Conc. of DA
                        Length                   in HP
            n            (mm)           ([micro]g x [g.sup.-1])

26-Feb-03   12   111.58 [+ or -] 4.19   283.67 [+ or -] 62.69
03-Apr-03   10   112.50 [+ or -] 2.46   837.70 [+ or -] 250.84
23-May-03   10   107.80 [+ or -] 2.90   452.53 [+ or -] 202.18
03-Jul-03   11   108.70 [+ or -] 5.31   296.05 [+ or -] 114.60
30-Jul-03   12   113.83 [+ or -] 5.29   265.90 [+ or -] 56.09
03-Sep-03   12   109.75 [+ or -] 4.67   262.16 [+ or -] 111.62
25-Sep-03   12   113.42 [+ or -] 5.87   210.45 [+ or -] 99.96
12-Nov-03   12   114.64 [+ or -] 6.83   172.42 [+ or -] 95.67
04-Dec-03   12   111.08 [+ or -] 5.71   208.72 [+ or -] 46.59
11-Feb-04   12   112.92 [+ or -] 4.99   244.55 [+ or -] 103.88

                    Longline

                  Conc. of DA
                    in gonad
              ([micro]g x [g.sup.-1])

26-Feb-03      2.25 [+ or -] 0.03
03-Apr-03      7.10 [+ or -] 0.23
23-May-03      7.12 [+ or -] 0.30
03-Jul-03      2.24 [+ or -] 0.24
30-Jul-03      1.96 [+ or -] 0.98
03-Sep-03      2.58 [+ or -] 0.06
25-Sep-03      3.54 [+ or -] 0.26
12-Nov-03      3.09 [+ or -] 0.15
04-Dec-03      4.36 [+ or -] 1.12
11-Feb-04      3.76 [+ or -] 0.79

Values represent the mean [+ or -] standard deviation.