Peter H Santschi
email: santschi@tamug.edu

Sarah D Oktay
email: sarah.oktay@umb.edu

Luis Cifuentes
email: lcifuentes@ocean.tamu.edu

Carbon isotopes and iodine concentrations in a Mississippi River delta core re cording land use, sediment transport, and dam building in the river's drainage basin

Keywords: Mississippi River delta, organic carbon, sediment transport, land use, radiocarbon, C-13, iodine, salt marsh

, and significantly, includes unique features that had not previously been seen in the marine environment. These special features include a plutonium isotopic close-in fallout record that indicates a purely terrestrial source for these sediment particles and the elements associated with it, and a distinct iodine isotopic peak (as well as peaks for plutonium and cesium isotopes) that indicate little bioturbation in this core. Our carbon isotopic and iodine data can thus be compared to published records of changes in drainage basin land use, river hydrology, and hydrodynamic sorting of suspended particles to elucidate if these changes are reflected in nearshore sediments. This comparison suggests a significant contribution for organic carbon (OC) from C4 plants to these sediments during the 1950's to early 1960's. Relative older carbon isotopes, and episodically high iodine concentrations (up to 34 ppm) were observed during this time period that 1) indicate sediment deposition that is coincident with the times of major hydrological changes induced from dam and levee building in both the upper and lower reaches of the Mississippi River drainage basin, and 2) suggest episodic organic carbon deposition from wetland plant residues.

Introduction

The Mississippi River watershed is the largest of the contiguous United States, draining approximately 40% of its surface area. The Mississippi River is also by far the largest source of sediment and organic matter to the Gulf of Mexico. In its delta region, the potential exists for reconstructing the history of organic matter sources eroded from a large part of the U.S. drainage basins through analysis of particulate organic carbon, POC, in radioisotopically-dated sediment cores. There have been relatively few attempts to interpret organic carbon isotopic records in terms of its sources and in a historical context (e.g., [START_REF] Eadie | Records of nutrient-enhanced coastal ocean productivity in sediments from the Louisiana continental shelf[END_REF]. Since suspended sediment loads change in response to variations in hydrological conditions along the river, and suspended sediments reaching the delta and shelf regions of the Gulf of Mexico are sorted hydrodynamically [START_REF] Bianchi | Sources of terrestrially-derived organic carbon in lower Mississippi River and Louisiana shelf sediments: Implications for differential sedimentation and transport at the coastal margin[END_REF], comparing organic carbon source datum in Mississippi Delta area sediment cores with the drainage basin historical record will have to take into account both changes in hydrological conditions as well as hydrodynamic regimes.

Of particular interest are changes in hydrological conditions such as the construction of dams and levees that were installed to reduce flooding or accommodate increases in traffic on the river. As part of the New Deal, Roosevelt backed the construction of locks and dams in order to improve trade and transportation and provide work for Americans struggling to survive after the Depression. For instance, the Fort Peck dam was constructed on the Upper Missouri in Montana between 1933 and 1940, and at the time it was built was the 5 th largest dam in the world and is still one of the world's largest earth-filled dams. In 1953, flood control dam construction began again on the Missouri River and all dam construction was completed by 1967. The Missouri River which joins the upper Mississippi near St Louis has always been the primary contributor of sedimentary material to the Mississippi, but this total contribution was reduced by 70% after the construction of these dams [START_REF] Keown | Historic trends in the sediment flow regime of the Mississippi River[END_REF]. The average annual sediment load has decreased three-fold as measured at a station in St, Louis Missouri, with a reduction from the 1953 value of 3.19x10 8 tons per year to 1.1x10 8 tons per year in 1967. Dissolved silicate loads decreased as well due to removal behind newly constructed dams [START_REF] Carey | The role of the Mississippi River in Gulf of Mexico hypoxia[END_REF]. This large decrease in sediments from damming the western tributaries (Missouri and Arkansas rivers) was counterbalanced by a significant increase in sediment loads from the Ohio River resulting from deforestation and rowcrop farming [START_REF] Keown | Historic trends in the sediment flow regime of the Mississippi River[END_REF].

As a consequence of the reduced loads from the Missouri River, the sediment load determined at Tarbert Landing along the main stem of the Mississippi River flowing into the Gulf of Mexico decreased from 3.0x10 8 tons/yr from 1950-1962 to 1.6x10 8 tons/yr for the period 1970-1978. Concurrent to the changes in flood control dams for the Mississippi River in the upper drainage basin, Old River Control Structures were constructed to regulate the flow to the Atchafalaya River in order to partition the flow between the main stem and the Atchafalaya River and prevent rapid silting of navigation channels in the lower Mississippi River. They were completed and became operational in 1963.

According to [START_REF] Keown | Historic trends in the sediment flow regime of the Mississippi River[END_REF], approximately 75% of the total sediment discharged from the lower Mississippi River was derived from the Missouri River before the 1950's. Recent work has shown that bulk particle organic carbon (POC) derived from the dominant sources of grasslands in the Missouri River watershed has a lignin signature that is typical of non-woody angiosperm sources [START_REF] Onstad | Sources of particulate organic matter in rivers from the continental USA: lignin phenol and stable carbon isotope compositions[END_REF]. Erosion of soils of the Mississippi River drainage basin with extensive grasslands containing isotopically heavier C4 plants is a main contributor of POC in the Mississippi River [START_REF] Goni | Sources and contributions of terrigenous organic carbon to surface sediments in the Gulf of Mexico[END_REF]. Downcore variations of the δ 13 C signature of POC from a site located in the Mississippi River Delta region were previously interpreted as changes between marine productivity and terrestrial organic matter inputs [START_REF] Eadie | Records of nutrient-enhanced coastal ocean productivity in sediments from the Louisiana continental shelf[END_REF]. However, these data can be reinterpreted using the data of [START_REF] Goni | Sources and contributions of terrigenous organic carbon to surface sediments in the Gulf of Mexico[END_REF] by considering the contribution of terrestrial POC from C4 plants that has increased during the past century. Land use changed in the Mississippi River Basin from the predominantly grassland (C4)-forest settings of the early 19 th century to the current intensive use of over one third of the land in the basin for agricultural purposes [START_REF] Keown | Historic trends in the sediment flow regime of the Mississippi River[END_REF].

A mechanism has been proposed by [START_REF] Bianchi | Sources of terrestrially-derived organic carbon in lower Mississippi River and Louisiana shelf sediments: Implications for differential sedimentation and transport at the coastal margin[END_REF] that can explain the observations of POC deposition in riverine and nearshore regions of woody C3 plant derived carbon, and of C4 plant-derived POC further offshore in shelf and slope sediments. These authors suggested hydrodynamic sorting of POC in Mississippi River Delta regions and the shelf waters, primarily as a result of frequent resuspension of sediments in the river bed caused by changes in river discharge. This would cause fine-grained sediments to be winnowed out in the water column and transported downstream, while the larger sediments with woody material associated with it and with higher relative densities would selectively settle out in the lower river and inner shelf sediments.

Changes in erosion river beds near New Orleans have also occurred in the 1960s in response to changes in subsidence and river hydrology [START_REF] Galler | Biogeochemical implications of levee confinement in the lowermost Mississippi River[END_REF]. This has exposed peat deposits with radiocarbon ages of 2000-4000 years, and δ 13 C values of -24.6 to -25 ‰. [START_REF] Keppler | Organoiodine formation during humification in peatlands[END_REF] found that peatlands sequester large amounts of iodine with average concentrations of 5-36 mg/kg, therefore, one could recognize such layers in a sedimentary record by their unusually high iodine concentrations.

There are other organic matter sources with high iodine concentrations, however, such as salt marsh vegetation, which typically have heavy δ 13 C signatures due to C4 photosynthetic pathways [START_REF] Hoefs | Stable Isotope Geochemistry[END_REF][START_REF] Fuge | The geochemistry of iodine -a review[END_REF]. Thus, the combined use of carbon isotopes and iodine concentrations become promising tools to differentiate peat from salt marsh organic matter sources.

Here, we will compare organic carbon isotopic and iodine records that are imprinted into a previously described Mississippi River Delta sediment core [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF]. This nearshore (60 m water depth) sediment core from the Mississippi River delta region is unique in that it has preserved the terrestrial close-in plutonium bomb fallout record that has not been documented in any previously published marine record. Early US bomb tests at the Nevada test site conducted in the early 1950's were low-yield and near-ground, moving large radioactive dust clouds from Nevada across the continental USA, which received fallout with low 240 Pu/ 239 Pu ratios of 0.05 and lower. While marine sediment Pu isotopic ratios below the sediment surface have only documented global bomb fallout 240 Pu/ 239 Pu ratios of 0.18, the sedimentary record of Pu isotopes by [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF] is, to our knowledge, the only marine record of the terrestrial close-in fallout from the Nevada test site. Thus, the low Pu isotopic ratio deeper in this sediment core is strong evidence that Pu and associated elements in this sediment core from this Mississippi River delta site are mainly from terrestrial sources, with minimal marine input. Furthermore, the iodine isotopic record from bomb fallout contains a sharp 1963 fallout peak that is preserved in this sediment core material, and thus clearly indicates a record with little alteration by benthic bioturbation or iodine diagenesis.

Most importantly, this sediment core allowed us to compare the sedimentary organic carbon and iodine signals with recorded changes in drainage basin land use, river hydrology, and hydrodynamic sorting of suspended particles. Since coastal areas are subject to organic carbon from different sources with different ages, the interpretation of any carbon isotopic record is often a challenging task, and requires additional isotopic and elemental records (e.g., Santschi et al., 2001a). The fallout isotopic records (e.g., 239 Pu/ 240 Pu, 137 C, 129 I/ 127 I, and 210 Pb xs ) that are available from the work of [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF] thus provide a unique opportunity to investigate the historical record of terrestrial organic matter inputs into the Mississippi River delta region through the additional analyses of organic carbon isotopes ( 13 C, 14 C).

Methods and experimental approaches

Sampling

Two sediment cores from the Mississippi River Bight were taken during the 1993 Texas A&M University research cruise 93P13 (aboard the R/V Pelican), approximately 24 km due west of the mouth of the South West Pass of the Mississippi River (Figure 1). This location was chosen in order to procure sediment cores with predictably high sedimentation rates based on their location within the plume of particulate material that exits the Mississippi River as can be clearly seen in Figure 1. The core location in the Mississippi River Delta region are: core 93P13 #2 at 28°55.5' N, 89° 40.6' W in about 60 m water depth in the vicinity of Southwest Pass, of which the radiochemical results are described in [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF], and carbon isotopic results described here), and parallel core 93P13 #1 from the same general site, of which the persistent inorganic and organic pollutants, as well as accompanying radiochemical results are described in Santschi et al. (2001).

Analytical techniques

Isotopic analyses of δ 13 C and ∆ 14 C were conducted on dry sediment core material. Five 500-600 mg sediment aliquots were analyzed for ∆ 14 C by the Accelerator Mass Spectrometry (AMS) facility at the Lawrence Livermore National Laboratory (LLNL), with relative standard deviations of ±5-10‰ (corresponding to errors in radiocarbon ages of 50-100 years). δ 13 C was measured on 100 mg aliquots of several depths throughout the core via conventional mass spectrometry, according to [START_REF] Cifuentes | Stable and nitrogen isotope biogeochemistry in the Delaware estuary[END_REF] and [START_REF] Guo | Isotopic and elemental characterization of colloidal organic matter from the Chesapeake Bay and Galveston Bay[END_REF]. The precision and accuracy of the d 13 C analysis was better than 0.1 ‰, as determined from replicate analyses of standards and samples [START_REF] Guo | Cycling of dissolved organic matter in the Middle Atlantic Bight as revealed by carbon isotopic ( 13 C, 14 C) signatures[END_REF]; actual 1 standard deviation values are given in Table 1. Organic and inorganic carbon concentrations were obtained using a Perkin Elmer CHN/S analyzer on 0.5 g sediment aliquots. The relative standard deviation of the particulate organic carbon analysis was ± 1% (relative) as determined from replicate analyses of standards and samples [START_REF] Guo | Isotopic and elemental characterization of colloidal organic matter from the Chesapeake Bay and Galveston Bay[END_REF], which would result in a standard deviation of 0.01 % at 1 % OC. Stable iodine (I 127 ) concentrations, previously published in [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF], were measured via ICP-MS using either a Sciex/Perkin Elmer ELAN 500 or a Hewlett Packard model 4500 instrument, after extraction of iodine from the sediments using an alkali leach and fusion method (Nishizumi et al., 1983). The sediment samples were placed in alumina oxide crucibles with a saturated NaOH solution and dried overnight. Next, concentrated Na 2 O 2 was added and the mixture is heated in a muffle furnace, first at 300°C for 30 minutes and then at 600°C for 2 hours. De-ionized water, 1M NaHSO 3 and 50% H 2 SO 4 were added to help dissolve the majority of the material. Finally, the sample was centrifuged and the supernatant decanted for iodine processing [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF].

Analytical results are shown in Table 1. As δ 13 C data show only gradual changes, this allowed interpolations for missing data points that were used to compare with • 14 C values.

Results and discussion

The 210 Pb xs and 239,240 Pu and/or 240 Pu/ 239 Pu geochronology of this sediment core makes it possible to determine average sedimentation rates for certain depth intervals, and to assign dates at three specific depths throughout the core: 1993 (surface), 1963 (at 21 cm; denoting the bomb fallout peak) and 1953 (at 59 cm; beginning of significant fallout from atmospheric testing of atomic bombs). Corresponding dates between these event horizons were assigned via linear interpolation using the calculated sedimentation rate for each portion. 210 Pb xs dating indicates a break in sedimentation rates in the early 1960's, as the sedimentation rate decreased from 2 cm/yr before that time to 0.7 cm/yr in recent years [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF], in accordance with a decrease in sediment supply at the same time.

Radiocarbon and δ 13 C data (Figure 2) from organic carbon (OC) in this delta sediment core that was previously dated using plutonium isotopic ratios and 210 Pb xs [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF] shows not only a minimum (Figure 2a) in 14 C and maximum (Figure 2b) in δ 13 C, but also a maximum in total iodine in layers dated as 1957 (Figure 3). Iodine in suspended and sinking sediments is mostly from geochemically modified erosional inputs in response to higher river flow rates [START_REF] Oktay | 129 I and 127 I transport in the Mississippi River[END_REF]. 14 C data from shelf environments receiving multiple sources of organic carbon are more difficult to interpret than those from delta regions of large rivers such as the Mississippi River. For example, a changing 14 C signal from bulk POC can be a result of a variable mixing ratio of old riverine and/or shelf organic carbon into a marine organic carbon background source (e.g., Palos Verdes shelf; Santschi et al., 2001a).

While δ 13 C peaks in about 1957, and 14 C shows a minimum at about the same time, 240 Pu/ 239 Pu ratios are steadily decreasing in sediments older than 1963, and 137 Cs required decay correction to the time of deposition because of transport and drainage basin effects [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF]. Most important for lack of iodine mobility and lack of bioturbation, 129 I/ 127 I ratios exactly track the Pu isotopic ratios (Figure 5 of [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF], despite multiple sources. This indicates that the 129 I, 239 Pu and 240 Pu and other bomb fallout nuclides were from Mississippi River drainage basin soils, deposited from close-in tropospheric fallout originating from the Nevada test sites in the 1950's [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF]. Therefore sediments and POC in this section of the core are clearly of terrestrial origin.

Thus, the observations of changing sedimentation rate, coupled to the change in carbon isotopic signatures of sedimentary organic carbon can be interpreted relative to decreases in sediment supply to the delta caused by anthropogenic modification of the river flow regime, as well as changes in organic carbon sources.

Interestingly, core 93P13#1, taken from a nearby site (Santschi et al., 2001b), showed similar sedimentation rates over the past 50-100 years based on 210 Pb xs and 239,240 Pu peaks, i.e., 0.7 cm/yr, but did not show decreasing 240 Pu/ 239 Pu ratios below the peak. This suggests that the depositional environment near the site our core (93P13 #2) was taken was uneven, possibly with bottom flows of river sediment fingering out from the delta region. Inventories of particlereactive 239,240 Pu in both cores from this site were enhanced over those expected from atmospheric deposition, which is to be expected for a river delta region. While Pu isotope inventories in sediments off the Mississippi River Delta are elevated and decrease with increasing water depth [START_REF] Yeager | Sediment accumulation and radionuclide inventories ( 239,240 Pu, 210 Pb and 234 Th) in the northern Gulf of Mexico, as influenced by organic matter and macrofaunal density[END_REF], this elevated Pu inventory in our core, together with the unique signature of lower 240 Pu/ 239 Pu ratios in subsurface sediments, also agrees with our contention of significant inputs of terrestrial material to the area our sediment core was taken.

Iodine concentrations in our core peak near a depth of 40 cm (dated to around 1957), and periodically below, with significant offsets of these few data points from the trend line of iodine vs. OC concentration (Figure 4). While the trend line shows molar I/C ratios of about 5x10 -5 , the few high points with higher iodine concentrations have molar I/C ratios of about 20x10 -5 . No I/C data is available from Mississippi River wetlands. However, both ratios appear somewhat higher than reported I/C ratios in peat bogs from Southern Chile (e.g., 1x10 -5 ; [START_REF] Keppler | Organoiodine formation during humification in peatlands[END_REF]. This suggests episodic deposition of organic material from a high iodine content, relatively low carbon (e.g. in comparison to algae) source such as peat or marsh plant residues. Organic carbon from such a source, while old, indicates C4 or CAM (crassulacean acid metabolism) origin, as δ 13 C values for this section of the core are relatively heavy (-21.2 ‰), rather than -24 to -30 ‰ for the isotopically lighter C3 plants. Furthermore, in the 1956-1993 data set, 14 C-age and (interpolated) δ 13 C linearly and significantly correlate (Figure 5, Table 1), suggesting a mixing behavior of at least two distinct carbon sources. While a correlation between 14 C -ages and δ 13 C values had been described previously by [START_REF] Gordon | Sources and distribution of terrigenous organic matter delivered by the Atchafalaya River to sediments in the northern Gulf of Mexico[END_REF] for nearshore sediments near the Atchafalaya River, albeit with significantly more depleted δ 13 C values ranging from -21 to -27 ‰, characteristic for a large part of the shelf, their range of δ 13 C values is much larger (i.e., -21 to -27‰), and their linear correlation (i.e., slope and intercept) is significantly different. Nonetheless, the points in our correlation still fall within the range of values [START_REF] Gordon | Sources and distribution of terrigenous organic matter delivered by the Atchafalaya River to sediments in the northern Gulf of Mexico[END_REF] observed.

It might be possible that peat residues dated by 14 C to be several thousand years old, exhibiting isotopically depleted δ 13 C signatures (~ -25 ‰) that had been observed in eroding river beds near New Orleans [START_REF] Galler | Biogeochemical implications of levee confinement in the lowermost Mississippi River[END_REF] could be the source material. However, because δ 13 C values in our core peak at -21.2 ‰ and not -25 ‰, this possibility can be ruled out. Since δ 13 C values of -21.2 ‰ are coincident with higher iodine values, this suggests that our 13 C record indicates episodic deposition of more 13 C-and iodine-enriched wetland deposits rather than peat residues from old river beds deposited during the 1950's. Wetland plants and their residues are known to contain more isotopically heavy carbon [START_REF] Hoefs | Stable Isotope Geochemistry[END_REF] that are also enriched in iodine [START_REF] Fuge | The geochemistry of iodine -a review[END_REF]. Thus, our sedimentary record suggests that eroded organic carbon from wetlands has episodically been eroded and deposited into these delta sediments during those particular times.

While [START_REF] Goni | A reassessment of the sources and importance of land-derived organic matter in surface sediments from the Gulf of Mexico[END_REF] considered the possibility of marsh-derived organic matter as a source of the terrigenous organic matter they observed in Gulf of Mexico sediments, they dismissed this possibility for a number of reasons. They argued that the subsidence of the deltaic plain and surrounding lands should make them a sink and not a source of sediments, and they expected that marsh sediments should be younger in 14 C-age than the old sediments they observed on the shelf and slope. However, they only considered current conditions, and obviously, hydrological conditions during the 1950's and early 1960's were different. Also, while our data suggests that the OC originates from older wetland deposits that must have periodically been deposited in the delta sediments at 60 m water depth, these layers with highest iodine concentrations appear to account for only a minor fraction of the sedimentary deposits.

The significant on-shore -off-shore gradient in δ 13 C values on the Louisiana -Mississippi shelf, with δ 13 C values of -25‰ in Mississippi and Atchafalaya Rivers and near-shore sediments, and -22 to -20 ‰ in outer shelf and slope surface sediments, has been explained by hydrodynamic sorting of POC, with C3 plant material being heavier and depositing closer to shore, and C4 plant residues associated with finer-grained particles that are being transported further off-shore [START_REF] Bianchi | Sources of terrestrially-derived organic carbon in lower Mississippi River and Louisiana shelf sediments: Implications for differential sedimentation and transport at the coastal margin[END_REF]. Obviously, this general pattern does not differentiate contributions from different sources of C4 plant residues, e.g., from grass or marsh lands. Only in combination with iodine signatures, is it possible to distinguish deposits of iodine-enriched wetland-derived POC, which have only small isotopic deviations in δ 13 C values from those of C4 plants.

Conclusions

The carbon isotope and iodine data from a Mississippi River delta core correlates not only with changes in land use and decreases in sediment and OC deposition since the early 1960s, but also, shown here for the first time, with episodic organic carbon deposition from older wetland plant residues (which are known to contain high iodine concentrations). This was especially pronounced for sediments deposited in the late 1950's, when sediment deposition rates at this site were higher. The δ 13 C maximum at the same depth also indicates a more significant contribution for OC from C4 plants during the 1950's to early 1960's, coincident with the times of major hydrological changes both in the upper and lower reaches of the Mississippi River drainage basin. Most recently, hurricanes Rita and Katrina allowed a study of their effects on wetlands in Lousiana [START_REF] Turner | Wetland sedimentation from hurricanes Katrina and Rita[END_REF], which concluded that hurricanes nourish rather than erode wetlands. Recent sediments in our core contain younger (1560 years) and isotopically lighter OC (-21.96 ‰), suggesting a lesser influence of wetland carbon deposits, in accordance with the increased channelization of the Mississippi and Atchafalaya Rivers in recent times. These findings, even though only documented in one sediment core, are significant as they allow extrapolation to other large rivers that have been dammed and channelized in recent times. However, more work needs to be carried out on inputs from wetlands and the use of 239 Pu/ 240 Pu, δ 13 C and I/C ratios in sediment cores as tracers for inputs from different terrestrial environments.

Table 1. Carbon isotopic results from core 93P13 #2, along with % OC and iodine data from [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF]. -: not determined; *: interpolated.

Depth

Year of deposition OC (±0.01) Core location in Gulf of Mexico: core 93P13 #2 = 28°55.5' N, 89° 40.6' W in about 60 m water depth [START_REF] Oktay | The 129 Iodine Bomb Pulse Recorded in Mississippi River Delta Sediments: Results from Isotopes of I, Pu, Cs, Pb, and C. Geochim[END_REF], with new carbon isotopic results described here. Figure 4. Total iodine concentrations as a function of % OC, demonstrating a trend line of increasing iodine with increasing organic carbon concentrations, with the outliers consisting of data points from the layers that were deposited episodically and that were inferred to contain peat-OC.

Figure 1 .

 1 Figure captions

Figure 2 .

 2 Figure 2. Downcore variations of 14 C (a) and δ 13 C (b).

Figure 3 .

 3 Figure 3. Percent organic carbon (OC) and total 127 Iodine concentration (ppm) as a function of depth.

Figure 5 .

 5 Figure 5. Linear relationship between 14 C age (years) and δ 13 C (‰) for data points between 1950-1993.

Fig. 1

 1 Fig.1

Fig. 2

 2 Fig.2

Fig. 3

 3 Fig.3

Fig. 4

 4 Fig.4

Fig. 5

 5 Fig. 5

Acknowledgements

The ∆ 14 C results were analyzed by the Accelerator Mass Spectrometry (AMS) facility at the


Adriana De Abreu Corrêa

Jonas Dutra

Albarnaz 1a

Vanessa Moresco

; Carlos

Rogério Poli

Adriano Luiz Teixeira

Cláudia Maria Oliveira

Simões 2a

Célia Regina

Monte Barardi
email: cbarardi@ccb.ufsc.br

Oyster Depuration

Keywords: Oysters Crassostrea gigas, depuration, Salmonella enterica serovar Typhimurium, PCR

The State of Santa Catarina produces the greatest quantity of edible mollusks in Brazil. To guarantee sanitary quality, mollusk cultures should be monitored for contamination by pathogenic microorganisms. A self-purification or "depuration" system that eliminates Salmonella enterica serovar Typhimurium contamination from oysters has been developed and evaluated. The depuration process occurred within a closed system, in which 1,000 L of water was recirculated for 24h. The water was sterilized with ultraviolet (UV) light, chlorine, or both together. Oysters (Crassostrea gigas) artificially contaminated with S. Typhimurium were harvested every six hours. Samples of oyster tissue were excised and both the presence and numbers of bacteria were determined. Combined UV light and chlorine treatments resulted in total elimination of bacteria within 12h. Polymerase chain reaction detected bacteria in water exposed to the three treatments. This pioneering study is the first of its kind in Brazil and represents a major contribution to commercial mollusk culture in this country.

Introduction:

In Brazil, large amounts of treated, and sometimes untreated, human wastewaters are discharged into marine environments in highly populated coastal areas, resulting in localized increases in pathogen concentrations, which in turn serve as sources for transmission of many diseases [START_REF] Lemarchand | Molecular biology and DNA microarray technology for microbial quality monitoring of water. Critical Reviews Microbiology[END_REF]. Filter-feeding mollusks accumulate and concentrate microorganisms when grown in polluted waters, including human pathogenic bacteria and viruses. Contaminated mollusks can represent significant health risks when consumed raw or lightly cooked (Butt, Aldridge, & Sander, 2004;[START_REF] Lee | Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink TM NH microwell plate sandwich hybridization[END_REF]. The enteric bacteria Salmonella can readily contaminate the fauna in marine environments, especially molluscan shellfish, and for this reason, oysters and mussels are associated with worldwide salmonelosis outbreaks [START_REF] Lee | Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink TM NH microwell plate sandwich hybridization[END_REF][START_REF] Martinez-Urtaza | Influence of Environmental Factors and Human Activity on the Presence of Salmonella Serovars in a Marine Environment[END_REF][START_REF] Vazquez-Novelle | Eight-hour PCR-based procedure for the detection of Salmonella in raw oysters[END_REF]. Controlled self-purification ("depuration") of shellfish is a method that reduces the levels of bacteria present in mollusk meat, thus decreasing the potential for infections associated with shellfish consumption. Depuration is a commercial process where shellfish are placed in tanks filled with clean seawater for several hours to purge contaminants from their tissues by filtering [START_REF] Croci | Effects of Depuration of Molluscs experimentally contaminated with Escherichia coli, Vibrio cholerae O1 and Vibrio parahaemolyticus[END_REF][START_REF] Rodrick | Molluscan Shellfish Depuration[END_REF][START_REF] Marino | Uptake of Escherichia coli, Vibrio cholerae non-o1 and Enterococcus durans by, and depuration of mussels (Mytilus galloprovincialis)[END_REF].

There are two general types of mollusk shellfish depuration plants: flow-through or recirculating (closed) system. Both depuration processes are effective, both the flow-through system requires a near-by, reliable, but consistently clean source of seawater. In the closed depuration system, seawater is recirculated for at least 24h, and during this cycle, the water must be chemically (chlorine and/or ozone) or physically (UV irradiation) treated to eliminate 4 microbial contamination. The advantage of the closed system is that it uses vastly less amounts of water (Richards, 1988;[START_REF] Power | Differential Depuration of Poliovirus, Escherichia coli, and Coliphage by the Common Mussel, Mytilus edulis[END_REF]Dore & Lees, 1995).

The State of Santa Catarina is a major producer of mollusks in Brazil due to excellent geographical conditions for marine organism cultures, especially bivalve mollusks, such as the oyster species Crassostrea gigas [START_REF] Souza | Custo de Produção da Ostra Cultivada[END_REF][START_REF] Oliveira Neto | Diagnóstico do cultivo de moluscos em Santa Catarina[END_REF]. In recent years, however, increased contamination of mollusk culture habitats by sewage discharged from coastal communities has lead to contamination of cultured mollusks by bacterial and viral pathogens [START_REF] Coelho | Hepatitis A vírus detection in oysters (Crassostrea gigas) in Santa Catarina State, Brazil, by reverse transcription-polymerase chain reaction[END_REF][START_REF] Rigotto | Detection of adenoviruses in shellfish by means of conventional PCR, nested-PCR and integrated cell culture PCR (ICC/PCR)[END_REF][START_REF] Sincero | Detection of Hepatites A virus (HVA) in Oysters (Crassostrea gigas)[END_REF]. The increasing pollution of the coastal waters of Santa Catarina requires monitoring of cultured bivalve mollusks to ensure health safety standards. While depuration is not yet an established practice in Brazil, it is used in many other countries, such as European countries and even in United States according to the water quality. To expand commercial bivalve mollusk culture in Brazil, a safe product with high commercial value must be guaranteed.

In this study, the efficacy of a commercial oyster depuration system was evaluated. The system was designed by an aquaculture company (Blue Water Aquaculture, Florianopolis, Santa Catarina State, Brazil). Oysters (Crassostrea gigas) artificially contaminated with Salmonella enterica serovar Typhimurium were used, and the efficacy of the system was evaluated through the detection of this pathogen by microbiological and molecular methods.

Material and methods

Bacterial strain, oysters and seawater: S. Typhimurium (ATCC 14028) was used to contaminate oysters and was kindly provided by the Laboratory of Medical Microbiology of the Universidade Federal de Santa Catarina. The bacterial strain was propagated in brain heart infusion (BHI, Oxoid, Basingstoke, Hampshire, U.K.) broth and xylose-lysine-deoxycholate agar (XLD, Oxoid). Liquid cultures were obtained by overnight growth at 37 ºC. Bacterial suspensions were quantified by direct plate counting. The Pacific oysters (C. gigas) were harvested from an oyster culture farm located in the city of Florianopolis, Santa Catarina (SC), Brazil. Seawater was collected from the same location.

Depuration System: The depuration (UV/Chlorine system) system was designed by the company Blue Water Aquaculture (Florianopolis, SC). The depuration plant was placed in the same oyster farm where the oysters were harvested. The depuration unit consisted of a 1,000 litre (L) water tank, with 200 dozen oysters, in 19 perforated plastic baskets, distributed in four floors. The water was circulated by a pump at a rate of 2,200 L/h. The sterilizing system consisted of one 25W UV (254 nm) low-pressure tube, a chlorine metering pump, a sand filter, and a refrigeration system. The perforated plastic baskets were kept in a plastic basin with a grid base, to avoid recontamination by particulate matter released from the oysters. The temperature of the depuration system was maintained at 19ºC during all experiments in order to prevent the mollusks from spawning.

Artificial Contamination:

Prior to the depuration experiment, oysters were placed in an aquarium (55 x 45 x 30 cm) containing 30 L of seawater. A fresh, overnight culture of S.

Typhimurium was added to the aquarium to a final concentration of approximately 1,000 CFU/ml. The oysters were allowed to bio-accumulate the seeded bacteria for 2 h. After this period, the oysters were removed from the aquarium and their shells were washed in potable water plus 0.5% chlorine for 10 min. At this point, three oysters were retained as an undepurated control. The remaining oysters were labeled with red ink and immersed in the depuration tank together with uncontaminated oysters.

Depuration Assay: Depuration assays were undertaken over a 24-h period. Oysters were removed at 6, 12, 18 and 24 h for analysis. Water temperature was monitored routinely. Three treatments were conducted: chlorine (1ppm) alone, UV irradiation (254 nm) alone, and chlorine plus UV irradiation. Each treatment was repeated three times, and for each sample 6 period, one sample (three oysters artificially contaminated) per level was collected randomly.

After the collection, each sample was put into a plastic bag and stored at 4°C. At the end of the depuration assay, the samples were transported to the laboratory for the analyses. In each depuration assay, uncontaminated samples were collected before and after as negative controls. At the end of each depuration assay, the water was discharged into the sewage system. The experiment was devised to obtain depurated shellfish in the shortest period of time and at the lowest cost to local producers.

Analytical procedures:

Bacterial analysis: Following artificial contamination and depuration, oyster's shells were opened aseptically using a sterile shucking knife under aseptic conditions. The digestive tissue was dissected and then processed in a homogenizer (Ultra-Turrax, model T25 basic, IKA Labortechnik). The tissues of three oysters (one sample) were pooled and weighed. To quantify the bacterial concentration, one gram of homogenate was diluted in 9 ml of nonselective lactose broth (1:10 dilution). The time zero sample (undepurated tissue) was subjected to subsequent decimal serial dilutions (1:100 and 1:1000) and the depurated samples, were subjected to 1:10 dilution. Portions of each dilution (0.1mL) were spread onto plates of selective XLD agar medium. The CFU/g of samples was counted after overnight growth at 37 ºC.

The first dilution of each sample was incubated overnight at 37 ºC in selective XLD liquid medium to confirm the presence of S. Typhimurium, when present in amounts below the plate detection limit.

DNA extraction from contaminated oyster tissues

After incubation in liquid medium, a 1.5 ml aliquot was centrifuged at 1,400 × g for 5 min in a microcentrifuge (Eppendorf 5415 C, Hamburg, Germany). The pellets were suspended in 567 µL of TE buffer (10 mM Tris-HCl, pH 7.0, 1 mM EDTA), 30 µL of 10 % SDS (sodium dodecyl sulfate) and 6 µL of proteinase K (20 U/mg) (Invitrogen, Carlsbad, CA, USA), and incubated at 56 °C for 1 h. After that, 80 µL of 10 % CTAB (cetyltrimethylammonium bromide) and 100 µL of 5 M NaCl were added, gently homogenized and incubated at 65°C for 10 min. They were subsequently extracted with an equal volume of phenol-chloroformisoamyl alcohol (25:24:1). After centrifugation (12,000 × g, 5 min, RT), the aqueous phase was transferred to a fresh microtube and the same procedure was repeated with one volume of chloroform-isoamyl alcohol (24:1). Isopropanol precipitation was carried out for 10 min at RT. Resulting pellets were washed with chilled 75% (v/v) ethanol, suspended in 200 µL of TE buffer and stored at -20°C until used [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF]. The pellets were allowed to dry for 10 min, suspended in 100 µL of TE buffer, and stored at -20 º C. DNA derived from pure cultures of S. Typhimurium was used as positive control in all experiments.

Oligonucleotides primers:

The primer set used in this study was previously described by Cocolin, Manzano, Cantoni, & Comi (1998) (Salm3 5'-GCT GCG CGC GAA CGG CGA AG-3' and Salm4 5'-TCC CGG CAG AGT TCC CAT T-3'), and amplified a 387 bp genome region of Salmonella spp. These primers were derived from the invA gene sequence (Genbank accession number M90846), essential for Salmonella invasiveness into ephitelial cells [START_REF] Galan | Molecular and functional characterization of the Salmonella invasion gene invA: homology of invA to members of a new protein family[END_REF].

PCR amplification and DNA analysis: PCR amplifications were performed in a final volume of 50 µl, containing 3 µl of DNA template, 1X Buffer (10 mM Tris-HCl pH 9.0; 50 mM KCl, 2 mM MgCl 2 ), 0,2 mM of each deoxynucleotide (Invitrogen Life Technologies, Carlsbad, Ca, USA), 20 pmol of each primer and 0.5 units of Taq DNA polymerase (Biotools, Madrid, Spain). Amplifications were carried out in a Thermocycler (Techne Flexigene).

Initial denaturation was at 94 o C for 1 min, followed by 30 cycles of denaturation at 94 º C for 30 s, annealing at 62 º C for 30 s and extension at 72 º C for 2 min, with a final extension at 72 º C for 10 min. Negative (water) and positive controls (purified S. Typhimurium DNA) were used in all experiments. Amplification products were separated by electrophoresis of 10 µl of the final reaction mixture on 1.5 % agarose gel, at 100 V. The agarose gel was visualized on UV light after ethidium bromide (1 µg/ml) staining.

Statistics analysis:

The statistical analyses were conducted using GraphPad Prism 4.

Statistical differences in the numbers of bacteria per level of the depuration system across the different treatments were determined by ANOVA and non-linear regression analyses. PCR results were analyzed using the exact Fisher's Test with a p value of < 0.05.

Results and Discussion:

Prior to the start of the purification process, S. Typhimurium counts in artificially contaminated oysters ranged from 15,000 to 20,000 CFU/g of digestive tissue.

Samples collected before and after the purification period, and samples not artificially contaminated were always negative for the presence of S. Typhimurium, indicating that cross contamination due to contact with contaminated samples presented in the same basket did not occur.

Ultraviolet irradiation (254 nm) and chlorine (1 ppm) treatments, separately, were effective in reducing the numbers of S. Typhimurium, but did not totally eliminate the bacteria from all four levels (data not shown). According to [START_REF] Son | Behavior of pathogenic bacteria in the oyster, Crassostrea commercialis, during depuration, re-laying and storage[END_REF], UV irradiation treatment for water depuration was effective in reducing high levels S. Typhimurium to low levels, on a laboratory scale in glass tanks (20 L). The reduction in numbers of bacteria accomplished here by UV and chlorine treatments was consistent with previously published studies on oysters and mussels purification [START_REF] Rowse | Effects of water temperature and salinity on elimination of Salmonella charity and Escherichia coli from Sidney Rock Oysters (Crassostrea commercialis)[END_REF]Murprhee & Tamplin, 1991;[START_REF] Doré | Behavior of Escherichia coli and Male-Specific Bacteriophage in Environmental Contaminated Bivalve Molluscs before and after Depuration[END_REF][START_REF] Croci | Effects of Depuration of Molluscs experimentally contaminated with Escherichia coli, Vibrio cholerae O1 and Vibrio parahaemolyticus[END_REF]. Ultraviolet irradiation and chlorine treatment together, however, effectively eliminated S. Typhimurium from all levels after 12 hrs (Figure 1). While plate counts showed that bacteria were eliminated from all levels by 12 hrs, S.

Typhimurium was detected up to 24 hrs (Table 1).

In general, the effectiveness of the three disinfection methods was similar for each of the four levels of the depuration system. Linear regression analyses of each treatment revealed that the decrease in the number of CFU of bacteria per gram of oyster tissue was closely associated with purification time. The R 2 value was approximately 0.9 for all three treatments on all four levels (Table 2). The closed depuration system has a major advantage because it uses vastly less water than flow-through system. The major disadvantage is that the water must be disinfected as it is recirculated (Richard, 2001;[START_REF] Rodrick | Molluscan Shellfish Depuration[END_REF]. Treatment with chlorine is one of the methods for disinfecting polluted waters. Although it is effective against enteric bacteria, it is not very effective against bacterial spores, viruses and protozoa [START_REF] Oppenheimer | Testing the equivalency of ultraviolet light and chlorine for disinfection of wastewater to reclamation standards[END_REF][START_REF] Monarch | The infuence of different disinfectants on mutagenicity and toxicity of urban wastewater[END_REF]. Moreover, health risks due to mutagenic and/or carcinogenic agents produced during chlorine treatment (Koivunem & Heinonem-Tanski, 2005) raise concerns about the exclusive use of chlorine in depuration systems.

Ultraviolet irradiation is an effective disinfectant and it is used widely in water purification systems [START_REF] Caslake | Disinfection of contaminated water by using solar irradiation[END_REF]Mamane-Grawetz & Linden, 2004). According to Koivunem & Heinonem-Tanski (2005), UV irradiation eliminates enteric bacteria, viruses, bacterial and protozoan spores in water, without the production of toxic products or other chemical residues. The disadvantage of UV irradiation is that, under certain conditions, some microorganisms inactivated by UV may be able to repair DNA damage and continue to grow [START_REF] Lubello | Comparison between PAA/UV and H 2 O 2 /UV disinfection for wastewater reuse[END_REF]. In some organisms, particularly bacteria, the mutagenic effects of short wavelength UV can be repaired after exposure to long wavelength light, especially visible light (320-400 nm) [START_REF] Upadhyaya | The Design and Comparison of Continuous and Pulsed Ultraviolet Reactors for Microbial Inactivation in Water[END_REF]. Purification systems that use UV lamps for disinfection must filter water through sand to eliminate suspended material, because large suspended particles in the water limit the penetration of UV rays, and thus reduce the effectiveness of this treatment [START_REF] Richards | Enteric Virus contamination of foods through industrial practices: a primer on intervention strategies[END_REF].

The combination of chlorine and UV irradiation both increased the efficiency of disinfection and decreased the time required to complete the depuration process. The two methods had a synergistic effect because they inflict different types of damage in microorganisms (Koivunem & Heinonem-Tanski, 2005). Chlorine treatment damage cell walls and membranes, as well as enzymatic and transporting systems, while UV irradiation causes DNA damages, and with repair mechanisms overloaded, the microorganisms die [START_REF] Lubello | Comparison between PAA/UV and H 2 O 2 /UV disinfection for wastewater reuse[END_REF].

In this study, S. Typhimurium was detected in oyster tissues by PCR, even though no bacteria were detected by plate counts. Detection of DNA by PCR in the samples that did not contain viable bacteria was most probably due to the fact that bacteria that have been inactivated or killed by chlorine and/or UV irradiation may be re-ingested and bioaccumulated by mollusks.

If DNA from these dead bacteria is not completely degraded, molecular analyses, such as PCR, may still react with this DNA, resulting in false positive results. Therefore, quantification of bacteria using the plate count method is essential to detect the viability of the microorganisms present in the oyster's tissues. In a study that was similar to this one, [START_REF] Croci | Effects of Depuration of Molluscs experimentally contaminated with Escherichia coli, Vibrio cholerae O1 and Vibrio parahaemolyticus[END_REF] evaluated depuration of mussels that had been artificially contaminated with E. coli, V. cholerae O1, and V. parahaemolyticus. The effectiveness of the depuration process was monitored using the Most Probable Number (MPN) methodology, and PCR was used only as a confirmatory test for the Vibrio positive samples.

The results obtained in the present study are the first data in Brazil regarding the depuration dynamics of S. Typhimurium in oysters using a closed system. Most countries that produce bivalve mollusks for export markets have strict regulations for monitoring depuration systems and operating procedures to ensure quality control of the purification process. Monitoring

Figures legends Figure 1 .

 legends1 Figures legendsFigure1. Elimination of S. Typhimurium from oysters after treatment with UV irradiation and

Table 2 :

 2 Correlation between decontamination treatment and time for all locations within the depuration system. Decreases in the numbers of CFU/g oyster were correlated with time of treatment for each location within the depuration system. R 2 values from each linear regression, location and treatment indicate a significant correlation between decontamination and time for all locations and treatments.

	Water Treatment	Level	R 2
		1st	0.8710
		2nd	0.8562
	Chlorine 1ppm	3rd	0.8565
		4th	0.8610
		1st	0.8515
		2nd	0.8724
	UV	3rd	0.8562
		4th	0,8822
		1st	0.8669
		2nd	0.8829
	Chlorine 1ppm/UV	3rd	0.8622
		4th	0.8885

Acknowledgements: This work was accomplished with the financial support of CNPq, FAPESC (FCTP 845), Blue Water Aquaculture Ltda., and Aquaculture and Fishes Special Clerkship of the Brazilian Federal Government. The first author thanks to CAPES/MEC for her postgraduate research fellowship, and to Ford Foundation for a travel grant to attend the Ecological Society of America Meeting, in Merida, Mexico, 2006, January. We thank Dr. David B. Levin from University of Victoria, Canada, and Dr. Jack Littlepage from the Division of Technology and International Development at the Centre for Global Studies, University of Victoria, for the kind review of this manuscript.

temperature and salinity of the water during the purification cycle is also recommended (European Communities, 1991;National Shellfish Sanitation Program, 2005). The Brazilian legislation does not regulate depuration systems, and only the presence of fecal coliforms in culture waters and the presence of Salmonella spp. in the mollusk meat are currently regulated (ANVISA, 2001;CONAMA, 2005).

Conclusion:

The bivalve mollusk culture in the State of Santa Catarina is growing rapidly. Countries that produce bivalve mollusks for export markets have strict regulations for bacterial contamination to ensure a safe, high quality product. The purification system designed by Blue Water Aquaculture (Florianopolis, SC, Brazil) was effective for the purification of oysters artificially contaminated by S. Typhimurium. The combination of UV irradiation and chlorine treatments eliminated all bacteria by 12 hrs, as determined by plate counts.

Quantification of bacteria by plate counts proved to be a more reliable method of evaluating the effectiveness of the depuration treatments, as PCR detected the presence of bacterial DNA (but not viable bacteria) in oysters in all three treatments up to 24 hrs. The depuration can increase the oyster commercial value for the producer, from around U$ 2,00 (actual market price) to U$ 4,00. Actually, the final cost of the depuration system is around U$ 7.000,00, and the small and average farmers can afford it.