Vibrio vulnificus - Coastal Bend Bays & Estuaries Program

Vibrio vulnificus Monitoring in Recreational Waters Final Report Publication CBBEP – 54 Project Number – 0624 March 2008

Principal Investigators Joanna Mott, Ph.D., Professor of Biology, Gabriel Ramirez, and Gregory Buck, Ph.D., Assistant Professor, Biology and Biomedical Sciences Department of Life Sciences Texas A&M University-Corpus Christi, 6300 Ocean Drive, Corpus Christi, TX 78412

Submitted to: Coastal Bend Bays & Estuaries Program 1305 N. Shoreline Blvd., Suite 205 Corpus Christi, TX 78401 The views expressed herein are those of the authors and do not necessarily reflect the views of CBBEP or other organizations that may have provided funding for this project.

Vibrio vulnificus Monitoring in Recreational Waters

Final Report

Report prepared by: Joanna Mott, Ph.D. Professor of Biology, Gabriel Ramirez, and Gregory Buck, Ph.D. Assistant Professor, Biology and Biomedical Sciences Department of Life Sciences Texas A&M University-Corpus Christi, 6300 Ocean Drive, Corpus Christi, TX 78412

CBBEP Project Manager: Jim Bowman Coastal Bend Bays and Estuaries Program, Inc. 1305 N. Shoreline, Suite 205 Corpus Christi, Texas 78401

TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................................... iii LIST OF TABLES ........................................................................................................................ v ACKNOWLEDGEMENTS ........................................................................................................ vii EXECUTIVE SUMMARY ............................................................................................................ 1 INTRODUCTION.......................................................................................................................... 3 Induction of Viable but Non-Culturable State................................................................. 7 Culture Media .......................................................................................................................... 8 Objectives ................................................................................................................................ 9 METHODS................................................................................................................................... 10 Sites ......................................................................................................................................... 10 Field Collection..................................................................................................................... 18 Laboratory Analysis ............................................................................................................ 19 Statistical Analysis .............................................................................................................. 20 RESULTS.................................................................................................................................... 21 Water Quality Parameters.................................................................................................. 21 Microbiological Analysis.................................................................................................... 29 Statistical Analysis .............................................................................................................. 42 CONCLUSIONS ......................................................................................................................... 51 RECOMMENDATIONS............................................................................................................. 53 LITERATURE CITED ................................................................................................................ 54 APPENDIX .................................................................................................................................. 59

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LIST OF FIGURES Figure 1: Location of sample site in upper Laguna Madre on Bird Island Basin at PINS. Google Earth 2006………………………………………………………………...12 Figure 2: Location of sample site in Nueces Bay at North end of US Highway 181. Google Earth 2006………………………………………………………………...13 Figure 3: Location of sample site in Corpus Christi Bay at south end of Cole Park. Google Earth 2006…......................................................................................14 Figure 4: Location of sample site in Redfish Bay at the Morris and Cummings Cut. Google Earth 2006.........................................................................................15 Figure 5: Location of sample site at Copano Bay Causeway at SH35. Google Earth 2006…………………………………………………………………………………16 Figure 6: Location of sample site in Copano Bay off FM136 near Bayside. Google Earth 2006......................................................................................................17 Figure 7: Enterococcus sp. means at sites in south Texas coastal waters from August 2006 through July 2007..................................................................................32 Figure 8: Numbers of V. vulnificus at Bird Island, Nueces Bay and Cole Park sites from August 2006 to July 2007…………………………………………………………34 Figure 9: Numbers of V. vulnificus at Redfish Bay, Copano Bay and Bayside sites from August 2006 to July 2007…………………………………………………………34 Figure 10: Water temperature, salinity, and dissolved oxygen values versus numbers of V. vulnificus at Bird Island from August 2006 through July 2007.............................................................................................................37 Figure 11: Water temperature, salinity, and dissolved oxygen values versus numbers of V. vulnificus at Nueces Bay from August 2006 through July 2007.............................................................................................................38 Figure 12: Water temperature, salinity, and dissolved oxygen values versus numbers of V. vulnificus at Cole Park from August 2006 through July 2007..............39 Figure 13: Water temperature, salinity, and dissolved oxygen values versus numbers of V. vulnificus at Redfish Bay from August 2006 through July 2007..............40

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Figure 14: Water temperature, salinity, and dissolved oxygen versus numbers of V. vulnificus at Copano Bay Causeway from August 2006 through July 2007……………………………………………………………………………….41 Figure 15: Water temperature, salinity, and dissolved oxygen values versus numbers of V. vulnificus at Copano Bay near Bayside from August 2006 through July 2007.............................................................................................................42 Figure 16: Numbers of V. vulnificus vs. enterococci at Bird Island, Nueces Bay and Cole Park sites from August 2006 to July 2007………………………………43 Figure 17: Numbers of V. vulnificus vs. enterococci at Redfish Bay, Copano Bay and Bayside sites from August 2006 to July 2007………………………………...43 Figure 18: Water temperature versus V. vulnificus at sites in south Texas coastal waters from August 2006 through July 2007...............................................48 Figure 19: Salinity versus V. vulnificus at sites in south Texas coastal waters from August 2006 through July 2007...................................................................49 Figure 20: Dissolved oxygen versus V. vulnificus at sites in south Texas coastal waters from August 2006 through July 2007...........................................................50

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LIST OF TABLES Table 1: Reported V. vulnificus infections in Texas for the years 2000-2006 by type of exposure (Texas Department of State Health Services, 2008 customized report)………………………………………………………………………………...4 Table 2: Texas Coastal Bend study sites and their coordinates …………........ ………10 Table 3: Historical physical water quality parameter ranges from Bird Island, Nueces Bay, Cole Park, Redfish Bay, Copano Bay, and Bayside for 1969 – 2006 (TCEQ, 2007 http://www.tceq.state.tx.us/compliance/monitoring/crp/data/samplequery.html) ........................................................................................................................11 Table 4: Physical water quality parameters from Bird Island, Nueces Bay, and Cole Park for August 2006 through July 2007........................................................22 Table 5: Physical water quality parameters from Redfish Bay, Copano Bay, and Bayside for August 2006 through July 2007...................................................23 Table 6: Physical water quality parameter statistics from sites in south Texas coastal waters for August 2006 through July 2007.....................................................24 Table 7: Physical water quality parameter ranges from sites in south Texas coastal waters for August 2006 through July 2007.....................................................25 Table 8: Rainfall: one day and one week prior to sampling at Bird Island, Nueces Bay, and Cole Park for August 2006 through July 2007.........................................27 Table 9: Rainfall: one day and one week prior to sampling at Redfish Bay, Copano Bay, and Bayside for August 2006 through July 2007...................................28 Table 10: Numbers of V. vulnificus and Enterococcus sp. at Bird Island, Nueces Bay, and Cole Park for August 2006 through July 2007 (CFU/100mL; average of two samples)…………………………………………………………………….30 Table 11: Numbers of V. vulnificus and Enterococcus sp. at Redfish Bay, Copano Bay, and Bayside for August 2006 through July 2007 (CFU/100mL, average of 2 samples)…………………………………………………………………………….31 Table 12: Mean Enterococcus sp. and V. vulnificus concentrations (CFU/100mL) at sites in south Texas coastal waters for August 2006 through July 2007........32

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Table 13: Analysis of V. vulnificus data using a two-way ANOVA: Tests of BetweenSubjects Effects Dependent Variable: LOG10CFU/100mL V. vulnificus……………………………………………………………………………33 Table 14: Correlation matrix for V. vulnificus vs physical water quality parameters from sites in south Texas coastal waters for August 2006 through July 2007................................................................................................................44 Table 15: Statistical comparison of V. vulnificus levels at sites, analyzed using the Tukey HSD: Multiple Comparisons Dependent Variable: LOG10CFU/100ML………………………………………………………………..45 Table 16: Multiple Regression analysis for levels of V. vulnificus: Model Summary….46 Table 17: ANOVA from multiple regression analysis for levels of V. vulnificus…………46 Table 18: Coefficients with P-values from multiple regression analysis for levels of V. vulnificus……………………………………………………………………………47

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ACKNOWLEDGEMENTS

Funding for this project was provided by the Coastal Bend Bays and Estuaries Program. Field sampling and laboratory analyses were conducted by Research/Field/Technical Personnel in the Environmental Microbiology Laboratory at Texas A&M UniversityCorpus Christi (Gabriel Ramirez, Nina Bishop, Stephen Capuyan, Stephanie Ely and Megan Mahlke) with supervision and oversight by Amanda Smith, Research Specialist, and the P.I.s. Thanks to Bridgette Froeschke for assistance in formatting and statistical analyses for this report.

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Vibrio vulnificus Monitoring in Recreational Waters Joanna Mott, Ph.D. and Gregory Buck, Ph.D.

EXECUTIVE SUMMARY Vibrio vulnificus is a common halophilic organism in coastal waters of some parts of the U.S. It is a well documented pathogen and is the leading cause of death in the U.S. related to seafood consumption. This species is also responsible for wound infections in persons who have had contact with marine waters. In the Coastal Bend region there have been recent cases of this pathogen infecting fishermen and causing death or severe illness and loss of limbs. There is no ongoing bacteria monitoring for this species in Texas coastal waters. In order to determine the distribution of the pathogen, for future education of the public, this study examined levels of V. vulnificus in Coastal Bend estuarine waters in relation to other environmental parameters such as temperature, salinity/conductivity, and dissolved oxygen. The objective of this study was to determine the occurrence and distribution of the pathogen, V. vulnificus in south Texas coastal waters by establishing baseline data on V. vulnificus occurrence and abundance at six sites in the CBBEP area, monitoring seasonal variation in V. vulnificus at each site over a one year period and evaluating certain factors that may be associated with elevated levels of V. vulnificus. e.g. temperature, salinity, enterococci. Due to the lack of data on V. vulnificus populations in Coastal Bend area waters, site selection was made with a goal of including several bays with different characteristics. The staff and management at CBBEP selected six public-use beaches, fishing areas, and/or recreational waters for the project. These included sites in the Laguna Madre (Bird Island Basin), Corpus Christi Bay (Cole Park), Nueces Bay, Copano Bay and Redfish Bay. Overall consideration was given to accessibility and safety. Water sampling was conducted at each site monthly for one year from August 2006 to July 2007 to provide seasonal data. V. vulnificus is known to enter a viable but nonculturable (VBNC) state during colder months in cooler waters. For South Texas waters where temperatures remain high, seasonal variation may differ. Field sampling procedures documented in the TCEQ Surface Water Quality Monitoring Procedures Volume 1: Physical and Chemical Monitoring Methods for Water, Sediment and Tissue (December 2003) were followed for collection of water samples and measurement of field parameters. Two water samples were collected from each site using sterile, one liter screw-cap polypropylene bottles. Field parameters were measured with an YSI water quality multiprobe instrument. Field data sheets were used to document field parameters. V. vulnificus and enterococci were isolated and enumerated following the U.S. FDA Bacteriological Analytical Manual Chapter 9. Vibrio. Online May, 2004, (http://www.cfsan.fda.gov/~ebam/bam-9.html), and U.S. EPA Method

1600 for enterococci, respectively. There is no one-step method to identify and enumerate V. vulnificus. Presumptive isolates were obtained by filtration and plating on VVA (V. vulnificus agar) and mCPC (modified cellobiose-polymyxin B-colistin) agar. Isolates from VVA were confirmed using a molecular technique described in the U.S. FDA Bacteriological Analytical Manual and FDA/Gulf Coast Seafood Laboratory Protocol (Vv-ISSC) 11/02 in which plating for colony isolation is followed by colony hybridization with a specific alkaline phosphatase-labeled gene probe that targets the cytolysin gene, vvhA. Baseline data on the occurrence and abundance of V. vulnificus in the CBBEP area was collected. V. vulnificus was ubiquitous in the marine waters sampled and was found throughout the year, being isolated from each site each sampling event at levels as high as 3.97 X 104 CFU/100mL. Numbers of V. vulnificus varied seasonally over the 12 month period with highest levels generally found from August through October/November. However, the bacteria were isolated in low numbers throughout the winter months at all six sites. The highest numbers of V. vulnificus were found at Nueces Bay in June 2007 with the second highest water temperature (27.4°C) and second lowest salinity (23.48 ppt) at this site. Numbers of V. vulnificus were positively correlated with water temperature and negatively correlated with dissolved oxygen levels. Multiple regression analyses indicated that levels of V. vulnificus were primarily influenced by water temperature and salinity, with these two parameters accounting for 48.2% of the variability in the concentration of V. vulnificus. The other 51.8% of the variability in the levels of V. vulnificus was caused by unknown factors. Thus, it appears that no single parameter is primarily responsible for the levels of V. vulnificus in south Texas coastal waters. Enterococci levels ranged from 95% presumptive identification of V. vulnificus from mCPC plates (DePaola et al., 1994). V. vulnificus agar (Kaysner and DePaola, 2004) is considered less selective than mCPC agar (Masad and Oliver, 1987), but was designed to be used in conjunction with the VVAP oligonucleotide probe for identification (Wright et al., 1993). By this method, cellobiose fermentation generates presumptive yellow colonies surrounded by a yellow halo. Direct detection from Gulf Coast oysters has been achieved on VVA agar and then confirmed with the VVAP probe (DePaola et al., 1997).

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The U.S. Food and Drug Administration (FDA) in its Bacteriological Analytical Manual (BAM) describes two molecular methods to confirm V. vulnificus isolates on the two aforementioned selective media following filtration (Kaysner and DePaola, 2004). The first involves colony isolation on selective media followed by confirmation using polymerase chain reaction (PCR). The second method involves plating to achieve colony isolation and then using colony hybridization with a DNA probe to confirm colonies. The DNA probe, alkaline phosphatase-labeled and referred to as VVAP, has been employed in multiple studies to confirm the identification of environmental and clinical isolates (Cerda-Cuellar et al., 2001; DePaola et al., 1997; Hoi et al., 1998a; Wright et al, 1993; Wright et al., 1996). The VVAP probe was derived from gene sequencing of the V. vulnificus cytolysin structural gene, vvhA (Wright et al., 1993). It was proven to be very selective because it hybridized with DNA from each of the strains tested and did not display any false-positives with non-V. vulnificus strains. Objectives The objective of this study was to determine the incidence and distribution of V. vulnificus in south Texas estuarine waters. Specific objectives of this study were to: 1. Establish baseline data on V. vulnificus occurrence and abundance at six sites in the CBBEP area. 2. Monitor seasonal variation in V. vulnificus at each station over a one year period. 3. Compare V. vulnificus levels in relation to other environmental parameters measured at each station e.g. temperature and salinity. In addition, levels of V. vulnificus were compared to those of the fecal indicator, Enterococcus sp. to assess whether levels of Enterococcus (routinely monitored) can provide an indication of V. vulnificus levels in surface waters. The project objective was to collect data that complies with TCEQ rules for surface water quality monitoring programs and water quality assessments. The specific objective of this project was to collect the necessary water quality monitoring data to evaluate certain factors that may be associated with elevated levels of V. vulnificus. These data include bacteria (V. vulnificus and enterococci) and routine field parameters.

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METHODS All methods followed the approved Quality Assurance Project Plan (QAPP) for this study (Mott and Buck, 2006). Sites Due to the lack of data on Vibrio vulnificus populations in Coastal Bend area waters site selection was made with a goal of including several bays with different characteristics. The staff and management at CBBEP selected six public-use beaches, fishing areas, and/or recreational waters for the project. These areas included sites in the Laguna Madre (Bird Island Basin), Corpus Christi Bay (Cole Park), Nueces Bay, Copano Bay and Redfish Bay. Coordinates are shown in Table 2 and site locations in Figures 1-6. Overall consideration was given to accessibility and safety. Historical ranges in water quality parameters at the six sampling sites from 1969 to the present are shown in Table 3 (TCEQ, 2007). Bird Island Basin is located in the upper Laguna Madre at PINS (Padre Island National Seashore) (Figure 1). It is used as a fishing area and other recreational activities include windsurfing, kayaking, and camping. TCEQ data show salinities have reached as high as 52 ppt at this site, usually in warmer months. Nueces Bay is a shallow bay that receives freshwater from Nueces River and exchanges saline water with Corpus Christi Bay. The sampling site was located in Nueces Bay at the north end of US Highway 181 (Figure 2). Nueces Bay is primarily used as a recreational fishing area. Water temperatures have ranged from a low of 4°C in winter to 32°C in summer. Salinity levels range from a low of 9 to 42 ppt, due to the freshwater inflow from the Nueces River. Table 2: Texas Coastal Bend study sites and their coordinates. Site Bird Island Basin Nueces Bay Cole Park Redfish Bay Copano Bay Causeway Copano Bay near Bayside

Coordinates 27°28'10.01" N 97°18'29.05" W 27°50'34.19" N 97°23'42.08" W 27°46'02.48" N 97°23'02.61" W 27°53'02.51" N 97°07'02.50" W 28°07'59.66" N 97°00'29.69" W 28°03'51.17" N 97°13'12.33" W

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Table 3: Historical physical water quality parameter ranges from Bird Island, Nueces Bay, Cole Park, Redfish Bay, Copano Bay, and Bayside for 1969 – 2006 (TCEQ, 2007 http://www.tceq.state.tx.us/compliance/monitoring/crp/data/samplequery.html) Location

Range

Bird Island

high low

Temp (°C) 32 13

Conductivity Salinity (μmhos/cm) (ppt) 76000 52 42000 27

DO (mg/L) 10 4

pH (s.u.) ~8 ~8

Secchi (m) 0.8 0.4

Nueces Bay

high low

32 4

63000 4000

42 9

17 4

8 7

> 0.55 0.1

Cole Park

high low

29 13

63000 40000

42 25

10 4

~8 ~8

> 0.72 0.15

Redfish Bay

high low

32 10

60000 1000

39 7

12 4

9 6

> 15 0.1

Copano Bay

high low

30 9

37000 2000

24 1

13 5

~8 ~8

>1 0.03

Bayside

high low

34 4

56000 1000

34 1

16 2

9 7

1 0.03

The Corpus Christi Bay sampling site was located at the south end of Cole Park at what is known as Oleander Point (Figure 3). Recreational activities at Cole Park include windsurfing and fishing. Water temperatures range from 13 to 29° C depending on season, while salinity levels are between 25 and 42 ppt. The Redfish Bay sampling site was located at the Morris and Cummings Cut by Stedman Island (Figure 4). Redfish Bay is primarily used by fishermen. Water temperatures are between 10 and 32°C. Salinity levels range from 7 to 39 ppt. Two sites in Copano Bay were sampled. One sampling site was located at the north end of Copano Bay at Copano Bay Causeway at SH35 (Figure 5). Water temperature ranges from 9 to 30°C. Salinity levels range from a low of 1 to 24 ppt. The second sampling site in Copano Bay was located at the south end of the bay off FM136 near Bayside and receives freshwater inflow from the Aransas River resulting in a range of salinity from 1 to 34 ppt (Figure 6). Microbiological analyses were conducted at the Texas A&M University-Corpus Christi Environmental Microbiology Laboratory.

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FIG 1. Location of sample site in upper Laguna Madre on Bird Island Basin at PINS. Google Earth 2006.

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FIG 2. Location of sample site in Nueces Bay at North end of US Highway 181. Google Earth 2006.

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FIG 3. Location of sample site in Corpus Christi Bay at south end of Cole Park. Google Earth 2006.

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FIG 4. Location of sample site in Redfish Bay at the Morris and Cummings Cut. Google Earth 2006.

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FIG 5. Location of sample site at Copano Bay Causeway at SH35. Google Earth 2006.

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FIG 6. Location of sample site in Copano Bay off FM136 near Bayside. Google Earth 2006.

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Field Collection Sampling was conducted monthly for a one-year period from August 2006 to July 2007 to provide seasonal data. Vibrio vulnificus is known to enter a viable but not culturable (VBNC) state during colder months, in cooler waters (Oliver et al., 1995). For south Texas waters where temperatures remain high, seasonal variation may differ. Field sampling procedures documented in the TCEQ Surface Water Quality Monitoring Procedures Volume 1: Physical and Chemical Monitoring Methods for Water, Sediment and Tissue (December 2003) were followed for the collection of water samples and measurement of field parameters. Standard field data sheets (as approved in the QAPP) were used to document field parameters. Abiotic factors (water temperature, salinity, conductivity, dissolved oxygen, pH, and turbidity) were measured at each station during each sampling event using a YSI water quality multiprobe instrument Model 6820 and a Secchi disk. Probe parameters were measured in situ when possible, but when that was not possible or unsafe, the TCEQ Surface Water Quality Monitoring Procedures Volume 1: Physical and Chemical Monitoring Methods for Water, Sediment and Tissue (December 2003) protocol for field measurements from a bucket was followed. Calibration of the YSI mulitprobe instrument followed the manufacturer’s instructions. Precipitation data were obtained through the National Oceanic and Atmospheric Administration (NOAA) website (www.noaa.com). Other observations (e.g. weather conditions, water appearance, odor, wind intensity, direction, etc.) were also recorded on the field data sheets (Appendix). Two water samples were collected from six sampling stations at several public use beaches, fishing areas, and/or recreational waters within the CBBEP study area including Bird Island Basin in upper Laguna Madre, Nueces Bay, Cole Park in Corpus Christi Bay, Redfish Bay, and two stations in Copano Bay. Samples were collected using sterile, screw-cap, one liter polypropylene bottles which were cleaned and autoclaved prior to each use. At Bird Island, Nueces Bay, Cole Park, and near Bayside in Copano Bay, water samples were collected from the shoreline by wading out to a depth of ~0.6 m. Two samples were taken using sterile, one liter polypropylene bottles by submerging one bottle at a time under water to a depth of ~0.3 m and uncapping the bottle under water until the bottle was filled to ~2.5 cm from the top; the cap was immediately replaced while the bottle was under water. At Redfish Bay and Copano Bay Causeway, water samples were taken from a pier or bridge, respectively, using a sterile bucket. At Redfish Bay, water samples were taken at the end of the pier at the Morris and Cummings Cut. At Copano Bay Causeway, water samples were taken halfway down the bridge (~400 m). A sterile bucket was rinsed three times with ambient water before collecting the water sample. The bucket was completely submerged under water to collect the sample. The water sample was then poured from the bucket into two sterile one liter polypropylene bottles.

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The one liter sampling bottles containing the water samples were transported in ice chests with ice (to ensure samples were maintained between 1OC and 4OC) to the TAMU-CC environmental laboratory for analysis within the required holding time (six hours plus two hours lab time). Bottles were labeled with an indelible, waterproof marker. Label information included the site identification, the date and time of sampling. A standard TCEQ approved Chain of Custody form was completed with collector signature and date/time collected. On arrival at the laboratory the TAMU-CC Laboratory Manager or a trained lab analyst checked times of collection to ensure holding times had not been exceeded. An additional bottle of water of the same specifications as the sample bottles was included in the ice chest and was used to check temperature of the samples on arrival in the laboratory. Laboratory Analysis Procedures for laboratory analyses were in accordance with the most recently published edition of Standard Methods for the Examination of Water and Wastewater, the latest version of the TCEQ Surface Water Quality Monitoring Procedures Volume 1: Physical and Chemical Monitoring Methods for Water, Sediment and Tissue (December 2003), 40 CFR 136, or other reliable procedures acceptable to TCEQ. Exceptions to this included analyses and sample matrices for which no regulated methods exist. Enterococci were isolated and enumerated using EPA Method 1600: Membrane Filter Test Method for Enterococcus in Water U.S. EPA (1997, 2000, 2002). Following filtration of three volumes of water using 0.45µ membrane filters, each filter was placed onto a plate containing the selective mEI (membrane-Enterococcus Indoxyl-β-DGlucoside agar) medium and then incubated at 41OC for 24 hours. After incubation all colonies with a blue halo were counted as Enterococcus. Verifications of colonies were performed as outlined in the method protocol. (http://epa.gov/waterscience/methods/biological/1600enterococcus.pdf). V. vulnificus isolations were performed by filtering multiple different volumes of each water sample (0.45µ membrane filters). Enumeration following the U.S. FDA Bacteriological Analytical Manual Chapter 9. Vibrio. Online May, 2004, http://www.cfsan.fda.gov/~ebam/bam-9.html, using the FDA/Gulf Coast Seafood Laboratory Protocol. (Vv-ISSC) 11/02 (U.S. FDA, 2002), with plating onto VVA (Vibrio vulnificus agar) followed by colony-blot hybridizations with a specific alkaline phosphatase-labeled gene probe (VVAP) to confirm isolates. A positive result for V. vulnificus was indicated by a purple colony blot, whereas a negative result was colorless, yellow, or light brown. Comparison plating was performed using mCPC (modified cellobiose-polymyxin B-colistin) agar (a more specific medium for V. vulnificus, but which may inhibit certain strains). Detailed laboratory QC requirements were followed as described in the QAPP for the project. Laboratory bacteriological duplicates were performed on samples from the sample bottle on a 10% basis. Results of bacteriological duplicates were evaluated by

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calculating the logarithm of each result and determining the range of each pair. Performance limits were used to determine the acceptability of duplicate analyses. The quality control for bacteriological membrane filtration methods followed TCEQ Surface Water Quality Monitoring Procedures Volume 1: Physical and Chemical Monitoring Methods for Water, Sediment and Tissue (December 2003). For each membrane filter test, sterility of the media, petri dishes, membrane filters, dilution water and apparatus were checked using sterile water. If colonies appear on the blank then all data from samples filtered after the blank were discarded. A blank was run at the start and end of each group of samples analyzed. In cases where extremely high levels of bacteria were present in the sample, the blank run at the end of the group should have less than 1% of the colonies on the sample filter. Additional method specific QC requirements were performed (e.g., positive controls, negative controls) as specified in Section 9020 B. Standard Methods for the Examination of Water and Wastewater (21st Edition, 2005), American Public Health Association (APHA), American Water Works Association (AWWA), and Water Environment Federation (WEF) 2005. Recommended positive and negative control cultures for enterococci were used as per Standard Methods Table 9020:V (Enterococcus faecalis American Type Culture Collection (ATCC) 29212 and Streptococcus salivarius ATCC 13419). Recommended positive and negative controls for V. vulnificus were used as per the U.S. FDA Online 2004 Method for V. vulnificus and U.S. FDA. 2002. MPN Procedure for the enumeration of Vibrio vulnificus using gene probe for identification. FDA/Gulf Coast Seafood Laboratory Protocol. (Vv-ISSC) 11/02. Positive controls included V. vulnificus (ATCC 27562 and 33817) and negative controls were V. alginolyticus (ATCC 17749) V. mimicus (ATCC 33653), and V. parahaemolyticus (ATCC 17802). The requirements for these samples, their acceptance criteria, and corrective action are method-specific. Statistical Analysis The statistical program SPSS (Statistical Package for the Social Sciences) version 12.0 for Windows was used to analyze the data. A two-way analysis of variance (ANOVA) was conducted to determine whether there was a statistically significant difference in V. vulnificus populations isolated using VVA versus mCPC media and between the sites sampled. The Tukey HSD test was used to determine which sites differed in V. vulnificus populations. V. vulnificus numbers were log transformed to achieve normality. A multiple regression analysis was completed, and Pearson’s coefficient of correlation was also used to detect correlations between numbers of V. vulnificus as confirmed by gene probe, and each environmental factor at 95% and 99% confidence levels.

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RESULTS Water Quality Parameters A total of 144 water samples were collected from the six sites. Water temperatures ranged from 9 to 31°C from August 2006 through July 2007 at all of the sites (Tables 4 and 5). Between the November and December 2006 sampling events, water temperatures dropped by 5 - 8°C from 20 - 22°C to 13 - 16°C. For Bird Island, Nueces Bay, and Cole Park water temperatures increased 8 to 10°C between the March and April 2007 sampling events from 15 - 16°C and remained > 24° C through July 2007. At Redfish Bay, Copano Bay causeway, and Copano Bay near Bayside, water temperatures increased from 9 - 12°C to 18 - 21°C between the February and March 2007 sampling events followed by a cool front, which dropped water temperatures by 4°C to 17 - 18°C during the April 2007 sampling event. Water temperatures then increased by 8 to 10°C during the May 2007 sampling event and remained greater than 26°C through July at Redfish Bay and both Copano Bay sites. Average water temperatures were between 22 and 23°C at each of the sites sampled (Table 6). The highest water temperature (31°C) occurred at Redfish Bay during the August 2006 sampling event, while the lowest water temperature (9°C) occurred at Copano Bay near Bayside in February 2007 (Table 5). Copano Bay near Bayside had the widest water temperature range of the six sites sampled (21°C) (Table 7). There was a broad range in salinity at each of the sites sampled. Nueces Bay and Cole Park had similar average salinities of 32 – 33 ppt (Table 6). Salinity levels were between 31 and 41 ppt at Nueces Bay and Cole Park for 8 of 12 sampling events (Table 4). The highest salinities at each of these sites were 41.5 – 41.8 ppt in October 2006 (Table 4). Both sites in Copano Bay had salinity levels between 1 and 20 ppt with the exception of three sampling events that exceeded 20 ppt at the Copano Bay causeway (September 2006, 23.6 ppt; December 2006, 22.2 ppt; and January 2007, 20.6 ppt, respectively). The lowest salinities for the two sites in Copano Bay were 0.89 and 0.62 ppt in July 2007 presumably related to rainfall (see later) (Table 5). The widest range in salinity occurred at Bird Island and Redfish Bay (26.9 - 52.7 ppt and 12.4 - 37.8 ppt, respectively) (Table 7). Salinity at Redfish Bay ranged from 19.8 – 37.8 ppt with the exception of July 2007 (12.3 ppt). Bird Island had the highest mean salinity of all the sites (39.7 ppt) reaching 52.7 ppt in October 2006 (Table 4). Salinity levels changed throughout the study at each site, and even at Bayside which had the smallest range in salinity, the range was 16 ppt (range 0.62 to 16.69 ppt) (Table 7). Bayside had the lowest salinities overall of the sites sampled (Table 5). Since conductivity is strongly dependent on salinity, both parameters varied similarly (Tables 4 and 5).

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Table 4: Physical water quality parameters from Bird Island, Nueces Bay, and Cole Park for August 2006 through July 2007.

Location Bird Island

Date

8/15/2006 9/6/2006 10/4/2006 11/8/2006 12/6/2006 1/3/2007 2/7/2007 3/7/2007 4/4/2007 5/9/2007 6/6/2007 7/5/2007 Nueces Bay 8/15/2006 9/6/2006 10/4/2006 11/8/2006 12/6/2006 1/3/2007 2/7/2007 3/7/2007 4/4/2007 5/9/2007 6/6/2007 7/5/2007 Cole Park 8/15/2006 9/6/2006 10/4/2006 11/8/2006 12/6/2006 1/3/2007 2/7/2007 3/7/2007 4/4/2007 5/9/2007 6/6/2007 7/5/2007

Temp Conductivity (°C) (μmhos/cm) 27.76 72920 26.26 62630 27.38 76008 21.34 63960 13.19 63210 13.91 52060 15.18 46800 15.55 54770 25.13 61990 24.59 60390 26.71 58320 26.84 41980 28.51 62800 24.86 61410 26.33 61074 21.82 51390 14.67 53660 13.12 43600 16.26 47070 15.68 49920 24.46 48670 24.71 45260 27.4 37180 26.06 31590 29.45 63400 27.27 57930 27.32 62021 22.59 52410 15.38 53970 14.2 51550 15.19 45730 16.43 47870 24.07 50110 25.42 45370 28.07 44950 27.89 39770

Salinity (ppt) 47.27 42.18 52.67 43.29 42.53 34.24 30.44 36.28 41.72 40.51 38.88 26.89 39.24 41.28 41.5 33.81 35.44 28.09 30.65 32.72 31.77 29.29 23.48 19.64 38.88 38.57 41.82 34.56 35.69 33.87 29.67 31.23 32.83 29.36 28.98 25.29

DO (mg/L) 6.28 4.68 4.49 3.59 7.61 9.95 9.2 5.37 4.19 8.53 5.83 7.05 5.88 6.07 4.21 6.8 6.71 9.48 7.48 5.48 5.16 7.93 6.75 8.44 6.95 6.74 4.48 7.13 6.39 9.61 9.65 5.58 5.93 8.68 6.81 7.75

pH Secchi Disk (s.u.) (m) 8.39 0.35 8.41 0.375 8.39 >0.46 8.39 >0.45 8.25 >0.66 8.13 >0.51 8.32 >0.49 8.07 >0.57 7.73 >0.48 7.57 >0.51 7.8 >0.48 7.52 >0.51 8.04 0.175 8.05 0.175 8.12 0.33 8.13 >0.55 8.16 0.32 8.2 >0.42 8.37 0.31 7.96 0.275 7.9 0.1 7.77 0.3 7.78 0.225 7.51 0.35 8.03 0.35 8.01 0.5 8.01 >0.52 8.12 >0.72 8.13 >0.66 8.12 0.41 8.01 >0.54 8.11 >0.6 7.96 0.15 7.87 0.3 7.88 0.455 7.62 0.275

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Table 5: Physical water quality parameters from Redfish Bay, Copano Bay, and Bayside for August 2006 through July 2007.

Location Redfish Bay

Copano Bay

Bayside

Date 8/23/2006 9/13/2006 10/11/2006 11/15/2006 12/13/2006 1/11/2007 2/14/2007 3/21/2007 4/11/2007 5/16/2007 6/13/2007 7/11/2007 8/23/2006 9/13/2006 10/11/2006 11/15/2006 12/13/2006 1/11/2007 2/14/2007 3/21/2007 4/11/2007 5/16/2007 6/13/2007 7/11/2007 8/23/2006 9/13/2006 10/11/2006 11/15/2006 12/13/2006 1/11/2007 2/14/2007 3/21/2007 4/11/2007 5/16/2007 6/13/2007 7/11/2007

Temp Conductivity Salinity (°C) (μmhos/cm) (ppt) 31.35 57060 37.78 27.44 54400 35.92 26.21 48043 31.55 22.37 47250 30.77 16.24 47570 31.01 15.6 45240 29.32 12.25 31840 19.85 22.09 40720 26.08 18.5 31800 19.87 26.22 45920 29.73 29.48 38440 24.31 29.15 20740 12.34 30.4 35500 19.95 27.09 37390 23.63 26.34 30096 19.2 21.04 29550 18.31 14.13 35260 22.24 14.83 32900 20.62 12.48 21720 13.08 21.61 29490 18.27 17.03 19920 11.93 27.68 17240 10.11 30.1 19750 11.68 29.46 1768 0.89 30.2 30080 16.69 26.27 26290 16.05 24.96 15140 8.8 20.57 23050 13.96 15.6 27440 16.9 15.61 22690 13.74 9.71 16610 9.75 21.43 3740 1.98 17.26 15600 9.15 27.17 16940 9.92 30.19 15410 8.92 29.64 1257 0.62

DO (mg/L) 6.16 5.99 7.76 5.8 11.62 7.03 10.16 4.91 9.27 8.19 7.01 6.34 5.88 5.67 6.91 7.57 13.36 7.72 10.75 5.28 10.81 9.4 8.7 7.54 5.07 6.2 7.49 8.27 11.98 8.15 12.01 6.9 10.79 9.21 7.97 7.04

pH (s.u.) 8.25 8.08 8.15 8.09 8.03 7.87 8 7.97 8.07 7.85 8.09 8.25 8.02 7.98 7.85 7.99 8.09 7.75 8.17 7.8 8.05 8.05 8.01 8.45 7.93 7.72 7.94 7.95 7.98 8.04 8.25 8.02 8.01 7.81 8.02 8.39

Secchi (m) 0.3 0.72 0.8 0.19 >1 0.75 0.59 0.525 0.55 0.75 0.95 0.8 0.3 0.82 1.05 >1.05 >0.85 0.75 0.73 0.4 0.7 0.55 0.55 0.25 0.575 >0.42 >0.55 >0.48 >0.46 >0.46 0.15 0.03 0.25 0.35 >0.48 0.2

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Table 6: Physical water quality parameter statistics from sites in south Texas coastal waters for August 2006 through July 2007.

Location Bird Island

Statistic (N=12) Mean Std Dev

Temp Conductivity Salinity (°C) (μmhos/cm) (ppt) 21.99 59586.50 39.74 5.83 9771.60 7.07

DO (mg/L) 6.40 2.08

pH (s.u.) 8.08 0.34

Secchi Disk (m) 0.49 0.08

Nueces Bay Mean Std Dev

21.99 5.51

49468.67 9561.62

32.24 6.70

6.70 1.48

8.00 0.23

0.29 0.12

Cole Park

Mean Std Dev

22.77 5.83

51256.75 7175.35

33.40 4.82

7.14 1.57

7.99 0.15

0.46 0.17

Redfish Bay Mean Std Dev

23.08 6.23

42418.58 10326.47

27.38 7.28

7.52 1.98

8.06 0.13

0.66 0.24

Copano Bay Mean Std Dev

22.68 6.69

25882.00 10238.11

15.83 6.48

8.30 2.42

8.02 0.18

0.67 0.26

Bayside

22.38 6.76

17853.92 8833.75

10.54 5.32

8.42 2.20

8.01 0.18

0.37 0.17

Mean Std Dev

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Table 7: Physical water quality parameter ranges from sites in south Texas coastal waters for August 2006 through July 2007.

Location

Range

Bird Island

high low

Temp Conductivity Salinity (°C) (μmhos/cm) (ppt) 27.76 76008.00 52.67 13.19 41980.00 26.89

DO (mg/L) 9.95 3.59

pH (s.u.) 8.41 7.52

Secchi Disk (m) >0.66 0.35

Nueces Bay high low

28.51 13.12

62800.00 31590.00

41.50 19.64

9.48 4.21

8.37 7.51

>0.55 0.10

Cole Park

high low

29.45 14.20

63400.00 39770.00

41.82 25.29

9.65 4.48

8.13 7.62

>0.72 0.15

Redfish Bay high low

31.35 12.25

57060.00 20740.00

37.78 12.34

11.62 4.91

8.25 7.87

>1.00 0.19

Copano Bay high low

30.40 12.48

37390.00 1768.00

23.63 0.89

13.36 5.28

8.45 7.75

>1.05 0.25

Bayside

30.20 9.71

37400.00 1257.00

16.69 0.62

12.01 5.07

8.39 7.72

0.58 0.03

high low

Dissolved oxygen (DO) levels ranged from 3.6 to 13.4 mg/L at all sites during the sampling period (Tables 4 and 5). At Bird Island, Nueces Bay, and Cole Park, DO levels ranged from 3.6 to 10 mg/L (Table 7). DO levels ranged from 4.9 to 13.4 mg/L at Redfish Bay and both sites at Copano Bay (Table 7). Average DO levels were between 6.4 and 8.4 mg/L at each of the sites sampled (Table 6). The highest DO level (13.36 mg/L) occurred at Copano Bay causeway during the December 2006 sampling event, while the lowest DO level (3.59 mg/L) occurred at Bird Island during the November 2006 sampling event (Tables 4 and 5). Overall, DO levels were highest at the two Copano Bay sites and lowest at Bird Island and Nueces Bay (Tables 4 and 5). pH means at all sites were between 7.99 and 8.08 s.u. and ranged from 7.51 – 8.45 s.u. throughout the study (Tables 6 and 7). Measurements of turbidity using Secchi disk ranged from 0.03 to > 1.05 m (Tables 4 and 5). Secchi disk measurements ranged from 0.1 to > 0.72 m at Bird Island, Nueces Bay, and Cole Park (Table 7). At Redfish Bay and Copano Bay Causeway, Secchi disk measurements ranged from 0.19 to > 1.05 m (Table 7). At Bayside, Secchi disk measurements ranged from 0.03 to 0.58 m (Table 7). Mean Secchi disk measurements were between 0.29 and 0.67 m at each of the sites sampled (Table 6). The highest Secchi disk measurement (> 1.05 m) occurred at Copano Bay causeway during the November sampling event, while the lowest Secchi disk measurement (0.03 m)

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occurred at Bayside during the March sampling event (Table 5). Overall, turbidity was lower at Redfish Bay and Copano Bay causeway sites and highest at the Nueces bay site (Table 6). Significant rainfall (over one inch, 2.5 cm) occurred the week prior to the September 2006 sampling event at Redfish Bay, Copano Bay, and Bayside, and there was also significant rainfall the week prior to July 2007 sampling event at each of the sites sampled (Tables 8 and 9). Rainfall data was gathered from the National Oceanic and Atmospheric Association. Nearly half an inch of rain (1.25 cm) occurred the day before sampling Redfish Bay, Copano Bay, and Bayside in September 2006 and nearly two and a half inches (6.25 cm) of rain occurred during the week prior to sampling (Table 9). There was three and a half inches (8.75 cm) of rain the day before sampling at Bird Island, Nueces Bay, and Cole Park in July 2007 and just over fourteen and a quarter inches (35.63 cm) of rain during the week prior to sampling (Table 8). There was over three and a half inches (8.75 cm) of rain during the week prior to sampling at Redfish Bay, Copano Bay, and Bayside in July 2007 (Table 9). The lowered salinity at each site in July 2007 was presumably related to the preceding heavy rainfall (Tables 4 and 5). Less than an inch (