CICEET Progress Report for the period 9/01/08 Through 2/15/09

Project Title: Validation of a Real-Time, Field Deployable Biosensor, for the Detection and Quantification of Polycyclic Aromatic Hydrocarbons (PAHs) in Aquatic Systems
Principal Investigator(s): Michael A. Unger
Additional Investigator(s): Stephen L. Kaattari, Erin S. Bromage
Project Start Date: September 1, 2007

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Tasks to meet objectives
1) Improve precision and accuracy of Biosensor instrument: We will work in close contact with the instrument manufacturer (Sapidyne) to improve performance of the instrument with hydrophobic organic compounds. Silanization and DMSO solvent have been proposed as possible options. The instrument will also be tested for accuracy and precision under field conditions to evaluate any issues with temperature or humidity changes on performance.

2) Conduct field validation trials of the sensor in the Elizabeth River: Newly established end users will be providing groundwater and additional river sampling opportunities over the next several months. EPA is interested in the potential for real-time monitoring of remedial dredging so we will coordinate this trial.

3) Develop additional antibodies with great specificity for alkyl-PAH detection: New antibody development will be a high priority over the next six months. A new antibody that recognizes two ring PAH (described here) will need to be fused and put into production soon. Additional haptens will be developed to induce alkyl PAH recognition.

4) Further develop multi-compound detection with the biosensor using multiple antibodies for simultaneous detection of mixtures: As new antibodies become available these techniques will be refined to better quantify PAH and relate this to totals and individual compounds as determined by GC-MS.

5) Disseminate results: A presentation at the SETAC conference will occur in November. A publication describing one of the new antibodies and methods for PAH antibody development is now in draft form and will be submitted soon.

Progress on Tasks
a. Improve precision and accuracy of Biosensor instrument: A new cleaning protocol and sampling sequence was developed for the biosensor using DMSO. This has proven effective at eliminating cross contamination issues from high concentration samples that interfered with precision and accuracy of the instrument.

b. Conduct field validation trials of the sensor in the Elizabeth River: We have completed two field trials of the sensor, one in the Elizabeth River, VA and one in New Bedford, MA. We have also conducted a validation trial of the sensor for measuring PAH concentrations during toxicity experiments. Results of these trials are presented in this progress report.

c. Develop additional antibodies with great specificity for alkyl-PAH detection: We currently have several new antibodies under development at VIMS and in the Bromage lab at U Mass Dartmouth. Specificity testing of one of the VIMS antibodies is presented in this report. Four antibodies are in development in the Bromage lab, which demonstrate a good degree of specificity for their targets. These include, 2 alkylated naphthalenes, alkylated phenanthrene, and biphenyl. Fusions have been performed and detailed specificity screening is currently being conducted. If successful, these antibodies will be used in the second deployment of the sensor at Dartmouth.

d. Further develop multi-compound detection with the biosensor using multiple antibodies for simultaneous detection of mixtures: This has been delayed until the additional antibodies are further evaluated for performance and available for multi-analyte detection.

e. Disseminate results: Results have been presented at international and local meetings and published in the peer-reviewed literature.

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal? Please explain.
New difficulties with interfering fluorescence in particulate material from the Elizabeth River have demonstrated the need for a viable filtration technique in the field. This will be a major research focus over the next couple of months. We need rapid, consistent filtration techniques that are repeatable in the laboratory for large volume samples for GC-MS validation of filtered samples that does not interfere with PAH quantification. A protocol will be developed to determine if filtering is needed in the field. In many cases, the filtering may not be required.

Collaborations with new end users (academia, USFW, EPA) for toxicity testing and analysis of PAH in water at a Superfund creosote site will expand our field validation efforts.

Dissemination activities during this reporting period (please include the number of participants where applicable).
Project related presentations/poster sessions at workshops/conference
The following was presented at the SETAC meeting in Tampa, FL, November 2008: Lab and field evaluation of an Inline Biosensor for water-soluble PAHs

Manuscripts published or submitted for publication
Manuscript in press and now available on line:
Spier, C. R., E. S. Bromage, T. M. Harris, M. A. Unger and S. L. Kaattari. (In press) The development and evaluation of monoclonal antibodies for the detection of polycyclic aromatic hydrocarbons. Analytical Biochemistry, (2009), doi:10.1016/j.ab.209.01.020

Manuals, Protocols (submitted and in progress)
The dual-analyte detection protocol is on hold until additional antibodies are available to evaluate the technique in a realistic manner. Those antibodies are under development (described here) and should be available very soon. As we only currently have a single monoclonal antibody specific for an oil compound (Spier et al 2009) we have been unable to address the dual analyte 'oil' sensor in this current period. However, a second project that is running in the Bromage Laboratory (U Mass Dartmouth) is examining the concentrations of two antigens in animal tissue homogenates. We have successfully utilized the biosensor to perform this task and have achieved very good results; we expect that it will take only minor modifications to adapt the protocol for dual analyte oil detection when the additional monoclonal become available.

Outreach Activities
"An update on CICEET funded biosensor research at VIMS: working with industry and government partners" (Unget et al.) VIMS/William and Mary/Industry Partnership Meeting, VIMS. February 13, 2008. Approximately 40 individuals from academia, industry and federal agencies were present.

"Sensors in Bioscience from Research to Products Symposium" (Kator et al.) Hampton Roads Research Partnership. Sulfolk, VA Feb. 17, 2009.

Contact with End Users
Continued discussions about collaboration and validation of the Biosensors have occurred with Joe Rieger, Director of Watershed Restoration, Elizabeth River Project; Randy Sturgeon, US EPA Region III Superfund Site; and Ross Worsham, Atlantic Wood Industries. Regulatory concerns for the dredging project have been resolved and Biosenor monitoring of the dredge project at Money Point in the Elizabeth River is planned for June 2009.

A new application of the Biosensor to monitor contaminant concentrations during aquatic toxicity testing was explored with other Virginia Institute of Marine Science researchers/end users (Chris Prosser and Wolf Vogelbein). Monitoring of a preliminary toxicity test was performed and the results are presented in this report. Monitoring of additional experiments is planned for the near future.

As a result of the presentation at SETAC 2008, USFW personnel contacted us at the Tampa meeting to discuss the possible application of the biosensor at a creosote contaminated Superfund site in Virginia. We met with USFW (Anne Condon, John McCloskey) on February 25th to learn more about the site and possible applications for the sensor. A plan was developed to conduct a site visit at the end of March and deploy the sensor to sample ambient waters for PAH contamination derived from creosote. The sensor will be used to find elevated PAH concentrations and then validation samples will be collected for GC-MS analysis. Logistics are being worked out by USFW with EPA and landowners to facilitate this effort in late March.

Patent, Copyright, Invention Disclosure Activity
The PI's and student (Spier) have filed an additional disclosure statement with William and Mary describing a new antibody that is being evaluated as part of this research.

Student activity (e.g. theses, dissertations, etc.) on the project (please identify students as graduate or undergraduate)
The dissertation research of one doctoral graduate student, Candace Spier, is directly tied to objectives of this research project. Her dissertation "The Detection of Petroleum-Derived Thiophenes in Environmental Samples Using Immunochemical Techniques" is focused on the development and validation of antibodies to detect petroleum derived PAH and thiophenes in environmental samples. Her assistantship and tuition is currently funded by sources other than this grant but much of her research is supported by this project.

Ms. Spier presented results from our CICEET research at the 2008 SETAC meeting in Tampa, FL and is first author on our recent publication.

Difficulties
Field testing of the Biosensor on the Elizabeth River under severe conditions (ambient temperatures in 35-40 °F range) caused problems with pumping solutions in the Biosensor. Once moved into the truck on site and warmed, the fluidics in the system worked routinely. Another difficulty was encountered when particulate material in the ambient river water contained fluorescence that interfered with detection of the antibody response in the Biosensor. This is the first time we experienced that type of interference from particulate material in natural samples. Samples brought back to the laboratory were filtered and measured 1.2 µg/L total 3-5 ring PAH by the biosensor. GC-MS analysis showed 1.1 µg/L for the same sample. New field/lab filtration protocols are a new objective for the next period as will be further field testing before monitoring a dredge event with ERP at the Elizabeth River in June. This interference may be a seasonal or site-specific phenomenon. We will develop protocols to determine if filtration is needed in the field at all times.

Data Generated to date
Biosensor analysis of PAH in water samples from a toxicity experiment, October 2008. A new application of the Biosensor was recently demonstrated with toxicology researchers (C. Prosser, W. Vogelbein) at VIMS. The biosensor enabled the researcher to monitor PAH concentrations during a 12-hour exposure period to evaluate disappearance of the toxicant before renewal at 12 hours. Standard analytical methods didn't allow this level of resolution due to effort and expense. Good correlation between the sensor and later HPLC analysis was found. Slightly higher values reported by the biosensor at 6 and 12 hours may be due to metabolites not quantified by HPLC. This application of the biosensor technology allows more accurate dose measurements during toxicity testing because of the finer resolution possible by continuous sampling.

On December 17, 2008, the first trial field deployment of the sensor took place in Clark Cove, New Bedford MA. Turbidity readings on this day were in excess of 9 feet, (i.e. clear water). The sensor was deployed from a jetty (supplied with power) over a 12 hours period, using the DBT antibody, to examine the stability of the sensor in field conditions. A single line was deployed at a 1.5 m surface depth and a second line was placed at the bottom of a 10 L tank containing the seawater sourced from the same location. Samples from both lines were taken every hour and quantified against the standards. Over the 12-hour period we only observed minor fluctuations (+/- 0.2 µg/L) in samples obtained from the first line and no variation in samples obtained from the control tank (Figure 1). Field samples were below, or slightly above, our detection limit (0.1µg/L) indicating little PAH contamination. At 12.30pm the control vessel was spiked with 100µg of dibenzothiophene. The subsequent samples from the control tank demonstrated an increasing BDT concentration until the 4pm reading when it stabilized. From this first deployment we have determined that in the absence of significant particulates (silt, plankton, etc), the sensor can perform reliably without any fouling or sample line blockage. A second deployment is scheduled for early March (weather and ice dependent) on the opposite side of New Bedford Peninsula (New Bedford Harbor). We expect to see significant PAH contamination in these samples and we will validate the sensor readings with traditional chemical validation.

The evaluation of a new antibody under development at VIMS. A plot demonstrating that an antibody in mouse sera is selective for biphenyl and only 3-ring PAHs, while not recognizing the 2-, 4-, or 5-ring PAHs tested here. Our currently used antibody (Spier et al, 2009) recognizes 3-5 ring PAH. Further development of a monoclonal from this serum is underway and it will be evaluated as a dual analyte method soon.

Monitoring PAH concentration on the shore of the Elizabeth River, VA, February 16, 2009. Ambient temperatures in 35-40 °F range caused problems with pumping solutions in the Biosensor. Once moved into the truck on site and warmed, the fluidics in the system worked routinely. Background particulate material in the ambient river water contained fluorescence that interfered with detection of the antibody response in the Biosensor. This is the first time we experienced that type of interference from particulate material. Samples brought back to the laboratory were filtered and measured 1.2 &mig/L total 3-5 ring PAH by the biosensor. GC-MS analysis showed 1.1 µg/L for the same sample. New field/lab filtration protocols are a new objective for the next period as will be further field testing before monitoring a dredge event in June.

Project Objectives for Next Reporting Period

Objectives
1) Improve precision and accuracy of Biosensor instrument including evaluation of filtration techniques.
2) Conduct field validation trials of the sensor:

1. In the Elizabeth River,
2. At New Bedford, Mass,
3. Monitor a laboratory toxicity experiment
4. Monitor a creosote Superfund site.

Work Plan to Meet Objectives
1) Improve precision and accuracy of Biosensor instrument: We will develop a filtering protocol for field and laboratory samples. This will require a rapid small volume field technique as well as an equivalent method for large volume validation samples in the laboratory.

2) Conduct field validation trials of the sensor in the Elizabeth River: Trials with end users are scheduled for the Elizabeth River in June, USFW in March and VIMS toxicity research scientists in April. Additional trials in New Bedford Harbor will also take place once conditions permit.

3) Develop additional antibodies with great specificity for alkyl-PAH detection: New antibody development is a continuing high priority over the next six months. Preliminary evaluation of sera shows several promising antibodies under development that should be available soon as monoclonals.

4) Further develop multi-compound detection with the biosensor using multiple antibodies for simultaneous detection of mixtures: As new antibodies become available these techniques will be refined to better quantify PAH and relate this to totals and individual compounds as determined by GC-MS.

5) Disseminate results: A presentation at the SETAC conference will occur in November 2009. New publications describing field validation and multi-analyte detection are planned for submission in 2009. Field validations will be performed in collaboration with potential end-users of the technology.

Dissemination Objectives for next reporting period
One of the PIs (Unger) has been invited to participate in the Coastal Response Research Center (CRRC), University of New Hampshire (UNH) and NOAA Office of Response and Restoration (ORR) Workshop:"Research & Development Priorities: Oil Spill Workshop" on March 17 ­ 19, 2009 at the University of New Hampshire. During the 2 _ day meeting, participants will review the innovative research that has occurred since the 2003 report and identify remaining gaps and future needs to improve response. The new technology development funded by this CICEET grant has important potential application for oil spill detection and analyses as well as application in oil spill related toxicity research.

We plan to make presentations at SETAC 2009 in New Orleans, November 2009. Additional peer-reviewed publications showing multi-analyte detection and field application of the Biosensor for PAH analyses are planned for submission in 2009.

Overall Project Timeline Update

Expenditures
All expenditures are in the range expected for the work accomplished so far. Additional costs for supplies and field-testing of the sensor are expected in the next period and are accounted for in the budget.

End User Advisor Feedback
End User Advisor: Joe Rieger, Director of Watershed Restoration
Organization: The Elizabeth River Project
Location: 475 Water Street, Suite 103
Portsmouth, Virginia 23704
Phone number: 757-399-7487
E-mail: jrieger@elizabethriver.org

The Elizabeth River Project and Living River Restoration Trust are non-profit organizations which working to remediate contaminated sediments in the Southern Branch of the Elizabeth River. The Southern Branch has some the highest PAH sediment concentrations in the Chesapeake Bay which are the result of defunct creosote facilities and urban stormwater runoff. One such creosote site is at Money Point where Elizabeth River Project and the Trust are working to clean up. The remedial option for Money Point is to dredge over 75,000 cy of PAH contaminated sediments. However, during dredging contamination will be released into the water column and currently we are not aware of technologies that can give us real time results in the field. The biosensor being developed will be used to help us better understand the amount of contamination entering the water column.

Has anything changed about this project's potential applicability since the last reporting period?
Since the Elizabeth River Project's last report much has happen to forward a large scale restoration which will include dredging up to 800 cubic yards of contaminated sediments at Money Point. The Elizabeth River Project has received all regulatory permits and is currently working to hire a construction contractor in the next two months. It is anticipated that the dredging of contaminated sediments, between 45 ppm- 2000 ppm, could begin as early as late June 2009. The pilot scale dredging project will provide VIMS with an ideal sampling event to field test their sensor. The data which is collected during this event will provide the Elizabeth River Project along with regulatory agencies real-time data to determine the amount of dissolved PAHs which might be re-suspended during an environmental dredging project. Current technologies, that we are aware of, cannot produce this real-time data during a dredging project.

During the summer of 2009, The Elizabeth River Project will work directly with VIMS to coordinate field monitoring activities during dredging and the data will be used by the design engineer Earth Tech/AECOM in the final design of the larger 75,000cy dredging project anticipated at Money Point in 2010-2011. The data will guide the design by provide data on how effective silt curtains and booms are at controlling dissolved PAH's in the water column during dredging.

PI Response to End User Advisor Feedback
No specific questions to address. The ERP partnership with industry, government agencies and academia has provided us with the ideal opportunity to obtain field samples and demonstrate the new technology to a variety of end users. The planned monitoring of the dredging event has been a useful goal for our research as it is guiding the field validation portion of our research. The instrument manufacturer has planned modifications to the instrument based on problems encountered in our field trials for this application.

Additional Comments from Chris Prosser, Virginia Institute of Marine Science Researcher, about conducting PAH measurements with the Biosensor during PAH toxicity experiments:

The ability to use real time quantification techniques for measuring contaminant concentration would be an invaluable tool for my future research experiments. Currently samples collected are run through a 35 minute cycle using high performance liquid chromatography (HPLC), followed by manual integration of chromatograms and concentration calculations. The nature of this method can allow for unintended error to bias results. Repetitive sampling of specific volumes, addition of internal standards and manual peak integrations are areas where human error can occur. Long run times and high sample numbers can also result in loss through volatilization as samples wait to be run. The biosensor technology reduces human error, and eliminates virtually all wait time between sample runs. The successful application of this technology results in more plentiful concentration data, lower cost, more efficient time use, and reduced experimental error.