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Technical Workgroups :: Biosensors and Biomarkers

Effect of Mississippi River Diversion on Carbon and Nitrogen Loading, Water Quality, Microbial Ecology, and Hypoxia in the Wetlands of Gulf of Mexico
Dr. Ramaraj Boopathy, Nicholls State University Department of Biological Sciences

This project monitors the water quality, in terms of carbon and nitrogen loading and microbial population in the water column as it influences hypoxia in the wetlands, of lower Barataria-Terreobonne Estuary. Specific objectives were:

  1. To continue to monitor the water quality and microbial loads in the water columns of the wetland.
  2. To monitor physico-chemical changes including pH, salinity, temperature, dissolved oxygen, nitrate, phosphate, ammonia, and total organic carbon in the bay in many sampling locations through various seasons.
  3. To study the influence of organic pollution and nitrate from fertilizer run-off on hypoxia in the lower reaches GoM estuary.

Water quality was monitored at five different sites, namely, Point Legard Bend, Coffee Bay, Hackberry Bay, Pelican Point, and Independent Island, in the Barataria Bay. Monthly water samples were collected and water quality was analyzed for Chemical Oxygen Demand (COD), pH, turbidity, salinity, temperature, Dissolved Oxygen (DO), nitrate, nitrite, ammonia, phosphate, fecal coliform bacteria, and total heterotrophic bacteria. The data indicates that there is a seasonal difference as well as difference among various sites. The carbon and nitrogen loadings were higher than other sites in two of the tested areas, Hackberry Bay and Independent Island. The source of this pollution in Hackberry Bay is a cattle farm nearby; due to runoff of manure from cattle increased the carbon and nitrogen concentration in this site. At Independent Island, the reason for increased nitrogen and carbon concentration is the presence of a bird-sanctuary nearby. There is a localized hypoxia in these waters. We continue to monitor the water quality and we have a good data set for the past four years on these five sites in the lower reaches of Barataria bay. One interesting observation was the presence of antibiotic resistant bacteria such as E. coli and Pseudomonas in the water columns of Barataria bay. We isolated and identified several antibiotic resistant enteric bacteria in the water samples. Some of these bacteria are also salt tolerant which survived at high salinity conditions up to 55 ppt. This observation will have significant impact on oyster farming in the bay. The Louisiana Department of Human and Health Services closes the oyster beds for harvesting if the E.coli count is 14 MPN FC/100 ml sample, and this water quality standard is set based solely on the fact that the E. coli cannot survive high salinity.

Monitoring Reproductive Biomarkers of Fish and Amphibians in the Barataria-Terrebonne Wetlands Restoration Area
Dr. Gary Lafleur, Nicholls State University Department of Biological Sciences
Dr. Lois Nelson, Nicholls State University Department of Biological Sciences

The objective of this project was to monitor reproductive health in a subset of the aquatic organisms in the Barataria-Terrebonne Estuary combining the following surveys:

  1. Behavioral biomarkers of reproduction including frog-call surveys to document which species are courting, the seasonal duration of their activity, and annual variability in courtship.
  2. Anatomical biomarkers of reproduction including the gonadosomatic index and gonadal histology of fish and amphibians to document reproductive seasons and energy investments
  3. Molecular biomarkers of reproduction including the expression of female-specific proteins such as vitellogenin, and male-specific proteins such as the androgen receptor in order to test whether cross-gender gene expression may be occurring.

We have established five survey sites within the BTES ranging from the saltwater habitats of the Isled Dernieres Barrier Island Refuge to the upper reaches of the freshwater swamps near Bayou Chevreuil and Lake Des Allemands. At these sites we have collected a diverse group of aquatic animals ranging from fish to amphibians, and we have documented reproductive health using the above combination of observations.

Impacts of Hypoxia on White and Brown Shrimp (Penaeus spp.)
Dr. Enmin Zou, Nicholls State University Department of Biological Sciences

In recent years, the occurrence of environmental hypoxia has become commonplace in the warm seasons in the northern Gulf of Mexico. The depletion of oxygen in the waters of the northern Gulf of Mexico comprises a serious environmental stress to the aquatic animals, such as the brown shrimp, Penaeus aztecus, an important commercial crustacean, inhabiting in this environment. Besides the stress from hypoxia, the brown shrimp is subject to the pollution of polycyclic aromatic hydrocarbons (PAHs) from gas and oil productions in the northern Gulf of Mexico. The PAHs enter water column and sediments via a number of routes, such as through produced water discharges and discharges from drilling operations, the natural seepage of reservoired oil and gas, and oil spills. Therefore, the brown shrimp in the Gulf of Mexico is faced with two stressors, hypoxia and PAHs pollution. Acute exposure to naphthalene at 2.0 mg/L was found to significantly decrease the oxyregulating capacity of Penaeus aztecus. An ensuing consequence of a decrease in oxyregulating ability is that a hypoxic condition that would otherwise be tolerable to shrimps in the clean environment would become a stress to the animals that are simultaneously subject to PAH pollution. Therefore, the shrimps become more susceptible to hypoxia in the presence of the PAH compound naphthalene. The implication of this finding is that PAH contamination is an important factor in determining the responses of biota of the northern Gulf of Mexico to environmental hypoxia. Aquatic animals are known to respond to environmental hypoxia by increasing the ventilation rate. It has been postulated that hypoxia may increase the accumulation of a toxicant in an aquatic organism due to a higher water flow over the gills. Our results show that Hypoxia has no effect on bioaccumulation in the brown shrimp. Enzymatic analysis reveals that the absence of a significant effect of hypoxia on naphthalene bioaccumulation is due to the increased metabolism of this compound. Current research is focused on determining whether hypoxia and naphthalene have an effect on molting of the penaeid shrimp.

Pharmaceuticals and Personal Care Products (PPCPs), From the Mississippi River to the Gulf of Mexico
Dr. Guangdi Wang, Xavier University of Louisiana Department of Chemistry

The overall goal of this project is to monitor the occurrence and fate of persistent organic pollutants in the Mississippi River (MR) and Gulf of Mexico (GoM) estuary by detecting and quantifying compounds of concern. The compounds studied are 16 polycyclic aromatic hydrocarbons (PAHs), 28 polychlorinated biphenyls (PCBs), and 12 pharmaceuticals and personal care products (PPCPs). We monitored each of these in Mississippi river, Lake Pontchartrain, Bayou St. John, and Gulf of Mexico waters and particulate matter. The data show that most of PAHs were detected in all surface waters. Most of the PCBs monitored were not detected in surface waters. Of the 12 PhACs monitored, 9 were detected in New Orleans surface waters.

Biosensors and Platform Development
Dr. Diane Blake, Tulane University Department of Biochemistry
Dr. Robert Blake, Xavier University of Louisiana College of Pharmacy
R. George Rey, COTS Technology, LLC

The long-term goal of this project is to develop highly sensitive and specific immunosensors to monitor selected organic contaminants in both fresh and saltwater samples. A suite of advanced biosensors is envisioned that will enable both portable, on-site field tests and autonomous, real-time sampling and testing capabilities. Toward that end, our laboratories have initiated the development of both a hand-held immunosensor and an antibody-based biosensor to be deployed on both point-source monitoring stations (buoys) and autonomous underwater vehicles (AUVs) for analysis of pollutants in the Mississippi River/Gulf of Mexico estuary. In collaboration with Sapidyne Instruments, Inc, Dr. Diane Blake’s lab has developed varying sizes of immunosensors for use in the lab and in the field. Each sensor can detect a variety of human pollutants, including ethylenediaminetetraacetic acid (EDTA) and caffeine with changeable antibody cartridges. R. George Rey of COTS Technology is working with the Blakes’ labs to place the immunosensor onto AUVs that would be deployed from a research vessel to test in areas that are hard to reach by other means.

Trace Gases of Microbial Flora as a Biomarker of Pollution
Dr. Ramaraj Boopathy, Nicholls State University

Department of Biological Sciences The relationship between microbial trace gas production in sediments and pollution was investigated using sediments collected from the wetlands of Barataria-Terrebonne Estuary (BTE) in Louisiana. The results indicated the production of various gases such as CO2, methane, hydrogen sulfide, and nitrous oxide in various sampling sites. The trace gas composition varied from site to site. There was a linear relationship between the nitrate concentration in the sediment and the production of nitrous oxide, which indicated the denitrification activity in the sediment. Based on these data, we were able to track the possible source for nitrate run-off from a cattle farm near the site. The results also indicated a possible connection between nitroaromatic compounds such as nitrophenol, nitrobenzene contamination and the production of hydrogen sulfide in the sediment due to specific activity of sulfate reducing bacteria. Some microbial gases, namely nitrous oxide and hydrogen sulfide, could be used as a biomarker to indicate certain contaminant and specific microbial activity in the sediments.

Molting in Fiddler Crabs, Uca pugilator, as a Biomarker of Pollution
Dr. Enmin Zou, Nicholls State University Department of Biological Sciences

Aquatic environments are increasingly contaminated with various organic pollutants. Many, especially the organochlorine compounds, are highly hydrophobic and can readily accumulate in the tissues of crustaceans at concentrations much higher than those in the water column and sediment. Molting is an important physiological process for crustaceans because it not only allows for growth and development of these animals bearing a rigid, confining exoskeleton, but is also tied to metamorphosis during the early stages of the life cycle and reproduction during the adult stage. Considering the importance of molting in the life of crustaceans and for the purpose of regulation of anthropogenic chemicals, it is necessary to develop an assay that can be used to identify xenobiotics capable of interfering with molting of crustaceans. The objective of this project is to develop a screening assay that utilizes the activity of chitinase, an ecdysteroid-regulated enzyme, as the endpoint to detect molt-interfering activities of xenobiotics. To prove that chitinase activity is a reliable marker for the action of the molting hormone in Uca pugilator, we first examined whether activity of chitinase in the epidermis is inducible by the molting hormone. Two injections of 20-hydroxyecdysone at a dosage of 25 μg/g live weight resulted in a two-fold increase in epidermal chitinase activity (P < 0.001), suggesting that chitinase activity in the epidermis of Uca pugilator is regulated by the molting hormone. In the next experiment, we screened for molt-interfering activity of nine xenobiotics using epidermal chitinase activity as the endpoint. Of five organochlorine pesticides, only o,p’-DDT at 0.2 mg/l significantly inhibited chitinase activity in the epidermis of Uca pugilator by 32% after a seven-day exposure (P < 0.05). Although changes in enzymatic activity were not statistically significant, a seven-day exposure of fiddler crabs to kepone at 0.5 mg/l and methoxychlor at 0.2 mg/l (P < 0.1 in both cases), nonetheless, resulted in a 23% and 25% decrease in epidermal chitinase activity, respectively. Atrazine at 1.0 mg/l (P > 0.05), TBT at 1.0 mg/l, methoprene at 1.5 mg/l, permethrin at 5.0 μg/l (P > 0.05 for these three chemicals), dieldrin at 0.1 mg/l and p,p’-DDT at 0.1 mg/l (P > 0.05 for both chemicals) had no effect on epidermal chitinase activity. Neither acetone nor ethanol was found to have an effect on epidermal chitinase activity in Uca pugilator.