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Tulane/Xavier UniversityHazardous Materials in Aquatic Environments

of the Mississippi River BasinQuarterly Project Status Report

(October 1, 1993 - December 31,1993)

Administrative Activities

We continued to seek improvement in our methods of communication and interactionsto support the inter-disciplinary, inter-university collaborators within this program. Inaddition to the defined collaborative research teams, there is increasing interaction amonginvestigators across projects. For example there has been improvement in thecoordination of sampling and analysis among the projects. The formation of sampling andanalytical committees and the project presentations at the monthly P.I. meetings have beenfundamental to this process.

The co-directors of the project participated in the Office of Technology Exhibit at DOE-Headquarters November 29 and 30 and at the Germantown on December 2. Threeundergraduate students (2 from Xavier, 1 from Tulane) who participated in an internshipprogram at Oak Ridge National Laboratory this past summer visited the exhibits at DOEHeadquarters. They also met with Isiah Sewell and Susan Prestwich at Germantown. Allof these students have indicated that the exposure to the internship program and the visithave provided them with new perspectives on career opportunities in energy relatedresearch. At the time, one of the two biology majors had begun work in a master's atTulane's School of Public Health, and the other was applying to graduate programs inPublic Health at universities outside the State. The engineering student who began lastsummer with a lot of indecision regarding his plans after the completion of his B.S. hasdecided to go on to graduate school in either civil or environmental engineering.

Planning for the second year of the project has included the development of our internalrequest for proposals, and refining the review process for selection of proposals forfunding. Additionally, an external panel (see attached listing) has been selected to reviewthe proposals and make recommendations regarding funding and program orientation forthe second year.

Planning has gotten underway for the academic poster session to be held Monday, February7, 1994. The poster session will serve as a site visit for the DOE Program Officer assigned tothe project. The session will provide an opportunity for all participating investigators tohighlight their on-going research supported by the project.

The poster session format wa_ chosen for several reasons. 1) The format is believed to bemore conducive to exchange o_ information than the extremely brief 5-10 minutepresentations. 2) The format allows us to address a larger audience. Announcementsregarding the session will be sent to selected State and local environmentally relatedagencies and organizations, other universities around the State, related industry contactsand interested Federal contacts (U.S. Department of Forestry, EPA and Army Corp of

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Engineers). 3) The session is scheduled the day before the review panel meets. Thisformat will allow the reviewers to meet investigators and to evaluate the progress of theprojects that are requesting continued funding. 4) The session also will provide aneducational opportunity to students. Both undergraduate and graduate students will beencouraged to the attend the session. Graduate students working on funded projects mayparticipate in the poster session itself. 5) The focus on poster preparation will promotemany discussions and exchanges of data among the investigators.

DISCLAIMER

This report was prepared as an account of work sponsored by an agency of the United StatesGovernment. ?4either the United States Governmentnor any agency thereof, norany of theiremployees, makes any warranty, express or implied, or assumes any legal liability or responsi-bility for the accuracy, completeness, or usefulness of any information, apparatus, product, orprocess disclosed, or representsthat its use would not infringe privately owned rights. Refer-ence herein to any specific commercial product, process, or service by trade name, trademark,manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recom-mendation, or favoring by the United States Government or any agency thereof. The viewsand opinions of authors expressed herein do not necessarily state or reflect those of theUnited States Governmentor any agency thereof.

HAZARDOUS MATERIALS IN AQUATIC ENVIRONMENTSOF THE MISSISSIPPI RIVER BASIN

TULANE/XA VIER EM PROJECT

1994 REVIEW PANEL

William Schutte*

DOE - Office of Research and Development

Norman Cutshall*

Deputy DirectorOffice of Environmental Technology DevelopmentOak Ridge National Laboratory

David Norriss*

Crescent Technology, Inc.

Glenn Paulson- Director*

Armour College of Engineering & ScienceIllinois Institute of Technology

Charles Kidd*

Associated Vice President for Environmental ProgramsFlorida A&M University

Maureen LichtveldATSDR

Charles Turner

University of El Paso

I ilill i il II III i I i li

EDUCATION PROPOSAL REVIEWERS

Isiah SeweU

Department of Energy

Jeanette JonesAlabama A&M University

Joan MillerAssociated Western Universities

t I I

Collaborative Cluster Projects

Project Title: Biologica! Fate and Transport of Toxic and Hazardous Materials

Principal Investigator:. A.A. Abdelghani, ScD

All subclusters reported having obtained some results from research conducted during thisquarter. Most of these findings will be presented in the DOE Tulane/Xavier poster sessionon February 7, 1994.

I. Ecology Subcluster:Fish, invertebrates, sediment and water samples were taken in the third quarter from theMississippi River near Buras, LA and Devil's Swamp at the northern-most end of Devil'sSwamp Lake. Once again, the samples were shared by several investigators in theBiological Fate group. Dry. Bill Hartley of Tulane and Rani Thiyagarajah of Xavier andassociates performed health assessments on the fish specimens. They report interestingtumorous growths on at least one of the species of catfish from Devil's Swamp Lake.Stomachs were removed from the fish and delivered to Dr. Peter Martinat of Xavier for

analysis of stomach contents. The data on feeding are being used to determine theimportant links in the aquatic food chain. D.r Martinat is identifying invertebrates frombottom sediments to elucidate the ecology of organisms in fishes' diets. After dissection,fish carcasses are being retained in a frozen state together with samples of water andsediments for analysis of inorganic and organic contaminants by Dr. Abdelghani. To date,the analysis has revealed unsafe levels of lead (Pb) in samples of fish and crayfish fromDevil's Swamp. The electrofishing boat ordered for the project was delivered in Decemberand will be used in sampling in coming months.

Drs. Leonard Thien, Erik Ellgaard and Margaret Devall have been actively coring cypresstrees in Bayou Trepagnier, Devil's Swamp and Cat Island on the Mississippi River nearBayou Sara. A total of 91 cores have been taken to date. The cores have been mounted andsanded, and their collection coordinates logged into a computer. They are now undergoingcross-dating analysis at the U.S. Forest Service, and heavy metals analysis on ICP/AA inthe Coordinated Instrumentation Facility. Sally Spahn, a graduate student of Tom Sherry,has established feeding sites for a large colony of wading birds near Bush, Louisiana andhas collected samples of feathers and droppings from five species from the colony. Sallyhas also made trips to Devil's Swamp to observe feeding by birds and collect samples ofpotential prey items. Samples of feathers, egg shell, droppings and prey items from bothsites are awaiting contaminant analysis.

II. Biomarkers Subcluster:The Biomarker Subcluster is evaluating specific biomarkers of toxic effects in fish and frogswith focus on cancer and disease, developmental effects, neurotoxicity andimmunotoxicity. Biomarker evaluation of these endpoints will be conducted in the fieldand validated in laboratory "in vivo" aquatic animal models.

Fish were evaluated by Drs. Hartley and Thiyagarajah. The total lengths of channel catfish(lctalurus punctatus)ranged from 19.5-28.5 cm, and the yellow bullheads (Ictalurus natalis)

ranged from 27-39 cm. Dr. Thiyagarajah examined the histological preparations of varioustissues from 10 channel catfish and 4 yellow bullheads.

The protozoan parasites (epitheliocystis, trichodina, ambiphrya, henneguya,myxosporidians) were frequently found in gills. Monogenean flukes were also seen in thegills of channel catfish. Two of the four yellow bullheads had liver telangiectasis where theabnormally dilated sinusoids contained large amounts of blood. The same bullheads alsohad a suspected benign neoplasm "Cavernous hernangioma." In addition, all four yellowbullheads had a lesion which could be an early lesion of spongiosis hepatis. These lesionswere characterized by dilated sinusoids devoid of blood and a dilated perisinusoidal spacecontaining pale eosinophilic flocculent material. There was no damage to the endotheliallining. The multilocular cystic structures were seen around the larger blood vessels andalso along the sinusoids. These lesions were somewhat similar to those described in ratsand in other fish species after exposing to chemical carcinogens, and also in fish collectedfrom polluted rivers. In addition two bullheads had amyloidosis in the livers, and in thefollicular artery of the spleen. Initial results on the pathological evaluation of garfish andcarp from Devil's Swamp have resulted in identification of several lesions. In garfishlesions included inflammation of muscle tissue, high density melanin macrophage centersin the liver and inflammation of the pancreas. In carp, lesions included ectopic thyroidtissue in the kidney, telangiectasis of the gill and periductal inflammation of the liver andvascular elements. Approximately fifty fish representing nine different other species fromDevil's Swamp are under histopathologic evaluation.

Dr. Ide, Dr. Tompkins and Dr. Homer have continued to explore the normal neurologicaland immunological parameters in frogs from Devil Swamp contaminants. Wild caughtRana .catesbiana (N=5) from the banks of Devil's Swamp Lake had normal immunefunction as measured by PHA and CON A mitogen stimulation of T-cells. The animalswere of robust size and appeared healthy. Immunocytochemistry for neural IL-1 1_and theglial fibrillary acidic protein (GFAP) appeared normal in the brain. Both of these proteinshave been shown to be upregulated during exposure to heavy metals such as lead.

In laboratory studies related to field studies, spawnings from the laboratory frog, Xenopus)aegy._viswere obtained from mating pairs stimulated with hormone. Early cleavage stageembryos were placed in 6 bowls (50 embryos/bowl). Two bowls contained water fromDevil's Swamp Lake, 2 contained water plus sediments from the lake, and 2 bowlscontained laboratory water. All groups showed normal development and good swimmingbehavior through feeding tadpole stages when the experiment was terminated.

Since mercury is one of the major contaminants of the Clinch River system at Oak Ridge,and since mercury is also a contaminant found in Devils' Swamp, we chose to focus onhow low levels of mercury equivalent to those found in areas of Devil's Swamp mightinfluence frog physiology and development. Expected endpoints of these studies are toisolate specific biomarkers of exposure to levels of mercury found in the environment.Cultured splenocytes from adult Xenopus laevis laboratory frogs were treated with methylmercury. Mitogen assays were inhibited by concentrations of methyl mercury from 0.25 to50 ppb. Concentrations above 50 ppb were lethal to the cultured cells.

Pilot studies using approximately 250 embryos showed that methyl mercury in lowconcentrations caused mortality and severe developmental defects in Xenopus laevis

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embryos. Animals treated with 5 ppb showed some changes in mortality after day 5. Somesurvivors at 50 to 500 ppb showed morphological changes such as bent anterior-posteriorbody axis, shorter tail, disorganized posterior somites, and in some extreme cases,proximal-distal duplication of the digestive tract, and ventralization of the eyes. Mortalitystudies will be extended to include the analysis of levels of methyl mercury in both treatedwater and in the embryos themselves. Our prediction is that more severely affectedanimals will show greater uptake, perhaps earlier.

In another experiment, embryos were placed 25 to a bowl at morula stage. By day 3 oftreatment, control animals were swimming vigorously in response to changes in ambientillumination (an escape response) or to tactile stimulation with a fine hair. Embryostreated at 25 ppb showed a sluggish response, even though no overt changes inmorphology had yet occurred. Embryos treated at 50 ppb barely responded, showing anoccasional tail flip after strong tactile stimulation. To determine if lack of escape behavioris due to neurological or muscular deficiencies as a consequence of treatment with methylmercury, a spawning of unresponsive mutant Xenopus embryos was obtained and treatedwith either 0_g/L, l_g/L, 5_g/L, 25_g/L or 50_g/L, methyl mercury respectively.Unresponsive is a recessive mutation which renders embryos incapable of movement upthrough the first 5 days of development (Reinschmidt et al. 1984; Dudek, Ide andTompkins, 1987). Paralysis is due to lack of excitation- contraction coupling within themuscle fiber. Hindbrain swimming pattern generator circuits as well as neuromuscularsynapses function normally (Dudek, Ide and Tompkins, 1987). Since the embryo isnaturally paralyzed placement of an intracellular electrode within a single muscle fiber canbe accomplished without chemical anesthesia. Recordings done in embryos treated with 50ppb methyl mercury showed healthy muscle cells as gauged by robust resting potentials ofup to 90 mv negative. However, neither changes in ambient light nor direct tactilestimulation produced excitatory junctional potentials (ejp's) as in control embryos. Inembryos treated with 25 ppb methyl mercury, some ejp's were evident, but were of smallersize and duration. The pattern of ejp activity was also of shorter duration. Thus our initialhypothesis is that the primary focus of methyl mercury effects on escape circuitry appearsto reside in the brain and spinal cord, and not in the muscle fiber itself.

Dr. Hartley is exposing embryos of the Japanese medaka fish (Oryzias lati ep_.e__to severalwater samples from Devil's Swamp. Concurrently the field water samples being used inthese developmental studies are being analyzed for pollutants.

Dr. Huang has recently optimized experimental conditions for the analysis of esterase inthe brain and liver of channel catfish.

Drs. Hill, Howard and Phadtare have reviewed the literature for hexachlorobutadiene and

they have found out that the metabolites of the parent compound hexachlorobutadiene(HCBD) are thought to elicit the toxic effect observed subsequent to HCBD exposure.Various intermediates have been elucidated and molecular modeling calculations on themore reactive species have been initiated. Second level calculations should be completedshortly.

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IH. Exposure SubcJuste_a_Uptake/Metabolism/Plan_ts:Members of the Lemnaceae (the duckweeds) are of worldwide distribution andconsiderable ecological value. Almost any permanent standing surface water, in eitherurban or rural locations, will contain one or more genera. They are prominent links inmany food chains and therefore their ability to take up and accumulate heavy metals is ofimportance because of potential damage to ecosystems in general, but more specifically, tohuman health. However, the ability to take up and concentrate metal ions also haspotential value (Clark et al., 1981, Mo et. al., 1988). It is possible that the plants can be usedto scavenge deleterious ions, present in surface waters at low concentrations so that theycan subsequently be disposed of in more environmentally satisfactory ways.

Our DOE grant has examined certain of the above possibilities. The acute toxidties ofarsenic, cadmium, chromium, lead, thorium, and uranium have been determined withregard to the vegetative reproduction of axenic cultures of Lemna _, grown underdefined conditions in the lab. Growth curves depicting % frond increase vs. concentrationof metal have been constructed (see for example, Figs. 1.1 & 1.2)* and can be used todetermine EC50's. The use of an ion selective cadmium electrode has shown that, over a

7-clay growth period, approx 20 L. gibba fronds (i.e., _ few) were able to reduce theconcentrations of cadmium (starting from 5.0 x 10-5M) in their growth medium by half

(Fig. 1.3)*.With a greater number of plants, cadmium was reduced to less than detectablelevels (Fig. 1.4)*. Similar results were obtained from uranium (supplied as uranyl nitrate)and assayed by fluorescence spectroscopy. Growth media of Lemna containing 100~M

uranyl shows a strong emission peak at 500 nm when excited at 320 nm (Fig 1.5)*, after 7days of growth (20 fronds) this peak can no longer be seen (Fig. 1.6)* These results, thoughpreliminary, provide strong evidence that the premise upon which this proposal is based,is sound. Lemna, clearly, takes up and accumulates cadmium and uranyl ions; weantidpate the same will be true for the other ions under investigation.

The acute toxicities of several heavy metals, with regard to the vegetative reproduction ofLemna gibba (duckweed), grown in sterile culture, have been determined. The growth datafor cadmium, lead, chromium, uranyl, arsenic and thorium are complete. Growth curvesdepicting, % frond increased vs. different concentrations of each heavy metal have beenconstructed and will now be used to determine EC10's and EC50's. Field studies at BayouTrepagnier have ceased since the cold weather prohibits duckweed growth but will resumein the spring. Lab-grown plants were placed at Bayou Trepagnier in specially constructedcontainment pens and returned to the lab for heavy metal accumulation studies. "Wild-type" duckweed, water and sediment samples were collected from the same field site.

Initial studies have begun on the accumulation and metabolism of organic pollutants byduckweed. The use of the HPLC-radiochromatography detector is indispensable for thesemetabolism studies. To date we have shown that butanol, aniline and tetrachloroethylene(TCE) are accumulated and metabolized by duckweed. Butanol disappears from the growthmedia over a two week growth period and is converted into a less polar metabolite whichis retained in the plant. TCE is taken up by the plant and metabolized to a much morepolar compound which is then transported back into the growth media. Aniline is alsometabolized to two m_re polar compounds. One of the compounds is transported backinto the growth media and the other is retained in the plant.

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Crayfish and fiddler crabs have been used to determine the influence of metal ions(specifically cadmium) on hormonally regulated color changes in crustaceans. There _s asignificant color change in crabs exposed to cadmium (10 ppm) with the animals becominglighter in color. The sinus gland complex in the eyestalk is the source of a pigmentdispersing hormone in crustaceans. Studies are underway to determine if the pigmentdispersing hormone is not being released or if the amount of hormone is simply loweredon exposure to cadmium. Eyestalk and brain tissue are being processed for histologicalstudies to determine if there is any effect of cadmium at the cellular level. Polymerasechain reaction (PCR), amplification studies of putative metallothionein gene homologshave been initiated. Four crayfish metallothionein sequences have been expressed in E.col____i.Whether all four are indeed coded for in the crayfish is under investigation.

The uptake of Cd +2 and Pb +2 into zeolite X is currently being investigated using ISE

techniques. It was found that Cd +2 efficiently exchanges for three out of four Na + sites insodiozeolite X - even with a thousand-fold excess of Na + excess of Na + in solution.

Studies involving competitive exchange in the presence of dications (e.g. Ca +2) arecurrently in progress.

The application of volatile organosilanes to chemically seal zeolite pores is also beingstudied. The reaction of phenylsilane with protio-zeolite X leads to formation of a silicate"plug" at very low temperatures. The mechanism of this reaction is currently being probed

by MS, FTIR and 29Si CPMAS solid-state NMR.

The derivitization of minerals to enhance their ability to absorb and immobilize heavy

metals is still progressing. We have intercalated ammonium phosphate into the clay

mineral kaolinite. Heating the intercalate to 250oc leads to a condensation reaction theirreversibly binds the phosphate to the aluminosilicate layers. It was determined that this

mineral uptakes Cd +2 readily from water (0.1 g of ion-exchanger reduces the concentration

of Cd +2 in 30 ml of water from 65 ppm to 11 ppm, very rapidly). The derivitized kaolinperforms three time better than the parent mineral in this respect. However, the

equilibrium concentration of Cd +2 was unacceptably high. We therefore synthesized asecond ion-exchange material, Na2[LiAI2(OH)6(PO4)], based on a layered aluminum oxide

mineral. This was prepared by reaction of LiAI2(OH) 7 with disodium phosphate. Under

the same conditions as the kaolin ion-exchangers, this material reduced Cd +2concentrations below the detection limit of the ICP. As well, we are searching for layeredor porous three-dimensional sulfur-containing species which can bind heavy metals ashighly-insoluble sulfide phases. Currently, NH4-intercalated TiS 2, KCu4S 3 and

(NH4)Cu7S 4 are being pursued.

An extensive literature search in the areas of toxicology in relation to channel catfish wasdone. Preliminary studies commenced after successfully setting up an aquarium. Ahatchery has also been identified for a regular supply of channel catfish. Fish collected fromthe hatchery are allowed to acclimatize to laboratory conditions and used as controls in ourstudies.

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Preliminary studies were conducted on the behavioral activity and the vascular system ofcatfish. We have also studied the blood glucose levels of channel catfish from Devil'sSwamp, the average blood glucose level is 83 mg/dl. We are currently analyzing the bloodlevels of cholesterol, triglyceride and insulin in the channel catfish using Sigma kits.

Preliminary studies were also conducted on the acetylcholinesterase activity of the brain inchannel catfish. Acetylcholinesterase is an appropriate parameter for monitoringbiological effects. The direct target of organophosphate and carbamate insecticides is a well-known enzyme, acetylcholinesterase (ACHE). The role of this enzyme is essential in thecorrect transmission of the nerve impulse since AChE is responsible for hydrolysis of thechemical mediator acetylcholine which propagates the impulse (Massoulie and Bon, 1982).Acetylcholine released at synapses is quickly inactivated by ACHE, but inhibition of thisenzyme leads to an accumulation of the mediator which thus maintains the excitation.This situation generally causes tetany, paralysis and finally death. The importance of thisenzyme for monitoring purpose is thus considerable. However, a lack of information oninducer pollutant sites required us to determine the critical area of monitoring, hence weplan to carry out monitoring brain acetylcholinesterase activity in channel catfish in theDevil's Swamp site an area proved to be highly contaminated with heavy metals,hydrocarbons, organophosphates, etc.

In our present investigation the yellow bullhead brains were received from Dr.Thiyagarajah. The control fish were obtained from hatchery and acclimatized in thelaboratory for approximately 2 weeks. The activity of brain acetylcholinesterase wasdetermined by the Ellman method (Ellman and Courtney, 1961).

The results of the present study indicates a significant inhibition of brainacetylcholinesterase in the fish of Devil's Swamp as compared with the control fisheswhich were maintained in the laboratory. These valuable results suggests that theinhibition of AChE in Devil's Swamp fish is due to the pollutants which are present inDevil's Swamp water. Further investigation is under progress to see which toxicant ismore responsible to inhibit AChE in catfish.

b. Toxicity and Uptake/Aquatic OrganismsDr. Abdelghani will continue to conduct short term toxicity testing on crayfish and Bluegillsunfish. Short term toxicity tests (Bioassays) are used to evaluate acute toxicities ofchemicals to aquatic organisms (catfish, bluegill fish, crayfish, etc.) and microorganisms.Mortality is used as the end point to determine the response to a certain toxicant. Testresults are expressed as 96 hour lethal concentration (LC50). Both refer to the

concentration or dose which kills fifty percent of test animals at the end of 96 hour period.(American Public Health Association -1992).

Acute tests can also be used to determine toxicant concentrations for intermediate and longterm tests.

An acute bioassay is conducted in two phases:

• Phase I: This includes the range finding to explore the approximate concentrationsto be used in actual test (Phase II). Usually organisms are exposed to differentconcentrations (logarithmic ratio: 0.1, 1.0, 10, 100, 1000 ppm, etc.). This range must

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include the concentration which kills all test organisms and the other concentrationwhich kills none.

• Phase II: Animals are exposed to different concentrations ranging from the onewhich kills all test animals, and the other which kills none and 3-5 others inbetween.

Bio_assays.on Crayfish and Bluegill Fish:

(These species were selected because of their presence in Devil's Swamp. There is apossibility of their exposure to elevated concentrations of chemicals in the environment.They are also of value Locommercial and sport fishing interests.)(1) Toxicity of Arsenic:

While arsenical compounds are readily absorbed by plants and soils, and may betransformed to different chemical species, they find their way to the aquaticecosystem and may exert a range of toxic effects depending on their chemical form.

The toxicological effect of arsenical compounds are determined by the form andquantity in which the arsenicals are administered. Arsenic exists in four valencestates: gas form (-3), metalloid (0), arsenite (+3) and arsenate (+5). Organic trivalentarsenic is 10 to 15 times more toxic than the organic pentavalent compounds.Organic pentavalent arsenicals are 180 times less toxic than the inorganic trivalentarsenicals. (Abdelghani, et al)

The present bioassay study describes the acute lethal toxicity of Arsenic Trioxide (+3inorganic). The toxicity of other forms of arsenic on fish and crayfish is in progress.

A. Crayfish Studies5000 live adult crayfish of mixed species and sex (procambarus sp) werepurchased from a local seafood dealer. Crayfish were declawed to minimizepredation and were placed in 50 gallon, all glass aquariums filled withdechlorinated tap water. Plastic coated chicken wire was coiled and placed ineach aquarium for zoning purposes and to provided ample livingrequirements for the crayfish during acclimation. Crayfish were adapted tolaboratory conditions for three weeks. They were fed commercial QuakerOats every day during acclimation. Food was stopped three days prior to theexperiment and during exposure. Crayfish weighed about 20 grams each. Astatic bioassay procedure (Standard Methods 1990) were performed todetermine the lethal concentration which kills fifty percent of experimentalanimals. The LC50's for arsenic, cadmium and mercury for crayfish and fishare shown in Table 1.

B. Bluegill Sunfish (2" Long) (Lapomis macroch!rus)Six thousand bluegills were obtained from the Natchitoches Hatchery inLouisiana. Fish were acclimated to laboratory conditions for three weeks.They were fed fish flakes during acclimation. Food was stopped three daysprior and during the test.. Same bioassay procedures were followed ascrayfish. Results are shown in Table 1.

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Table 1: 96 Hour Acute Toxicities of Mercury, Arsenic and Cadmium to Bluegill Fishand Crayfish (mg/L)

Aquatic.Organism Toxicant

.............. Mercury' ..... Arsenic cadmiumChloride Chloride Chloride

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Fish 0.5 1310 13.0

Crayfish 6.5 45.0 ss.o '_ -..................

Table 1 illustrates the toxicity of mercury, arsenic and cadmium to Bluegill fish andcrayfish. Table shows an order of toxicity as follows:

A. Fish Hg > As and Cd

B. Crayfish Hg > As > Cd

Mercury showed the highest toxicity (LC50 = 0.5 and 6.5 mg/l to fish and crayfishrespectively).

Arsenic and cadmium had the same 96 hour LC50 in fish LC50 13 mg/1). Arsenic was twiceas toxic as cadmium to crayfish (LC50 = 45 and 85 mg/l).

We have isolated and indentified microbes from water and sediment samples collectedfrom Devil's Swamp and Bayou St. John (Harrison Ave. bridge and Orleans Ave. bridgeareas). Organisms tentatively identified by the API 20E system are:

Entetrobacter AerogenesSalmonella enteritidis/Salmonella subgenus I, II, IVPseudomonas CepaciaProvidencia Stuartii

Klebsiella OxytocaAcinetobacter Calcoaceticus Var. Anitartusl(lebsiella Ozanenae

These cultures will be employed during future studies to assess bioavailability of variousspeciation of selected compounds of study and to factor in half-life projections. Ultimatelyresults will factor in fate and transport evaluations.

B. The total organic carbon (TOC) analyzer has been repaired for subsequent testing ofsediments and water samples to assist in characterization of the study sites.

Presentations

Hartley, W.R., Mizell, M., Thiyagarajah, A., Nelson C., and Sikorski, K. 1994. Sentinels ofcarcinogenic risk in a contamined Mississippi River Basin Swamp. Resident aquaticvertebrates and developing medaka embryos. To be presented at the 85th annual meetingof the American Association for Cancer Research, San Francisco, California, April 10-13.

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Thiyagarajah, A., Hartley, W.R., Mizell, M., and Thimmaiah, W. 1994. Vascular lesions inyellow bullheads (Ictalurus nata!is) collected from Devil Swamp Lake Louisiana. To bepresented at the 1st international symposium on Ecosystem Health and Medicine: Newgoals for Environmental Management, Ottawa, Ontario, Canada, June 19-22.

Summaries of technical progress were presented at the November and December PImeetings.

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Project Title: Assessment of Mechanisms of Metal-Induced Reproductive Toxicity inAquatic Species as a Biomarker of Exposure

Principal Investigator:. Mary Bitner Anderson

Field Studies: Catfish fingerlings were placed in pens in Bayou Trepagnier to assess theability of these fish to survive in two of the five evaluated sites (sites 3 and 4, suspectinflow sites in the central region of the Bayou). After a 4-week period, no fish survived ateither site. We are currently in the process of repeating this in situ exposure and willcollect specimens after one, two and three weeks of exposure for analytical evaluation ofmetal and hydrocarbon accumulation. Samples of sediment and water were collected atsites 1, 3, 4, and 7 for a second evaluation of the metals and hydrocarbons present. Thesesamples are currently being analyzed. These are also the sites which members of otherclusters are being taken to conduct their studies. In December, Steve Adams took Dr. ElliotHomer to Bayou Trepagnier for collection of specimens.

Laboratory Studies: Last quarter, crayfish were exposed in the laboratory to either 150 ppblead or 150 ppb chromium for four wee,_:s,which continued into this quarter with 7-weekexposures to the metals. As at the end of the 4-week exposures, the crayfish were weighed,hemolymph collected, and the crayfish were sacrificed. Hepatopancreases, gills, muscletissue and gonads were weighed, and then collected for either metal analyses orhistological study.

Ovarian and hepatopancreas weights are expressed as percent body weight resulting incalculation of the ovarian index and the hepatopancreas index. Calculations are as follows:

Index No. = Organ weight X 100Body weight

Results after 7 weeks of metal-exposure are as follows:FEMALES:

Body Wts (_ Ovarian Index HepatoDancreas Index

Control (N=5) 20.53 + 3.04 3.24 + .37 3.43 + .24Lead (N=8) 18.47 + 0.97 3.55 + .50 4.02 + .18Chromium (N=10) 18.64 + 1.18 3.25 + .37 3.68 + .28

Values are reported as mean + SEM.

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There are no significant differences among the groups. After four weeks of exposure theovarian index was significantly higher for the lead group than for the control orchromium groups.

The ovaries at the time of autopsy were all very dark in color reflecting the mature state ofthe oocytes within. The ovarian index indicates that oocyte development correspondswith the late vitellogenic stage; in fact, one crayfish in each experimental group and thecontrol group had laid her eggs which were attached to the abdomen. Histological study ofthe ovaries is currently in progress from both the 4- and 7-week exposure studies.

MALES:

,Body wts (_ Testicular Index* Hepatopancreas Index

Control (N=5) 25.60 _+3.62 .017 + .002 3.60 +_.44Lead (N=3) 17.13 + 0.82 .073 + .058 2.96 + .56Chromium (N=3) 16.37 + 1.15 .054 + .002 2.83 + .56

Values are reported as mean + SEM.*For each group only two testes were recovered. The N is not large enough forstatistical analyses of testicular index. Additional studies well be conducted toaddress any changes in the testicular index.

There are no significant differences in the hepatopancreas index. Histological study of thetestes is currently in progress for both the 4- and 7-week exposure periods. The techniquefor spermatophore collection has been established. Spermatophores are collected fromcrayfish by electrical stimulation. Adult male crayfish are stimulated with a 12V electricalstimulus applied to the based of the fifth pair of walking legs. The sperm in the extrudedspermatophores may then be analyzed.

Histological study of the hepatopancreas is nearing completion. The hepatopancreasconsists of three lobes with a central lumen into which the tubular structures of the

hepatopancreas open. The tubes of the hepatopancreas appear to be lined by a stratified orpseudostratified epithelium with several morphologically different cells. Some cellscontain large amounts of a very basophilic material, which appears to be secretory material.Some areas contain cyst-like structures containing few cells and a flocculent type ofmaterial suggesting that these were fluid filled structures. There is very little connectivetissue associated with the hepatopancreas. Endothelial lined vascular channels are seenrunning through the organ. There are no obvious differences between thehepatopancreases of males and females. In one 4-week lead exposed male, anaccumulation of hemosiderin-like material was observed in the hepatopancreas. Thehepatopancreases of 7-week exposed crayfish are sectioned but have not been stained.These slides should demonstrate if any metal accumulation is really occurring in thehepatopancreases of metal-exposed crayfish.

Tissues from both the 4- and 7-week metal exposed crayfish as well as the controls havebeen microwave digested and lead and chromium concentrations in the hemolymph,hepatopancreas, gonads, gills, and muscle are currently being analyzed by atomicabsorption spectrometry to determine the metal concentrations in these organs which isexpected to be completed by January 21.

14

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Project Title: Bioremediation Of Selected Contaminants In Aquatic Environments Of TheMississippi River Basin

Principal Investigator: Sanjoy K. Bhattacharya

The Devil's Swamp area was visited again. Work has continued on 1) purification ofmutant proteins, 2) microorganism isolation from contaminated soils and 3) fungienrichment. Selected toxicity tests were begun.

SamplingWater and sediment samples were collected from Devil's Swamp on December 2 andDecember 21, 1993. Samples on December 2 were collected from the southern part ofDevil's Swamp Lake and from the central part of Devil's Swamp. Samples collected onDecember 21 were from the northern part of Devil's Swamp Lake as far north as can bereached without crossing Rollin's pipeline.

ExperimentalWork has continued on protein engineering methods to reshape the substrate binding siteof the cytochrome P450BM-3(BM-3) from Bacillus megaterium with a goal of producingnovel isozymes capable of oxidizing selected organic compounds contaminating Devil'sSwamp. Site-directed mutagenesis was used to introduce amino acid substitutions into thefirst eight amino acid residues at the terminal end of the protein, and thus far 169 mutantshave been constructed, some of which have multiple amino acid substitutions.

Isolation of microorganisms from contaminated soils continue. Standard dilution platingwith selected media containing anti-bacterial agents were used to enrich for fungi. Twentyspecies of white rot fungi and three molds have been analyzed for the ability to decolorizethe polymeric dye, R-481. Ability to decolorize this dye is dependent on lignin-degradingenzymes, so R-481 is a useful model compound for screening studies.

Sediment samples from Devil's Swamp and Bayou St. John were cultivated for anaerobicbacteria to be used in CCI 4 toxicity experiments, and several cultures were isolated andidentified. Pseudomonas cepacia appears to be the predominant organism in thesesamples.

Sediment samples from Bayou St. John have been analyzed for trace metals at selecteddepths. The upper layer of sediment at the Orleans bridge showed a much higher ratio oflead to zinc in a June, 1993 sampling; this was not the case with October, 1993 samples, orwith any of the samples at other depths. Cadmium levels were over two orders ofmagnitude less than lead or zinc, -600 ppm for Pb and Zn vs. ~ 2ppm for Cd.

Serum bottle toxicity tests on hexachloro-l,3-butadiene and hexachlorobenzene indicatetoxicity levels between 1 and 10 ppm.

Serum bottles were used for Anaerobic Toxicity Assays on glucose-enrichment, non-sulfateanaerobes, and lactate enrichment sulfate reducing bacteria. Compounds used to spike theserum bottles were carbon tetrachloride, methylene chloride, and chloroform.

15

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EquipmentThe gas chromatograph was received and set up. Several items of laboratory equipmentand field equipment to facilitate sampling in the Devil's Swamp area have been received,including a global-positioning system, an inflatable dinghy, an ouLboard motor, and waterand sediment sampling equipment.

The Xavier ICP was updated with an ultrasonic nebulizer and state-of-the-art software.

MiscellaneousA summary of the technical progress of this cluster was presented at the December 8, 1993Principal InvesLigator meeting at Xavier UniversiLy.

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Project Title: Pore-Level Flow, Transport, Agglomeration and Reaction Kinetics ofMicroorganisms

]

Principal Investigator: Lisa J. Fauci

Goals

Understanding the detailed pore-level behavior of microorganisms through porous mediais essential for the development of effective in situ bio-remediation strategies. We aredeveloping integrated experimental and computational models of the pore-level behaviorof microorganisms. Our models will include the detailed analysis of convection anddiffusion within the pores and the convection and chemotactic responses of swimmingmicroorganisms to the local concentration gradients. Additionally, these models willinclude microbial adhesion to each other and the surrounding pore structure, and thereaction kinetics of these organisms to the toxic contaminant.

Accomplishments1) Experiments

We are continuing our experimental investigations of chemotaxis through smallpores constructed of glass capillary tubes. These studies are being conducted by Mr.Zewen Liu under the guidance of Dr. Kyriakos Papadopoulos. Thus far, theapparatus has been constructed, and image analysis techniques are being used totrack individual E. Coli bacteria as they progress through a field of constant nutrientgradient.

2) Coordinated analytical approachThis semester, Drs. Fauci, Gaver, and Dillon met regularly to discuss thesimulations performed on the mathematical models developed last quarter. Thesemodels couple fluid dynamics, contaminant transport and microorganism motilityand adhesion. These coupled non-linear partial differential equations are complex.Thus far, we have successfully formulated and computed concentration fieldestimates of contaminants within single pores using a finite-differenceapproximation of the convection-diffusion equation. Presently, Dr. Dillon iscoupling the contaminant transport equation to the fluid flow equations. Thecomputational solution of the fluid equations have been developed and validatedby Dr. Fauci. Coupling these two physical mechanisms permit us to incorporate

16

complex particle/structure interactions and thus allow us to model the flow andagglomeration of bacteria in individual pores. Our initial simulations using thiscoupled algorithm involve multiple particles in a pore moving under fluidconvection and chemotaxis. These initial simulations have allowed us to identifymodeling difficulties that have necessitating model reformulation. Specifically,with the help of Dr. Charles Peskin, we have redefined the motility-force applied toeach particle.

3) Student involvement and trainin_vTwo undergraduates, Ms. Stephanie Kute of Tulane University and Ms. AntoinetteDiaz of Xavier University, were employed full-time during the summer as researchassistants. They both worked at Tulane, and coded the numerical solution of acontaminant reaction-diffusion in both two and three spatial dimensions. Ms. Kuteused a Silicon Graphics Personal Iris graphics workstation to visualize the results.

Two graduate students, Mr. Zewen Liu of Tulane University and Mr. Dean Bottinoof Tulane have also been working on aspects of this project. As described above,Mr. Zewen Liu, a Chemical Engineering graduate student, has conducted ourexperimental studies. Mr. Dean Bottino, a Mathematics graduate student, hasstarted preliminary numerical investigations of contaminant transport.

4) Consultin;_ arrangementsv v ',During the last quarter, three experts in related fields have served as consultants onthis project. In October, Dr. Aaron L. Fogelson, a professor of mathematics at theUniversity of Utah, visited Tulane. Dr. Fogelson has ongoing collaboration withDr. Fauci, and is an expert in the computational modelling of cell transport andadhesion in biological flows. During this visit, Dr. Fogelson made available hiscomputer code, which simulates platelet aggregation. This code will beincorporated into our aggregation models. On December 6, Dr. Richard Ewing,Dean of Sciences at Texas A&M, visited our research group. Dr. Ewing heads a DOEconsortium whose goals include bioremediation computational modelling fromthe micro- to macro-scale. In addition, Dr. Charles S. Peskin, a professor ofmathematics at the Courant Institute of Mathematical Sciences, visited from12/8/93 - 12/11/93. Dr. Peskin is a world-renowned expert in the computationalbiological fluid mechanics. In fact, he pioneered the computational approachesused in our studies.

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Project Title: Natural and Active Chemical Remediation of Toxic and Radioactive Metalsin Aquatic Environments

Principal Investigator:. Gary McPherson

The most significant accomplishment of this quarter is the installation of the inorganicanalysis instrumentation ICP spectrometer and graphite furnace AA spectrometer) at theCIF satellite facility in Dinwiddie Hall (Tulane). Both instruments which are automatedhave been operational since early December. A substantial number of samples fromseveral cluster groups including this one have been examined. A preliminary survey ofthe metal contents (using the ICP) of over 100 sediment samples taken from the Barataria

17

Basin during the summer has been completed. The preparation procedure used in thesurvey analysis (microwave digestion HNO3/HF) completely dissolves the samples. Thesesediments has already been analyzed for acid volatile sulfide (AVS). Several of thesamples have been singled out for further study using the more sensitive AA instrument.

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Project Title: Expert Geographical Information System For Assessing HazardousMaterials In Aquatic Environments

Principal Investigator:. J.L. Regens

This cluster consists of two discrete elements. Project Element #1 develops and applies GIS-based approaches to decision support for environmnetal restoration by delineatingpotential exposures and health risks associated with radioactive elements in soils andsediments at the Rocky Flats Plant and profiling contemporary and historicaldemographic/land use patterns at Sandia National Laboratories. Project Element #2develops ESS software for surface water and ground water contaminants in the MississippiRiver Basin. The project teams successfully completed a series of activities during the rhirdquarter. Primary accomplishments for each Project Element sre summarized below.

P___,roiectElement #1Project Element #1 consists of a team from the Tulane School of Public Health, Tulane

Department of Sociology, and Xavier University. The team successfully negotiated a two-pronged effort demonstrating the utility of GIS-based approaches as decision support aidsfor environmental restoration at major DOE facilities.

• Agreements were developed to use IHSS 199 and IHSS 200 from OU3 (Rocky FlatsPlant) and baseline data to profile contemporary and historical demographic/land usepatterns (Sandia National Laboratories) as field application sites. Emphasis was placedon (1) the start-up of the Entergy Spatial Analysis Research Laboratory located in the J.Bennett Johnston Health and Environmental research Building st Tulane UniversityMedical Center to provide a central facility for data management and analyis and (2)establishment of working relationships with major facilities in the DOE complex toconduct a meaningful ER research program.

• Accomplishments include the initial acquisition and/or installation of computerequipment, GIS hardware and software, ORACLE database manager, and relatedsoftware.

• The research team also conducted a thorough literature review that resulted in thecompilation of an annotated bibliography on GIS applications of potential relevance toenvironmental restoration and waste management.

• Contact was made with the Department of Energy Comprehensive Epidemiologic DataResource program (CEDR) to obtain information about access to CEDR data sets onworkers.

• Presentations of the conceptual approach and capabilities were made at Rocky Flats andSandia National Laboratories following initial site visits during the second quartercoordinated through EM-50.

• Contacts also were established with the U.S. Army Corps of Engineers, NationalBiological Survey, Agency for Toxic Substances Disease Registry, and U.S.Environmental Protection Agency.

18

• Dr. Regens was successful in obtaining a commitment from Entergy Corporation tomake a gift to Tulane University designating the spatial analysis laboratory as theEntergy Spatial Analysis Research Laboratory.

• One technical paper drawing on the Project Element #1 efforts was accepted forpresentation at Waste Management '94.

Project Element #.2Project Element #2 consists of faculty from the Tulane School of engineering. Afterexamining the various research aspects in the initial phase of the project (Year 1), adecision was made to focus on two specific areas of flow and transport modeling:

(1) saturated ground water flow and single phase transport, and(2) dynamic surface water flow of soluble compounds.The programs MODFLOW and MT3D were selected as the ground water flow and

dissolved contaminant transport models to be implemented in the EGIS package to bedeveloped in Years 2 and 3. Copies of the programs were obtained and compiled. Drawingon the experience of the principal investigators, a cursory review of publicly available flowand transport models resulted in a two-level approach. When data is sufficient to justifythe expense and complexity of three-dimensional analysis a combination of MODFLOWand MT3D will be used to determine the fate of single phase transport within a saturatedaquifer. If data set is sparse,/i or the area is too large, then a two-dimensional flow andtransport model called GWTRANS will be used. Copies of the programs were obtainedand compiled on personal computers. All three models have been obtained and arecurrently running on computer facility of the Department of Civil and EnvironmentalEngineering (CEE)at Tulane.

EPA's Water Quality Analysis Simulation Program (WASP) was chosen as the dynamicsurface water transport model. The latest version of the program was obtained from EPAand/i has also been loaded on the CEE computer facility. The program was selected becauseof its flexibility and its wide-spread use. Another model, called Hydrologic SimulationProgram-FORTRAN (HSPF), is currently being evaluated to determine if it would moreeasily, or more accurately, predict tributary and non-point source loads for the WASPmodel. A dynamic flow model called DAFLOW is operating as the flow model. The linkbetween the flow and transport model is still under development.

Some preliminary work has been accomplished on the Uptown GIS personal computerfadlity. Several software packages, including the expert system shell CLIPS by NASA,FORTRAN and C compilers by Microsoft Corp., and Autocad by Autodesk Corp., wereacquired through departmental resources and installed on the Uptown GIS personalcomputers. The expert system shell LEVEL5by Information Builders, Inc. and FoxProdatabase management software by Microsoft Corp. were also added to the software librarythrough a separate grant from the US Army Corps of Engineers. Researchers from the CEEand the Geology Departments at Tulane have received as a grant fi'om Landmark GraphicsCorp. a comprehensive graphics package for geophysical, seismic, cartographic, reservoirand geo-data management valued at over $250,000. This package will be loaded on the Sunserver of the Uptown facility for use in the future GIS research by the two departments.

A PC based GIS system for East Jefferson Parish was developed using the availableequipment and software. An electronic map of the greater New Orleans area, whichincludes Jefferson and Orleans Parishes, was digitized from a paper copy using a desktop

19

Summagraphics MM series digitizer and the CAD software Autocad. The map containedseveral layers each displaying similar features. The Mississippi River, lake Pontchartrain,runoff drains and navigation canals were all included in one layer. A second layerdisplayed the locations of 750 soil boring logs obtained by the researchers from the USArmy COE and two local geotechnical engineering firms. Other layers contained citystreets, major water crossings and other geographical features. Soil data from the boringlogs was stored in database file. Programs were developed to enter, sort, retrieve, anddisplay soil data based on location on the map through menu-driven interfaces. Thisprototype system is similar in concept to the proposed EGIS.

Contacts have been established with several organizations involved GIS research such asthe DOE, New Orleans District (NOD) of the Corps of Engineers (COE), COE WaterwaysExperiment Station in Vicksburg (WES), Orleans Parish, and the Geography Department atthe University of New Orleans (UNO). Several visits were made by the engineeringfaculty in Year 1 to these facilities which contributed to the formulation of the work planfor Years 2 and 3.

• Advantages and disadvantages of different hardware and software used in thesefacilities were recognized in the selection of equipment and software for the Tulanefacilities.

• Some "hands-on" experience was gained by the engineering faculty by using the GISfacility at UNO. The UNO UNIX Sun station based facility has been a recognized byESRI/i as a formal training and research center for ARC/INFO since 1990.Demonstrations of some of the locally developed GIS applications at UNO indicated theneed for an integrated software interface between the different components to enhancethe user interface with the GIS.

• Agreement was reached to share geotechnical information currently available in theCOE database of soil Boring Logs Data Management (BLDM) software developed for theComputer Applications in Geotechnical Engineering (CAGE) program at WES.

Two technical papers were submitted for publication in the ASCE journal (under review)and an international conference in France (accepted) on use of expert systems indeveloping soil site investigation, and on reliability of soil properties obtained fromlaboratory and field tests./iA third paper is under preparation. Future research proposalswill be submitted to outside sources to extend the study to other parts of the Rio GrandeValley.

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Education Project

Project Title: Enhancement of Environmental Education at Tulane and XavierUniversities

Principal Investigator. Sr. Monica Loughlin

Curriculum DevelopmentThe Tulane LAS committee has outlined a curriculum for an environmental sciencecoordinate major. To do this, the current environmental curriculum was reviewed and arevised set of requirements for the coordinate major in environmental studies wasdeveloped for all appropriate departments in LAS. Curriculum gaps were identified in theexisting program and new courses were proposed to fill these gaps. Each department wascontacted individually for suggestions and recommendations.

Meetings were held with selected admissions officers, key faculty members and deans aswell as with other relevant personnel to solicit their input and support for the newcoordinate major. A strategic plan for LAS, "A Vision for Environmental Studies atTulane University," was drafted in late fall 1993.

The Tulane University Department of Civil and Environmental Engineering hasdeveloped a new curriculum leading to a BS degree in Environmental Engineering. Thecurriculum is offered to the sophomore class of 1993. Currently at least 10 students havejoined the Environmental Engineering program with high interest among women andAfrican-American students. This program is available to Xavier students who follow the 3+ 2 track to obtain a BS degree in physics from Xavier and a BS in environmentalengineering from Tulane.

Xavier University's plans include efforts to establish a minor in Environmental Studies,and a track in Environmental Science within the Science Departments. The mechanismfor accomplishing these have been identified. Key faculty have been approached regardingthe development of new courses which will make up the new academic programs.Through faculty mini-grants, the development of new courses, the infusion ofenvironmental topics into existing courses and the development of new faculty, havebeen supported and will continue to get support until the academic programs are fullydeveloped.

In November, Dr. O'Connor made a presentation to the Xavier University College of Artsand Sciences Faculty Assembly on the environmental education activities sponsoredunder the DOE Tulane/Xavier partnership. Some of the topics discussed during thepresentation include faculty workshops on curriculum development, the LIFE ScholarsProgram, faculty development through mini-grants, and environmental coursedevelopment and course infusion.

Fa_l.ty DevelopmentIn October, guidelines for faculty mini-grants awards were developed and distributed to allXavier faculty. The mini-grants are intended to provide opportunities for faculty toconduct activities which will enhance the environmental content of Xavier University's

21

overall curriculum. Eight proposals were submitted in October, one in November and twoin December. The proposed activities ranged from course development and courseinfusion to pre-college educational outreach and faculty workshops. The Faculty Mini-Grants Committee reviewed proposals in November and December (each time for theprevious month) and has approved four in November and one in December.

As a result of their mini-grant awards, Sr. Stephanie Henry (Chemistry) held anintroductory environmental educational workshop for a youth community center in BelleGlade, FI., Dr. Duplantier (Communications) has begun work on developingenvironmental educational materials with instructors of selected courses, Dr. Allen(Communications) has started developing his proposed course in environmentalcommunications, Dr. Jesalva (Chemistry) was able to attend a workshop on chemicaltoxicology, to learn more on the topic to enable her to develop a course, and Ms. Mesa(Mathematics) started the infusion of environmental topics into Xavier's Calculus 3course. In October, the Survey of Environmental Chemistry course, developed by Sr.Stephanie Henry, received departmental and administrative approval. The course will beoffered in Spring Semester, 1994.

Two new courses were developed at Tulane for undergraduate programs and are open toall undergraduate students at Tulane and Xavier. Dr. Bhattacharya has developed anundergraduate course, CVEN 207, Introduction to Environmental Studies, open to allfreshman and sophomore students. The course is designed to cover basic aspects of waterand air pollution and ecosystems. As an introductory course with no prerequisites, it isopen to both technical (science and engineering) and non-technical students. Anotherundergraduate course, CHEM 250, Environmental Chemistry, was developed by Dr. AllenApbett and will be taught in the Spring Semester, 1994. Procedures for implementingrelevant administrative tasks such as cross registration between the Universities andstudent recruitment have been defined and implemented.

Student Develooment=

The Xavier Environmental Education Committee (XEEC) met twice during the quarter tomake final recommendations on students who will receive environmental scholarshipsand to discuss planned faculty workshops on curriculum development. A total of 35scholarship applications from Xavier students, including typed essays, transcripts,curriculum vitae, and three letters of recommendations, were received by the FallSemester deadline date. Four Xavier students with interest in pursuing an environmentalcareer were selected to receive LIFE scholarships and one student was given a LIFEinternship. The students selected to receive scholarships were Lawrence Carter(sophomore, chemistry), Camille Fouche (junior, political science), Tamara Mosby (junior,philosophy), and Temperance Smiley (sophomore, biochemistry). Lauren Nicolas (junior,mass communications) was selected to receive the LIFE internship. All students arecurrently doing environmental research under selected faculty mentors.

The Environmental Programs Office held an Environmental Scholars Colloquium onNovember 12, 1993, attended by faculty, staff, students and parents. Recipients ofenvironmental scholarships were announced and presented with certificates of award. Atthe Symposium, four environmental scholars gave short presentations on their researchprojects. Officers and members of the Environmental Club were recognized by the Clubadvisors, Sr. Stephanie Henry and Mrs. Croscina Crockett. The agenda also included an

22

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invited guest speaker from the Environmental Protection Agency who gave a brief talk on_he importance of student participation in environmental research. A receptionimmediately following the ceremony was held.

Other Activitie_Starting in October, the Environmental Programs Office resumed publication of theEnviroNews, a monthly newsletter about campus environmental activities. Three issues,October, November and December, have been written, printed and distributed to all Xavierfaculty and staff.

During this quarter, Tulane and Xavier Universities hosted three visits and seminars onenvironmental topics for faculty and students. On November 17, Dr. Norman Cutshall, ascientist/manager at Oak Ridge National Laboratory, visited the universities. He spokewith faculty about the opportunities for Xavier and Tulane students to participate inresearch projects at the Oak Ridge National Laboratory and provided a list of eight otherscientists at Oak Ridge willing to present seminars on research project ranging fro 'bioremediation to GIS mapping. Dr. Charles Allen, a scientist/project manager at .,JttellePacific Northwest Laboratory, gave a seminar on November 19 on problems regarding thecleanup of wastes at Hanford. On December 10, 1993, Paul Ryan, Professor of Video atSavannah College of Art and Design, held a seminar entitled "Video Mind, Earth Mind:Art, Communication, Ecology." In this seminar, Professor Ryan discussed how to usevideo as a tool for environmental consensus-building.

The Center for Environmental Programs also coordinated the visits of Michele Diamond,Coordinator of Student Recruitment for the Graduate Programs in the School of Public and

' Environmental Affairs at Indiana University, and Dr. Norman Eder, Vice President ofPublic Affairs at Oregon Graduate Institute of Science and Technology (OGI). Mrs.Diamond spoke with various department chairmen about opportunities for Xavierstudents in Indiana University's Master of Science in Environmental Science (MSES) andMaster of Public Affairs (MPA) degree programs. Dr. Eder visited Yavier University onMonday, December 13, 1993 to discuss the University's possible participation in theNational Alliance of Minorities in Engineering and Science (NAMES), a partnership beingdeveloped between OGI, the Battelle Pacific Northwest Laboratory (PNL), the SavannahRiver Site (SRS) and selected minority academic institutions.

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I

Initiation Projects

Project Title: Heavy Metal Immobilization In Mineral Phases

Principal Investigator: Allen W. Apblett

We are developing methodology whereby toxic metals may be encapsulated andimmobilized in a very durable ceramic material. This waste form will be designed suchthat the pollutants are locked in the same crystalline mineralogical phases in which theyoccur naturally (i.e. materials that have a demonstrated ability to sequester the metals forbillions of years).

One target wasteform is the mineral sphlaerite (ZnS) which has the ability to form solidsolutions with heavy metal sulfides. We have prepared ethyl xanthate complexes of mostcommon heavy metal ions as well as those of innocuous metal ions ( e.g. Na +, Ca2+,

Mg2+) and determined that the relative solubility of the complexes allows the separationof the toxic species from the benign ones. Furthermore, the complexes decompose atextremely low temperatures (ca. 120oc) to the corresponding metal sulfides. The xanthatecomplexes may potentially be used in two ways to accomplish entrapment of heavy metalsin sphlaerite;

(1) zinc ions may be added to contaminated water and it and the heavy metals maybe co-precipitated by addition of potassium ethylxanthate to the solution,(2) separated xanthate precipitates and zinc bis(ethylxanthate) may be dissolved ina high-boiling organic solvent (e.g. xylene) and the subsequent solution refluxedto deposit a homogeneous sulfide ceramic.

We have demonstrated that both approaches work for encapsulation of cadmium insphlaerite and are currently investigating the treatment of a simulated wastestream fromwet lime-gypsum flue gas desulfurization plants

Another mineral wasteform that has demonstrated utility for encapsulation andimmobilization of all types of toxic metal ions is Synroc. This material is based onnaturally occurring materials (hollandite, BaAI2Ti6016, perovskite, CaTiO3, andzirconolite, CaZrTi207) and is used for high level radioactive waste. Any process for theincorporation of high level waste must satisfy two crSteria. First consolidation to a near-theoretically dense ceramic must occur below 1200oc. Otherwise volitalization ofradioactive elements (i.e. cesium, ruthenium, technetium, and uranium) or melting toless durable glassy phases might occur. Second, it is important to keep the amount ofreactive oxygen present in the system low to avoid the formation of water-soluble higheroxides of the transition metals or actinides (e.g. CaUO4). Since, typically radwaste containsnitrate salts of the metals and this anion is a very strong oxidizing agent at elevatedtemperatures the avoidance of oxidation of the metals has usually required addition of aredox buffer (e.g. titanium metal) to the preceramic powder. Unfortunately, this addsfurther complexity to the final wasteform and leads to deviation from the idealstoichiometry. We have found a means to circumvent this problem. Simulated PW-4bhigh level radioactive waste was mixed with nitrate salts of the elements required for the

.......• ............................................... ..............................._,.,......... . ........ _ _ ....

Synroc matrix in aqueous solution. The water was removed from the system and theresidue was suspended by stirring in glacial acetic acid. An excess of acetaldehyde wasadded to the mixture which was then heated at reflux for 12 hours. During that time, thenitrate salts were converted to acetates due to oxidation of the acetaldehyde by nitrate. Theremoval of the organic solvents from the mixture by rotary evaporator yielded a deep-redglassy solid that was completely amorphous to X-rays indicating intimate mixing of theradwaste and ceramic-matrix metal ions. This material decomposed and sintered uponheating to the usual Synroc phases without the necessity of a redox buffer since acetateprovides a neutral (or slightly reducing) atmosphere upon its decomposition. Such aprocess could also be useful for other concentrated wastestreams that contain oxoanions.

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Project Title: A Pilot Study of the Applicability of Polarography to Exposure andBioremediation Problems in Aquatic Systems

Principal Investigator: K.J. Bundy

During the present quarter, word was received that the manuscript "Polarographic TraceLevel Analysis Can Be Applied to the Detection of Environmental Contaminants" wasaccepted for publication in the Proceedings of the Annual Meeting of the Society forEnvironmental Geochemistry and Health.

Progress in the present quarter has mainly been concerned with analysis of water,sediment, and frog samples obtained for us by Dr. Hank Bart on his sampling expeditionsto Devil's Swamp. We use a series of macrofilters: 290 ptm, 105 Ixm, 20 I.tm, and 1 lira, toseparate both the aqueous and sediment fractions into their various moieties. Thisseparates out coarse particulates and organic matter, fine particulates (clay particles), andwater (perhaps containing suspended particles below 1 I.tm in diameter).

So far, our analysis has centered on lead detection. No lead has been found in the water inthe aqueous phase. If it were present, we should be able to polarographically detect it atabout 3 ppb levels. The inference is that many of the possible lead salts are insolubleand/or that lead ions have a tendancy to adsorb on suspended matter. Analysis of theparticulate moieties shows that significant amounts of lead are present in each moiety ofwater and sediment. For example, 20.2 ppm was noted in the fine particulate sedimentcompartment.

The method we are using for extraction of lead follows ASTM D3974-81 Standard Practicefor Extraction of Trace Elements from Sediments. It consists of element leaching with HCIand HNO3. This process for acid digestion is not as severe as others available, e.g. ASTMD4698-87 Standard Practice for the Total Digestion of Sediment Samples for ChemicalAnalysis of Various Metals, which involves extraction using hydrofluoric, nitric, andperchloric acids. However, it was selected because it perhaps will yield a somewhat betterestimate of the amount of lead which could be potentially bioavailable. In other words itprobably more closely matches the digestive juices. Our research would suggest that apossible exposure mode for frogs in Devil's Swamp is swallowing of water containingparticulates, leaching of lead from particulates by gastric juices, and uptake via intestinalabsorption.

25

Besides testing of water and sediment samples, we also have analyzed tissue from frogstaken from Devil's Swamp, although at this time our results are preliminary. Availableliterature(I) indicates that of all tissues in _: ,tog, that which concentrates lead mostextensively is bone. Two frog femurs have been ashed and assayed for lead. However,none was found in either case. At this time the interpretation of this result is unclear.Several hypotheses would be: 1)othe frogs were taken from areas of the swamp lesspolluted than the locations where the water and sediment were taken and thus theanimals suffered minimal exposure to lead, 2). the bogs were exposed to lead in theenvironment but that little of it was intestinally absorbed, or 3) the literature cited is notapplicable because different frog species could behave differently regardingbioaccumulation and different lead bearing pollutants could be metabolized differently.

The last part of hypothesis 3 would mean lead could be stored in other organs in greateramounts than in bone. We will be examining this possibility shortly. If no lead is foundin Devil's Swamp frogs, this will mean that we will have to rethink which animal speciesshould be used in our laboratory studies planned for the future.

In this quarter three principal investigator meetings have been attended: at the TulaneSchool of Public Health, Tulane Uptown campus, and at Xavier. At the first meeting Dr.Bundy gave a presentation on the work to date on this project. He also attended a meetingof the Sampling Committee, of which he is a member. The latter part of the quarterlyreporting period was spent interacting with investigators in the "Natural and ActiveRemediation of Toxic and Radioactive Metals in Aquatic Environments" cluster and in the"Assessment of Mechanisms of Metal-Induced Reproductive Toxicity in Aquatic Species asa Biomarker of Exposure" cluster to prepare proposals in which polarographic testing isused to investigate problems of interest to these research groups.

Reference:

1. R. Eisler, 1988. Lead Hazards to Fish, Wildlife, and Invertebrates: a Synoptic Review, U.S. Fish Wildl. Serv. Biol. Rep. 85(1.14).

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Project Title: An Interactive, Hypermedia Cultural Ecology Model of RiskCommunication about Hazardous Waste Remediation for Scientists,Administrators and Students

Principal Investigator: Stephen Duplantier

The PI collected information on cultural, archaeological, historical, biological, ecological,toxicological, risk communication, and socio-economic processes in the Mississippi Riverbasin study area. Emphasis was placed on St. James and St. John civil parishes in the firstiteration of the educational multimedia software product. The material collected was inthe form of reports, printed information, photographs, maps, drawings, and video tape.The information was scanned or digitized into a Macintosh computer and authored intoan interactive series of HyperCard© "stacks."

The CPU used was a Macintosh 840AV with 16 megabytes of RAM and a 500 megabyte harddrive. An Apple Color OneScanner was used to transfer the printed and typed material

26

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into word processing files. The software used for optical character recognition wasTypeReader 1.0. The same scanner was used to digitize photographs and halftones. Thescanner controller software used was Ofoto. Once scanned, the images were processed forcolor and visual effect with Adobe Photoshop 2.5.

The PI spent a total of 22 hours videotaping natural social and cultural scenes andprocesses in the study area. Selected pats of this videotape were digitized using a SigmaMovieMovie Nubus slot board. Screen edit was the software used for the capture and JPEGcompression of the video images. Adobe Premiere was used to edit some of the digitalvideo footage.

The other investigators (Calef and Watson) supplied the PI with texts in organizationalcommunication and environmental ethical issues. This material was converted from EX_format using an Apple file converter application.

HyperCard and HyperGASP, an application which works along with HyperCard, were thetwo applications used to perform the multimedia authoring of the information.HyperGASP uses the "colorize HC" XCMD to add a color layer to HyperCARD. At the endof the authoring, HyperGASP was stripped out of the stacks, leaving the program able torun in a player version of HyperCard.

The final product is fully a interactive multimedia product which can be used by interestedpeople from ages 10+. Advanced users will find it just as informative as younger ones.The random branching pathways taken by users determine what information is presentedto them. The synergistic advantage of having the totality of cultural and natural ecologyfacts, images, sounds, and movies all in one place makes the software very powerful.

, . ,,

Project Title: Bioenvironmental Analytical Support Services for DOE Clusters

Principal Investigator:. William J. George,

Efforts this quarter by the cluster have advanced the capabilities of the analytical laboratoryfacilities and have begun generating data ,or DOE projects as itemized below.

1. Two full-time technicians assisted by supervisory personnel have placed intoservice the new Perkin-Elmer 4100L furnace atomic absorption spectrometer (AA)and CEM microwave digester which was installed in Dinwiddie Hall on the mainTulane campus this quarter. Personnel have received training and have becomefamiliar with the operation of both the software and hardware and have establishedthe necessary dishwashing, sample storage, and other preliminary protocolsnecessary to place the lab in operation.

2. AA methods for the analysis of lead in digested tissues, blood, and urine specimenswere set up. The methods were confirmed by standard curves, independentcertified controls, sample spikes, and characteristic mass calculations. Thesepreliminary methods verifications are time-consuming, but now that they arecomplete it is anticipated that analysis of provided specimens will proceed rapidly.

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3. Two hundred thirty-four crayfish tissues (gills, tails, carapace, liver, hepatopancreas,and gonads) from laboratory exposure experiments from the DOE ReproductiveCluster (Anderson, George) were digested in preparation for AA analysis. Adigested "known" lead spiked tuna control was also prepared and verified for theanalysis of these specimens.

4. The cluster technicians have assisted Mr. Aigno, a graduate student working for Dr.Gonzales in Chemical Engineering, by setting up an AA method for the analysis oflead in clay digests, and have assisted him in the processing of samples and inbecoming familiar with the operation of the AA.

5. Our cluster has acquired a loaner AA which has been out of service for several yearsfrom another department in the Medical School. We are working with the Perkin-Elmer Company to install this instrument in the CBR Building and areinvestigating the feasibility of upgrading the software and adding an autosampler tothe instrument using non-DOE Toxicology Laboratory funding.

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Project Title: Evaluation of the Carcinogenic, Reproductive, and DevelopmentalEffects of Mixtures of Contaminants on the Medaka Fish (Oryzias latipes)

Principal Investigator: William Hartley

The objective of this grant is to primarily investigate the toxicity of chemical mixtures inthe Mississippi River Basin. The specific mixture in which this grant is concerned iscadmium, a heavy metal, and trichloroethylene, an industrial solvent. The medaka fish(Oryzias latipes) is used to assess the toxicity of the chemical mixture.

The tollowing is a brief list and discussion of what has been accomplished up to the presenttime:

The medaka colony at the Tulane School of Public Health has been adequately expanded tofill the need for present and future experimentation.

An extensive literature search was completed on cadmium. The literature search alsoincluded a computer search of all major chemical, toxicological and biological databasesavailable.

Preliminary experimentation was completed to determine the ability of the medakaembryo to grow in an extremely low water hardness environment. The conclusionsreached were the desired hardness for water would be achieved by dilution of the embryorearing solution to retain essential nutrients.

Exposure experimentation using cadmium has been performed. The experiments whichhave been completed involved assessment of the developmental toxicity of cadmium onthe Oryzias latipes (Medaka) embryo. 4 groups of 30 medaka embryo (n=120) were exposedto nominal cadmium concentrations of 0.0, 2.0, 4.0 and 8.0 mg Cd/L for 96 hours.Additionally, 5 groups of 5 medaka embryo were exposed to nominal cadmiumconcentration of 0.0, 15.0, 30.0, 60.0 mg Cd/L and 1.0 gm Cd/L for 96 hours.

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The results of the experiments focuses specifically on the physiological and morphological,(sublethal) effects of the cadmium upon the development of the medaka embryo.Additionally, stage development as a function of time was observed. Preliminaryconclusions are as follows.

• Retarded or delayed stage development of the Oryzias latipes embryo was observed incadmium exposed groups. Approximately 90% of the embryo which experienceddelayed stage development in the first exposure (n=120, c= 8, 4, 2, mg Cd/L) died shortlyafter delayed stage development was observed. The second exposure (n=25, c= 1.0 gmCd/L, 60, 30, 15 mg Cd/L) showed a slight increase of survival of embryo whichexperienced delayed stage development due to aqueous cadmium exposure.

• Exposure of Oryzias latipes embryos to aqueous cadmium were observed as inhibitingthe proper progression of many early development parameters including improper orabnormally shaped cleavage planes, retarded development of the blastula, failure ofblastula to flatten and advance into gastrulation, and improper formation of early andlate gastrula.

• Physiological development observations revealed a decrease of heartrate duringadvancement of the exposed embryo. The trend of reduction of heartrate at the thirdday of development was apparent in both exposure ranges.

.. Morphological abnormalities which were observed included improper formation ofthe vascular system, improper coloration of blood, macrencephaly, irregular spinalflexure, unusual clefts in yolk sac and early cranium, improper pigmentation of trunk,failure of embryo trunk to expand to full embryonic axis, and abnormal bifurcation ofembryonic axis.

All specimens from experimentation were fixed in 10% neutral buffered formalin and areawaiting histopathological study.

_ tII I III i II Ill ii I I II i l:

Project Title: A Coupled Chemo-Enzymatic Technique To Remove AromaticContaminants From Aqueous Streams

Principal Investigator:. Vijay John

Aromatics are major organic pollutants found in aqueous waste streams. Phenols, forexample, are prevalent in waste streams from coal conversion processes, and are generatedduring coal pretreatment steps prior to combustion. Our research expands on a noveltechnique that we have developed to detoxify waste streams of these carcinogens.

Concept and Status of WorkThe technique that we have developed is based on a fairly recent and novel enzymaticapproach to remove phenolics from waste streams (Klibanov et al., 1983; Nakamoto andMachida, 1992). Here an oxidative enzyme, peroxidase, is used to couple phenolicsresulting in a polymer that is water-insoluble. The polymer is thus precipitated out ofsolution; in addition it is relatively nontoxic compared to the monomer. Our approach

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• 1 j

expands on this technique to include not just phenolics, but a variety of other aromatics.Essentially, the method consists of hydroxylating aromatics to the corresponding phenolsand subsequently using the peroxidase to couple the phenols and remove them fromsolution.

The Fenton reaction (Walling, 1975) involves the oxidation of Fe(II) to Fe(III) and the

consequent formation of a very reactive hydroxyl radical (OH_i ) as shown in equation (1)of the following mechanism.

(1) H202 + Fe2+ --> Fe3+ + HO" + OH fi

(2) HOfi + RH ---> RHOH _i

(3) RHOH_i --> ROH + H_i

(4) 2 RHOH fi ---> R- R + 2H20

The hydroxyl radical transforms the aromatic, RH (e.g. benzene) to the hydroxylated

radical, RHOH _i (Equation 2). Subsequently the hydroxylated radical is converted to thephenol (Equation 3)_ ,_ to the dimeric species (e.g. biphenyl) through equation 4. Thedimeric species (biphenyl) is insoluble in water and precipitates out.

The novelty of our approach is the subsequent coupling of the phenolic species using theoxidative enzyme, horseradish peroxidase. In work-to-date, we have shown the feasibilityof benzene removal through this coupled process. As the table illustrates, at equimolarlevels of H202 (Runs 1 and 2), the Fenton reaction leads to about 50% conversion ofbenzene almost exclusively to phenol and biphenyl. The phenol remains in solution whilebiphenyl is precipitated out. With an excess of H202 (Run 3) almost complete conversionof benzene is obtained, with some conversion to higher oxidation products (equation 7).

.........

Run Fe(lI) H2G? Benzene Benzene Phenol Biphenyl# (raM) (raM) (mM) Conversion (%) (%)

(%)

1 2.13 6.4 6.4 48 39 5, _- ,L ,, .,, ,i i, i ,i ,

2 6.4 6.4 6.4 57 38 14,,,. ..... ,,, , , , , ,,,,. ,, i. i ,, ,J|.l=:

3 6.4 19.2 6.4 82 54 10

i , i ,i

We have found that the phenol remaining in solution can subsequently be completely andefficiently removed by enzymatic polymerization.

References

Klibanov, A.M., Tu, T., Scott, K.P., Science, 221,259(1983).

Nakamoto, S., Machida, N., Water Research, 26, 49(1992).

Walling, C., Acc. Chem. Res., 8, 125(1975).

3O

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Project Title: Genetically Engineered Microorganisms: Aromatic HydrocarbonBiodegradation Genes From Rhodococcus

Principal Investigator:. Kevin J. Kendall

The fourth Quarter of this project has been spent sequencing a fragment of DNA fromRhodococcus sp. ATCC 19070 that may contain genes similar to metabolic genes found inPseudomonas putida. Work has continued to identify and isolate additional fragments ofDNA from Rhodococcus sp. ATCC 19070 that bear strong resemblance to DNA fromPseudomonas putida.

We have found that, despite the fact that Rhodococci are very different bacteria fromPseudomonas, both of our strains of Rhodococci contain some DNA that is very similar toDNA in Pseudomonas. In particular, there is very strong similarity to Pseudomonas DNAthat encodes enzymes required to metabolize benzoic acid, an intermediate in thebiodegradation of toluene. This similarity was found using the technique of Southern blotanalysis. This suggests that we may be able to use Pseudomonas DNA to clone toluenebiodegradation genes from Rhodococcus.

We have constructed a gene bank of Rhodococcus DNA cloned in E. colt and haveidentified two clones that contains sequences homologous to the Pseudomonas putida TOLplasmid. It is possible that these clones contain genes that encode enzymes similar to theones utilized by Pseudomonas to degrade aromatic hydrocarbons. These clones arecurrently being characterized and sequenced.

We are currently identifying additional clones that contain DNA that is similar toPseudomonas. These clones will also be sequenced to identify the enzymes that areencoded by the DNA. Clones encoding biodegradation enzymes will then be analyzed inmore detail as a first step towards creating genetically manipulated strains of Rhodococcuswith enhanced biodegradation potential.

I_1... I .... _. . . / "]1IlII .._'.. I.., .... ,11 , ,, ..... I , ,, ..... I I IIIl/I I1,,1,,111{li i I I LIft [ I ,l [_:] I I| tl_ ti J_ I

Project title: Laser Ablation/Ionization Studies Related to the Removal of NuclearMaterials from Metal Surfaces

Principal Investigator:. Brent Koplitz

Overview

As a research initiation project, we use our existing laser and mass spectrometer technology tostudy laser ablation/ionization processes that are relevant to the removal of nuclear materialfrom metal surfaces. Methods to understand and improve laser ablation studies as well ascontainment issues related to the ejected material are being studied. The benefit for DOE is animproved understanding of laser ablation processes. If successful, these studies will assist laserclean-up efforts where laser ablation is concerned. In fact, one of our approaches to particlecontainment, laser ionization of the ablated material, complements existing suction-type removamethods.

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6, J if.

Pro_essA new experimental apparatus for conducting ablation/ionization experiments is now working iour laboratory. Methyl iodide has been photoionized with 193 nm radiation, and we are in theprocess of calibrating the time-of-flight mass spectrometer. Experiments involving laser ablationare now underway.

Project Title: Asymmetric PVDF Pervaporation Membranes for the Removal of OrganicContaminants from Waste Water

Principal Investigator:. Peter N. Pintauro

ObjectiveTo fabricate and test a new class of asymmetric polyvinylidene fluoride (PVDF)pervaporation membranes for the selective removal of non-polar organic contaminantsfrom waste water.

Polyvinylidene fluoride (PVDF) membranes were investigated for the removal ofchlorinated aromatic hydrocarbons from water in a pervaporation separation process. A"wet cast" procedure was employed to fabricate the membranes. A solution of 14 wt.%PVDF, 13 wt.% dimethylacetamide, and 73 wt.% acetone was prepared and heated to 48-

50°C for 0.5-1.0 hour. The polymer solution was then spread on a wetted glass plate to adepth of 250 I_n and allowed to air dry for 12 minutes at an air temperature of 23-25oc anda relative humidity of 45-55%. The plate and film were then immersed in three differentprecipitation baths: 15 minutes immersion in a solution of 50 vol.% water/40 vol.%acetone/10 vol. % dimethylacetamide maintained at 15°C, 15 minutes immersion in asecond bath of 60 vol.% water/40 vol.% acetone (at 15°C), and a final 30 minute treatment

in a pure water bath at 15oc. After t