ISSN: 0098-4590 Q u Vorida Scientist F(o F(p3 Number Autumn, 2003 Volume 66 CONTENTS Suwanee River Humic Substances: Evidence of Humics in Salt Water Thomas Manning, Stacy Strickland, Amy Feldman, Infrared Studies of Chlorination of Tice Umberger, Derek Lovingood, Mamadou Coulibay, John Elder, and Lyn Noble 253 Effects of Supplemental Feeding on Key Deer Behavior in Urban Areas Roel R Lopez, Nova J Silvy, Philip A Frank, and Brian L Pierce 267 Evaluating the Relative Effects of Life History Stages in the Conservation of the American Crocodile (Crocodylus acutus) in Florida Paul M Richards 273 Record of the Escolar Chino, Scombrops oculatus (Poey, 1860) off the U.S Atlantic Coast, with Comments on its Taxonomy Ramon Ruiz-Carus, Richard E Matheson, Jr., and Frederic E Vose 287 New Record of the Endangered Lakeside Sunflower, Helianthus First carnosus (Asteraceae), From Putnam County, Florida Marc C Minno and Cecil Slaughter 291 Do Gopher Tortoises at Archbold Biological Station, a Long-Protected Area in Florida, Have Upper Respiratory Tract Disease? Jennifer C Zipser and Kyle G Ashton 294 Human Intestinal Parasitic Infections in Villages Located Around Lake Atitlan in Guatemala Camille Z Bentley, Joel Litter S Spalter, Elisa L Ginter, and Harold E Laubach 300 Gender Composition and Maternal Behavior in the Southern Short-Tailed Shrew, Blarina carolinensis (Insectivora) Fred Punzo 308 Sediment Contamination Survey on St Marks National Wildlife Refuge Jon M Hemming and Michael S Brim 314 Acknowledgment of Reviewers 323 u FLORIDA SCIENTIST Quarterly Journal of the Florida Academy of Sciences Institute © by the Florida Academy of Sciences, Inc 2003 Martin Co-Editor: Mrs Barbara B Martin for Environmental Studies, Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620-5250 Phone: (813) 974-2374; e-mail: dmartin@chumal.cas.usf.edu Business Manager: Dr Richard L Turner Department of Biological Sciences, Florida Institute of Technology, 150 West University Boulevard, Melbourne, Florida 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Barry University Miami Shores, FL 33161 Program Chair: Engineering, Inc Hays Barry University Miami Shores, FL 33161-6695 Published by The Florida Academy of Sciences, Inc Printing by Allen Press, Inc., Lawrence, Kansas M 2G04 Scientist^^n^ Florida QUARTERLY JOURNAL OF THE FLORIDA ACADEMY OF SCIENCES Dean F Barbara Martin, Editor Volume 66 B Martin, Co-Editor Number Autumn, 2003 Environmental Chemistry INFRARED STUDIES OF SUWANNEE RIVER HUMIC SUBSTANCES: EVIDENCE OF CHLORINATION OF HUMICS IN SALT WATER Thomas Manning* \ Stacy Strickland \ Amy Feldman 0) 0) Tice Umberger \ Derek Lovingood Mamadou Coulibay 0) (2) John Elder and Lyn Noble (3) , ( l , , , ( ' 'Department of Chemistry, Valdosta State University, Valdosta, Georgia 31698 '^'Department of Biology, Valdosta State University, Valdosta, GA 31698 '"Chemistry and Pharmacy Department, Sunderland University, Sunderland England Abstract: Uumic Substance (HS) samples were collected from twenty-five Suwannee River These sites included the Okeefenokee delta where the river empties used to we into the Swamp, several springs and and the the Gulf of Mexico Fourier Transform Infrared Spectroscopy (FT-IR) is categorize the relative abundance of the functional groups of these samples In salt-water samples identified a relatively intense C-CI vibration indicating the chlorination of the humic substance also measured the vibrational spectra of humic acid with several concentrations of Na4 P along sites tributaries, HN0 KOH , We and over twenty-eight days to optimize our extraction procedure by minimizing condensation reactions Key Words: Humic, fulvic, humic substances, infrared, IR, FT-IR, Suwannee River Humic substances (HS), the product of plant molecular weight component, and to is soluble in water at dark brown values of pH, is all is into the lowest characterized by a yellow to tan color in solution pH's in color in the and animal decay, are divided humic acid (HA), and humin FA, three classifications: fulvic acid (FA), HA is insoluble below a aqueous phase Humin black in color, and is found * Corresponding author (tmanning@valdosta.edu) 253 at the is pH of two and is brown insoluble in water at bottom of all rivers, streams, oceans, FLORIDA SCIENTIST 254 and lakes The carbon content of FA [VOL 66 lower than is HA and humin, and the mass percent of oxygen decreases with the progression from fulvic acid to humic acid and onto humin (Pettersson et al., 1994; Schulten, 1996) HS are macromolecules that aggregate and are comprised of cellulose, lignin, amino acids, peptides, sugars and contain significant quantities of the functional groups phenols, amines, carbonyls, A humic acid model proposed by Schulten (1996) has the C 66.69%, O 25.96%, 6.09% H, and N 1.26% The International Humic Substances Society (IHSS) uses humic acid and fulvic acid from the Suwannee River at Fargo, GA as its and carboxylates chemical formula of C308H335O90N5 giving mass percentages of al., 1994; Manning et al., 2000; Ouatinane et al., 2000; Rasyid The molar mass of FA from the Suwannee River (Fargo, GA) was reported to be 1000-1500 amu and molar mass of HA from Fargo was reported to be 5000-10000 amu (Thurman et al., 1982) When Ouatmane and coworkers (2000) used FTIR to study HA, they found several reoccurring spectral features including an O-H group, an aliphatic CH stretch, the presence of C=C, C=0, and/or C=0 of standard (Averett et 1992) et al., conjugated ketones, deformation of -1 cm with aromatic COOH that CH observed in HS amides, spectral evidence indicated C-H , in list does not represent a complete list of potential vibrations Suwannee River Humic Substances (SRHS), such more complete vibrations so a and CH groups, and correlated spectral features below 1290 C=C, COOH and CH C-O of aryl ether, and C-O and OH of groups Their could be found quinones or table of functional groups are presented (Table CN or C-Cl and frequencies that can be 1) Dupuy and Douay (2001) studied the behavior of heavy metals in soils that complex humics by infrared spectroscopy Klucakova and coworkers (2000) characterized Humic Acids (HA) and fulvic acids (FA) by their ability to complexes with inorganic and organic molecules capable of causing the cross-linking form effect of by FT-IR Lynch and Smith (1992) reviewed inconsistencies in -1 cm regions of the IR spectra of humic acids assigning features in the 1000-1200 either to carbohydrate or to siliceous components Yonebavashi and coworkers (1989) studied forty humic acids from various types of soils by *H spectroscopy Baes and Bloom (1989) measured spectra of HA and NMR FA and IR from peat were obtained using Diffuse Reflectance IR Fourier transform (DRIFT) spectros- KBr copy and Fourier Transform-IR (FTIR) spectra using In measuring the IR spectra of that HS, it is pellets important to use an extraction procedure does not damage the molecular structure In performing these extractions, wanted to avoid condensation reactions that may we compounds, combine with occur Carbonyl carboxylic compounds, phenols, imines, and aromatic compounds all each other and among themselves to form larger molecules For example, carbonyl condensations, or aldol condensations, involve the presence of a carbonyl, an enolate, and a strong base The enolate, a carbonyl compound electrophile, attacks the carbonyl that the aldol to give a p- However, the aldol product may give an ot,p-unsaturated carbonyl compound This is due to the fact hydroxy ketone or aldehyde also known further react to that acts as an group under nucleophilic addition condensation is among carbonyl compounds, as an aldol an equilibrium process The condensation reactions carboxylic compounds, phenols, imines, and aromatic MANNING ET AL.— HUMIC ACID STUDIES No 2003] Table found Correlation table of infrared frequencies for functional groups that could potentially be humic substances in Wavenumber (cm Functional group C-Cl 255 600-800 (aliphatic) 1200-1300 (bend) P-0 1100-925 C=S 1200-1050 C-C 1200 C-N Phenolic OH bending And C-0 Sharp 1050-1100 C-Cl (Aryl) CH -C1 Notes ') Strong 1200 Usually strong 1200 Strong 1410-1310 Strong stretch P=0 1300-1250 C-N C-0 1360-1250 Alcohols (1220-1040) Ethers (1300-1000) Aromatic C=C 1650-1450 N-H -CN 1620-1510 Strong 1600 Usually strong c=c c=o 1660 1710 (aldehydes) Very strong 1715 (ketones) 1730-1700 (carbox acids) 1735 (Esters) 1655 (amides) COOH C=N Just S-H 2600-2550 1725-1680 Sp C-H 3000-2800 O-H Sp C-H Sp C-H Alcohol O-H Amine N-H N-H stretch 3000 3100-3000 Acid Strong above 2200 Usually strong Weak Broad 3300 3300 Broad 3300 Broad with spikes Medium Medium Medium 1° 3500 and 3400 2° 3500-3310 =N-H 3400-3300 compounds can take place with the addition of a catalyst to produce a large molecule and a smaller molecule, usually water or alcohol The most in these reactions is a strong base such as NaOH, or or combination of strong acids can also be used for Because various isolation processes involving solutions, we sought HS KOH, common some condensation HS reactions involve either strong acid or basic a long-term (several weeks) study to identify took place that would alter the catalysts used however, a strong acid if any reactions structure Past work by Hoekstra and coworkers (1999) has shown that humics can become chlorinated under natural conditions They reported the natural formation of 4-chlorophenol, 2,4-2,5- and 2,6-dichlorophenol, and 2,4,5-trichlorpenol in four FLORIDA SCIENTIST 256 a [VOL 66 Alapaha River - 0.4 0.3- Si c - - 0.1 aJ L-^—s Wavenum b Suwannee V_ 2900 2400 1900 1400 900 40 ber (1/cm 3400 3900 ) River bank 0.45 0.1 - j\Ji A I \—J—' 400 900 J ^-> 1400 1900 2400 2900 W avenum bers (cm \ \v 3400 390 " ) Gulf of Mexico 2.5 0.5 400 900 1400 1900 Wavenum 2400 2900 ber (cm ) 3400 ' 3900 MANNING ET AL.— HUMIC ACID STUDIES No 2003] selected areas of a rural with a solution of Douglas Na 37 Cl and fir forest 257 Here the humic layer was spiked in situ covered by an enclosure, and incubated for one year Chlorinated phenols were formed naturally from organic matter and inorganic novo synthesis or chloroperoxidase-catalyzed chloride presumably by either de While the conditions chlorination Suwannee River generated naturally when humics found are different than those show basin, the study does in the lower can be that chlorinated aromatics are in the presence of chlorides Recently additional studies on the interaction of humic and fulvic acids with chlorine and chloride in soils were published (Casey, 2002; Singer, 1999) These compounds studies confirm that organochlorine transfer of chlorine from inorganic are present in soil and identify a net An abundance forms with weathering to organic compounds by hydrogen humic materials is widespread and catalytic peroxidase facilitates the chlorination of aromatic peroxide in may soils They claim that chlorination in account for organochlorine concentrations found in unpolluted environments Organochlorine compounds attract the attention of environmental scientists because of their toxicity and carcinogenic potential in humans Materials and Methods for —A Mattson Fourier Transform measurements Before using FT-IR all select conditions for extraction that would minimize any was used Infrared Spectrometer (FT-IR) HS"s from the Suwannee River, we sought to study structural damage to properly to the naturally occurring organic matter Infrared spectroscopy has long been used as a tool to qualitatively identify various HS's (Yates, 1999; Klavins, 1999; Francioso structure aspects (carbonyls, carboxylates, etc.) of Na^P^Oy, 0.05 M 1% HN0 20% HN0 , 0.05 , Na4P of sodium azide to prevent the growth of microorganisms These solutions were saturated with cially available HA (Sigma Aldrich) was spectroscopically analyzed ditions We then made IR would have on HS We the HS did this to examine what impact, commer- left if on the IR transmission card any, extreme acid or base con- over a various time periods SRHS While our extractions with acids and bases of by analyzing longer time scales we hoped to FT-IR could detect these changes insensitive 7, transmission measurements of the seven samples was dried and 21, and 28 days In each case the water at 14, et al., M M KOH, and M KOH (100 mL each) were prepared They were treated with low levels 1998) Solutions of deionized ultra filtered (DIUF), were conducted in short time scales (minutes), amplify any destructive reactions taking place so the We found substantial concentration acids or bases and identified appropriate acid structural (