Ligninolytic Enzymes and Phytotoxic Effects Following Growth of Strains of Pleurotus in  MWI Fly Ash Contaminated With PCDD/Fs

M. F. Dos Reis – Gorny and L.R. Durrant, Food Science Department, Food Engineering Faculty, Campinas State University,  Campinas-SP, Brazil.

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) are produced by incineration processes and as by-products during chemical reactions. These compounds have high toxicity and genotoxic potential. The purpose of this work was to determine the production of ligninolytic enzymes by three Pleurotus strains, following their growth in medium containing PCDD/Fs and also to evaluate the toxicity produced. The fungi were inoculated in Erlenmeyer’s flasks containing 50 mL of medium composed by  minerals, thiamin and yeast extract. On the third day of growth, 2.5 g; 3.5 g or 12.5 g of MWI fly ashes having  high concentrations of PCDD/Fs, were added and the culture flasks were sealed and oxygenated. Following seven days samples consisting of the whole flasks were collected and then after each seven days, up to 42 days. Oxygenation was also carried out every seven days. The supernatants were collected and used for the determination of lignin- and manganese-peroxidases (LiP and MnP), and laccase, and also for toxicity levels. Control samples consisting of uninoculated flasks were also run and triplicates were used for each assay. Enzyme activities were determined by spectrophotometric assays. The toxicity levels were determined using a bioassay employing lettuce seeds. Results were expressed as % of phytotoxic effect: [(lengths of roots after growth in water – length of roots after growth on tested compound)x100/ lengths of roots after growth in water].

No fungal growth was observed when 12.5 g of fly ashes were added. When 2.5 g were used, low levels of LiP, Lacase e MnP, were detected, regardless of the strain. When Pleurotus sp. BCCB 068 was grown in medium containing 3.5 g of ashes, high lacase activities (20U/L), was detected up to the 7^th day. Lower activities were observed for P. sajor caju 020 (2.5 U/L – 14^th).  MnP was produced by all fungi in all the days in which it was assayed. The highest  MnP activities were  detected on the 28^th  day for P. BCCB 068 (20 U/L), P. sajor caju 020 (15/U/L) e  P. tailandia (10 U/L). Pleurotus BCCB 068 was the only strain able to produce a supernatant showing a good decrease in the phytotoxic effect, i.e., 35.90 % following 42 days of growth. The other two strains showed no increase in the toxicity of medium, since 67.58 % and 74.51% of phytotoxic effect were obtained for the supernatants of P. tailandia  and P. sajor caju 020, respectively. Our results indicate that Pleurotus sp. BCCB 068 produced metabolites, which were less toxic to the lettuce seeds than the other two strains, and that the higher levels of lacase and MnP produced by it may play an important role in the degradation of PCDD/Fs.

We acknowledge FAPESP for financial support.

Using Microarrays to Determine Genetic Biomarkers in Largemouth Bass Exposed to Environmental Contaminants

Patrick Larkin , EcoArray, Alachua, FL
Tara Sabo-Attwood, Interdisciplinary Program in Pharmacology and Therapeutics, University of Florida, Gainesville, FL
Kevin J. Kroll, Biotechnology Program, University of Florida, Gainesville, FL
Nancy D. Denslow, Biotechnology Program, University of Florida, Gainesville, FL and Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL

The purpose of this study was to determine the specific expression profile of 132 genes, some of which are estrogen responsive, in largemouth bass (Micropterus salmoides) following exposure to estradiol (E₂), or to two hormonally active compounds 4-nonylphenol (4-NP) and 1,1-dichloro-2, 2-bis (p-chlorophenyl) ethylene (p, p’-DDE) using microarray technology.  Gene expression profiles were also examined in livers of female and male largemouth bass throughout their reproductive cycle.  A custom built, targeted array containing many genes that are estrogen responsive, was used for the experiment.  The results of the exposure experiments showed that LMB exposed to E₂ and 4-NP had similar, but not identical genetic signatures for the genes examined, some of which are known to be estrogen-responsive genes.  The differences suggest that 4-NP may have additional modes of action that are independent of the estrogen receptor.  We have also shown that exposure of male LMB to p, p’-DDE results in an increase in some estrogen-responsive genes.  But in female LMB, the observed changes were a down-regulation of the normally up-regulated estrogen responsive genes.  Other genes were also down-regulated.  For the seasonal study, fish were housed in outdoor holding pens and were sampled throughout the year.  As expected, gene expression patterns in the livers of male and female bass are different, especially for genes that are involved in oogenesis.  Genes that change in females include those that encode vitellogenin and the zona radiata proteins, among others.  The up-regulation of these genes coincided with the peak of plasma estradiol and peak plasma vitellogenin protein in these animals.    The results from these experiments suggest that gene arrays can be used in order to gain a better understanding of critical windows in reproduction in largemouth bass, and to assess sensitive life stages for exposure to various pollutants.

Hazard Assessment of Pesticides and Metals in Sediment from South Florida Canals

Gary M. Rand, Florida International University, Ectoxicology & Risk Assessment, 3000 N.E. 151st Street, N. Miami, FL 33181, Tel: 305-919-5869, Fax: 305-919-5887
John F. Carriger, Florida International University, Ectoxicology & Risk Assessment, 3000 N.E. 151st Street, N. Miami, FL 33181, Tel: 305-919-5869, Fax: 305-919-5887

A lower tier hazard assessment of pesticides and metals in sediment of South Florida canals was conducted for background information about compounds and canal sites that may be of potential risk to aquatic organisms.  Later tier work included a probabilistic risk assessment for sites and compounds of concern.  In this second tier, chemicals were assessed both singly and as mixtures utilizing the msPAF approach for concentration and response addition based on the compounds' toxic mode of action to benthic invertebrates.  A total of 36 sites are currently sampled by the South Florida Water Management District (SFWMD) for pesticide monitoring and over 60 pesticides and degradation products are analyzed. The SFWMD contaminant database was used for this assessment.  For the lower tier assessment,  sediment quality criteria were obtained from the National Oceanic and Atmospheric Administration NOAA) and Threshold Effects Levels (TELs) in sediment were used for screening benchmarks.  The majority of TEL exceedences came from DDT and its metabolites, DDD and DDE.  These violations were found in sediment from Lake Okeechobee to the Water Conservation Areas to the southern Everglades.  Canal sites bordering Lake Okeechobee also had violations for metals and organics.  Adjacent to the southern Everglades, sites on or near C-111 had more metal violations than any other region assessed.  Several organic compounds were found to have exceedences around canal structures (S-6 and S-7) near the Water Conservation Areas of the Northern Everglades.  Sites on or near the Caloosahatchee River had violations for metals and organics.  Over a dozen compounds did not have sediment quality criteria  available.

Reliability of Numerical Sediment Quality Benchmarks Compared to Sediment Characterization Data as Indicators of Suitability for Dredged Material Disposal 

Ann B. Shortelle, MACTEC Engineering and Consulting, Inc., Gainesville FL
Glenn Schuster, USACE - Jacksonville District, Jacksonville FL 
Bryan Cotter, PPB Laboratories, Gainesville FL

Evaluations under Section 103 of the Marine Protection, Research, and Sanctuaries Act of 1972 consider potential dredged material proposed for disposal in Ocean Dredged Material Disposal Sites for maintenance dredging projects.  The determination of suitability for ocean disposal is based upon physical, chemical, and biological evaluations.  Currently, a tiered testing procedure is used for evaluating the potential dredged material.  Our prior study concluded that for the Florida and Caribbean sites evaluated, no requests for open ocean disposal could have been permitted based upon screening criteria alone, and both false positive and false negative results were common for the metals evaluated.  Results from the tiered toxicity testing result in significantly fewer toxic responses than suggested by simple but conservative screening values.  These site-specific data, together with results for dredge material testing conducted in 2002, are statistically evaluated to extend last year's evaluation to organic constituents in sediments.  Results are comparable to the small subset of metals evaluated previously, and show that some sediment quality benchmarks for both organic and inorganic constituents are unreliable predictors of bulk sediment toxicity.  Thus, site-specific data enable ocean disposal of dredged materials with significantly more confidence that adverse impacts will not occur. 

Top