Determining the Efficacy of Selected Conks of the Aphyllophorales Family for use as Heavy Metal Biomonitors.

1. Determining the Efficacy of Selected Conks of the Aphyllophorales Family for use as Heavy Metal Biomonitors. James G. Wells Departments of Biology and Chemistry & Biochemistry SUNY College at Oneonta

2. What makes an organism an effective biomonitor? • The organism in question must be able to uptake and/or sequester the contaminant of interest. • The species cannot be a rare species due to a need for a good geographical range and out of consideration for conservation efforts. • The organism must show the contaminant levels in a way as to be indicative of the levels throughout the environment. • The organism cannot degrade or deteriorate the contaminant in question. • The species must be able to be sampled in a statistically sound methodology, i.e. no bias, no hypervariability, no mobility, etc..

3. Conks and Polypores

4. Nature of Research: The research project that was carried out had two distinct parts. • First was the collection of conks from various host trees in forest communities of Otsego and Tioga counties noting date, host, coordinates (both lat/longitude and UTC), species identity, and in some cases number of sporocarps present. These collected conks were then cleaned of dirt and debris on outer surfaces with double distilled 17.9 M water and a stiff bristle brush. After cleaning the conks were dried in an oven at 95°C for two days then sealed in plastic bags until time of analysis.

5. Nature of Research con’t: The second part of the research was the analysis of the conks for selected heavy metals and the interpretation of the data. • Digest a ~0.6 g section of the conk excluding outer surfaces with the aid of 10 mL concentrated nitric acid and microwave assisted digestion carried out in a pressurized Teflon® bomb. • Dilute the solution left after digestion to 100 mL with 17.9 MΩ water and analyze the solution for Cu, Cd, Pb, U, and Sr using ICP-AES. • All digestion and analysis was checked for quality and accuracy through use of National Institute of Standards and Technology standard reference materials and independent quality control checks.

6. InductivelyCoupledPlasma-AtomicEmissionSpectroscopy (ICP-AES) • The solutions of samples/standards are pumped to a nebulizer which mists a fine spray into the plasma. • The plasma, which is at 6,000-10,000°C, excites atoms in the sample/standard and the spectrometer measures the wavelength and emission intensity of the light emitted from the selected elements. • The ICP-AES takes the emissions from the samples and uses the equation from the linear regression analysis of the standards to compute a concentration for the samples. • The machine does 3 replicates for every measurement so mean, standard deviation, and percent relative standard deviation are reported.

7. Copper Emission peak from 1997 Tube Layer of G. applanatum

8. Calibration curve for Strontium R2= 0.998987

9. Digestion Method Validation • Validation for the efficiency of the microwave digestion system in getting all of the metals in a sample into solution was checked through the digestion and analysis of NIST SRM 1573a tomato leaves. The results are displayed in the table below.

10. Metals of Interest Copper (Cu)- This metal, although known as a essential micronutrient for many organisms, is toxic to both plant and fungal species in higher concentrations. Cadmium (Cd)- Cadmium is one of the most toxic heavy metals due to its observed toxicity at ppm concentrations to organisms ranging from microbes to human beings. Lead (Pb)- Known to have been a probable cause of the demise of the roman empire, lead is a dangerous metal throughout the natural world having many deleterious effects in a multitude of organisms. Strontium (Sr)- While normal strontium isn’t deleterious to health and is present in most natural systems, its primary radioisotope Sr-90 is uptaken and incorporated into bone structure in place of calcium. Uranium (U)- Present in most natural systems, this metal can also be radioactive and is harmful when present in large amounts.

11. Toxicity of Metals of Interest

12. Where do these metals come from? The metals of interest to this research are all present at some levels throughout most environments. So where do the higher amounts that are toxic come from? • Via atmospheric deposition as the result of varying human activities. Fossil fuel power plants and smelters are two examples. • By uptake of organisms due to remobilization of the metals in ionic form as the result of their release from binding and/or chelating complexes in the soil from the effects of acidic deposition. Again stemming from, and a direct result of, anthropogenic activities.

13. Efficacy for use and metal concentration by species Piptoporus betulinus (the Birch polypore) Range: Wide range across temperate zones, limited by host tree: Betula sp. Morphology: Annual sporocarp compromised of a thin tube layer with a considerably thicker body layer. much softer and pliable than others. Physiology: Produces a carbonizing brown rot of the sapwood.

14. Piptoporus betulinus (the Birch polypore) Due to host consideration, morphology, and unknowns concerning physiology P. betulinus appears to have very little benefit to be used as a biomonitor for heavy metals in the environment.

15. Fomes fomentarius (the tinder polypore) Range: This conk has a wide range worldwide in temperate and subtropical regions. Morphology: The sporocarp of this species is perennial producing new spores yearly from the same tube layer so no tube layer stratification is formed. Physiology: This fungus is a perennial white rot species that feeds on both the sapwood and heartwood of dead and living tress.

16. Fomes fomentarius (the tinder polypore) Due to what appears to be extreme variability the benefits of the morphology and physiology of this fungus are cancelled ruling this perennial, woody conk not fit for use as a biomonitor.

17. Pleurotus ostreatus (the Oyster mushroom) Range: This is a widespread fungus found worldwide in both temperate and tropical zones. Morphology: While it is neither a polypore or a conk, this fungus produces an annual sporocarp. To say it isn’t woody is a vast understatement. Physiology: This is a white rot fungus that attacks the heartwood of both living and, mainly, dead trees.

18. Pleurotus ostreatus (the Oyster mushroom) The use of this species as a biomonitor could have possible use, but the PI believes that this species could better be used as an indicator of the degree of safety in harvesting and consuming edible wild mushrooms. The alarmingly high amount of Cd present in the mushroom by dry weight helps with this inference .

19. Daedaleopsis confragosa(the thin-walled maze polypore) Range: It has a wide range worldwide and is very oppurtunistic as far as host tree choices go. The PI received one sample of this species from a vine! Morphology: This polypore is often found in groups on the same tree. The sporocarps can be quite variable in their structure and layer depths. Physiology: A carbonizing brown rot fungi, this fungi directly attacks only the sapwood of living or soon to die trees.

20. Daedaleopsis confragosa(the thin-walled maze polypore) Daedaleopsis confragosa, beside being a mouthful to say, appears to have very little use as a biomonitor for heavy metal contamination. Even though it is very common, the lack of distinction within the sporocarp coupled with the extreme variability of metal concentrations and the nature of its growth and physiology make this species not an acceptable biomonitor.

21. Trametes versicolor (Turkey tail) Range: This is the polypore for temperate deciduous forests. This is a cosmopolitan species that is found worldwide and is extremely common. Morphology: The sporocarps often fuse together either laterally or vertically. They are thin and form only two discernible layers. Physiology: This is a white rot species of fungus that infects and digests the heartwood of living and dead trees.

22. Trametes versicolor ( Turkey tail) While the widespread growth and nature of digestion hint that this species would be a good biomonitor, the sporocarp morphology rules this polypore out for use as a heavy metal biomonitor.

23. Ganoderma applanatum( the Artists’ conk) Range: This is a widespread and fairly common conk. It is found worldwide and the only places that are not apt to be this fungi’s home are the artic extremes. Morphology: The sporocarp produces distinct stratified yearly tube layers with some specimens actually reaching 20-30 years in age. Physiology: G. applanatum is a species of white rot fungi. It infects trees and digests the heartwood.

24. Ganoderma applanatum ( the Artists’ conk) The 2001 layer is statistically different from the Cap and 2002 layers at the 95% Confidence Interval. Variability of the metal concentrations in the separate layers of G. applanatum was tested. The results showed low variability in the same layer but differing concentrations in different layers. This means that theoretically the distinct layers can be analyzed to gain a qualitative chronological record for metal deposition in an area! Potentially, this species is a suitable biomonitor!

25. Analysis of G. applanatum

27. Pro’s: Is widespread and fairly common. 2+ sporocarps formed at one site. Can be re-sampled in later years after new sporocarp growth. Woody composition is likely to resist leaching. Tube layer stratification allows for chronological data instead of just current data. Is a species with probable considerable secondary heavy metal uptake from host. Con’s: Is woody and requires a chisel or prybar for sampling. Bulky and large so storage could be an issue. People collect them for art or some are just destroyed because of their nature. Requires a need for some knowledge of fungal morphology for dissection of tube layers. Ganoderma applanatum ( the Artists’ conk) Pro’s and Con’s for biomonitor use.

28. Conclusions: • Out of all specimens sampled only Ganoderma applanatum appears to have use as a possible biomonitor for heavy metal deposition. • The high variability of both metal concentrations and morphology among the Aphyllophorales leads the PI to think that research focused only on a specific Genus would yield much more viable and statistically sound data. • There can be no distinction between atmospheric deposition and secondary uptake from the host without further testing in a controlled laboratory environment. • Stratified sporocarps appear more viable than non-stratified sporocarps for use as biomonitors. • Conks and polypores seem to be better biomonitors than other fungal types due to woody structure and perennial nature. • Further testing on G. applanatum needs to be done before submission of this work for publication.

29. Hopes for the future: • Preliminary results lead the PI to think that further testing of G. applanatum will show the viability of this conk as a chronometric biomonitor. • Hopefully this method will join dendroanalysis, concentric rings of bivalve shells analysis, and analysis of the earbones of sturgeon as an addition to the developing suite of chronometric biomonitors. • M.Sc. Or Ph.D. for the PI whom plans to carry this type of research throughout academic career.

30. Acknowledgements: • Dr. Vogler and Dr. Schaumloffel for their endorsement, support and ideas. • Dr. Marr for use of the “vintage” polypore specimens. • Dean Merilan, Dr. Chaing and the Department of Chemistry and Biochemistry • Dr. Pietraface and the Department of Biology • The Senate Research Committee for project funding • Christine Barnes and Don Polley for materials support • Tom Volk and the Boston Mycological Club for their websites of fungi images • NSF-MRI Grant #EAR0215734 for the acquisition of the ICP-AES and microwave digestion system