1. Phytoremediation of Arsenic Contaminated Soils Using Chinese Brake Fern Maria Silva Soil and Water Science Department University of Florida

2. Arsenic  A nthropogenic activities account for most As contamination in soils and water;  A s is carcinogenic  R emediation of As contaminated sites has become an important issue  Current remediation technologies are expensive and environmental disruptive.

3. The use of plants to remove, contain, or render harmless environmental pollutants. Phytoremediation Plant biomass Uptake Plant species Contaminant concentration

4. Use of accumulating plants to remove metals from soil by concentrating them in harvestable parts.Phytoextraction

5. Chinese Brake fern (Pteris vittata L)  N atural ability to tolerate, accumulate, and translocate high concentrations of As  A s high as 2.3% in fronds  R apid growth rate  P erennial  H igh biomass  C apable of taking up both organic and inorganic As

6. Although the fern shows great potential to be used in Phytoremediation, many questions still need to be answered!!!

7. Research objectives 1.D etermine the effectiveness of Chinese Brake fern (CBF) in continuously remove As from soils over time; 2.I nvestigate the impacts of CBF on As mobilization and redistribution in the rhizosphere soil 3.D etermine the NPK levels for optimum As removal by the fern 4.E valuate the effect of plant maturity on As accumulation by the fern.

8. Experiment 1 Effectiveness of Chinese Brake fern on arsenic removal over time HYPOTHESES HYPOTHESES: 1. C hinese Brake fern is capable of continuously removing As from soils; 2. A rsenic availability in soil will decrease as more available As is taken up by Chinese Brake 3. C hanges in plant available As can be predicted by partitioning the soil As-pools using a sequential fractionation procedure

9. Materials and Methods Experimental setup  Completely Randomized Design  Six As-contaminated soils  One plant per pot containing 4 kg soil  Four replications  Controls without plants Sampling  Harvest fronds 2-3 times a year  Collect soil with plant harvest

10. Experimental Soils

11. Analyses  Aboveground biomass  Total As concentrations (soil and plant) Hot block digestion system (EPA Method 3050A) Graphite furnace AAS  Arsenic fractionation in soil Wenzel et al. (2001)

12. FractionsExtracting solutionExtraction condition SSR* As-N As-N: Non-specifically- bound (NH 4 ) 2 SO 4 0.05M4h shaking, 20 o C1:25 As-S As-S:Specifically- bound ( NH 4 )H 2 PO 4 0.05M16h shaking, 20 o C 1:25 As-A As-A:Amorphous hydrous oxide-bound NH 4 -oxalate buffer (0.2 M) pH 3.25 4h shaking, 20 o dark 1:25 As-C As-C:Crystalline hydrous-oxide-bound NH 4 -oxalate buffer (0.2 M) + ascorbic acid (0.1M) pH 3.25 30 min in a water basin at 96 o C with light 1:25 As-R As-R:ResidualHNO 3 /H 2 O 2 Hot block digestion 1:50 SSR=soil to solution ratio As fractionation procedure As availability

13. Experiment 2 Arsenic mobilization and redistribution in the rhizosphere of two ferns HYPOTHESES HYPOTHESES: 1. M ore roots of CBF will be developed in arsenic- rich soil than arsenic-free soil; 2. C hanges in the rhizosphere of CBF will be greater than those of Boston fern, a non- hyperaccumulator.

14. Materials and Methods Experimental setup  Completely Randomized Design  Factorial scheme (2 x 2)  2 plants (CBF and Boston fern)  2 soils (contaminated and non-contaminated)  1 plant/pot containing 2.5kg of soil  4 replications  Controls without plants Sampling  Harvest after 8 weeks of growth  Collect rhizosphere and bulk soil

15. Rhizopot Rhizosphere soil Bulk soil 45um

16. Analyses Plant  Frond and root biomass  Frond and root As concentration  Root length density  Root area density Soil (rhizosphere and bulk)  water-soluble As  Total As  pH and DOC  As fractionation

17. Experiment 3 Interactive effects of N, P, K and As on plant arsenic uptake HYPOTHESES HYPOTHESES: 1. As accumulation affects plant nutrient requirements; 2. M aximum plant As removal can be achieved through optimum application of NPK ;

18. Materials and Methods Experimental setup  4 factor- 5 level central composite design  N = 60 to 300 ppm;  P = 30 to 150 ppm;  K = 30 to 300 ppm;  As = 50 to 200 ppm;  one plant/pot with 1.5 kg of soil  Three replications Sampling  Harvest after 8 weeks of growth  Soil

19. Analyses Plant  Frond and root biomass  Frond and root As concentration  Frond and root macro and micronutrients Soil (rhizosphere and bulk)  Total As concentrations  pH and DOC

20. Experiment 4 Arsenic removal by Chinese brake fern using plants of different maturity HYPOTHESES HYPOTHESES: 1.P lant maturity affects its arsenic removal capability; 2. M aximum plant As removal can be achieved using optimum plant age ;

21. Materials and Methods Experimental setup  Completely Randomized Design  One plant per pot with 2.5 kg of soil  Plants: 45 days, 8, 10 and 16 month-old  One As contaminated soil (150 mg kg -1 )  Four replications Sampling  Harvest after 12 weeks of growth  Bulk and rhizosphere soil

22.  Plant biomass  As concentrations in soil and plant  Soil pH and DOC in the rhizosphere and bulk soil Analyses

23. Preliminary Results

24. Frond biomass in the first harvest Initial frond biomass = 8g Experiment 1

25. Fern As concentration in the first harvest

26. Arsenic removed from soils by fern after one harvest

27. Changes in Soil Arsenic fractions after first harvest As-N non-specifically bound, As-S specifically bound, As-A amorphous Fe and Al As-C crystalline Fe and Al As-R residual

28. Clean soil As soil Experiment 2 Clean soil As soil Chinese brake fern Boston fern

29. Plant biomass, As concentration & removal after 8 weeks of growth Fern specie BiomassAs concentration As remediated (%) Frond RootFrondRoot --- g plant -1 ----- --mg kg -1 ----- Non-contaminated soil CBF 6.331.980.582.65- Boston 2.460.860.100.30- Contaminated soil CBF 5.541.844321161.03 Boston 1.750.6019.798.00.04

30. Water-soluble As a aa C B A

31. Soil pH aa a A B B

32. Summary-1 1.CBF grew well in all six soils with 195- 360% biomass increase after 4 months; 2.CBF was effective in removing As from the soils, with 4- 9% reduction after one harvest; 3. The majority of soil As was associated with the amorphous fraction

33. Summary-2 4.Compared to Boston fern, CBF accumulated greater biomass and greater arsenic in the fronds; 5. Water-soluble As was lower in the rhizosphere of CBF than Boston fern; 6. pH was greater in the rhizosphere of CBF than Boston fern;

34. Special Thanks Advisor- Dr. Lena Ma Dr. Jorge Santos Committee members: Dr. Commerford Dr. Rhue Dr. Stamps Dr. Bonzongo Tom Luongo Trace metal biogeochemistry group Sponsorship – CAPES

35. Thank you