of Persistent Organic : Mechanistic studies and Field Application

Jason C. White Head, Department of Analytical Chemistry The Connecticut Agricultural Experiment Station Persistent Organic Pollutants (POPs) in • They persist for decades • Global distribution • Likely mutagenic, estrogenic, carcinogenic effects • , biomagnification • Other remediation strategies are ineffective due to high degree of sequestration; should not facilitate EPA Dirty Dozen their remediation Aldrin DDT/DDE

Dieldrin Dioxins Endrin

Furans Heptachlor Lindane

Mirex PCBs Toxaphene Uptake and translocation of weathered p,p’-DDE by selected species

3000 Mustard Cucurbita pepo ssp pepo Clover Rye 2500 S. squash W. squash Zucchini 2000 Pumpkin

1500 -DDE (ng/g) 1000 p,p' Cucurbita pepo ssp 500 ovifera

0 Soil Soil Soil Soil Soil Soil Leaf Leaf Leaf Leaf Soil Fruit Fruit Fruit Fruit Roots Roots Roots Roots Roots Roots Stems Stems Stems Stems Shoots Shoots Shoots

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p,p' system V p,p' system

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52 y R of root to soil (values are 10

2.25 1.50 0.75 0.00 % Phytoextracted % Phytoextraction (% removal) of weathered by plant species. Numbers in green indicate mass ratio by selectedplant species of chlordanefrom soil Uptake andtranslocation by selectedplant species of chlordanefrom soil Uptake andtranslocation Chlordane (ng/g) 21000 24000 2500 5000 7500 0 olRoots Soil Stems Leaves Soil Zucchini Cucumber Spinach Corn Fruit DDX in the xylem sap of 3 cucurbits

20.0 Zucchini DDE DDT Squash 15.0 Cucumber

10.0 DDD

5.0

1.5

1.0

0.5

Xylem sap ConcentrationXylem (ng/mL) 0.0

35 Zucchini 30 Cucumber Squash 25

20

15

10 DDE Flow (ng/h)

5

0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Root mass (g, dry weight) CAES Research on the C. pepo ssp pepo system • Focused on two separate lines of investigation: 1. Optimizing remediation- Field/pot studies in an effort to further enhance contaminant uptake (cropping strategy, surfactants, organic chelators or TPAs, fungal inocula, intercropping) 2. Mechanistic studies- hydroponic, rhizotron, grafting, breeding, and molecular studies to elucidate governing processes. The C. pepo ssp pepo system

• Mechanistically, you need to consider two areas • Ex planta- Release and uptake of a highly sequestered residue from soil to roots - Plant exudates are released to facilitate acquisition from soil. Species vary widely in exudation. Greater amounts of low molecular weight organic acids result in soil matrix deconstruction - This disruption in soil structure inadvertently increases the bioavailability of weathered POPs • In planta- Translocation of a hydrophobic residue from roots to shoots. - Hydrophobic contaminants must move with water through root tissue and cross into the vascular cylinder. Should be minimal but some plants are clearly more effective than others. Zucchini exude more organic acids, whether you look directly at the root surface (left) or in the pore water (right)

20.0 Zucchini DDE DDT Squash 15.0 Cucumber

10.0 DDD

5.0

1.5

1.0

0.5

Xylem sap Concentration sap Xylem (ng/mL) 0.0

12000

8000 10000

6000 8000

6000 4000

4000

2000 2000 Total Organic Acid (picomoles) Total Organic Acid (picomoles) Total Organic 0 0 Zucchini Squash Cucumber Control Zucchini Squash Cucumber Control

* Organic acids summed are citric, malic, succinic, tartaric, malonic Accumulation of chlordane components from soil by shoots of intact or grafted zucchini and cucumber plants (units are raw area counts)

120 16 Zucchini * Cucumber * 12 80 * Chordanes 8 + HEPX 40 * * 4 Transnonachlor + Cis-chlordane ND*ND 0 0 + Trans-chlordane Zucchini homograft Cucumber homograft + MC5a/MC5b 60 12

40 8

20 4

0 0 ND ND Zucchini shoot on cucumber root Cucumber shoot on zucchini root 6 25

20 4 15

10 2 Mattina et al., 2007 5

ND 0 ND 0 Differential uptake of weathered p,p’-DDE by 2 subspecies of Cucurbita pepo (ssp pepo and ssp ovifera)

Average BCF Average BCF 12 Stems pepo- 7.2 Roots pepo- 5.4 10 ovifera- 2.4 ovifera- 0.54 10 8 8 6 BCF 6

BCF 4 4 2 2

0 0 Raven Magda Zephyr Sunray Magda Raven Seneca Zephyr Costata Sunray Starship Seneca Costata Revenue Sunburst Starship Goldrush Early Pro Early Patty pan Patty Revenue Sunburst Goldrush Early ProEarly Patty pan Patty Black zuke Black Hybrid jack Hybrid Butter scal. Black zuke Black Hybrid jack Hybrid Butter scal. Black magic Black Hybrid butter Hybrid Black beauty Black Black magic Yellow Crook Yellow Hybrid butter Hybrid Black beauty Black Yellow Crook Yellow Gurneys pride Gurneys Gurneys pride Gurneys Hybrid crescent Hybrid Hybrid crescent Hybrid Stem DDE bioconcentration factors and % DDE phytoextracted for 3 cultivars each of two C. pepo subspecies

10 Zucchini Non-zucchini summer squash Zucchini Non-zucchini summer squash (C. pepo ssp pepo) (C. pepo ssp ovifera) 1.0 (C. pepo ssp pepo) (C. pepo ssp ovifera)

8 0.8

6 0.6

4 0.4 Stem DDE BCF Stem DDE

2

% DDE phytoextracted % 0.2

0 0.0 r r a h n y a h n y y k y t k t t t s e s e t t o a h o a h t u v a t u v a r p o r p o s a r s a r d e P d e P o l R C o l R C Z Z o o C . C . G Y G Y Traditional Breeding Studies- DDE-accumulating zucchini and non- accumulating squash are in the same subspecies. What will be the abilities of the hybrids? A 16 Stem

12 C bioconcentration C 8 factors (BCFs)

Stem DDE BCF and % DDE 4 B phytoextracted 0 Parents F1 Hybrids by parental and Zucchini (C. pepo ssp pepo) Squash ( C. pepo ssp ovifera) A Hybrid Zucchini (ssp pepo x ssp ovifera) F1 hybrid Hybrid Squash (ssp ovifera x ssp pepo) 1.6 zucchini and 1.2 C squash

0.8 D

% Phytoextracted 0.4 B

0.0 Parents F1 Hybrids A Zucchini Squash (C. pepo ssp pepo) (C. pepo ssp ovifera) % DDE 1.5 Phytoextracted and

B B Stem BCFs by 1.0 parental, F1 hybrid, B and F1 Backcrossed 0.5 % Phytoextracted % A zucchini and squash A (preliminary data) 0.0 l s l d s i s d ta r i s o ta r n b r n o b r e y c r e y c k r a h k c a h 1 c P a 1 Zucchini Squash P a A F F B B 1 (C. pepo ssp pepo) (C. pepo ssp ovifera) 1 F 15 F

B B 10 B Stem BCF

5 C A

0 l s l d s i s a d ta r i s o t r n b r n o b r e y c r e y c k r a h h k c a 1 c P a P 1 F a B F

B 1 1 F F Uptake of DDE by zucchini and squash exposed to 120 ng/mL DDE under batch hydroponic conditions for 96 hours

1000 A Exposed to Non-exposed DDE Controls 800

600

400

B B 200 B Stem DDE content (ng/g) Stem DDE content

0 Zucchini Squash Zucchini Squash Zucchini and squash that were or were not exposed to DDE were subjected to differential transcript analysis and subtractive hybridization techniques. Subtracted cDNAs were cloned into a TA cloning vector and expression in E. coli via colony array analysis was employed (Fig. 1). Figure 1. A. B..

Figure 2. SHOOT ROOT Blast search on isolated differentially expressed cDNAs Zucchini Squash Zucchini Squash DDE DDE DDE DDE includes sequence homology - + - + - + - + match to “phloem protein I” (PP1) and a . PP1 Cyt P450 Semi-quantitative RT PCR Actin Actin confirmed differential regulation (Fig. 2). List of DDE induced subtracted cDNA clones from zucchini Description Accession E value Max % Functions Number Identity Confirmation of upregulation of zucchini 2-Oxoisovalerate dehydrogenase (2 NM_203027 3e-73 79% subtracted cDNAs by RT-PCR analysis OVD) Aspartate aminotransferase (AAT) XM_002515409 5e-108 85% Amino acid DDE Brassinosteroid Insensitive 1 associated XM_002532844 1e-104 80% defense – + receptor kinase (Brassino. Inh) UK 1 a/b-binding protein -1 DQ471302 1e-76 84%

chlorophyll a/b-binding protein -2 XM_002284820 4e-97 83% photosynthesis UK 3

Cystatin (Cystatin like) ABF94587 3e-07 53% cystein UK 4 proteinase inhibitor UK 5 Cysteine Proteinase (Cys Pro) AJ415386 3e-59 82% signaling and abiotic stresses Brassino. Insensitive DNA binding protein with bHLH XP_002521010 3e-32 45% unknown domain (bHLH) Glucosylceramidase ( Gluc. Cera.) NM_124368 1e-09 81% Lipid Ze Epo. metabolism Protein phsophatase -2a XM_002513094 2e-60 84% Involved in Rub. Activase phosphorylation 50S ribosomal protein L5 (L5 Ribo 50S) XM_002511867 7e-51 79% Unknown Cystatin like Ribulose activase EF506513 1e-122 82% photosynthesis Phosphatase 2A Putative senescence-associated protein BAB33421 4e-44 79% Stress tolerance AAT Unknown protein 1 (UK 1) XP_002527893 0.004 48% Unknown bHLH Unknown protein 2 (UK 2) BT009458 8e-27 100% Unknown Chl a/b 1 Unknown protein 3 (UK 3) AY942801 4e-33 77% Unknown

Unknown protein 4 (UK 4) XM_002532526 3e-48 80% Unknown Chl a/b 2

Unknown protein (predicted protein XM_002519497 1e-52 82% Unknown Cys Pro. mahagunin like) (UK 5) Unknown protein (predicted protein with U01964 1e-23 83% Unknown chlorophyll a/b-binding) (UK 6) 2 OVD Unknown protein (Predicted protein XP_002531567 4e-09 49% Unknown with DNA binding domain (UK 7) Unkown predicted protein (UK 8) XM_002309035 1e-31 78% Unknown L5 Ribo 50S

Zeaxanthin epoxidase (Ze. Epo.) DQ641126 2e-98 90% abiotic stress ACT2 control tolerance Conclusions • C. pepo ssp pepo uniquely accumulates significant quantities of weathered POPs from soil • POP accumulation is dependent on both contaminant properties and plant genotype • Soil amendments such as organic acids, surfactants, and fungal inocula can increase POP extraction but effects are cultivar- and treatment-specific • Although C. pepo ssp pepo has a unique organic acid exudation profile (as a nutrient acquisition strategy), the differences do not explain the observed POP accumulation • Based on hydroponic, grafting, and molecular data, it appears that a unique protein in C. pepo ssp pepo tissue permits POP movement into the vascular cylinder • Investigations elucidating the nature of this transport system are underway Acknowledgements • Dr. Om Parkash (UMass), Dr. MaryJane Mattina, Dr. Brian Eitzer, Dr. Martin Gent, Dr. Neil Schultes • Dr. Zakia Parrish, Dr. Mehmet Isleyen, Dr. Xiaoping Wang, Dr. Wen-Yee Lee • Lydia Wagner, Bill Iannucci-Berger, Craig Musante, Terri Arsenault • Funding from US EPA STAR, USDA CSREES