1

Methods for

measuring plant

uptake of chemicals:

lab and field

Modeling plant uptake of

chemicals and application in

science and engineering

Course no 12906

17-21 August 2015, DTU

Why are you making the

measurement?

• Risk assessment (ecological and human)

– Concentrations in specific plant compartments

• Biomonitoring

– Mechanism of uptake (root, foliar?)

– Relate plant to environmental concentrations

• Phytoremediation

– Removal

– Change in concentration/time, degradation

• Support model development

What are you measuring?

• Plant concentrations

– Leaves, fruit, shoot, tree cores

• Exposure media concentration

– Groundwater, soil, air (gaseous/particulate)

• Transformation products

• Supporting information

– Transpiration, growth rate, light intensity, etc.

– pH, organic carbon content, dissolved vs. bound

General considerations • Chemical properties

– Volatile/non-volatile, neutral/ionizable, stability

• Chemical analysis – GC, GC/MS, HPLC, LC/MS, LSC (14C-labeled)

• Exposure type and duration

• Uptake/fate endpoint(s) being measured – BCF, TSCF, distribution in plant

• Plant species

• Plant growth environment – Field, greenhouse, growth chamber, bench top

• Controls

Plant considerations • Species

– Unique requirements (size, time to harvest, etc.)

• Exposure concentration-toxicity

• Growth/exposure media (not always same)

– Hydroponics, soil, sand, soil/sand etc.

• Light

– Photoperiod (often16hr light/8hr dark)

– Intensity (not often specified or measured)

• Humidity, temperature

• Nutrients

– Variants of Hoagland’s solution

• Oxygen, CO2

Chemical considerations

• Exposure

– Concentration

• Constant, varies with time

– Duration

• Properties of test chemical

– Hydrophobicity (KOW)

– Volatility (vapor pressure, Henry’s constant)

– Charge

– Chemical and biological stability

2

Experimental design • Exposure media

– Hydroponics

• Water, sand-water, glass beads-water

– Soil

– Air

• Chemical properties

– Hydrophobicity (KOW)/aqueous solubility

– Volatility (Henry Law Constant)

– Biological or abiotic stability

• Distinguish between root and foliar uptake

Goals of laboratory methods

• Avoid experimental artifacts

– Use appropriate controls

– Sufficient replication

– Consider chemical and biological variability

• Generate laboratory data that can be

accurately extrapolated to field

Trade-offs between collecting data in lab or field settings?

Advantage: Better control of environmental and confounding variables

Disadvantage: Lack of external validity (the extent to which study results can

be generalized to other situations) Use Models

Case studies • Hydroponics

– Sulfolane & DIPA (high S, low H, low degradability)

– Benzene (mid S, high H, high degradability

– TCE (mid S, high H, low degradability)

– Nonylphenol (low S, low H, mid degradability)

• Soil

– Nonylphenol (low S, low H, mid degradability)

• Air

– Hydrocarbon and chlorinated solvents (low S, high

H, mixed degradability)

Sulfolane and Diisopropanolamine

S= 870 g/L,

log Kow = -0.86

H (dimensionless) = 7E-6

Slow biodegradability

pKa = 9.1 DIPA

S= 1000 g/L,

log Kow = -0.77

H (dimensionless = 7E-8

Slow biodegradability

Sulfolane

Question

1)Are the high concentrations of sulfolane measured

in field plant samples “reasonable”?

(note: High uptake not predicted by Briggs model)

Nutrient solution

DO 2-5 ppm

Temp. 22±3˚C

pH = 7

Duration = 50 days

(40 mg/L)

(20 mg/L)

Initial experimental set up

3

Final plant concentrations (mg/kg dry weight)

DIPA

Roots

Sulfolane

Exposure: (20 mg/L) /(40 mg/L)

46

48

143

5310

17

17.9

7.5

1.6

Question

1) Are the concentrations of sulfolane measured in

field plant samples reasonable?

Answer

1) High field uptake also observed in laboratory.

(Suggests Briggs model not universally true)

Benzene

S = 1780 mg/L

KOW = 2.13

H (dimensionless) = 0.23

Biodegradable (aerobically)

• Question

– What are the main removal mechanisms in a

constructed wetlands located in Alberta, CA?

Plant uptake?

Degradation (DO < 1 mg/L)?

Volatilization?

(Phragmites (common reed) representative plant)

14C Organic Traps(EGBE)

N2

Gas

Dosing/SamplingNeedle

14CO2 Traps(1M KOH)

To VacuumPump

[DO] = < 1 mg/L

pH = 6 to 8

Experimental System

Root/Foliar

Seal

• Flow-through hydroponic/gravel system

• Flow differential to minimize leaks

• Sealed above root zone

• Benzene added manually

Water Level

Flow = 10 mL/min Flow = 25-30 mL/min

Plant uptake studies: DIPA & sulfolane

4

Transpiration

Gas Sampling

N2

Gas

14C % Distribution

Planted: 82%

Unplanted: 78%

Poisoned: 91%

TOTAL RECOVERY

3.5

2.8

1.2

Root Zone Solution

45

60

88

Organic Traps

30

14

0.05

CO2 Traps

2.4 roots

0.05 leaves

0.6 stems

Plants

• Question

– What are the main removal mechanisms in

constructed wetlands?

Answer

Volatilization and degradation

Trichloroethylene

• Question

– Can we accurately measure a TSCF for TCE for

predicting plant uptake during phytoremediation?

S = 1280 mg/L

Log KOW = 2.42

H (dimensionless) = 0.39

Low biodegradability

Using TSCF to predict uptake

Plant Uptake = (TSCF)(CC)(T)

TSCF = transpiration stream concentration factor

CC = chemical concentration in GW

T = Transpiration (200 - 1400 L/m2-yr)

Burken and Schnoor appartus

Burken, J. G. and J. L. Schnoor (1998). "Predictive relationships for uptake of organic contaminants by

hybrid poplar trees." Environmental Science & Technology 32(21): 3379-3385

0.8-1.1 L/min

Root zone O2 supplied by

headspace

5

Schroll, R. and I. Scheunert. 1992. A laboratory system to determine separately

the uptake of organic chemicals from soil by plant roots and by leaves after

vaporization. Chemosphere 24(1): 97-108.

10 mL/min

Hexachlorobenzene

450 mL/min

45 mL/min

McCardy, McFarlane, Gander. 1990. The transport of 2,3,7,8-TCDD in

soybean and corn. Chemosphere 21(3):359-376.

Orchard, B. J., W. J. Doucette, J. K. Chard and B. Bugbee (2000). "Uptake of trichloroethylene by hybrid

poplar trees grown hydroponically in flow-through plant growth chambers." Environmental Toxicology and

Chemistry 19(4): 895-903.

TCE uptake-growth chamber

Photo-TCE

• Question

– Can we accurately measure a TSCF for TCE

for predicting plant uptake during

phytoremediation?

– Answer

• Maybe

6

0

0.2

0.4

0.6

0.8

1

-1 0 1 2 3 4 5log Kow

Tra

nsp

irati

on

Str

eam

Con

cen

trati

on

Facto

r

Orchard et al. 2000

Chard, 1999

Burken & Schnoor 1998

TSCF

Davis et al. 1998

Variability of TSCF for TCE

Davis et al. 1998

No “standard” method, variation in exposure system,

duration, analysis

Dettenmaier et al., 2009

Variety of chemicals

• Question

– Can we reproduce the Briggs model using

pressure chamber method?

• 25 Chemicals

• 14 Volatiles (headspace/GC/MS)

• 2 semi-volatiles (LLE/GC/MS)

• 9 14C-labeled (LSC) (1 volatile, 8 semi-volatiles)

• Ranged in log Kow -0.77 to 5

• Used in previous plant uptake studies

Results-pressure chamber

Ave TSCF vs log Kow 25 chemicals

Dettenmaier, EM., Doucette, WJ., Bugbee, B. 2009. Chemical Hydrophobicity and

Uptake by Plant Roots. Environ. Sci. Technol. 43 (2):324-329.

0

0.2

0.4

0.6

0.8

1

-2 -1 0 1 2 3 4 5 6

log Kow

TS

CF

model thick roots only with fine roots Briggs regression

TSCF vs log Kow predicted (Trapp 2006)

“The model predicts that polar, non-volatile compounds will

effectively be transported from soil to fruits, while lipophilic

compounds will preferably accumulate from air into fruits.”

7

? Method: Intact plants, hydroponics

Plant: barley

Plant age: 10 days

Exposure duration: 24-48 hrs

Water transpired: Average 1 mL/day

Root weight: 0.1 g/plant

Method: Pressure chamber

Plants: Tomato and Soybean

Plant age: 21- 75 days

Exposure duration: 5-48 hrs, steady state

Water “transpired”: 300-3000 mL

Root weight: 20-110 g

Reported TSCF Values

Literature Pressure Chamber TSCF

> TSCF

Volatilization

Metabolism

Faster, less costly,

more reproducible? Distribution

TSCF Pressure chamber vs. intact plants

Nonylphenol • log Kow = 4.48

• This study, log Kow = 3.28

• S = 5.4 mg/L

• H (dimensionless) = 4x10-4

• t1/2= 28 to 104 days

(soil and surface water)

Question: Is there significant translocation of NP move

from roots to shoot? Any mineralization?

Experimental System

Design

Ethylene glycol

monoethyl ether 1 M KOH

8

A

D C

B

[NP] = 0.07 mg/L [NPE4] = 0.3 mg/L [NPE9] = 0.4 mg/L

14C

Parent

Compound

-Foliar tissue

-Roots

-Nutrient solution

-Organic traps

-CO2 traps

Foliar

tissue-

Roots-

Nonylphenol

Steam distillation/HPLC

SFE/HPLC-fluorescence

NPE4, NPE9, phenol

SFE/HPLC-fluorescence

14C-labeled NP, NPE4, NPE9,

phenol

Combustion/LSC

Plant Tissue

Analysis

Foliar region:

NP 1.3%

NPE4 4.0%

NPE9 7.8%

Phenol 8.6%

Roots:

NP 73.5%

NPE4 54.1%

NPE9 41.7%

Phenol 51.9%

Hydroponic solution:

NP 17.0%

NPE4 20.1%

NPE9 32.2%

Phenol 3.4%

Trapping solution:

VOC CO2

NP 0.2% 1.2%

NPE4 0.0% 0.9%

NPE9 0.1% 1.1%

Phenol 0.1% 18.2%

Distribution of 14C

Foliar tissue

2%

None

detected

Root tissue

98%

4 - 40 µg/kg NP (dry wt.)

14C

NP

14C

NP

Distribution of 14C

& NP in plant

tissue after 99

days.

Question: Is there significant translocation of NP move

from roots to shoot? Any mineralization?

Answer: Little translocation of 14C into foliar tissue and

ittle or no mineralization was observed.

Nonylphenol

• Question: Does NP associated with land

applied biosolids persist in the soil

environment and contaminate plants?

• log Kow = 4.48

• This study, log Kow = 3.28

• S = 5.4 mg/L

• H (dimensionless) = 4x10-4

• t1/2= 28 to 104 days

(soil and surface water)

9

NP experimental design

10

• Question:

– Does NP associated with land applied

biosolids persist in the soil environment and

contaminate plants?

• Answer

– 10 % mineralization, minimal plant uptake

Trichloroethylene

• Question

– Will fruit from trees growing over TCE

contaminated groundwater contain TCE?

S = 1280 mg/L

Log KOW = 2.42

H (dimensionless) = 0.39

Low biodegradability

Risk Assessment

Elevated Stand

[14C]TCE/H2O

Reservoir

[

Leaves, Branches, Fruit

Trunk

Soil

Roots

Irrigation Water

Sampling and analysis

Combustion/LSC-14C

Headspace GC/MS-TCE

Greenhouse fruit uptake photo C3

B2

C1

A1

D2

B2

D1

A3

D3

B3

C2

A2

E1

F1

A: 5 mg/L apple

B: 500 mg/L apple

E: Control apple

F: Control peach C: 5 mg/L peach

D: 500 mg/L peach G: Control apple (2)

H: Control peach (2)

Apple & peach photo

11

Average [14C] data 2nd yr

high/low (µg/kg fresh wt)

Comparing Peaches to Peaches

Fruit flesh

44 / 0.6

Leaves

260 / 3.2

Branches

560 / 8.4

Elevated Stand

Irrigation

water (µg/L)

690/ 5.3

Fruit peel

Fruit flesh

Branches

Leaves*

Irrigation

water

67

483

64

562

1210 259

626 606

*No statistical difference

Elevated Stand

171 days 14C [TCE]

exposure

Elevated Stand

220 days 14C [TCE]

exposure

TCE <0.1

TCE <0.1

TCE <0.1

TCE 690

Comparing Apples to Peaches

average [14C] 2nd yr (µg/kg fresh wt)

23 44

TCE <0.1

Control apple trees

(no sulfolane added)

Treatment apple trees

(100 ppm sulfolane added)

Sulfolane in apple trees

Leaves: 3700 mg/kg

30-day

exposure:

55 mg/L

Apple: 16 mg/kg

Xylem

Volatilization (glycoside metabolite,

trichloroethanol)

(TCE, sulfolane)

(TCE)

Tentative Hypothesis

Phloem

sulfolane

TCE

Volatile organic compounds

(VOC) • Aromatic hydrocarbons

– Benzene, toluene, xylene

• Chlorinated solvents

– Trichloroethylene, tetrachloroethylene

Question: Could house plants be used to

monitor indoor air concentrations of VOCs?

Static Headspace Study

12

Air Sampling

•Constant flow Sampling Pumps

•Tenax® Sorbent tubes

•Thermal desorption GC/MS

Plant Sampling

•Leaves collected by gloved hand

•Headspace GC/MS

Flow Through Chamber Diagram

Flow Through Chamber (Ficus)

0

0.2

0.4

0.6

0.8

1

1.2

0 50 100 150 200 250 300 350 400

Co

nce

ntr

atio

n R

atio

Time (min)

PCE

Blank Chamber

Soil and Pot

Ficus

0

0.2

0.4

0.6

0.8

1

1.2

0 50 100 150 200 250 300 350 400

Co

nce

ntr

atio

n R

atio

Time (min)

m-Xylene

Blank Chamber

Soil and Pot

Ficus

Question: Could house plants be used to

monitor indoor air concentrations of

VOCs?

Answer: Probably for initial screening and

monitoring trends over time.

OCSPP 850.4800: Plant Uptake

and Translocation Test

• US EPA Office of Chemical Safety and

Pollution Prevention

• Primary endpoints:

– Concentrations of free parent compound,

metabolites, soluble residues, and bound

residues in pooled plant organs and whole plants

• Laboratory

– Hydroponic, soil

• Field

Table 2. Summary of Test Conditions for Plant Uptake and Translocation Test

Test duration To provide sufficient biomass (or to allow gas exchange measurements), or until fruit or seeds are mature.

Substrate Quartz sand or glass beads. Hydroponic system may also be used. (For pesticides, natural or synthetic soil may also be used.)

Nutrients Watered with nutrient solution (half-strength modified Hoagland’s medium)

Temperature 25/20 C (daytime/nighttime) ± 3 C (applicable to growth chambers)

Relative humidity 70/90% (daytime/nighttime) ± 5% (applicable to growth chambers)

Carbon dioxide 350 ± 50 μmol/m2/sec (applicable to growth chambers)

Light quality Fluorescent or representative of natural sunlight

Light intensity 350 ± 50 μmol/m2/sec

Photoperiod 16 hours light: 8 hours dark for all species except soybean with 11 hours light: 13 hours dark prior to flowering

Watering Bottom watering as needed, using nutrient solution

Test chamber (pot) size Varies with plant species selected.

Number of organisms per test chamber

Typically, 1 - 4 seedlings of one species per pot

Number of replicate chambers per test treatment

6 (minimum)

Number of organisms per test treatment

6 - 24 (minimum)

Test treatment levels Minimum of 3 treatment levels plus appropriate controls

13

Minimum acceptable criteria?

Chemical (name, CAS #); plant species identified;

Units explicitly defined;

Appropriate chemical analysis: exposure medium & plant;

Chemical conc. in exposure media (beginning, during, end);

No apparent toxicity to the plant;

Amount of water transpired; plant mass, growth rate;

Reasonable growth conditions (i.e. light and nutrients);

Exposure medium properties (e.g. organic carbon content

in the soil, pH for ionogenic organics);

Composition of plant compartments (i.e. lipid & water %);

Appropriate controls;

USE “PROBE OR STANDARD” COMPOUNDS?

Summary

• Easier to control exposure and other

variables in laboratory

• Difficult to simulate field conditions in lab

– Light, humidity, duration of experiment,

changes over time

– Anticipate impact of key variables

– Maximize information collected

– Information not considered study focus may

be critical in modeling/understanding

mechanism