Background Phytoremediation potential of the novel Atrazine, 2 - - PDF document

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Phytoremediation potential of the novel atrazine tolerant Lolium multiflorum and studies on the mechanisms involved

Merini et al 2009 Merini et al., 2009

Presented by: Ellen Sherck & Rhiannon Fox

Background

• Atrazine, 2‐chloro‐4‐

(ethylamino)‐6‐ (isopropylamino)‐s‐ triazine (ATZ)

• Cyanuric chloride treated

Cyanuric chloride treated with ethylamine and isopropyl amine

• Inhibits photosynthesis

by binding to the plastoquinone‐binding protein in Photosystem II

Applications and Extent

• 76 million pounds

applied annually in U.S.

• 2nd‐largest selling

pesticide worldwide

• Over 6,000 tons applied

annually in Argentinian Pampa region

• Other uses:

landscaping, forestry, golf courses, etc.

Environmental and Health Risks

• Most common

herbicide or pesticide contaminant of ground and surface water

• Effects on wildlife

– Contributes to amphibian declines (Rohr et al., 2004) E d i di t i

(Capel & Larson, 2001)

• Highly mobile and can

travel in precipitation (Thurman & Cromwell, 2000)

• Half life up to 120 days

– Endocrine disruptor in fish and amphibians – Neural damage and pregnancy loss in rodents

• Effects on humans

– Fertility loss in men (Swan et al., 2003)

Other Remediation Strategies

• Bioaugmentation ‐ inoculating soils with a

microorganism that can catabolize ATZ

• Addition of killed and stabilized suspensions of

Escherichia coli cells engineered to Escherichia coli cells engineered to

• verexpress the enzyme atrazine

chlorohyrolase (Strong et al. 2000)

• All are effective, but expensive and often slow

Phytoremediation!

• Challenges

– Atrazine inhibits photosynthesis – Remediating plant cannot interfere with main crop

• Objectives:
• Objectives:

– Assess the tolerance of annual ryegrass (Lolium multiflorum) to agronomic doses of atrazine – Examine the mechanisms involved in Atrazine tolerance – Evaluate its potential for phytoremediation

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Lolium mutiflorum

• Annual ryegrass that

grows in Argentina

• Already used for

intercropping with corn (Carruthers et al., 2000)

• One of the best adapted

ryegrass species

• Ability to acquire

tolerance to herbicides

Atrazine Tolerance in Plants

• Genetic mechanisms

– Point mutation in psbA gene  changes in D1 protein of PSII  A to G transition confers resistance

• Enzymatic mechanisms

– The cytochrome P450 detoxification pathway  detoxes and eliminates endogen metabolites & xenobiotics  commonly confers Atrazine resistance

• Non‐enzymatic mechanisms

– Benzoxazinone hydroxylation of atrazine confers Atrazine resistance in some plants

Methods – Tolerance Assay

• Seeds were sterilized and germinated in semisolid

sterile media amended with a 2,000 mg / kg atrazine solution

• Atrazine concentration ranged from 1 mg / kg (equal

to 2x the agronomic application rate) to 50 mg / kg to 2x the agronomic application rate) to 50 mg / kg (heavy industrial contamination) with two intermediate concentrations

• Triplicates of each seed type sown and incubated in a

culture chamber

• Growth of plants were recorded regularly –

harvested at 21 days

Results: Atrazine Tolerance

• Able to grow and

germinate in presence

• f 1 mg kg⁻¹ of Atrazine

(more than 2X heavy field application)

• Able to germinate

without external source

• f carbon making it a

good candidate for phytoremediation

Methods – Phytoremediation Assay

• Microcosms of clean (i.e. no atrazine) soils placed in

growing chambers. – Control = no seeds – Experimental = 10 seeds each

• At 3rd leaf stage, atrazine added  concentration of

1 mg / kg of soil

• Triplicates sampled at 0, 7, 14, and 21 days after

herbicide application

• Plants harvested, tested for chlorophyll a and b

content and enzymatic activity

• Soil metabolites tested by HPLC

Results: Phytoremediation

• After 21 days atrazine

degradation in soil microcosms was 20% higher with L. multiflorum plants

• During first week

During first week degradation rate was 2.5X higher than natural soil attenuation capacity

• Higher degradation rates

than studies using other plants (Singh et al., 2004)

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Methods ‐ Genetics

• PCR DNA extraction performed on aerial parts
• f the plants
• psbA gene had not been previously sequenced

in L multiflorum in L. multiflorum

• Used taxonomically similar species to design

primers for amplification of psbA gene in L. multiflorum

• PCR fragments were purified and sequenced

Results: Genetic Mechanisms

• Sequencing of psbA gene

– Mutation in psbA encoding the substitution of amino acids signifies resistance to photosynthesis inhibitors herbicides – No substitution in amino acid serine in D1 protein

• If replaced by glycine confers resistance to triazinic

herbicides

– In the other 16 amino acids there was only one substitution of tyrosine by threonine

• herbicide tolerance is under investigation

Methods – Enzymatic Mechanisms

• L. multiflorum response to atrazine + 1‐

aminobenzotriazole (ABT)

• Sterilized seeds sown in nutrient solution.
• At day 14, 4 treatments: control, ABT, atrazine,

and atrazine + ABT

• After 7 days of treatment, solutions replaced

by fresh nutrient solution

• After another 21 days, survival index and

shoot length were recorded for each treatment

Results: Enzymatic Mechanisms

• Atrazine and 1‐aminobenzotriazole (ABT)

– What was the significance of cytocrome P₄₅₀ to atrazine resistance in L. multiflorum?

Results: Enzymatic Mechanisms

• No significant

difference in plants with exposure to ABT

• r ATZ compared

to control

• Significant

difference between ATZ and ATZ + ABT

Methods – Non‐Enzymatic Mechanisms

• Benzoxazinones (Bx) were extracted from

seedlings (roots and shoots)

• Extract was dissolved in a buffer containing 1

mg / L atrazine mg / L atrazine

• Control solution without extract was

processed the same way

• Samples collected at 0, 30 min, 1, 6, and 24 h

and analyzed by HPLC to determine atrazine degradation and presence of metabolites

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Results: Non‐enzymatic mechanisms

• Benzoxainones (Bx) extraction and analysis

– After plant material extraction analysis of Bx was performed – HPLC analysis on the samples from atrazine HPLC analysis on the samples from atrazine hydrolysis tests showed no difference with respect to control after 24hrs of incubation at 120 r.p.m. and 25° C – Bx presence in plant tissues does not necessarily signify Atrazine tolerance

Conclusions

• Atrazine tolerance in L. multiflorum is

conferred by the P₄₅₀ enzymatic capacity to

• xidatively deactivate the herbicide (increased

metabolism via P₄₅₀) metabolism via P₄₅₀)

• Benzoxazinones were found in L. multiflorum

but a correlation between these alleochemicals and atrazine tolerance was not

• bserved

Conclusions

• L. multiflorum is a promising candidate for

field scale phytoremediation!

– Ability to germinate & grow in above agronomic doses of Atrazine doses of Atrazine – Elevated herbicide removal capacity – High initial degradation rate (Important for avoiding Atrazine run‐off) – Already proven agronomic benefits from intercropping

References

• Ackerman, F., 2007. The economics of Atrazine. International Journal of

Occupational and Environmental Health, 13:441‐449

• Capel, P. and S. Larson, 2001. Effect of scale on the behavior of atrazine in surface
• waters. Environ. Sci. Tech., 35(4): 648‐657.
• Carruthers, K., Prithiviraj, B., Fe, Q., Cloutier, D., Martin, R.C., Smith, D.L., 2000.

Intercropping corn with soybean, lupin and forages: yield component responses. European Journal of Agronomy 12, 103–115

• Rohr J R A A Elskus B S Shepherd P H Crowley T M McCarthy J H
• Rohr, J. R., A. A. Elskus, B. S. Shepherd, P. H. Crowley, T. M. McCarthy, J. H.

Niedzwiecki, T. Sager, A. Sih and B. D. Palmer. Multiple Stressors and Salamanders: Effects of an herbicide, food limitation, and hydroperiod. Ecol. Appl. 14*4)

• Strong, L. C., H. McTavish, M. J. Sadowsky, and L. P. Wackett. 2000. Field‐scale

remediation of atrazine‐contaminated soil using recombinant Escherichia coli expressing atrazine chlorohydrolase. Environmental Microbiology 2:91‐98.

• Thurman, E. and A. Cromwell, 2000. Atmospheric transport, deposition, and fate of

triazine herbicides and their metabolites in pristine areas at Isle Royale National

• Park. Environ. Sci. Tech., 34: 3079‐3085.
• USDA Forest Service Species Index. Available:

http://www.fs.fed.us/database/feis/plants/graminoid/lolmul/all.html Accessed: 01/22/2012