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Permit application 11-052-101rm 

received from ArborGen

Field testing of genetically engineered 

Eucalyptus grandis X Eucalyptus 

urophylla

Final Environmental Assessment

April 2012

Biotechnology Regulatory Services 

Agency Contact

Cynthia Eck, Document Control Officer 

USDA, APHIS, BRS 

4700 River Road, Unit 147 

Riverdale, MD 20737-1237 

Phone: (301) 734-0667

Fax: (301) 734-8669 

Cynthia.A.Eck@aphis.usda.gov 

____________________________________

The U.S. Department of Agriculture (USDA) prohibits 

discrimination in all its programs and activities on the basis of race, 

color, national origin, sex, religion, age, disability, political beliefs, 

sexual orientation, or marital or family status. (Not all prohibited 

bases apply to all programs.) Persons with disabilities who require 

alternative means for communication of program information 

(Braille, large print, audiotape, etc.) should contact USDA’S 

TARGET Center at (202) 720–2600 (voice and TDD).

To file a complaint of discrimination, write USDA, Director, Office of 

Civil Rights, Room 326–W, Whitten Building, 1400 Independence 

United States

Department of 

Agriculture

Marketing and 

Regulatory 

Programs

Animal and 

Plant Health 

Inspection 

Service
Page 2 of 91

Avenue, SW, Washington, DC 20250–9410 or call (202) 720–5964 

(voice and TDD). USDA is an equal opportunity provider and 

employer.

____________________________________

Mention of companies or commercial products in this report does not 

imply recommendation or endorsement by the U.S. Department of 

Agriculture over others not mentioned. USDA neither guarantees 

nor warrants the standard of any product mentioned. Product names 

are mentioned solely to report factually on available data and to 

provide specific information.

____________________________________

This publication reports research involving pesticides. All uses of 

pesticides must be registered by appropriate State and/or Federal 

agencies before they can be recommended. 

____________________________________

CAUTION: Pesticides can be injurious to humans, domestic animals, 

desirable plants, and fish or other wildlife—if they are not handled or applied 

properly. Use all pesticides selectively and carefully. Follow recommended

practices for the disposal of surplus pesticides and pesticide containers.
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TABLE OF CONTENTS

I. SUMMARY................................................................................................................................... 5

II. PURPOSE AND NEED.............................................................................................................. 6

A. PROPOSED ACTION................................................................................................................... 6

B. PURPOSE OF THIS ENVIRONMENTAL ASSESSMENT................................................................ 7

C. NEED FOR THIS ACTION........................................................................................................... 8

D. PURPOSE AND DESCRIPTION OF THE RESEARCH ................................................................... 8

III. AFFECTED ENVIRONMENT ................................................................................................ 8

IV. ALTERNATIVES.................................................................................................................... 13

A. NO ACTION – DENY THE PERMIT .......................................................................................... 14

B. PREFERRED ALTERNATIVE – ISSUE THE APHIS PERMIT.................................................... 15

V. ENVIRONMENTAL CONSEQUENCES.............................................................................. 15

A. NO ACTION.............................................................................................................................. 15

B. PREFERRED ALTERNATIVE.................................................................................................... 16

C. THE DEGREE TO WHICH THE POSSIBLE EFFECTS ARE HIGHLY UNCERTAIN OR INVOLVE 

UNIQUE OR UNKNOWN RISKS ........................................................................................ 32

D. CUMULATIVE EFFECTS .......................................................................................................... 35

E. RISKS TO THREATENED AND ENDANGERED SPECIES .......................................................... 37

F. IMPACTS ON UNIQUE CHARACTERISTICS OF PROTECTED AREAS ...................................... 37

G. OTHER CONSIDERATIONS...................................................................................................... 37

H. CONCLUSION........................................................................................................................... 39

VI. LISTING OF AGENCIES AND PERSONS CONSULTED............................................... 39

VII. REFERENCES....................................................................................................................... 41

APPENDIX I: STATUS OF EXISTING FIELD TESTS ALLOWED TO FLOWER ............ 47

APPENDIX II: HYDROLOGY CONSIDERATIONS FOR PLANTED 

EUCALYPTUS SUBMITTED BY ARBORGEN LLC.................................................... 49

APPENDIX III: USDA FOREST SERVICE ASSESSMENT OF IMPACTS ON 

HYDROLOGY.................................................................................................................... 60

APPENDIX IV: THREATENED AND ENDANGERED SPECIES ANALYSIS.................... 69
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APPENDIX V: PROXIMITY OF RELEASE SITES TO PROTECTED AREAS.................. 82

APPENDIX VI: PROPOSED SUPPLEMENTAL PERMIT CONDITIONS FOR 

MOVEMENT...................................................................................................................... 84

APPENDIX VII: PROPOSED SUPPLEMENTAL PERMIT CONDITIONS FOR 

RELEASE............................................................................................................................ 86
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I. Summary

United States Department of Agriculture (USDA), Animal Plant Health Inspection 

Service (APHIS) has prepared an environmental assessment (EA) in response to a 

confined environmental release and movement permit application (APHIS Number 11-

052-101rm) received from ArborGen Inc. (ArborGen) to allow the interstate movement, 

planting and flowering of genetically engineered (transgenic) Eucalyptus trees at six 

confined field site locations in Alabama, Florida, Mississippi, and South Carolina. A 

total of 14.7 acres (all 6 sites combined) is being requested by the applicant. These plants 

are a clone1 coded EH1 derived from a hybrid of Eucalyptus grandis X Eucalyptus 

urophylla and have been genetically engineered with different constructs than the trees 

previously permitted for environmental release by APHIS. The purpose of the confined 

environmental release is for ArborGen to assess the effectiveness of introduced gene 

constructs which are intended to confer cold tolerance; to test the efficacy of genes to 

alter lignin biosynthesis; to test the efficacy of genes to alter growth; and to test the 

efficacy of genes designed to alter flowering. In addition the trees have been engineered 

with a selectable marker gene which confers resistance to the antibiotic kanamycin. 

ArborGen has also requested under this permit application that all genetically engineered 

Eucalyptus trees authorized under permits 09-070-101rm and 11-041-101rm be 

incorporated into this new permit and allowed to flower. Trees will also be moved 

interstate under this permit from ArborGen’s nursery and greenhouse facilities in South 

Carolina to the field test sites identified in the permit application for planting.

In 2010, APHIS completed and EA and FONSI for a permit application from ArborGen 

to authorize the planting, field testing and flowering of a GE Eucalyptus hybrid clone 

engineered to express various genes on 28 confined field site locations in the southeastern 

United States, including Alabama, Florida, Georgia, Louisiana, Mississippi, South 

Carolina, and Texas (http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf) and 

(http://www.aphis.usda.gov/brs/aphisdocs/08_014101r_ea.pdf). Pertinent and current 

information available in the 2010 EA and FONSI has been incorporated by reference into 

this EA. 

APHIS has issued ArborGen permits authorizing the environmental release of transgenic 

Eucalyptus trees, some of which allowed flowering and some of which have not. 

ArborGen has grown Eucalyptus under permits 06-325-111r, 08-011-106rm, 08-014-

101rm, 08-039-102rm, 08-151-101r, 09-070-101rm and 11-201-103r. For permits 06-

325-111r, 08-011-106rm, 08-014-101rm, 08-151-101r and 11-201-103r the trees have 

been allowed to flower. Permits 06-325-111r, 08-011-106rm, 08-014-101rm were issued 

by APHIS upon completion of an EA and subsequent FONSI 

(http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf) and 

(http://www.aphis.usda.gov/brs/aphisdocs/08_014101r_ea.pdf). Permit 06-325-111r 

expired on June 27, 2010 and was renewed under 10-112-101r. Permit 08-039-102r 

expired on April 25, 2011 and was renewed under 11-041-101rm. Permit 08-011-106rm 

is active and will expire on May 12, 2013. Permit 08-014-101rm is active and will expire

 1 Clone – as defined in horticulture and forestry means is a population of genetically identical plants that 

has been derived from one individual. Despite popular use of the word, a clone is not an individual.
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on May 12, 2013. Permit 08-151-101r expired June 30, 2011and was incorporated into 

08-014-101rm. Permit 09-070-101rm is active and will expire on May 11, 2012. Permit 

11-201-103r is active and will expire on July 28, 2014.

There are currently six active permits under which ArborGen is authorized by APHIS to 

grow GE Eucalyptus which include 32 unique locations within 7 States: 08-011-106rm, 

08-014-101rm, 09-070-101rm, 10-112-101r, 11-041-101rm and 11-201-103r. No 

plantings at any of the 32 locations are authorized by APHIS to exceed 20 acres in size. 

Trees are allowed to flower under four permits: 08-011-106rm, 08-014-101rm, 10-112-

101r and 11-201-103r (two of the locations in Berkeley and Dorchester Counties, SC

trees are not authorized to flower). Under permits 09-070-101rm and 11-041-101rm trees 

are not authorized to flower. As of September 2011, ArborGen has reported to APHIS 

that they are growing a total of approximately 67 acres of trees on 18 of the 32 permitted 

locations.

 

ArborGen has submitted a new permit application 11-052-101rm to APHIS requesting 

approval for environmental release of hybrid Eucalyptus trees at 6 locations in the States 

of Alabama, Florida, Mississippi, and South Carolina encompassing a total of 14.7 acres 

(the subject of this EA). Five of these locations currently have active APHIS permits 

(08-011-106rm, 08-014-101rm, 09-070-10rm, 10-112-101r, and 11-041-101rm) for 

environmental release of GE Eucalyptus hybrids in Alabama, Florida, Mississippi, and 

South Carolina. The sixth site in South Carolina has been listed as a holding site for 

transgenic trees in previous APHIS permits and notifications and is a new location for the 

release of GE Eucalyptus. ArborGen is requesting that trees be allowed to flower at four 

locations in Alabama, Florida and Mississippi. At the two locations in Berkeley and 

Dorchester Counties, SC, ArborGen has requested to release trees in containers and have 

indicated they will not allow these trees to flower at these locations. The size of each 

individual confined field test sites identified in ArborGen’s permit application ranges 

from 0.5 to 7.7 acres, which is less than the 20 acres analyzed in APHIS EA and 

subsequent FONSI prepared for Permits 08-011-106rm, 08-014-101rm and 10-112-101r

(http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf) and 

(http://www.aphis.usda.gov/brs/aphisdocs/08_014101r_ea.pdf). 

II. Purpose and Need

A. Proposed Action

The proposed action is for APHIS to approve ArborGen permit application request (11-

052-101rm) and issue a confined environmental release and interstate movement permit 

for a hybrid of Eucalyptus grandis X Eucalyptus urophylla with supplemental permit 

conditions (see Appendices VI and VII). The movement authorization would be valid for 

one year and the release for a three-year period. The permit will need to be renewed by 

ArborGen and subsequently approved by APHIS to allow the transgenic Eucalyptus

plants to remain in the ground beyond the three-year time period specified in the permit 

application.

APHIS would issue a confined environmental release and interstate movement permit to 

ArborGen in accordance with 7 CFR part 340 to allow the interstate movement, planting, 
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field testing and flowering of a GE Eucalyptus hybrid clone (Eucalyptus grandis x

Eucalyptus urophylla) engineered to express various genes at 6 confined field site 

locations within in the States of Alabama, Florida, Mississippi, and South Carolina 

encompassing a total of 14.7 acres. The permit would allow all the trees except the trees 

on two of the sites in South Carolina to flower. Trees would also be authorized to be 

moved interstate from ArborGen’s greenhouse and nursery facilities in Berkeley, 

Charleston and Dorchester Counties in South Carolina to the field test locations. All 

genetically engineered Eucalyptus trees authorized under permits 09-070-101rm and 11-

041-101rm would be incorporated into this new permit and allowed to flower. 

B. Purpose of this Environmental Assessment

The purpose of this EA is to assess the potential environmental impacts on the human 

environment associated with APHIS responding to a confined environmental release and 

interstate movement permit application (11-052-101rm) received from ArborGen in 

February 2011 to authorize the planting of GE Eucalyptus hybrid clone to support 

proposed field research studies on six research sites in Alabama, Florida, Mississippi, and 

South Carolina. The permit application requests APHIS to allow all the trees except the 

trees on two of the sites in South Carolina to flower. The purpose of the confined 

environmental release is for ArborGen to assess the effectiveness of introduced gene 

constructs which are intended to confer cold tolerance; to test the efficacy of genes to 

alter lignin biosynthesis; to test the efficacy of genes to alter growth; and to test the 

efficacy of genes designed to alter flowering. In addition the trees have been engineered 

with a selectable marker gene which confers resistance to the antibiotic kanamycin. 

ArborGen has also requested under this permit application that all genetically engineered 

Eucalyptus trees authorized under permits 09-070-101rm and 11-041-101rm be 

incorporated into this new permit and allowed to flower. New trees will also be moved 

interstate from ArborGen’s nursery and greenhouse facilities located in South Carolina to 

the release locations identified in the permit application where they will be planted.

This EA was conducted pursuant to: (1) The National Environmental Policy Act of 1969 

(NEPA), as amended (42 U.S.C. § 4321 et seq.), (2) regulations of the Council on 

Environmental Quality for implementing the procedural provisions of NEPA (40 CFR 

parts 1500-1508), (3) USDA regulations implementing NEPA (7 CFR part 1b), and (4) 

APHIS' NEPA Implementing Procedures (7 CFR part 372). Generally, issuances of 

permits for confined field trials of regulated articles are categorically excluded under 

APHIS NEPA Implementing Procedures. However, if APHIS determines that a confined 

field release of genetically engineered organisms may have the potential to significantly 

impact the quality of the human environment, as those terms are defined in 40 CFR 

1508.27 and 1508.14, an environmental assessment may be prepared, pursuant to 7 CFR 

§ 372.5(d) to determine if an Environmental Impact Statement is warranted. In this case, 

APHIS has prepared this EA specifically to address potential environmental impacts 

associated with new genetic constructs not previously reviewed or permitted for 

environmental release and interstate movement by APHIS and with allowing existing 

plantings of genetically engineered Eucalyptus trees permitted by APHIS to flower.
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C. Need for This Action

APHIS- Biotechnology Regulatory Services (BRS) mission is to protect America’s 

agriculture and the environment using a dynamic and science-based regulatory 

framework that allows for the safe development and use of GE organisms. APHIS’ 

regulations in 7 CFR part 340, which were promulgated pursuant to authority granted by 

the plant pest provisions of the Plant Protection Act, as amended, (7 U.S.C. 7701–7772), 

regulates the introduction (importation, interstate movement, or release into the 

environment) of certain GE organisms and products. The Plant Protection Act directs the 

USDA to facilitate imports and interstate commerce in agricultural products in ways that 

will reduce, to the extent practicable, the risk of dissemination of plant pests. Under 

APHIS regulations, the APHIS Administrator has authority to regulate any organism or 

product altered or produced through genetic engineering that the Administrator 

determines is a plant pest or has reason to believe is a plant pest. When APHIS receives 

an application for a permit for environmental release and movement, the application is 

evaluated to determine whether the environmental release and movement, with 

appropriate conditions imposed, can be carried out while preventing the dissemination 

and establishment of plant pests. The receipt of a permit application to introduce a 

genetically engineered organism requires a response from the Administrator:

Administrative action on applications. After receipt and review by 

APHIS of the application and the data submitted pursuant to paragraph 

(a) of this section, including any additional information requested by 

APHIS, a permit shall be granted or denied (7 CFR 340.4(e)).

The applicant has provided the required information associated with this request in the 

permit application. This information has been reviewed and analyzed in this EA. 

D. Purpose and Description of the Research

The purpose of the ArborGen research is to assess the efficacy of the introduced cold 

tolerance genes, genes to alter lignin biosynthesis, genes to control flowering, and genes

to alter the growth rate in Eucalyptus. According to the applicant, genetically engineered 

cold tolerant Eucalyptus would enable the production of this hardwood species for 

pulping and for biofuel applications in managed plantation forests in the southeastern 

U.S. The confined release of the trees in different areas of the southeast U.S. will allow 

the applicant to obtain data on performance of the transgenic trees and the efficacy of the 

inserted genes in a wide variety of environments. It is important that trees be planted in 

locations in Florida because this is the only location where a comparison between the 

transgenic and non-transgenic control trees can be made. In other locations the trees may 

be damaged by cold which makes a true comparison difficult to impossible.

III. Affected Environment

Biology of Eucalyptus and Status in the United States
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The genus Eucalyptus belongs to family Myrtaceae (subfamily: Leptospermoideae) 

which includes over 700 species. Eucalyptus is native to Australia with the exception of 

some species that are native to the Timor Islands (Groves 1994, Ladiges 1997). There 

are no wild relatives of Eucalyptus that occur naturally in the United States. An overview 

of the biology of Eucalyptus grandis has been published by the US Forest Service 

(Meskimen and Francis 1990). Eucalyptus has been planted as an ornamental species in 

the extreme southern United States where mild winters will allow some species to grow. 

Eucalyptus normally propagates in its native range via seeds. It does not spread 

vegetatively like other trees such as poplar or willow. In the United States it is usually 

propagated and sold commercially as rooted stem cuttings. 

There have been numerous attempts to grow Eucalyptus as a commercial forest tree in the 

southeastern United States, but due to its sensitivity to cold temperatures, these attempts 

have not met with success. It is only grown in commercial plantations in central and 

southern Florida, where it normally survives freezing temperatures which are rare and 

usually not severe. Eucalyptus is adapted to live in the mild arid and semi-arid climate of 

Australia. Severe freezing events that can occur in the southern United States have 

limited its establishment as a commercial forest tree. There are plantations of Eucalyptus 

grandis and E. amplifolia currently grown in south central Florida as short rotation 

energy crops and for mulch production (Stricker et al. 2000, Rockwood et al. 2004). 

These trees are generally planted in areas where severe freezing events are rare.

The species hybrid E. grandis x E. urophylla (also known as E. urograndis) that 

ArborGen wishes to allow to mature and flower under this permit has not been 

categorized as invasive. The E. grandis x E. urophylla hybrid has been grown for forty 

years in South America and during this time there has been no evidence of invasiveness 

by into natural forest areas which are growing as part of an integrated land management 

system (Luis Silva, International Paper Company, Brazil, comment to docket APHIS2008-0059). The University of Florida Institute of Food and Agricultural Sciences 

(IFAS) recently completed a review of the potential invasiveness of E. urograndis and 

found that it is not likely to be invasive and can be a recommended species for planting: 

http://plants.ifas.ufl.edu/assessment/pdfs/wra/Eucalyptus%20urograndis_WRA.pdf

See also the chart at: 

http://plants.ifas.ufl.edu/assessment/pdfs/concl_genus_Feb2011.pdf. Because it is not 

predicted to be invasive, no specific management practices are recommended for this 

species, unlike other species of Eucalyptus grown in Florida (see: 

http://plants.ifas.ufl.edu/assessment/pdfs/results_feb2011.pdf.

Numerous species of Eucalyptus were introduced into California during that State’s early 

history (see Santos: http://wwwlibrary.csustan.edu/bsantos/euctoc.htm), and some of 

these species have become established. Two of these, E. globulus (Tasmanian blue gum) 

and E. camaldulensis (Red gum) are now categorized as invasive by the California 

Invasive Plant Council (http://www.cal-ipc.org/ip/inventory/weedlist.php). Neither of 

these species is being proposed to be planted at the permitted field site.

Traits Engineered into Eucalyptus
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ArborGen LLC wishes to field test genetically engineered (transgenic) Eucalyptus trees 

during which time the trees may flower. These plants are a clone coded EH1 derived 

from a hybrid of Eucalyptus grandis X Eucalyptus urophylla. These have been 

genetically engineered with different constructs. The purpose of the field trials is to test 

the effectiveness of different genes to confer cold tolerance, alter lignin biosynthesis, or 

alter growth rate along with testing the efficacy of the Barnase gene designed to alter 

fertility. In addition the trees have been engineered with a common selectable marker 

gene (nptII) which confers resistance to the antibiotic kanamycin. With the exception of 

the C-Repeat Binding Factor (CBF) gene Barnase gene, and nptII gene, all genes are 

claimed as Confidential Business Information (CBI). 

Confined Field Test Locations

The confined field tests would take place on land controlled by ArborGen or through 

private contracts established by ArborGen for field testing. The exact locations are 

claimed as CBI and have been submitted as part of the APHIS permit application. Under 

the permit application submitted by ArborGen, there are six research sites where trees 

would be planted. A total of 14.7 acres (all 6 sites combined) is being requested by the 

applicant. Five of these locations currently have active APHIS permits (08-011-106rm, 

08-014-101rm, 09-070-10rm, 10-112-101r, and 11-041-101rm) for environmental release 

of GE Eucalyptus hybrid clone (Eucalyptus grandis x Eucalyptus urophylla) in Alabama, 

Florida, Mississippi, and South Carolina. The remaining site in Dorchester County, 

South Carolina has been listed in previous APHIS permits/notifications as a holding area 

for transgenic trees. ArborGen is requesting that trees be allowed to flower at four 

locations in Alabama, Florida and Mississippi. At the two locations in Berkeley and 

Dorchester Counties, SC, they have requested to release trees in containers and have 

indicated they will not allow these trees to flower at these locations. The size of each 

individual confined field test sites identified in ArborGen’s permit application ranges 

from 0.5 to 7.7 acres, which is less than the 20 acres analyzed in APHIS EA and 

subsequent FONSI prepared for Permits 08-011-106rm, 08-014-101rm and 10-112-101r 

(http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf) and 

(http://www.aphis.usda.gov/brs/aphisdocs/08_014101r_ea.pdf). For the five research 

sites that are currently authorized by APHIS to plant GE Eucalyptus hybrid clone 

(Eucalyptus grandis x Eucalyptus urophylla), ArborGen has indicated in their permit 

application that any new planting authorized by APHIS under 11-052-101rm would not

expand the current field site locations beyond the areas currently authorized by APHIS. 

See Table 1 which shows the proposed field test locations and acreage.

All the confined field test sites listed in the permit application by ArborGen are either on 

privately owned managed plantation forests and agricultural farm lands or experimental 

research stations managed by academic institutions and industry, and have been in 

managed agricultural production for more than 5 years. The standard agricultural and 

silvicultural practices for land preparation, planting, irrigation, and harvesting of plants 

have been routinely used on these sites. Sites that include managed pastures have had 

intense activity including the use of heavy machinery for general upkeep, irrigation, 

fertilization, controlled grazing and management of grasses. Standard silvicultural

practices would be used at these sites for the duration of the field tests. Surveys 
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conducted by the applicant at each of these locations indicate that there are not any old 

growth forests or undisturbed natural areas in the immediate surroundings of the test 

sites. ArborGen has indicated that the trees would be planted on individual research sites 

ranging from 0.5 up to 7.7 acres, depending on the location with planting density ranging 

from 300 - 600 trees per acre2

. An acre is about the size of a football field.

Table 1. Environmental release locations requested under permit application 11-052-

101rm including existing Eucalyptus permits at each location.

Location Existing Permit(s) Flowering allowed Acreage requested 

under the new 

permit

Baldwin County 

Alabama

10-112-101r Yes 2.0

Escambia County 

Alabama

08-011-106rm Yes 2.0

Pearl River County 

Mississippi

08-011-106rm Yes 2.0

Berkeley County 

South Carolina*

08-011-106rm

08-014-101rm

09-070-10rm

11-041-101rm

No 0.5

Dorchester County 

South Carolina*

New location for 

Eucalyptus

No 0.5

Highlands County 

Florida

08-014-101rm

09-070-10rm

11-041-101rm

Yes

No

No

7.7

Total 14.7

*Both locations in South Carolina are fenced areas for holding plants in containers prior 

to planting where no flowering is allowed. 

Baldwin County Alabama Site:

This location has been an agricultural research station for more than 20 years. The 

location has been used for managed production of annual agricultural crops and forest 

trees. Approximately 3.55 acres of field trials of genetically modified Eucalyptus trees of 

some lines in the permit are being grown under APHIS issued permits 08-011-106rm 

(0.75 acres) and 10-112-101r (2.8 acres). These trees are authorized by APHIS to flower. 

The oldest of these trees were planted in August 2006. Similar to the current permit

authorized by APHIS, site preparation would involve herbicide application, subsoiling, 

and planting of trees in flat beds. The surrounding areas of the test site consist of field 

plantings of agricultural crops, experimental forest trees and an abandoned pecan 

 2 Planting density typically refers to the number of trees per acre. Planting densities can vary greatly 

depending upon the tree species and the environment, but densities of short rotation hardwood trees in the 

southeastern US are typically in the range of 300–800 trees per acre. Therefore sites ranging from 10 to 20 

acres can have from 3000 to 16,000 total trees planted in the ground. Twenty acres, as defined by forest 

plantation standards in the southeast, is considered a small planting.
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orchard. Under the new permit application, APHIS could authorize up to 2.0 acres of 

field tests to be established and flower at this location (at around 300 - 600 trees per acre) 

over the next three years and would not expand the current field site location beyond the 

area currently authorized by APHIS.

Escambia County Alabama Site:

This location had previously been used as an intensely managed pasture for more than 5

years. The test site is currently planted with grasses suitable for cattle grazing. 

Approximately 0.5 acres of field trials of genetically modified Eucalyptus of some lines 

identified in the permit authorized by APHIS are being grown under permit 08-011-

106rm. These trees are authorized by APHIS to flower. The oldest of these trees were 

planted in July 2007. Similar to the current permit authorized by APHIS, site preparation

would involve herbicide application to remove existing grasses, subsoiling, preparation 

for possible irrigation, and planting of the test trees in flat beds. The surrounding areas of 

the test site consist of greater than 30 years-old slash pine, and a re-forested area with 

mixed stands of pine and hardwood species. Under the new permit application, APHIS 

could authorize up to 2.0 acres of field tests to be established and flower at this location

(at around 300 - 600 trees per acre) over the next three years and would not expand the 

current field site location beyond the area currently authorized by APHIS.

Pearl River County Mississippi Site:

This location has been an agricultural research station for more than 5 years. The location 

has been used for conducting research experiments with agricultural crops and grasses. 

The test site has been used for experimental planting of grasses. Approximately 3 acres of 

field trials of genetically modified Eucalyptus trees of some lines identified in the permit 

authorized by APHIS are being grown under permit 08-011-106rm. These trees are 

authorized by APHIS to flower. The oldest of these trees were planted in October 2007. 

Similar to the current permit authorized by APHIS, site preparation would involve 

herbicide application to remove existing grasses, subsoiling, preparation for possible 

irrigation installation, and planting of trees in flat beds. The surrounding areas of the test 

site consist of a grape research farm, mixed stands of hardwoods and pine, and a

residential area. Under the new permit application, APHIS could authorize up to 2.0 acres 

of field tests to be established and flower at this location (at around 300 - 600 trees per 

acre) over the next three years and would not expand the current field site location 

beyond the area currently authorized by APHIS..

Berkeley County South Carolina Site:

This is an extension of a greenhouse facility that has been used for acclimatization of 

transgenic and non-transgenic Eucalyptus tree plants for more than 8 years. Genetically

modified Eucalyptus trees are being grown in containers under APHIS permits 08-011-

106rm, 08-014-101rm, 09-070-10rm and 11-041-101rm. These trees are not authorized 

by APHIS to flower. Similar to the current permit authorized by APHIS, the proposed 

0.5 acre release site is located adjacent to greenhouse facilities and is surrounded by 

hardwoods and pine plantations and would not expand the current field site location 
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beyond the area currently authorized by APHIS. This site is a secure fenced holding area 

where trees growing in containers are transferred from the greenhouse to the out-of-doors

for acclimatization prior to field planting. Trees will not be allowed to flower at this 

location. 

Dorchester County South Carolina Site:

This location is an extension of a greenhouse facility and was previously cleared in 2010 

for development as a light industrial park, prior to which it had been a managed pine 

plantation for more than 15 years. The surrounding area consists of man-made ponds, 

hardwoods and pine plantations. This site is a secure fenced holding area where trees 

growing in containers are transferred from the greenhouse to the out-of-doors for 

acclimatization prior to field planting. Trees will not be allowed to flower at this 

location. This is a new location that was not assessed in the previous EA. However, this 

site has previously been listed as a holding area for trees in containers in previous APHIS 

permits and notifications for other transgenic tree species. 

Highlands County Florida Site:

This location has been used for field trials of transgenic Eucalyptus trees for at least 5 

years. Approximately 5.2 acres of field trials of genetically modified Eucalyptus trees are 

being grown under APHIS permits 08-014-101rm, 09-070-101rm and 11-041-101rm at 

this location. Trees that are being grown under permit 08-014-101rm are authorized by 

APHIS to flower. The oldest of these trees were planted in July 2006. Similar to the 

current permit authorized by APHIS, site preparation would involve herbicide 

application, plowing, and planting of trees in flat beds. The test site is surrounded by a 

citrus production area and second-growth pine and hardwood forests. Under the new 

permit application, APHIS could authorize up to 7.7 acres of field tests to be established 

and flower at this location (at around 300 - 600 trees per acre) over the next three years

and would not expand the current field site location beyond the area currently authorized 

by APHIS. ArborGen has also requested under this permit application that all genetically 

engineered Eucalyptus trees authorized under permits 09-070-101rm and 11-041-101rm

at this site location to be incorporated into this new permit and allowed to flower.

Interstate Movement

As requested in the permit application, APHIS could authorize the movement of 

genetically engineered Eucalyptus trees from nursery and greenhouse sites in Berkeley, 

Charleston, and Dorchester Counties in South Carolina to the four field release locations 

identified in the permit application that would allow flowering. As specified in the 

permit and supplemental permit conditions (see Appendix VI), labeled container-grown 

trees would be placed in an enclosed vehicle (trailer or a van) and transported to the 

destination test sites.

IV. Alternatives
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This EA analyzes the potential environmental consequences of APHIS’ response to a 

confined environmental release and interstate movement permit application (APHIS 

Number 11-052-101rm) received from ArborGen to allow the movement to, planting and 

flowering of genetically engineered Eucalyptus trees at six confined field site locations in 

Alabama, Florida, Mississippi, and South Carolina. A total of 14.7 acres (all 6 sites 

combined) is being requested by the applicant. These plants are a clone3 coded EH1 

derived from a hybrid of Eucalyptus grandis X Eucalyptus urophylla and have been 

genetically engineered with different constructs than the trees previously permitted for 

environmental release by APHIS. The purpose of the confined environmental release is 

for ArborGen to assess the effectiveness of introduced gene constructs which are 

intended to confer cold tolerance; to test the efficacy of genes to alter lignin biosynthesis; 

to test the efficacy of genes to alter growth; and to test the efficacy of genes designed to 

alter flowering. In addition the trees have been engineered with a selectable marker gene

which confers resistance to the antibiotic kanamycin. ArborGen has also requested under 

this permit application that all genetically engineered Eucalyptus trees authorized under 

permits 09-070-101rm and 11-041-101rm be incorporated into this new permit and 

allowed to flower.

Under APHIS regulations, the Administrator must either grant or deny permits properly 

submitted under 7 CFR part 340. Based upon the permit applications submitted by 

ArborGen, two alternatives are considered and analyzed in this EA: (1) deny the permit 

and (2) approve ArborGen permit application request and issue the APHIS permit. 

A. No Action – Deny the Permit

Under the No Action alternative APHIS would deny the permit application (11-052-

101rm) submitted by ArborGen. ArborGen would not be authorized to move and plant 

the GE Eucalyptus trees with new constructs and allow them to reach maturity and 

flower. Confined field release under permits 09-070-101rm and 11-041-101rm would

continue to not allow flowering. 

There are currently six active permits under which ArborGen is authorized by APHIS to 

grow GE Eucalyptus which include 32 unique locations within 7 States: 08-011-106rm, 

08-014-101rm, 09-070-101rm, 10-112-101r, 11-041-101rm and 11-201-103r. No 

plantings at any of the 32 locations are authorized by APHIS to exceed 20 acres in size. 

Trees are allowed to flower under four permits: 08-011-106rm, 08-014-101rm, 10-112-

101r and 11-201-103r (two of the locations in Berkeley and Dorchester Counties, SC 

trees are not authorized to flower). Under permits 09-070-101rm and 11-041-101rm trees 

are not authorized to flower. As of September 2011, ArborGen has reported to APHIS 

that they are growing a total of approximately 67 acres of trees on 18 of the 32 permitted 

locations. Under the No Action alternative, ArborGen could continue to grow GE 

Eucalyptus as authorized by APHIS under these six active permits. 

 3 Clone – as defined in horticulture and forestry means is a population of genetically identical plants that 

has been derived from one individual. Despite popular use of the word, a clone is not an individual.
Page 15 of 91

B. Preferred Alternative – Issue the APHIS Permit

The Preferred Alternative is to approve ArborGen permit application request (11-052-

101rm) and issue the APHIS confined field release and movement permit for a hybrid of 

Eucalyptus grandis X Eucalyptus urophylla with supplemental permit conditions (see 

Appendices VI and VII). The movement authorization would be valid for one year and 

the release would be valid for a three-year period. The permit will need to be renewed by 

ArborGen and subsequently approved by APHIS to allow the transgenic Eucalyptus

plants to remain in the ground beyond the three-year time period specified in the permit 

application. 

Under this alternative, APHIS would issue a confined environmental release permit to 

ArborGen in accordance with 7 CFR part 340 to allow the movement, planting, field 

testing and flowering of a GE Eucalyptus hybrid clone engineered to express various 

genes at 6 confined field site locations within in the States of Alabama, Florida, 

Mississippi, and South Carolina encompassing a total of 14.7 acres. The permit would 

allow all the trees except the trees on two of the sites in South Carolina to flower. All 

genetically engineered Eucalyptus trees authorized under permits 09-070-101rm and 11-

041-101rm would be incorporated into this new permit and allowed to flower. This 

permit would authorize ArborGen to move and plant the GE Eucalyptus trees with new 

constructs and allow them to reach maturity and flower. Under this alternative, the 

applicant would be allowed to gather data on performance of the transgenic trees over a 

multiyear period and the efficacy of the genes in a wide variety of environments for 

multiple years. This alternative would allow the safe development and use of GE 

organisms under the mission of BRS.

V. Environmental Consequences

A. No Action

Under the No Action alternative APHIS would deny the permit application (11-052-

101rm) submitted by ArborGen. ArborGen would not be authorized to move, or plant the 

GE Eucalyptus trees with new constructs identified in their permit application and allow 

them to reach maturity and flower. GE Eucalyptus trees would continue to be planted 

and grown as authorized by APHIS permits (08-011-106rm, 08-014-101rm, 09-070-

10rm, 10-112-101r, and 11-041-101rm) in the southeastern United States including the 5 

environmental release locations in Alabama, Florida, Mississippi, and South Carolina that 

are identified in ArborGen’s permit request. Confined field release under permits 09-

070-101rm and 11-041-101rm would continue to not allow flowering. 

There would be no environmental impacts associated with the new constructs identified 

in the permit application since the trees containing these new constructs would not be 

authorized by APHIS for interstate movement or environmental release. Under this 

alternative, the applicant would not be allowed to gather data on performance of the 

transgenic trees over a multiyear period and the efficacy of the genes in a wide variety of 

environments for multiple years. 
Page 16 of 91

Potential impacts associated with Permits 08-011-106rm, 08-014-101rm and 10-112-101r 

would not change from those analyzed in the 2010 EA and FONSI prepared for these 

permits (http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf) and 

(http://www.aphis.usda.gov/brs/aphisdocs/08_014101r_ea.pdf). As specified in the 

permit conditions for Permits 09-070-101rm and 11-041-101rm, trees at these sites are 

not authorized to flower and therefore would be required to be cut down prior to maturity. 

Preventing flower formation would prove impossible over time because the trees grow

too tall and too many flowers are produced to remove manually. Subsequently, these site 

locations could be replanted in short rotation GE Eucalyptus field tests if approved by 

APHIS, or the sites could be returned to other forest tree production, agriculture/forestry 

research, pasture, or other agricultural activities. Intense management activity including 

the use of heavy machinery for land preparation, general upkeep, irrigation, and 

fertilization for the management of tree plantings and grasses and standard silvicultural 

practices would continue to be used at these sites.

B. Preferred Alternative

Under this alternative, APHIS would issue a confined environmental release permit to 

ArborGen in accordance with 7 CFR part 340 to allow the movement, planting, field 

testing and flowering of a GE Eucalyptus hybrid clone engineered to express various 

genes at 6 confined field site locations within in the States of Alabama, Florida, 

Mississippi, and South Carolina encompassing a total of 14.7 acres with supplemental 

permit conditions (see Appendices VI and VII) for the requested three-year period. The 

permit would allow all the trees except the trees on two of the sites in South Carolina to 

flower. All genetically engineered Eucalyptus trees authorized under permits 09-070-

101rm and 11-041-101rm would be incorporated into this new permit and allowed to 

flower. 

Five of these six locations currently have active APHIS permits (08-011-106rm, 08-014-

101rm, 09-070-10rm, 10-112-101r, and 11-041-101rm) for environmental release of GE 

Eucalyptus hybrids in Alabama, Florida, Mississippi, and South Carolina.. The 

remaining site in Dorchester County, South Carolina has been listed in previous APHIS 

permits/notifications as a holding area for GE trees. In respect to the five locations that 

currently have active APHIS permits; the new permit application (11-052-101rm) does 

not expand the field sites beyond what is current authorized by APHIS or density 

(number) of trees that could be planted. The holding area in Dorchester County will 

remain as a holding area for trees. Adding Eucalyptus trees to the site will not change 

any existing conditions. As identified in the new permit application, GE Eucalyptus trees 

with the new constructs would be released at the 5 existing locations in addition to the 

trees currently approved by APHIS for environmental release. The new permit 

application does not expand the number of trees or total acreage at any of the 5 confined 

release sites currently approved by APHIS. Similar to current permits, if approved by 

APHIS, the trees would remain in the ground for at least 3 years and most likely until 

maturity because the applicant has indicated in their permit application that they are 

planning to renew the permits prior to expiration. If the renewal permits are approved by 
Page 17 of 91

APHIS, the trees could be allowed to stay in the ground until maturity or when normally 

harvested (age 7-9). The standard silvicultural practices for land preparation, planting, 

irrigation, and harvesting of trees would continue to be routinely used on these sites. 

Potential Environmental Impact of the Preferred Alternative

Interstate Movement of the trees under permit

Under this Alternative, APHIS could authorize the interstate movement of transgenic 

Eucalyptus trees in accordance with the regulatory requirements identified in 7 CFR 

340.4, 340.7 and 340.8, including specific permit conditions (see Appendix VI) assigned 

to this permit that would prevent the dissemination of the trees into the environment. As 

specified in the supplemental permit conditions, ArborGen would be moving trees in 

accordance with an APHIS approved variance that only allows trees to be moved in 

labeled containers within an enclosed vehicle. For the time period between 2005 to 2010, 

APHIS issued218 permits and notifications that authorized the interstate movement of 

forest trees. Of these 218 authorized movements, there was one compliance incident 

associated with a shipment of plants via mail. This compliance incident was associated 

with a movement that did not have similar APHIS approved variances as those that would 

be required for ArborGen to move GE Eucalyptus trees under this permit request. From 

2005 to 2010, no compliance incidents of authorized release were reported to APHIS for 

moving GE Eucalyptus trees using similar APHIS approved variances that would be 

required to move trees under this permit request.

Considering the specific permit conditions that must be adhered to by the permit 

requirements identified in 7 CFR 340.4, 340.7 and 340.8, the conditions specified under 

the APHIS approved variance, the proposed supplemental permit conditions for interstate

movement of the regulated article, and past compliance history of similar types of 

movements, APHIS considers the possibility of unintended exposure from moving GE 

Eucalyptus trees under this Alternative to be negligible to non-existent.

Alteration in Susceptibility to Disease or Insects – Potential of the Eucalyptus to 

Harbor Plant Pests

Overall impacts on disease or insect susceptibility would be similar to the No Action 

Alternative. 

As presented in the permit application submitted by ArborGen:

• There has been no intentional genetic change in these plants to affect their 

susceptibility to disease or insect damage. 

• None of the genes being engineered into the Eucalyptus plants are expected to 

alter the susceptibility of the transgenic Eucalyptus plants to disease or insect 

damage. 

There might be a concern that altered lignin could lead to an increase in insect or disease 

susceptibility, but the results so far with this particular gene do not indicate that this is the 
Page 18 of 91

case (see below). As prescribed in the supplemental permit conditions assigned to these 

permits, periodic monitoring of the field plots will allow the detection of any unexpected 

infestation by plant disease organisms or animal pests. The permittee is required to 

report any such unanticipated effects to APHIS under the terms of the permit - see 7 

CFR 340.4(f)(10)(ii). 

Although the trees originated from New Zealand, the trees were propagated in sterile 

tissue culture and were free of pests upon importation into the U.S. prior to their 

introduction. All materials were handled in accordance with the USDA–APHIS 

requirements for import and quarantine under a USDA–APHIS PPQ Post-entry 

quarantine permit.

Expression of the Gene Products, New Enzymes, or Changes to Plant Metabolism -

Risk of the Gene Products on the Environment

Overall impacts of genes for the selectable marker, cold tolerance, altered flowering,

altered lignin and altered growth would be similar to the No Action Alternative. These 

same types of genes are currently being tested in GE Eucalyptus trees and other species 

being field tested under current APHIS notifications or permits for confined field release.

Gene used as selectable marker 

The kanamycin resistance selectable marker gene (nptII) engineered into the trees is 

generally accepted as being safe (Fuchs et al. 1993) and has been used in thousands of 

field tests with no evidence that it has led to an increase in plant pest characteristics. This 

gene does not alter the expression of a gene product or change plant metabolism in such a 

way that it would be expected to cause risk to the environment. In a number of instances, 

plants transformed with this gene are no longer subject to the plant pest provisions of the 

Plant Protection Act and 7 CFR part 340 (e.g. corn, petition 01-137-01p; rapeseed, 

petition 01-206-02p; cotton, petition 95-045-01p; and papaya, petition 96-051-01p). 

Genes conferring cold tolerance

The C-Repeat Binding Factor (CBF) genes are transcription factors that belong to the 

AP2/EREBP family of DNA binding proteins (Riechmann and Meyerowitz 1998) and 

like other transcription factors act as control switches for the coordinated expression of 

other genes in defined metabolic pathways. CBF protein recognizes and binds to a coldand drought-responsive DNA regulatory sequence designated as the C-repeat 

(CRT)/dehydration-responsive element (DRE) (Baker et al. 1994), (YamaguchiShinozaki and Shinozaki 1994), which is found in the promoter regions of many coldinducible genes (Maruyama et al. 2004).

A common observation across experiments in which CBF genes are overexpressed in 

transgenic plants is that constitutive expression of CBF negatively impacts a number of 

other traits (Hsieh et al. 2002). In potato, for example, constitutive expression of 

Arabidopsis CBF genes using the CaMV35S promoter was associated with smaller 

leaves, stunted plants, delayed flowering, and reduction or lack of tuber production (Pino 
Page 19 of 91

et al. 2007). In contrast, CBF genes under the control of a cold-induced promoter, rd29A 

(Yamaguchi-Shinozaki and Shinozaki 1993, Kasuga et al. 1999), (Narusaka et al. 2003), 

increased freezing tolerance to the same level as constitutive expression (about 2 °C, or 

~3 °F) while restoring growth and tuber production to the levels similar to wild-type 

plants (Pino et al. 2007). In the rd29A controlled CBF plants the same level of freezing 

tolerance as the CaMV35S versions was observed after only a few hours of exposure to 

low but non-freezing temperatures. These results suggest that using a stress-inducible 

promoter to direct CBF transgene expression could significantly improve freeze tolerance 

without negatively impacting other agronomically important traits. In the case of these 

Eucalyptus trees, the CBF gene is under the control of a cold inducible promoter which 

causes the gene to be expressed under cold temperatures, thus mitigating the potential of 

reduced growth by overexpression. Under this promoter the trees exhibit normal plant 

growth. The CBF gene is not expected to produce any toxic substances and is not 

expected to alter the characteristics of the engineered plants other than imparting 

tolerance to cold temperatures.

There are also five other genes intended to confer cold tolerance in the permit application 

that are claimed as CBI. These are all genes derived from other plants. These genes do 

not alter the expression of a gene product or change plant metabolism in such a way that 

it would be expected to cause risk to the environment. These genes in Eucalyptus or 

other plants have not produced unanticipated phenotypes that would indicate there have 

been changes to plant metabolism leading to increase plant pest characteristics.

Gene for altered fertility 

The barnase gene has been engineered into other crops that have been previously 

reviewed and addressed in multiple environmental assessments by APHIS. Male sterile 

corn (USDA APHIS petitions for deregulation 95-288-01p, 97-342-01p and 98-349-01p), 

rapeseed (petitions 98-278-01p and 01-206-01p) and chicory (petition 97-148-01p) have 

been reviewed by APHIS and are no longer subject to the plant pest provisions of the 

Plant Protection Act and 7 CFR part 340. There is no reason to believe that the function 

and expression of this gene will be any different from the plants in which it has been 

previously assessed. There were no toxicity or allergenicity issues found with this gene 

in previous FDA reviews (See BNF Nos. 31, 32, 45, 57 and 66 at: 

http://www.cfsan.fda.gov/~lrd/biocon.html).). The presence of this gene is likely to 

reduce the ability of the trees to produce progeny and thus further reduce the likelihood 

of the release of the regulated article into the environment. In greenhouse tests using 

tobacco and an early flowering model Eucalyptus (E. occidentalis), the applicant has 

found that the barnase gene has demonstrated 100% efficacy in preventing pollen 

formation. In developing flower buds from field grown transgenic Eucalyptus lines 

containing this cassette, 90% of lines showed complete pollen ablation. Recent 

observations from the replicated field study conducted in Alabama under permit 06-325-

111r (renewed as 10-112-101r) and Florida under permit 08-151-101r (now covered 

under 08-014-101rm) confirmed that cold tolerant translines grown in these field test did 

not produce any pollen (see Appendix I). 
Page 20 of 91

Genes for altered lignin

There are three genes identified in the permit application to alter lignin. These genes 

have been engineered into other crops that have been previously released into the 

environment under both APHIS notifications and permits. There might be a concern that 

altered lignin could lead to an increase in insect or disease susceptibility since lignin is 

often associated with resistance to insects and disease organisms. Changes in the lignin 

biosynthetic pathway could modify interactions between plants and potential pathogens 

or insect pests on many levels: from the structural integrity of the whole plant to 

responses at the cellular level and at the interface between the plant and potential pest or 

beneficial microorganism (Funnell and Pedersen 2006). Pederson et al. (2005) surveyed 

the literature examining the impact of reduced lignin on plant fitness in a number of 

species and concluded that there are strong interactions among lignin reducing genes, the 

genetic background in which they are placed, and the environment in which the resulting 

plant lines are grown. Taken as a whole, it appears that reducing lignin content of crop 

plants can negatively impact their agricultural fitness. However, when evaluating 

individual events of reduced lignin, effect on agricultural fitness may be neutral or even 

positive (Pedersen et al, 2005, Funnell and Pedersen, 2006). To date the results of field 

tests with these particular genes have shown no differences in plant pest susceptibility. 

Growth measurements have indicated that trees had normal to a moderately reduced 

growth phenotype. The trees have also been visually inspected on a monthly basis for the 

presence of any insect and disease damage and these observations found that there have 

been no differences in insect or diseases occurrence in the transgenic lines compared to 

the control trees. However, if during the tests there is evidence of increase disease or 

insect susceptibility, the applicant is required to report this to APHIS. The permittee is 

required to report any such unanticipated effects (including excessive mortality or 

morbidity) to APHIS under the terms of the permit - see 7 CFR 340.4(f)(10)(ii).

Genes for altered growth

There are four genes identified in the permit application intended to alter growth 

characteristics of the trees that are all claimed as CBI. These are all genes derived from 

Eucalyptus and other plants. These genes do not alter the expression of a gene product or 

change plant metabolism in such a way that it would be expected to cause risk to the 

environment. These genes have been field tested in Eucalyptus or other plants and have 

not produced unanticipated phenotypes that would indicate there have been changes to 

plant metabolism leading to increase plant pest characteristics. Some of these genes 

being examined by ArborGen in the transgenic Eucalyptus have been associated with an 

increase in growth along with an increase in flowering or seed yield. If this were to occur 

in these transgenic Eucalyptus, ArborGen is required to report this as an unusual 

occurrence and must report this as a part of the conditions for field testing.

Non-coding sequences
Page 21 of 91

The transgenic Eucalyptus also contains non-coding regulatory sequences4 derived from 

plants and plant pathogens. The non-coding regions of the plant pathogens will not result 

in the production of an infectious entity or cause plant disease symptoms. None of these 

sequences are expected to pose a plant pest risk.

Method of transformation

The genes were transferred to Eucalyptus via well-characterized laboratory techniques 

that utilize DNA sequences from Agrobacterium tumefaciens to transfer introduced genes 

into the chromosome of the recipient plant (see reviews by (Zambryski 1988, Klee and 

Rogers 1989) A. tumefaciens is a bacterial plant pathogen that can cause crown gall 

disease on a wide range of dicotyledonous plant species. Although some of the DNA 

sequences used in the transformation process were derived from the A. tumefaciens, the 

genes that cause crown gall disease are first removed, and therefore the recipient plant 

does not have crown gall disease. Following transformation, the bacteria are eliminated 

from the transformed plant tissue, and the DNA sequences introduced into the plant are 

maintained and inherited as any other genes of the plant cell.

Alteration in Weediness characteristics – Potential of the Engineered Eucalyptus to 

be Invasive.

The potential of the engineered Eucalyptus to be weedy and become invasive was 

covered in previous EAs and response to comments for APHIS permit 06-325-111r 

(http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf) and permits 08-011-

106rm, 08-014-101rm (http://www.aphis.usda.gov/brs/aphisdocs/08_014101r_ea.pdf) 

which are herein incorporated by reference.

The hybrid Eucalyptus EH1 used to produce the transgenic trees has not been shown to 

be weedy or invasive in the U.S. An assessment has been conducted on the weediness or 

invasiveness potential of the hybrid by The University of Florida IFAS (see the section

above Biology of Eucalyptus and Status in the United States). In that assessment it was 

concluded that it is not likely to be invasive and can be a recommended species for 

planting. None of the genes introduced into the Eucalyptus hybrid code for traits that 

would be expected to make the GE hybrids more weedy or invasive. The genes 

introduced to affect cold tolerance could make the engineered Eucalyptus more adapted 

to cold temperatures in the southern United States, but this trait alone would not impart 

invasive or weediness characteristics (Kolar and Lodge, 2001) to the engineered plants. 

The genes to affect the growth of the trees would not be expected to increase 

invasiveness or weediness unless they also affect seed release or seed production. The 

trees could be considered weedy or invasive if they were to produce many seedlings that 

were readily spread away from the field test sites. Where the non-engineered hybrid 

Eucalyptus (EH1) has been grown in Brazil, on an estimated 400,000 acres planted over 

 4 A non-coding sequence is the strand of DNA that does not carry the information necessary to make a 

protein. In this case the non-coding sequences are strands of DNA such as promoters and terminators that 

drive the expression of the gene but do not result in the formation of a protein, which is the product of the 

gene. Therefore promoters and terminators, by themselves, cannot result in the production of a diseasecausing entity.
Page 22 of 91

15 years, there has been no indication that large numbers of seedlings are being produced 

and are becoming invasive from the commercial plantations (L. Pearson, ArborGen pers. 

comm. and Luis Silva, International Paper Company, Brazil, comment to docket APHIS2008-0059). If the genes to alter growth were to have any effect on seed production, 

ArborGen is required to report any unusual occurrences, such as increased flowering or 

seed production to APHIS during the field testing period.

Eucalyptus generally has difficulty establishing without human intervention, even in 

warmer climates. Eucalyptus is intolerant of shade or weedy competition. In order to 

successfully germinate and establish, Eucalyptus seed need contact with bare mineral soil 

and the removal of competing plants, either as a result of human intervention or naturally 

following a fire event (Meskimen and Francis 1990, Bell and Williams 1997). The areas 

surrounding the field release sites would not be readily conducive to the establishment of 

seedlings because they are managed or unmanaged areas where other plant species are 

growing. So any seeds that attempted to germinate would face competition and would 

likely be unsuccessful. The addition of the cold-tolerance genes are not expected to 

affect the reproductive biology such as seed production or vegetative reproduction 

capabilities. The gene introduced to alter lignin biosynthesis would also not be expected 

to affect seed production or vegetative reproduction capabilities. If the genes that affect 

growth have any effect of seed or flower production this must be reported to APHIS. 

The selectable marker gene, when used previously, did not contribute to weediness or 

invasive properties of the genetically engineered plants. The gene for altered fertility 

should not contribute to weediness or invasive properties and should reduce the ability of 

the trees to produce progeny. None of the traits introduced into the transgenic Eucalyptus

will compromise the ability to control these plants as weeds.

In addition, the supplemental permit conditions assigned to this permit will limit the 

reproductive capabilities of this GE Eucalyptus outside the confined field trial locations.

Possibility of Gene Flow Within the Field Test

All of the trees in the test plots, including control non-transgenic trees, have the same 

parental genotype EH1. The high level of self-incompatibility in Eucalyptus (Campinhos 

et al. 1998, Pound et al. 2002) is expected to significantly reduce the potential for 

crossing5 (gene flow) within the test plots. Seed set from any self-pollination is expected 

to be poor, and the vigor of any selfed progeny is also expected to be greatly reduced. In 

experiments conducted in Brazil and Alabama, the control self-pollinated seed obtained 

from this genotype had abnormal morphology and failed to germinate (ArborGen, 

unpublished results). In recent field releases allowed to flower in Alabama and Florida, 

ArborGen has observed a low level of seed production. Controlled seed germination 

studies have been conducted with seed capsules collected over three years from field 

 5 When plants or trees “cross” the male pollen from one tree can pollinate (fertilize) the female ovule (or 

egg) on the same tree or on another tree. Unlike animals, some plants can fertilize themselves when the 

pollen and ovule are produced on the same tree. In this case all the trees are genetically identical (i.e. the 

same clone)(see footnote 1). Eucalyptus has a built-in mechanism that will inhibit self-fertilization. So 

these GE trees are likely to exhibit reduced fertility and reduced numbers of viable seed compared to fully 

sexually compatible Eucalyptus trees since they are genetically identical individuals.
Page 23 of 91

trials that have been allowed to flower. Results have indicated that either no, or a very 

low number of viable seeds, are produced in the transgenic as well as in the control trees, 

most likely as a result of limited self-fertilization by pollen from the fertile control trees. 

Regular volunteer monitoring of six different trials over 2-5 years have further

confirmed the absence of any seeded volunteers in or around the field tests. No seedlings 

have been found established beneath the trees or in the surrounding areas (Appendix I). 

Even if seed are produced in the test, several factors in the biology of Eucalyptus would 

limit the potential for seed dissemination. Although Eucalyptus seed is very light and

small, it is not adapted to wind dispersal and consequently the dispersal of seed is very 

limited, generally being confined within a radius of twice the tree or canopy height 

(approximately 50 meters for a 25 meter tall tree at harvest age)(Cremer 1977, Gill 1997, 

Linacre and Ades 2004). Another consequence of the very small size of Eucalyptus

seeds is that they have very limited reserves and are intolerant of shade or weedy 

competition. In order to successfully germinate and establish, Eucalyptus seed needs 

contact with bare mineral soil and lack of competition either as a result of human 

intervention or naturally following a fire event (Meskimen and Francis 1990, Bell and 

Williams 1997). Eucalyptus plantations are typically established using rooted plantlets 

because of poor establishment using direct seeding methods. Even for the rooted plants, 

competition control is recommended for several months after planting to ensure good 

survival (Meskimen and Francis 1990). Therefore, there is limited possibility that 

volunteer seedlings could become established in any unmanaged areas that may be close 

to the site. However, if they were to appear, the supplemental permit conditions for the 

permit will require that all volunteers be reported to APHIS, found and destroyed to 

prevent any spread of trees from the field release site.

Eucalyptus seeds do not have any dormancy barriers to prevent germination of volunteer 

seeds (Grose 1960, Wellington 1989, Gill 1997) and seed viability and storage of 

Eucalyptus seeds in soil are less than one year (Gill 1997). The Eucalyptus species that 

have become invasive in California (E. camaldulensis and E. globulus) are particularly 

adapted to a Mediterranean climate subject to summer fog, which is conducive to seed 

germination in those species (Sellers 1910). This type of climate does not exist in the 

Southeastern U.S. In the event that any viable seeds are produced and are deposited on in 

an area conducive to germination from this field trial, these seeds would be expected to 

germinate within 7 – 14 days (Meskimen and Francis 1990). In accordance with the 

supplemental permit conditions for these permits, the bordering fields within 100 meters 

from the edge of the trials will be monitored every six months for germinating seedlings

by the applicant. This distance is twice the 50 meter distance that seeds would be 

expected to be dispersed from a tree at harvest age (Cremer 1977, Gill 1997, Linacre and 

Ades 2004). If transgenic seedlings are observed they will be destroyed by the applicant 

either by uprooting or by spraying with EPA approved herbicides (e.g., glyphosate or 

other herbicides to which these trees are susceptible) and APHIS will be notified of their 

occurrence.
Page 24 of 91

Possibility of Gene Flow Outside of the Field Test

Eucalyptus is adapted for insect pollination, with bees being the predominant vector

(Pacheco et al. 1986, Pacheco 1987, House 1997). Under ideal conditions of humidity 

and temperature, viable Eucalyptus pollen can only be found within approximately 100 

meters from the edge of nearest tree stand (Peters et al. 1990, Linacre and Ades 2004). 

Pacheco (1987) verified that bees (Apis spp.) are the most effective pollinators of 

Eucalyptus, with activity increasing up to 100 meters from the beehive, and decreasing 

after this distance. de Assis (1996) indicated that the minimum distance to prevent 

undesirable pollen contamination of seed producing areas is approximately 300 meters. 

Even if bees were to transport pollen farther distances from the field test sites, there are 

no sexually compatible species nearby with which they could cross and produce offspring 

(see description of the field test sites below).

There could be two possible routes of gene flow outside of the confined field test to other 

Eucalyptus species trials. One could be with nearby transgenic Eucalyptus field test trees 

and the other could be with other nearby non-transgenic Eucalyptus species trials.

Transgenic trials being conducted by the applicant under currently approved APHIS 

notifications and permits, of the same hybrid Eucalyptus variety EH1, are planted 

adjacent to or within the proposed field test plot locations at the sites in Escambia and 

Baldwin Counties in Alabama, Pearl River County in Mississippi; and Highlands County

in Florida, and Berkeley County in South Carolina. The applicant has indicated that they 

are not aware of any commercial plantings of sexually compatible Eucalyptus species

within 1000 meters of the proposed test plot location at any of these sites. Therefore, 

based upon the limited distance that viable pollen is likely to occur outside a tree stand, it 

is highly unlikely that gene flow would occur outside of the confined field test sites at 

these locations. The two field trials in Alabama and Florida that have allowed these 

hybrids to flower under an APHIS permit are producing low numbers of viable seeds (see 

Appendix I). Based upon monitoring conducted from the applicant, no volunteer 

seedlings have been observed at these two field trials to date.

There are other species of cold-hardy Eucalyptus that can possibly be grown in the 

Southeast U.S. These species include E. neglecta, E. niphophila, E. pauciflora, E. 

camphora, E. nova-anglica, E. macarthurii, E. gunnii and E. cinerea. These could occur 

in the same states as the proposed field trials . Among these species, E. cinerea, also 

known as the silver dollar tree or Argyle Apple, is the most popular species grown for its 

ornamental foliage (http://www.australiaplants.com/Eucalyptus_cinerea.htm). These and 

other ornamentals are likely to be grown as specimen trees (one or few grown together) 

and not part of large-scale plantations. 

The transgenic Eucalyptus hybrids that will be grown in the proposed field trials are not 

likely to be sexually compatible with any of the cold hardy species listed above. For 

example, E. grandis and E. urophylla, for which hybrids have been generated in directed 

breeding programs, are in the Salignae and Resiniferae series, respectively, of section 

Transversaria (http://plantnet.rbgsyd.nsw.gov.au/cgi-bin/eucclass.pl?gn=Eucalyptus). In 

contrast, E. cinerea, and other cold hardy species mentioned above are far removed 
Page 25 of 91

genetically from the genotype used in this field trial on the evolutionary scale and reside 

within different Series and Sections of genus Eucalyptus (see

http://plantnet.rbgsyd.nsw.gov.au/cgi-bin/eucclass.pl?gn=Eucalyptus for details on 

sections and series in Eucalyptus). Even among the closely related species of Eucalyptus, 

hybridization rates are generally very low (Volker 1995). The published literature 

supports the fact that natural hybridization among distantly related species within genus 

Eucalyptus is rare and hybrid inviability increases with increasing taxonomic distance 

between parents (Potts and Dungey 2004). Where hybridization is possible, it often 

requires significant human intervention in directed breeding/crossing efforts. Potts and 

Dungey (2004) make reference to the high degree of inviability in F1 hybrids (offspring). 

Inviability of these offspring may be expressed at germination, in the nursery and even 

after planting in the field. Slower germination of hybrid seed often occurs, along with 

reduced survival of germinants in the nursery, and many seedlings have abnormal 

phenotypes. Griffin et al. (1988) surveyed natural and manipulated hybrids in the genus 

Eucalyptus and discussed the challenges of developing even human-made hybrids from 

such wide crosses (in this case E. grandis and E. globulus in sections Transversaria and 

Maidenaria, respectively), with only 4.4% of seed germinating and only 3.2% of these 

producing trees that were worthy of further evaluation. To achieve the development of 

viable hybrids sometimes hundreds of hand pollinations must be made to find a viable 

hybrid that will grow normally. An example of the procedures required to make these 

wide-cross hybrids is given in Barbour and Spencer (2000). 

A further barrier to potential crossing between the transgenic trees with ornamental E. 

cinerea and other species is the expected differences in flowering times between species

(Gore and Potts 1995, Potts et al. 2003). For example, E. cinerea flowers in spring, while 

the transgenic hybrid genotype used in this test initiates flowers in early summer with 

expected maturation in mid to late summer. In the United States, ArborGen data indicate 

that flowering of the clone being tested occurs in the summer.

Based on the above information, there is little if any significant risk for outcrossing to or 

from other Eucalyptus species because: 1) to date the trees that have been allowed to 

flower have shown no mature pollen formation; 2) other species that are or could be 

grown in the area are unlikely to be compatible; 3) it is unlikely that flowering time in 

other species will overlap with the hybrid used in this test and; 4) hybrids, in the event 

that they could form, would be expected to be of very poor vigor.

Possibility of Vegetative Propagation / Persistence Outside of the Field Test

Overall impacts on the possibility of vegetative propagation and persistence outside the 

field test location would be similar to the No Action Alternative. 

Unlike some other hardwood forest trees, Eucalyptus does not spread in the environment 

via natural abscissions of branches, or cladoptosis. The asexual propagation of shoots via 

rooted cuttings requires specific environmental conditions such as a greenhouse or a high 

humidity environment (Hartney 1980), so it is highly unlikely that any shoots that fall or 

that are removed from the trees would propagate themselves in the wild. 
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Suckering (production of shoots from subterranean roots) does not occur in this 

Eucalyptus hybrid. Regrowth of shoots from stumps of felled trees is common and this 

practice, known as coppicing, is used to regrow trees in a plantation after harvest

(Meskimen and Francis 1990). In accordance with supplemental permit conditions this 

regrowth will be managed in this confined field test at termination by devitalizing any 

sprouts that form from the stumps of harvested trees using EPA registered herbicide

treatments.

There could be a concern that seeds of the hybrid could be widely distributed by severe 

storms such as hurricanes or tornadoes. The Eucalyptus hybrid that is being grown in 

these proposed field tests produces mature capsules in February and seed fall is shortly 

after this. Therefore seed release is in late winter / early spring and well outside out of 

the normal hurricane season which occurs between June and November 

(http://www.nhc.noaa.gov/). The probability that other storm events might distribute 

seeds that could survive is extremely low. There would have to be a number of events 

that would have to combine to have any consequence: seed happens to be shedding at the 

time a severe storm rolls through a field test site, the seed happens to land on a suitable 

seed bed of bare soil with no weeds, the site is then not disturbed either by plowing or 

herbicide treatment, and moisture conditions are suitable for a germinating seedling to 

survive and grow. Research and experience have shown that long distance dispersal of 

Eucalyptus seeds and seedling establishment is very rare. Forsyth et al (2004) point out 

that “...in most parts of the world where Eucalyptus have invaded, they seldom spread 

considerable distances from planting sites, and their regeneration is frequently sporadic.” 

Richardson (1998) indicates “Eucalypts are also represented on many national or regional 

weed lists from other parts of the world. Despite this, they have not been nearly as 

successful in invading alien environments as other widely planted trees such as pines and 

legumes. Many eucalypts produce large quantities of seeds, so their lack of success as 

invaders is rather puzzling.” This could likely be due to the fact that Eucalyptus seeds 

are very small, have very limited reserves and are intolerant of shade or weedy 

competition. In order to successfully germinate and establish, Eucalyptus seed needs 

contact with bare mineral soil and lack of competition either as a result of human 

intervention or naturally following a fire event (Meskimen and Francis 1990, Bell and 

Williams 1997). In Brazil where E. grandis, E. urophylla and their hybrids have been 

grown since the 1960’s and are now planted on several thousand hectares, there is no 

evidence that wind borne seeds are spreading the trees beyond managed plantations. 

Over 70,000 hectares of the hybrid has been planted extensively by International Paper, 

who developed EH1, with no evidence of invasiveness (Luis Silva, International Paper 

Company, Brazil – comment to the docket to the EA for permits 08-011-106rm and 08-

014-101rm). In these environments Eucalyptus obviously does not behave like other 

windblown seeds of grasses, for example, which can be pioneering species. Therefore 

the likelihood of significant escape and successful establishment of seeds from the field 

test sites due to storms is negligible. APHIS has determined that the field test will be 

confined and storms are not expected to compromise this confinement.
Page 27 of 91

Potential of the Eucalyptus in the Field Tests to Become an Invasive Species that 

Threatens Native Plant and Animal Communities.

There could be a concern that adding the cold tolerance trait would make the engineered 

Eucalyptus more adaptive and invasive in the southeastern U.S. It has been hypothesized 

that engineered traits such as cold tolerance could significantly affect the engineered 

variety’s ability to propagate, survive, and impact native ecosystems. 

There is no evidence to date that the untransformed clone of the Eucalyptus hybrid in 

these permits is weedy or invasive in the U.S. (L. Pearson, ArborGen pers. comm.) None 

of the genes introduced into Eucalyptus code for traits that would be expected to make 

the plants more weedy or invasive. The genes introduced to affect cold tolerance could 

make the engineered Eucalyptus more adapted to cold temperatures in the southern 

United States, but this trait in and of itself would not impart invasive or weediness 

characteristics (Kolar and Lodge 2001) to the engineered plants.

There may also be a concern that adding genes that affect growth could make the 

engineered Eucalyptus more invasive if they increased the growth rate. However, simply 

increasing growth without affecting other traits would not lead to increased invasiveness. 

If the genes that affect growth were to lead to an increase in seed production or release of 

more seed then this could have an impact on its invasive or weedy potential. If there 

were to be a significant increase in the number of flowers or seeds produced in these 

tests, this would be seen as an unusual occurrence and must be reported to APHIS as a 

part of the conditions of the permit. An increase in seed production would not 

necessarily lead to an increase in invasiveness unless the seeds were to find a suitable 

environment for germination.

As discussed in previous sections of this EA, there are multiple mechanisms in place that 

would prevent these Eucalyptus hybrids from establishing themselves in the wild. Since 

only one clone is being planted, viable seed set is likely to be limited due to selfincompatibility. In accordance with supplemental permit conditions, monitoring of seed 

set and seedling viability will be required by the applicant during these confined field 

tests so that the extent of seed production and seedling establishment can be determined. 

In addition, altered fertility leading to the lack of viable pollen development has been 

engineered into the trees. The addition of the cold-tolerance genes are not expected to 

affect reproductive biology such as seed production or vegetative reproduction 

capabilities. The gene introduced to alter lignin biosynthesis would also not be expected 

to affect seed production or vegetative reproduction capabilities. If the genes introduced 

to alter growth were to lead to an increase in seed production this would be reported to 

APHIS as a part of the conditions of the permit. The selectable marker gene, when used 

previously, did not contribute to weediness or invasive properties of the genetically 

engineered plants. The gene for altered fertility should not contribute to weediness or 

invasive properties and should reduce the ability of the tree to produce progeny. 

In unlikely event that seeds are formed and seedlings are produced, none of the traits 

introduced into the transgenic Eucalyptus will compromise the ability to control these 

plants so spread of seedlings and trees from the field test sites is highly unlikely. 
Page 28 of 91

Volunteers can be readily identified and controlled. Should any hybridization and viable 

seed production occur, in accordance with supplemental permit conditions, the

monitoring for and removal of volunteers within 100m from the edge of transgenic test 

plot by the applicant would effectively eliminate any seedlings that may be produced. If 

transgenic seedlings are observed they will be destroyed by the applicant either by 

uprooting or by spraying with EPA approved herbicides (e.g., glyphosate or other 

herbicides to which these trees are susceptible) and APHIS will be notified of their 

occurrence.

Impact on Existing Agricultural Practices

Overall impacts on existing agricultural practices would be similar to the No Action 

Alternative. 

The establishment and growth of these small confined field tests will not have any impact 

on existing agricultural practices because they are solely for research purposes. Current 

agricultural practices will essentially remain unchanged. As identified by the applicant, 

the field sites that are being proposed under these permits have been used as forest tree 

plantations, pastures, or for forestry and agriculture research and are specifically designed 

for field testing crop plants or forest trees. 

Potential Impacts to Wildlife 

Native floral communities

The field sites in the permit applications are located in Baldwin and Escambia Counties,

Alabama; Highlands County, Florida; Pearl River County, Mississippi; and Berkeley and 

Dorchester Counties, South Carolina. These sites are a mixture of pasture, crop lands and 

forested areas. The two locations in South Carolina are holding areas for plants in 

containers. In the unlikely event that viable seeds are formed, these areas are unsuitable 

for the establishment of the Eucalyptus hybrid clone. As discussed in previous sections 

of this EA, Eucalyptus is intolerant of shade or weedy competition. In order to 

successfully germinate and establish, Eucalyptus seed need contact with bare mineral soil 

and the removal of competing plants, either as a result of human intervention or naturally 

following a fire event. With the exception of the field test area, the agricultural areas 

surrounding the field sites are not conducive to the establishment of Eucalyptus. The 

surrounding agricultural and tree crops would provide a shady canopy and competition 

for light and other resources that would impede seedling establishment of Eucalyptus. 

The inhospitable conditions for seed germination, in combination with the supplemental 

permit conditions established for these permits, will make the establishment of 

Eucalyptus in the surrounding area highly unlikely. Should any hybridization and viable 

seed production occur, in accordance with supplemental permit conditions, the

monitoring for and removal of volunteers within 100m from the edge of transgenic test 

plot by the applicant would effectively eliminate any seedlings that may be produced. If 

transgenic seedlings are observed they will be destroyed by the applicant either by 

uprooting or by spraying with EPA approved herbicides (e.g., glyphosate or other 
Page 29 of 91

herbicides to which these trees are susceptible) and APHIS will be notified of their 

occurrence.

Terrestrial animals

The most likely animals to encounter the transgenic Eucalyptus trees in this confined

field trial would be browsing mammals (e.g., deer), burrowing animals (such as rodents), 

and leaf consuming insects (considered plant pests). In the event of consumption of plant 

material or seeds by other animals, the gene products produced by the selectable marker 

gene and genes of interest do not produce any toxin or have any similarity to known 

toxins (see Section above on - Risk of the Gene products on the Environment). 

Therefore APHIS concludes that the Eucalyptus hybrid would have no adverse impacts 

on any native vertebrate or invertebrate animal species.

Aquatic organisms

Eucalyptus germinates in areas of bare mineral soils and would not be expected to 

establish in aquatic or riparian environments. There is no expectation of toxicological 

effects on any aquatic organism due to the ingestion of the transgenic plant material in 

this confined field trial (see Section above on - Risk of the Gene products on the 

Environment). Therefore, APHIS concludes that the Eucalyptus hybrid would have no 

adverse impacts on any aquatic species.

Potential Impacts by Fire

Overall impacts of fire would be similar to the No Action Alternative.

Most Eucalyptus communities in Australia have evolved in the presence of periodic fire, 

and fires are an integral part of the Eucalyptus ecosystem (Ashton 1981, Gill 1997). 

Many Eucalyptus species are known to be highly flammable and depending upon the 

species, location and age, they can be very resistant or susceptible to fire damage (Gill 

1997). Eucalyptus fires can be very hot and move rapidly. The bark catches fire readily, 

and deciduous bark streamers tend to carry fire into the canopy and to disseminate fire 

ahead of the main front (Ashton 1981, Skolmen and Ledig 1990, Esser 1993). Other 

features of Eucalyptus that promote fire spread include heavy litter fall, flammable oils in 

the foliage, and open crowns bearing pendulous branches, which encourages maximum 

updraft (Esser 1993, Gill 1997). In the U.S., there have been reports of significant fires 

in California and many have been blamed on the widespread planting of Eucalyptus. 

Fuel buildup occurs very rapidly in unmanaged bluegum Eucalyptus stands in California

which has led to significant forest fires. The buildup of litter and dead grass are primary 

responsible for the spread of these fires (see Santos: 

http://wwwlibrary.csustan.edu/bsantos/euctoc.htm).). The Forest Service indicates that 

fuel reduction programs and the establishment of firebreaks in Eucalyptus plantings can 

reduce wildfire hazard (Esser 1993). 

There is a historical risk of forest fire in the southeastern U.S., however, the probability 

that these confined field trials will increase the risk and severity of forest fires in their 
Page 30 of 91

respective locations is very small. These new plantings are small (none greater than 7.7

acres) and they will be managed by the applicant to prevent litter buildup. Under all of 

the Eucalyptus permits authorized for planting by ArborGen, no location is planted with 

more than 20 acres. Authorization of this permit will not increase that acreage at any 

release location. These sites are also physically isolated from nearby plantations. If they 

were to catch fire, the fires would likely be readily contained. The sites are managed to 

reduce the risk of fire spreading to or from the study areas by maintaining a firebreak 

between the test plots and adjacent forested areas. Depending on local conditions at each 

site the firebreak may be a road, a cultivated strip or a plowed fire line.

Potential Impacts to Human Health

Overall impacts on human health would be similar to the No Action Alternative.

During the comment period for the EA prepared for permit 06-325-111r, there were 

concerns expressed that Eucalyptus field tests could be a source of Cryptococcus 

neoformans gattii. APHIS conducted a thorough review of C. neoformans gattii and the 

possibility that the field tests could pose a risk to human health ((APHIS 2004) EA and 

response to comments for permit 06-325-111r 

(http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf)) and concluded that the 

field trial would not lead to a higher incidence of C. gattii in the U.S. and therefore 

should not pose an unnecessary risk to human or animal health.

C. neoformans gattii is a fungal pathogen that is hosted on a variety of species of 

Eucalyptus as well as other tree species (Upton et al. 2007). It causes systemic fungal 

infections in humans, leading to fungal meningitis and death (Datta et al., 2009). C. 

neoformans gattii has been found on a number of Eucalyptus hosts, some of which are 

being grown in commercial plantations and imported and exported for ornamental use. 

People have contracted and died from cryptococcosis in India, Africa, Taiwan, South 

America and California (Datta e al, 2009). C. neoformans gattii infections are found 

particularly in AIDS patients due to their weak immune systems (Chaturvedi et al, 2005). 

Infections with this fungus are rare in those with fully functioning immune systems. For 

this reason, C. neoformans gattii is sometimes referred to as an opportunistic fungus. 

There was an outbreak of cryptococcal disease on the eastern portion of Vancouver 

Island, British Columbia in 1999 (Datta et al., 2009). The disease was previously only 

known to occur in tropical or semi-tropical climates.

 

It is unlikely that the trees that are the subject of the proposed field release can be a 

source that might introduce the pathogen into the U.S because the trees were derived 

from sterile tissue culture lines. The transgenic Eucalyptus started as a hybrid developed 

in Brazil. In Brazil, small pieces of the tissue derived from the hybrid were put into sterile 

tissue culture and sent to New Zealand for transformation. The transformed lines were 

sent to the U.S as sterile tissue culture lines that were inspected by APHIS Plant 

Protection and Quarantine inspectors prior to entry into the U.S. C. gattii spores readily 

germinate in culture. If C. gattii spores were present in the tissue culture, contamination, 

would be evident and the affected lines would be discarded prior to regeneration of trees 

for introduction into the environment. Another reason it is unlikely that spores could be 
Page 31 of 91

or were ever present in the hybrid lines used in the proposed confined field trial is that in 

the Eucalyptus species where C. gattii is associated, the pathogen is primarily found 

colonizing the bark or decaying wood in hollows of older trees (Kidd et al, 2007). The 

tissue culture used for the Eucalyptus hybrid was not derived from woody tissue nor was 

woody tissue generated during tissue culture. Because the trees were derived from 

tissues that are not known to be a source of the spores and were derived from sterile 

tissue culture lines that by all appearances were free from any fungal contamination, there 

is a negligible risk that the hybrid trees used in the field trial could be or have been 

contaminated with C. gattii. 

The risk that these field trials will result in a higher incidence of the fungus in the U.S. 

and thereby pose a risk to human health is considered to be negligible for the following 

reasons. First, there is not a clear association between E. grandis or E. urophylla and C. 

gattii. Second, there is no reason to believe that the genetic modification of the hybrids 

will alter the association of the trees with C. gattii. Third, the scale of the field tests is 

miniscule compared to the vast expanses of native trees that could potentially harbor the 

pathogen. Based on the above considerations we have concluded that an increase of 

additional acreage planted to Eucalyptus would not impact the likelihood that these field 

trials should lead to a higher incidence of C. gattii in the U.S. and therefore should not 

pose an unnecessary risk to human health. 

Transfer of Genetic Information to Organisms with which it Cannot Interbreed -

Horizontal Gene Transfer to Other Organisms

Overall impacts on horizontal gene transfer would be similar to the No Action 

Alternative.

Horizontal gene transfer (HGT) is any process in which an organism incorporates genetic 

material from another organism without being the offspring of that organism. HGT is a 

common phenomenon among bacteria but is not common between higher organisms

(Keese 2008). HGT and expression of DNA from these Eucalyptus hybrid plant species 

to bacteria is unlikely to occur. First, many genomes (or parts thereof) have been 

sequenced from bacteria that are closely associated with plants including Agrobacterium

and Rhizobium (Kaneko et al. 2000, Wood et al. 2001, Kaneko et al. 2002). There is no 

evidence that these organisms contain genes derived from plants. Second, in cases where 

review of sequence data implied that horizontal gene transfer occurred, these events are 

inferred to occur on an evolutionary time scale on the order of millions of years (Koonin 

et al. 2001, Brown 2003). Third, transgene DNA promoters and coding sequences are 

optimized for plant expression, not prokaryotic (i.e., bacterial) expression. Thus even if 

horizontal gene transfer occurred, proteins corresponding to the transgenes are not likely 

to be produced. Fourth, many common transgenes used in plant biotechnology are 

derived from bacteria commonly found in the environment. The FDA has evaluated 

horizontal gene transfer from the use of selectable marker genes and concluded that the 

likelihood of transfer of such genes from plant genomes to microorganisms in the 

gastrointestinal tract of humans or animals, or in the environment, is remote 

http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocume
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nts/Biotechnology/ucm096135.htm. Therefore APHIS concludes that horizontal gene 

transfer poses no environmental risk.

C. The Degree to Which the Possible Effects are Highly Uncertain or 

Involve Unique or Unknown Risks

Potential Effects of Growing Eucalyptus on Soil Hydrology

Eucalyptus is recognized as having impacts on hydrology and large widespread plantings 

could have potential impacts on hydrology in the southeastern United States (Farley et al. 

2005a). Since large plantings of Eucalyptus have not been grown in many parts of the 

southeastern US (other than southern Florida) the potential impacts of such plantings on 

hydrology are unknown. In preparing the EA for permits 08-011-106rm and 08-014-

101rm APHIS requested additional information on potential impacts of hydrology from 

ArborGen and also consulted with the USDA Forest Service to assess the potential 

impacts of planting Eucalyptus on hydrology. The additional information supplied by 

ArborGen and the Forest Service are included as Appendices II and III. The document 

supplied by the Forest Service represents only their opinion on the potential impacts of 

these field tests on hydrology and does not represent the position of the USDA on the 

pros and cons of deploying Eucalyptus as a biofuel, bioenergy or fiber crop.

The Forest Service indicates that planting large-scale Eucalyptus plantations may 

potentially lower the water table, and affect groundwater recharge and stream flow 

dynamics. Eucalyptus is very efficient at using water. It can produce more biomass per 

unit water consumed than native southeastern pines; however, their extremely rapid 

biomass production has proportionally higher transpiration costs and therefore greater 

water use. The Forest Service has estimated that a mature Eucalyptus plantation growing 

in southwest Georgia could potentially transpire 882 mm per year, exceeding all other 

forest types on average by a factor of 2.5. Eucalyptus transpiration could exceed that of 

pine plantations by a factor of 1.6, and previous pasture land by a factor of 3.5. The 

comparison with agricultural crops is more variable where Eucalyptus transpiration may 

be greater or lesser than that of crop plants depending on the crop, the growing season, 

and the management practices.

Eucalyptus has a dimorphic rooting pattern which means that it has surface roots that 

draw water from the surface as well as deep roots which draw water from deep within the 

soil. The mean maximum rooting depth for Eucalyptus ranges between 15 and 40 

meters, which is a characteristic of a dimorphic rooting pattern (Canadell et al. 1996a). 

In contrast, mean maximum rooting depths of pine plantation (P. taeda and P. elliottii) 

and grass species are 3 meters and 2.6 meters, respectively (Canadell et al. 1996a). 

According to the Forest Service, conversion to Eucalyptus on sites where the water tables 

are less than 10 meters will likely lower down-slope water tables via direct means (i.e., 

direct use of ground water by deep roots), affect groundwater-aquifer dynamics, and 

result in evapotranspiration rates that exceed precipitation input, as have been reported 

for this species in other locations (Calder et al. 1997a).
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Recent research suggests that Eucalyptus plantations would reduce stream flow more 

than pine plantations, and could potentially eliminate low flows. In a review of more 

than 20 catchment6 conversion studies, Farley and others (Farley et al. 2005a) showed 

that converting existing vegetation to Eucalyptus plantations reduced stream flow by 20% 

more than converting it to a pine plantation. This review also showed that the loss of low 

flows were more complete for Eucalyptus plantations compared to pine plantations 

(100% vs. ~80% reduction of low flows). Elimination of low stream flows could have 

important ramifications for threatened and endangered aquatic species, such as the gulf 

strain striped bass, and species of endemic freshwater mussels (Golladay et al. 2004a, 

Couch and McDowell 2006b). 

Due to a lack of available data in the southeastern U.S. on planting Eucalyptus, it is 

difficult to determine the significance of the effects on hydrology if large acreage of 

Eucalyptus were to be planted. The Forest Service has indicated that collection of data 

and modeling will be useful to determine the long-term impacts of planting large 

acreages of the genus. The Forest Service has also pointed out that the significance of the 

impact on groundwater and stream flow will depend greatly on the area extent, size, and 

spatial distribution of the plantations. For example, a few small (less than 10 hectares, 

i.e. approximately 25 acres) and well-dispersed plantations may only have very localized 

impacts and negligible impacts at the watershed scale.

The field test sites requested in the permit application are well dispersed and are limited 

in size (none are greater than 7.7 acres). Under all of the Eucalyptus permits authorized 

for planting by ArborGen, no location is planted with more than 20 acres and it is 

anticipated that they are not likely to have significant impacts on hydrology. 

Authorization of this permit will not increase that acreage. At the request of APHIS, 

ArborGen has supplied data indicating the maximum size of each of the plantings at each 

site, the individual watersheds where the plantings occur, the area of the watershed, how 

much of the watershed will be occupied by the field tests, the location of the closest 

primary and secondary streams, and the location of any critical habitat for Federally listed 

threatened and endangered species within the watershed. 

Using the 8 digit HUC (Hydrologic Unit Code) as the Watershed to be analyzed, the data 

provided by ArborGen show that none of the sites in the permit application occupy more 

than 0.005% of any given watershed. The closest critical habitat for an aquatic species 

(such as a fish and mussel) is 6 kilometers at one location and ranges from 6 to 90 km for

any of the sites having any proximity to habitats that could be impacted. There are no 

nearby threatened or endangered plant species that could be impacted by hydrological 

effects (see also Appendix IV). Any effects would be very localized on existing nearby 

agricultural and forestry plantings. Therefore APHIS concludes that while the effects on 

hydrology, including the watershed and aquifers, are unknown and uncertain for very 

large plantings of Eucalyptus, these small-scale field tests are unlikely to have any 

significant negative impacts on hydrology and on native flora and fauna.

 6 A catchment or drainage basin is an extent of land where water from rain or snow-melt drains downhill 

into a body of water, such as a river, lake, reservoir, estuary, wetland, sea or ocean. The drainage basin 

includes both the streams and rivers that convey the water as well as the land surfaces from which water 

drains into those channels, and is separated from adjacent basins by a drainage divide.
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Potential Allelopathic Effects of Eucalyptus

Allelopathy refers to “any process involving secondary metabolites produced by plants, 

microorganisms, viruses and fungi that influence the growth and development of 

agricultural and biological systems” (See: http://www.international-allelopathysociety.org/main/home/main.php). Allelochemicals from plants are released into the 

environment by exudation from roots, leaching from stems and leaves, or decomposition 

of plant material. Allelopathy can have both negative and positive impacts on the 

environment (Eljarrat and Barceló 2001, Xuan et al. 2005, Kohli et al. 2006). There has 

been increased research activity in this area, one of which is taking advantage of plants

that produce allelopathic compounds in developing agroforestry and sustainable 

agriculture systems (Kohli et al. 2006, Narwal 2006). Allelopathy has been 

demonstrated in many commercially important tree species including Acacia, Ailanthus 

Eucalyptus, Juglans, Quercus, Leucaena, Pinus, Picea, Aibes, Populus and Acer; and has 

been demonstrated in agronomic crops such as rye, wheat and alfalfa (Nandal et al. 1994, 

Ferguson and Rathinasabapathi 2003, Reigosa and Gonzáles 2006, Mallik 2008).

There have been extensive studies conducted on allelopathy in Eucalyptus and there are 

several comprehensive reports and review articles on this genus (Ong 1993, Sunder 1995)

(Nandal et al. 1994, Davidson 1995, White 1995). Eucalyptus species are known to 

produce chemical compounds that are required by the plant for defense against herbivores 

and pathogens. There are several studies in the literature that demonstrate the negative, 

positive and neutral allelopathic interaction of Eucalyptus species and their hybrids with 

other crop plants (Sanginga and Swift 1992, Khan et al. 2004, Espinosa-Garcia et al. 

2008). These interactions vary greatly depending upon the crop species and conditions 

under which they are grown. There is inconclusive data as to whether these compounds 

produced by Eucalyptus are exclusively responsible for allelopathic influence on 

understory vegetation in Eucalyptus plantations. Most allelopathic studies in Eucalyptus 

species have involved laboratory experiments with extracts obtained from different plant 

parts or leaf litter to investigate allelopathic effects on seed germination and growth in 

potted plants. These laboratory bioassays and pot culture studies may or may not be 

applicable to field conditions. The perceived allelopathic effects observed in the field on 

growth of understory or adjacent intercropped food crops could also result from 

competition for water, nutrients and light.

Allelopathy tends to be an inexact science and many studies in allelopathy are 

inconclusive and difficult to interpret due to potential interactions with other aspects of 

the environment. For example in a recent study, (Nandal and Dhillon 2005) tested the 

allelopathic effects of poplar (Populus deltoides) leaf extracts on germination and growth 

of ten wheat varieties under laboratory conditions. They reported that lower concentration 

of leaf extracts from poplar had stimulatory effects on root length in all wheat varieties 

whereas higher concentrations adversely affected germination and seedling growth of 

some of the wheat varieties tested. In a field experiment, the performance of all ten 

wheat varieties was also evaluated under four different poplar spacings in an agrisilviculture system. Although the grain yield of wheat varieties was significantly lower 

under all spacings of poplar compared to controls, yields increased significantly with 
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increased spacing of poplar, possibly due to reduced competition for light and nutrients. 

However, no correlation was found between the laboratory bioassay using leaf extracts 

and the field studies.

In a recent study, the allelopathic interaction of Eucalyptus grandis, E. urophylla and E. 

grandis x urophylla on the germination and early growth of four annual crops (maize, 

bean, watermelon and squash) was investigated (Espinosa-Garcia et al. 2008). Soil 

samples were collected from different soil horizons and at varying distances from 

Eucalyptus trees growing at the plantation edge and used for growth studies in pots. The 

dried soil samples used for growth studies were also analyzed for total soluble phenolics 

present in the soil. The study showed that soil samples from different plantations had 

differential effects ranging from no effect, to slightly inhibitory, to a stimulatory effect on 

germination and radicle7 growth of test crops. Among the three Eucalyptus species tested, 

the soil samples from E. grandis x urophylla plantations had an inhibitory effect on 

germination of maize, bean and watermelon but had a stimulatory effect on squash. The 

soil from E. grandis plantations had an inhibitory effect on squash. The total soluble 

phenolics varied in different soil samples but did not explain the differential effects on 

the test crops. The authors concluded that soil samples collected from plantations of 

Eucalyptus species contained allelochemicals that affected germination and early growth 

of some annual crops but such effects could be avoided by planting crops at a distance of 

15 meters away from the edge of plantations.

Even though the Eucalyptus under this permit could demonstrate allelopathic properties, 

the presence of any allelochemicals is not going to make the Eucalyptus planted under 

these permits more invasive or present a plant pest risk. Since all these field tests are 

confined and limited in size, any allelopathic effects should be small. As a standard 

silvicultural practice, herbicides will also be used within the field test sites and any of 

their effects on understory vegetation will be as severe or more severe than any 

allelopathic effects. In the future, should any negative allelopathic or other competitive 

interactions be observed under field conditions outside of the immediate field tests sites, 

these could be mitigated by adjusting the tree spacing, irrigation and fertilization 

practices or by planting the field tests at least 15 meters away from any agronomic crops 

or sensitive areas. Any unusual observations at the field test sites are to be reported to 

APHIS under the supplemental permit conditions of the permit; including any indications 

of allelopathic effects.

D. Cumulative Effects

As identified in the permit application, the field test sites in this permit application have 

been in agricultural or forest research, or in agricultural production or forest tree 

plantations for 5 to 15 years. Therefore the land has been in continuous agricultural or 

forest tree production at all the proposed field sites for at least 5 years prior to these

proposed releases and it is reasonably foreseeable that if the permit were not issued that 

the sites would continue to be maintained under similar agriculture or forestry 

 7 The radicle is the first part of a seedling (a growing plant embryo) to emerge from the seed during the 

process of germination. It is an embryonic root.
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production. Five of these six locations identified in the permit application currently have 

active APHIS permits (08-011-106rm, 08-014-101rm, 09-070-10rm, 10-112-101r, and 

11-041-101rm) for environmental release of GE Eucalyptus hybrids in Alabama, Florida, 

Mississippi, and South Carolina. In respect to the five locations that currently have active 

APHIS permits; the new permit application (11-052-101rm) does not expand the field 

sites beyond what is current authorized by APHIS or density (number) of trees that could 

be planted. With regard to the Dorchester County, South Carolina site, this location has 

previously been listed as a holding area for GE trees in previous permits/notifications. 

This new permit, which allows the release of GE Eucalyptus at this location does not 

change any conditions applied to that site. The Dorchester County site will remain a 

holding area for GE trees in containers where flowering is not allowed.

As identified in the new permit application, GE Eucalyptus trees with the new constructs 

would be released at the 5 existing locations in addition to the trees currently approved by 

APHIS for environmental release. The new permit application does not expand the 

number of trees or total acreage that could be planted at any of the 5 confined release 

sites currently approved by APHIS. It is also reasonably foreseeable that the applicant 

may request to further extend the permit for this environmental release for additional 

years beyond the 3 years indicated in the pending permit to observe the growth of these 

trees to maturity. Moreover, APHIS has received a petition for a determination of 

nonregulated status of a subset of these transgenic Eucalyptus trees, however, the 

environmental effects of that petition will be analyzed in a separate NEPA document. 

The temporary change from agricultural crops to a tree crop may result in a temporary 

change in resident animal and plant species, but after harvest and termination of the 

proposed permits, it is reasonably foreseeable that the land will return to agriculture or be 

replanted to tree production or research. At the end of the field test, transgenic plant 

material will be removed from the test site and/or destroyed in accordance with 

supplemental permit conditions established for these permits. The effect of propagation, 

cross-breeding, and invasiveness on listed species or native ecosystems was not 

considered a potential impact because of the supplemental permit conditions that prevents 

these events from occurring. Fire risk to TES was not considered a potential impact 

because the small size of the field trial combined with the unlikelihood of TES presence 

within the proposed test area. Likewise, the effect on hydrology was not considered an 

impact because of the small size of the field trial combined with the distance from water 

sources that listed species rely upon. In addition, the small size of the field plots and the 

distance between test sites spread over thousands of miles indicate no cumulative effects 

would result from APHIS issuing the proposed field release permits. Therefore the only 

past, present, and reasonably foreseeable actions associated with the locations for the 

proposed releases under permit are those related to agricultural or forest tree production. 

Based on the analysis provided in the EA, APHIS has determined that there are no past, 

present, or reasonably foreseeable actions that would aggregate with effects of the 

proposed action to create cumulative impacts or reduce the long-term productivity or 

sustainability of any of the resources (soil, water, ecosystem quality, biodiversity, etc.) 

associated with the release sites or the ecosystem in which they are situated. No 

resources will be significantly impacted due to cumulative impacts resulting from the 

proposed action. 
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E. Risks to Threatened and Endangered Species

APHIS analyzed the potential for effects from the preferred alternative of this EA on 

federally listed threatened and endangered species (TES) and species proposed for listing, 

as well as designated critical habitat and habitat proposed for designation, as required 

under Section 7 of the Endangered Species Act. APHIS worked with the United States 

Fish and Wildlife Service to obtain species lists and information and critical habitat 

information for the proposed field site locations. After analyzing the potential for any 

effect, APHIS has reached a determination that the proposed environmental release and 

interstate movement of GE Eucalyptus trees will have no effect on federally listed 

threatened or endangered species or species proposed for listing, and no effect on 

designated critical habitat or habitat proposed for designation. Consequently, 

consultation with the United States Fish and Wildlife Service is not required for the 

action described in the preferred alternative of this EA. Appendix IV includes the BRS 

analysis of threatened and endangered species in the areas of the field releases.

F. Impacts on Unique Characteristics of Protected Areas

Appendix V includes the BRS analysis of the release locations for their proximity to State 

and Federal protected areas. Based on this analysis, issuance of the permit to allow 

flowering of these additional constructs is not expected to impact unique characteristics 

of protected areas such as park lands, wetlands, wild and scenic areas, or ecologically 

critical areas. 

None of the release sites are within protected areas. Common forestry practices that 

would be carried out in the cultivation of the engineered Eucalyptus are not expected to 

deviate from current practices. The trees will be grown on agricultural land currently 

suitable for the field testing of forest trees. There are no proposed major ground 

disturbances; no new physical destruction or damage to property; no alterations of 

property, wildlife habitat, or landscapes; and no prescribed sale, lease, or transfer of 

ownership of any property. This action is limited to issuance of the permit. This action 

would not convert land use to nonagricultural use. Standard forestry or agricultural 

practices for land preparation, planting, irrigation, and harvesting of plants would be used 

on the permitted sites, including the use of EPA registered pesticides. Applicant’s 

adherence to EPA label use restrictions for all pesticides will mitigate potential impacts 

to the human environment. 

G. Other Considerations

Consideration of Executive Orders, Standards and Treaties Relating to 

Environmental Impacts.

Executive Order (EO) 12898, "Federal Actions to Address Environmental Justice in 

Minority Populations and Low-Income Populations," requires Federal agencies to 

conduct their programs, policies, and activities that substantially affect human health or 

the environment in a manner so as not to exclude persons and populations from 
Page 38 of 91

participation in or benefiting from such programs. It also enforces existing statutes to 

prevent minority and low-income communities from being subjected to 

disproportionately high and adverse human health or environmental effects.

EO 13045, “Protection of Children from Environmental Health Risks and Safety Risks,” 

acknowledges that children may suffer disproportionately from environmental health and 

safety risks because of their developmental stage, greater metabolic activity levels, and 

behavior patterns, as compared to adults. The EO (to the extent permitted by law and 

consistent with the agency’s mission) requires each Federal agency to identify, assess, 

and address environmental health risks and safety risks that may disproportionately affect 

children. 

Each alternative was analyzed with respect to the above EO 12898 and 13045. The 

human health and environmental impacts of the action alternatives are presented in 

Section V of this EA. No human health or environmental effects were identified for any 

of the action alternatives that would have a disproportionate adverse effect or that would 

exclude a particular group of persons or populations, including minority and low-income 

populations, or children, from expected benefits.

EO 13112, “Invasive Species”, states that federal agencies take action to prevent the 

introduction of invasive species and provide for their control and to minimize the 

economic, ecological, and human health impacts that invasive species cause. As 

presented in Section V of this EA, the hybrid species of Eucalyptus being grown is not 

considered an invasive species and does not establish itself without human intervention. 

Based on historical experience with the Eucalyptus in these field tests, the engineered 

plant is not expected to have an increased invasive potential. 

Executive Order 12114, “Environmental Effects Abroad of Major Federal Actions”

requires Federal officials to take into consideration any potential environmental effects 

outside the U.S., its territories and possessions that result from actions being taken. 

APHIS has given this due consideration and does not expect an environmental impact 

outside the United States should APHIS choose any of the two alternatives. These 

confined field tests are being conducted in the continental U.S. and would not be 

expected on have environmental effects outside of the U.S.

Migratory Bird Treaty Act, 1918 as amended and Executive Order 13186. Migratory 

birds include all native wild birds found in the United States except the house sparrow, 

starling, feral pigeon, and resident game birds such as pheasant, grouse, quail, and wild 

turkeys. A reference list of migratory game birds is found in Title 50, Code of Federal 

Regulations, Part 10. The Migratory Bird Treaty Act makes it unlawful for anyone to 

kill, capture, collect, possess, buy, sell, trade, ship, import, or export any migratory bird, 

including feathers, parts, nests, or eggs. Executive Order 13186 “Responsibilities of 

Federal Agencies to Protect Migratory Birds” requires Federal officials to consider the 

impacts of planned actions on migratory bird populations and habitats for all planning 

activities. APHIS has determined that it is reasonable to assume that the activities at the 

field test sites such as planting, collecting samples and eventual harvest of the trees 
Page 39 of 91

should have no adverse impact on migratory birds since they would not be expected to

nest or permanently inhabit these types of field test sites.

Consistency of Proposal with other Environmental Requirements:

The proposal is believed to be consistent with other environmental requirements. This 

environmental assessment was prepared in accordance with: (1) The National 

Environmental Policy Act of 1969 (NEPA), as amended (42 U.S.C 4321 et seq.); (2) 

regulations of the Council on Environmental Quality for implementing the procedural 

provisions of NEPA (40 CFR parts 1500-1508); (3) USDA regulations implementing 

NEPA (7 CFR part 1b); and (4) APHIS’ NEPA Implementing Procedures (7 CFR part 

372).

H. Conclusion

As outlined under the Purpose and Need sections of this document, this EA was prepared 

by APHIS to evaluate the potential impacts to the human environment resulting from the 

inclusion of new constructs in field test releases of flowering Eucalyptus, which could 

potentially lead to a lack of confinement of the field tests and other impacts to the 

environment. APHIS has evaluated the permit application to determine whether the 

environmental release and interstate movement, with appropriate conditions imposed by 

APHIS, can be carried out while preventing the dissemination and establishment of plant 

pests. After preparing this EA, APHIS has concluded that because there is no increase in 

the number of sites or acreage where trees will be allowed to reach maturity and flower, 

over those already authorized by APHIS to flower under permits 08-011-106rm, 08-014-

101rm, 10-112-101r and 11-201-103r, there is no substantially greater risk of loss of 

confinement and risk to the environment. Based on the analysis and information 

provided in this EA and supporting permit application, the new genes that are engineered 

into the trees should also not pose any greater risk of loss of confinement and risk to the 

environment. The addition of new genes to increase cold tolerance, alter lignin and 

growth should not compromise the ability of these to remain confined field tests. 

Therefore, APHIS concludes that the releases will remain as confined field tests and that 

the release and interstate movement of genetically engineered trees will not pose a 

significant plant pest risk. In addition, no threatened and endangered species or critical 

habitat would be impacted by allowing the trees reach maturity and flower.

VI. Listing of Agencies and Persons Consulted

James M. Vose - USDA-Forest Service Coweeta Hydrologic Laboratory, Otto, NC

Chelcy R. Ford - USDA-Forest Service Coweeta Hydrologic Laboratory, Otto, NC

Jody Smithen – US Fish and Wildlife Service – Daphne, Alabama Field Office

Kathy Chapman - US Fish and Wildlife Service - Coastal Georgia Field Office

James Harris – US Fish and Wildlife Service – Lacombe, Louisiana Field Office

Laura Zimmerman - US Fish and Wildlife Service – Charleston, South Carolina Field 

Office
Page 40 of 91

Caroline Stahaller - US Fish and Wildlife Service - Panama City, Florida Field Office

Brad Rick - US Fish and Wildlife Service - Vero Beach, Florida Field Office

Candice Martino - US Fish and Wildlife Service - Jacksonville, Florida Field Office
Page 41 of 91

VII. References

APHIS. 2004. The emergence and colonization of Cryptococcus gattii in British 

Columbia. Emerging disease notice. December 22, 2004. Available online at:

http://www.aphis.usda.gov/vs/ceah/cei/taf/emergingdiseasenotice_files/cryptococ

cal_disease_britishcolumbia.htm.

Ashton, D. H. 1981. Fire in tall open-forests (wet sclerophyll forests). Pages 339-366 in

A. M. Gill, R. H. Groves, and I. R. Noble, editors. Fire and the Australian biota. 

The Australian Academy of Science, Canberra City, ACT.

Baker, S. S., K. S. Wilhelm, and M. F. Thomashow. 1994. The 5'-region of Arabidopsis 

thaliana cor15a has cis-acting elements that confer cold-, drought- and ABAregulated gene

expression. Plant Mol Biol. 24:701-713.

Barbour, E. L. and N. Spencer. 2000. The potential of a crossing technique for 

interspecific hybridization between E. globulus and E. dunnii. Pages 390-394 in

Hybrid Breeding and Genetics of Forest trees” Proceedings of QFRI/CRC_SPF 

Symposium, 9-14 April 2000 

Bell, D. T. and J. E. Williams. 1997. Eucalyptus ecophysiology. Pages 168-196 in J. E. 

Williams and J. Woinarski, editors. Eucalyptus Ecology: Individuals to 

Ecosystems. Cambridge University Press, Cambridge.

Brown, J. R. 2003. Ancient horizontal gene transfer. Genetics 4:121-132.

Calder, I. R., P. T. W. Rosier, K. T. Prasanna, and S. Parameswarappa. 1997. Eucalyptus 

water use greater than rainfall input-a possible explanation from southern India. 

Hydrology and Earth System Sciences 1:249-256.

Campinhos, E. N., I. Peters-Robinson, F. L. Bertolucci, and A. C. Privets. 1998. 

Interspecific hybridization and inbreeding effect in seed from Eucalyptus grandis 

x E. urophylla clonal orchard in Brazil. Genet. Mol. Biol. [online], 21, No. 3. 

Available from: <http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-

47571998000300014 &lng=en&nrm=iso>. 

Canadell, J., R. B. Jackson, J. R. Ehleringer, H. A. Mooney, O. E. Sala, and E. D. 

Schulze. 1996. Maximum rooting depth of vegetation types at the global scale. 

Oecologia 108:583-595.

Chaturvedi, S., M. Dyavaiah, R. A. Larsen, and V. Chaturvedi. 2005. Cryptococcus gattii 

in AIDS patients, southern California. Emerging Infectious Diseases 11:1686-

1692.

Couch, C. A. and R. J. McDowell. 2006. Flint River Basin Regional Water Development 

and Conservation Plan. Georgia Dept. of Natural Resources, Environmental 

Protection Division. Page 242.

Cremer, K. W. 1977. Distance of seed dispersal in Eucalyptus estimated from seed 

weight. Australian Forest Research 7:225-228.

Datta, K., K. H. Bartlett, and K. A. Marr. 2009. Cryptococcus gattii: Emergence in 

Western North America: Exploitation of a Novel Ecological Niche. 

Interdisciplinary Perspectives on Infectious Diseases 2009:1-8.

Davidson, J. 1995. Ecological aspects of Eucalyptus plantations.in Proceedings of the 

Regional Expert Consultation on Eucalyptus. FAO Regional Office for Asia and 

the Pacific, Bangkok.
Page 42 of 91

de Assis, T. F. 1996. Melhoramento genético do eucalipto. Informe Agropecuário, Belo 

Horizonte (Brazil) 18:32-51.

Eljarrat, E. and D. Barceló. 2001. Sample handling and analysis of allelochemical 

compounds in plants. Trends in Analytical Chemistry 20:584-590.

Espinosa-Garcia, F. J., E. Martinex-Herandez, and A. Quiroz-Flores. 2008. Allelopathic 

potential of Eucalyptus spp plantations on germination and early growth of annual 

crops. . Allelopathy Journal 21:25-37.

Esser, L. L. 1993. Eucalyptus globulus. In: Fire Effects Information System. U.S. 

Department of Agriculture, Forest Service - Rocky Mountain Research Station, 

Fire Sciences Laboratory (Producer) - Available: 

http://www.fs.fed.us/database/feis/.

Farley, K., J. E., and J. R.B. 2005. Effects of afforestation on water yield: a global 

synthesis with implications for policy. Global Change Biology 11:1565-1576.

Ferguson, J. J. and B. Rathinasabapathi. 2003. Allelopathy: How plants suppress other 

plants. University of Florida HS944 http://edis.ifas.ufl.edu/hs186.

Forsyth, G. G., D. M. Richardson, et al. 2004. A rapid assessment of the invasive status

of Eucalyptus species in two South African provinces. South African Journal of 

Science 100: 75-77.

Fuchs, R. L., J. E. Ream, B. G. Hammond, M. W. Naylor, R. M. Leimgruber, and S. A. 

Berberich. 1993. Safety assessment of neomycin phosphotransferase II (NPTII) 

protein. Nature Biotechnology 11:1543-1547.

Funnell, D. L. and J. F. Pedersen. 2006. Reaction of Sorghum Lines Genetically

Modified for Reduced Lignin Content to Infection by Fusarium and Alternaria 

spp. Plant Disease 90: 331-338.

Gill, A. M. 1997. Eucalyptus and Fires: Interdependent or Independent. Pages 151-167 in

J. E. Williams and J. Woinarski, editors. Eucalyptus Ecology: Individuals to 

Ecosystems. Cambridge University Press, Cambridge.

Golladay, S. W., P. Gagnon, M. Kearns, J. M. Battle, and D. W. Hicks. 2004. Response 

of freshwater mussel assemblages (Bivalvia:Unionidae) to a record drought in the 

Gulf Coastal Plain of southwestern Georgia J. N. Am. Benthol. Soc. 23:494-506.

Gore, P. and B. M. Potts. 1995. The genetic control of flowering time in Eucalyptus 

globulus, E. nitens and their F1 hybrids. Pages 241-242 in Eucalyptus Plantations: 

Improving Fiber Yield and Quality, Proc CRCTHF - IUFRO Conference. CRC 

for Temperate Hardwood Forestry, Hobart. Australia, 19 -24 Feb.

Griffin, A. R., I. P. Burgess, and L. Wolf. 1988. Patterns of Natural and Manipulated 

Hybridisation in the Genus Eucalyptus L'Herit - A review. Australian J. Botany 

36:41-66.

Grose, R. J. 1960. Effective seed supply for natural regeneration of Eucalyptus 

delegatensis. Journal of Australian Pulp and Paper Industry Association 13:131-

147.

Groves, R. H., editor. 1994. Australian vegetation. Cambridge University Press, 

Cambridge.

Hartney, V. J. 1980. Vegetative propagation of the eucalypts. Australian Forest Research 

10:191-211.

House, S. M. 1997. Reproductive biology of eucalypts. Pages 30-56 in J. E. Williams and 

J. Woinarski, editors. Eucalyptus Ecology: Individuals to Ecosystems. Cambridge 

University Press, Cambridge.
Page 43 of 91

Hsieh, T. H., J. T. Lee, P. T. Yang, L. H. Chiu, Y. Y. Charng, Y. C. Wang, and M. T. 

Chan. 2002. Heterology expression of the Arabidopsis C-repeat/dehydration 

response element binding factor 1 gene confers elevated tolerance to chilling and 

oxidative stresses in

transgenic tomato. Plant Physiol. 129:1086-1094.

Kaneko, T., Y. Nakamura, S. Sato, E. Asamizu, T. Kato, S. Sasamoto, A. Watanabe, K. 

Idesawa, A. Ishikawa, K. Kawashima, T. Kimura, Y. Kishida, C. Kiyokawa, M. 

Kohara, M. Matsumoto, A. Matsuno, Y. Mochizuki, S. Nakayama, N. Nakazaki, 

S. Shimpo, M. Sugimoto, C. Takeuchi, M. Yamada, and S. Tabata. 2000. 

Complete Genome Structure of the Nitrogen-fixing Symbiotic Bacterium 

Mesorhizobium loti. DNA Research 7:331-338.

Kaneko, T., Y. Nakamura, S. Sato, K. Minamisawa, T. Uchiumi, S. Sasamoto, A. 

Watanabe, K. Idesawa, M. Iriguchi, K. Kawashima, M. Kohara, M. Matsumoto, 

S. Shimpo, H. Tsuruoka, T. Wada, M. Yamada, and S. Tabata. 2002. Complete 

genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium 

japonicum DNA Research 9:189-197.

Kasuga, M., Q. Liu, S. Miura, K. Yamaguchi-Shinozaki, and K. Shinozaki. 1999. 

Improving plant drought, salt, and freezing tolerance by gene transfer of a single 

stressinducible

transcription factor. Nature Biotechnology 17:287-291.

Keese, P. 2008. Review Article: Risks from GMOs due to horizontal gene transfer. 

Environ. Biosafety Res. 7: 123-149.

Khan, E. A., M. A. Khan, H. K. Ahmad, and K. F. U. 2004. Allelopathic effects of 

Eucalyptus leaf extracts on germination and growth of cotton 

(Gossypiumhirsutum). Pakistan J. Weed Sci. Res. 10:145-150.

Kidd, S. E., Y. Yat Chow, S. Mak, P. J. Bach, H. Huiming Chen, A. O. Hingston, J. W. 

Kronstad, K. H., and K. H. Bartlett. 2007. Characterization of Environmental 

Sources of the Human and Animal Pathogen Cryptococcus gattii in British 

Columbia, Canada, and the Pacific Northwest of the United States. . Applied and 

Environmental Microbiology 73:1433-1443.

Klee, H. J. and S. G. Rogers. 1989. Plant gene vectors and genetic transformation: plant 

transformation systems based on the use of Agrobacterium tumefaciens. Pages 1-

23 in I. K. Vasil, editor. Cell Culture and Somatic Cell Genetics of Plants. 

Academic Press, Orlando, FL.

Kohli, R. K., D. R. Batish, and H. P. Singh. 2006. Allelopathic interactions in 

agroecosystems. Pages 465-493 in R. Reigosa, N. Pedrol, and L. Gonzáles, 

editors. Allelopathy: A Physiological Process with Ecological Implications. 

Springer.

Kolar, C.S. and D. M. Lodge. 2001. Progress in invasion biology: predicting invaders. 

Trends in Ecology and Evolution 16:199-204.

Koonin, E. V., K. S. Makarova, and L. Aravind. 2001. Horizontal gene transfer in 

prokaryotes: Quantification and classification. Annual Review of Microbiology 

55:709-742.

Ladiges, P. Y. 1997. Phylogenetic history and classification of Eucalyptus. Pages 16-29 

in J. E. Williams and J. Woinarski, editors. Eucalyptus Ecology: Individuals to 

Ecosystems. Cambridge University Press, Cambridge.
Page 44 of 91

Linacre, N. A. and P. K. Ades. 2004. Estimating isolation distances for genetically 

modified trees in plantation forestry. Ecological Modeling 179:247-257.

Mallik, A. U. 2008. Allelopathy in forested ecosystems.in R. S. Zeng, A. U. Mallik, and 

S. M. Lou, editors. Allelopathy in sustainable agriculture and forestry. Springer.

Maruyama, K., Y. Sakuma, M. Kasuga, I. Y., M. Seki, H. Goda, Y. Shimada, S. Yoshida, 

K. Shinozaki, and K. Yamaguchi-Shinozaki. 2004. Identification of coldinducible downstream genes of the Arabidopsis DREB1A/CBF3 transcriptional 

factor using two

microarray systems. Plant Journal 38:982-993.

Meskimen, G. and J. K. Francis. 1990. Rose Gum Eucalyptus.in R. M. Burns and B. H. 

Honkala, editors. Silvics of North America: Volume 2. Hardwoods. Agriculture 

Handbook 654. U.S. Department of Agriculture, Forest Service, Washington, DC.

Nandal, D. P. S., S. S. Bisla, S. S. Narwal, and J. C. Kaushik. 1994. Allelopathic 

Interactions in Agroforestry Systems Pages 93-130. in S. S. Narwal and P. Tauro, 

editors. Allelopathy in Agriculture and Forestry. Scientific Publishers, Jodhpur, 

India.

Nandal, D. P. S. and A. Dhillon. 2005. Allelopathic effects of poplar (Populus deltoides 

Bartr Ex Marsh): an assessment on the response of wheat varieties under 

laboratory and field conditions. Fourth World Congress in Allelopathy,, Wagga 

Wagga, NSW Australia.

Narusaka, Y., K. Nakashima, Z. K. Shinwari, Y. Sakuma, T. Furihata, H. Abe, M. 

Narusaka, K. Shinozaki, and K. Yamaguchi-Shinozaka. 2003. Interaction between 

two cis-acting elements, ABRE and DRE, in ABA-dependent expression of 

Arabidopsis rd29A gene in

response to dehydration and high-salinity stresses. Plant J. 34:137-148.

Narwal, S. S. 2006. Allelopathy in ecological sustainable agriculture.in M. J. Reigosa, N. 

Pedrol, and L. Gonzáles, editors. Allelopathy: A Physiological Process with 

Ecological Implications. Springer.

Ong, C. K. 1993. On the difference between competition and allelopathy. Agroforestry 

Today 5:12-14.

Pacheco, I. A. 1987. Polinização de Eucalyptus saligna Smith (Myrtaceae) por Apis 

mellifera L. 1758 (Hymenoptera, Apidae). Universidade de São Paulo, Piracicaba.

Pacheco, I. A., P. Y. Kageyama, F. M. Wiendl, and E. B. Filho. 1986. Estudo da 

dispersão de pólen de Eucalyptus saligna Smith por abelhas Apis mellifera L. 

utilizando-se o radiofósforo. IPEF Piracicaba (Brazil 34:47-52.

Pedersen, J. F., K. P. Vogel, and D. L. Funnell. 2005. Impact of reduced lignin on plant 

fitness. Crop Science 45:812-819.

Peters, G. B., J. S. Lonie, and G. F. Moran. 1990. The breeding systems, genetic diversity 

and pollen sterility in Eucalyptus pulverulenta, a rare species with small disjunct 

population. Australian J. Botany 38:559-570.

Pino, M. T., J. S. Skinner, E. J. Park, Z. Jeknić, H. P.M., M. F. Thomashow, and T. H. 

Chen. 2007. CBF3/DREB1A expression and plays a central role in stress 

tolerance in

Arabidopsis. Use of a stress inducible promoter to drive ectopic AtCBF 

expression improves potato freezing tolerance while minimizing negative effects 

on tuber yield. Plant Biotechnol J. 5:591-604.
Page 45 of 91

Potts, B. M., R. C. Barbour, A. B. Hingston, and V. R.E. 2003. Turner Review: the risk 

of genetic pollution of native Eucalyptus gene pools. Australian J. Botany 51:1-

25.

Potts, B. M. and H. S. Dungey. 2004. Interspecific hybridization of Eucalyptus: Key 

issues for breeders and geneticists. New Forest 27:115-138.

Pound, L. M., M. A. B. Wallwork, B. M. Potts, and M. Sedgley. 2002. Selfincompatibility in Eucalyptus globulus ssp. globulus (Myrtaceae). Australian 

Journal of Botany 50:365-372.

Reigosa, M. J. and L. Gonzáles. 2006. Forest Ecosystems and Allelopathy. Pages 451-

463 in R. Reigosa, N. Pedrol, and L. Gonzáles, editors. Allelopathy: A 

Physiological Process with Ecological Implications. Springer.

Riechmann, J. L. and E. M. Meyerowitz. 1998. The AP2/EREBP family of plant 

transcription

factors. . Biol Chem 379:633-646.

Richardson, A. D. 1998. Forestry Trees as Invasive Aliens. Conservation Biology 12:

18-26.

Rockwood, D. L., D. R. Carter, and J. A. Stricker. 2004. Commercial Tree Crops for 

Phosphate Mine Lands, Fourth Year Cumulative Progress Report - Prepared for 

Florida Institute of Phosphate Research FIPR Project Number 99-03-141R. 

University of Florida.

Sanginga, N. and M. J. Swift. 1992. Nutritional effects of Eucalyptus litter on the growth 

of maize (Zea mays). Agriculture, Ecosystems and Environment 41:55-65.

Sellers, C. H. (1910). Eucalyptus: its history, growth, and utilization, A.J. Johnston -

Original from Harvard University. 93 pp.

Skolmen, R. G. and F. T. Ledig. 1990. Bluegum Eucalyptus.in R. M. Burns and B. H. 

Honkala, editors. Silvics of North America: Volume 2. Hardwoods. Agriculture 

Handbook 654. U.S. Department of Agriculture, Forest Service, Washington, DC. .

Stricker, J., D. L. Rockwood, S. A. Segrest, G. R. Alker, R. M. Prine, and D. R. Carter. 

2000. Short rotation woody crops for Florida. Pages 15-23 in Proc. 3rd Biennial 

Short Rotation Woody Crops Operations Working Group Conference, October 

10-13, 2000, Syracuse, NY.

Sunder, S. 1995. The Ecological, Economic and Social Effects of Eucalyptus. in

Proceedings of the Regional Expert Consultation on Eucalyptus. FAO Regional 

Office for Asia and the Pacific, Bangkok.

Upton, A., J. A. Fraser, S. E. Kidd, C. Bretz, K. H. Bartlett, J. Heitman, and K. A. Marr. 

2007. First Contemporary Case of Human Infection with Cryptococcus gattii in 

Pugent Sound: Evidence for Spread of the Vancouver Island Outbreak. . Journal 

of Clinical Microbiology 45:3086-3088.

Volker, P. W. 1995. Evaluation of Eucalyptus nitens x globulus for commercial forestry. 

Pages 222-225 in Eucalyptus Plantations: Improving Fiber Yield and Quality, 

Proc CRCTHF - IUFRO Conference. CRC for Temperate Hardwood Forestry, 

Hobart. Australia. 19-24 Feb 

Wellington, A. B. 1989. Seedling regeneration and population dynamics of eucalyptus. 

Pages 155-167 in J. C. Noble and R. A. Bradstock, editors. Mediterranean 

Landscapes in Australia: Mallee Ecosystems and their Management. CSIRO, 

Melbourne.
Page 46 of 91

White, K. J. 1995. Silviculture of Eucalyptus Plantings – Learning from the Region 

(Australia).in Proceedings of the Regional Expert Consultation on Eucalyptus. 

FAO Regional Office for Asia and the Pacific, Bangkok.

Wood, D., J. Setubal, R. Kaul, D. Monks, J. Kitajima, V. Okura, Y. Zhou, L. Chen, G. 

Wood, A. J. N., L. Woo, Y. Chen, I. Paulsen, J. Eisen, P. Karp, S. Bovee, D., P. 

Chapman, J. Clendenning, G. Deatherage, W. Gillet, C. Grant, T. Kutyavin, R. 

Levy, M. Li, E. McClelland, C. Saenphimmachak, Z. Wu, P. Romero, D. Gordon, 

S. Zhang, H. Yoo, Y. Tao, P. Biddle, M. Jung, W. Krespan, M. Perry, B. GordonKamm, L. Liao, S. Kim, C. Hendrick, Z. Zhao, M. Dolan, F. Chumley, S. Tingey, 

J. Tomb, M. Gordon, M. Olson, and E. Nester. 2001. The Genome of the Natural 

Genetic Engineer Agrobacterium tumefaciens C58. Science 294:2317-2323.

Xuan, T. D., S. Tawata, T. D. Khanh, and I. M. Chung. 2005. Decomposition of 

Allelopathic Plants in Soil. J. Agronomy and Crop Science 191:162-171.

Yamaguchi-Shinozaki, K. and K. Shinozaki. 1993. Characterization of the expression of 

a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its 

promoter in

transgenic plants. Mol. Gen. Genet. 23:331-340.

Yamaguchi-Shinozaki, K. and K. Shinozaki. 1994. A novel cis-acting element in an 

Arabidopsis gene is involved in responsiveness to drought, low-temperature, or 

high-salt

stress. Plant Cell 6:251-264.

Zambryski, P. 1988. Basic processes underlying Agrobacterium mediated DNA transfer 

to plant cells. Annual Review of Genetics 22:1-30.
Page 47 of 91

APPENDIX I: Status of Existing Field Tests Allowed to Flower

The applicant has been authorized by APHIS to allow transgenic Eucalyptus trees flower 

under APHIS permits 06-325-111r (renewed as 10-112-101r); 08-151-101r (now covered 

under 08-014-101rm); 08-011-106-rm and 08-014-101rm. The following is a summary 

of information that has been collected by the applicant and provided to APHIS as part of 

their annual reporting requirements under the existing permits that allow flowering.

All transgenic and non-transgenic trees in field tests covered under Permit 06-325-111r 

(renewed as 10-112-101r), spanning 6.2 acres in Baldwin County, Alabama produced 

flowers in the late summer months of years 2007 to 2010. The field test from Permit 08-

151-101r (now covered under 08-014-101rm) spanning 1.4 acres in Highlands County, 

Florida produced mature flowers on all transgenic and non-transgenic trees in the late 

summer months of 2008, to 2010. Three field tests flowered in the late summer months 

of 2010 under Permit 08-011-106-rm; the field test spanning 0.75 acre in Baldwin 

County, Alabama; the field test spanning 0.2 acres in Evans County, Georgia; and the 

field test spanning 0.7 acre in Jasper, County Texas. A field test covered under permit 08-

014-101rm in Gadsden County, Florida spanning 0.2 acres flowered in 2010 as well. 

Where flowering occurred in these tests, several hundred to a few thousand flowers were 

estimated on each tree. For the field tests in Alabama, Georgia, and Texas that flowered 

in summer of 2010; the transgenic trees produced more flowers per tree compared to the 

non-transgenic controls as a result of significant cold damage to non-transgenic trees. In 

Florida, there was no difference in the number of flowers produced by both transgenic 

and non-transgenic control trees of the same parental genotype. Observations from the 

replicated field study conducted in Alabama under permit 06-325-111r (renewed as 10-

112-101r) and Florida under permit 08-151-101r (now covered under 08-014-101rm) 

confirmed that cold tolerant translines grown in these field test did not produce any 

pollen. The results to date have shown that the barnase gene cassette that has been 

engineered into these trees is effective at preventing pollen formation.

Mature (but not yet opened) capsules have been collected from non-transgenic and 

transgenic trees in the following field tests: Baldwin County, Alabama under Permit 06-

325-111r (renewed as 10-112-101r) in January 2008 and 2009; Highlands County, 

Florida under Permit 08-151-101r (now covered under 08-014-101rm) in January 2009 

and 2010; Evans County, Georgia; and Jasper County, Texas under Permit 08-011-106rm 

in January 2011. 

Replicate capsule samples were collected from transgenic trees and non-transgenic 

controls in the field tests. For the January 2008 collection only from Baldwin County, AL 

(Permit 06-325-111r - renewed as 10-112-101r), capsules from a subset of transgenic 

trees plus non-transgenic controls were dried in the laboratory and allowed to open to 

evaluate the presence of seed or seed like structures in the capsules. Approximately 100 

capsules for each of the two replicate samples were analyzed. Microscopic examination 

of the material inside the capsules did not show any seed or seed like structures in 

capsules of either non-transgenic or transgenic lines. Controlled germination studies of 

the material extracted from the capsules did not produce any germinating seeds. 
Page 48 of 91

Mature seed capsules, prior to opening, were sampled from select trees in three field tests 

in Baldwin County, Alabama (Permit 06-325-111r - renewed as 10-112-101r); and trees 

from the single field test in Highlands County, Florida (Permit 08-151-101r - now 

covered under 08-014-101rm) in early March 2009. Samples consisted of approximately 

70 to 100 capsules collected from different positions in the crown. The capsules were 

returned to ArborGen’s greenhouse facility where they were dried and contents of the 

capsules were extracted and stored at 4°C. A controlled germination test was conducted 

using approximately 0.1 g of the extracted contents of each sample spread on moist filter 

paper in a standard Petri dish. Open pollinated seed of EH1 obtained from Brazil were 

germinated as control seedlings for comparison.

Of the samples collected and analyzed from trees in Baldwin County, AL, approximately 

4% of the samples showed a low level of germination while approximately 83% of the 

samples collected from trees in the field trial in Highlands County, FL, showed 

germination, including samples from both transgenic and non-transgenic control trees. 

For both transgenic and non-transgenic tree samples analyzed, the applicant observed 2-8 

seedlings for each 0.1g sample plated for germination. In the literature, for the same 

sample size tested for E. grandis 31 to 65 seedlings have been reported. The applicant 

indicates that, as expected from limited self-pollination that may have occurred in these 

trees, the number of viable seeds produced in these tests is much lower than would be 

expected for open pollinated mixed stands of Eucalyptus.

Mature seed capsules, collected prior to opening, were sampled from the single field test 

in Highlands County, Florida in early March 2010 (Permit 08-151-101r - now covered 

under 08-014-101rm). As expected from self-fertilization, a very low number of viable 

seeds (less than 1) per capsule were observed for transgenic lines and non-transgenic 

controls trees tested compared to the number of viable seeds ( ~25 per capsule) expected 

from an open pollinated mixed stands of Eucalyptus.

The controlled seed germination studies with seed capsules collected over three years 

(2008 to 2010) from field trials allowed to flower have indicated that either no, or a very 

low number of viable seeds are produced in translines and control EH1 trees compared to 

what would be expected for an open-pollinating mixed stand of Eucalyptus. These data 

are consistent with the hypothesis that limited self-pollination can occur from viable 

pollen produced by the non-transformed control trees. The applicant has also not 

observed any volunteer seedlings in or around the test sites where trees have flowered 

and produced seed capsules. Volunteer monitoring and reporting the presence of 

volunteers is required in the supplemental permit conditions.

Similar collections of seed capsules have also occurred during 2011 in field tests from 

Evans County, Georgia and Jasper County, Texas. The seed has not yet been analyzed.

Monthly field test monitoring observations have not identified any differences in diseases 

and insects or other non-target organisms between the transgenic and non-transgenic trees 

in the field test.
Page 49 of 91

APPENDIX II: Hydrology considerations for planted 

Eucalyptus submitted by ArborGen LLC

Submitted by ArborGen. LLC to USDA APHIS BRS in support of consideration for

approval of permits for field trials of Eucalyptus at multiple sites.

August 12, 2008

Introduction

The relevant scientific literature and conclusions drawn by experts in the field of

hydrology, ecology, and plantation management on the hydrology of Eucalyptus

plantations are discussed in this document. Extensive research on hydrology has been

conducted in countries where large plantations of Eucalyptus have been established for

many years, including India, China, South Africa and Brazil, as well as in its native

Australia. The FAO, in response to the criticisms and concerns expressed about

Eucalyptus plantations, has developed several expert reviews on the ecological impacts of

Eucalyptus (discussed below).

The main hydrological concerns voiced against Eucalyptus plantations are that they

deplete water supplies. The authors of an early FAO report (Poore and Fries, 1985) noted

that these same criticisms would apply equally to any other plantation tree species, and

that society tends to judge more harshly forestry crops relative to agricultural crops. As

stated in Poore and Fries, 1985, “... most crops in many parts of the world are of foreign

origin (wheat, maize, rice, potatoes, manioc, rubber, oil palm, coconut and many others.

No one is surprised either that the soil under agricultural crops becomes depleted if these

are continuously cropped without adding fertiliser. But both of these features are

considered grounds for criticism in forestry.” Ironically, Eucalyptus evolved to be water

efficient as the Australian continent itself became more dry (Davidson, 1995). In fact

Eucalyptus uses less water per unit weight of biomass produced than do other kinds of

trees (Chaturvedi, 1987) and many agricultural crops (Davidson, 1995).

It is also important to take into account the breadth of the genus Eucalyptus, often

referred to broadly as eucalypts, where different species have different characteristics

which may prove detrimental or beneficial under different situations. There are several

different ecological situations globally in which eucalypts may be planted: in place of

existing closed forest; in place of other natural vegetation such as savannah, scrub or

grassland; on degraded or waste land either as a potential crop or to assist in the control

of erosion or salinity; within agricultural land as shelter belts or as components of

agroforestry systems, or as intensively managed crops for wood production. It is

important to understand the particular application in order to evaluate the potential

ecological impact of Eucalyptus in a scientific manner. For example, the effect on soil

moisture content and the water table can only satisfactorily be judged with reference to

the pre-existing conditions before the establishment of the planting of Eucalyptus. In the

case of field trials of Eucalyptus requested in ArborGen’s permit applications, all sites are

on land previously managed for forestry, agricultural production or maintained as pasture
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land.

The Water Cycle

In considering the potential hydrological impacts of Eucalyptus it is important to put

these in the context of the water cycle. Poore and Fries (1985) provide a good overview

of the water cycle (see Figure 1 in Poore and Fries, 1985, or see Figure 1-1 in NRC,

2008). One key variable is the amount of rain that is intercepted by the canopy and is

then evaporated back into the atmosphere. As a result, such intercepted water does not

contribute to water in the soil. Of the water that does reach the soil some of this is

absorbed while some fraction runs off the surface or is evaporated. A certain amount of

water is maintained in the soil layer against the forces of gravity (called the 

‘fieldcapacity’ and dependent on soil texture and organic content) while any excess 

drains to the water table, the level at which the soil is permanently saturated. Depending 

on the depth of the water table it may be accessed by deep rooting plants such as trees. 

Even without roots that reach to the water table, plants may be able to access water from

deeper, wetter soil layers through capillary action, depending on the soil type, where

there can actually be an upward movement of water.

Through normal transpiration plant roots take up available water which is transported

through the stem to the leaves, the majority of which is lost to the atmosphere.

Evapotranspiration is the total water returned to the atmosphere through transpiration and

evaporation from the ground, bodies of water, plus intercepted water in the canopy. The

relative rates of evapotranspiration and precipitation are often compared in assessments

of hydrological systems. Where precipitation exceeds evapotranspiration then there is a

net water gain to groundwater or downstream systems. Where evapotranspiration

exceeds precipitation then the available water resources may be depleted. It is important

to understand, as pointed out in Poore and Fries (1985), that water loss is a “price that

plants must pay for growth”. When stomata in plant leaves close then photosynthesis and

growth both cease. In general terms, the rate of growth or biomass production in a plant

is proportional to its water use. Consequently fast growing trees, of any species, use

large quantities of water. As described below, Eucalyptus is actually more efficient in

terms of water used per gram of biomass produced than many other tree species.

Hydrology of Eucalyptus – Key Literature Reviews

There has been extensive literature published on the hydrology of Eucalyptus. Google

Scholar for example lists over 60,000 hits for the keywords ‘Eucalyptus’ and ‘water’. It

is therefore not possible to provide summaries of the entire breadth of the literature.

Several review articles are available including reviews sponsored by FAO that assessed

the ecological impacts of Eucalyptus plantations, including analysis of the impact of

Eucalyptus on hydrology. We provide here a summary of these reviews together with

data from some specific reports (see below) where the hydrology of Eucalyptus has been

studied in detail. FAO has also released two annotated bibliographies (FAO, 2002a,

2002b) that collate and summarize publications on environmental, social and economic

impacts of Eucalypts, and which include many references to water use. While there are

specific examples and geographic regions where Eucalyptus (and other trees) can
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negatively impact hydrology, in general, the literature indicates that Eucalyptus can be

grown in a sustainable manner and that its associated water use is not a major ecological

threat. Most of the reviews indicate that soil and water characteristics of the site should

be taken into consideration when establishing and maintaining a Eucalyptus plantation, in

the same way that would be appropriate for plantings of agricultural crops.

The first FAO review was published in 1985 by Poore and Fries. At that time the authors

suggested that there were relatively few existing studies in several important areas

including hydrology (Lima, 1984; Poore and Fries, 1985). Where comparative studies

showed that for dry alpine conditions the water regime for Eucalyptus did not differ from

adjacent grasslands (see Lima, 1984), this was attributed to Eucalyptus’ ability to control

the rate of transpiration, an evolutionary adaptation for survival of drought stress which is

often typical of the rainfall regimens of their native habitats. For deep soils and higher

rainfall Eucalyptus plantations might be expected to reduce streamflow or groundwater

recharge but that this is comparable to these same effects in pine plantations. In contrast,

the water intercepted and re-evaporated by the foliage, and therefore not available to the

soil, is less for Eucalyptus when compared to pines, due in part to the near vertical

orientation of leaves in Eucalyptus (Whitehead and Beadle, 2004). It was concluded that

the conditions of a particular site need to be taken into account as well as balancing local

demands for forest products and water.

By 1993, at an FAO sponsored regional Expert Consultation on Eucalyptus (White et al,

1995) more information was available about hydrology and Eucalyptus plantations.

These experts presented their experiences with Eucalyptus plantations from Asia. The

report recognized the potential benefits of Eucalyptus and noted that many of the

criticisms of the species were based on inappropriate government policies on

afforestation or social concerns rather than the biology of the trees themselves (see also

Casson, 1997). Calder et al (2004) highlights that many early policies were based on

public misconceptions about the impacts of forests on water. With regard to hydrological

effects on intercropping with other species, the experts in the FAO report concluded that

while Eucalyptus can have negative effects in drier climates, in regions where rainfall is

above 1,200 mm/year this is not expected to be a problem. The report suggests that for

Eucalyptus plantings in those regions where water is scarce or demanded by other

sectors, biomass production could be adjusted to match the amount of water available, for

example by planting fewer trees per unit area or by thinning existing plantations.

As part of this expert consultation Sunder (1995) reported that the overall use of water by

Eucalyptus is limited to the total rainfall of the area, in the absence of access of the tree to

the water table. He concluded that there is an equilibrium between rainfall and

evapotranspiration in Eucalyptus and that this does not differ significantly from other

trees. As an example, monthly evapotranspiration of an E. globulus plantation in

Portugal was the same as that of a natural open stand of cork oak (Quercus suber) with a

developing understory of shrubs (de Almeida and Riekerk, 1990). Patil (1995) reported

data on water consumption at sites in India, which although high in Eucalyptus, was the

most efficient in terms of water consumed per gram of biomass produced (see also Silva

et al 2004). In fact, water use efficiency in Eucalyptus actually increases with greater

water availability (Stape et al, 2004a, 2004b). Patil (1995) also noted that there were no
Page 52 of 91

hydrological impacts of Eucalyptus on adjacent crops at these sites. White (1995) stated

that large plantings of Eucalyptus may reduce water yield and lower water tables but this

varies from one situation to another and most importantly can be mitigated through

management practices such as changes in tree stocking regimes. The environmental

considerations of Eucalyptus are the same as those for agricultural crops. Davidson

(1995) noted that drawing water from shallow or deep wells to supply high water

demanding crops such as rice or cotton can have a greater impact on drawing down water

tables than fast growing tree plantations. He also concluded that many potential adverse

effects are reversible, as noted earlier by Poore and Fries (1985).

A review of the environmental issues of Eucalyptus plantations in Brazil was published

by Oak Ridge National Laboratory (Couto and Betters, 1995). This report summarized

that the hydrology of Eucalyptus plantations was comparable to other tree plantations or

natural forest cover and that any effects would largely depend on management practices.

Numerous studies demonstrate that forest cover and any changes in this alter water yield:

reducing forest cover typically increases water yield and vice versa (Bosch and Hewlett,

1982; Sahin and Hall, 1996).

More recent reviews support the points made above. Binkley and Stape (2004) contend

that very large tree plantations must address similar issues of sustainability as seen in

agriculture. They refer to the many hundreds of trials that have been conducted in Brazil,

with particular reference to a very large watershed project conducted in collaboration

with Aracruz Cellulose Company (reported by Almeida et al 2007, described in more

detail below). Binkley and Stape conclude that in semi-arid environments afforestation

with any species of trees may increase water use, lower ground water levels and reduce

streamflow. Given appropriate silvicultural management however, wood production

should face no barriers to sustainability.

Whitehead and Beadle (2004) provided a comprehensive review of the physiological

regulation of water use in Eucalyptus. These species have evolved several mechanisms

to allow them to cope with drought conditions in their native habitats. These include

dynamic changes in leaf area index (LAI), arrangement of leaves, high stomatal

sensitivity to air saturation deficit, osmotic manipulation to maintain turgor in leaves, as

well as an ability to form deep roots. Maximum potential rates of photosynthesis are high

in Eucalyptus compared to other broad-leaved trees, but actual rates are often much less

because of water limitations. Some examples are noted where Eucalyptus plantings have

led to reductions in yields of water catchments. Conversely, the high water usage by

Eucalyptus may be valuable in purposefully lowering water tables to reduced potential

salinity problems. It is therefore important to assess productivity and water use in

relation to climate variables, nutrient supply and options for silvicultural management,

and careful matching of species to sites where available water may be limited. One of the

physiological responses of Eucalyptus to limited water noted by Whitehead and Beadle is

to reduce LAI, thus although Eucalyptus are evergreen species there can be large seasonal

changes in LAI in response to dry seasons. Similar observations were made in reduced

LAI along a gradient of water availability by Ares and Fownes (2000). The root systems

of Eucalypts are dimorphic, with widely spreading lateral root systems below the surface

plus a deep tap root system. In a plantation of 7 year old Eucalyptus trees in Brazil the
Page 53 of 91

tap root extended to a depth of about 2.5m (Almeida and Soares, 2003) consistent with

other observations (see Srivastava et al, 2003). When artificially stressed, by using

plastic sheets on the soil surface to prevent rain entering the soil, young Eucalyptus

developed roots 8 m or greater in depth. Under other conditions water was utilized from

soil below the root zone by upward movement from wetter levels. In considering these

physiological adaptations Whitehead and Beadle conclude that in the case of South

Africa, where planted Eucalyptus replaced native grasslands, the decreased water yields

resulted from increased transpiration in the evergreen and deep rooted Eucalyptus during

the dry season compared to the seasonally dormant grasses. It is well established that

forests have greater evapotranspiration than grasslands (Zhang et al, 1999).

Specific Hydrology Issues for Planted Eucalyptus:

Eucalyptus Afforestation and Hydrology.

In those cases where Eucalyptus has been shown to have negative impacts on hydrology

this has been associated with afforestation, most notably of lands where trees were

previously absent. Typically, these are areas of low rainfall that are normally dominated

by grasses. Under these conditions afforestation with different species of trees, including

Eucalyptus, has led to changes in the water balance including lowering of water tables

and restricting stream flows. Calder and colleagues have published several reports on

afforestation efforts in India including examples where deep-rooted Eucalyptus were able

to tap into water resources not previously utilized by short-rooted species (Calder et al ,

1997), but also describes cases where water use by Eucalyptus was comparable to

indigenous forests at some sites (Calder, 1994). Similar studies of native grasslands have

documented negative impacts of Eucalyptus on the water balance in South Africa (Lesch

and Scott, 1997; Scott and Lesch, 1997; Scott et al, 1998) and Argentina (Jobbagy and

Jackson, 2004; Engel et al, 2005; Nosetto, 2005). In many of these cases other

introduced trees including pines had similar impacts and particularly in South Africa

impacts on water balance result from a wide variety of introduced species (Le Maitre et

al, 2000; 2002).

These examples contrast with the experience in Brazil where there has been extensive

reforestation with Eucalyptus over many decades. Much of this literature is in

Portuguese but often abstracts are published in English. Lima and colleagues have

published a number of reports that analyzed potential impacts of both Eucalyptus and

pine plantations on the cerrado grasslands in Brazil. Lima et al (1990) showed that in the

region there was adequate rainfall to meet the evapotranspiration demands of Eucalyptus.

A comparison of 6-year old Eucalyptus and pine plantings showed comparable levels of

evapotranspiration during the dry season (May through September) as herbaceous

vegetation (Lima and Freire, 1976). In these trials Eucalyptus actually showed greater

interception than pine (Lima, 1976) and contrasts with references above, but likely

reflected the greater average height of the Eucalyptus at 13.4 m compared to an average

of 6 m for pine.

Similarly, an examination of the water balance of Eucalyptus plantations in China were

not considered to be deleterious for water supplies (Lane et al 2004). While
Page 54 of 91

evapotranspiration exceeded precipitation in the dry season, water storages were

replenished during the wet season.

Comparison of Water Use by Eucalyptus with other Tree Species.

In addition to the reports cited above many authors have concluded that the hydrological

impacts of Eucalyptus are comparable to and should be viewed in the context of other

tree species (see for example Myers et al, 1995; Wullschleger et al, 1998).

One of the largest studies comparing Eucalyptus and native trees conducted to date has

been a catchment area in Brazil of over 280 hectares (owned by Aracruz Cellulose S.A.)

consisting of 190 ha of planted hybrid Eucalyptus and almost 90 ha of native Atlantic

rainforest, that was analyzed over a period of six years. Average precipitation at this site

was 1147 mm, which is similar or less than the sites listed in this permit (range from

~1160 mm in Glades County Florida to almost 1750 mm in Escambia County, Alabama).

Mean high temperature at the Aracruz site was 32.6 C (~91 F) for February (the summer

season in the southern hemisphere), again, comparable to mean high temperatures in the

summer for the sites in this permit. Data from the studies of this catchment area in Brazil

indicated that evapotranspiration was strongly influenced by precipitation (Almeida et al,

2007). In an unusually dry year evapotranspiration was about half that compared to when

water was readily available. In this dry year evapotranspiration exceeded precipitation

but conversely in wetter years evapotranspiration was much less than precipitation. Over

the length of the study evapotranspiration was ~95% of precipitation. This adjustment in

response to varying conditions and water availability was indicative that these hybrid

trees exert strong stomata control and utilize water according to its availability. In a

series of studies in this same area conducted over a period of 8 years Almeida and Soares

(2003, text in Portuguese with abstract and figure legends in English) examined a number

of other hydrological parameters. Stomatal conductance was steady over several months

with adequate water and then dropped significantly as available water dropped and the

predawn leaf water potential (Ψ) increased, again demonstrating strong stomatal control.

Rainfall interception by the Eucalypts averaged ~11% compared to ~24% in the native

forest and water availability (at a depth of 2.5m) is almost identical in the native forests

and the Eucalyptus plantations during the wet summer months but is less in the area with

Eucalyptus during the drier winter. The authors attribute this to the deeper roots systems

(>5m) of the native trees accessing water at deeper levels, while the Eucalyptus (with

roots only to ~2.5m) are limited to the available water in the shallower levels. Finally,

the authors compared the ratio of evapotranspiration and precipitation (ET/P) of the

planted Eucalyptus with the native forest. In years with normal precipitation ET/P was

comparable for both the Eucalyptus and native forest. In years with less than normal

precipitation the native forest had higher ET/P (that is, evapotranspiration was much

greater than precipitation) compared to the Eucalyptus. Based on their data, the authors

suggest that the native forest has a greater consumption of water relative to the

growth/harvest cycle of Eucalyptus, since in the first few years after planting

transpiration in the plantation is much less than the native forest.

Competition for Resources between Eucalyptus and Adjacent Crops
Page 55 of 91

There has been speculation that water use by Eucalyptus could have a negative impact on

water resources available for adjacent vegetation or agricultural crops. Such issues have

been extensively researched in relation to widespread agroforestry systems (reviewed by

Nuberg 1998, and Schroth, 1999). There are important tradeoffs between the positive

effects from windbreaks and shelter belts versus potential competition for light, nutrients

and water resources. Such effects typically occur within 1 to 2 tree-heights (50 to 100

feet for a 50 foot tall tree, Nuberg, 1998) and can be attributed to direct competition by

roots for available soil moisture. Often this can be managed by root pruning to reduce

the area occupied by the tree roots. Impacts attributed to Eucalyptus depend on specific

site conditions, and as with other concerns, there are examples where no negative impact

on adjacent agricultural crops were observed (e.g. Patil, 1995). At some sites this could

be attributed to deeper rooted trees versus shallow rooting crops utilizing water from

different soil profiles. Finally, tree planting has been proposed as a mitigation strategy

where rising water levels increase salinity and reduce crop yields (Hatton and George,

2001).

Summary and Conclusions:

Many studies report that water use in Eucalyptus is comparable to other tree species.

There are some cases where afforestation with Eucalyptus (or other tree species) has lead

to reduced water run-off and supply of streams or changes in water table levels,

especially in regions with limited rainfall. However, in many well documented cases

Eucalyptus plantations do not have any significant negative impacts on hydrology.

Where there have been purported negative impacts, these often reflect more complex

issues such as socioeconomic and land ownership disputes rather than the physiology of

Eucalyptus itself. A key finding of many experiments has been that Eucalyptus is highly

effective in regulating its water consumption relative to available supplies and regulates

its growth accordingly. Based on numerous comparisons that have been made between

the potential hydrological impacts of Eucalyptus and other tree species, we do not expect

that the Eucalyptus trials planted under these permits would be any more impactful on

local hydrology than planting other fast growing trees species.

References:

Almeida, A.C. and Soares, J.V. (2003). Comparison of water use in Eucalyptus

grandis plantations and Atlantic rainforest in eastern coast of Brazil. Revista Arvore

27(2): 159-170.

Almeida, A.C., Soares, J.V., Landsberg, J.J. and Rezende, G.D. (2007). Growth and

water balance of Eucalyptus grandis hybrid plantations in Brazil during a rotation for

pulp production. Forest Ecology and Management, 251:10-21

Ares, A. and Fownes, J.H. (2000) Productivity, nutrient and water-use efficiency of

Eucalyptus saligna and Toona ciliata in Hawaii. Forest Ecology and Management

139:227-236

Binkley,D. and Stape, J. L. (2004). Sustainable Management of Eucalyptus
Page 56 of 91

Plantations in a Changing World. In: Borrelho, N., et al., Eucalyptus in a Changing

World Proc. IUFRO Conference, Aviero, 11-15, October 2004.

Bosch, J.M. and Hewlett, J.D. (1982). A review of catchment experiments to

determine the effect of vegetation changes on water yield and evapotranspiration.

Journal of Hydrology, 55:3-23.

Calder I.R., Rosier, P.T.W., Prasanna, K.T. and Parameswarappa, S. (1997).

Eucalyptus water use greater than rainfall input-a possible explanation from southern

India. Hydrology and Earth System Sciences, 1:244-256.

Calder, I., Amezaga, J., Aylward, B., Bosch, J., Fuller, L., Gallop, K., Gosain, A.,

Hope, R., Jewitt, G., Miranda, M., Porras, I. and Wilson, V. (2004). Forest and Water

Policies. The need to reconcile public and science perceptions. Geologica Acta, 2:157-

166

Calder, I.R. (1994). Eucalyptus, Water and Sustainability: A Summary Report. ODA

Forestry Series No. 6, United Kingdom Office of Development Assistance, London.

Casson, A. (1997). The controversy surrounding eucalypts in social forestry

programs of Asia. National Center for development Studies. Working Paper 97/1

Chaturvedi, A.N. (1987). Silvicultural requirements of Eucalyptus for small farms.

In Proc. International Workshop Multipurpose tree species for small farms. Winrock

International Institute for Agricultural Development USA, International Development

Research Centre of Canada.

Couto, L. and D. R. Betters. (1995). Short-rotation Eucalypt Plantations in Brazil:

Social and Environmental Issues, prepared for the Biofuels Feedstock Development

Program, ORNL/TM-12846, Oak Ridge National Laboratory, Oak Ridge, Tennessee.

Available on-line at: http://bioenergy.ornl.gov/reports/euc-braz/toc.html

Davidson, J., (1995). Ecological aspects of Eucalyptus plantations. In: Proceedings

of the Regional Expert Consultation on Eucalyptus, 4-8 October, 1993, Vol. 1, FAO

Regional Office for Asia and the Pacific, Bangkok. Available on-line at:

http://www.fao.org/docrep/005/ac777e/ac777e00.htm#Contents

de Almeida, P.A. and Riekerk, H. (1990). Water balance of Eucalyptus globulus and

Quercus suber forest stands in south Portugal. For. Ecol. Manage., 38: 55-64.

Engel V., Jobbagy, E.G., Stieglitz, M., Williams, M. and Jackson, R.B. (2005).

Hydrological consequences of Eucalyptus afforestation in the Argentine Pampas. Water

Resour. Res., 41, W10409

FAO (2002a). Annotated bibliography on environmental, social and economic

impacts of Eucalypts (Spanish Version), Forest Plantations Working Papers, Working

Paper 17 (S). Palmberg, C., Forest Resources Development Service, Forest Resources

Division. FAO, Rome (unpublished).
Page 57 of 91

FAO (2002b). Annotated bibliography on environmental, social and economic

impacts of Eucalypts (Spanish Version), Forest Plantations Working Papers, Working

Paper 16. Forest Resources Development Service, Forest Resources Division. FAO,

Rome (unpublished).

Hatton,T. and George, R. (2001). The Role of Afforestation in Managing Dryland

Salinity. In Plantations, Farm Forestry and water: Workshop Proceedings RIRDC

Publication No. 01/20. Namibar, E.K.S. and Brown, A.G. (eds).

Jobbagy, E.G. and Jackson, R.B. (2004). Groundwater use and salinization with

grassland afforestation. Global Change Biology 10:1299–1312

Lane, P.N.J., Morris, J., Zhang, N., Guangyi, Z., Guangyi, Z., Guoyi, Z. and Daping,

X. (2004). Water balance of Eucalyptus plantations in southeastern China. Agricultural

and Forest Meteorology, 124: 253-267

Le Maitre, D.C., Versfeld, D.B. and Chapman, R.A. (2000). The impact of invading

alien plants on surface water resources in South Africa: A preliminary assessment. Water

SA 26:397-408

Le Maitre, D.C., van Wilgen, B.W., Gelderblom, C.M., Bailey, C., Chapman, R.A.

and Nel, J.A. (2002). Invasive alien trees and water resources in South Africa: case

studies of the costs and benefits of management. Forest Ecology and Management

160:143–159

Lesch. W., and Scott, D.F. (1997). The response in water yield to the thinning of

Pinus radiata, Pinus patula and Eucalyptus grandis plantations. Forest Ecology and

Management 99:295-307

Lima, W. de P. (1976). Interceptação da Chuva em Povoamentos de Eucalipto e de

Pinheiro. IPEF, n.13, p.75-90. (Portuguese with English abstract)

Lima, W.P. (1984). The Hydrology of Eucalypts Forests in Australia - A Review.

IPEF n.28, p.11-32

Lima, W. de P. and Freire, O. (1976). Evapotranspiração em Plantações de Eucalipto

e de Pinheiro, e em Vegetação Natural. IPEF, n.12, p.103-117. (Potuguese with English

abstract)

Lima, W.D. Zakia, M.J.B. Libardi, P.L. and Filho, A. (1990). Comparative

evapotranspiration of Eucalyptus, pine and natural vegetation measured by the soil water

balance method. IPEF International Piracicaba. 1:5-11.

Myers, B.J. Theiveyanathan, S. O’Brien, N.D. and Bond, W.J. (1995). Growth and

water use of Eucalyptus grandis and Pinus radiata plantations irrigated with effluent.

Tree Physiology, 16:211-219.
Page 58 of 91

Nosetto, M.D., Jobbagy, E.G. and Paruelo, J.M. (2005). Land-use change and water

losses: the case of grassland afforestation across a soil textural gradient in central

Argentina. Global Change Biology (2005) 11, 1101–1117

NRC (2008). Hydrologic Effects of a Changing Forest Landscape (Prepublication

copy available on-line at http://www.nap.edu/catalog/12223.html)

Nuberg, I.K. (1998). Effect of shelter on temperate crops: a review to define research

for Australian conditions. Agroforestry Systems 41:3–34.

Patil, V. (1995). Local Communities and Eucalyptus - An Experience in India. In:

Proceedings of the Regional Expert Consultation on Eucalyptus, 4-8 October, 1993, Vol.

1, FAO Regional Office for Asia and the Pacific, Bangkok. Available on-line at:

http://www.fao.org/docrep/005/ac777e/ac777e00.htm#Contents

Poore, M.E.D. and Fries, C. (1985). The Ecological Effects of Eucalyptus. FAO

Forestry Paper No. 59

Sahin, V. and Hall, M.J. (1996). The effects of afforestation and deforestation on

water yield. Journal of Hydrology 178:293-309

Schroth , G. (1999). A review of belowground interactions in agroforestry, focusing

on mechanisms and management options. Agroforestry Systems 43:5–34.

Scott, D.F. and Lesch, W. (1997). Streamflow responses to afforestation with

Eucalyptus grandis and Pinus patula and to felling in the Mokobulaan experimental

catchments, South Africa. Journal of Hydrology 199:360-377

Scott, D.F., Le Maitre, D.C. and Fairbanks D.H.K. (1998). Forestry and streamflow

reductions in South Africa: A reference system for assessing extent and distribution.

Water SA 24:187-200

Silva, W., Sediyama, T., Silva, A., and Cardoso, A. (2004). Consumption and Water

Use Efficiency Index by E. citriodora and E. grandis plants cultivated in pots with three

different water contents in the soil jointly with Brachiaria brisantha populations. Foresta,

32 (3): 325-335. (Potuguese with English abstract)

Srivastava, R,J., Kumar, A. and Prasad, K. (2003). Studies on Soil Moisture

Variations Under Eucalyptus Plantations. XII World Forestry Congress, 2003, Quebec

City, Canada. Available on-line at: http://www.fao.org/DOCREP/ARTICLE/WFC/

XII/0500-B2.HTM

Stape, J.L., Binkley, D. and Ryan, M.G. (2004a). Eucalyptus production and the

supply, use and efficiency of use of water, light and nitrogen across a geographic gradient

in Brazil. Forest Ecology and Management 193:17–31

Stape, J.L. Binkley, D. Ryan, M.G. and Gomes, A.N. (2004b). Water use, water
Page 59 of 91

limitation, and water use efficiency in a Eucalyptus plantation. Bosque 25:35–41.

Sunder, S. (1993). The Ecological, Economic and Social Effects of Eucalyptus. In:

Proceedings of the Regional Expert Consultation on Eucalyptus, 4-8 October, 1993, Vol.

1, FAO Regional Office for Asia and the Pacific, Bangkok

White, K.J. (1995). Silviculture of Eucalyptus Plantings – Learning from the Region

(Australia). In: Proceedings of the Regional Expert Consultation on Eucalyptus, 4-8

October, 1993, Vol. 1, FAO Regional Office for Asia and the Pacific, Bangkok

White, K., Ball, J. and Kashio, M. (eds) (1995). Proceedings of the Regional Expert

Consultation on Eucalyptus, 4-8 October, 1993, Vol. 1, FAO Regional Office for Asia

and the Pacific, Bangkok

Whitehead, D. and Beadle, C.L. (2004). Physiological regulation of productivity and

water use in Eucalyptus: A review. Forest Ecology and Management. 193:113-140

Wullschleger, S. Meinzer, F. and Vertessy, R. (1998). A Review of Whole Plant

Water Use Studies in Trees. Tree Physiology, 18: 499-512.

Zhang, L., Dawes, W.R. and Walker, G.R (1999). Predicting the effect of vegetation

changes on catchment average water balance. Cooperative Research Center for

Catchment Hydrology Technical report 99/12
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APPENDIX III: USDA Forest Service assessment of impacts 

on hydrology

This document was prepared by C. R. Ford and J. M. Vose in response to the document 

titled “Hydrology considerations for planted Eucalyptus” submitted by ArborGen LLC to 

USDA APHIS BRS in support of consideration for approval of permits for field trials of 

Eucalyptus at multiple sites 

Executive Summary

We reviewed the materials provided by ArborGen and synthesized the literature on water 

use by Eucalyptus and other vegetation in the southeastern US. Based on these materials 

and our best professional judgment, we provide the following assessments: 

1. Water use efficiency (WUE) is not a good metric to evaluate impacts on hydrology

From a hydrologic standpoint, total water use (transpiration + interception) is a 

more appropriate metric to assess hydrologic impacts. A species may have high 

WUE (defined by ArborGen as volume of wood produced per amount of water 

required), but still transpire and intercept a significant amount of water. 

2. Annual Et losses by Eucalyptus hybrid plantations planted in the southeast US will 

greatly exceed Et by other native southeastern forest types 

Our review of the literature and estimate of Eucalyptus transpiration suggests that 

water use is at least 2-fold greater than most other native forests in the 

southeastern US. 

3. If Eucalyptus invades native forests, forest water use will increase

Due to a combination of physiological and structural characteristics, Eucalyptus

will use more water than most native species regardless of whether it is planted or 

invades native forests.

4. Afforestation from existing vegetation into Eucalyptus plantations reduces stream 

flow more so than afforestation to pine plantations

Our review of the literature suggests that stream flow will be about 20% lower in 

Eucalyptus plantations vs. pine plantations. 

5. Planting Eucalyptus hybrid plantations will lower the water table, and affect 

groundwater recharge and stream flow dynamics

The combination of shallow and deep roots typical of Eucalyptus species has the 

potential to impact both surface and groundwater hydrology.

6. It is unlikely that lower stocking levels will be an acceptable management practice 

to reduce hydrologic impacts of Eucalyptus plantations.

High biomass production requires fully stocked stands. Reducing stocking to 

minimize hydrologic impacts is likely to counter the benefits of planting fast 

growing Eucalyptus.
Page 61 of 91

Possible impacts of Eucalyptus hybrid plantations on 

southeastern US hydrology

Water use efficiency (WUE) is not a good metric to evaluate impacts on hydrology 

From a physiological standpoint, water use efficiency is defined as the ratio of the moles 

of carbon fixed to the moles of water lost. WUE is a leaf-level metric. The Hydrology 

document prepared by ArborGen provided a ratio of liters of water consumed to grams of 

biomass produced. While these ratios provide good information regarding the 

transpirational cost of biomass production, they do not incorporate information on the 

magnitude of evapotranspirational losses, nor do they integrate stand management effects 

(e.g., planting density, rotation age). Hence, WUE is a poor metric to evaluate the effects 

of Eucalyptus on water resources. For example, Eucalyptus can produce more biomass 

per unit water consumed than native southeastern pines; however, their extremely rapid 

biomass production has proportionally higher transpirational costs and hence greater 

water use. Better metrics of evaluating the impacts of Eucalyptus hybrid plantations on 

hydrology exist. In order of scale, these are evapotranspiration (ET, mm H2O yr-1

), 

transpiration (Et, mm H2O yr-1), and whole-tree water use (Q, kg H2O day-1). 

Evapotranspiration (ET) integrates water loss by Et, interception (Ei), and soil 

evaporation (Es), and is often estimated at the landscape scale using precipitation input 

minus stream flow output on paired-catchments (P-Ro). The net effects of greater 

evapotranspiration losses are reduced soil moisture, reduced groundwater depth and 

recharge, and reduced stream flow. These parameters can also be used to evaluate 

impacts on hydrology.

Annual Et losses by Eucalyptus hybrid plantations planted in the southeast US will 

greatly exceed Et by other southeastern forest types 

Previous studies have quantified annual Et from various southeastern US forested and 

crop lands (Table 1). Native pine plantations consume nearly twice the water consumed 

by longleaf pine savannas, but only marginally more than mature upland hardwood 

forests. In contrast, a mature Eucalyptus plantation (age 5, 1111 trees ha-1

, LAI of 6 m2

m-2

) growing in southwest GA could potentially transpire 882 mm yr-1, exceeding all 

other forest types on average by a factor of 2.5. The Hydrology document prepared by 

ArborGen states that the proposed sites are on land previously managed for forestry, 

agricultural production or maintained as pasture land. In these cases, we may expect 

Eucalyptus Et to exceed that of previous pine plantations by a factor of 1.6, and previous 

pasture land by a factor of 3.5. The comparison with agricultural crops is more variable; 

Eucalyptus Et may be greater or lesser than crop Et, depending on the crop, the growing 

season, and the management practices.
Page 62 of 91

Table 1

Vegetation type

Mean 

transpiration 

(mm yr-1) Reference

Longleaf pine savanna 244 (Ford et al. 2008)

Old field 250 (Stoy et al. 2006)

Oak-pine-hickory forest 278 (Oren and Pataki 2001)

Upland oak forest 313 (Wullschleger et al. 2001)

Mixed pine hardwood 355 (Phillips and Oren 2001)

Mixed pine hardwood 442 (Stoy et al. 2006)

Planted loblolly pine 490 (Stoy et al. 2006)

Mixed pine hardwood 523 Schafer and others 2002

Slash pine flatwoods 563 (Powell et al. 2005)

Eucalyptus hybrid plantation 882 Estimated for SW GA in average climate 

and rainfall year from model published in 

(Mielke et al. 1999)

Cotton (non-irrigated, annual) 392 (Howell et al. 2004)

Strawberries (irrigated, 7-

month crop, 5-month fallow)

1397 (Clark 1994, Allen et al. 1998)

Watermelon (irrigated, 3-

month crop, 9-month fallow)

237 (Allen et al. 1998, Shukla et al. 2007)

Eucalyptus has much higher stomatal conductance (gs) in humid environments 

compared to native species

The Hydrology document prepared by ArborGen states that Eucalyptus has evolved 

several mechanisms that allows it to cope with drought conditions in their native habitats, 

including high stomatal sensitivity to air vapor pressure deficit (VPD). Across many taxa 

of plants, two main gs responses to VPD exist (Figure 1). Both strategies regulate gs (and

thus transpiration) according to allowable variation in leaf water potential. The benefit of 

having a high δ, is having a high conductance in humid environments (gsref). The 

southeastern US is a relatively humid environment, with average daily VPD values 

around 1.5 kPa (Ford et al. 2004). In this humid environment, we can expect that 

Eucalyptus hybrid plantations will have stomatal conductance rates that are roughly 

double the conductance rates of native southeastern pine species. This is one mechanism 

that confers a greater transpiration rate of the former compared to the latter. 
Page 63 of 91

Figure 1: Stomatal 

conductance response to 

VPD for pine and Eucalyptus

(Oren et al. 1999)

If Eucalyptus invades native forests, forest water use will increase

In general, species that evolved in arid climates have a more sensitive gs response to VPD 

than those in more mesic environments (McDowell et al. 2008). When comparing water 

use of native species to plant species that have invaded a system (invasives), a recent 

meta-analysis across all biomes shows that stomatal conductance and photosynthesis are 

significantly greater in the invasive species compared to any of the native species in the 

system (Cavaleri and Sack 2008). Specifically, for systems that had been invaded by tree 

life-forms, stand level transpiration was significantly greater compared to un-invaded 

systems (Cavaleri and Sack 2008).

Afforestation from existing vegetation into Eucalyptus plantations reduces stream

flow more so than afforestation to pine plantations

The Hydrology document prepared by ArborGen states that afforestation with any tree 

species may reduce stream flow; and that while Eucalyptus plantations might reduce 

stream flow, the reduction would be comparable to the reduction by pine plantations. 

Recent research suggests that Eucalyptus plantations would reduce stream flow more 

than pine plantations, and more importantly, Eucalyptus plantations could eliminate low 

flows. In a review of more than 20 catchment conversion studies, Farley and others 

(2005b) showed that converting existing vegetation to Eucalyptus plantations reduced 

stream flow by 20% more than converting it to a pine plantation. This review also 

showed that the loss of low flows were more complete for Eucalyptus plantations 

compared to pine plantations (100% vs. ~80% reduction of low flows).

In perennial streams throughout the southeast which have base flows sustained by 

subsurface flow from the water table (or unconfined aquifers), elimination of low flows 

VPD (kPa)

0 1 2 3 4 5 6

gs (mmol m-2 s-1)

0

100

200

300

400

500

600

700

800

Eucalyptus

δ = -194

gs

ref = 328

Pine

δ = -81

gs

ref = 144
Page 64 of 91

may have important ramifications for threatened and endangered aquatic species, such as 

the gulf strain striped bass, and thee species of endemic freshwater mussels (Golladay et 

al. 2004b, Couch and McDowell 2006a).

Planting Eucalyptus hybrid plantations will lower the water table, and affect 

groundwater recharge and stream flow dynamics

The Hydrology document prepared by ArborGen states that afforestation with any tree 

species may lower ground water levels. This is highly dependent on subsurface flow 

patterns, local hillslope hydrology, and species-specific rooting patterns. For example, in 

sites where the water table can be recharged laterally, if roots extend to the water table, 

then stomatal conductance and transpiration can be maintained even when water in the 

upper soil layers is insufficient to maintain transpiration. If Eucalyptus hybrid plantations 

mine water from the saturated zone (i.e., water table), groundwater recharge could be 

reduced. The southeastern Coastal Plain is characterized in many places by karst geology 

in which groundwater from the semi-confined Upper Floridan Aquifer (UFA) is 

hydraulically connected to the water table (surface water) (Opsahl et al. 2007). Mean 

water table depths typically range 3–8 m (Ford et al. 2008). The mean maximum rooting 

depth for Eucalyptus is 15 m, characteristic of its dimorphic rooting pattern; in contrast, 

mean maximum rooting depths of pine plantation (P. taeda and P. elliottii) and grass 

species are 3 m and 2.6 m, respectively (Canadell et al. 1996b). The average age of 

groundwater in the UFA is ~20 years (Happella et al. 2006) and groundwater is regularly 

recharged by surface water in this region (Opsahl et al. 2007). Conversion to Eucalyptus

on sites with water tables <10 m will likely lower down-slope water tables via direct 

means (i.e., direct use of ground water by deep roots), affect groundwater-aquifer 

dynamics, and result in ET rates that exceed precipitation input, as have been reported for 

this species in other locations (Calder et al. 1997b).

The Hydrology document prepared by ArborGen states that the high water usage 

by Eucalyptus may be valuable in purposefully lowering water tables to reduced potential 

salinity problems. Eucalyptus has been used to afforest areas and lower the saline 

groundwater in highly weathered landscapes (e.g., AUS). This application is not relevant 

to the southeastern US, as soils are not saline. Furthermore, receding groundwater levels 

in the UFA are being replaced in coastal areas by saltwater (i.e., saltwater intrusion) 

(Andersen et al. 2006). Thus, lowering the water table, and the groundwater levels in the 

UFA would not reduce salinity problems (as stated in the Hydrology document), and may 

actually exacerbate them. 

Key Point: The significance of the impact on groundwater and stream flow will depend 

greatly on the area extent, size, and spatial distribution of the plantations. For 

example, a few small (i.e., < 10 ha) and well dispersed plantations may only have very 

localized impacts and negligible impacts at the watershed scale.

Management of Eucalyptus as coppice stands will affect water use of future rotations

Management practices may create a perennial root stock in Eucalyptus plantations. If 

Eucalyptus plantations are managed as coppice stands, the remaining mature, deeply-
Page 65 of 91

penetrating root stock may be able to supply the second rotation stems with more water 

resources for use than similar sized stems in their first rotation (Swift and Swank 1981). 

It is unlikely that lower stocking levels will be an acceptable management practice to 

reduce hydrologic impacts of Eucalyptus plantations

Some of the reports cited in the ArborGen document discuss the potential for altering 

management practices to minimize the impacts of intensively managed Eucalyptus

plantations on hydrology. The most viable option for reducing hydrologic impacts is to 

manage stocking (“stocking” is a term to describe the how much of the site is occupied 

by the species of interest; stand basal area expressed in m2 stem area hectare-1 is often 

used as a measure of stocking). Water use is highly regulated by stand leaf area and 

reducing basal area will result in lower stand leaf area. Empirical research (Douglass and 

Swank 1972) at Coweeta has shown that stand basal area needs to be reduced by at least 

15 % before any impact on stream flow is obtained. Large and sustained increases in 

stream flow typically require significant reductions in stocking because trees growing in 

more open conditions will increase transpiration rates in response to changes in 

micrometeorological conditions in the tree crown. 

From a practical standpoint, it is unlikely that lower stocking levels will be an 

acceptable management practice for Eucalyptus plantations because one of the primary 

objectives of growing Eucalyptus is to maximize biomass production -- this requires fully 

stocked stands. 

Monitoring Impacts on Hydrology

Several options are available to monitor the impacts of Eucalyptus plantations on 

hydrology. Options include direct or indirect measurements of impacts, and vary in 

complexity and cost. The ArborGen document provided basic information on the 

hydrologic cycle and its components so these will not be repeated here. 

Indirect Measurements:

Transpiration & Interception

Instrumentation required = rain gauges, throughfall collectors, sap flow sensors

PROS: direct measure of change in water use component on hydrologic cycle

CONS: does not directly measure impacts on stream flow or groundwater; 

expensive, high maintenance

Soil Moisture

Instrumentation = TDR probes and data loggers (automated); soil probe for 

gravimetric (manual)

PROS: easy to implement, relatively inexpensive

CONS: does not directly measure impacts on stream flow or groundwater

Direct Measurements:

Groundwater Depth
Page 66 of 91

Instrumentation = access wells & pressure transducers (automated); access wells 

(manual)

PROS: direct measure of impacts; reliable

CONS: expensive 

Stream flow

Instrumentation = flumes & data loggers (automated); pressure transducer, rating 

curve, stream survey

PROS: direct measure of impacts; reliable

CONS: expensive, requires stream, expertise

Regardless of the monitoring approach chosen, the monitoring design will require 

a suitable control for comparison. Ideally, the plantation site(s) and the control site(s) 

would be measured for at least 1 to 2 years prior to be being planted, and then both sites 

would be measured for the duration of the monitoring period. We recommend that the 

monitoring period begin at plantation establishment and continue through canopy closure 

(approximately 5 years). 

Additional analyses on groundwater dynamics and linkages with aquatic ecosystems 

are required

We recommend that APHIS solicit input from experts on groundwater hydrology (e.g., 

from USGS) to assess the potential impacts on groundwater recharge and associated 

dynamics. In addition, our analysis suggests that stream flow will be reduced by at least 

20% relative to pine plantations and perhaps even greater reductions will be observed 

relative to native ecosystems. We recommend that APHIS solicit input from aquatic 

ecologists to assess the potential impacts on aquatic ecosystems and associated species. 
Page 67 of 91

References

Allen RG, Pereira LS, Raes D, Smith M. 1998. Crop evapotranspiration - Guidelines for

computing crop water requirements. FAO - Food and Agriculture Organization of

the United Nations. Paper 56.

Andersen PF, Mercer JW, White Jr. HO. 2006. Numerical modeling of salt-water

intrusion at Hallandale, Florida. Ground Water 26: 619–630.

Calder IR, Rosier PTW, Prasanna KT, Parameswarappa S. 1997. Eucalyptus water use

greater than rainfall input-a possible explanation from southern India. Hydrology

and Earth System Sciences 1: 249–256.

Canadell J, Jackson RB, Ehleringer JR, Mooney HA, Sala OE, Schulze ED. 1996.

Maximum rooting depth of vegetation types at the global scale. Oecologia 108:

583-595. <Go to ISI>://A1996VX11000001

Cavaleri M, Sack L. 2008. Native vs. invasive plant water use at multiple scales: A global

meta-analysis. Presented at the Ecological Society of America Annual Meeting in

Milwaukee, WI. August 3–8.

Clark GA. 1994. Water requirements for drip irrigated strawberries in south central

Florida. Agricultural and Biological Engineering Department, Florida

Cooperative Extension Service, Institute of Food and Agricultural Sciences,

University of Florida. AE253.

Couch CA, McDowell RJ. 2006. Flint River Basin Regional Water Development and

Conservation Plan. Georgia Dept. of Natural Resources, Environmental

Protection Division. p. 242.

Douglass JE, Swank WT. 1972. Streamflow modification through management of eastern

forests. USDA Forest Service, Southeastern Forest Experiment Station. Research

Paper SE-94.

Farley K, Jobbagy E, Jackson RB. 2005. Effects of afforestation on water yield: a global

synthesis with implications for policy. Global Change Biology 11: 1565–1576.

Ford CR, Goranson CE, Mitchell RJ, Will RE, Teskey RO. 2004. Diurnal and seasonal

variability in the radial distribution of sap flow: predicting total stem flow in

Pinus taeda trees. Tree Physiol. 24: 951-960.

Ford CR, Mitchell RJ, Teskey RO. 2008. Water table depth affects productivity, water

use, and the response to nitrogen addition in a savanna system. Can. J. For. Res.

38: 2118–2127.

Golladay SW, Gagnon P, Kearns M, Battle JM, Hicks DW. 2004. Response of freshwater

mussel assemblages (Bivalvia:Unionidae) to a record drought in the Gulf Coastal

Plain of southwestern Georgia. Journal of the North American Benthological

Society 23: 494-506.

Happella JD, Opsahl S, Top Z, Chanton JP. 2006. Apparent CFC and 3H/3He age

differences in water from Floridan Aquifer springs. J. Hydrol. 319: 410–426.

Howell TA, Evett SR, Tolk JA, Schneider AD. 2004. Evapotranspiration of full-, deficitirrigated,

and dryland cotton on the northern Texas high plains. Journal of

Irrigation an Drainage Engineering 130: 277–285. doi:10.1061/(ASCE)0733-

9437(2004)130:4(277)

McDowell N, Pockman WT, Allen CD, Breshears DD, Cobb N, Kolb T, Plaut J, Sperry J, West A, 

Williams DG, Yepez EA. 2008. Tansley review: Mechanisms of plant
Page 68 of 91

survival and mortality during drought: why do some plants survive while others

succumb to drought? New Phytol. 178: 719–739.

Mielke MS, Oliva MA, deBarros NF, Penchel RM, Martinez CA, Almeida ACd. 1999.

Stomatal control of transpiration in the canopy of a clonal Eucalyptus grandis

plantation. Trees- Structure and Function 13: 152-160.

Opsahl SP, Chapal SE, Hicks DW, Wheeler CK. 2007. Evaluation of ground-water and

surface-water exchanges using streamflow difference analyses. Journal of the

American Water Resources Association 43: 1132–1141.

Oren R, Pataki DE. 2001. Transpiration in response to variation in microclimate and soil

moisture in southeastern deciduous forests. Oecologia 127: 549-559.

Oren R, Sperry JS, Katul GG, Pataki DE, Ewers BE, Phillips N, Schafer KVR. 1999.

Survey and synthesis of intra- and interspecific variation in stomatal sensitivity to

Vapour pressure deficit. Plant Cell Environ. 22: 1515-1526.

Phillips N, Oren R. 2001. Intra- and inter-annual variation in transpiration of a pine

forest. Ecol. Appl. 11: 385–396.

Powell TL, Starr G, Clark KL, Martin TA, Gholz HL. 2005. Ecosystem and understory

water and energy exchange for a mature, naturally regenerated pine flatwoods

forest in north Florida. Can. J. For. Res. 35: 1568-1580.

Shukla S, Jaber F, Srivastava S, Knowles J. 2007. Water use and crop coefficient for

watermelon in southwest Florida. Institute of Food and Agricultural Sciences

(IFAS), University of Florida. Report No. WRP-LY-0009 submitted to Southwest

Florida Water Management District

Stoy P, Katul G, Siqueira M, Juang J, Novick K, McCarthy HR, Oishi AC, Umbelherr J,

Kim H, Oren R. 2006. Separating the effects of climate and vegetation on

evapotranspiration along a successional chronosequence in the southeastern US.

Global Change Biology 12: 2115–2135.

Swift LW, Swank WT. 1981. Long term responses of streamflow following clearcutting

and regrowth. Hydrological Sciences Bulletin 26: 245–256.

Wullschleger SD, Hanson PJ, Todd DE. 2001. Transpiration from a multi-species

deciduous forest as estimated by xylem sap flow techniques. For. Ecol. Manage.

143: 205-213.
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APPENDIX IV: Threatened and Endangered Species Analysis

Congress passed the Endangered Species Act (ESA) of 1973, as amended, to prevent extinctions 

facing many species of fish, wildlife, and plants. The purpose of the ESA is to conserve 

endangered and threatened species and the ecosystems on which they depend as key components 

of America’s heritage. To implement the ESA, the U.S. Fish and Wildlife Service (USFWS) 

works in cooperation with the National Marine Fisheries Service (NMFS); other Federal, State, 

and local agencies; Tribes; non-governmental organizations; and private citizens. Before a plant or

animal species can receive the protection provided by the ESA, it must first be added to the 

Federal list of threatened and endangered wildlife and plants.

A species is added to the list when it is determined by the USFWS/NMFS to be endangered or 

threatened because of any of the following factors:

• The present or threatened destruction, modification, or curtailment of its habitat or range;

• Overutilization for commercial, recreational, scientific, or educational purposes;

• Disease or predation;

• The inadequacy of existing regulatory mechanisms; and

• The natural or manmade factors affecting its survival.

Once an animal or plant is added to the list, in accordance with the ESA, protective measures 

apply to the species and its habitat. These measures include protection from adverse effects of 

Federal activities. Section 7 (a)(2) of the ESA requires that Federal agencies, in consultation with 

USFWS and/or the NMFS, ensure that any action they authorize, fund, or carry out is not likely to 

jeopardize the continued existence of a listed species or result in the destruction or adverse 

modification of designated critical habitat. It is the responsibility of the Federal agency taking the 

action to assess the effects of their action and to consult with the USFWS and NMFS if it is 

determined that the action “may affect” listed species or critical habitat. 

The threatened and endangered species analysis was accomplished by considering the possibility of 

effects on all listed species including species proposed for listing and designated critical habitat 

including habitat proposed for designation within the county where the test site is located. 

Normally consideration would be limited to only those species and critical habitat identified by the 

United States Fish and Wildlife Service and/or National Marine Fisheries Service to be likely found 

in the area of the field tests. However, because the locations are declared by the applicant to be 

confidential business information, APHIS instead obtained species lists and critical habitat 

information for the entire county where the field tests are to occur. Although it is very clear that 

there could be no effect on some species (e.g. West Indian manatee) all species on the list and all 

critical habitat in each county are included in the discussion below. 

The following resources were used in the analysis:

US Fish and Wildlife Service Endangered Species Homepage: http://www.fws.gov/endangered

US Fish and Wildlife Service Critical Habitat portal: http://criticalhabitat.fws.gov/crithab/

ArcGis Explorer with critical habitat metadata supplied by US Fish and Wildlife Service 

County species lists from FWS field offices in Alabama, Mississippi, South Carolina and Florida

obtained from http://www.fws.gov/offices/
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South Carolina Heritage Trust Database

Telephone contacts with USFWS Field Office personnel

Discussions with property owners of field test sites 

1. Baldwin County, AL

This location has been an agricultural research station for more than 20 years. The location has 

been used for managed production of annual agricultural crops and forest trees. Approximately 

3.55 acres of field trials of genetically modified Eucalyptus trees of some lines in the permit are 

being grown under issued permits 08-011-106rm (0.75 acres) and 10-112-101r (2.8 acres) under 

which these trees are allowed to flower. The oldest of these trees were planted in August 2006. Site 

preparation will involve herbicide application, subsoiling, and planting of trees in flat beds. The 

surrounding areas of the test site consist of field plantings of agricultural crops, experimental forest 

trees and an abandoned pecan orchard. Under the new permit up to 2.0 acres of field tests could be 

established at this location (at around 300 - 600 trees per acre) over the next three years.

Seventeen TES animals and one TES plant are listed in Baldwin County (list accessed from FWS 

website 8/30/2011). The TES Animals include loggerhead sea turtle (Caretta caretta), green sea 

turtle, (Chelonia mydas), Alabama red-belly turtle (Pseudemys alabamensis), Kemp's ridley sea 

turtle (Lepidochelys kempii), Hawksbill sea turtle (Eretmochelys imbricata, Leatherback sea turtle 

(Dermochelys coriacea), Alabama beach deermouse (Peromyscus polionotus ammobates), Perdido 

Key beach deermouse (Peromyscus polionotus trissyllepsis), piping plover (Charadrius melodus), 

West Indian manatee (Trichechus manatus), wood stork (Mycteria americana), gulf sturgeon 

(Acipenser oxyrinchus desotoi), Alabama sturgeon (Scaphirhynchus suttkusi), southern clubshell

(Pleurobema decisum), Alabama inflated heelsplitter mussel (Potamilus inflatus), and eastern 

indigo snake (Drymarchon corais couperi). The listed plant is American chaffseed (Schwalbea 

americana). 

The American chaffseed occurs on sandy peat, sandy loam, acidic, seasonally moist to dry soils. It 

is generally found in habitats described as open, moist pine flatwoods, fire-maintained savannas, 

ecotonal areas between peaty wetlands and xeric sandy soils, and other open grass-sedge systems. 

According to Jody Smithen (contacted February 20, 2008) of the Daphne Field Office USFWS, the 

only location this plant is known to be in the county is in the northeast corner, far from the release 

site. The plant has no critical habitat listed in the action area. 

The six turtle species (loggerhead sea turtle, green sea turtle, Alabama redbelly turtle, Kemp’s 

ridley sea turtle, Hawksbill sea turtle and the leatherback sea turtle), the gulf and Alabama 

sturgeons, and the West Indian manatee occur in aquatic habitats, and their habitat systems (bays, 

lagoons, salt marshes, creeks, ship channels, and other saltwater and freshwater environments) do 

not overlap with the trial site. The inflated heelsplitter mussel occurs in the Tombigbee and Black 

Warrior rivers. The southern clubshell mussel occurs in major streams of the Mobile river basin. 

The closest stream where these mussels could possibly occur is at least a half a mile away from the 

release site so their habitat does not overlap with the trial site. The wood stork primarily inhabits 

wetland systems notably cypress or mangrove swamps and would not use the field test site. The 

piping plover uses sparsely vegetated dunes and coastal beaches in southern Baldwin County, also 

far away from the field site (about 60 miles). The two mouse species listed above (Alabama beach 
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mouse and Perdido Key Beach deermouse) are found only in coastal dune areas and Perdido Key 

Beach, respectively, where they feed on sea oats, bluestems, and a variety of insects. Both habitats 

are located 65+ miles from the proposed field trial (distance estimated using ArcGis Explorer). The 

Eastern indigo snake is known to inhabit a wide range of habitats (agriculture fields, pine flatwoods, wet depressions, stream bottom thickets and margins of swamps). It appears to be very rare 

in Baldwin County where a case has been reported in an unknown location (US Forest Service). 

According to Jody Smithen (contacted February 20, 2008) of the Daphne Field Office USFWS, the 

species has not been documented in the county for many years, but there are occasionally 

unsubstantiated reports. They do not feel there is any concern. Although it is highly unlikely that 

the species would be found at the site, the applicant will provide all workers with identifying 

characteristics of the snake and instructions on what to do if the species is encountered. These 

measures are a variation of standard protective measures the USFWS uses when they have reached 

a “may affect” determination for construction sites.

Critical Habitat: Most of the TES animals within the county area use inshore or wetland systems 

most of which are concentrated essentially in the southern and southeastern coastal beaches of 

Baldwin County. There is critical habitat listed for the Perdido Key beach deermouse, the Alabama 

beach mouse and the piping plover. There is no designated critical habitat in the immediate 

environment of the field test. The closest critical habitat (for the Alabama Beach mouse, Perdido 

Key Beach deermouse and the piping plover) is about 26 miles away. The gulf sturgeon has 

proposed critical habitat but this does not occur in Baldwin County. (http://crithab.fws.gov/

accessed 8/31/2011)

Conclusion: No federally listed threatened or endangered species or species proposed for listing 

are likely to be found at the release site. If they were to enter the site, their presence would be 

fleeting as the habitat is either not suitable or does not contain constituent elements required by the 

species. Field activities will result in no changes to the habitat used by any listed species or species 

proposed for listing. The site is not within or near designated critical habitat or habitat proposed for 

designation. Therefore, the action will have no effect on listed species or species proposed for 

listing and would not affect designated critical habitat or habitat proposed for designation.

 

2. Escambia County, AL

This location had previously been used as an intensely managed pasture for more than 5 years. The 

test site is currently planted with grasses suitable for cattle grazing. Approximately 0.5 acres of 

field trials of genetically modified Eucalyptus of some lines in the permit are being grown under 

issued permit 08-011-106rm under which these trees are allowed to flower. The oldest of these 

trees were planted in July 2007. Site preparation will involve herbicide application to remove 

existing grasses, subsoiling, preparation for possible irrigation, and planting of the test trees in flat

beds. The surrounding areas of the test site consist of greater than 30 years-old slash pine, and a reforested area with mixed stands of pine and hardwood species. Under the new permit up to 2.0 

acres of field tests could be established at this location (at around 300 - 600 trees per acre) over the 

next three years.

Five TES animals are listed for Escambia County (list accessed from FWS website 8/31/2011). 

The animals are gulf sturgeon, (Acipenser Oxyrinchus desotoi), red-cockaded woodpecker 
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(Picoides borealis), wood stork (Mycteria Americana), gray bat (Myotis grisescens) and eastern 

indigo snake (Drymarchon corais couperi). The listed plant is American chaffseed (Schwalbea 

americana). The USFWS has recently proposed the listing of several mussel species (Federal 

Register / Vol. 76, No. 192 / 10/04/11), six of which occur in Escambia County. These are the 

Alabama pearlshell (Margaritifera marrianae), round ebonyshell (Fusconaia rotulata), southern 

kidneyshell ((Ptychobranchus jonesi), Choctaw Bean (Villosa Choctawensis), Narrow Pigtoe 

(Fusconaia Escambia), and the Fuzzy Pigtoe (Pleurobema Strodeanum). The gulf sturgeon occurs 

in the Gulf of Mexico and spawns in freshwater rivers. It will not be affected by the field test since 

the closest river is over 3.5 miles away (as measured via ArcGis Explorer). The red cockaded 

woodpecker inhabits old growth forests, primarily longleaf pine. It could visit the field test site but 

would not nest there. The wood stork primarily inhabits wetland systems notably cypress or 

mangrove swamps and would not find the field test site hospitable. The gray bat might visit the 

field test location but would not nest there. The eastern indigo snake is highly unlikely to be found 

at the site, however, the applicant will provide all workers with identifying characteristics of the 

snake and instructions on what to do if the species is encountered. The American chaffseed occurs 

on sandy peat, sandy loam, acidic, seasonally moist to dry soils. As noted above it is generally 

found in habitats described as open, moist pine flatwoods, fire-maintained savannas, ecotonal areas 

between peaty wetlands and xeric sandy soils, and other open grass-sedge systems and wound not 

occur in the vicinity of the field test site. None of the proposed mussel species would be found in 

the release site. Mussels are found on the bottoms of streams and rivers. The closest known 

location of any of these species is the Conecuh River, approximately 3.5 miles away. 

Critical Habitat: The designated critical habitat for the gulf sturgeon includes the Escambia River 

System in Santa Rosa and Escambia counties, Florida and Escambia, Conecuh, and Covington 

counties, Alabama. The establishment of the field test site would not impact this habitat. It is 

about 3.5 miles away from the Conecuh river (http://crithab.fws.gov/ accessed 8/31/2011). There is 

also proposed critical habitat for a number of freshwater mussel species in Escambia County 

(Federal Register / Vol. 76, No. 192 / 10/04/11). Six of these habitats occur in Escambia County. 

These are Alabama pearlshell (Margaritifera marrianae), round ebonyshell (Fusconaia rotulata), 

southern kidneyshell ((Ptychobranchus jonesi), Choctaw Bean (Villosa Choctawensis), Narrow 

Pigtoe (Fusconaia Escambia), and the Fuzzy Pigtoe (Pleurobema Strodeanum). The proposed 

critical habitat for these species in Escambia County occurs in Burnt Corn Creek, and in the lower 

Escambia and Conecuh rivers, and is limited to the creek and river channels up to the ordinary 

high-water line. The closest CH to the release location is about 3.5 miles away in the Conecuh 

river. 

Conclusion: No federally listed threatened or endangered species or species proposed for listing 

are likely to be found at the release site. If they were to enter the site, their presence would be 

fleeting as the habitat is either not suitable or does not contain constituent elements required by the 

species. Field activities will result in no changes to the habitat used by any listed species or species 

proposed for listing. The site is not within or near designated critical habitat or habitat proposed for 

designation. Therefore, the action will have no effect on listed species or species proposed for 

listing and would not affect designated critical habitat or habitat proposed for designation.

3. Highlands County, FL
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This location has been used for field trials of transgenic Eucalyptus trees for at least 5 years. 

Approximately 5.2 acres of field trials of genetically modified Eucalyptus trees are being grown 

under issued permits 08-014-101rm, 09-070-101rm and 11-041-101rm at this location. The oldest 

of these trees were planted in July 2006. Current and future site preparation involves herbicide 

application, plowing, and planting of trees in flat beds. The test site is surrounded by a citrus 

production area and second-growth pine and hardwood forests. Under the new permit up to 4.0 

acres of additional tests could be established under this permit. 

Fifteen TES animals, nineteen TES plants, and one TES lichen are listed for Highlands County (list 

accessed from FWS website 9/1/2011).. The animals are: American alligator (Alligator 

mississippiensis), wood stork (Mycteria americana), Audubon’s crested caracara (Caracara 

cheriway), red-cockaded woodpecker (Picoides borealis), Florida scrub-jay (Aphelocoma 

coerulescens), Florida grasshopper sparrow (Ammodramus savannarum floridanus), Everglade 

snail kite (Rostrhamus sociabilis plumbeus), ivory-billed woodpecker (Campephilus principalis), 

Whooping crane (Grus americana) (non-essential experimental population), Florida panther (Puma 

concolor coryi), Puma (Puma concolor – similarity of appearance), West Indian manatee 

(Trichechus manatus), Eastern indigo snake (Drymarchon corais couperi), bluetail mole skink 

(Eumeces egregius lividus) and sand skink (Neoseps reynoldsi).

The plants are: Florida bonamia (Bonamia grandiflora), pygmy fringetree (Chionanthus 

pygmaeus), pigeon wings (Clitoria fragrans), short-leaved rosemary (Conradina brevifolia), Avon 

Park hare-bells (Crotalaria avonensis), Garrett’s mint (Dicerandra christmanii), scrub mint 

(Dicerandra frutescens), scrub buckwheat (Eriogonum longifolium var. gnaphalifolium), snakeroot 

(Eryngium cuneifolium), highlands scrub hypericum (Hypericum cumulicola), scrub blazingstar 

(Liatris ohlingerae), Britton's bear-grass (Nolina brittoniana), papery whitlow-wort (Paronychia 

chartacea ssp. Chartacea), Lewton's polygala (Polygala lewtonii), wireweed (Polygonella 

basiramia), sandlace (Polygonella myriophylla), scrub plum (Prunus geniculata), Carter's mustard 

(Warea carteri), and Florida ziziphus (Ziziphus celata).

The lichen is: Florida perforate cladonia (Cladonia perforate).

The American alligator (Alligator mississippiensis) is no longer biologically endangered or 

threatened; however, it is listed by USFWS as Threatened throughout its entire range due to 

similarity of appearance to other endangered or threatened crocodilians. The wood stork (Mycteria 

Americana) primarily inhabits wetland systems notably cypress or mangrove swamps and would 

not find the field test site hospitable. The crested caracara (Caracara cheriway) is associated with 

open country; dry prairie with scattered cabbage palms, wetter prairies, and to some extent also 

improved pastures and sometimes wooded areas having associated limited areas of open grassland. 

The center of range is the Kissimmee Prairie, an area of shallow ponds and sloughs with scattered 

hummocks of live oaks and cabbage palms. The red cockaded woodpecker (Picoides borealis) 

inhabits old growth forests, primarily longleaf pine. The Florida scrub-jay (Aphelocoma 

coerulescens) prefers oak scrub on white, drained sand, in open areas without a dense canopy 

associated with Palmetto, sand pine and rosemary. This includes scrub with no canopy, sandpine 

scrub, scrubby flatwoods, and coastal scrub. The Florida grasshopper sparrow (Ammodramus 

savannarum floridanus) prefers dry prairie with stunted saw palmetto and dwarf oaks, bluestems 

and wiregrass and unimproved cattle pastures. Its habitat is maintained by periodic fires. The snail 
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kite (Rostrhamus sociabilis plumbeus) prefers large, open freshwater marshes and lakes with 

shallow open waters. The ivory-billed woodpecker (Campephilus principalis) formerly occurred in 

the southeastern United States and Cuba and has declined to extinction or near extinction. It once 

occupied swampy forests, especially large bottomland river swamps of coastal plain and 

Mississippi Delta and cypress swamps of Florida, in areas with many dead and dying trees. It 

would not occur in an agricultural environment such as the field test site. Whooping crane (Grus 

americana) prefers freshwater marshes and wet prairies. It nests in dense emergent vegetation 

(sedge, bulrush) in shallow ponds, freshwater marshes, wet prairies, or along lake margins, within 

large expanses of undisturbed wilderness. The cranes listed in this county are an experimental 

reintroduction of whooping cranes in Florida initiated in 1993 to establish a non-migratory 

population. This is an experimental, non-essential population. Any of the bird species could 

potentially visit the field test site but would not nest there. The Florida panther (Puma concolor

coryi) generally occurs in heavily forested areas in lowlands and swamps, also upland forests in 

some parts of range; areas with adequate deer or wild hog population. Habitats include tropical 

hammocks, pine flatwoods, cabbage palm forests, mixed swamp, cypress swamp, live oak 

hammocks, sawgrass marshes, and Brazilian pepper thickets. It depends on large contiguous 

blocks of wooded habitat, though interspersed fields and early successional habitats may be 

beneficial through their positive effect on prey populations. Its day-use sites typically are dense 

patches of saw palmetto surrounded by swamp, pine flatwoods, or hammock. It would not occur in 

the trial area due to the openness and continued presence of humans in the area. The West Indian 

manatee (Trichechus manatus) occurs in shallow coastal waters, rivers, bays and lakes; none of 

which are close to any of this release location. For the Eastern indigo snake (Drymarchon cosair 

couperi) – according to Candice Martino (904-232-2580 ext. 129) Section 7 Endangered Species 

biologist from the Jacksonville, FL Field Office, (contacted February 25, 2008) the species is 

seldom seen but could be anywhere. However, the habitat at the release site would not be suitable. 

The historic and continuous use of the release site and the surrounding area as a citrus grove and 

Eucalyptus field trials makes it extremely unlikely that the species would be found in the area. 

Therefore, the appropriate determination would be “no effect.” Although it is highly unlikely that 

the species would be found at the site, the applicant will provide all workers with identifying 

characteristics of the snake and instructions on what to do if the species is encountered. These 

measures are a variation of standard protective measures the USFWS uses when they have reached 

a “may affect” determination for construction sites.

The bluetail mole skink (Eumeces egregius lividus) inhabits sand pine-rosemary scrub or, less 

frequently, longleaf pine-turkey oak association sandhills. It occupies localized pockets of 

sufficient leaf litter and moisture to provide abundant food and nesting sites. The sand skink 

(Neoseps reynoldsi) occurs only on Florida's central ridges, at elevations of 27 m or more. It 

inhabits loose sands of sand pine-rosemary scrub, less often longleaf pine-turkey oak sandhills or 

turkey oak barrens adjacent to scrub, especially high pine-scrub ecotones. It was determined that 

the release site is within a geographic area where these two skink species are found. According to 

Brad Rick, (contacted February 27 and 28, 2008) of the Vero Beach Field Office USFWS, sand 

skinks and bluetail mole skinks are found in scrub habitat with areas of open sand. The literature 

indicates that skinks are sometimes found in active and abandoned citrus groves and the applicant 

confirmed that sandy soils are predominant in the area. It was decided to have the applicant 

conduct a survey of the species using USFWS protocols to determine if the species is present. The 

protocols were provided by the Vero Beach Field Office. The USFWS protocol recommends that 
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surveys be conducted between March 1 and May 15 as this is an ideal time to observe evidence of 

the skinks. A coverboard survey was conducted over a one month period from March 18 to April 

15, 2008. The coverboards were checked weekly on March 25, April 1, April 8 and April 15. No 

evidence indicating the presence of sand skinks or bluetail mole skinks was observed and they are 

presumed absent. Therefore, the appropriate determination would be “no effect.”

For the plants, the Florida bonamia (Bonamia grandiflora) grows in natural clearings of bare 

ground and invades disturbed areas of open sand. Although not common, it is often locally 

abundant where there is little or no shade from trees or shrubs. It is locally abundant on deep, 

white, dry sands of ancient dunes and sandy ridges in clearings or openings of scrub habitat on the 

Central Ridge of Florida. Pygmy fringetree (Chionanthus pygmaeus) is generally found in xeric, 

coarse white sand of scrub/oak scrub areas found at the southern end of the Central Florida Ridge. 

It is also found occasionally in longleaf pine-turkey oak vegetation, high pineland, dry hammocks, 

and transitional habitats. Pigeon wings (Clitoria fragrans) is widely scattered in undisturbed 

clearings of xeric sandhill and scrub communities on well-drained upland soils. It is typically 

found in undisturbed clearings in scrub areas but also occurs in very open scrub as well. Shortleaved rosemary (Conradina brevifolia), is found in white sands of sand pine-oak scrub with 

scattered overstory of sand pine and scrub oak. Avon Park hare-bells (Crotalaria avonensis) 

occurs in upland habitats (scrub and sandhill), often along trails. It grows in full sun or partial 

shade provided by characteristic scrub shrubs or sand pine. Garrett’s mint (Dicerandra 

christmanii) occurs in openings in sand pine-oak scrub on yellow soils of the Central Florida Ridge. 

Scrub mint (Dicerandra frutescens) occurs in well-drained soils of scrub or sandhill vegetation. It 

is locally abundant in and around the sand pine-evergreen oak scrub, where it may occur in the low 

shrub layer or in open stands, clearings, or adjacent sandy places. It is not found in areas cleared 

for pasture, or areas in which wholesale site preparation has taken place. Scrub buckwheat 

(Eriogonum longifolium var. gnaphalifolium) is long-lived, slow growing and flowers and 

reproduces primarily after fires or other disturbances (e.g. logging, mowing) that increase light 

availability. It prefers dry pinelands, sandhills, and scrub (longleaf pine-turkey oak, scrub oaks) 

and is more commonly found in transition habitats between scrub and high pine and in turkey oak 

barrens than in either dense scrub or open high pine. Snakeroot (Eryngium cuneifolium) is 

generally found in areas of open sand, including blowouts and other highly disturbed soil surfaces, 

such as road shoulders. It occurs in exposed sunny openings; areas in scrub, especially rosemary 

scrub. Highlands scrub hypericum (Hypericum cumulicola) occurs in patches of open, nutrientpoor sand within oak and rosemary scrub. It is often associated with reindeer lichen (Cladonia

spp.) and snakeroot (Eryngium cuneifolium). Scrub blazingstar (Liatris ohlingerae) occurs in 

openings in oak-rosemary scrub and sand pine scrub. Britton's bear-grass (Nolina brittoniana) 

occurs in deep, fine-textured, well-drained sands of sand pine-evergreen oak scrub or longleaf pineturkey oak sandhills. Nolina is entirely dependent on fire or some other mechanism to maintain an 

open successional stage in scrub or sandhills. Papery whitlow-wort (Paronychia chartacea ssp. 

Chartacea) is a sand scrub that occurs on ancient dunes in the lake region, in white sand clearings 

or blowouts. Lewton's polygala (Polygala lewtonii) occurs in sandhills characterized by longleaf 

pine and low scrub oaks, including low turkey oak woods, and transitional sandhill/scrub habitats. 

This species occasionally inhabits powerline clearings or new roadsides. Wireweed (Polygonella 

basiramia) is restricted to bare patches within sand pine-evergreen oak scrub vegetation. It grows 

on areas of bare sand within sand pine (Pinus clausa) and Florida rosemary (Ceratiola ericoides). 

Sandlace (Polygonella myriophylla) occurs in areas of sand pine scrub and ancient sand dunes. 
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Scrub plum (Prunus geniculata) has a very narrow range and small widely scattered populations. It 

frequently forms small colonies of several plants but may grow as solitary individuals. It grows in 

deep, yellow sands of longleaf pine-turkey oak sandhill and white, excessively leached, winddeposited soils of evergreen scrub oak-sand pine scrub. Carter's mustard (Warea carteri) occurs in 

sandy clearings in sand scrub and sandhills; scattered overstory of sand; longleaf or slash pine and 

scrub oaks. Florida ziziphus (Ziziphus celata) is a scrub that occurs on gently rolling hills with 

vegetation dominated by Carya floridana and Quercus species. It prefers open, sunny areas. The 

Perforate Reindeer Lichen (Cladonia perforate) occurs in sandy openings in stabilized sand dunes 

with Florida scrub vegetation. It is often associated with Ceratiola. None of the plants and the 

lichen listed above would find the field test site as suitable habitat and would not be present given 

the historic and continuous use of the release site and the surrounding area as a citrus grove and 

research area used for growing Eucalyptus.

Critical Habitat: There is proposed critical habitat listed for the West Indian manatee but none of 

the proposed habitat occurs in this county. The whooping crane population in this county is a nonessential experimental population and does not have critical habitat (http://crithab.fws.gov/

accessed 9/1/2011).

Conclusion: No federally listed threatened or endangered species or species proposed for listing 

are likely to be found at the release site. If they were to enter the site, their presence would be 

fleeting as the habitat is either not suitable or does not contain constituent elements required by the 

species. Field activities will result in no changes to the habitat used by any listed species or species 

proposed for listing. The site is not within or near designated critical habitat or habitat proposed for 

designation. Therefore, the action will have no effect on listed species or species proposed for 

listing and would not affect designated critical habitat or habitat proposed for designation

4. Pearl River County, MS

This location has been an agricultural research station for more than 5 years. The location has been 

used for conducting research experiments with agricultural crops and grasses. The test site has been 

used for experimental planting of grasses. Approximately 3 acres of field trials of genetically

modified Eucalyptus trees of some lines in the permit are being grown under issued permit 08-011-

106rm under which these trees are allowed to flower. The oldest of these trees were planted in 

October 2007. Current and future site preparation involves herbicide application to remove existing

grasses, subsoiling, preparation for possible irrigation installation, and planting of trees in flat beds. 

The surrounding areas of the test site consist of a grape research farm, mixed stands of hardwoods 

and pine, and a residential area. Under the new permit up to 2.0 acres of field tests could be 

established at this location (at around 300 - 600 trees per acre) over the next three years.

There are six TES animals listed in this county and one TES plant (list accessed from FWS website 

9/1/2011). The animals are the ringed map turtle (Graptemys oculifera), gopher tortoise (Gopherus 

polyphemus), Louisiana black bear (Ursus a. luteolus), gulf sturgeon (Acipenser oxyrhynchus 

desotoi), inflated heelsplitter (Potamilus inflatus) and red-cockaded woodpecker. The listed plant 

is Louisiana quillwort (Isoetes louisianensis). 
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The ringed map turtle (Graptemys oculifera) inhabits wide rivers with strong currents, adjacent 

white sand beaches, and an abundance of basking sites in the form of brush, logs, and debris. The 

field test will not impact this aquatic species which occurs in the Pearl River system –

approximately 11 miles from the field test site. The gopher tortoise (Gopherus polyphemus) 

inhabits dry sand ridges dominated by pine and areas maintained by fire. It is common in longleaf 

pine forests, but its numbers have decreased with the replacement of longleaf pine forests with 

loblolly pine forests. This field test is located in an agricultural research station that would be an 

inhospitable environment for the gopher tortoise. According to James Harris (contacted February 6 

and 7, 2008), Supervisory Wildlife Biologist with the USFWS in Lacombe, LA, the species is 

found in a large geographic area that includes the release site. However, the species is not found 

everywhere within this geographic area. The species is not likely to be on the site because of its 

location on a facility used for many years as an agricultural research station. The research facility 

was contacted to determine if gopher tortoises have been observed at the facility. The farm 

manager has not observed the species at the facility, and no sightings have been reported to him 

during his nine years as manager. He is familiar with the species and has seen them at another 

location about 7-8 miles from the release site. Another employee contacted has worked at the site 

for over 35 years and has never seen a gopher tortoise at the facility but did observe one 

approximately ten years ago about 1⁄4 mile from the facility. The applicant surveyed the site for the 

presence of gopher tortoise burrows on January 29, 2008 and none were found. Considering the 

use of the facility, testimony of the facility employees, and the negative result of the survey, it can 

be concluded that the species is not present now and would be unlikely to use the site while it 

operates as an agricultural research station. The Louisiana black bear (Ursus a. luteolus) prefers 

bottomland forests with diverse food resources, including a variety of hard-mast-producing species. 

Its habitat includes remote areas with little or no human activity so it would not likely be found at 

the site. The red cockaded woodpecker (Picoides borealis) inhabits old growth forests, primarily 

longleaf pine. It might visit the field test site but would not nest there. The gulf sturgeon, the 

inflated hellsplitter mussel, and the Louisiana quillwort occur in aquatic environments so would not 

be affected by the field test. 

Critical habitat: The gulf Sturgeon has designated critical habitat in this county in the Pearl River 

system. The Pearl River is about 11 miles from the field test site. Tributaries of the Pearl River 

system are approximately 3.5 and 7.7 miles from the test site (http://crithab.fws.gov/ accessed 

9/1/2011).

Conclusion: No federally listed threatened or endangered species or species proposed for listing 

are likely to be found at the release site. If they were to enter the site, their presence would be 

fleeting as the habitat is either not suitable or does not contain constituent elements required by the 

species. Field activities will result in no changes to the habitat used by any listed species or species 

proposed for listing. The site is not within or near designated critical habitat or habitat proposed for 

designation. Therefore, the action will have no effect on listed species or species proposed for 

listing and would not affect designated critical habitat or habitat proposed for designation.

5. Berkeley County, SC

This is an extension of a greenhouse facility that has been used for acclimatization of transgenic 

and non-transgenic plants for more than 8 years. The 0.5 acre release site is located adjacent to 
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greenhouse facilities and is surrounded by hardwoods and pine plantations. This site is a secure 

fenced holding area where trees growing in containers are transferred from the greenhouse to the

out-of-doors for acclimatization prior to field planting. Trees will not be allowed to flower at this 

location.

Five threatened or endangered animals and three endangered plants are listed in Berkeley County

(list accessed from FWS website 9/1/2011). The TES animal species are shortnose sturgeon 

(Acipenser brevirostrum), frosted flatwoods salamander (Ambystoma cingulatum), red-cockaded 

woodpecker (Picoides borealis), West Indian manatee (Trichechus manatus), wood stork (Mycteria 

americana). The TES Plants are pondberry (Lindera melissifolia), Canby's dropwort (Oxypolis 

canbyi) and American chaffseed (Schwalbea Americana). 

Canby’s dropwort is an herbaceous perennial whose existing populations are maintained mainly 

through asexual reproduction. This species is strongly clonal, reproducing vegetatively by means 

of stoloniferous rhizomes. It has been found in a variety of habitats, including cypress ponds, 

grass-sedge dominated Carolina bays, wet pine savannahs, shallow pineland ponds and cypresspine swamps or sloughs. The largest and most vigorous populations reported occur in open bays or 

ponds which are flooded throughout most of the year and which have little or no canopy cover. It 

grows in soils with a medium to high organic content, high water table, that are deep, poorly 

drained, and acidic. The pondberry occurs in similar locations, in wetland habitats such as 

bottomland and hardwoods in the interior areas, and the margins of sinks, ponds and other 

depressions in the more coastal sites. The plants generally grow in shaded areas but may also be 

found in full sun. The American chaffseed occurs on sandy peat, sandy loam, acidic, seasonally 

moist to dry soils. It is generally found in habitats described as open, moist pine flatwoods, firemaintained savannas, ecotonal areas between peaty wetlands and xeric sandy soils, and other open 

grass-sedge systems. The site in Berkeley County is a fenced research plot/holding area which 

would be very inhospitable to these species. Laura Zimmerman (843-727-4707 ext. 226) 

(contacted February 14 and 20, 2008) of the Charleston Field Office of the USFWS states that she 

does not believe the species would be likely to be in the area. According to the species’ recovery 

plan, most known occurrences are on US Forest Service land and the only two occurrences on 

private land are not near the release site. A check of the SC Heritage Trust Database did not 

identify any occurrences in the area of the release. 

The shortnose sturgeon occurs in rivers and estuaries. The West Indian manatee occurs in shallow 

coastal waters, rivers, bays and lakes; none of which are close to this release location. The red 

cockaded woodpecker inhabits old growth forests, primarily longleaf pine and might visit the field 

test site but would not nest there. The wood stork primarily inhabits wetland systems notably 

cypress or mangrove swamps and would not find the field test site hospitable. The frosted 

flatwoods salamander inhabits longleaf or slash pine forests lying between drier land upslope and 

wetlands and seasonally inhabits wet pine flat-woods with vernal pools. Discussions with Laura 

Zimmerman (843-727-4707 ext. 226) (contacted February 14 and 20, 2008) of the Charleston Field 

Office of the USFWS indicate that the species is not known to be in the area of the release. Known 

populations in the county are far from the release site in the Francis Marion National Forest. A 

check of the SC Heritage Trust Database did not identify any occurrences in the area of the release.
Page 79 of 91

Critical Habitat: There is critical habitat for the frosted flatwoods salamander in Berkeley County. 

This is located in the Francis Marion National Forest which is about 19 miles away from the release 

location (http://crithab.fws.gov/ accessed 9/1/2011).

Conclusion: No federally listed threatened or endangered species or species proposed for listing 

are likely to found at the release site. If they were to enter the site, their presence would be fleeting 

as the habitat is either not suitable or does not contain constituent elements required by the species. 

Field activities will result in no changes to the habitat used by any listed species or species 

proposed for listing. The site is not within or near designated critical habitat or habitat proposed for 

designation. Therefore, the action will have no effect on listed species or species proposed for 

listing and would not affect designated critical habitat or habitat proposed for designation.

6. Dorchester County, SC

This is an extension of a greenhouse facility that is used for acclimatization of transgenic and nontransgenic plants. This location was previously cleared in 2010 for development as a light industrial 

park, prior to which it had been a managed pine plantation for more than 15 years. The surrounding 

area consists of man-made ponds, hardwoods and pine plantations. This site is a secure fenced

holding area where trees growing in containers are transferred from the greenhouse to the out-ofdoors for acclimatization prior to field planting. Trees will not be allowed to flower at this 

location.

Five threatened or endangered species (TES) are listed in Dorchester County (TES list dated March 

2010 obtained from FWS website 8/16/11). The TES animal species are shortnose sturgeon 

(Acipenser brevirostrum), red-cockaded woodpecker (Picoides borealis) and wood stork (Mycteria 

americana). The TES Plants are pondberry (Lindera melissifolia) and Canby's dropwort (Oxypolis 

canbyi). 

Canby’s dropwort is an herbaceous perennial whose existing populations are maintained mainly 

through asexual reproduction. This species is strongly clonal, reproducing vegetatively by means 

of stoloniferous rhizomes. It has been found in a variety of habitats, including cypress ponds, 

grass-sedge dominated Carolina bays, wet pine savannahs, shallow pineland ponds and cypresspine swamps or sloughs. The largest and most vigorous populations reported occur in open bays or 

ponds which are flooded throughout most of the year and which have little or no canopy cover. It 

grows in soils with a medium to high organic content, high water table, that are deep, poorly 

drained, and acidic. The pondberry occurs in similar locations, in wetland habitats such as 

bottomland and hardwoods in the interior areas, and the margins of sinks, ponds and other 

depressions in the more coastal sites. The plants generally grow in shaded areas but may also be 

found in full sun. The site in Dorchester County is a fenced research plot/holding area which would 

be very inhospitable to these species. It is located in an industrial park that was established a year 

ago so no new land is being disturbed.

The shortnose sturgeon occurs in rivers and estuaries. The red cockaded woodpecker inhabits old 

growth forests, primarily longleaf pine and might visit the release site but would not nest there. 

The wood stork primarily inhabits wetland systems notably cypress or mangrove swamps and 

would not find the field test site hospitable.
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Critical Habitat: There is no critical habitat or proposed critical habitat list for Dorchester County 

(http://criticalhabitat.fws.gov/crithab/ accessed 8/16/11)

Conclusion: No federally listed threatened or endangered species or species proposed for listing 

are likely to found at the release site. If they were to enter the site, their presence would be fleeting 

as the habitat is either not suitable or does not contain constituent elements required by the species. 

Field activities will result in no changes to the habitat used by any listed species or species 

proposed for listing. The site is not within or near designated critical habitat or habitat proposed for 

designation. Therefore, the action will have no effect on listed species or species proposed for 

listing and would not affect designated critical habitat or habitat proposed for designation.

7. Interstate Movement

APHIS considered the possible effects that interstate movement of GE Eucalyptus trees authorized 

by APHIS could potentially have on threatened or endangered species and their critical habitat. 

APHIS could authorize the interstate movement of transgenic Eucalyptus trees in accordance with 

the regulatory requirements identified in 7 CFR 340.4, 340.7 and 340.8, including specific permit 

conditions (see Appendices VI) assigned to this permit that would prevent the dissemination of the 

trees into the environment. As specified in the supplemental permit conditions, ArborGen would 

be moving trees in accordance with an APHIS approved variance that only allows trees to be 

moved in labeled containers within an enclosed vehicle. For the time period between 2005 to 2010, 

APHIS issued 218 permits and notifications that authorized the interstate movement of forest 

trees. Of these 218 authorized movements, there was one compliance incident associated with a 

shipment of plants via mail. This compliance incident was associated with a movement that did not 

have similar APHIS approved variances as those that would be required for ArborGen to move GE 

Eucalyptus trees under this permit request. From 2005 to 2010, no compliance incidents of 

unauthorized release were reported to APHIS for moving GE Eucalyptus trees using similar APHIS 

approved variances that would be required to move trees under this permit request.

Conclusion: Considering the specific permit conditions that must be adhered to by the permit 

requirements identified in 7 CFR 340.4, 340.7 and 340.8, the conditions specified under the APHIS 

approved variance, the proposed supplemental permit conditions for interstate movement of the 

regulated article, and past compliance history of similar types of movements, APHIS has 

determined that the movement of GE Eucalyptus trees in accordance with APHIS approved 

supplemental permit conditions will have no effect on listed species or species proposed for listing 

and would not affect designated critical habitat or habitat proposed for designation.

Overall Conclusions

The field test sites in these permit applications have been in agricultural or forest research, or in 

agricultural production or forest tree plantations for from 5 to 15 years. No federally listed 

threatened or endangered species or species proposed for listing are likely to be found at any of the 

release sites. If they were to enter the site, their presence would be fleeting as the habitat is either 

not suitable or does not contain constituent elements required by the species. Field activities will 

result in no changes to habitat used by any listed species or species proposed for listing. The sites 
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are not within or near designated critical habitat or habitat proposed for designation. Potential 

effects from moving trees would be negligible to non-existent due to the applicants adherence to 

regulatory requirements identified in 7 CFR 340.4, 340.7 and 340.8, including specific permit 

conditions (see Appendix VI) assigned to this permit that would prevent the dissemination of the 

trees into the environment. Therefore, the action will have no effect on listed species or species 

proposed for listing and would not affect designated critical habitat or habitat proposed for 

designation. Because of this no effect determination, consultation and/or the concurrence of the 

USFWS and/or the NMFS are not required. 
Page 82 of 91

APPENDIX V: Proximity of release sites to Protected Areas

The proposed release locations were accessed for their proximity to State and Federal protected 

areas and any potential foreseeable impacts to these areas. The analysis used the Protected Areas 

Database (PAD-US 1.1) (http://www.databasin.org/protected-center/features/PAD-US-CBI) loaded 

into ArcGis Explorer. Proximity to the protected areas was determined using distance measuring 

tools in ArcGis Explorer. 

Baldwin County, AL 

Bon Secour National Wildlife Refuge is located in Baldwin County

http://www.fws.gov/bonsecour/. The Bon Secour National Wildlife Refuge comprises provides 

habitat for the endangered Alabama beach mouse and serves as a nesting site for loggerhead, and 

includes the Kemp’s Ridley sea turtle, the Perdido Key beach mouse and the piping plover. The 

Baldwin County release location is approximately 21 miles away from the Bon Secour National 

Wildlife Refuge. Another protected area located in Baldwin County is the Mobile-Tensaw Delta 

Wildlife Management Area. http://www.dcnr.state.al.us/publiclands/stateLands/foreverwild/FWTracts/mobile-tensawComplex/. It is located approximately 16 

miles from the field test location.

Escambia County, AL

Conecuh National Forest is located in Escambia County http://www.stateparks.com/conecuh.html.

Clear-cut in the 1930s, the Conecuh was reforested with slash pine that reduced the number of 

nesting trees for the endangered red-cockaded woodpecker. The forest is currently undergoing a 

reforestation from slash pine to the native longleaf. In time, this should increase the number of redcockaded woodpeckers as the trees mature. The Escambia County release location is 

approximately 8.5 miles from the western edge of the forest. The Perdido River Wildlife 

Management Area is approximately 35 miles away

http://myfwc.com/viewing/recreation/wmas/cooperative/Perdido-River. The northern border of 

Blackwater River State Forest in Florida is located about a mile away from the release location

http://www.fl-dof.com/state_forests/blackwater_river.html. Blackwater River State Forest is 

known for its longleaf pine/wiregrass ecosystem, which, in combination with the Conecuh National 

Forest to the north and Eglin Air Force Base to the south, is the largest contiguous ecological 

community of this type in the world.

Highlands County, FL

Wales Ridge National Wildlife Refuge occurs in Highlands County

http://www.fws.gov/lakewalesridge/. The release location is about 7 miles from one section of the 

refuge. The refuge contains twenty-two different threatened or endangered species of plants. 

Highlands Hammock State Park is also located in this county and the field test site is adjacent to 

one border of the State park. http://floridastateparks.org/highlandshammock/. This park is known 

for its old-growth hammock, cypress swamps and offers a number of recreational activities. 
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Pearl River County, MS

Bogue Chitto National Wildlife Refuge occurs in Pearl River County

http://www.fws.gov/boguechitto/. The refuge’s bottomland forests contain sweetgum-water oak 

stands interspersed with bald cypress-tupelo brakes and overcup oak-water hickory stands. It 

includes habitat for a number of endangered species. The release site is approximately 9 miles 

away from the eastern boundary of the refuge. 

Berkeley County, South Carolina.

Berkeley County, Georgia contains parts of the Francis Marion National Forest which provides 

habitat for black bear, alligator, and the endangered red-cockaded woodpecker

http://www.fs.usda.gov/scnfs/. The field test site is located approximately 15 miles from Francis 

Marion National Forest.

Dorchester County, SC.

Dorchester County contains Colonial Dorchester State Historic Site 

http://www.southcarolinaparks.com/park-finder/state-park/725.aspx and Givhans Ferry State Park

http://www.southcarolinaparks.com/park-finder/state-park/1219.aspx. Both of these areas are 

protected recreational areas maintained by the State of South Carolina. The field test site is located 

approximately 5 miles from Givhans Ferry State Park and approximately 7 miles from the Colonial 

Dorchester State Historic Site.
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APPENDIX VI: Proposed Supplemental Permit Conditions for 

Movement

For Movement of Eucalyptus grandis x Eucalyptus urophylla under permit 11-052-101rm

1. BRS should be notified in writing of any proposed changes to the permit application (or 

approved permit) including for example changes in movement protocols, additional transgenic 

lines or constructs, new destinations, or amount introduced. Changes usually require 

amendments to the permit and must be pre-approved by BRS. Requests should be directed to 

Regulatory Permit Specialist, USDA APHIS BRS, Biotechnology Permit Services, 4700 River 

Road, Unit 147, Riverdale, Maryland 20737.

2. The regulated article is to be shipped in containers as specified in the Title 7 Code of Federal 

Regulation Section 340.8 (7 CFR 340.8) unless a variance request has been reviewed and 

approved by APHIS/BRS. BRS Notes that the trees would be moved under a previously 

approved Variance 07-014.

3. While in storage all regulated articles covered under this permit are to be kept in a locked 

storage facility with limited access to only authorized personnel. Storage containers must be 

identified as containing a genetically engineered regulated article. At least one sign stating 

"Authorized Personnel Only" must be posted in each area where the regulated article is stored.

4. Upon completion of research, all regulated articles (except those retained for future studies) 

should be rendered non-viable by an appropriate method (e.g., heat or steam sterilization, 

bleach treatment, etc.).

5. There is to be no further distribution of this regulated article under this permit without prior 

approval from State (intrastate movement) and Federal regulatory officials (interstate 

movement).

6. APHIS/BRS and/or APHIS/PPQ personnel may conduct inspections of facilities and/or records 

at any time.

7. Reporting an Unauthorized or Accidental Release

1. According to the regulation in 7 CFR § 340.4(f)(10)(i), APHIS shall be notified orally 

immediately upon discovery and notified in writing within 24 hours in the event of any 

accidental or unauthorized release of the regulated article.

- For immediate verbal notification, contact APHIS BRS Compliance Staff at (301) 734-5690 and 

ask to speak to a Compliance and Inspection staff member. Leave a verbal report on voicemail if 

the phone is not answered by a Compliance Officer.

- In addition, in the event of an emergency in which you need to speak immediately to APHIS 

personnel regarding the situation, you may call:

The APHIS/BRS Regional Biotechnologist assigned in the region where the field test occurs:
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For Western Region, contact the Western Region Biotechnologist at (970) 494-7513

or e-mail: BRSWRBT@aphis.usda.gov

For Eastern Region, contact the Eastern Region Biotechnologist at (919) 855-7622 or e-mail: 

BRSERBT@aphis.usda.gov

Or

The APHIS State Plant Health Director for the state where the unauthorized release occurred. The 

list of APHIS State Plant Health Directors is available at:

http://www.aphis.usda.gov/services/report_pest_disease/report_pest_disease.shtml.

or

http://pest.ceris.purdue.edu/stateselect.html

2. Written notification should be sent by one of the following means:

By e-mail:

BRSCompliance@aphis.usda.gov

By mail:

Biotechnology Regulatory Services (BRS)

Regulatory Operations Program

USDA/APHIS

4700 River Rd. Unit 91

Riverdale, MD 20737

3. Additional instructions for reporting compliance incidents may be found at

http://www.aphis.usda.gov/biotechnology/compliance_incident.shtml

8. No person shall move a regulated article interstate unless the number of the limited permit 

appears on the outside of the shipping container.
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APPENDIX VII: Proposed Supplemental Permit Conditions for 

Release

For Release of Eucalyptus grandis x Eucalyptus urophylla under permit 11-052-101rm

1. The test sites and adjacent land within 100 meters shall be monitored for any volunteer 

Eucalyptus plants every 6 months during the field test (as indicated in the permit) and for one 

year after completion of the field test, during which time any volunteer plants will be destroyed 

before they flower. During the monitoring period following completion of the field test, the site 

will not be planted with Eucalyptus, so that any volunteer seedlings that emerge can be easily 

identified. If volunteers or stump sprouts are still emerging at the end of the first year, a second 

year will be added to the monitoring period to ensure that no shoots are continuing to be 

produced. (2) Please note that transportation of all test and plant materials to and from the field 

test location must be done in accordance with APHIS/USDA regulations outlined in "Container 

requirements for the movement of regulated articles", 7CFR 340.8(b) unless a shipping 

container variance has been approved by APHIS-BRS.

2. Any unusual flowering or seed set much be reported to APHIS as an unusual occurrence. If 

there is an overabundance of flowers or seeds produced this must be reported as soon as 

observed.

3. BRS should be notified in writing of any proposed changes to the permit application (or 

approved permit) including for example confinement protocols, transgenic lines or constructs, 

release locations, acreage, etc. Changes usually require amendments to the permit and must be 

pre-approved by BRS. Requests should be directed to Regulatory Permit Specialist, USDA 

APHIS BRS, Biotechnology Permit Services, 4700 River Road, Unit 91, Riverdale, Maryland 

20737.

4. Any regulated article introduced not in compliance with the requirements of 7 Code of Federal 

Regulation Part 340 or any standard or supplemental permit conditions, shall be subject to the 

immediate application of such remedial measures or safeguards as an inspector determines 

necessary, to prevent the introduction of such plant pests. The responsible party may be subject 

to fines or penalties as authorized by the Plant Protection Act (7 U.S.C. 7701-7772).

5. This Permit does not eliminate the permittee's legal responsibility to obtain all necessary 

Federal and State approvals, including for the use of: (1) any non-genetically engineered plant 

pests or pathogens as challenge inoculum; (2) plants, plant parts or seeds which are under 

existing Federal or State quarantine or restricted use; (3) experimental use of unregistered 

chemical; and (4) food or feed use of genetically engineered crops harvested from the field 

experiment.

6. APHIS/BRS and/or an APHIS/PPQ personnel may conduct inspections of the test location, 

facilities, and/or records at any time.
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7. Harvested plant material may not be used for food or animal feed unless it is first devitalized 

and approved for such use by the U.S. Food and Drug Administration; and for plantincorporated protectants, a tolerance for the pesticide must first be established by the U.S. 

Environmental Protection Agency.

8. Reporting an Unauthorized or Accidental Release

1. According to the regulation in 7 CFR § 340.4(f)(10)(i), APHIS shall be notified orally 

immediately upon discovery and notified in writing within 24 hours in the event of any 

accidental or unauthorized release of the regulated article.

- For immediate verbal notification, contact APHIS BRS Compliance Staff at (301) 734-5690 

and ask to speak to a Compliance and Inspection staff member. Leave a verbal report on 

voicemail if the phone is not answered by a Compliance Officer.

- In addition, in the event of an emergency in which you need to speak immediately to APHIS 

personnel regarding the situation, you may call:

The APHIS/BRS Regional Biotechnologist assigned in the region where the field test occurs:

For Western Region, contact the Western Region Biotechnologist at (970) 494-7513

or e-mail: BRSWRBT@aphis.usda.gov

For Eastern Region, contact the Eastern Region Biotechnologist at (919) 855-7622 or e-mail: 

BRSERBT@aphis.usda.gov

Or

The APHIS State Plant Health Director for the state where the unauthorized release occurred.

The list of APHIS State Plant Health Directors is available at:

http://www.aphis.usda.gov/services/report_pest_disease/report_pest_disease.shtml.

or

http://pest.ceris.purdue.edu/stateselect.html

2. Written notification should be sent by one of the following means:

By e-mail:

BRSCompliance@aphis.usda.gov

By mail:

Biotechnology Regulatory Services (BRS)

Regulatory Operations Program

USDA/APHIS

4700 River Rd. Unit 91

Riverdale, MD 20737

3. Additional instructions for reporting compliance incidents may be found at 

http://www.aphis.usda.gov/biotechnology/compliance_incident.shtml

9. Reporting Unintended Effects:

According to the regulation in 7 CFR § 340.4(f)(10)(ii), APHIS shall be notified in writing as

soon as possible but within 5 working days if the regulated article or associated host organism 

is found to have characteristics substantially different from those listed in the permit application 

or suffers any unusual occurrence (excessive mortality or morbidity, or unanticipated effect on 

non-target organisms).

Written notification should be sent by one of the following means:
Page 88 of 91

By e-mail:

BRSCompliance@aphis.usda.gov

By mail:

Biotechnology Regulatory Services (BRS)

Regulatory Operations Program

USDA/APHIS

4700 River Rd. Unit 91

Riverdale, MD 20737

10. Reports and Notices:

Send notices and all reports (CBI and CBI-deleted or non-CBI copies) to BRS by e-mail, mail, 

or fax.

BRS E-mail:

BRSCompliance@aphis.usda.gov

BRS Mail:

Animal and Plant Health Inspection Service (APHIS)

Biotechnology Regulatory Services (BRS)

Regulatory Operations Program

4700 River Rd. Unit 91

Riverdale, MD 20737

BRS Fax:

Regulatory Operations Program

(301) 734-8910

a. Planting or Environmental Release Report

Planting reports must be submitted to BRS by the 15th of the month following the month in

which the environmental release was started and must include the following data:

- Permit number

- Regulated article

- State

- County

- Location Name (Unique ID)

- GPS coordinates of the planting

- Planting Unique ID

- Planting Start Date

- Total acreage of the regulated article planted or otherwise released

- List of all constructs planted

(This list is optional in the planting report but must be included in the Annual and Field Test 

Reports)

b. Annual Report

Within 30 days after the anniversary date (one-year increments from the effective date listed on 

the permit) an Annual Report must be submitted to APHIS. If the permit has been amended, the 

Annual Report is due 30 days from the anniversary date (one year increments from the effective 
Page 89 of 91

date) of the ORIGINAL permit. FAILURE TO SUBMIT ANNUAL REPORTS MAY 

RESULT IN REVOCATION OF THE PERMIT. The Annual Report shall reflect the current 

status and observations to date for each location. It shall include:

- Permit number

- State

- County

- Location Name(s)

- Location Unique ID(s)

- Any plantings that occurred at each location

- GPS coordinates for each planting

- Size of the plantings (in acres) at each location

- Phenotypic designations (all constructs that were planted)

- Include an accounting of the total acreage that remains in the ground

- Include a detailed map of the plantings

- Indicate if any of the planted material was destroyed before harvest

If so provide the Pre-Harvest destruction completion date and describe how the preharvested material was destroyed

- Indicate if any of the planted material was harvested

If so provide the harvest completion date

- Describe how the harvested material was terminated

- If the material was terminated in the field and not removed from the field, provide the date the 

field test was completely terminated and describe the method of termination

- If the material was removed from the field and terminated off site describe how it was 

disposed and provide the date of off site destruction.

- If material was removed from the field and placed in storage, provide the amount of material 

that was stored and provide a description of the storage location

- Describe any other disposition methods that may be applicable

- Describe any deleterious effects on plants, non-target organisms, or the environment

- Describe methods of observations and resulting data and analyses

- Indicate if you have submitted any of the following:

1. A report on the accidental or unauthorized release of the regulated article;

2. A report that characteristics of the permitted species are substantially different from those 

listed in the application; or

3. A report of any unusual occurrence

Also include any reports of any overabundance of flowering or seed set that occurred and how 

this was handled.

We encourage the inclusion of other types of data if the applicant anticipates submission of a 

petition for determination of non-regulated status for their regulated article.

APHIS considers these data reports as critical to our assessment of plant pest risk and 

development of regulatory policies based on the best scientific evidence. Failure

by an applicant to provide data reports in a timely manner for a field trial may result in the 

withholding of permission by APHIS for future field trials.

c. Field Test Report
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Within six months after the expiration date of the permit, the permittee is required to submit a 

Field Test Report. Field Test Reports provide the final status and observations at each location 

and must include:

- Permit number

- State

- County

- Location Name(s)

- Location Unique ID(s)

- Any plantings that occurred at each location

- GPS coordinates for each planting

- Size of the plantings (in acres) at each location

- Phenotypic designations (all constructs that were planted)

- Indicate if any of the planted material was destroyed before harvest

If so provide the Pre-Harvest destruction completion date and describe how the pre-harvested 

material was destroyed

- Indicate if any of the planted material was harvested

If so provide the harvest completion date

- Describe how the harvested material was terminated

- If the material was terminated in the field and not removed from the field, provide the date the 

field test was completely terminated and describe the method of

termination

- If material was removed from the field and terminated off site describe how it was disposed 

and provide the date of off site destruction.

- If material was removed from the field and placed in storage, provide the amount of material 

that was stored and provide a description of the storage location

- Describe any other disposition methods that may be applicable

- Describe any deleterious effects on plants, non-target organisms, or the environment

- Describe methods of observations and resulting data and analyses

- Indicate if you have submitted any of the following:

1. A report on the accidental or unauthorized release of the regulated article;

2. A report that characteristics of the permitted species are substantially different from those 

listed in the application; or

3. A report of any unusual occurrence

Also include any reports of any overabundance of flowering or seed set that occurred and how 

this was handled.

We encourage the inclusion of other types of data if the applicant anticipates submission of a 

petition for determination of non-regulated status for their regulated article.

APHIS considers these data reports as critical to our assessment of plant pest risk and 

development of regulatory policies based on the best scientific evidence. Failure

by an applicant to provide data reports in a timely manner for a field trial may result in the 

withholding of permission by APHIS for future field trials.

d. Volunteer Monitoring Report

The report must include:
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- Permit number

- State

- County

- Location Name(s)

- Location Unique ID(s)

- Dates when the field site and perimeter fallow zone were inspected for volunteers

- Number of volunteers observed

- Any actions taken to remove or destroy volunteers

The final monitoring report is due no later than three months from the end of the volunteer 

monitoring period.