Page 1 of 98 

# Permit applications 08

- 011

- 1 0 6rm
and 08

- 014

- 101rm received from
ArborGen LLC

Field testing of genetically engineered Eucalyptus grandis X Eucalyptus urophylla

Final Environmental Assessment April 2010

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). United States Department of Agriculture

Marketing and Regulatory Programs

Animal and
Plant Health Inspection
ServicePage 2 of 98 To file a complaint of discrimination, write USDA, Director, Office of Civil Rights, Room 326

– W, Whitten Building, 1400 Independence 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 d oes 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 d ata 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 injur ious 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.Page 3 of 98 TABLE OF CONTENTS

I. SUMMARY

................................ ................................ ................................ ................................ ... 4

II. PURPOSE AND NEE D

................................ ................................ ................................ .............. 5

A. P ROPOSED A CTION

................................ ................................ ................................ ................... 5

B. P URPOSE OF THIS E NVIRONMENTAL A SSESSMENT

................................ ................................ 5
C. N EED FOR T HIS A CTION

................................ ................................ ................................ ........... 6

D. P URPOSE AND D ESCRIPTION OF THE R ESEARCH

................................ ................................ ... 6

III. AFFECTED ENVIRO NMENT

................................ ................................ ................................ 6

IV. ALTERNATIVES

................................ ................................ ................................ .................... 16

A. N O A CTION

– D ENY THE P ERMIT

................................ ................................ .......................... 17

B. P REFERRED A LTERNATIVE

– I SSUE THE APHIS P ERMIT

................................ .................... 17

V. ENVIRONMENTAL CO NSEQUENCES

................................ ................................ .............. 18

A. N O A CTION

................................ ................................ ................................ .............................. 18

B. P REFERRED A LTERNATIVE

................................ ................................ ................................ .... 18
C. C UMULATIVE E FFECTS

................................ ................................ ................................ .......... 33

D. T HE D EGREE TO W HICH THE P OSSIBLE E FFECTS ARE H IGHLY U NCERTAIN OR I NVOLVE U NIQUE OR U NKNOWN R ISKS

................................ ................................ ........................ 34

E. R ISKS TO T HREATENED AND E NDANGERED S PECIES

................................ .......................... 37

F. O THER C ONSIDERATIONS

................................ ................................ ................................ ...... 38
G. C ONCLUSION

................................ ................................ ................................ ........................... 39

VI. LISTING OF AGEN CIES AND PERSONS CON SULTED

................................ ............... 40

VII. REFERENCES

................................ ................................ ................................ ....................... 40

APPENDIX I: STAT US OF EXISTING FIELD TESTS ALLOWED TO FLO WER

............ 46

APPENDIX II: HYDROLO GY CONSIDERATIONS FO R PLANTED
EUCALYPTUS SUBMITTED BY ARBORGE N L LC

................................ .................... 48

APPENDIX III: USDA F OREST SERVICE ASSESS MENT OF IMPACTS ON
HYDROLOGY

................................ ................................ ................................ .................... 59

APPENDIX IV: THREATE NED AND ENDANGERED S PECIES ANALYSIS

.................... 68

APPENDIX V: SUPPLEME NTAL PERMIT CONDITIO NS

................................ ................... 94Page 4 of 98 

## I. Summary

Unite d States Department of Agriculture ( USDA ), Animal Plant Health Inspection Service ( APHIS ) has prepared a n environmental assessment (EA) in response to confined
environmental release permit applications (APHIS Number 08

- 011

- 106rm and 08

- 014

- 101rm) received from ArborGen LLC (ArborGen) to continue research on genetically engineered ( transgenic ) Eucalyptus trees currently permitted by APHIS

, to allow the plant ing of additional trees

, and to allow genetically engineered ( GE ) Eucalyptus trees to
flower at confined field site locations . These plants are a clone 1 coded EH1 derived from a hybrid of Eucalyptus grandis X Eucalyptus urophylla and have been genetically engineered with different constructs. The purpose of the confined environmental release
is for Arbo r Gen to assess the effectiveness of gene constructs which are intended to confer cold tolerance; to test the efficacy of a gene introduced to alter lignin biosynthesis;
and to test the efficacy of a gene designed to alter fertility . In addition the trees have been engineered with a selectable marker gene which confers resistance to the antibiotic kanamycin.

ArborGen has been granted several permits for transgenic Eucalyptus trees, some of
which allow flowering and some of which do not allow flo wering. ArborGen previously applied for a permit (06

- 325

- 111r) requesting that transgenic Eucalyptus trees be allowed to flower at a confined field site in Alabama on 1.1 acres. The permit was granted following the completion of an EA and reaching of a FONSI ( http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf ). This permit was subsequently amended to allow flowering on an additional 5.1 acres at the same location bringing the total acres at this site allowed to flower to 6.2 acres. ArborGen has also been granted permit 08

- 151

- 101r to allow flowering of transgenic Eucalyptus on 1.4 acres on a confined field trial site in Florida. Under permits 06

- 325

- 111r and 08

- 151

- 101r, a t otal of 7.6 acres of trees at 2 sites are authorized by APHIS to flower. These two permits were issued for three years and will expire on June 27, 2010 and June 30, 2011repectively. ArborGen has been allowed to plant trees under a different permit 08

- 039

- 102rm, but they cannot flower under this permit which expires April 25, 2011. All multiyear field trials are issued under three

- year permits, and if applicants wish to continue the field trials thay can renew these permits for an additional 3 years.

ArborGen has submitted two new permit applications 08

- 014

- 101rm and 08

- 011

- 106rm, for planting and growing trees on 28 sites and is also requesting that trees on 27 of these sites be allowed to flower (the subject of this EA). The original request was to plant 29 sites and allow 28 to flower, however, one location was removed from permit application

08

- 014

- 101rm, which reduced the number to 28 sites

, with 27 allowed to flower. The 28 sites include those already authorized for planting under permit 08

- 039

- 102rm, As of April 2010, ArborGen is currently growing approximately 37 acres of the transgenic Eucalyptus hybrid on 15 sites under APHIS permit 08

- 039

- 102rm that would be allowed to flower under the two new permits if approved. These two new permits ( 08

- 014

- 101rm
and 08

- 011

- 106rm ) combined would allow flowering on up to 330 acres across 27

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.Page 5 of 98 locations. The size of each confined field test site in these new permit applications ranges from 0.5 to 20 acres.

T he genes are the same as th ose in permit s 06

- 32 5

- 111r and 08

- 151

- 101r, with the addition of a gene aimed at al tering lignin biosynthesis that is being tested on some of the sites. The status of the field tests permitted under permits 06

- 325

- 111r and 08

- 151

- 101r is included in Appendix I.



## II. Purpose and Need

A. Proposed Action

The proposed action is for APHIS, Biotechnology Regulatory Services (BRS) to issue

two confined environmental release permits to allow the planting, field testing and flowering of a GE Eucalyptus hybrid clone engineered to exp ress various genes on 2 8
confined field site locations in the southeastern United States . The genes are intended to confer increased tolerance to cold temperatur es , alter fertility and alter lignin biosynthesis

. In addition there is a gene used as a sele ctable marker that confers no other
benefit to the GE Eucalyptus trees

.


### B. Purpose of this Environmental Assessment

The purpose of this EA is to assess potential environmental impacts of field research studies being conducted on 2 8 research sites in Alaba ma, Florida

, Georgia

, Louisiana

, Mississippi, South Carolina, and Texas . Two permit applications were received by

APHIS

– BRS in January 2008 from ArborGen to continue research on GE Eucalyptus hybrid trees originally planted under APHIS authorized notifica tions, to plant additional trees, and to allow all the trees except the trees on one of the sites in South Carolina to flower. These are permit application numbers 08

- 011

- 106rm and 08

- 014

- 101rm.

This EA was conducted pursuant to : (1) The National Environ mental 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, issuance s of permit s 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 ma y be prepared, pursuant to 7 CFR § 372.5(d) to determine if an Environmental Impact Statement is warranted

. In this case,

an EA has been prepared because the permittee intends to allow GE Eucalyptus trees to grow under an APHIS confined environmental rel ease permit for 7 years to allow the

trees to reach maturity and flower. The actions described in the permit applications
involve the confined field release of transgenic Eucalyptus grandis x Eucalyptus urophylla into the environment. APHIS has prepared this EA to address concern s that
the increased number of locations and size of the releases that would be allowed to flower could potentially result in the unconfined release of pollen and/or seed into thePage 6 of 98 environment

. Other impacts such as hydrology, al lelopathy and fire are also addressed in
this EA

.



### C. Need for This Action

USDA

- APHIS

- 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 Protection Act, as amended, (7 U.S.C. 7701

– 7772), regulates the introduction (importation, interstate movement, or
release into the e nvironment) 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 pest s. 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 r eceives an application for a permit for environmental release, the application is evaluated to determine whether the environmental release, with appropriate conditions imposed, can be carried out while preventing the dissemination and establishment of plan t 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 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 and gene to alter lignin biosynthesis 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. In addition, the applicant is researching mechanisms for altered fertility . 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.



## III. Affected Environment

Biology of Eucalyptus and S tatus in the United StatesPage 7 of 98 The genus Eucalyptus belongs to family Myrtaceae (subfamily: Leptospermoideae) which includes over 700 species. Eucalyptus is native to Austr alia 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 b iology 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 commer cial 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. 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 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 f orty 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 ( L uis S ilva , International Paper

Co mpany , Brazil , comment to docket APHIS

- 2008

- 0059 )

. O ne of the parents of the hybrid, E. grandis has been shown to be naturalized in the State of Florida ( http://www.florida.plantatlas.usf.edu ). In South Africa

, E. grandis has been shown to be
an invasive species (Forsyth et al., 2004). It is also naturalized in both New Zealand (New Zealand Plant Conservation Network 2005 : http://www.nzpcn.org.nz/exotic_plant_life_and_weeds/we ed_list.asp

. ) and Ecuador ( http://i3n.iabin.net/participants/ecuador.html ). Daehler (1998) listed E. grandis as an invader of natural areas. These and other data were recently used in the Unive rsity of
Florida’s IFAS Assessment of Non

- native Plants in Florida’s Natural Areas. The

a ssessment conclusion for E. grandis is that it is now: "Predicted to be invasive: recommend only under specific management practices that have been approved by the
IFAS Invasive Plant 2 Working Group" ( http://plants.ifas.ufl.edu/assessment/predictive_response_forms.html ). S pecific management practices for four specific cultivars of E. grandis have b een approved by the Working Group ( http://plants.ifas.ufl.edu/assessment/

-- see Approved, Specif i ed and

Lim i ted Uses link ). The University of Florida IFAS has not made an assessment of t he E. grandis and E. urophylla hybrid.Page 8 of 98 Numerous species of Eucalyptus were introduced into California during that State’s early history (see Santos: http://wwwlibrary.csustan.edu/bsantos/euc toc.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 E ngineered into Eucalyptus

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 genetica lly engineered with different constructs. The purpose of the field trials is to test the effectiveness of the CBF gene which is intended to confer cold tolerance and to test the efficacy of the Barnase gene designed to alter fertility. In a small set of experiments the CBF and Barnase genes are also being tested in combination with genes introduced to alter lignin biosynthesis (claimed as CBI). In addition the trees have been engineered
with a common selectable marker gene ( nptII ) which confers resistanc e to the antibiotic kanamycin.

Confined Field Test Locations

The confined field tests are taking place on land controlled by ArborGen or through contracts for field testing. The exact locations are claimed as Confidential Business Information ( CBI ) and have been submitted as part of the APHIS permit application

. Under the two permits, there are 28 sites where trees have been planted or will be planted, and on 27 of these sites the trees will be allowed to flower. See below for the
States and Counties in which these 28 sites are located. An additional site in South Carolina is a holding area for plants in pots and trees. Trees will be held there for planting and will not be allowed to flower.

All the confined field test sites listed in this permit application are either on privately owned managed plantation forests and agricultural farm lands or experimental research stations managed by academic institutions and industry. 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, i rrigation, fertilization, controlled grazing and management of grasses. Standard silvicultural practices will be used at these sites for the duration of the field tests. Surveys conducted by the applicant at each of these locations indicate that there ar e not any old growth forests or undisturbed natural areas in the immediate surroundings of the test sites. The trees will be planted from 0.5 up to 20 acres, depending on the location. In the case of these tests the planting density will be from 300

- 60 0 trees per acre 2

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 . An acre is about
the size of a football field.Page 9 of 98 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. Site preparation involved 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. Approximately 6.2 acres of existing field trials of genetically modif ied Eucalyptus of the same clone (EH1) are being grown under issued permit # 06

- 325

- 111r under which these trees are allowed to flower. T he oldest of these trees
at this site a re now entering their fifth growing season.

Up to 8,000 containerized trees, including non

- transgenic trees could be transferred to this field test site and planted in field plots of up to 10 acres (at around 300

- 600 trees per acre)
over the next thre e years. An additional f ield trial of the same clone (EH1) was established at
this site on approximately 0.8 acres now covered under permit 08

- 039

- 102rm

. This trial

would be transferred to permit # 08

- 011

- 106rm to allow flowering.

Escambia County Alabama Site:

This location had previously been used as an intensely managed pasture for more than 5 years and was planted with grasses suitable for cattle grazing. Site preparation involved herbicide application to remove existing grasses, subsoiling, p reparation for possible irrigation, and planting of the test trees in flat beds. The surrounding areas of the test site consist of experimental forest trees ; approximately 30 year

- old slash pine and a re

- forested area with less than 7 year

- old mixed stand s of pine and hardwood species.

Up to 8,000 containerized trees, including non

- transgenic trees could be transferred to this field test site and planted in field plots of up to 10.3 acres over the next three years. The existing f ield trials of the same c lone (EH1) were planted at this site on approximately 0.5
acres that are now covered under Permit #08

- 039

- 102rm. These trials w ould be transferred to permit # 08

- 011

- 106rm to allow flowering.

Evan s County Georgia Site:

This location has been a commerci al nursery for forest seedling production for over 30 years. Site preparation involved herbicide application, subsoiling and planting of trees in flat beds. The areas surrounding the test site consist of nursery beds of forest tree seedlings, experimental forest trees, agricultural crops and mixed stands of hardwood and pine.

Up to 4,000 containerized trees, including non

- transgenic trees, could be transferred to this field test site and planted on field plots of up to 5 acres over the next three years. A field trial of the same clone (EH1) was planted on approximately 0.2 acres at this site that is now covered under BRS permit # 08

- 039

- 102rm. This trial w ould be transferred to permit # 08

- 011

- 106rm to allow flowering.

acres can have from 3000 to 16,000 total trees pla nted in the ground. Twenty acres, as defined by forest plantation standards in the southeast, is considered a small planting.Page 10 of 98 Saint Landry’s Parrish Louisiana Sit e:

This location has been an experimental agricultural farm for more than 25 years, used for conducting research experiments with soybean, cotton and wheat. Site preparation involved herbicide application, subsoiling and planting of trees in flat beds. T he areas surrounding the test site consist of agricultural fields of rice, sugarcane and millet.

Up to 8,000 containerized trees, including non

- transgenic trees could be transferred to this field test site and planted in field plots of up to 11.7 acres over the next three years. F ield
trials of the same clone (EH1) were planted at this site on approximately 1.9 acres under
Permit #08

- 039

- 102rm. All tests except 0.2 acr es were subsequent ly terminated

. The remaining 0.2 acres w ould be transferred to permit # 08

- 011

- 106rm to allow flowering.

Marshall County Mississippi Site:

This location has been an agricultural research station for more than 50 years, used for
conducti ng research experiments with agricultural crops and grasses. The test site was previously used for experimental planting of grasses. Site preparation involved herbicide application, subsoiling and planting of trees in flat beds. The areas surrounding the t est site consist of experimental forest trees, agricultural fields, and less than 5 year

- old pine plantations.

A field test of the same clone (EH1) was established at this site on 0.5 acres under permit 08

- 039

- 102rm. This test was subsequent ly terminated . No additional plantings are currently anticipated at this site. Additional tests, if planted at this site, would be allowed to flower

under permit # 08

- 011

- 106rm

.

Pearl River County Mississippi Site:

This location has been used as an agricultural resea rch station for more than 5 years for conducting research experiments with agricultural crops and grasses. The test site was used for experimental planting of grasses. Site preparation involved herbicide application to remove existing grasses, subsoiling , preparation for irrigation installation, and planting of
trees in flat beds. The areas surrounding the test site consist of a grape research farm, mixed stands of hardwoods and pine, and a residential area.

Up to 4,000 containerized trees, including n on

- transgenic trees could be transferred to this field test site and planted in field plots of up to 7 acres over the next three years. The existing

f ield trials of the same clone (EH1) were established at this site on approximately 3.0 acres
that are now covered under permit 08

- 039

- 102rm. These trials w ould be transferred to permit # 08

- 011

- 106rm to allow flowering.

Bamberg County South Carolina Site:

This location has been a managed forest plantation for more than 14 years. The location has been speci fically used for short

- rotation planting of hardwoods and softwood trees for forestry research. Standard silvicultural practices for site preparation, irrigation, fertilization, planting and harvesting have been used at this location. Similar practices wil l be used for the additional field tests to be established at this site. The areas surrounding the test site consistPage 11 of 98 of experimental forest trees, young pine plantations, mixed stands of hardwoods and pine, and agricultural fields. There are experimental t est plots of non

- transgenic cold

- hardy E. macarthurii, E. benthamii, E. viminalis, E. badjensis, and E. dorrigoensis planted at least 1000 meters from the test plot location.

Up to 4,000 containerized trees, including non

- transgenic trees could be transf erred to this field test site and planted in field plots of up to 8.3 acres over the next three years. Field
trials of the same clone (EH1) were established at this site on 3.5 acres under permit 08

- 039

- 102rm. All tests except 0.2 acres were subsequent ly terminated. The remaining 0.2 acres

w ould be transferred to permit # 08

- 011

- 106rm to allow flowering.

Berkeley County South Carolina Site 1:

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

- trans genic plants for more than 7 years. The 0.5 acre release site is located adjacent to 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 th e out

- of

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

Berkeley County South Carolina Site 2:

This location has been a managed forest plantation for more than 7 years. The location has been specifically used for short rotation planting of cottonwood and Eucalyptus Hybrid (EH1) for forestry research. Site preparation involved herbicide application, subsoiling, drip irrigation installation, and planting of trees in flat beds. The test site is located adjacent to greenhouse facilities and is surrounded by pine plantations.

Field trials of the same clone (EH1) were established at this site on 0.5 acre s under permit

08

- 039

- 102rm. These tes ts were subsequently terminated. No additional plantings are currently anticipated at this site. Additional tests, if planted at this site, would be allowed to

flower under permit # 08

- 011

- 106rm

.

Charleston County Sou th Carolina Site:

This location has been a managed forest plantation for more than 10 years. The location has been specifically used for short

- term planting of hardwoods and softwood trees for forestry
research. Standard silvicultural practices for sit e preparation, irrigation, fertilization, planting and harvesting were used at this location. Similar practices were used for the additional field
tests established at this site. The areas adjacent to the field test site include young mixed

stands of har dwoods and pines.

Field trials of the same clone (EH1) were established at this site on 3.1 acres under permit 08

- 039

- 102rm. All tests were subsequently terminated. No additional trials are currently planned for this site. Additional tests, if planted a t this site, would be allowed to flower under permit # 08

- 011

- 106rm

.

Mar l boro County South Carolina Site:Page 12 of 98 This location has been a commercial nursery for forest seedling production for over 30 years. Site preparation involved herbicide application, subsoiling and planting of trees in flat beds. The surrounding areas consist of field plantings of agricultural crops, nursery beds of forest tree seedlings and less than 30 year

- old mixed hardwood and pine plantations.

A field trial of the same clone (E H1) was established at this site on 0.3 acre under permit 08

- 039

- 102rm. This test was subsequent ly terminated. No additional trials are currently planned
for this site. Additional tests, if planted at this site, would be allowed to flower under permit

# 08

- 011

- 106rm

.

Hardin County Texas Site:

This location has been a managed forest plantation for more than 30 years. The location consists of mixed hardwood tree plantations planted using standard silvicultural practices and was harvested by the owner in 2 004. The test site is within the larger harvested area and was

re

- bedded by the owner for planting. Site preparation included herbicide application and sub

soiling. The areas surrounding the test site consist of mixed hardwood stands and managed

lobloll y pine plantations. There are experimental test plots of non

- transgenic cold

- hardy Eucalyptus species, E. macarthurii, E. benthamii, and E. viminalis planted within 100 m of the transgenic test plot location.

Up to 16,000 containerized trees, including non

- transgenic trees, could be transferred to this field test site and planted in field test plots of up to 20 acres over three years. A field trial of the same clone (EH1) was established at this site on 19.7 acres und er permit 08

- 039

- 102rm

. This trial w ould be transferred to permit # 08

- 011

- 106rm to allow flowering.

Jasper County Texas Site 1:

This location has been a managed pine plantation for more than 25 years. Previous plantings were cultivated in beds using standard silvicultural practices. The existing pine plantation at this site was harvested by the owner in 2007 and re

- bedded for planting. The test site is within the larger harvested and bedded site prepared by the site owner. Further site preparation involved herbicide application, subsoi ling and planting of trees in flat beds. The areas surrounding the test site consist of harvest age pine plantations.

Up to 16,000 containerized trees, including non

- transgenic trees, could be transferred to this field test site and planted in field tes t plots of up to 20 acres over three years. A field trial of the same clone (EH1) was planted at this site on approximately 0.7 acres under BRS permit #

08

- 039

- 102rm. This trial w ould be transferred to permit # 08

- 011

- 106rm to allow flowering.

Jasper County Texas Site 2:

This location has been in agriculture for more than 30 years. The test site was used for pasture for the past 10 years. Site preparation involved herbicide application to remove existing grasses, subsoiling, preparation for irrigation installation, and flat plantings of trees. The areas surrounding the test site consist of natural stands of mixed pine and hardwoods.Page 13 of 98 Up to 8,000 containerized trees, including non

- transgenic trees, could be transferred to this
field test site and planted in field test plots of up to 10 acres over three years. A field trial of the same clone (EH1) was established at this site on 1.0 acre under permit 08

- 039

- 102rm

. This trial w ould be transferred to permit # 08

- 011

- 106rm to allow flowering.

Jefferson County Texas Site:

This location has been used for managed agricultural production of rice for more than 5 years. Site preparation will involve herbicide application, subsoiling and planting of trees in flat beds. The surrounding areas of the test site consist of rice plantations. Up to 8,000 containerized trees, including non

- transgenic trees, could be transferred to this field test site and planted in field test plots of up to 10 acres over three years

Newton County Texas Site 1:

This loca tion has been a managed loblolly pine plantation for at least 30 years. The previous plantings were cultivated in beds using standard silvicultural practices and the areas have recently been harvested. Site preparation has or will include herbicide appli cation, plowing, and planting of trees in raised or flat beds. The areas surrounding the test sites consist of managed loblolly pine plantations and/or mixed hardwood stands. Up to 16,000 containerized trees, including non

- transgenic trees, could be tran sferred to this field test site and planted in field test plots of up to 20 acres over three years.

Newton County Texas Site 2:

This location has been a managed loblolly pine plantation for at least 30 years. The previous plantings were cultivated in be ds using standard silvicultural practices and the areas have recently been harvested. Site preparation has or will include herbicide application, plowing, and planting of trees in raised or flat beds. The areas surrounding the test sites consist of

manage d loblolly pine plantations and/or mixed hardwood stands. Up to 16,000 containerized trees, including non

- transgenic trees, could be transferred to this field test site and planted in field test plots of up to 20 acres over three years.

Newton County Texas Site 3:

This location has been a managed loblolly pine plantation for at least 30 years. The previous plantings were cultivated in beds using standard silvicultural practices and the areas have recently been harvested. Site preparation has or will include herbicide application, plowing, and planting of trees in raised or flat beds. The areas surrounding the test sites consist of managed loblolly pine plantations and/or mixed hardwood stands. Up to 16,000 containerized trees, including non

- transge nic trees, could be transferred to this field test site and planted in field test plots of up to 20 acres over three years.

Newton County Texas Site 4:

This location has been a managed loblolly pine plantation for at least 30 years. The previous plantin gs were cultivated in beds using standard silvicultural practices and the areas have recently been harvested. Site preparation has or will include herbicide application, plowing, and planting of trees in raised or flat beds. The areas surrounding the tes t sites consist ofPage 14 of 98 managed loblolly pine plantations and/or mixed hardwood stands. Up to 16,000 containerized trees, including non

- transgenic trees, could be transferred to this field test site and planted in field test plots of up to 20 acres over three years.

Bay County Florida Site:

This location was used as an intensely managed pasture for more than 15 years, and was planted with grasses suitable for cattle grazing. Site preparation involved herbicide application to remove existing grasses, subsoiling, preparation for possible irrigation installation, and planting of trees in flat beds. The areas surrounding the test site consist of experimental forest trees, agricultural crops and less than 25 year

- ol d hardwoods and pine.

Up to 4,000 containerized trees, including non

- transgenic trees, could be transferred to this site and planted in field test plots of up to 5 acres over the next three years. F ield trials of the same clone (EH1) were planted at thi s site on approximately 1.35 acres of test plots under permit #08

- 039

- 102rm. All tests except approximately 0.2 acre were subsequently

terminated. The remaining 0.2 acres w ould be transferred to permit # 08

- 014

- 101rm to allow flowering.

Columbia County Florida Site:

This location has been a managed pine plantation for more than 20 years. Previous plantings were cultivated in beds using standard silvicultural practices. This area of the tract was burned in a fire in May 2007. After the fire, the area was raked and bedded by the site owner in preparation for re

- planting. The test site is within the larger harvested and bedded area, which is surrounded by existing pine plantations and additional harvested tracts. Up to 16,000 containerized trees, inclu ding non

- transgenic trees, could be transferred to this site and planted in field test plots of up to 20 acres over the next three years.

Gadsden County Florida Site 1 :

This location has been used as an agricultural research station for more than 10 year s for conducting research experiments on agricultural crops. Standard silvicultural practices were used for site preparation, including herbicide application, plowing and planting of trees in
raised or flat beds. The areas surrounding the test site consi st of mixed pine

- hardwood forests and pine plantations as well as research plantings of agricultural and horticultural

crops. There is an experimental plot of non

- transgenic Eucalyptus species including E. grandis, E. amplifolia , and E. camaldulensis at l east 1000 meters away from the transgenic
test location.

Up to 8,000 containerized trees, including non

- transgenic trees, could be transferred to this site and planted in field test plots of up to 10 acres over the next three years. A field trial of
the same clone (EH1) was planted at this site on approximately 0.2 acres under permit # 08

- 039

- 102rm. This trial w ould be transferred to permit # 08

- 014

- 101rm to allow flowering.

Gadsden County Florida Site 2:

This location has been an agricultural research station for more than 10 years. The field has been fallow for approximately seven years. Standard silvicultural practices will be used forPage 15 of 98 site preparation, including herbicide application, plowing and plan ting of trees in raised or
flat beds. The areas surrounding the test site consist of mixed pine

- hardwood forests and pine plantations, as well as research plantings of agricultural and horticultural crops. Up to 12,000 containerized trees, including non

- transgenic trees, could be transferred to this site and planted in field test plots of up to 15 acres over the next three years.

Glades County Florida Site:

Due to changes in experimental plans this site will not be used for field trials of these

trees a nd was removed from the permit application per the applicant ’ s request

.

Highlands County Florida Site:

This location was previously used for managed production of citrus for at least 15 years. The planting area at this location had been used for field trials of transgenic Eucalyptus for more than 6 years. Site preparation involved herbicide application, plowing, and planting of trees
in flat beds. Areas surrounding the test site consist of less than 5 year

- old second

- growth pine and hardwood with mixed grasses. Field trials of 1.4 acres of some of EH1 translines are being grown under issued permit # 08

- 151

- 101r under which these trees are allowed to
flower. These trees are now entering their fifth growing season. An additional 2.3 acres of tests of different lines with the same EH1 hybrid are being grown under permits 08

- 039

- 102rm (2.0 acres) and 09

- 070

- 101rm (0.3 acres)

.

Up to 8,000 containerize d trees, including non

- transgenic trees, could be transferred to this site and planted in field test plots of up to 10 acres over the next three years.

Marion County Florida Site:

This location has been used as an agricultural research station for more t han 5 years for conducting research experiments on agricultural crops. Site preparation involved herbicide application, plowing, and planting of trees in flat beds. Areas surrounding the test site consist of agricultural fields and plantings of horticult ural crops. An experimental test of non

transgenic E. amplifolia is planted approximately 200 meters from the test plot location. In addition, a field test of 0.3 acres of different lines with the same EH1 hybrid was planted at this site under permit 9

- 0 70

- 101rm.

Up to 8,000 containerized trees, including non

- transgenic trees, could be transferred to this site and planted in field test plots of up to 10 acres over the next three years. A field trial of
some of the same lines of EH1 clone was establishe d at this site on 0.9 acres under permit 08

- 039

- 102rm. This trial w ould be transferred to permit # 08

- 014

- 101rm to allow flowering.

Taylor County Florida Site 1:

T his location has been a managed pine plantation for over 20 years. The previous plantings were cultivated in beds using standard silvicultural practices. The existing pine plantations at these sites were harvested by the owner and prepared for re

- planting. Additional site preparation involved herbicide application, plow ing to remove stumps, and planting of treesPage 16 of 98 in raised beds. Areas surrounding these test sites consist of managed stands of pine plantations.

Up to 16,000 containerized trees, including non

- transgenic trees, could be transferred to this site and planted in field test plots of up to 20 acres over the next three years. A field trial of the same clone (EH1) was planted at this site on approximately 4.3 acres under permit # 08

- 039

- 102rm. This trial w ould be transferred to permit # 08

- 014

- 101rm to allow flower ing.

Taylor County Florida Site 2:

This location has been under managed pine plantations for over 20 years. The previous plantings were cultivated in beds using standard silvicultural practices. The existing pine plantations at these sites were harvest ed by the owner and prepared for re

- planting. Additional site preparation involved herbicide application, plowing to remove stumps, and planting of trees in raised beds. Areas surrounding these test sites consist of managed stands of pine plantations.

Up to 16,000 containerized trees, including non

- transgenic trees, could be transferred to this site and planted in field test plots of up to 20 acres over the next three years. A field trial of the same clone (EH1) was planted at this site on approximate ly 3.7 acres under permit # 08

- 039

- 102rm. This trial w ould be transferred to permit # 08

- 014

- 101rm to allow flowering.

Taylor County Florida Site 3 :

This location has been under managed pine plantations for over 20 years. The previous plantings were cultivated in beds using standard silvicultural practices. The existing pine plantations at these sites were harvested by the owner and prepared for re

- planting. Additional site preparation involved herbicide application, plow ing to remove stumps, and planting of trees in raised beds. Areas surrounding these test sites consist of managed stands of pine plantations.

Up to 16,000 containerized trees, including non

- transgenic trees, could be transferred to this site and planted in field test plots of up to 20 acres over the next three years. A field trial of the same clone (EH1) was planted at this site on approximately 1.3 acres under permit # 08

- 039

- 102rm. This trial w ould be transferred to permit # 08

- 014

- 101rm to allow flo wering.



## IV. Alternatives

This EA analyzes the potential environmental consequences of a proposal for APHIS to
issue two confined environmental release permit s to ArborGen to allow the planting, field testing and flowering of a GE Eucalyptus hybrid clone engineered to express various genes on 2 8 confined field site locations in the southeastern United States. Specifically

, t hese two permits would authorize ArborGen to continue research on GE Eucalyptus hybrid trees allowed to be planted under APHIS author ized notifications and permits on
28 sites , to plant additional trees, and to allow all the trees except the trees on one of the
sites in South Carolina to flower. Therefore 27 of 28 sites originally allowed to be planted with the GE Eucalyptus would be allowed to flower. Based upon the permitPage 17 of 98 application s submitted by ArborGen, t wo alternatives are considered and analyzed in this EA: (1) deny the permit s and (2) issue the APHIS permit s

.



### A. No Action

– Deny the Permit

Under APHIS

– BRS regulat ions, the Administrator must either grant or deny permits properly submitted under 7 CFR part 340. For the purposes of this EA , the No Action alternative would be the denial of permit applications 08

- 011

- 106rm and 08

- 014

- 101rm.

Transgenic Eucalyptus tree s ( hybrid of Eucalyptus grandis X Eucalyptus urophylla ) have been previously approved for planting under APHIS authorized notifications, and permits. ArborGen has been allowed to plant trees on 28 sites under permit 08

- 039

- 102rm that are requested under t he two new permit application s to allow flowering; and

t wo field test s ites totaling 7.6 acres have been authorized by APHIS to flower under permits 06

- 325

- 111r and 08

- 151

- 101r. All three permits are still active. Under the No Action Alternative, the transgenic Eucalyptus plants ( hybrid of Eucalyptus grandis X Eucalyptus urophylla ) currently approved by APHIS for confined field release under permit 08

- 039

- 102rm will continue to not be allowed to fl ower. The trees could remain in the field, but the applicant will be required to either remove developing flowers or remove the trees from the field test if removing flowers becomes too difficult. Under this alternative, the applicant would not be allowe d 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. (Permits 06

- 325

- 111r (as amended) and 08

- 151

- 101r , both of which already allow flowering, will remain in eff ect until they expire (unless they are renewed under a new permit). These two permits are not affected by the issuance of permits 08

- 014

- 101rm and 08

- 011

- 106rm. )



### B. Preferred Alternative

– Issue the APHIS Permit

The APHIS

- preferred alternative is to iss ue the APHIS confined field release permits for

a hybrid of Eucalyptus grandis X Eucalyptus urophylla with supplemental permit
conditions (Appendix V) for the requested three

- year period. The permits will need to be
renewed and subsequently approved by APHIS to allow the transgenic plants to remain in the ground beyond th e 3 year time period. Under this alternative, APHIS would issue
two confined environmental release permit s to ArborGen to allow the planting, field testing and flowering of a GE Eucalyptus hybrid clone engineered to express various genes on 2 8 confined field site locations in the southeastern United States. Specifically, these two permits would authorize ArborGen to continue research on GE Eucalyptus hybrid trees originally planted under APHIS authorized notifications, to plant additional trees, and to allow all the trees except the trees on one of the sites in South Carolina to
flower. Under this alternative, GE Eucalyptus hybrid trees would be allowed to g row and flower where the applicant can gather data on performance of the transgenic trees over a multiyear period and the efficacy of the genes in a wide variety of environments. This
alternative would allow the safe development and use of GE organisms un der the mission
of BRS.Page 18 of 98 

## V. Environmental Consequences

A. No Action

Under the no action alternative, the applicant would not be allowed to let the trees flower

on 2 7 sites currently authorized by APHIS for planting under permit 08

- 039

- 102rm . At
this tim e 15 of the 2 7 sites have been planted with Eucalyptus hybrid trees

. The applicant could continue to plant trees under permit 08

- 039

- 102rm, but w ould not allow the trees to
flower. Preventing flower formation would prove impossible over time because the trees

g row too tall and too many flowers are produced to remove manually. Therefore the
trees on these sites under 08

- 039

- 102rm would likely be cut down prior to maturity. The Eucalyptus trees currently planted under APHIS permits would remain in the gro und but would likely be cut down within 2 or 3 years to prevent flowering. At each of the field sites the locations could remain planted in short rotation Eucalyptus field tests if approved by APHIS under a notification or permit, or the sites could be re turned to other forest tree production or agriculture/forestry research. Some sites could be returned to pasture or other agricultural activities. Intense activity including the use of heavy machinery for land preparation, general upkeep, irrigation, and fertilization for the management of tree plantings and grasses would continue. For forest tree plantings, standard silvicultural practices would continue to be used at these sites

.



### B. Preferred Alternative

Under this alternative, the applicant would be allowed to let GE Eucalyptus trees produce flowers and to plant more trees. This means that the trees would remain in the ground for at least 3 years and most likely longer since the applicant has indicated t hat they are plan ning to renew the permits. If the renewal permits are approved by APHIS , t he 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, pla nting, irrigation, and
harves ting of trees would continue to be routinely used on these sites.



### Potential Environmental Impact of the Preferred Alternative

Alteration in Susceptibility to Disease or Insects

– Potential of the Eucalyptus to
H arbor P lant P ests

Overall impacts on disease or insect susceptibility would be similar to the no action
alternative.

As presented in the permit application submitted by Arbo r Gen :

**•** T here has been no intentional genetic change in these plants to affect their

suscep tibility to disease or insect damage.Page 19 of 98 **•** All of the genes have been previously tested in Eucalyptus in existing field tests and the permittee has observed no changes in the incidence of pests, beneficial insects or pathogens between the transgenic and non

- t ransgenic controls.

**•** 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 damag e.

There might be a concern that altered lignin could lead t o an increase in insect or disease susceptibility, but the results so far with this particular gene do not indicate that this is the case ( see below). As prescribed in the supplemental permit conditions assigned to these permits

, periodic monitoring of the fie ld 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).

Althoug h 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 i mport and quarantine under a USDA

– APHIS PPQ Post

- entry quarantine permit.

Expression of the G ene P roducts, N ew E nzymes, or C hanges to P lant M etabolism

- Risk of the Gene P roducts on the Environment

Overall impacts o f genes for the selectable marker, cold tolerance, altered flowering and altered lignin would be similar to the no action alternative. These same types of genes are currently being used in GE Eucalyptus trees being field tested under current APHIS notifications or pe rmits for confined field release. T he same genes are being expressed
whether the trees are allowed to flower or not to flower.

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 gen e 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 have been deregulated by APHIS (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 cold

-Page 20 of 98 and drought

- responsive DNA regulatory sequence designated as the C

- repeat (CRT)/dehydration

- responsive element (DRE) (Baker et al. 1994)

, (Yamaguchi

Shinozaki and Shinozaki 1994) , w hich is found in the promoter regions of many cold

inducible genes (Maruyama et al. 2004)

.

A common observation across experiments in which CBF genes are overexpressed in
transge nic 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 wa s associated with smaller leaves, stunted plants, delayed flowering, and reduction or lack of tuber production (Pino 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 sa me 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

ca uses 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 e xpected to alter the characteristics of the engineered plants other than imparting tolerance to cold temperatures. 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 t o the environment. These genes are in a number of previous and existing field tests and 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 and granted non

- regulated status by APHIS. There is no rea son to believe
that the function a nd 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 pollenPage 21 of 98 formatio n. 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 being conducted in Alabama under the approved BRS permit (BRS # 06

- 325

- 111r

- a1) confirm that cold tolerant trees grown at the site and allowed to flower did not produce any viable pollen (see also Appendix I).

Gene for altered lignin

This gene has been engineered into other crops that have been previously re leased into the environment under both notifications and permits. The gene engineered into the plants in these field tests has been previously tested in ArborGen field trials for more than
two years. There might be a concern that altered lignin could lea d to an increase in insect or disease susceptibility since lignin is often associated with resistance to insects and disease organisms (Pederson et al. 2005) , but the results of field tests with this particular gene have shown no differences in plant pest susceptibility. Growth measurements have indicated that trees containing this gene had normal to a moderately reduced growth phenotype. The trees were also visually inspected on a monthly bas i s for the presence of any insect and disease damage and t hese observations found that there have been no differences in insect or diseases occurrence in the transgenic lines compared to t he
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 mort ality or morbidity) to APHIS under the terms of the permit

- s ee 7 CFR 340.4(f)(10)(ii).

Non

- coding sequences

The transgenic Eucalyptus also contains non

- coding regulatory sequences 3 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 b y (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 gal l disease. F ollowing transformation, the bacteria are eliminated

3 A non

- coding sequence is the strand of DNA that does not carry the information necessary to make a protein. In this case the n on

- 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 re sult in the production of a disease

causing entity.Page 22 of 98 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 E ngineered Eucalyptus to
be I nvasive.

The potential of the engineered Eucalyptus to be weedy and become invasive was
covered in a previous EA and response to comments for APHIS permit 06

- 325

- 111r ( http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf ) and is herein incorporated by reference.

T he hybrid Eucalyptus EH1 used to produce the transgenic trees has not been shown t o be weedy or invasive in the U.S (L. Pearson, ArborGen pers. comm

. ) , but n o formal assessment has been conducted on its weediness or invasiveness potential. None of the genes introduced into Eucalyptus code for traits that would be expected to make the G E 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 characteristi cs (Kolar and Lodge, 2001) to the engineered plants. The trees would 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 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 L uis S ilva

, International Paper Co mpany , Brazil , comment to docket APHIS

- 2008

- 0059)

.

Eucalyptus generally has difficulty establishing without human intervention, even in
warmer climates. Eucalyptus is intolerant of shade or weedy competition. In order to

su ccessfully 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 n ot 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 vegetat ive reproduction capabilities. The selectable marker gene, when used previously, did not contribute to weediness or invasive properties of the genetically engineered plant s. The gene for altered fertility should not contribute to

w eediness or invasive pr operties 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 these permits will limit the reproductive capabilities of this GE Eucalyptu s outside the confined field trial locations

.Page 23 of 98 Possibility of Gene Flow W ithin 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 crossing 4 (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. However, no seedlings have been found established beneath the trees or in the surrounding areas (Appe ndix 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 dis persal 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 rese rves 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 ev ent (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
surviv al (Meskimen and Francis 1990) . The refore there is li mited 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 these

two permit s 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 Califo rnia are particularly adapted to a Mediterranean climate subject to summer fog, which is conducive to seed germination in those species ( http://ucce.ucdavis.edu/datastore/detailreport.cfm?usernumber=48&surveynumber=182 )

. 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,

4 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 24 of 98 t he bordering fields within 100 meters from the edge of the trial s 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.

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 2 004)

. Pacheco ( 1 987) 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 tri a ls being conducted by the applicant under approved under current BRS notifi cations 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; Evans County in Georgia; Saint Landry’s Parish, Louisiana; Pe arl River County in Mississippi; Jasper County in Texas; and Bay, Highlands, Taylor and Gadsden (Site 1) Counties in Florida. The applicant has indicated that they are not aware of any commercial plantings of compatible Eucalyptus species within 1000 mete rs 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 a t these locations

. T he
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 the se two field trials to date.

**•** At all test sites in Berkeley, Charleston, and Marlboro Counties in SC; Marshall County in MS; Jefferson and Newton Counties in Texas; Columbia and Gadsden (except Site 1) Coun ties in Florida, there are no known existing transgenic or non

transgenic Eucalyptus field trials. The applicant is not aware of any commercial plantings of compatible Eucalyptus species within 1000 meters of the proposedPage 25 of 98 test plot location at these sites

. Therefore, based upon the limited distance that viable pollen (if it were to be produced) 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.

**•** At the Bamb erg County South Carolina site

, there are t ransgenic trials, covered under permit # 08

- 039

- 102rm, of the same hybrid Eucalyptus variety EH1 planted adjacent to the proposed field test plot location at this site. There are also experimental test plots of non

- transgenic cold

- hardy Eucalyptus species ( E.
macarthurii

, E. benthamii

, E. viminalis

, E. badjensis , and E. dorrigoensis ) planted by the applicant within 1000 meters of the proposed test plot location. These cold hardy Eucalyptus species are highly unl ikely to be reproductively compatible with the hybrid Eucalyptus variety because of distant phylogenetic relationship and asynchronous flowering. E. grandis and E. urophylla , for which hybrids have been generated in directed breeding programs, are in the Salignae and Resiniferae series, respectively, of S ection Transversaria ( http://plantnet.rbg syd.nsw.gov.au/cgi

- bin/eucclass.pl?gn= Eucalyptus ). In

contrast, E. macarthurii

, E. benthamii

, E. viminalis

, E. badjensis , and E. dorrigoensis are far removed genetically from the genotype used in this field trial on the evolutionary scale and reside withi n different Series and in the Section
Maidenaria of genus Eucalyptus ( see http://plantnet.rbgsyd.nsw.gov.au/cgi

bin/eucclass.pl?gn= Eucalyptus for details on sections and seri es in Eucalyptus ). 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)

. In accordance with the supplemental permit conditions, t he applicant will monitor flower development in both the tra nsgenic trial and the non

- transgenic trial to determine if there is any overlap in the occurrence of mature flowers. If overlaps are observed the applicant is required to report their findings to APHIS.
In the unlikely event that viable pollen from the non transgenic Eucalyptus trees
fertilize s the transgenic trees, an y seed dispers al i s expected to be within 100 m of the test pl ot. Should any hybridization and viable seed production occur, in accordance with supplemental permit conditio ns, the monitorin g for and removal of volunteers within 100 m from the edge of transgenic test plot by t he applicant
would effectively eliminate any seedlings that may be produced

.

**•** At the Hardin County Texas site

, there are t ransgenic trials, covered unde r permit #

08

- 039

- 102rm, of the same hybrid Eucalyptus variety EH1 planted adjacent to the proposed field test plot location at this site. E xperimental test plots of non

transgenic E. macarthurii , E. benth a mii and E. viminalis are planted within 100
meters of the proposed test plot location. T h ese cold

- hardy Eucalyptus species

are unlikely to be reproductively compatible with the hybrid Eucalyptus variety EH1 because of distant phylogenetic relationship and asynchronous flowering. E. grandis and E. urophylla , for which hybrids have been generated in directed breeding programs, are in the Salignae and Resiniferae series, respectively, of

S ection Transversaria ( http://plantnet.rbgsyd.nsw.gov.au/cgi

bin/eucclass.pl?gn= Eucalyptus ). In contrast, E. macarthurii , E. benthamii and E.Page 26 of 98 viminalis are far removed genetically from the genotype used in this field trial on the evolutionary scale and reside within a different Series and in the Section
Maidenaria of genus Eucalyptus ( see http://plantnet.rbgsyd.nsw.gov.au/cgi

bin/eucclass.pl?gn= Eucalyptus for details on sections and series in Eucalyptus ). 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 b etween parents (Potts and Dungey 2004)

. In accordance with the supplemental permit conditions, t he applicant will
monitor f lower development in both the transgenic trial and the non

- transgenic trial to determine if there is any overlap in the occurrence of mature flowers. If overlaps are observed the applicant is required to report their findings to APHIS.
In the unlikely ev ent that viable pollen from the non transgenic Eucalyptus trees
fertilize s the transgenic trees, any seed dispersal is expected to be within 100 m of the test plot. Should any hybridization and viable seed production occur, in accordance with supplemental permit conditions, the monitoring for and removal
of volunteers within 100 m from the edge of transgenic test plot by the applicant
would effectively eliminate any seedlings that may be produced.

**•** At the Marion County Florida site

, a transgenic trial, covered under Permit # 08

- 039

- 102rm, of the same hybrid Eucalyptus variety EH1 is planted at the proposed field test plot location. A n experimental test of non

- transgenic E. amplifolia is planted approximately 200 meters from the proposed test plot locati on. This Eucalyptus species is unlikely to be reproductively compatible with the hybrid Eucalyptus variety EH1 because of its distant phylogenetic relationship and asynchronous flowering. Phenology of the Eucalyptus hybrid used in the transgenic trial (m ature flowers in mid to late summer) and E. amplifolia (mature flowers in the spring) indicates that there would not be any overlap in flowering
times. As noted above

, viable pollen dispersal is generally limited to within 100

m of the tree stand with the minimum distance to prevent undesirable pollen contamination of seed producing areas set at approximately 300 m

. All trees to be planted in this proposed field trial have been shown in previous trial s to exhibit
the reduced fertility tra it (see Appendix I) . Results from these studies have shown
that the reduced fertility trait is highly effective in preventing the formation of

mature viable pollen. Based upon this information, the hybrid Eucalyptus variety

EH1 are not expected to successfully fertilize the non transgenic E. amplifolia

trees. Observations will be made by the applicant on developing flowers in the

test site to verify that these t rees do have reduced f ertility

. The applicant will monitor flower development in both the transgenic trial and the non

- transgenic trial to determine if there is any overlap in the occurrence of mature flowers. In the unlikely event that viable pollen from the non transgenic E. amplifolia trees could fertilize the transgenic trees, any seed dispersal is expected to be within 100

m of the test plot. 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 t he applicant
would effectively eliminate any seedlings that may be produced.Page 27 of 98 **•** At the Gadsden County Florida Site 1

, a transgenic trial, covered under Permit #
08

- 039

- 102rm, of the same hybrid Eucalyptus variety EH1 is planted at the proposed field test plo t location. T here are experimental plantings of non

transgenic Eucalyptus species including E. grandis

, E. amplifolia , and E.
camaldulensis which are at least 1000 meters away from the transgenic test
location. Therefore, based upon the limited distance that viable pollen (if it were to be produced) 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. In addition, even though t he hybrid genotype used in the se studies for transformation may be compatible with E. grandis trees grown in Florida, E. grandis produces mature flowers in the fall whereas the hybrid genotype used in these tests produces mature flowers in mid to late summer. Therefore, b ecause of asynchronous flowering, hybridization of transgenic trees with the E. grandis at
this site would be virtually negligible.

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 s tates 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 )

.

T he 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 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 ef forts. (Potts and Dungey 2004) make reference to the high degree of inviability in F 1 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 th e genus Eucalyptu s 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% o f 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 viablePage 28 of 98 hybrid that will grow normally. An example of the procedures required to ma ke 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 show n no mature pollen formation ; 2 ) other species that are or c ould 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 F ield T est

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 greenhou se 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 wil d.

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 t his 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.

Potential of the Eucalyptus i n the F ield T est s to B ecome an I nvasive S pecies that

T hreatens N ative P lant and A nimal C ommunities.

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.

T here is no evidence to date th at 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 makePage 29 of 98 the plants more weedy or i nvasive. 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.

As discussed in previous sections of this EA, t here are multiple mechanisms in place that would prevent these Eucalyptus hybrids from establishing themselves in the wild. Since only one clone is being p lanted, viable seed set is likely to be limited due to self incompatibility

. In accordance with supplemental permit conditions m onitoring of seed set and seedling viability will be required by the applicant during the se confined field
tests so that the ex tent 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 t he 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. 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 as weeds so spread of seedlings and trees from the fiel d test sites is highly unlikely. 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 fr om 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 herbicid es (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 indentified by the applicant,

t he field sites that are being proposed under th ese permit s have been used as forest tree plantations, pastu res, or for forestry and agriculture research and are specifically designed for field testing crop plants or forest trees.

Potential Impacts to WildlifePage 30 of 98 Native floral communities

The field sites in the permit applications are located in Bamberg, Ber keley

, Charleston, and Marlboro counties, South Carolina; Evans county, Georgia; Baldwin and Escambia counties, Alabama; St. Landry's Parish, Louisiana; Marshall and Pearl River counties, Mississippi; Hardin Jasper, Jefferson and Newton cou n ties, Texas; and Bay, Columbia, Gadsden, Highlands, Marion, and Taylor counties, Florida. The se sites are a mixture of pasture, crop lands and forested areas. In the unlikely event that viable seed s 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 compet ing 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 co nditions 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 b y 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.

Terrestrial animals

The most likely animals to encounter the transgenic Eucalyptus trees i n this confi ned
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 sim ilarity 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 inverte brate animal species.

Aquatic o rganisms

Eucalyptus germinates in areas of bare mineral soils and would not be expected to establish in aquatic or riparian environments. T here 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.Page 31 of 98 Potential I mpacts by F ire

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 fi res 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

, the y 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
ahea d of the main front (Ashton 1981) (Skolmen and Ledig 1990 ) (Esser 1 993) . 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 planti ng of Eucalyptus

. F uel buildup occurs very rapidly in unmanaged bluegum Eucalyptus stands in California which has lead 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

f uel reduction programs and the establishment of firebreaks in Eucalyptus plantings can reduce wildfire haz ard

. (Esser 1993)

.

There is a historical risk of forest fire in the southeastern U .S., however , the probability
that these confined field t rials will increase the risk and severity of forest fires in their respective locations is very small. T hese plantings are small (none greater than 20 acres ) and they will be managed by the applicant to prevent litter buildup. The se 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 I mpacts t o H uman H ealth

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 Cryptococcu s 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 w ell as other tree species ( Upton et al. 2007 ) . It causes systemic fungalPage 32 of 98 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 c ryptococc osis in India, Africa, Taiwan, South
America and California (Datta e al, 2009)

. C. neoformans 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 is sometimes referred to as an opportunistic fungus. There

was an out break 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 s ubject 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 li nes 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 affecte d lines would be discarded prior to regeneration of trees for introduction into the environment. Another reason it is unlikely that spores could be 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) . T he
tissue culture used for the Eucalyptus hybrid was not derived from woody tissue nor was woody ti ssue 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 con sidered 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 G enetic I nformation to O rganisms w ith w hich it C annot I nterbreed

- Horizontal Gene Transfer to Other Organisms

Overall impacts on horizontal gene transfer would be similar to the no action alternative.Page 33 of 98 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 o f 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, tr ansgene 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, m any 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 nts/Biotechnology/ucm096135.htm

. Therefore APHIS concludes that horizontal gene transfer poses no environmental risk.



### C. Cumulative Effects

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 50 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 th ese proposed release s 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 production. It is a lso 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 the deregulation 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 fi eld test, transgenic plant material will be removed from the
test site and / or destroyed in accordance with supplemental permit conditions established for these permits

. Therefore the only past, present, and reasonably foreseeable actions
associated with t he location s for the proposed release s under permit are those related to agricultural or forest tree production. APHIS has determined that there are no past, present, or reasonably foreseeable actions that would aggregate with effects of the proposed acti on to create cumulative impacts or reduce the long

- term productivity or sustainability of any of the resources (soil, water, ecosystem quality, biodiversity, etc.)Page 34 of 98 associated with the release site s or the ecosystem in which they are situated. No

resources will be significantly impacted due to cumulative impacts resulting from the proposed action.



### D. The Degree to Which the Possible Effects are Highly Uncertain or Involve Unique or Unknown Risks

Potential Effects of G row ing Eucalyptus on Soil H ydrology

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. 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 Serv ice 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 deploy ing Eucalyptus as a biofuel, bioenergy or fiber crop.

The Forest Service indicates that p lanting large

- scale Eucalyptus plantations may potentially lower the water table, and affect groundwater recharge and stream flow dynamics

. Eucalyptus is very effic ient 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 transpirational costs and therefore greater

water use. The F orest Service ha s 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 pl antations 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 Eucalyptu s is 15 m eters

, which is a
characteristic of a dimorphic rooting pattern . In contrast, mean maximum rooting depths of pine plantation ( P. taeda and P. elliottii ) and grass species are 3 m eters and 2.6 m eters

, respectively (Canadell et al. 1996a) . According to the Forest S ervice, c onversion to Eucalyptus on sites where the water tables are less than 10 m eters 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 evapotr anspiration rates that exceed precipitation input, as have been reported for this species in other locations (Calder et al. 1997a)

.Page 35 of 98 Recent research suggests that Eucalyptus plantations would reduce stream flow more than pine plantations, and could potenti ally eliminate low flows. In a review of more
than 20 catchment 5 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 Eu calyptus , 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 F orest S ervice 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 h ectares, 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 under the two permits are well dispersed and a re limited in size (none are greater than 20 acres) and it is anticipated that they are not likely to have significant impacts on hydrology. At the request of APHIS, ArborGen has supplied data indicating the maximum size of each of the plantings at each s ite, 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 Fede rally 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 occupy more than 0.03% 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
enda ngered 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,
includi ng 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.

Potential A llelopathic E ffects of Eucalyptus

5 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

.Page 36 of 98 Allelopathy refers to “ any process involving secondary metabolites produced by plants, microorganisms, viruses and fungi that influence the growth and development of

a gricultural and b iological s ystems ” ( See: International Allelopathy Foundation

- http://www.allelopathy

- journal.com/allelopathy.aspx ). 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 negati ve and positive impacts on the environment (Eljarrat and Barcel 

### ó 2001, Xuan et al. 2005, Kohli et al. 2006) . There has been increased research ac tivity in this area, one of which is taking advantage of plant s 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 R athinasabapathi 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 pla nt 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 t o 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 all elopathic 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 ge rmination 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 agri

silviculture system. Although the grain yield of whea t varieties was significantly lower under all spacings of poplar compared to controls, yields increased significantly with increased spacing of poplar, possibly due to reduced competition for light and nutrients.Page 37 of 98 However, no correlation was found between t he 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, waterme lon 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 use d 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

germ ination and radicle 6 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 planta tions 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 Eucalyp tus 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. S ince all these field tests are
confined and l imited 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 allelopath ic 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 fertilizati on 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.



### 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 will

6 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.Page 38 of 98 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. Other Considerations

Consideration of Executive Orders, Standards and Treaties R elating 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 participation in or benefiting from such programs. It also enforces existing statutes to prevent minority and low

- income communities from bei ng 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 he alth 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 id entify, 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 al ternatives 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 populati ons, 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, t he 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 ha ve 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 impactPage 39 of 98 outside the United States should APHIS choose any of the two alternatives. These
confined field tests are being conducted in the cont inental 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 sparr ow, 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 fo r 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 samp les and eventual harvest of the trees
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 pr oposal 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).



### G. Conclusion

As outlined under the Purpose and Need section s of this document, this EA was prepared
because it was necessary for APHIS to evaluate the potential environmental impacts resulting from the increased number of locations and size of the 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 applications to determine whether the environmental release, with appropriate conditions imposed, can
be carried out while preventing the dissemination and establishment of plant pests. After preparing this draft EA

, APHIS has concluded that even though there is an increase in the
number of sites where trees will be allowed to reach maturity and flower, o ver those already allowed to flower under permits 06

- 325

- 111r and 08

- 151

- 101r, there is no substantially greater risk of loss of confinement and risk to the environment. APHIS
concludes that the releases will remain a s confined field test s and that the genetically engineered trees will not pose a significant plant pest risk. In addition, APHIS concludes that granting permits will not significantly affect the quality of the human environment. No threatened and endangered species or critical habi t at should be impact ed by letting the trees reach maturity and flower at the increased number of locations.Page 40 of 98 

## 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
Caroline Stahaller

- US Fish and Wildlife Service

- Panama City, Florida Field Office
Brad Rick

- US Fish and Wildlife Service

- Vero Beach, Florida Fie ld Office
Candice Martino

- US Fish and Wildlife Service

- Jacksonville, F lorida Field Office



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incompatibility in Eucalyptus globulus ssp. globulus (Myrtaceae). Australian Journal of Botany 50 :365

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Honk ala , editors. Silvics of North America: Volume 2. Hardwoods. Agriculture Handbook 654. U.S. Department of Agriculture, Forest Service, Washington, DC.

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Melbourne. White, K. J. 1995. Silviculture of Eucalyptus Plantings

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- 30.Page 46 of 98 

## APPENDIX I: Status of E xisting F ield T ests A llowed to F lower

The applicant has been a llowed to let transgenic Eucalyptus trees flower under APHIS permits 06

- 325

- 111r

- a2 and 08

- 151

- 101r. These trees have the same genetic constructs
as those Eucalyptus hybrid trees under APHIS consideration in the two permit applications submitted by ArborG en

. The following is a summary of information that has been collected by the applicant and provided to APHIS as part of their annual reports under the existing permits that allow flowering.

All transgenic and non

- transgenic trees in these field tests

, spanning 6.2 acres in Alabama and 1.4 acres in Florida, produced mature flowers in late August

- early September of 2007
and/or 2008. On the 25

- 30 foot tall trees in these tests there were estimated to be several
thousand flowers on each tree. No differenc es were noted in flower formation in transgenic trees compared to non

- transgenic controls (the same parental genotype).

In January 2008, mature (but not yet opened) capsules were collected from non

transgenic and transgenic trees under permit 06

- 325

- 111r

. Replicate samples were collected from transgenic trees and non

- transgenic controls in two separate blocks. 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 eith er non

- transgenic or transgenic lines. Controlled germination studies of the material extracted from the capsules did not produce any germinating seeds . O bservations from the replicated field study being conducted in Alabama under permit No. 06

- 325

- 111r confirm that cold tolerant lines grown in this field test also did not produce any pollen. The results to date have shown that the barnase gene that has been engineered into these trees is effective at preventing pollen formation.

After the initial draft EA was published, the applicant submitted annual reports for permits (06

- 325

- 111r

- a2 and 08

- 151

- 101r) as required under the supplemental
conditions

. T he transgenic and non

- transgenic trees planted under these permits in Baldwin County, Alabama and Highlan ds County, Florida were previously allowed to
flower. The following is a summary of this report.

In Alabama, the transgenic trees produced several hundred to a few thousand flowers per
tree whereas the non

- transgenic controls had much fewer flowers as a result of significant cold damage. In Florida, there was no difference in the number of flowers produced by both transgenic and non

- transgenic trees

. Mature s eed capsules , prior to opening, were sampled from select trees in three field tests in Baldwin Co unty, Alabama and trees from
the single field test in Highlands County, Florida in early March 2009

. Samples consisted of approximately 70 to 100 capsules collected from different positions in the

crown. T he capsules were returned to ArborGen’s greenhous e facility where they were
dried and contents of the capsules were extracted and stored at 4°C. A controlled germination test was conducted using a pproximately 0.1 g of the extracted contents ofPage 47 of 98 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 seeds showed a l ow level of germina tion while approximately 83% of the samples collected from trees in the field trial in Highlands County, FL, showed germina tion , including samples from both transgenic and non

- transgenic control trees. For both transgenic and non

- transg enic 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 expect ed from limited self pollination that may have occurred in these

trees, the number of seedlings observed in these tests is much lower than would be expected for open pollinated seeds.

The controlled germination tests from samples collected in spring 2009 from field trials

a t two sites indicate that a very low level of seedlings can be produced from these trees.

T he number of seedlings produced is significantly less than what would be expected for

an open

- pollinating mixed stand of Eucalyptus

. So far t he 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 site s

. Volunteer monitorin g and reporting the presence of volunteers is required in the supplemental permit conditions.

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 48 of 98 

## 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 depl ete 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 c rops. 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 bec omes 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 Euca lyptus , 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 thePage 49 of 98 case of field trials of Eucalyptus requeste d in ArborGen’s permit applications, all sites are on land previously managed for forestry, agricultural production or maintained as pasture
land.

The Water Cycle

In considering the potential hydrological impacts of Eucalyptus it is important to put
thes e 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 amou nt of water is maintained in the soil layer against the forces of gravity (called the ‘fieldcapacity’ and dependant on soil texture and organic content) while any excess
drains to the water table, the level at which the soil is permanently saturated. Depen ding
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 so il 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 hydrolo gical 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 underst and, 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 summ arize publications on environmental, social and economicPage 50 of 98 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 negatively impact hydrology, in g eneral, 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 cons ideration 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 ther e 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 grasslan ds (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 hig her
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
afforestatio n 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
eff ects 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 planta tions.

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 bioma ss produced (see also SilvaPage 51 of 98 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 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 t ree 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 gr eater 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 depe nd 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 parti cular 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 s pecies 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 lowe ring 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 wh ere 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 d ry seasons. Similar observations were made in reduced LAI along a gradient of water availability by Ares and Fownes (2000). The root systemsPage 52 of 98 of Eucalypts are dimorphic, with widely spreading lateral root systems below the surface plus a deep tap root syste m. In a plantation of 7 year old Eucalyptus trees in Brazil the 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 th e 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 ad aptations 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 seaso n 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 dominat ed by grasses. Under these conditions afforestation with different species of trees, including Eucalyptus , has lead to changes in the water balance including lowering of water tables and restricting stream flows. Calder and colleagues have published severa l 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) an d 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 Engl ish. 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

veg etation (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.Page 53 of 98 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 evapotranspiration exceeded precipitation in the dry season, water storage s 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 Arac ruz 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 list ed 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 m ean 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 stud ies 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
fo rest 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 precipi tation (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, evapo transpiration 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 yea rs after planting transpiration in the plantation is much less than the native forest.Page 54 of 98 Competition for Resources between Eucalyptus and Adjacent Crops

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 f rom 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 competiti on 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 n egative 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 propose d 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 w here 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 fi nding 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:

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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, WorkingPage 56 of 98 Paper 17 (S). Palmberg, C., Forest Resources Development Service, Forest Resources Division. FAO, Rome (unpublished).

FAO (2002b). Annota ted 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 Geo rge, 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 u se 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. Agricultura l 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
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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. (1 997). The response in water yield to the thinning of
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- A Review. IPEF n.28, p.11

- 32

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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
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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 ” s ubmitted by ArborGen LLC to USDA APHIS BRS in support of consideration for approval of permits for field trials of Eucalyptus at multiple sites

E

xecutive 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 as sessments:

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 tr anspire and intercept a significant amount of water.

2. Annual E t losses by Eucalyptus hybrid plantations planted in the southeast US will greatly exceed E t 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 structu ral 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 afforestat ion 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.Page 60 of 98 High biomass production requires fully stocked stands. Reducing stocking to minimize hydrologic impacts is likely to counter the benefits of planting fast growing Eucalyptus

.


## Possible impacts of Euc alyptus 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 mo les of water lost. WUE is a leaf

- level metric . T he 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 wat er 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 me trics of evaluating the impacts of Eucalyptus hybrid plantations on hydrology exist. In order of scale, these are evapotranspiration (ET, mm H 2 O yr - 1 ), transpiration ( E t , mm H 2 O yr - 1 ), and whole

- tree water use ( Q , kg H 2 O day - 1 ). Evapotranspiration (ET) integrates water loss by E t , interception ( E i ), and soil evaporation ( E
s ), and is often estimated at the landscape scale using precipitation input minus stream flow output on paired

- catchments ( P

- R
o ). 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 E t losses by Eucalyptus hybrid plantations planted in the so utheast US will greatly exceed E t by other southeastern forest ty pes Previous studies have quantified annual E t 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 plant ation (age 5, 1111 trees ha - 1 , LAI of 6 m 2

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. T he H ydrology document prepared by
ArborGen state s 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 E t to exceed that of previous pine plantations by a factor of 1.6, and previous pasture land by a factor of 3.5. The co mparison with agricultural crops is more variable; Eucalyptus E t may be greater or lesser than crop E t , depending on the crop, the growing season, and the management practices.Page 61 of 98 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 G A 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 a l. 1998, Shukla et al. 2007)

Eucalyptus has much high er stomatal conductance ( g s ) 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 g s responses to VPD exist (Figure 1). Both strategies regulate g s (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 ( g s ref). 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 62 of 98 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 g s 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 greate r 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 plantation s T he H ydrolog y document prepared by ArborGen states that afforestation with any t ree species may reduce stream flow ; and that while Eucalyptus plantations might reduce

stream fl ow , 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 VPD (kPa) 0 1 2 3 4 5 6gs(mmol m-2s-1)

0 100 200 300 400 500 600 700 800

Eucalyptus

**δ** = -194

g s ref = 328 Pine

**δ** = -81

g s ref = 144Page 63 of 98 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

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

T he H ydrology document prepared by ArborGen state s 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 lateral ly, 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 t his 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)

. T he H ydrology document prepared by ArborGen state s that the high water usage by Eucalyptus may be valuable in purposefully lowe ring 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 sali ne. Furthermore, receding groundwater levels in the UFA are being replaced in costal 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 signifi cance 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.Page 64 of 98 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

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 impac ts of Eucalyptus plantations Some of the reports cited in the A r borGen 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 m 2 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 tra nspiration 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 pr imary 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 stre am 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 s tream flow or groundwaterPage 65 of 98 Direct Measurements : Groundwater Depth 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 bot h 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

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– 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 defic it. 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 energ y 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. Hydro logical 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.Page 68 of 98 

## APPENDIX IV: Threatened and Endangered Specie s Analysis

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 wi thin 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. humpback whale) all species on the list and all critical habitat in each county are included in the discussion below.

The analysis was originally completed in early 2008, and updated in February 2009 to reflect changes to species’ regulatory status. After receiving comments in response to the draft EA, it was decided to review the entire analysis to ensure that it was up to date and satisfies the requirements
of Section 7 (a) (2) of the Endangered Species Act. This was completed in April 2010. Species lists and critical habitat for each county were verified with USFWS field offices to ensure accuracy. The review resulted in the addition of some new species to the county lists, and the removal of others from consideration. The Glades County, Florida site was not included in the revised analysis because the applicant removed this location from the permit applications. There are no significant changes to the potential for species to be affected by the action, or the likelihood of any species to
be found in the test sites.

The following resources were used in the analysis: US Fish and Wildlife Service Endangered Species Homepage: http://www.fws.gov/endangered/
US Fish a nd Wildlife Service ECOS system: http://ecos.fws.gov/ecos_public/index.do NatureServe Explorer: http://www.natureserve.org/explorer/index.h tm Google Earth with critical habitat metadata supplied by US Fish and Wildlife Service County s pecies lists from FWS field offices in Alabama, Georgia, Louisiana, Mississippi, South Carolina, Texas and Florida. South Carolina Heritage Trust Database Telephone contacts with USFWS Field Office personnel Discussions with property owners of field test sites

Baldwin County, AL (AL

- BAL

- 01)

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 6.2 acres of field trials of genetically modified Eucalyptus Hy brid (EH1) trees of some of the lines in the permit request are being grown under issued permit # 06

- 325

- 111r under which these trees are allowed to flower. The oldest of these trees at this site are now entering their fifth growing season.
An additional 0.75 acres of tests with some of these lines are being grown under permit 08

- 039

- 102rm. Site preparation involved herbicide application, subsoiling, and planting of trees in flatPage 69 of 98 beds. The surrounding areas of the test site consist of field plantings o f agricultural crops, experimental forest trees and an abandoned pecan orchard.

Fourteen TES animals and one TES plant are listed in Baldwin County. The TES Animals include

l oggerhead sea turtle ( Caretta caretta ), green sea turtle, ( Chelonia mydas ), Alab ama red

- belly turtle ( Pseudemys alabamensis ), Alabama beach deermouse ( Peromyscus polionotus ammobates ), Perdido Key beach deermouse ( Peromyscus polionotus trissyllepsis ), piping plover ( Charadrius
melodus ), least tern ( Sternula antillarum ), West Indian manatee ( Trichechus manatus ), wood stork ( Mycteria americana ), Kemp's ridley sea turtle ( Lepidochelys kempii ), gulf sturgeon ( Acipenser oxyrinchus desotoi ), Alabama sturgeon ( Scaphirhynchus suttkusi ), heavy pigtoe mussel ( Pleurobema taitianum ), and inflate d heelsplitter mussel ( Potamilus inflatus ). 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 ope n, 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 designated critical habitat.

The four turtle species (loggerhead sea turtle, green sea turtle, Alabama redbelly turtle and Kemp’s ridley sea turtle), the gulf and Alabama sturgeons, and the West Indian manatee occur in aquatic habitats, and their habitat systems (bays, lagoons, salt m arshes, creeks, ship channels, and other saltwater and freshwater environments) do 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 two mouse species listed above ( Alabama b each mouse and Perdido Key B each deer mouse ) 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 over 60+ mile s from the proposed field trial. The p iping plover uses sparsely vegetated dunes and coastal beaches in southern Baldwin County, also far away from the field site (about 60 miles ). The Least Tern breeds on seacoasts, beaches, bays, estuaries, lagoons, la kes, and rivers and rests on sandy beaches, mudflats, and salt

- pond dikes which are far away from the field test site.

Three species have been removed from the county list since the original TES effects analysis was completed in 2008. The FWS has deter mined that the species are so unlikely to be found in the county that it would be unreasonable for federal agencies to include them in TES analysis for projects within the county. These are the red

- cockaded woodpecker ( Picoides borealis ), the
reticulated f latwoods salamander ( Ambystoma bishopi ), and the Eastern indigo snake ( Drymarchon corais couperi ). However, we are retaining the information and findings from the earlier analysis
as follows: The red

- cockaded woodpecker could potentially visit the field tests but this species prefers mature pine stands as habitat for nesting. The reticulated f latwoods salamander uses the wet pine flat

- woods associated with ephemeral wetlands

. They typically breed in the low wetlands where they lay their eggs. After hat ching, they spend 11 to 18 weeks as larvae before metamorphosing into adults and leaving the wetland for higher ground to burrow into the soil. It is important that the area through which the species moves is vegetated with grasses. Reticulated
flatwood salamanders may move as far as 450 meters from their breeding sites. The areaPage 70 of 98 surrounding the release site out to beyond this distance has been intensively managed for over 20 years as an agricultural research station, making it unlikely that the species would find this habitat
useable. The Eastern indigo snake is known to inhabit a wide range of habitats (agriculture fields, pine flat

- woods, wet depressions, stream bottom thickets and margins of swamps). It appears to be very rare in Baldwin County wher e 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 u nsubstantiated reports. They do not feel this field trial poses concern for this species. 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 H abitat : M ost 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 b each d eermouse and the

p iping plover

. Notice of designation of critical habitat for the reticulated flatwood salamander (final rule) was published in the Federal Register on February 10, 2009 ( http://edocket.access.gpo. gov/2009/pdf/E9

- 2403.pdf ). There is no designated critical habitat for this species in Alabama . The closest critical habitat (for the Perdido Key Beach d eer mouse and the

p iping p lover ) is about 65 miles away. The gulf sturgeon has critical habitat in Al abama but this does not occur in Baldwin County.

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
fleetin g 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.

Escambia County, AL (AL

- ESC

- 01)

This location had previously been used as an intensely managed pasture for more than 5 years and was planted with grasses suitable for cattle grazing. Site preparation involved herbicide application to remove existing gras ses, subsoiling, preparation for possible irrigation, and planting of the test trees in flat beds. Field tests with some of the lines in these permit requests were established in 2007 (0.3 acres) and 2008 (0.2 acres) and are currently covered under permit 08

- 039

- 102rm. The surrounding areas of the test site consist of experimental forest trees, ~ 30 year

- old slash pine and a

re

- forested area with less than 7 year

- old mixed stands of pine and hardwood species.

Three TES animals are listed for Escambia Cou nty. The animals are gulf s turgeon, ( Acipenser Oxyrinchus desotoi ), r ed

- cockaded w oodpecker ( Picoides borealis ) and wood stork ( Mycteria

a mericana ) . The g ulf s turgeon 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. T he red cockaded woodpecker inhabits old growth forests, primarily longleaf pine. It could visit the fieldPage 71 of 98 test site but would not nest there. The wood stork primarily inhabits wetland sy stems notably cypress or mangrove swamps and would not find the field test site hospitable.

Critical Habitat: The designated critical habitat for the gulf sturgeon includes the Escambia River System in Santa Rosa and Escambia counties, Florida and Escamb ia, 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 R iver.

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 wil l 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.

Evans County, GA (GA

- EVA

- 01)

This location has been a commercial nursery for forest seedling production for over 30 years. Site

prepara tion involved herbicide application, subsoiling and planting of trees in flat beds. A field test of 0.2 acres of some of the lines included in these permit requests were established under permit

08

- 039

- 102rm in 2008. The areas surrounding the test site c onsist of nursery beds of forest tree seedlings, experimental forest trees, agricultural crops and mixed stands of hardwood and pine.

There are four TES animals listed for Evans County. There are no listed plants. The animals are the Eastern indigo snak e ( Drymarchon corais couperi ), wood stork ( Mycteria americana ), red

cockaded woodpecker ( Picoides borealis ), and frosted flatwoods salamander ( Ambystoma cingulatum ).

The listed birds (red

- cockaded woodpecker and wood stork) could potentially visit the f ield test site
but would not nest there. The red

- cockaded woodpecker nests in old growth longleaf pine. The wood stork nests in marshes, floodplain lakes, and swamps. The flatwoods salamander ( Ambystoma cingulatum ) was a species that required further ana lysis because it typically inhabits longleaf or slash pine forests lying between drier land upslope and wetlands and seasonally inhabits wet pine
flat

- woods with vernal pools. Originally it was associated with a unique community of longleaf pine/wire gras s, but much of this habitat is now replaced by slash pine plantations. According to Kathy Chapman (contacted February 22 and 26, 2008) of the Coastal Georgia Field Office, there should not be an effect on the species because there would be no change to th e habitat. However, she suggested verifying that there is no suitable breeding habitat near the release site. She provided information on life history and suitable habitat for the species. Viewing the site using Google Earth does not readily identify an y suitable breeding habitat. To ensure that there is no suitable habitat in the area, BRS provided the species information to the applicant and in early March 2008, the applicant conducted a breeding habitat survey of the area within 450 meters (1476 feet ) of the release site. No suitable habitat was found. For the eastern indigo snake ( Drymarchon couperi ) according to Kathy Chapman (contacted February 22 and 26, 2008) of the Coastal Georgia Field Office, the species is associated with the gopher tortois e in sandhill areas with wetlands. However,Page 72 of 98 land use is a determining factor on the likelihood of either species being present. The site has been used as a commercial nursery for forest seedling production for over 30 years. She indicated that
she was n ot concerned with the site because there would be no change to the habitat. 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 o n 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: There is no designated critical habit at or habitat proposed for designation in the
county.

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

fl eeting 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.

St. L andry’s Parish, LA (LA

- SLP

- 01)

This location has been an experimental agricultural farm for more than 25 years, used for conducting research experiments with soybean, cotton and wheat. Site preparation will involved herbicide application, subsoiling and planting of trees in flat beds. Field tests of some of the lines included in these permit requests were established during 2006, 2007 and 2008 on 1.9 acres total, now covered under permit 08

- 039

- 102rm. Approximately 1.7 acres were subsequently terminated

. The areas surrounding the test site consist of agricultural fields of rice, sugarcane and millet.

There are two TES animals listed for St. Landry’s Parish; the pallid sturgeon ( Scaphirhynchus
albus ) and the Louisiana black bear ( Urus americanus luteolu s ). There are no listed plants.

The pallid sturgeon occurs in larger channels of the Mississippi

- Missouri river system. The field
test site is approximately 24 miles away from the Mississippi river, so would not be impacted by
the field test. The Louis iana black bear ( Ursus a. luteolus ) prefers bottomland forests with d iverse 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.

Critical Habitat: There is no designated critical habitat or habitat proposed for designation in the parish

. The Louisiana black bear does have critical habitat in adjoining Pointe Coupee parish, but
this is over 10 miles from the release site.

Conc lusion: 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 co ntain 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 forPage 73 of 98 d esignation. 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.

Marshall County, MS (MS

- MAR

- 01)

This location has been an agricultural research station for more than 50 years, used for conducting research experiments with agricultural crops and grasses. The test site was previously used for experimental planting of grasses. Site preparation involve d herbicide application, subsoiling and planting of trees in flat beds. A field test of 0.5 acres of some of the lines included in these permit requests were established in 2007, now under permit 08

- 039

- 102rm. This test was subsequently
terminated. The areas surrounding the test site consist of experimental forest trees, agricultural fields, and less than 5 year

- old pine plantations.

There are no listings for TES animals or plants in the county.

Critical Habitat: There is no designated critical habitat or habitat proposed for designation in the
county

.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the release site. Field activities will result in no changes to the hab itat 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 a nd would not affect designated critical habitat or habitat proposed for designation.

Pearl River County, MS (MS

- PRC

- 01)

This location has been used as an agricultural research station for more than 5 years for conducting research experiments with agricul tural crops and grasses. The test site was used for experimental planting of grasses. Site preparation involved herbicide application to remove existing grasses, subsoiling, preparation for irrigation installation, and planting of trees in flat beds. Fie ld tests of some of the lines included in these permit requests were established in 2007 (2.0 acres) and 2008 (0.98 acres), now covered under permit 08

- 039

- 102rm. The areas surrounding the test site consist of a grape research farm, mixed stands of hardwo ods and pine, and a residential area.

There are five TES animals listed in this county and one TES plant . The animals are the r inged map turtle ( Graptemys oculifera ), g opher t ortoise ( Gopherus polyphemus ), Louisiana black bear ( Ursus a. luteolus ), g ulf s turgeon ( Acipenser oxyrhynchus desotoi ) and i nflated heelsplitter ( Potamilus inflatus ). The listed plant is Louisiana quillwort ( Isoetes louisianensis ).

The r inged map turtle ( Graptemys oculifera ) inhabits wide rivers with strong currents, adjacent
whi te sand beaches, and an abundance of basking sites in the form of brush, logs, and debris. The
field test will n o t impact this aquatic species which occurs in the Pearl River system

– approximately 11 miles from the field test site . The g opher t ortoise ( 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 fores ts withPage 74 of 98 loblolly pine forests. This field test i s locate d 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
fou nd 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 agricultur al 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 obse rve 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 employ ees, 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 forest s 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 gulf sturgeon, the inflated hellsplitt er 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.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at th e 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 aff ect designated critical habitat or habitat proposed for designation.

Bamberg County, SC (SC

- BAM

- 01)

This location has been a managed forest plantation for more than 14 years. The location has been specifically used for short

- rotation planting of hardwoo ds and softwood trees for forestry research. Standard silvicultural practices for site preparation, irrigation, fertilization, planting and harvesting have been used at this location. Similar practices will be used for the additional field tests to be

es tablished at this site. Several field tests of some of the lines included in these permit requests (3.5 acres total) were established from 2006 to 2008. These tests were covered under permit 08

- 039

- 102rm, and all but 0.2 acres were subsequently terminate d. The areas surrounding the test site
consist of experimental forest trees, young pine plantations, mixed stands of hardwoods and pine, and agricultural fields.

Three TES are listed in Bamberg County. The TES species listed are a plant, Canby’s dropwortPage 75 of 98 ( Oxypolis canbyi ), and two animals, the wood stork ( Mycteria americana) and the red cockaded woodpecker ( Picoides borealis )

.

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,

gr ass

- sedge dominated Carolina bays, wet pine savannahs, shallow pineland ponds and cypress

pine 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 Lisa Matthews Memorial Bay is a 52 acre site in Bamberg County which was given to the South Carolina Native Plants Society by the Nature Conservancy for the purpose of preserving Canby's d ropwort ( Oxypolis canbyi ). This depression wetland is apparently a remnant Carolina Bay which is being restored and expanded to protect this endangered species. The location of the field test under this permit is a managed forest research area and does not provide the proper habitat for the species.

The wood stork inhabits riparian areas with lagoons and shallow water. It lives in freshwater situations: marshes, swamps, lagoons, ponds, flooded fields and brackish wetlands. It nests mostly in the upper parts of cypress trees, mangrov es, or dead hardwoods over water or on islands along streams or adjacent to shallow lakes and feeds in freshwater marshes, swamps, lagoons, ponds, flooded pastures and flooded ditches, and in depressions in marshes. The field trial location is
unsuitable habitat for the wood stork so it will be very unlikely to occur in this location. The red cockaded woodpecker inhabits old growth forests, primarily longleaf pine and might visit the field
test site but would not nest there.

Critical Habitat: There is no designated critical habitat or habitat proposed for designation in the
county.

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 sit e, 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 listin g. 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 pro posed for designation.

Berkeley County, SC (SC

- BER

- 01 and SC

- BER

- 02)

There are two release site locations in Berkeley County. One site is an extension of a greenhouse facility that has been used for acclimatization of transgenic and non

- transgenic plant s for more than 7 years. The release site is located adjacent to greenhouse facilities and is surrounded by hardwoods and pine plantations. The trees are held here on a temporary basis until field planting. The other location has been a managed forest p lantation for more than 7 years. The location has been specifically used for short rotation planting of cottonwood and Eucalyptus for forestry research. Site preparation involved herbicide application, sub soiling, drip irrigation installation,Page 76 of 98 and planti ng of trees in flat beds. The test site is located adjacent to greenhouse facilities and is surrounded by pine plantations. Field tests of some of the lines included in these permit requests were established in 2006 (0.3 acres) and 2007 (0.2 acres) that were covered under permit 08

- 039

- 102rm but subsequently terminated.

Five threatened or endangered species (TES) animals and three plants are listed in Berkeley County. The TES animal species are s hortnose s turgeon ( Acipenser brevirostrum ), frosted
flatwo ods s alamander ( Ambystoma cingulatum ), red

- cockaded w oodpecker ( Picoides borealis ), West Indian manatee ( Trichechus manatus ), and w ood s tork ( Mycteria americana ). The TES

p lants are p ondberry ( Lindera melissifolia ), Canby's d ropwort (Oxypolis canbyi ) and American

c haffseed ( Schwalbea a mericana ).

The shortnose sturgeon ( Acipenser brevirostrum ) occurs in rivers and estuaries. The West Indian

manatee (Trichechus manatus) occur s in shallow coastal waters, rivers, bays and lakes; none of
which are close to an y of th ese release location s . The red cockaded woodpecker ( Picoides
borealis ) inhabits old growth forests, primarily longleaf pine and might visit the field test site but
would not nest there. The wood stork ( Mycteria americana ) primarily inhabits wetlan d systems notably cypress or mangrove swamps and would not find the field test site hospitable. The frosted
flatwoods salamander ( Ambystoma cingulatum ) inhabits longleaf or slash pine forests lying between drier land upslope and wetlands and seasonally in habits wet pine flat

- woods with vernal pools. Discussions with Laura Zimmerman (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 in the Francis Marion National Forest

, far from the release site. A check of the SC Heritage Trust Database did not identify any occurrences in the area of the
release. One s ite in Berkeley County is a fenced research plot/holding area so it is highly unlikely that these animal species would occur at this location. The other site is a managed forest plantation for short rotation planting of cottonwood for forestry research. The bird species might visit the
location but would not nest ther e.

Canby’s dropwort (Oxypolis canbyi ) 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 fo und in a variety of habitats, including cypress ponds, grass

- sedge dominated Carolina bays, wet pine savannahs, shallow pineland ponds and cypress

- pine 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 ( Lindera melissifolia ) 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 chaff seed ( Schwalbea a mericana ) 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. The site in Berkeley County is a fenced research plot/hol ding area which would be very inhospitable to these species. Laura Zimmerman (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 toPage 77 of 98 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

.

Critical Habitat: Notice of designation of critical habitat for the frosted flatwood salamander (final rule) was published in the Federal Register on February 10, 2009 ( http://edocket.a ccess.gpo.gov/2009/pdf/E9

- 2403.pdf ). Unit FFS

- 6 is within Berkeley County.
This is located in the Francis Marion National Forest which is about 19

- 20 miles away from the
release location.

Conclusion: No federally listed threatened or endangered spec ies or species proposed for listing are likely to be found at these release sites. If they were to enter the sites, their presence would be fleeting as the habitat is either not suitable or does not contain constituent elements required by the species. F ield activities will result in no changes to the habitat used by any listed species or species proposed for listing. The sites are not within or near designated critical habitat or habitat proposed for designation. Therefore, the action will have no effe ct on listed species or species proposed for listing and would not affect designated critical habitat or habitat proposed for designation.

Charleston County, SC (SC

- CHA

- 01)

This location has been a managed forest plantation for more than 10 years. The location has been specifically used for short

- term planting of hardwoods and softwood trees for forestry research. Standard silvicultural practices for site preparation, irrigation, fertilization, planting and harvesting
were used at this location. Simila r practices were used for the additional field tests established at this site. Field tests of 3.1 acres of some of the lines included in the permit request were established during 2006 and 2007. These tests were covered under permit 08

- 039

- 102rm, and wer e all subsequently terminated. The test plots adjacent to the field test site include young mixed stands of hardwoods and pines. No additional trials are currently planned for this site.

Thirteen TES animals and 4 TES plants are listed for Charleston Co unty. The animal species are:

s hortnose s turgeon ( Acipenser brevirostrum ), West Indian manatee ( Trichechus manatus ), Kemp's ridley sea turtle ( Lepidochelys kempii ), l eatherback sea turtle ( Dermochelys coriacea ) and g reen sea turtle, ( Chelonia mydas ) , l oggerhead s ea turtle ( Caretta caretta ) , w ood s tork ( Mycteria a mericana ),

r ed

- cockaded w oodpecker ( Picoides borealis ), p iping p lover ( Charadrius melodus ), frosted

f latwoods s alamander ( Ambystoma cingulatum ) , finback whale ( Balaenoptera physalis ), humpback

w hale ( Megaptera novaengliae ), and right whale ( Balaena glacialis )

. The plant species are:

s eabeach a maranth ( Amaranthus pumilus ), Canby's d ropwort ( Oxypolis canbyi ), p ondberry ( Lindera melissifolia ) and American chaffseed ( Schwalbea americana ).

The s hort nose Sturgeon, West Indian manatee, Kemp's ridley sea turtle, l eatherback sea turtle

, g reen sea turtle, l oggerhead s ea turtle

, West Indian m anatee , finback whale, humpback whale, and right whale are all aquatic animals and would not be affected by the fiel d test. The birds (piping

p love r , r ed

- cockaded w oodpecker and w ood stork ) could all potentially visit the field test site but would not nest there. The piping plover nest along the coast. The red

- cockaded woodpecker nests in old growth longleaf pine. The wood stork nests in marshes

, floodplain lakes, and swamps

. The
frosted flatwoods salamander inhabits longleaf or slash pine forests lying between drier landPage 78 of 98 upslope and wetlands and seasonally inhabits wet pine flat

- woods with vernal pools. Discussions with Laura Zimmerman (contacted February 14 and 20, 2008) of the Charles ton 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 Santee Coastal Reserve. A check of the SC Heritage Trust Database did not identify any occurrences in the area of the release.

For the plants

, the s eabeach a maranth occurs in areas just above the high tide line on accreting
shorelines ; those where the beach is building up or expanding. Canby's d ropwort occurs in a variety of coastal plain communities, including pond cypress savannahs, the shallows and edges of

cypress and pond pine ponds, sloughs, and wet pine savannas. Pondberry is found in swamp and pond margins, sandy sinks, swampy depressions or wet flats that are subject to dry ing but the roots are submerged at times. American chaffseed is found in various sandy soil areas on the coastal plain; plants are usually found on margins of savannas and cypress ponds that are seasonally wet; best managed by prescribed fire. The locati on of the field test, which has been a managed forest plantation for more than 10 years, would not be hospitable habitat for any of these species.

Critical Habitat: There is designated critical habitat for the piping plover in Charleston County but
it is along the coast and over 15 miles from the field test. Notice of designation of critical habitat for the frosted flatwood salamander (final rule) was published in the Federal Register on February 10, 2009 ( http://edocket.access.gpo.gov/2009/pdf/E9

- 2403.pdf ). Unit FFS

- 7 is within Charleston County. This is located in the Santee Coastal Reserve which is over 50 miles away from the Charleston County site.

Conclusion: No federally listed t hreatened 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 req uired 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 ac tion will have no effect on listed species or species proposed for listing and would not adversely modify designated critical habitat or habitat proposed for designation.

Marlboro County, SC (SC

- MAR

- 01)

This location has been a commercial nursery for forest seedling production for over 30 years. Site preparation involved herbicide application, subsoiling and planting of trees in flat beds. A 0.3 acre field test of some of the lines included in these permit requests were established in 2007, now

cover ed under permit 08

- 039

- 102rm but subsequently terminated. The surrounding areas consist of field plantings of agricultural crops, nursery beds of forest tree seedlings and less than 30 year

- old mixed hardwood and pine plantations. No additional trials ar e currently planned for this site.

There are two TES animals listed for Marlboro County; the red

- cockaded w oodpecker ( Picoides
borealis ) and the shortnose sturgeon ( Acipenser brevirostrum ) . The listed plant is Canby's Dropwort ( Oxypolis canbyi ).Page 79 of 98 The r ed

- cockaded woodpecker nests in old growth longleaf pine not found at the location of the
field test

. The shortnose sturgeon occurs in rivers and estuaries . Little is known about the status of any of the populations in South Carolina. The closest body of water where the sturgeon could possibly live is about 5 miles away. Canby's dropwort occurs in a variety of coastal plain communities, including pond cypress savannahs, the shallows and edges of cypress and pond pine ponds, sloughs, and wet pine savann as. The location of the field test, which has been a managed

nursery for more than 3 0 years, would not be hospitable habitat for any of these species.

Critical Habitat: There is no designated critical habitat or habitat proposed for designation in the

c ounty.

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 pr oposed 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.

Hardin County, TX (TX

- HAR

- 01)

This location has been a managed forest plantation for more than 30 years. The location consists of mixed hardwood tree plantations planted using standard silvicultural practices and was harvested by the owner in 2004. The test site is within the larger harvested area and was re

- bedded by the owner for planting. Site preparation included herbicide application and sub

- soiling. A field test of 19.7 acres of some of the lines included in these permit requests were established under permit 08

- 039

- 102rm in 2009. The areas su rrounding the test site consist of mixed hardwood stands and managed loblolly pine plantations.

One TES animal and one TES plant are listed for Hardin County. The animal is the red

- cockaded woodpecker ( Picoides borealis ), and the plant is the Texas trailing phlox ( Phlox nivalis ssp. texensis).

The red

- cockaded woodpecker might visit the field test site but would not nest there since it prefers old growth pine forests, particularly longleaf pine. The Texas trailing phlox is endemic to the Pineywoo ds of the west gulf coastal plain of east Texas. It occurs in deep sandy soils in fire

maintained openings in upland longleaf pine savannas or post oak

- bluejack oak woodlands. Since this site has been under managed forest plantations for more than 30 yea rs this species would not
find the field test site a suitable habitat.

Critical Habitat: There is no designated critical habitat or proposed critical habitat listed for this
county.

Conclusion: No federally listed threatened or endangered species or s pecies 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 thePage 80 of 98 species. Field activit ies 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 s pecies or species proposed for listing and would not affect designated critical habitat or habitat proposed for designation.

Jasper County, TX (TX

- JAS

- 01 and TX

- JAS

- 02)

There are two locations in Jasper County Texas. The first site, TX

- JAS

- 01, has been a managed pine plantation for more than 25 years. Previous plantings were cultivated in beds using standard silvicultural practices. The existing pine plantation at this site was harvested by the owner in 2007
and re

- bedded for planting. The test site is within the larger harvested and bedded site prepared by the site owner. Further site preparation involved herbicide application, subsoiling and planting of trees in flat beds. A field test of 0.7 acre of some of the lines included in these permit reques ts were established under permit 08

- 039

- 102rm in 2008. The areas surrounding the test site consist of harvest age pine plantations.

The second site, TX

- JAS

- 02, has been in agriculture for more than 30 years. The test site was used for pasture for the pa st 10 years. Site preparation involved herbicide application to remove existing grasses, subsoiling, preparation for irrigation installation, and flat plantings of trees. A field test of 1.0 acre of some of the lines included in these permit requests wer e established under permit 08

- 039

- 102rm in 2008. The areas surrounding the test site consist of natural stands of mixed pine and
hardwoods.

Two threatened or endangered species (TES) animals and one plant are listed in Jasper County.
The animals are the red

- cockaded woodpecker ( Picoides borealis ) and the Louisiana black bear ( Ursus americanus luteolus ). The plant is Navasota l adies'

- tresses ( Spiranthes parksii ).

The red

- cockaded woodpecker ( Picoides borealis ) inhabits old growth forests, primarily lon gleaf pine and might visit the field test sites but would not nest there. The Louisiana black bear ( Ursus
americanus luteolus ) d epends on diverse, productive bottomland forest with diverse food resources, including a variety of hard

- mast

- producing species . High quality habitat includes remote areas with little or no human activity so it would not likely occur at either site. Navasota ladies'

- tresses ( Spiranthes parksii ) occurs at the m argins of post oak ( Quercus stellata ) woodlands in sandy loams along i ntermittent tributaries of rivers. The field test sites have none of these characteristics.

Critical Habitat: There is no designated critical habitat or habitat proposed for designation in the
county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at these release sites. If they were to enter the sites, 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 sites are not within or near designated critical habitat or habitat proposed for designation. Therefore, the action wi ll have no effect on listed species or species proposed for listing and would not affect designated critical habitat or habitat proposed for designation.Page 81 of 98 Jefferson County, TX (TA

- JEF

- 01)

This location has been used for managed agricultural production of rice for more than 5 years. Site preparation will involve herbicide application, subsoiling and planting of trees in flat beds. The surrounding areas of the test site consist of rice plantations.

There are six TES animals listed for Jefferson County. Th ere are no plants listed. The animals are

g reen t urtle ( Chelonia mydas )

, h awksbill turtle ( Eretmochelys imbricata imbricate ) , Kemp's ridley
sea turtle ( Lepidochelys kempii )

, l eatherback t urtle ( Dermochelys coriacea )

, l oggerhead sea turtle ( Caretta caretta ) and p iping p lover ( Charadrius melodus ).

All of the turtles occur in the open waters of the Gulf of Mexico or in s hallow coastal and estuarine

waters . The field test site is about 30 miles away from the Gulf. The p iping plover uses sparsely vegetated dunes and coastal beaches in southern Jefferson County, which is also at least 30 miles from the field test site. None of these species would be impacted by the field test.

Critical Habitat: There is critical habitat listed for the piping plover but it does not occur in this
county. The closest critical habitat is in neighboring Galveston County, approximately 40 miles
from the field test site.

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 h abitat 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.

Newton County, TX (TX

- NEW

- 01, TX

- NEW

- 02, TX

- NEW

- 03, TX

- NEW

- 04)

There are four locations in Newton County. All locations have been under managed loblolly pine plantati ons for at least 30 years. The previous plantings were cultivated in beds using standard silvicultural practices and the areas have recently been harvested. Site preparation has been or will include herbicide application, plowing, and planting of trees i n raised or flat beds. The surrounding areas of the test site consist of managed loblolly pine plantations and/or mixed hardwood stands.

Two TES animals are listed for Newton County. The animals are the red

- cockaded woodpecker ( Picoides borealis ), and Louisiana black bear ( Ursus americanus luteolus ). There are no plants listed for this county.

The red

- cockaded woodpecker might visit the field test site but would not nest there since it prefers old growth pine forests, particularly longleaf pine. The Louisiana black bear ( Ursus a. luteolus ) prefers bottomland forests with diverse food resources, including a variety of hard

- mast

- producing species. According to the Black Bear Recovery Plan ( http://www.bbcc.org/web/images/stories/information/pdf/FinalRestorationPlanwithFigures.pdf ) inPage 82 of 98 east Texas

, potentially occupied habitat may occur in at least four Counties: Cass, Shelby, Panola and Angelina Countie s. All of these locations are from fifty to over a hundred miles away from
these test sites. Louisiana black bear habitat includes remote areas with little or no human activity
so it is un likely to be found at the site.

Critical Habitat: The proposed c ritical habitat is located in Avoyelles, East Carroll, Catahoula, Concordia, Franklin, Iberia, Iberville, Madison, Pointe Coupee, Richland, St. Martin, St. Mary, Tensas, West Carroll, and West Feliciana Parishes, Louisiana. All of these locations are over a hundred miles away from these test sites.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the se release site s . If they were to enter the site s , their presence would be
fleeti ng 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 s are not within or n ear 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.

Bay Cou nty, FL (FL

- BAY

- 01)

This location was used as an intensely managed pasture for more than 15 years, and was planted with grasses suitable for cattle grazing. Site preparation involved herbicide application to remove existing grasses, subsoiling, preparati on for possible irrigation installation, and planting of trees in flat beds. Field tests of some of the lines included in these permit requests were established in 2007 (1.0 acre) and 2008 (0.35 acres), now covered under permit 08

- 039

- 102rm. All tests exc ept 0.18 acre were subsequently terminated. The areas surrounding the test site consist of experimental forest trees, agricultural crops and less than 25 year

- old hardwoods and pine.

Sixteen TES animals and 6 TES plants are listed for Bay County. The a nimals are gulf moccasinshell ( Medionidus penicillatus ), oval pigtoe ( Pleurobema pyriforme ), gulf sturgeo n ( Acipenser oxyrinchus ) l oggerhead t urtle ( Caretta caretta ), e astern i ndigo s nake ( Drymarchon

corais couperi ), piping p lover ( Charadrius melodus ), r ed

- cockaded w oodpecker ( Picoides
borealis ), West Indian m anatee ( Trichechus manatus ), Choctawhatchee b each mouse ( Peromyscus polionotus allophrys ), St. Andrews b each m ouse ( Peromyscus polionotus peninsularis ), reticulated

f latwoods salamander ( Ambystoma b ishopi ), g reen t urtle ( Chelonia mydas ), l eatherback t urtle ( Dermochelys coriacea ), h awksbill turtle ( Eretmochelys imbricata imbricate ), Kemp's r idley turtle ( Lepidochelys kempii ), and w ood stork, ( Mycteria americana ) . The plants are t elephus s purge ( Eupho rbia telephioides ), w hite b irds

- in

- a

- nest ( Macbridea alba ), Godfrey’s (v iolet ) b utterwort ( Pinguicula ionantha ), Florida skullcap ( Scutellaria floridana ), Harper's beauty, ( Harperocallis flava ) and Crystal Lake nailwort, ( Paronychia chartacea minima )

.

The gulf moccasinshell, oval pigtoe, gulf sturgeon, West Indian manatee, and the turtles (loggerhead, green, leatherback, hawksbill and Kemp's ridley) are all aquatic animals and would not be affected by the field test. The birds (piping plover, red

- cocka ded woodpecker and wood stork) could all potentially visit the field test site but would not nest there. The piping plover nest s along the coast which is about 20 miles away from the release site . The red

- cockaded woodpeckerPage 83 of 98 nests in old growth longleaf pine. The wood stork nests in marshes, floodplain lakes, and swamps. The two mouse species live along the coast in beach areas so would not be impacted by the field
release . For the eastern indigo snake, according to Caroline Stahaller (contacted Februa ry 20, 2008) of the Panama City Field Office USFWS, the species has not been positively identified in the county or anywhere else on the Florida panhandle in 15

- 20 years. Considering the area is currently managed as a pasture, a habitat where the species is unlikely to occur, there is no concern for the species at this site. 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
wh at 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 reticulated flatwoods salamander inhabits longleaf or sl ash pine forests lying between drier land upslope and wetlands and seasonally inhabits wet pine flat

- woods with ephemeral wetlands. They typically breed in the low wetlands where they lay their eggs. After hatching, they spend 11 to 18 weeks as larvae be fore metamorphosing into adults and leaving the wetland for higher ground to burrow into the soil. It is important that the area through which the species moves is vegetated with grasses. Management of the area with controlled burning is generally needed to provide quality flatwoods salamander habitat. Reticulated f latwoods salamanders may move as far as 450 meters from their breeding sites. Although the release site is not an area with known populations, and is at least 10 miles from the critical habi tat, Ms. Stahaller of USFWS suggested a closer look at the site to determine if the habitat would be suitable for the species. The property owner was forwarded survey protocol information provided by USFWS. In late March 2008 , the owner, (who is 64 and h as lived at the site his entire life) walked the site within 450 meters of the release. The owner was contacted on April 7, 2008 to discuss his observations. The owner looked for adult salamanders but did not find any, and does not recall ever seeing the m at the site. There were four naturally wet areas that were found. One was in the pasture itself. It was a small site of about 8 ft. by 10 ft. and is surrounded by an area that has been in continuous agricultural use for at least 60 years. The other t wo sites were heavily wooded areas surrounded by pasture and remain constantly wet in most years. They are approximately 250 yards from the release site. The fourth site is approximately 400 to 500 yards from the release site and is a 5 acre wetland that shares the same elevation with an adjacent creek and remains constantly wet, but does dry around the perimeter. Vegetation in the site is longleaf pine with a wiregrass groundcover

– habitat the species finds most suitable. Vegetation between this poten tial breeding site and the release site is pasture. It is unlikely that the flatwoods salamander would be using the release site for a number of reasons. First, the only possible breeding sites within 450 meters of the release remain constantly wet durin g most years, a condition that would not serve as breeding habitat. The five acre site where the perimeter dries could perhaps allow for breeding in some years but it is about at the farthest possible distance from the release site that the adults are kno wn to travel from their breeding area. Second, the sites are either surrounded by land that has been used for agricultural purposes for at least 60 years or is separated from the release site by such land. Activities associated with agricultural producti on, disking, plowing, application of pesticides etc. would make the habitat unsuitable for the species. In addition, none of this area is managed by controlled burning, a management practice that is generally used in areas where the species has viable pop ulations. The lack of suitable breeding habitat, combined with the ongoing and historic agricultural activities at the site, point to a conclusion that the reticulated flatwood salamander will not be present in the area of the release, nor will they be li kely to enter the site during the field trial. None of the six listed plant species would be expected to grow in the existing pasture. They grow in wiregrassPage 84 of 98 dominated longleaf pine savannahs, in the mesic flatwoods, on low sand ridges of pine

- scrub oak near the Gulf of Mexico or in wet prairies and seepage bogs.

This location is being converted from a pasture to a forest tree research plot. Based on the above evaluation, APHIS has determined that disturbance of the field site for the release (e.g. plow ing, removing vegetation, burning etc.) will not directly or indirectly affect a federally listed threatened or endangered species or species proposed for listing , or affect the habitat for these species

.

Critical Habitat: There is designated critical ha bitat for the Gulf sturgeon along the entire coast of Bay County. Critical habitat for the Choctawhatchee b each mouse , St. Andrew’s beach mouse, and the piping plover is along the coast in the Panama City area. The field test site is approximately 18

mil es away from the coast so would not impact these critical habitats

. Notice of designation of critical habitat for the reticulated flatwood salamander (final rule) was published in the Federal Register on February 10, 2009 ( http://edocket.access.gpo.gov/2009/pdf/E9

- 2403.pdf ). Unit RFS

- 6, subunit B is just beyond the northern border of Bay County in neighboring Washington County. The field test site is approximately 28 miles away from this location. Unit RFS

- 9 subunit A is located in neighboring Calhoun County approximately 10 miles from the site. There is propos ed
critical habitat listed for the West Indian m anatee but it does not occur in this county.

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 ent er 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 h abitat proposed for designation.

Columbia County, FL (FL

- COL

- 01)

This location has been under managed pine plantations for more than 20 years. Previous plantings were cultivated in beds using standard silvicultural practices. This area of the tract was burned in a fire in May 2007. After the fire, the area was raked and bedded by the site owner in preparation for

re

- planting. The test site is within the larger harvested and bedded area, which is surrounded by existing pine plantations and additional h arvested tracts.

Five TES animals are listed for Columbia County. The animals are oval pigtoe ( Pleurobema pyriforme ), gulf sturgeon ( Acipenser oxyrinchus ), eastern indigo snake ( Drymarchon corais couperi ), red

- cockaded woodpecker ( Picoides borealis ), an d wood stork ( Mycteria a mericana ). There are no plants listed for this county.

The oval pigtoe and gulf sturgeon are aquatic animals and would not be affected by the field test. The birds (red

- cockaded woodpecker and wood stork) could all potentially vi sit the field test site
but would not nest there. The red

- cockaded woodpecker nests in old growth longleaf pine. The wood stork nests in marshes, floodplain lakes, and swamps. For the eastern indigo snake, according to Caroline Stahaller (contacted Febru ary 20, 2008) of the Panama City Field Office USFWS, the species has not been positively identified in the Florida panhandle in 15

- 20 years.Page 85 of 98 Considering the area has been managed as a pine plantation for many years

, it is unlikely to be
found at this site . 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 variatio n of standard protective measures the USFWS uses when they have reached a “may affect” determination for construction
sites.

Critical Habitat: There is designated critical habitat for the Gulf sturgeon but it is not listed in this
county. There is no pr oposed critical habitat listed for this county.

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
fleetin g 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.

Gadsden Co unty, FL (FL

- GAD

- 01, FL

- GAD

- 02)

There are two planting sites at this location. One location, FL

- GAD

- 01, has been an agricultural research station for more than 10 years and has been used for conducting research experiments on agricultural crops. Standar d silvicultural practices were used for site preparation, including herbicide application, plowing and planting of trees in raised or flat beds. A field test of 0.2 acre of some of the lines included in these permit requests were established under permit 08

- 039

- 102rm in 2008. The areas surrounding the test site consist of mixed pine

- hardwood forests and pine plantations as well as research plantings of agricultural and horticultural crops.

The second location, FL

- GAD

- 02, has been an agricultural research station for more than 10 years. The field has been fallow for approximately seven years. Standard silvicultural practices will be used for site preparation, including herbicide application, plowing and planting of trees in raised or
flat beds. The area s surrounding the test site consist of mixed pine

- hardwood forests and pine plantations, as well as research plantings of agricultural and horticultural crops.

Ten TES animals and six TES plants are listed for Gadsden County. The animals are:

f

at t hreeridge mussel ( Amblema neislerii ), p urple b ankclimber mussel ( Elliptoideus sloatianus ),

s hinyrayed p ocketbook ( Hamiota subangulata ), o chlockonee m occasinshell ( Medionidus simpsonianus ), o val p igtoe ( Pleurobema pyriform e ), c hipola slabshell ( Elliptio chipolaensis)

, gulf

s turgeon ( Acipenser oxyrinchus desotoi ), w ood stork ( Mycteria americana ), r ed

- cockaded

w oodpecker, ( Picoides borealis ), Eastern i ndigo s nake ( Drymarchon corais couperi ) . The plants
are: Florida t orreya ( Torreya taxifolia ), Chapman's r hododendron ( Rhododendron chapmanii ),
American c haffseed ( Schwalbea americana ), f ringed c ampion ( Silene polypetala ), Miccosukee gooseberry ( Ribes echinellum ) and g entian pinkroot ( Spigelia gentianoides ).

The six mussel species (f at t hreeridge mussel

, p ur ple b ankclimber mussel

, s hinyrayed p ocketbook

, Ochlockonee moccasinshell

, oval pigtoe and chipola slabshell ), and the gulf sturgeon ( AcipenserPage 86 of 98 oxyrinchus desotoi ) are all aquatic animals and would not be affected by the field test. (See CH
info below on the gulf sturgeon). T he red cockaded woodpecker ( Picoides borealis ) inhabits old growth forests, primarily longleaf pine. It could visit the field test site but would not nest there. The wood stork ( Mycteria americana ) primarily inhabits wetland systems notably cypress or mangrove swamps and would not find the field test site hospitable. For the Eastern i ndigo s nake ( Drymarchon corais couperi ), according to Caroline St ahaller, (contacted February 20, 2008) of the Panama City Field Office USFWS, the spec ies has not been seen anywhere on the Florida panhandle in 15

- 20 years. She had no concern that the action could affect the species and agreed the action would have “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

.

None of the s ix listed plant species would be expected to grow at the field site/research station. For American chaffseed ( Schwalbea Americana ), according to the species recovery plan, the only
known extant population in the county is on private land. A site visit in 1994 found the area changed to a residential development. It is assumed that the population may be extirpated. For Chapman’s rhododendron ( Rhododendron chapanii ), according to the Federal Regi ster notice listing the species, there is only one population in Gadsden County. The population is on land owned by a paper company near the Gadsden

– Liberty county line, and is many miles from the release site. The Florida torreya ( Torreya taxifolia ) o ccurs on hardwood hammock slopes, ravines, and bluffs of the Apalachicola River region , u sually in steephead ravines (deep cuts made by erosion into coastal plain sediments). The ravines are much cooler and more moist than the land
surface above and harbo r remnants of the more temperate flora that existed in the region during the Tertiary ice ages . The fr inged c ampion ( Silene polypetala ) prefers well

- drained, sandy

- loam soils of deciduous woods, usually hillsides. The Miccosukee gooseberry ( Ribes echinellum ) is only known to exist on sites along east bank of Lake Miccosukee which is about 36 miles away from the
field test site. The g entian pinkroot ( Spigelia gentianoides ) is also listed in Gadsden County and specimens were once collected in the co unty, but presently the only population currently known to exist is at the Three Rivers State Recreation Area, Lake Seminole, Jackson County, FL. ( http://www.centerforplantconservation.org/ ). None o f these species would find the field test site
as suitable habitat.

Critical Habitat: The designated critical habitat for the gulf sturgeon includes the Apalachicola
River mainst r e a m, beginning from the Jim Woodruff Lock and Dam, Gadsden and Jackson

Coun ties, Florida, downstream to its discharge at East Bay or Apalachicola Bay, Franklin County, Florida. The establishment of the field test site would not impact this habitat. The field site is about 19 miles away from the Apalachicola river mainstream bel ow the Jim Woodruff Lock and

Dam. The mussel species have designated critical habitat, but the nearest to the release site is in the Ochlokonee River over 10 miles away.

Conclusion: No federally listed threatened or endangered species or species propose d for listing are likely to found at these release sites. If they were to enter the sites, 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 resu lt in no changes to the habitat used by any listed species or speciesPage 87 of 98 proposed for listing. The sites are not within or near designated critical habitat or habitat proposed for designation. Therefore, the action will have no effect on listed species or s pecies proposed for listing and would not affect designated critical habitat or habitat proposed for designation.

Highlands County, FL (FL

- HIG

- 01)

This location was previously used for managed production of citrus for at least 15 years. The planting are a at this location had been used for field trials of transgenic Eucalyptus for more than 6 years. Site preparation involved herbicide application, plowing, and planting of trees in flat beds.
Field trials of 1.4 acres of some of the lines included in thes e permit requests are being grown under issued permit # 08

- 151

- 101r under which these trees are allowed to flower. The oldest of these trees at this site are now entering their fifth growing season. An additional 2.3 acres of tests
of different lines wit h the same EH1 hybrid are being grown under permits 08

- 039

- 102rm and 09

- 070

- 101rm. Areas surrounding the test site consist of less than 5 year

- old second

- growth pine and hardwood with mixed grasses.

Fifteen TES animals, nineteen TES plants, and one TES l ichen are listed for Highland s County.
The animals are: American a lligator ( Alligator mississippiensis ), wood s tork ( Mycteria americana ),
Audubon’s crested c aracara ( Caracara cheriway ), red

- cockaded w oodpecker ( Picoides borealis ),
Florida s crub

- jay ( Aphe locoma coerulescens ), Florida grasshopper s parrow ( Ammodramus savannarum floridanus ), Everglade snail kite ( Rostrhamus sociabilis plumbeus ), i vory

- billed woodpecker ( Campephilus principalis ), Whooping crane ( Grus a mericana ) (non

- essential experimental population) , Florida p anther ( Puma concolor coryi ), Puma ( Puma concolor

– similarity of appearance), West Indian manatee ( Trichechus manatus ), Eastern i ndigo s nake ( Drymarchon corais couperi ), b luetail mole s kink ( Eumeces egreg ius lividus ) and sand s kink ( Neoseps reynoldsi ).

The plants are: Florida b onamia ( Bonamia grandiflora ), p ygmy f ringetree ( Chionanthus

pygmaeus ), p igeon w ings ( Clitoria fragrans ), short

- leaved r osemary ( Conradina brevifolia ), Avon
Park hare

- bells ( Crotala ria avonensis ), Garrett’s mint ( Dicerandra christmanii ), scrub m int ( Dicerandra frutescens ), scrub b uckwheat ( Eriogonum longifolium var. gnaphalifolium ), sn akeroot ( Eryngium cuneifolium ), highlands s crub hypericum ( Hypericum cumulicola ), scrub blazingstar ( Liatris ohlingerae ), Britton's b ear

- grass ( Nolina brittoniana ), p aper y w hitlow

- wort ( Paronychia chartacea ssp. Chartacea ), Lewton's p olygala ( Polygala lewtonii ), w ireweed ( Polygonella
basiramia ), sandlace ( Polygonella myriophylla ), s crub p lum ( Prunus geniculata ), Carter's m ustard ( Warea carteri ), and Florida z iziphus ( Ziziphus celata ).

The lichen is: Florida p erforate 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 sw amps 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 are as having associated limited areas of open grassland.Page 88 of 98 The center of range is the Kissimmee Prairie, an area of shallow ponds and sloughs with scattered hummocks of live oaks and cabbage palms. T he red cockaded woodpecker ( Picoides borealis ) inhabits old g rowth 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, s andpine 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 m aintained by periodic fires. The snail kite ( Rostrhamus sociabilis plumbeus ) prefers large, open freshwater marshes and lakes with shallow open waters. The ivory

- billed woodpecker ( Campephilus principalis ) f ormerly occurred in
the southeastern United Sta tes 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

wo uld not occur in an agricultural environment such as the field test site. Whooping crane ( Grus

a mericana ) prefers freshwater marshes and wet prairies. It nests in dense emergent vegetation (sedge, bulrush) in shallow ponds, freshwater marshes, wet prair ies, 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 experimen tal, 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, cypres s 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 popula tions. 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 ( Triche chus 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 Enda ngered 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 releas e 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 locali zed 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 orPage 89 of 98 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 Ri ck, (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 Be ach Field Office. The USFWS protocol recommends that 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. The cov erboards 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 du nes and sandy ridges in clearings or openings of scrub habitat on the Central Ridge of Florida. Pygmy f ringetree ( Chionanthus pygmaeus ) is generally found in xeric,
coarse white sand of scrub/oak scrub areas found at the southern end of the Central Florid a Ridge. It is also found occasionally in longleaf pine

- turkey oak vegetation, high pineland, dry hammocks, and transitional habitats. Pigeon w ings ( Clitoria fragrans ) is widely scattered in undisturbed clearings of xeric sandhill and scrub communities o n well

- drained upland soils. It is typically found in undisturbed clearings in scrub areas but also occurs in very open scrub as well. Short

leaved r osemary ( Conradina brevifolia ), is found in white sands of sand pine

- oak scrub with scattered overstory o f 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 m int ( 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 prepa ration has taken place. Scrub b uckwheat ( Erio gonum 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 pin e

- 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. S nakeroot ( Eryngium cuneifolium ) is generally found in areas of open sand, incl uding blowouts and other highly disturbed soil surfaces, such as road shoulders. It occurs in exposed sunny openings; areas in scrub, especially rosemary scrub. Highlands s crub hypericum ( Hypericum cumulicola ) occurs in patches of open, nutrient

poor sa nd 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 b ear

- grass ( Nolina brittoniana ) occurs in deep, fine

- textured, well

- drained sands of sand pine

- evergreen oak scrub or longleaf pine

turkey oak sandhills. Nolina is entirely dependent on fire or some other mechanism to maintain anPage 90 of 98 open successional stage in scrub or sandhills. Paper y w hitlow

- wort ( Paronychia chartacea ssp.
Chartacea ) is a sand scrub that occurs on ancient dunes in the lake region, in white sand cl earings or blowouts. Lewton's p olygala ( Polygala lewtonii ) occurs in sandhills characterized by longlea f 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.
Scrub p lum ( 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, excessiv ely leached, wind

deposited soils of evergreen scrub oak

- sand pine scrub. Carter's m ustard ( Warea carteri ) occurs in sandy clearings in sand scrub and sandhills; scattered overstory of sand; longleaf or slash pine and
scrub oaks. Florida z iziphus ( Ziziph us 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 dune s 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 non

essential experimental population and does not have critical habitat.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the release site. If the y 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 spec ies 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.

Marion County, FL (FL

- MAR

- 01)

This location has been used as an agricultural research station for more than 5 years for conducting research experiments on agricultural crops. Site preparation involved herbicide application, plowing, and planting of trees in flat beds. A field test of 0.9 acre of some of the lines included in these permit requests were established under permit 08

- 039

- 102rm in 2008. In addition, a field test of 0.3 acres of different lines with the same EH1 hybrid was planted in 2009 under permit 9

- 070

- 101rm. Areas su rrounding the test site consist of agricultural fields and plantings of horticultural

crops.

Seven TES animals and four TES plants are listed for Marion County. The animals are: sand skink ( Neoseps reynoldsi ), Eastern indigo snake ( Drymarchon corais co uperi ), wood s tork ( Mycteria

a mericana )

, Everglades s nail k ite ( Rostrhamus sociabilis plumbeus )

, r ed

- cockaded woodpeckerPage 91 of 98 ( Picoides borealis ) , Florida s crub

- jay ( Aphelocoma coerulescens )

, and West Indian m anatee ( Trichechus manatus ). The plants are: Florid a b onamia ( Bonamia grandiflora )

, l ongspurred m int ( Dicerandra cornutissima )

, s crub b uckwheat ( Eriogonum longifolium var. gnaphalifolium )

, and

Lewton's p olygala ( Polygala lewtonii ).

The Everglades snail kite ( Rostrhamus sociabilis plumbeus ) prefers large, open freshwater marshes and lakes with shallow open waters. T he red cockaded woodpecker ( Picoides borealis ) inhabits old growth forests, primarily longleaf pine. The Florida scrub

- jay ( Aphelocoma coerulescens ) prefers oak scrub on white, drained s and, 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 wood stork ( Mycteria a mericana ) primarily inhabits wetland
systems n otably cypress or mangrove swamps and would not find the field test site hospitable. Any of the four bird species could potentially visit the field test site but would not nest there. The West
Indian manatee 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 corais couperi )

– according to Candice Martino, Section 7 Endangered Species biologist from the Jacksonville, FL Field Office, (contacted Februar y 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 an agricultural research station makes it extrem ely 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 c haracteristics 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

. Also for t he sand skink ( Neoseps renoldsi )

– according to Candice Martino (contacted February 25, 2008), although the release site may be within a greater geographic area where the species may be found, the habitat
of the release site would not be suitable for the s pecies. The historic and continuous use of the release site and the surrounding area as an agricultural research station makes it extremely unlikely that the species would be found in the area. Therefore, the appropriate determination would be “no
effect ”.

For the plants, t he Florida b onamia ( 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. The l ongspurred m int ( Dicerandra cornutissima ) is scattered in openings (natural or artificial) in longleaf pine

- turkey oak scrub/sandhill or on low rises in slash pine

palmetto scrub. The s crub b uckwheat ( Eriogonum longifolium var. gnaphalifolium ) is an herbaceous perennial that occurs in dry pinelands, sandhills, and scrub (longleaf pine

- turkey oak,

s crub oaks )

. It 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. Lewton's p olygala ( 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. None of the four plant species listed above would find an agricultural research area as suitable habitat and would not be present given the historic and continuous use of the release site and the surrounding area as an agricultural research station

.Page 92 of 98 Critical Habitat: There is no designated critica l habitat or habitat proposed for designation in the
county. There is proposed critical habitat listed for the West Indian m anatee but it is not in this
county.

Conclusion: No federally listed threatened or endangered species or species proposed for lis ting 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 chan ges 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.

Taylor County, FL (FL

- TAY

- 01, FL

- TAY

- 02 and FL

- TAY

- 03)

There are three release site locations in Taylor County.

These locations have been under managed p ine plantations for over 20 years. The previous plantings were cultivated in beds using standard silvicultural practices. The existing pine plantations at these sites were harvested by the owner and prepared for re

- planting. Additional site preparation involved herbicide application, plowing to remove stumps, and planting of trees in raised beds. Approximately 9.3 acres of field tests of some of the lines included in these permits requests were established across these sites under permit 08

- 039

- 102rm in 2008 and 2009. Areas surrounding these test sites consist of managed stands of pine plantations.

There are ten TES animals listed for Taylor County. There are no liste d plants. The animals are:

Eastern i ndigo s nake ( Drymarchon corais couperi )

, w ood s tork ( Mycteria a mericana )

, p iping

p lover ( Charadrius melodus )

, r ed

- cockaded w oodpecker ( Picoides borealis )

, West Indian m anatee ( Trichechus manatus ) , Gulf s turgeon ( Acipenser oxyrinchus desotoi )

, g reen t urtle ( Chelonia mydas )

, l eatherback t urtle ( Dermochelys coriacea ) , Kemp's ridley sea turtle ( Lepidochelys kempii )
and l oggerhead sea turtle ( Caretta caretta ).

According to Caroline Stahaller, (contacted February 20, 2008) of the Panama City Field Office USFWS, the Eastern indigo snake ( Drymarchon c ouperi ) has not been seen anywhere on the Florida panhandle in 15

- 20 years. She had no concern that the action could affect the species. Although it is highly unlikely that the species would be found at the site, the applicant will provide
all workers w ith 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 s ites

. The birds (piping

p love r , r ed

- cockaded w oodpecker and w ood stork ) could all potentially visit the field test site but would not nest there. The piping plover nests along the coast which is seven or more miles away
from the field test sites. The re d

- cockaded woodpecker nests in old growth longleaf pine. The wood stork nests in marshes, floodplain lakes, and swamps. The remaining species: West Indian

m anatee ( Trichechus manatus ) , Gulf s turgeon ( Acipenser oxyrinchus desotoi )

, g reen t urtle ( Chelonia mydas )

, l eatherback t urtle ( Dermochelys coriacea ) , Kemp's ridley sea turtle ( Lepidochelys kempii ) and l oggerhead sea turtle ( Caretta caretta ) are all aquatic species and wouldPage 93 of 98 not occur anywhere close to the field test site. The closest body of water is t he Econfina river which is about 2.5 miles away from one of the test sites.

Critical Habitat: There is designated critical habitat listed for the piping plover in Taylor County. It is located along the coast in the southwest part of the county. It is approximately 15, 27 and 32 miles away from the three test sites. There is proposed critical habitat list ed for the West Indian

m anatee but it does not occur in this county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at these release sites. If they were to enter the sites, their p resence 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 si tes are 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 fo r designation.

Overall Conclusions

The field test sites in th ese permit application s have been in agricultural or forest research, or in agricultural production or forest tree plantations for from 5 to 50 years. No federally listed threatened or endange red 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 are 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. Because of this no effect determination, consultation and/or the concurrence of the USFWS and/or the NMFS ar e not required.Page 94 of 98 

## APPENDIX V: Supplemental Permit Conditions

For Release of Eucalyptus grandis x Eucalyptus urop hylla under permits 08

- 011

- 106rm and
08

- 014

- 101rm

(1) The test sites and adjacent land within 100 meters shall be monitored for any voluntee r 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 c ompletion 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 "Conta iner requirements for the movement of regulated articles", 7CFR 340.8(b) unless a shipping container variance has been approved by APHIS

- BRS.

(3) BRS should be notified in writing of any proposed changes to the permit application (or approved permit) incl uding 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 AP HIS 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 CFR p art 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 Ac t (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.

(7) Harvested plant material may not be used for food or animal feed unless it is first devitalized and approved for such us e by the U.S. Food and Drug Administration; and for plant

- incorporated protectants, a tolerance for the pesticide must first be established by the U.S. Environmental Protection Agency.Page 95 of 98 (8) The permittee shall provide all workers with identifying character istics of the threatened Eastern indigo snake and instructions on what to do if the species is encountered. This shall be done for release sites within the known range of the snake which include the following counties: Baldwin AL, Evans GA, Bay FL, Highla nds FL, Marion FL, Gadsden FL, Columbia, FL and Taylor FL.

( 9 ) Reporting an Unauthorized or Accidental Release 1. According to the regulation in 7 CFR 340.4(f)(10), 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 verba l 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

- ma il: 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_di sease.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) Compliance and Inspection Branch

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

( 10 ) Reporting Unintended Effects: According to the regu lation 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 anyPage 96 of 98 unusual occurrence (excessive mortality or morbidity, or unanticipated effect on non

- target organisms).

Written notification should be sent by one of the following means: By e

- mail: BRSCompliance@aphis.usda.gov By mail:
Biotech nology Regulatory Services (BRS) Compliance and Inspection Branch

USDA/APHIS
4700 River Rd. Unit 147 Riverdale, MD 20737

(1 1 ) 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

- m ail: BRSCompliance@aphis.usda.gov
BRS Mail: Animal and Plant Health Inspection Service (APHIS) Biotechnology Regulatory Services (BRS) Compliance and Inspection Branch
4700 River Rd. Unit 91 Riverdale, MD 20737

BRS Fax: Compliance and Inspection Branch (301) 734

- 8669

a. Planting Report Within 28 calendar days after planting, submit a report, in paper format or electronically, that includes the following information for each field test location: i. Permit number; ii. Regulated article;
iii. Release locat ion [provide state, county, internal identification number (if available), and either a single GPS coordinate as a reference point (center of plot or specify corner) or specific address]; iv. List of all constructs and specific transformed lines (event) pl anted; v. Total acreage of regulated article planted; vi. Total acreage of any border rows planted; vii. The actual planting date(s) If multiple plantings occur that are separated in time by more than a month, then a planting report is required within 28 d ays of each planting.

b. Annual Report Within 30 days after the anniversary date (one year increments from the effective date) an Annual Report must be submitted to APHIS. FAILURE TO SUBMIT ANNUAL REPORTS MAY

RESULT IN REVOCATION OF THE PERMIT. The Annual Report shall reflect the current statusPage 97 of 98 and observations to date for each location. It shall include the information submitted in the Planting Report, plus the following: i. An accounting of the acreage or number of plants per line (event) for each constr uct that remain in the ground; ii. A detailed map of the plantings; iii. Total remaining acreage (include acreage of border rows if appropriate); iv. The methods of observation; v. The resulting data and analysis regarding all deleterious effects on plants , non

- target organisms,
or the environment. This should include, but not be limited to, data on insect damage, disease susceptibility, gross morphology and any indications of weediness; vi. If any material was harvested, removed, or terminated or otherwise destroyed, a disposition table with the following information for each line (event) released should be provided: date(s) of harvest, removal, and/or termination; a formal record of how the regulated material was removed from the environment; what material and how much was harvested or removed and where it was transported, stored and further processed up to the time it is or was to be taken to a contained facility; and what was done to devitalize residual and/or harvested material at the location.

In this report also provide data documenting which trees produced flowers and which if any produced viable seeds. Also document seedling volunteer monitoring, including any volunteers
found and the method of devitalization.

c. Field Test Report
Within 6 months a fter the expiration date of the permit, the permittee is required to submit a Field Test Report. NOTE: If a new application is approved to continue the field test past its scheduled expiration date, an annual report should continue to be submitted until th e final expiration date, at which point the Field Test Report will be due after 6 months. Field Test Reports provide the final status and
observations at each location and shall include: i. List of all constructs and specific transformed lines (event) plan ted; ii. Planting date(s), and harvest dates if any material was harvested; iii. Total acreage of regulated article planted; iv. Total acreage of any border rows planted; vi. The methods of observation; vii. The resulting data and analysis regarding all de leterious effects on plants, non

- target organisms, or the environment. This should include, but not be limited to, data on insect damage, disease susceptibility, gross morphology and any indications of weediness. viii. A disposition table with the followin g information: Site name (or GPS), crop, harvest date(s), and disposition of harvested material. Date(s) of harvest, removal, and/or termination; a formal record of how the regulated material was removed from the environment; what material and how much was harvested or removed and where it was transported, stored and further processed up to the time it was taken to a contained facility; and what was done
to devitalize residual and/or harvested material at the location.

We encourage the inclusion of other t ypes 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 poli ciesPage 98 of 98 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. Flowering monitoring report In the locations where there are other species of eucalyptus within 1000 meters of the test plot s, if there is any overlap in flowering between the transgenic trial and the non

- transgenic trees, this must be reported to APHIS.

e . Monitoring Report
The fi nal monitoring report is due no later than 2 months from the end of the volunteer monitoring period. The report must include: i. Dates when the field location and perimeter fallow zone were inspected for volunteer plants; ii. Number of volunteers observed; iii. Any actions taken to remove or destroy volunteers.