Page 1 of 91 

# Permit applicatio n 11

- 052

- 101 rm received from ArborGen

Field testing of genetically engineered Eucalyptus grandis X Eucalyptus urophylla

Final Environmental Assessment April 201 2

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 comm unication of program information (Braille, large print, audiotape, etc.) should contact USDA’S TARGET Center at (202) 720

– 2600 (voice and TDD).

To file a complaint of discrimination, write USDA, Director, Office of Civil Rights, Room 326

– W, Whitten Building, 1400 Independence United States Department of Agriculture

Marketing and Regulatory Programs

Animal and Plant Health Inspection ServicePage 2 of 91 Avenue, SW, Washington, DC 20250

– 9410 or call (202) 720

– 5964 (voice and TDD). USDA is an equal opportunity provider and employer.

________________________________ ____ Mention of companies or commercial products in this report does not imply recommendation or en dorsement by the U.S. Department of Agriculture over others not mentioned. USDA neither guarantees nor warrants the standard of any product mentioned. Product names are mentioned solely to report factually on available data and to provide specific inform ation.

________________________________ ____ This publication reports research involving pesticides. All uses of pesticides must be registered by appropriate State and/or Federal agencies before they can be recommended.

________________________________ ____ CAUTION: Pesticides can be injurious to humans, domestic animals, desirable plants, and fish or other wildlife

— if they are not handled or applied properly. Use all pesticides selectively and carefully. Follow recommended practices for the disposal of surplus pesticides and pesticide containers.Page 3 of 91 TABLE OF CONTENTS

I. SUMMARY

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

II. PURPOSE AND NEE D

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

A. P ROPOSED A CTION

................................ ................................ ................................ ................... 6 B. P URPOS E OF THIS E NVIRONMENTAL A SSESSMENT

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

................................ ................................ ................................ ........... 8 D. P URPOSE AND D ESCRIPTION OF THE R ESEARCH

................................ ................................ ... 8

III. AFFECTED ENVIRO NMENT

................................ ................................ ................................ 8

IV. ALTERNATIVES

................................ ................................ ................................ .................... 13

A. N O A CTION

– D ENY THE P ERMIT

................................ ................................ .......................... 14 B. P REFERRED A LTERNATIVE

– I SSUE THE APHIS P ERMIT

................................ .................... 15

V. ENVIRONMENTAL CO NSEQUENCES

................................ ................................ .............. 15

A. N O A CTION

................................ ................................ ................................ .............................. 15 B. P REFERRED A LTERNATIVE

................................ ................................ ................................ .... 16 C. 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

................................ ................................ ........................ 32 D. C UMULATIVE E FFECTS

................................ ................................ ................................ .......... 35 E. R I SKS TO T HREATENED AND E NDANGERED S PECIES

................................ .......................... 37 F. I MPACTS ON U NIQUE C HARACTERISTICS OF P ROTECTED A REAS

................................ ...... 37 G. O THER C ONSIDERATIONS

................................ ................................ ................................ ...... 37 H. C ONCLUSION

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

VI. LISTING OF AGEN CIES AND PERSONS CON SULTED

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

VII. REFERENCES

................................ ................................ ................................ ....................... 41

APPENDIX I: STATUS O F EXISTING FIELD TES TS ALLOW ED TO FLOWER

............ 47

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

................................ .................... 49

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

................................ ................................ ................................ .................... 60

APPENDIX IV: THREATE NED AND ENDANGERED S PECIES ANALYSIS

.................... 69Page 4 of 91 APPENDIX V: PROXIMIT Y OF RELEASE SITES TO PRO TECTED AREAS

.................. 82

APPENDIX VI: PROPOS ED SUPPLEMENTAL PERM IT CONDITIONS FOR MOVEMENT

................................ ................................ ................................ ...................... 84

APPENDIX VII: PROPOS ED SUPPLEMENTAL PERM IT CONDITIONS FOR RELEASE

................................ ................................ ................................ ............................ 86Page 5 of 91 

## I. Summary

United States Department of Agriculture ( USDA ), Animal Plant Health Inspection Service ( APHIS ) has prepared a n environmental assessment (EA) in response to a confined environmental release and movement permit application (APHIS Number 11

- 052

- 101rm ) received from ArborGen Inc. (ArborGen) to allow the interstate movement, planting and flowering of genetically engineered ( transgenic ) Eucalyptus trees at six confin ed field site locations in Alabama, Florida, Mississippi, and South Carolina

. A total of 14.7 acres (all 6 sites combined) is being requested by the applicant. These plants are a clone 1 coded EH1 derived from a hybrid of Eucalyptus grandis X Eucalyptus

u rophylla and have been genetically engineered with different constructs than the trees previously permitted for environmental release by APHIS . The purpose of the confined environmental release is for Arbo r Gen to assess the effectiveness of introduced gene constructs which are intended to confer cold tolerance; to test the efficacy of genes to alter lignin biosynthesis; to test the efficacy of genes to alter growth; and to test the efficacy of gene s designed to alter flowering . In addition the trees ha ve been engineered with a selectable marker gene which confers resistance to the antibiotic kanamycin. ArborGen has also requested under this permit application that a ll genetically engineered Eucalyptus trees authorized under permits 09

- 070

- 101rm and 11

- 041

- 101rm be incorporated into this new permit and allowed to flower. Trees will also be moved interstate under this permit from ArborGen’s nursery and greenhouse facilities in South Carolina to the field test sites identified in the permit application for planting.

In 2010, APHIS completed and EA and FONSI for a permit application from ArborGen to authorize the planting, field testing and flowering of a GE Eucalyptus hybrid clone engineered to express various genes on 28 confined field site locations in the southeastern United States, including Alabama, Florida

, Georgia

, Louisiana

, Mississippi, South Carolina, and Texas ( http://www.aphis.usda.gov/brs/aphisdocs/06_32511 1r_ea.pdf ) and ( http://www.aphis.usda.gov/brs/aphisdocs/08_014101r_ea.pdf )

. Pertinent and current information available in the 2010 EA and FONSI has been incorporated by reference i nto this EA.

APHIS has issued ArborGen permits authorizing the environmental release of transgenic Eucalyptus trees, some of which allow ed flowering and some of which have not. ArborGen has grow n Eucalyptus under permits 06

- 325

- 111r, 08

- 011

- 106rm, 08

- 014

- 101rm, 08

- 039

- 102rm, 08

- 151

- 101r

, 09

- 070

- 101rm and 11

- 201

- 103r . For permits 06

- 325

- 111r, 08

- 011

- 106rm, 08

- 014

- 101rm

, 08

- 151

- 101r and 11

- 201

- 103r the trees have been allowed to flower. Permits 06

- 325

- 111r, 08

- 011

- 106rm, 08

- 014

- 101rm were issued by APHIS upon completion of an EA and subsequent FONSI ( http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf ) and ( http://www.aphis.usda.gov/brs/aphisdocs/08_014101r_ea.pdf ). Permit 06

- 325

- 111r expired on June 27, 2010 and was renewed under 10

- 112

- 101r. Permit 08

- 039

- 102r expired on April 25, 2011 and was renewed un der 11

- 041

- 101rm. Permit 08

- 011

- 106rm is active and will expire on May 12, 2013. Permit 08

- 014

- 101rm is active and will expire

1 Clone

– as defined in horticulture and forestry means is a population of genetically identical plants that has been derived from one individual. Despite popular use of the word, a clone is not an individual.Page 6 of 91 on May 12, 2013. Permit 08

- 151

- 101r expired June 30, 2011 and was incorporated into 08

- 014

- 101rm. Permit 09

- 070

- 101rm is active and will expire on May 11, 2012. Permit 11

- 201

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

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

g row GE Eucalyptus which include 32 unique locations with in 7 States : 08

- 011

- 106rm, 08

- 014

- 101rm, 09

- 070

- 101rm, 10

- 112

- 101r, 11

- 041

- 101rm and 11

- 201

- 103r

. No planting s at any of the 32 location s are authorized by APHIS to exceed 20 acres in size

. Trees are allowed to flower under four permits: 08

- 011

- 106rm, 08

- 014

- 101rm, 10

- 112

- 101r and 11

- 201

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

trees are not authorized to flower )

. Under permits 09

- 070

- 101rm and 11

- 041

- 101rm trees

are not authorized to flower. As of September 2011, ArborGen has reported to APHIS that they are growing a total of approximately 6 7 acres of trees on 1 8 of the 3 2 permitted locations.

ArborGen has submitted a new permit application 11

- 052

- 101rm to APHIS requesting approval for environmental release of hybrid Eucalyptus trees at 6 locations in the States of Alabama, Florida, Mississippi, and South Carolina encompassing a total of 14.7 acres (the subject of this EA). F ive of these locations curre ntly have active APHIS permits ( 08

- 011

- 106rm, 08

- 014

- 101rm, 09

- 070

- 10rm, 10

- 112

- 101r , and 11

- 041

- 101rm ) for environmental release of GE Eucalyptus hybrids in Alabama, Florida, Mississippi, and South Carolina. The sixth site in South Carolina has been listed as a holding site for transgenic trees in previous APHIS permits and notifications and is a new location for the release of GE Eucalyptus

. ArborGen is requesting that trees be allowed to flower at four loca tions in Alabama, Florida and Mississippi. At the two location s in Berkeley and Dorchester Count ies

, SC

, ArborGen has request ed to release trees in containers and have indicated they will not allow these trees to flower at these location s

. The size of each individual confined field test site s identified in ArborGen’s permit application ranges from 0. 5 to 7.7 acres , which is less than the 20 acres analyzed in APHIS EA and subsequent FONSI prepared for Permits 08

- 011

- 106rm, 08

- 014

- 101rm and 10

- 112

- 101r ( http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf ) and ( http://www.aphis.usda.gov/brs/aph isdocs/08_014101r_ea.pdf )

. 

## II. Purpose and Need

A. Proposed Action The proposed action is for APHIS to approve ArborGen permit application request (11

- 052

- 101rm) and issue a confined environmental release and interstate movement permit for a hybrid of Eucalyptus grandis X Eucalyptus urophylla with supplemental permit conditions ( see Appendices VI and VII). The movement authorization would be valid for one year and the release for a three

- year period. The permit will need to be renewed by ArborGen and subsequently approved by APHIS to allow the transgenic Eucalyptus plants to remain in the ground beyond th e three

- year time period specified in the permit application

.

APHIS would issue a confined environmental release and interstate move ment permit to ArborGen in accordance with 7 CFR part 340 to allow the interstate movement, planting,Page 7 of 91 field testing and flowering of a GE Eucalyptus hybrid clone ( Eucalyptus grandis x Eucalyptus urophylla ) engineered to express various genes at 6 confined field site locations within in the States of Alabama, Florida, Mississippi, and South Carolina encompassing a total of 14.7 acres

. The permit would allow all the trees except the trees on two of the sites in South Carolina to flower. Trees would also be a uthorized to be moved interstate from ArborGen’s greenhouse and nursery facilities in Berkeley, Charleston and Dorchester Counties in South Carolina to the field test locations. A ll genetically engineered Eucalyptus trees authorized under permits 09

- 070

- 1 01rm and 11

- 041

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



### B. Purpose of this Environmental Assessment The purpose of this EA is to assess the potential environmental impacts on the human environment associated with APHIS responding to a confined environmental release and interstate movement permit application (11

- 052

- 101rm) received from ArborGen in February 2011 to authorize the planting of GE Eucalyptus hybrid clone to support proposed field research studies on six resea rch sites in Alabama, Florida

, Mississippi, and South Carolina

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

trees on two of the sites in South Carolina to flower. The purpose of the confined environmental release is for ArborGen to assess the effectiveness of introduced gene constructs which are intended to confer cold tolerance; to test the efficacy of genes to alter lignin biosynthesis; to test the efficacy of gen es to alter growth; and to test the efficacy of genes designed to alter flowering . In addition the trees have been engineered with a selectable marker gene which confers resistance to the antibiotic kanamycin. ArborGen has also requested under this permi t application that a ll genetically engineered Eucalyptus trees authorized under permits 09

- 070

- 101rm and 11

- 041

- 101rm be incorporated into this new permit and allowed to flower. New trees will also be moved interstate from ArborGen’s nursery and greenhous e facilities located in South Carolina to the release locations identified in the permit application where they will be planted.

This EA was conducted pursuant to : (1) The National Environmental Policy Act of 1969 (NEPA), as amended (42 U.S.C. § 4321 et seq .), (2) regulations of the Council on Environmental Quality for implementing the procedural provisions of NEPA (40 CFR parts 1500

- 1508), (3) USDA regulations implementing NEPA (7 CFR part 1b), and (4) APHIS' NEPA Implementing Procedures (7 CFR part 372)

. Generally, 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 may be prepared, pursuant to 7 CFR § 372.5(d) to determine if an Environmental Impact Stateme nt is warranted

. In this case, APHIS has prepared this EA specifically to address potential environmental impacts associated with new genetic constructs not previously reviewed or permitted for environmental release and interstate movement by APHIS and wi th allowing existing planting s of genetically engineered Eucalyptus trees permitted by APHIS to flower

.Page 8 of 91 

### C. Need for This Action

APHIS

- Biotechnology Regulatory Services ( BRS ) mission is to protect America’s agriculture and the environment using a dynamic and science

- based regulatory framework that allows for the safe development and use of GE organisms. APHIS’ regulations in 7 CFR part 340, which were promulgated pursuant to a uthority granted by the plant pest provisions of the Plant Protection Act, as amended, (7 U.S.C. 7701

– 7772), regulates the introduction (importation, interstate movement, or release into the environment) of certain GE organisms and products. The Plant Pro tection Act directs the USDA to facilitate imports and interstate commerce in agricultural products in ways that will reduce, to the extent practicable, the risk of dissemination of plant pests. Under APHIS regulations, the APHIS Administrator has authori ty to regulate any organism or product altered or produced through genetic engineering that the Administrator determines is a plant pest or has reason to believe is a plant pest. When APHIS receives an application for a permit for environmental release an d movement , the application is evaluated to determine whether the environmental release and movement , with appropriate conditions imposed, can be carried out while preventing the dissemination and establishment of plant pests. The receipt of a permit appl ication to introduce a genetically engineered organism requires a response from the Administrator:

Administrative action on applications . After receipt and review by APHIS of the application and the data submitted pursuant to paragraph (a) of this section , including any additional information requested by APHIS, a permit shall be granted or denied (7 CFR 340.4(e)).

The applicant has provided the required information associated with this request in the permit application. This information has been review ed 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

, gene s to alter lignin biosynthesis , genes to control flowering, and genes to alter the growth rate in Eucalyptus . According to the applicant, genetically engineered cold tolerant Eucalyptus would enable the production of this hardwood species for pulping and for biofuel applications in managed plantation forests in the southeaster n U.S

. The confined release of the trees in different areas of the southeast U.S. will allow the applicant to obtain data on performance of the transgenic trees and the efficacy of the inserted genes in a wide variety of environments. It is important tha t trees be planted in locations in Florida because this is the only location where a comparison between the transgenic and non

- transgenic control trees can be made. In other locations the trees may be damaged by cold which makes a true comparison difficult to impossible.



## III. Affected Environment

Biology of Eucalyptus and S tatus in the United StatesPage 9 of 91 The genus Eucalyptus belongs to family Myrtaceae (subfamily: Leptospermoideae) which includes over 700 species. Eucalyptus is native to Australia wi th the exception of some species that are native to the Timor Islands (Groves 1994, Ladiges 1997) . There are no wild relatives of Eucalyptus that occur naturally in the United States. An overview of the biology of Eucalyptus grandis has been published by the US Forest Service (Meskimen and Francis 1990)

. Eucalyptus has been planted as an ornamental species in the extreme southern United States where mild winters will allow some species to grow. Eucalyptus normally propagates in its native range via seeds. It does not spread vegetatively like other trees such as poplar or willow. In the United States it is usually propagated and sold commercially as rooted stem cuttings.

There have been numerous attempts to grow Eucalyptus as a commercial forest tree in the southeastern United States, but due to its sensitivity to cold temperatures, these attempts have not met with success. It is only grown in commercial plantations in central and southern Florida, where it normally surviv es freezing temperatures which are rare and usually not severe. Eucalyptus is adapted to live in the mild arid and semi

- arid climate of Australia. Severe freezing events that can occur in the southern United States have limited its establishment as a com mercial 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 plant ed in areas where severe freezing events are rare.

The species hybrid E. grandis x E. urophylla (also known as E. urograndis ) that ArborGen wishes to allow to mature and flower under this permit has not been categorized as invasive. The E. grandis x E. ur ophylla 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 )

. The University of Florida Institute of Food and Agricultural Sciences (IFAS) recently completed a review of the potential invasiveness of E. urograndis and found that it is not likely to be invasive and can be a recommended species for planting: http://plants.ifas.ufl.edu/assessment/pdfs/wra/Eucalyptus%20urograndis_WRA.pdf See also the chart at: http://plants.ifas.ufl.edu/assessment/pdfs/concl_genus_Feb2011.pdf . Because it is not predicted to be invasive, no specific management practices are recommended for this species

, unlike other species of Eucalyptus grown in Florida (see: http://plants.ifas.ufl.edu/assessment/pdfs/results_feb2011.pdf

.
Numerous species of Eucalyptus were introduced into Californi a during that State’s early history (see Santos: http://wwwlibrary.csustan.edu/bsantos/euctoc.htm ), and some of these species have become established. Two of these, E. globulus (Tasmanian bl ue 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 i s being proposed to be planted at the permitted field site

.

Traits E ngineered into EucalyptusPage 10 of 91 ArborGen LLC wishes to field test genetically engineered (transgenic) Eucalyptus trees during which time the trees may flower. These plants are a clone coded EH1 derived from a hybrid of Eucalyptus grandis X Eucalyptus urophylla. These have been genetically engineered with different constructs. The purpose of the field trials is to test the effectiveness of different genes to confer cold tolerance , alter lignin biosynthesis, or alter growth rat e along with test ing the efficacy of the Barnase gene designed to alter fer tility. In addition the trees have been engineered with a common selectable marker

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

- Repeat Binding Factor ( CBF) gene Barnas e gene , and nptII gene, all genes are claimed as Confidential Business Information ( CBI )

.

Confined Field Test Locations

The confined field tests would take place on land controlled by ArborGen or through private contracts established by ArborGen for field testing. The exact locations are claimed as CBI and have been submitted as part of the APHIS perm it application . Under the permit application submitted by ArborGen , there are six research sites where trees would be planted. A total of 14.7 acres (all 6 sites combined) is being requested by the applicant. F ive of these locations currently have activ e APHIS permits ( 08

- 011

- 106rm, 08

- 014

- 101rm, 09

- 070

- 10rm, 10

- 112

- 101r , and 11

- 041

- 101rm ) for environmental release of GE Eucalyptus hybrid clone ( Eucalyptus grandis x Eucalyptus urophylla ) in Alabama, Florida, Mississippi, and South Carolina. The remaining site in Dorchester County, South Carolina has been listed in previous APHIS permits/notifications as a holding area for transgenic trees

. ArborGen is requesting that trees be allowed to flower at four locations in Alabama, Florida and Miss issippi. At the two locations in Berkeley and Dorchester Counties, SC, they have requested to release trees in containers and have indicated they will not allow these trees to flower at these locations. The size of each individual confined field test sit es identified in ArborGen’s permit application ranges from 0.5 to 7.7 acres, which is less than the 20 acres analyzed in APHIS EA and subsequent FONSI prepared for Permits 08

- 011

- 106rm, 08

- 014

- 101rm and 10

- 112

- 101r ( http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf ) and ( http://www.aphis.usda.gov/brs/aphisdocs/08_014101r_ea.pdf )

. For the five research sites that are currently authorized by APHIS to plant GE Eucalyptus hybrid clone ( Eucalyptus grandis x Eucalyptus urophylla )

, ArborGen has indicated in their permit application that any new planting authorized by APHIS under 11

- 052

- 101rm w ould not expand the current field site locations beyond the areas currently authorized by APHIS

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

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

p rivately owned managed plantation forests and agricultural farm lands or experimental research stations managed by academic institutions and industry

, and have been in managed agricultural production for more than 5 years

. The standard agricultural and silvicultural practices for land preparation, planting, irrigation, and harvesting of plants have been routinely used on these sites. Sites that include managed pastures have had intense activity including the use of heavy m achinery for general upkeep, irrigation, fertilization, controlled grazing and management of grasses. Standard silvicultural practices would be used at these sites for the duration of the field tests. SurveysPage 11 of 91 conducted by the applicant at each of these l ocations indicate that there are not any old growth forests or undisturbed natural areas in the immediate surroundings of the test sites. ArborGen h as indicated that t he trees would be planted on individual research sites ranging from 0. 5 up to 7.7 acres, depending on the location with planting density ranging from 300

- 600 trees per acre 2 . An acre is about the size of a football field.

Table 1. Environmental r elease locations requested under permit application 11

- 052

- 101rm including existing Eucalyptu s permits at each location. Location Existing Permit(s) Flowering allowed Acreage requested under the new permit Baldwin County Alabama 10

- 112

- 101r Yes 2.0

Escambia County Alabama 08

- 011

- 106rm Yes 2.0

Pearl River County Mississippi 08

- 011

- 106rm Yes 2.0

Berkeley County South Carolina* 08

- 011

- 106rm 08

- 014

- 101rm 09

- 070

- 10rm 11

- 041

- 101rm No 0.5

Dorchester County South Carolina* New location for Eucalyptus No 0.5

Highlands County Florida 08

- 014

- 101rm 09

- 070

- 10rm 11

- 041

- 101rm Yes No No 7.7

Total 14.7

*Both locations in South Carolina are fenced areas for holding plants in containers prior to planting where no flowering is allowed

.
Baldwin County Alabama Site:

This location has been an agricultural research station for more than 20 years. The l ocati on has been used for managed production of annual agricultural crops and forest trees. Approximately 3.55 acres of field trials of genetically modified Eucalyptus trees of some lines in the permit are being grown under APHIS issued permits 08

- 011

- 106rm (0. 75 acres) and 10

- 112

- 101r (2.8 acres) . These trees are authorized by APHIS to flower. The oldest of these trees were planted in August 2006. Similar to the current permit authorized by APHIS , s ite preparation would involve herbicide application, subsoilin g, and planting of trees in flat beds. The s urrounding areas of the test site consist of field plantings of agricultural crops, experimental forest trees and an abandoned pecan

2 Planting density typically refers to t he 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 si tes ranging from 10 to 20 acres can have from 3000 to 16,000 total trees planted in the ground. Twenty acres, as defined by forest plantation standards in the southeast, is considered a small planting.Page 12 of 91 orchard. Under the new permit application, APHIS could authorize u p to 2.0 acr es of field tests to be established and flower at this location (at around 300

- 600 trees per acre) over the next three years and would not expand the current field site location beyond the area currently authorized by APHIS

.
Escambia County Alabama Site:

This location ha d previously been used as an intensely managed pasture for more than 5 years. The test site is currently planted with grasses suitable for cattle grazing. Approximately 0.5 acres of field trials of genetically modified Eucalyptus of some lines identified in the permit authorized by APHIS are being grown under permit 08

- 011

- 106rm . T hese trees are authorized by APHIS to flower. The oldest of these trees were planted in July 2007. Similar to the current permit authorized by APHIS, s ite preparation would involve herbicide application to remove existing grasses, subsoiling, preparation for possible irrigation, and planting of the test trees in flat beds. The surrounding areas of the test site consist of greater than 30 years

- old slash pine , and a re

- forested area with mixed stands of pine and hardwood species. Under the new permit application, APHIS could authorize u p to 2.0 acres of field tests to be established and flower at this location (at around 300

- 600 trees per acre) over the next three years and would not expand the current field site location beyond the area currently authorized by APHIS

.
Pearl River County Mississippi Site:

This location has been an agricultural research station for more than 5 years. The location has been used for conducting research experiments with agricultural crops and grasses. The test site has been used for experimental planting of grasses. Approximately 3 acres of field trials of genetically modified Eucalyptus trees of some lines identified in the p ermit authorized by APHIS are being grown under permit 08

- 011

- 106rm . T hese trees are authorized by APHIS to flower. The oldest of these trees were planted in October 2007. Similar to the current permit authorized by APHIS, site preparation would involve h erbicide application to remove existing grasses, subsoiling, preparation for possible irrigation installation, and planting of trees in flat beds. The surrounding areas of the test site consist of a grape research farm, mixed stands of hardwoods and pine, and a residential area. Under the new permit application, APHIS could authorize u p to 2.0 acres of field tests to be established and flower at this location (at around 300

- 600 trees per acre) over the next three years and would not expand the current fie ld site location beyond the area currently authorized by APHIS

. .
Berkeley County South Carolina Site:

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

- transgenic Eucalyptus tree plants for more than 8 years. G enetically modified Eucalyptus trees are being grown in containers under APHIS permits 08

- 011

- 106rm

, 08

- 014

- 101rm

, 09

- 070

- 10rm and 11

- 041

- 101rm . T hese trees are not authorized by APHIS to flower. Similar to the current per mit authorized by APHIS, t he proposed 0.5 acre release site is located adjacent to greenhouse facilities and is surrounded by hardwoods and pine plantations and would not expand the current field site locationPage 13 of 91 beyond the area currently authorized by APHIS

. This site is a secure fenced holding area where trees growing in containers are transferred from the greenhouse to th e out

- of

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

Dorchester County South Carolina Site:

This location is an extension of a greenhouse facility and was previously cleared in 2010 for development as a light industrial park, prior to which it had been a managed pine plantation for more than 15 years. The surrounding area consists of man

- made ponds, hardwoods and pine plantations. This site is a secure fenced holding area where trees growing in containers are transferred from the greenhouse to th e out

- of

- doors for acclimatization prior to field planting. Trees will not be allowed to flower at this location. This is a new location that was not assessed in the previous EA

. However, this site has previously been listed as a holding area for trees in containers in previous APHIS permits and notifications for other transgenic tree species

.
Highlands County Florida Site:

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

. 2 acres of field trials of genetically modified Eucalyptus trees are being grown under APHIS permit s 08

- 014

- 101rm , 09

- 070

- 101rm and 11

- 041

- 101rm at this location

. T rees that are being grown under permit 08

- 014

- 101rm are authorized by APHIS to flower. The oldest of these trees were planted in July 2006. Similar to the current permit authorized by APHIS, site preparation would involve herbicide application, plowing, and planting of trees in flat beds. The test site is surrounded by a citrus production area and second

- growth pine and hardwood forests. Unde r the new permit application, APHIS could authorize u p to 7.7 acres of field tests to be established and flower at this location (at around 300

- 600 trees per acre) over the next three years and would not expand the current field site location beyond the area currently authorized by APHIS

. ArborGen has also requested under this permit application that a ll genetically engineered Eucalyptus trees authorized under permits 09

- 070

- 101rm and 11

- 041

- 101rm at this site location to be incorporated into this new pe rmit and allowed to flower.

Interstate Movement

As requested in the permit application, APHIS could authorize the movement of genetically engineered Eucalyptus trees from nursery and greenhouse sites in Berkeley, Charleston, and Dorchester Counties in South Carolina to the four field release locations identified in the permit application that would allow flowering. As specified in the permit and supplemental permit conditions ( see Appendi x VI), l abeled container

- grown trees would be placed in an enclosed vehicle (trailer or a van) and transported to the destination test sites.



## IV. AlternativesPage 14 of 91 This EA analyzes the potential environmental consequences of APHIS’ response to a confined environmental release and interstate movement permit application (APHIS Number 11

- 052

- 101rm) received from ArborGen to allow the movement to
, planting and flowering of genetically engineered Eucalyptus trees at six confined field site locations in Alabama, Florida, Mississippi, and South Carolina . A total of 14.7 acres (all 6 sites combined) is being requested by the applicant. These plants are a clone 3 coded EH1 derived from a hybrid of Eucalyptus gr andis X Eucalyptus urophylla and have been genetically engineered with different constructs than the trees previously permitted for environmental release by APHIS. The purpose of the confined environmental release is for ArborGen to assess the effectivene ss of introduced gene constructs which are intended to confer cold tolerance; to test the efficacy of genes to alter lignin biosynthesis; to test the efficacy of genes to alter growth; and to test the efficacy of genes designed to alter flowering . In addi tion the trees have been engineered with a selectable marker gene which confers resistance to the antibiotic kanamycin. ArborGen has also requested under this permit application that a ll genetically engineered Eucalyptus trees authorized under permits 09

- 070

- 101rm and 11

- 041

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

Under APHIS regulations, the Administrator must either grant or deny permits properly submitted under 7 CFR part 340. Based upon the permit application s submitted by A rborGen, t wo alternatives are considered and analyzed in this EA: (1) deny the permit and (2) approve ArborGen permit application request and issue the APHIS permit.



### A. No Action

– Deny the Permit

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

- 052

- 101rm ) submitted by ArborGen

. ArborGen would not be authorized to move and plant the GE Eucalyptus trees with new constructs and allow them to reach maturity and flower. C onfined field release under permits 09

- 070

- 101rm and 11

- 041

- 101rm w ould continue to not allow flower ing

.

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

grow GE Eucalyptus which include 32 unique locations with in 7 States: 08

- 011

- 106rm, 08

- 014

- 101rm, 09

- 070

- 101rm, 10

- 112

- 101r, 11

- 041

- 101rm and 11

- 201

- 103r

. No planting s at any of the 32 location s are authorized by APHIS to exceed 20 acres in size

. Trees are allowed to flower under four permits: 08

- 011

- 106rm, 08

- 014

- 101rm, 10

- 112

- 101r and 11

- 201

- 103r ( two of the loc ations in Berkeley and Dorchester Counties, SC

trees are not authorized to flower )

. Under permits 09

- 070

- 101rm and 11

- 041

- 101rm trees

are not authorized to flower. As of September 2011, ArborGen has reported to APHIS that they are growing a total of appr oximately 67 acres of trees on 18 of the 32 permitted locations. Under the No Action alternative, ArborGen could continue to grow GE Eucalyptus as authorized by APHIS under these six active permits.

3 Clone

– as defined in horticulture and forestry mea ns is a population of genetically identical plants that has been derived from one individual. Despite popular use of the word, a clone is not an individual.Page 15 of 91 

### B. Preferred Alternative

– Issue the APHIS Permit

The P referred A lternative is to approve Arbo r Gen permit application request (11

- 052

- 101rm) and issue the APHIS confined field release and movement permit for a hybrid of Eucalyptus grandis X Eucalyptus urophylla with supplemental permit conditions ( see App endices V I and VII ) . The movement authorization would be valid for one year and the release would be valid for a three

- year period. The permit will need to be renewed by ArborGen and subsequently approved by APHIS to allow the transgenic Eucalyptus plant s to remain in the ground beyond th e three

- year time period specified in the permit application

.

Under this alternative, APHIS would issue a confined environmental release permit to ArborGen in accordance with 7 CFR part 340 to allow the movement, planting, field testing and flowering of a GE Eucalyptus hybrid clone engineered to express various

genes at 6 confined field site locations within in the States of Alabama, Florida, Mississippi, and South Carolina encompassing a total of 14.7 acres . The permit would allow all the trees except the trees on two of the sites in South Carolina to flower. A ll genetically engineered Eucalyptus trees authorized under permits 09

- 070

- 101rm and 11

- 041

- 101rm would be incorporated into this new permit and allowed to flower. This permit would authorize ArborGen to move and plant the GE Eucalyptus trees with new constructs and allow them to reach maturity and flower. Under this alternative, the applicant would be allowed to gather data on performance of the transgeni c trees over a multiyear period and the efficacy of the genes in a wide variety of environments for multiple years. This alternative would allow the safe development and use of GE organisms under the mission of BRS.


## V. Environmental Consequences

A. No A ction

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

- 052

- 101rm) submitted by ArborGen

. ArborGen would not be authorized to move, or plant the GE Eucalyptus trees with new constructs identified in their permit application and allow them to reach maturity and flower. GE Eucalyptus trees would continue to be planted and grown as authorized by APHIS permits ( 08

- 011

- 106rm, 08

- 014

- 101rm, 09

- 070

- 10rm, 10

- 112

- 101r , and 11

- 041

- 101rm ) in the southeastern United States including the 5 environmental release locations in Alabama, Florida, Mississippi, and South Carolina that are identified in ArborGen’s permit request

. C onfined field release under permits 09

- 070

- 101rm and 11

- 041

- 101rm w ould continue to not allow flower ing.

There would be no environmental impacts associated with the new constructs identified in the permit application since the trees containing these new constructs would not be authorized by APHIS for interstate movement or environmental release. Under this

a lternative, the applicant would not be allowed to gather data on performance of the transgenic trees over a multiyear period and the efficacy of the genes in a wide variety of environments for multiple years.Page 16 of 91 Potential impacts associated with Permits 08

- 011

- 106rm, 08

- 014

- 101rm and 10

- 112

- 101r would not change from those analyzed in the 2010 EA and FONSI prepared for these permits ( http://www.aphis.usda.gov/brs/aphisdocs/06_325111 r_ea.pdf ) and ( http://www.aphis.usda.gov/brs/aphisdocs/08_014101r_ea.pdf )

. As specified in the permit conditions for Permits 09

- 070

- 101rm and 11

- 041

- 101rm, trees at these sites are not authorized to flower and therefore would be required to be cut down prior to maturity. Preventing flower formation would prove impossible over time because the trees g row too tall and too many fl owers are produced to remove manually. Subsequently, these site locations could be re planted in short rotation GE Eucalyptus field tests if approved by APHIS, or the sites could be returned to other forest tree production

, agriculture /forestry research

, p asture
, or other agricultural activities. Intense management activity including the use of heavy machinery for land preparation, general upkeep, irrigation, and fertilization for the management of tree plantings and grasses and standard silvicultural

prac tices would continue to be used at these sites

.



### B. Preferred Alternative

Under this alternative, APHIS would issue a confined environmental release permit to ArborGen in accordance with 7 CFR part 340 to allow the movement, planting, field testing and flowering of a GE Eucalyptus hybrid clone engineered to express various

genes at 6 confined field site locations within in the States of Alabama, Florida, Mississippi, and South Carolina encompassing a total of 14.7 acres with s upplemental permit conditions ( see Appendices V I and VII ) for the requested three

- year period

. The permit would allow all the trees except the trees on two of the sites in South Carolina to flower. A ll genetically engineered Eucalyptus trees authorized under permits 09

- 070

- 101rm and 11

- 041

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

F ive of these six locations currently have active APHIS permits ( 08

- 011

- 106rm, 08

- 014

- 101rm, 09

- 070

- 10rm, 10

- 112

- 101r , and 11

- 041

- 101rm ) for en vironmental release of GE Eucalyptus hybrids in Alabama, Florida, Mississippi, and South Carolina .. The remaining site in Dorchester County, South Carolina has been listed in previous APHIS permits/notifications as a holding area for GE trees

. In respect to the five locations that currently have active APHIS permits; the new permit application ( 11

- 052

- 101rm) does not expand the field sites beyond what is current authorized by APHIS or density (number) of trees that could be planted. The holding area in Dorchester County will remain as a holding area for trees. Adding Eucalyp t us trees to the site will not change any existing conditions. As identified in the new permit application, GE Eucalyptus trees with the new constructs would be released at the 5 existing locations in addition to the

trees currently approved by APHIS for environmental release . The new permit application does not expand the number of trees or total acreage at any of the 5 confined release sites currently approved by APHIS

. Similar to current permits, if approved by APHIS, the t rees would remain in the ground for at least 3 years and most likely until maturity beca u se the applicant has indicated in their permit application that they are plan ning to renew the permits prior to expiration

. If the renewal permits are approved byPage 17 of 91 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, plan ting, irrigation, and harves ting of trees would continue to be routinely used on these sites.



### Potential Environmental Impact of the Preferred Alternative

Interstate Movement of the trees under permit

Under this Alternative, APHIS could authorize the interstate movement of transgenic Eucalyptus trees in accordance with the regulatory requirements identified in 7 CFR 340.4, 340.7 and 340.8 , including specific permit conditions ( see Appendi x VI) assigned to this permit that would prevent the dissemination of the trees into the environment. As specified in the supplemental permit conditions, ArborGen would be moving trees in accordance with an APHIS approved variance that only allows trees to be moved in l abeled container s with in an enclosed vehicle. For the time period between 2005 to 2010

, APHIS issued 218 permits and notifications that authorized the interstate movement of forest trees. Of these 218 authorized movements, there was one compliance inciden t associated with a shipment of plants via mail . This compliance incident was associated with a movement that did not have similar APHIS approved variances as those that would be required for ArborGen to move GE Eucalyptus trees under this permit request. From 2005 to 2010, no compliance incidents of authorized release were reported to APHIS for moving GE Eucalyptus trees using similar APHIS approved variances that would be required to move trees under this permit request

.

Con sidering the specific permit conditions that must be adhered to by the permit requirements identified in 7 CFR 340.4, 340.7 and 340.8

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

mo vement of the regulated article, and past compliance history of similar types of movements, APHIS considers the possibility of unintended exposure from moving GE Eucalyptus trees under this Alternative to be negligible to non

- existent.

Alteration in Susce ptibility 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 N o A ction A lternative.

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 susceptibility to disease or insect damage.

• None of the genes being engineered into the Eucalyptus plants are expected to alter the susceptibility of the transgenic Eucalyptus pl ants 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 thePage 18 of 91 case ( see below). As prescribed in the supplemental permit conditions assigned to these permits

, periodic monitoring of the field plots will allow the detection of any unexpected infestation by plant disease organisms or animal pests. The permittee is required to report an y such unanticipated effects to APHIS under the terms of the permit

- see 7 CFR 340.4(f)(10)(ii).

Although the trees originated from New Zealand, the trees were propagated in sterile tissue culture and were free of pests upon importation into the U.S. prior to their introduction. All materials were handled in accordance with the USDA

– APHIS requirements for import and quarantine under a USDA

– APHIS PPQ Post

- entry quarantine permit.

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

- Risk of the Gene P roducts on the Environment

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

, altered lignin and altered growth would be similar to the N o A ction A lternative. These same types of genes are currently being tested in GE Eucalyptus trees and other species being field tested under current APHIS notifications or permits for confined field release.

Gene used as selectable marker

The kanamycin resistance selectab le 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 th e expression of a gene product or change plant metabolism in such a way that it would be expected to cause risk to the environment. In a number of instances, plants transformed with this gene are no longer subject to the plant pest provisions of the Plant Protection Act and 7 CFR part 340 (e.g. corn, petition 01

- 137

- 01p; rapeseed, petition 01

- 206

- 02p; cotton, petition 95

- 045

- 01p; and papaya, petition 96

- 051

- 01p).

Genes conferring cold tolerance

The C

- Repeat Binding Factor ( CBF) genes are transcription factors that belong to the AP2/EREBP family of DNA binding proteins (Riechmann and Meyerowitz 1998) and like other transcription factors act as control switches for the coordinated expression of other genes in defined metabolic pathways. CBF protein recognizes and binds to a cold

and drought

- responsive DNA regulatory sequence designated as the C

- repeat (CRT)/deh ydration

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

, (Yamaguchi

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

inducible genes (Maruya ma et al. 2004)

.
A common observation across experiments in which CBF genes are overexpressed in transgenic plants is that constitutive expression of CBF negatively impacts a number of other traits (Hsieh et al. 2002) . In potato, for example, constitutive expression of Arabidopsis CBF genes using the CaMV35S promoter was associated with smaller leaves, stunted plants, delayed flowering, and reduction or lack of tuber production (PinoPage 19 of 91 et al. 2007) . In contrast, CBF genes under the control of a cold

- induced promoter, rd29A (Yamaguchi

- Shinozaki and Shinozaki 1993, Kasuga et al. 1999)

, (Narusaka et al. 2003)

, increased freezing tolerance to the same level as constitutive expression (about 2 °C, or ~3 °F) while restoring growth and tuber production to the levels similar to wild

- type plants (Pino et al. 2007) . In the rd29A controlled CBF plants the same level of freezing tolerance as the CaMV35S versions was observed after only a few hours of ex posure to low but non

- freezing temperatures. These results suggest that using a stress

- inducible promoter to direct CBF transgene expression could significantly improve freeze tolerance without negatively impacting other agronomically important traits. In the case of these Eucalyptus trees, the CBF gene is under the control of a cold inducible promoter which causes the gene to be expressed under cold temperatures, thus mitigating the potential of reduced growth by overexpression. Under this promoter the trees exhibit normal plant growth. The CBF gene is not expected to produce any toxic substances and is not expected to alter the characteristics of the engineered plants other than imparting tolerance to cold temperatures.

There are also five other genes intended to confer cold tolerance in the pe rmit application that are claimed as CBI. These are all genes derived from other plants. These genes do not alter the expression of a gene product or change plant metabolism in such a way that it would be expected to cause risk to the environment. These genes in Eucalyptus or other plants have not produced unanticipated phenotypes that would indicate there have been changes to plant metabolism leading to increase plant pest characteristics.

Gene for altered fertility

The barnase gene has been engineered into other crops that have been previously reviewed and addressed in multiple environmental assessments by APHIS. Male sterile corn (USDA APHIS petitions for deregulation 95

- 288

- 01p, 97

- 342

- 01p and 98

- 349

- 01p), rapeseed (petition s 98

- 278

- 01p and 01

- 206

- 01p) and chicory (petition 97

- 148

- 01p) have been reviewed by APHIS and are no longer subject to the plant pest provisions of the Plant Protection Act and 7 CFR part 340

. There is no reason to believe that the function

a nd expressio n 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 e nvironment. In greenhouse tests using tobacco and an early flowering model Eucalyptus ( E. occidentalis ), the applicant has found that the barnase gene has demonstrated 100% efficacy in preventing pollen formation. In developing flower buds from field grown transgenic Eucalyptus lines containing this cassette, 90% of lines showed complete pollen ablation. Recent observations from the replicated field study conducted in Alabama under permit 06

- 325

- 111r (renewed as 10

- 112

- 101r ) and Florida under permit 08

- 151

- 101r (now covered under 08

- 014

- 101rm) confirmed that cold tolerant translines grown in these field t est did not produce any pollen (see Appendix I).Page 20 of 91 Gene s for altered lignin

There are three gene s identified in the permit application to alter lignin. These genes have been engineered into other crops that have been previously released into the enviro nment under both APHIS notifications and permits. There might be a concern that altered lignin could lead to an increase in insect or disease susceptibility since lignin is often associated with resistance to insects and disease organisms

. Changes in the lignin biosynthetic pathway could modify interactions between plants and potential pathogens or insect pests on many levels: from the structural integrity of the whole plant to responses at the cellular level and at the interface between the plant and pot ential pest or beneficial microorganism (Funnell and Pederse n 2006 ). Pederson et al. ( 2005) surveyed the literature examining the impact of reduced lignin on plant fitness in a number of species and concluded that there are strong interactions among lignin reducing genes, the genetic background in which they are placed, and the environment in which the resulting plant lines are grown. Taken as a whole, it appears that reducing lignin content of crop plants can negatively impact their agricultural fitn ess. However, when evaluating individual events of reduced lignin, effect on agricultural fitness may be neutral or even positive (Pedersen et al, 2005, Funnell and Pedersen, 2006 ). To date t he results of field tests with th ese particular gene s have shown no differences in plant pest susceptibility. Growth measurements have indicated that trees had normal to a moderately reduced growth phenotype. The trees have also been visually inspected on a monthly bas i s for the presence of any insect and disease dam age and t hese observations found that there have been no differences in insect or diseases occurrence in the transgenic lines compared to the control trees. However, if during the tests there is evidence of increase disease or insect susceptibility, the a pplicant is required to report this to APHIS. The permittee is required to report any such unanticipated effects (including excessive mortality or morbidity) to APHIS under the terms of the permit

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

Genes for altered growth

T here are four genes identified in the permit application intended to alter growth characteristics of the trees that are all claimed as CBI. These are all genes derived from Eucalyptus and other plants. These genes do not alter the expression of a gene pr oduct or change plant metabolism in such a way that it would be expected to cause risk to the environment. These genes have been field tested in Eucalyptus or other plants and have not produced unanticipated phenotypes that would indicate there have been changes to plant metabolism leading to increase plant pest characteristics. Some of these genes being examined by ArborGen in the transgenic Eucalyptus have been associated with an increase in growth along with an increase in flowering or seed yield. If this were to occur in these transgenic Eucalyptus , ArborGen is required to report this as an unusual occurrence and must report this as a part of the conditions for field testing.

Non

- coding sequencesPage 21 of 91 The transgenic Eucalyptus also contains non

- coding re gulatory sequences 4 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 pes t 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 gall disease. F ollowing transformation, the bacteria are eliminated from the transformed plant tissue, and the DNA sequences introduced into the plant are maintained and inherited as any other genes of the plant cell.

Alteration in Weediness characteristics

– Potential of the E ngineered Eucalyptus to be I nvasive.

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

- 325

- 111r ( http://www.aphis.usda.gov/brs/aphisdocs/06_325111r_ea.pdf ) and permits 08

- 011

- 106rm, 08

- 014

- 101rm ( http://www.aphis.usda.gov/brs/aphisdocs/08_014101r_ea.pdf ) which are herein incorporated by reference.

T he hybrid Eucalyptus EH1 used to produce the transgenic trees has not been shown to be weedy or invasive in the U.S . An assessment has been conducted on the weediness or invasiveness potential of the hybrid by The University of Florida IFAS ( see the section above Biology of Eucalyptus and Status in the United States )

. In that assessment it was concluded that it is not like ly to be invasive and can be a recommended species for planting . None of the genes introduced into the Eucalyptus hybrid code for traits that would be expected to make the GE hybrids more weedy or invasive. The genes introduced to affect cold tolerance c ould make the engineered Eucalyptus more adapted to cold temperatures in the southern United States, but this trait alone would not impart invasive or weediness characteristics (Kolar and Lodge, 2001) to the engineered plants. The genes to affect the grow th of the trees would not be expected to increase invasiveness or weediness unless they also affect seed release or seed production

. The

trees c ould be considered weedy or invasive if they were to produce many seedlings that were readily spread away from the field test sites

. Where the non

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

4 A non

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

- coding sequences are strands of DNA such as promoters and terminators that drive the expression of the gene but do not result in the formation of a protein, which is the product of the
gene. Therefore promoters and termin ators, by themselves, cannot result in the production of a disease

causing entity.Page 22 of 91 15 years, there has been no indication that large numbers of seedlings are being produced and are becoming invasive from the commercial plantations ( L. Pearson, ArborGen pers.

comm. and L uis S ilva , International Paper Co mpany , Brazil , comment to docket APHIS

2008

- 0059)

. If the genes to alter growth were to have any e ffect on seed production, ArborGen is required to re port any unusual occ u r rences, such as increased flowering or seed production to APHIS during the field testing period.

Eucalyptus generally has difficulty establishing without human intervention, even in warmer climates. Eucalyptus is intolerant of shade or weedy competition. In order to successfully germinate and establish, Eucalyptus seed need contact with bare mineral soil and the removal of competing plants, either as a result of human intervention or naturally following a fire event (Meskimen and Francis 1990, Bell and Williams 1997) . The areas surrounding the field release sites would not be readily conducive to the establishment of seedlings because they are managed or unmanaged areas where other plant species are growing. So any seeds that attempted to germinate would face compet ition 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 a lso not be expected to affect seed production or vegetative reproduction capabilities. If the genes that affect growth have any effect of seed or flower production this must be reported to APHIS. The selectable marker gene, when used previously, did not contribute to weediness or invasive properties of the genetically engineered plant s. The gene for altered fertility should not contribute to w eediness or invasive properties and should reduce the ability of the trees to produce progeny

. None of the trai ts introduced into the transgenic Eucalyptus will compromise the ability to control these plants as weeds.

In addition, the supplemental permit conditions assigned to th i s permit will limit the reproductive capabilities of this GE Eucalyptu s outside the c onfined field trial locations

.
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 5 (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 greatl y reduced. In experiments conducted in Brazil and Alabama , the control self

- pollinated seed obtained from this genotype had abnormal morphology and failed to germinate (ArborGen, unpublished results). In recent field releases allowed to flower in Alabama and Florida, ArborGen has observed a low level of seed production. Controlled seed germination studies have been conducted with seed capsules collected over three years from field

5 When plants or trees “cross” the male pollen from one tree can pollinate (fertilize) the female ovule (or egg) on the same tree or on another tree. Unlike animals, some plants can fertilize themselves when the pollen and ovule are produced on the same tree. In this case all the trees are genetically identical (i.e. the same clone)(see footnote 1). Eucalyptus has a built

- in mechanism that will inhibit self

- fertilization. So these GE trees are likely to exhibit reduced fertility and reduced numbers of viable seed compared to fully sexually compatible Eucalyptus trees since they are genetically identical individuals.Page 23 of 91 trials that have been allowed to flower . Results have indicated that eith er no, or a very low number of viable seeds, are produced in the transgenic as well as in the control t rees , most likely as a result of limited self

- fertilization by pollen from the fertile control trees.

Regular volunteer monitoring of six different tri als over 2

- 5 years have further confirmed the absence of any seeded volunteers in or around the field tests . N o seedlings have been found established beneath the trees or in the surrounding areas (Appendix I).

Even if seed are produced in the test, several factors in the biology of Eucalyptus would limit the potential for seed dissemination. Although Eucalyptus seed is very light and small, it is not adapted to wind dispersal and consequently the dispersal of s eed is very limited, generally being confined within a radius of twice the tree or canopy height (approximately 50 meters for a 25 meter tall tree at harvest age) (Cremer 1977, Gill 1997, Linacre and Ades 2004) . Another consequence of the very small size of Eucalyptus seeds is that they have very limited reserves and ar e intolerant of shade or weedy competition. In order to successfully germinate and establish, Eucalyptus seed needs contact with bare mineral soil and lack of competition either as a result of human intervention or naturally following a fire event (Meskimen and Francis 1990, Bell and Williams 1997)

. Eucaly ptus plantations are typically established using rooted plantlets because of poor establishment using direct seeding methods. Even for the rooted plants, competition control is recommended for several months after planting to ensure good survival (Meskimen and Francis 1990) . Therefore

, the re 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 the permit will require that all volunteers be report ed to APHIS

, found and destroyed to prevent any spread of trees from the field release site.

Eucalyptus seeds do not have any dormancy barriers to prevent germination of volunteer seeds (Grose 1960, Wellington 1989, Gill 1997) and seed viability and storage of Eucalyptus seeds in soil are less than one year (Gill 1997) . The Eucalyptus species that have become invasive in California ( E. cam aldulensis and E. globulus ) are particularly adapted to a Mediterranean climate subject to summer fog, which is conducive to seed germination in those species ( Sellers 1910)

. This type of climate does not exist in the Southeastern U.S. In the event that any viable seeds are produced and are deposited on in an area conducive to germination from this field trial , these seeds would be expected to germinate within 7

– 14 days (Meskimen and Francis 1990)

. In accordance with the supplemental permit conditions for these permits, 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.Page 24 of 91 Possibility of Gene Flow Outside of the Field Test

Eucalyptus is adapted for insect pollination, with bees being the predominant vector (Pacheco et al. 1986, Pacheco 1987, House 1997) . Under ideal conditions of humidity and temperature, viable Eucalyptus pol len can only be found within approximately 100 meters from the edge of nearest tree stand (Peters et al. 1990, Linacre and Ades 2004)

. Pacheco ( 1987) verified that bees ( Apis spp.) are the most effective pollinators of Eucalyptus , with activity increasing up to 100 meters from the beehive, and decreasing after this distance. de Assis (1996) indicated that the minimum distance to prevent undesirable pollen contamination of seed producing a reas 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 Eucaly ptus species trials.

Transgenic tri a ls being conducted by the applicant under currently approved APHIS notifications and permits, of the same hybrid Eucalyptus variety EH1

, are planted adjacent to or within the proposed field test plot locations at the sites in Escambia and Baldwin Counties in Alabama

, Pearl River County in Mississippi; and Highlands Count y in Florida

, and Berkeley County in S outh Carolina

. The applicant has indicated that they

are not aware of any commercial plantings of sexually compa tible Eucalyptus species within 1000 meters of the proposed test plot location at any of these sites. Therefore, based upon the limited distance that viable pollen is likely to occur outside a tree stand, it is highly unlikely that gene flow would occur o utside of the confined field test sites at 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.

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 )

. These and other ornamentals are likely to be grown as specimen tre es (one or few grown together) and not part of large

- scale plantations.

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

exam ple, 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 removedPage 25 of 91 genetically from the genotype used in this field trial on the evolutionary scale and reside within different Series and Sections of genus Eucalyptus ( see http://plantnet.rbgsyd.nsw.gov.au/cgi

- bin/eucclass.pl?gn= Eucalyptus for details on sections and series in Eucalyptus ). Even among the closely related species of Eucalyptus

, hybridization rates are generally very low (Volker 1995) . The published literature supports the fact that natural hybridization among distantly re lated species within genus Eucalyptus is rare and hybrid inviability increases with increasing taxonomic distance between parents (Potts and Dungey 2004) . Where hybridization is possible, it often requires significant human intervention in directed breeding/crossing efforts. Potts and Dungey (2004) make reference to the high degree of inviability in F 1 hybrids (offspring).
I nviability of these offspring may be expressed at germination, in the nursery and even after planting in the field. Slower germination of hybrid seed often occurs, along with reduced survival of germinants in the nursery, and many seedlings have abnormal phenotypes. Griffin et al. ( 1988) surveyed natural and manipulated hybrids in the genus 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% of these producing trees that were worthy of further evaluation. To achieve the development of viable hybrids sometimes hundreds of hand pollinations must be made to find a viable hybrid that will grow normally. An example of the procedures required to make these wide

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

.

A further barrier to potential crossing between the transgenic trees with ornamental E. cinerea and other species is the expected differences in flowering times between species (Gore and Potts 1995, Potts et al. 2003) . For example, E. cinerea fl owers 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 s ummer.

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 could be grown in the area are unlikely to be compatible; 3) it is unlikely that flowering time in other species will overlap with the hybrid used in this test and; 4) hybrids, in the event that they could form, would be expected to be of very poor vigor

.
Possibility of Vegetative Propagation / Persistence Outside of the F ield T est

Overall impacts on the possibility of vegetative propagation and persistence outside the field test location would be similar to the N o A ction A lternative.

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.Page 26 of 91 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.

There could be a concern that seeds of the hybrid could be widely distributed by severe storms such as hurricanes or tornadoes. Th e Eucalyptus hybrid that is being grown in these proposed field tests produces mature capsules in February and seed fall is shortly after this. Therefore seed release is in late winter / early spring and well outside out of the normal hurricane season which occurs between June and November ( http://www.nhc.noaa.gov/ ). The p robability that other storm events might distribute seeds that could survive is extremely low. T here would have to be a number of events that would have to combine to have any consequence: seed happens to be shedding at the time a severe storm rolls throu gh a field test site, the seed happens to land on a suitable seed bed of bare soil with no weeds, the site is then not disturbed either by plowing or herbicide treatment, and moisture conditions are suitable for a germinating seedling to survive and grow. Research and experience have shown that long distance dispersal of Eucalyptus seeds and seedling establishment is very rare. Forsyth et al (2004) point out that “...in most parts of the world where Eucalyptus have invaded, they seldom spread considerable distances from planting sites, and their regeneration is frequently sporadic.” Richardson (1998) indicates “Eucalypts are also represented on many national or regional weed lists from other parts of the world. Despite this, they have not been nearly as successful in invading alien environments as other widely planted trees such as pines and legumes. Many eucalypts produce large quantities of seeds, so their lack of success as invaders is rather puzzling.” This could likely be due to the fact that Eucalyptus seeds are very small, have very limited reserves and are in tolerant of shade or weedy competition. In order to successfully germinate and establish, Eucalyptus seed needs contact with bare mineral soil and lack of competition either as a result of human intervention or naturally following a fire event (Meskimen and Francis 1990, Bell and Williams 1997)

. In Br azil where E. grandis, E. urophylla and their hybrids have been grown since the 1960’s and are now planted on several thousand hectares, there is no evidence that wind borne seeds are spreading the trees beyond managed plantations. Over 70,000 hectares of the hybrid has been planted extensively by International Paper, who developed EH1, with no evidence of invasiveness (Luis Silva, International Paper Company, Brazil

– comment to the docket to the EA for permits 08

- 011

- 106rm and 08

- 014

- 101rm ) . In these en vironments Eucalyptus obviously does not behave like other windblown seeds of grasses, for example, which can be pioneering species. Therefore the likelihood of significant escape and successful establishment of seeds from the field test sites due to stor ms is negligible. APHIS has determined that the field test will be confined and storms are not expected to compromise this confinement.Page 27 of 91 Potential of the Eucalyptus 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 evid ence to date that the untransformed clone of the Eucalyptus hybrid in these permits is weedy or invasive in the U.S. (L. Pearson, ArborGen pers. comm.) None of the genes introduced into Eucalyptus code for traits that would be expected to make the plants more weedy or invasive. The genes introduced to affect cold tolerance could make the engineered Eucalyptus more adapted to cold temperatures in the southern United States, but this trait in and of itself would not impart invasive or weediness characterist ics (Kolar and Lodge 2001) to the engineered plants.

There may also be a concern that adding genes that affect growth could make the engineered Eucalyptus more invasive if they increased the growth rate . However, simply increasing growth without affecting other traits would not lead to increased invasiveness. If the genes that affect growth were to lead to an increase in seed production or release of more seed then this could have an impact on its inva sive or weedy potential. If there were to be a significant increase in the number of flowers or seeds produced in these tests, this would be seen as an unusual occurrence and must be reported to APHIS as a part of the conditions of the permit. An increas e in seed production would not necessarily lead to an increase in invasiveness unless the seeds were to find a suitable environment for germination.

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

incompatibility

. In accordance with supplemental permit conditions

, m onitoring of seed set and se edling viability will be required by the applicant during the se confined field tests so that the extent of seed production and seedling establishment can be determined. In addition

, altered fertility leading to the lack of viable pollen development has be en engineered into the trees. The addition of the cold

- tolerance genes are not expected to affect reproductive biology such as seed production or vegetative reproduction capabilities. The gene introduced to alter lignin biosynthesis would also not be exp ected to affect seed production or vegetative reproduction capabilities. If the genes introduced

to alter growth were to lead to an increase in seed production this would be reported to APHIS as a part of the conditions of the permit. The selectable mark er gene, when used previously, did not contribute to weediness or invasive properties of the genetically engineered plants. The gene for altered fertility should not contribute to weediness or invasive properties and should reduce the ability of the tree to produce progeny.

In unlikely event that seeds are formed and seedlings are produced, none of the traits introduced into the transgenic Eucalyptus will compromise the ability to control these plants so spread of seedlings and trees from the field test sites is highly unlikely.Page 28 of 91 Volunteers can be readily identified and controlled. Should any hybridization and viable seed production occur, in accordance with supplemental permit conditions, the monitorin g 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

uproo ting or by spraying with EPA approved herbicides (e.g., glyphosate or other herbicides to which these trees are susceptible) and APHIS will be notified of their

occurrence.

Impact on Existing Agricultural Practices

Overall impacts on existing agricultura l practices would be similar to the N o A ction A lternative.

The establishment and growth of these small confined field tests will not have any impact

on existing agricultural practices because they are solely for research purposes. Current agricultural practices will essentially remain unchanged

. As identified by the applicant,

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

Potential Impacts to Wildlife

Native floral communities

The field sites in the permit applications are located in Baldwin and Escambia C ounties, Alabama ; Highlands C ount y , Florida ; Pearl River County, Mississippi; and Berkeley and Dorchester Count ies , South Carolina

. The se sites are a mixture of pasture, crop lands and forested areas. The two locations in South Carolina are holding areas for plants in containers

. In the unlikely event that viable 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 competing plants, either as a result of human intervention or naturally following a fire event. With the exception of the field test area, the agricultural areas

surrou nding the field sites are not conducive to the establishment of Eucalyptus . The surrounding agricultural and tree crops would provide a shady canopy and competition for light and other resources that would impede seedling establishment of Eucalyptus

. The inhospitable conditions for seed germination, in combination with the supplemental permit conditions established for these permits , will make the establishment of Eucalyptus in the surrounding area highly unlikely

. Should any hybridization and viable

see d 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

trans genic seedlings are observed they will be destroyed by the applicant either by uprooting or by spraying with EPA approved herbicides (e.g., glyphosate or otherPage 29 of 91 herbicides to which these trees are susceptible) and APHIS will be notified of their

occurrence.

Terrestrial animals

The most likely animals to encounter the transgenic Eucalyptus trees in this 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 p roducts 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.

Potential I mpacts by F ire

Overall impacts of fire would be similar to the N o A ction A lternative.

Most Eucalyptus communities in Australia have evolved in the presence of periodic fire, and fires are an integral part of the Eucalyptus ecosystem (Ashton 1981

, Gill 1997)

. Many Eucalyptus species are known to be highly flammable and depending upon the species, location and age

, they can be very resistant or susceptible to fire damage (Gill 1997)

. Eucalyptus fires can be very hot and move rapidly. The bark cat ches fire readily, and deciduous bark streamers tend to carry fire into the canopy and to disseminate fire ahead of the main front (Ashton 1981

, Skolmen and Ledig 1990

, Esser 1993) . Other features of Eucalyptus that promote fire spread include heavy litter fall, flammable oils in the foliage, and open crowns bearing p endulous branches, which encourages maximum updraft (Esser 1993, Gill 1997)

. In the U.S., there have been reports of significant fires in California and many have been blamed on the widespread planting of Eucalyptus

. F uel buildup occurs very rapidly in unmanaged bluegum Eucalyptus stands in California which has led to significant forest fires. The buildup of litter and dead grass are primary responsible for the spread of these fires (see Santos: http://wwwlibrary.csustan.edu/bsantos/euctoc.htm )

. ) . The Forest Service indicates th at f uel reduction programs and the establishment of firebreaks in Eucalyptus plantings can reduce wildfire hazard (Esser 1993)

.

There is a historical risk of forest fire in the southeastern U .S., however , the probability that these confined field t rials will increase the risk and severity of forest fires in theirPage 30 of 91 res pective locations is very small. T hese new plantings are small (none greater than 7.7

acres ) and they will be managed by the applicant to prevent litter buildup. Under all of the Eucalyptus permits authorized for planting by ArborGen, no location is plant ed with more than 20 acres. Authorization of this permit will not increase that acreage at any release location

. 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 to H uman H ealth

Overall impacts on human health would be similar to the N o A ction A lternative.

During the comment period for the EA prepared for permit 06

- 325

- 111r, there were concerns expressed that Eucalyptus field tests could be a source of Cryptococcus neoformans gattii. APHIS conducted a thorough review of C. neoformans gattii and the possibility that the field tests could pose a risk to human health ( (APHIS 2004) EA and response to comments for permit 06

- 325

- 111r ( http://www.aphis.usda.gov/brs/aphisdocs /06_325111r_ea.pdf ) ) and concluded that the field trial would not lead to a higher incidence of C. gattii in the U.S. and therefore should not pose an unnecessary risk to human or animal health.

C. neoformans gattii is a fungal pathogen that is hosted on a variety of species of Eucalyptus as well as other tree species ( Upton et al. 2007 ) . It causes systemic fungal infections in humans, leading to fungal meningitis and death ( Datta et al., 2009 )

. C. neoformans gatti i 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 Califor nia (Datta e al, 2009)

. C. neoformans gattii infections are found particularly in AIDS patients due to their weak immune systems ( Chaturvedi et al, 2005)

. Infections with this fungus are rare in those with fully functioning immune systems. For this reason, C. neoformans gattii is sometimes referred to as an opportunistic fungus. There was an outbreak of cryptococcal disease on the eastern portion of Vancouver Island, British Columbia in 1999 (Datta et al., 2009) . The disease was previously only known to o ccur in tropical or semi

- tropical climates.

It is unlikely that the trees that are the subject of the proposed field release can be a source that might introduce the pathogen into the U.S because the trees were derived from sterile tissue culture lines . The transgenic Eucalyptus started as a hybrid developed in Brazil. In Brazil, small pieces of the tissue derived from the hybrid were put into sterile tissue culture and sent to New Zealand for transformation. The transformed lines were sent to the U.S a s sterile tissue culture lines that were inspected by APHIS Plant Protection and Quarantine inspectors prior to entry into the U.S. C. gattii spores readily germinate in culture. If C. gattii spores were present in the tissue culture, contamination, would be evident and the affected lines would be discarded prior to regeneration of trees for introduction into the environment. Another reason it is unlikely that spores could bePage 31 of 91 or were ever present in the hybrid lines used in the proposed confined field tria l 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 tissue generated during tissue culture. Because the trees were derived from tissues that are not known to be a source of the spores and were derived from sterile tissue culture lines that by all appearances were free from any fungal contamination, there is a negligible risk that the hybrid trees used in the field trial could be or have been contaminated with C. gattii

.
The risk that these field trials will result in a higher incidence of the fungus in the U .S. and thereby pose a risk to human health is considered to be negligible for the following reasons. First, there is not a clear association between E. grandis or E. urophylla and C. gattii. Second, there is no reason to believe that the genetic modifica tion 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 hav e 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 N o A ction A lternative.

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 gen es 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/GuidanceDocumePage 32 of 91 nts/Biotechnology/ucm096135.htm

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



### C

. The Degree to Which the Possible Effects are Highly Un certain 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 Unit ed States (Farley et al. 2005a)

. Since large plantings of Eucalyptus have not been grown in many parts of the southeastern US (other than southern Florida) the potential impacts of such plantings on hydrology are unknown. In preparing the EA for permits 08

- 011

- 106rm and 08

- 014

- 101rm APHIS requested additional information on potential impacts of hydrology from ArborGen and also consulted wit h the USDA Forest Service to assess the potential impacts of planting Eucalyptus on hydrology

. The additional information supplied by ArborGen and the Forest Service are included as Appendices II and III. The document supplied by the Forest Service represents only their opinion on the potential impacts of these field tests on hydrology and does not represent the position of the USDA on the pros and cons of deploying Eucalyptus as a biofuel, bioenergy or fib er 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 efficient at using water. It can produce more bio mass per unit water consumed than native southeastern pines; however, their extremely rapid biomass production has proportionally higher transpiration costs and therefore greater

water use. The F orest Service ha s estimated that a mature Eucalyptus plantat ion growing in southwest Georgia could potentially transpire 882 mm per year , exceeding all other forest types on average by a factor of 2.5. Eucalyptus transpiration could exceed that of pine plantations by a factor of 1.6, and previous pasture land by a factor of 3.5. The comparison with agricultural crops is more variable where Eucalyptus transpiration may be greater or lesser than that of crop plants depending on the crop, the growing season, and the management practices.

Eucalyptus has a dimorphic ro oting pattern which means that it has surface roots that draw water from the surface as well as deep roots which draw water from deep within the soil. The mean maximum rooting depth for Eucalyptus ranges between 15 and 40

m eters
, which is a characteristi c of a dimorphic rooting pattern (Canadell et al. 1996a)

. In contrast, mean maximum rooting depths of pine plantation ( P. taeda and P. elliottii ) and grass species are 3 m eters and 2.6 m eters , respectively (Canadell et al. 1996a)

. According to the Forest Service, 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 evapotranspiration rates that exceed pr ecipitation input, as have been reported for this species in other locations (Calder et al. 1997a)

.Page 33 of 91 Recent research suggests that Eucalyptus plantations would reduce stream flow more than pine plantations, and could potentially eliminate low flows. In a review of more than 20 catchment 6 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 f lows). Elimination of low stream flows could have important ramifications for threatened and endangered aquatic species, such as the gulf strain striped bass, and species of endemic freshwater mussels (Golladay et al. 2004a, Couch and McDowell 2006b)

.
Due to a lack of available data in the southeastern U.S. on planting Eucalyptus , it is difficult to determine the si gnificance 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 n egligible impacts at the watershed scale.

The field test sites requested in the permit application are well dispersed and are limited in size (none are greater than 7.7 acres) . Under all of the Eucalyptus permits authorized for planting by ArborGen, no l ocation is planted with more than 20 acres and it is anticipated that they are not likely to have significant impacts on hydrology. Authoriza t ion of this permit will not increase that acreage. At the request of APHIS, ArborGen has supplied data indicati ng the maximum size of each of the plantings at each site , the individual watersheds where the plantings occur, the area of the watershed, how much of the watershed will be occupied by the field tests, the location of the closest primary and secondary stre ams, and the location of any critical habitat for Federally listed threatened and endangered species within the watershed.

Using the 8 digit HUC (Hydrologic Unit Code) as the Watershed to be analyzed, the data provided by ArborGen show that none of the sites in the permit application occupy more than 0.0 05 % 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 prox imity to habitats that could be impacted. There are no nearby threatened or endangered plant species that could be impacted by hydrological effects (see also Appendix IV). Any effects would be very localized on existing nearby agricultural and forestry p lantings. Therefore APHIS concludes that while the effects on hydrology, including the watershed and aquifers, are unknown and uncertain for very large plantings of Eucalyptus , these small

- scale field tests are unlikely to have any significant negative im pacts on hydrology and on native flora and fauna.

6 A catchment or drainage basin is an extent of land where water from rain or snow

- melt drains downhill into a body of water, such as a river

, lake

, reservoir

, estuary
, wetland

, sea or ocean . The drainage basin includes both the streams and rivers that convey the water as well as the lan d surfaces from which water drains into those channels, and is separated from adjacent basins by a drainage divide

.Page 34 of 91 Potential A llelopathic E ffects of Eucalyptus

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: http://www.international

- allelopathy

society.org/main/home/main.php ). Allelochemicals from plants are released into the environment by exudation from roots, leaching from stems and leaves, or decomposition of plant material. Allelopathy can have both negative and positive impacts on the environment (Eljarrat and Barcel 

### ó 2001, Xuan et al. 2005, Kohli et al. 2006) . There has been inc reased research activity 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 alf alfa (Nandal et al. 1994, Ferguson and Rathinasabapathi 2003, Reigosa and Gonzáles 2006, Mallik 2008)

.
There have been extensive studies conducted on allelopathy in Eucalyptus and there are several comprehensive reports and review articles on this genus (Ong 1993, Sunder 1995) (Nandal et al. 1994, Davidson 1995, White 1995)

. Eucalyptus species are known to produce chemi cal compounds that are required by the plant for defense against herbivores and pathogens. There are several studies in the literature that demonstrate the negative, positive and neutral allelopathic interaction of Eucalyptus species and their hybrids wit h other crop plants (Sanginga and Swi ft 1992

, Khan et al. 2004

, Espinosa

- Garcia et al. 2008) . These interactions vary greatly depending upon the crop species and conditions under which they are grown. There is inconclusive data as to whether these compounds produced by Eucalyptus are exclusively responsible for allelopathic influence on understo ry 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 grow th in potted plants. These laboratory bioassays and pot culture studies may or may not be applicable to field conditions. The perceived allelopathic effects observed in the field on growth of understory or adjacent intercropped food crops could also resu lt 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 effect s 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 wh ereas higher concentrations adversely affected germination and seedling growth of some of the wheat varieties tested. In a field experiment, the performance of all ten wheat varieties was also evaluated under four different poplar spacings in an agri

silv iculture system. Although the grain yield of wheat varieties was significantly lower under all spacings of poplar compared to controls, yields increased significantly withPage 35 of 91 increased spacing of poplar, possibly due to reduced competition for light and nutri ents. However, no correlation was found between the laboratory bioassay using leaf extracts and the field studies.

In a recent study, the allelopathic interaction of Eucalyptus grandis, E. urophylla and E. grandis x urophylla on the germination and early growth of four annual crops (maize, bean, watermelon and squash) was investigated (Espinosa

- Garcia et al. 2008) . Soil samples were collected from different soil horizons and at varying distances from Eucalyptus trees growing at the plantation edge and used for gr owth studies in pots. The dried soil samples used for growth studies were also analyzed for total soluble phenolics present in the soil. The study showed that soil samples from different plantations had differential effects ranging from no effect, to sli ghtly inhibitory, to a stimulatory effect on germination and radicle 7 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 water melon but had a stimulatory effect on squash. The soil from E. grandis plantations had an inhibitory effect on squash. The total soluble phenolics varied in different soil samples but did not explain the differential effects on the test crops. The authors concluded that soil samples collected from plantations of Eucalyptus species contained allelochemicals that affected germination and early growth of some annual crops but such effects could be avoided by planting crops at a distance of 15 meters away from the edge of plantations.

Even though the Eucalyptus under this permit could demonstrate allelopathic properties, the presence of any allelochemicals is not going to make the Eucalyptus planted under these permits more invasive or present a plant pest risk . S ince all these field tests are confined and limited in size, any allelopathic effects should be small. As a standard silvicultural practice, herbicides will also be used within the field test sites and any of their effects on understory vegetation wil l be as severe or more severe than any allelopathic effects. In the future, should any negative allelopathic or other competitive interactions be observed under field conditions outside of the immediate field tests sites, these could be mitigated by adjus ting the tree spacing, irrigation and fertilization practices or by planting the field tests at least 15 meters away from any agronomic crops or sensitive areas. Any unusual observations at the field test sites are to be reported to APHIS under the supple mental permit conditions of the permit; including any indications of allelopathic effects.



### D . Cumulative Effects

As identified in the permit application, t he field test sites in this permit application have been in agricultural or forest research, or in agricultural production or forest tree plantations for 5 to 15 years. Therefore the land has been in continuous agricultural or forest tree production at all the proposed field sites for at least 5 years prior to th ese proposed release s and it is r easonably foreseeable that if the permit were not issued that the sites would continue to be maintained under similar agriculture or forestry

7 The radicle is the first part of a seedling (a growing plant embryo ) to emerge from the seed during the process of germination . It is an embryonic root.Page 36 of 91 production. F ive of these six locations identified in the permit application currently have active APHIS permits ( 08

- 011

- 106rm, 08

- 014

- 101rm, 09

- 070

- 10rm, 10

- 112

- 101r , and 11

- 041

- 101rm ) for environmental release of GE Eucalyptus hybrids in Alabama, Florida, Mississippi, and South Carolina. In respect to the five locations that currently have active APHIS permits; th e new permit application ( 11

- 052

- 101rm) does not expand the field sites beyond what is current authorized by APHIS or density (number) of trees that could be planted. With regard to the Dorchester County, South Carolina site, this location has previously been listed as a holding area for GE trees in previous permits/notifications. This new permit, which allows the release of GE Eucalyptus at this location does not change any conditions applied to that site. The Dorchester County site will remain a holdin g area for GE trees in containers where flowering is not allowed.

As identified in the new permit application, GE Eucalyptus trees with the new constructs would be released at the 5 existing locations in addition to the trees currently approved by APHIS for environmental release. The new permit application does not expa nd the number of trees or total acreage that could be planted at any of the 5 confined release sites currently approved by APHIS. It is also reasonably foreseeable that the applicant may request to further extend the permit for this environmental release for additional years beyond the 3 years indicated in the pending permit to observe the growth of these trees to maturity. Moreover, APHIS has received a petition for a determination of nonregulated status of a subset of these transgenic Eucalyptus trees, however, the environmental effects of that petition will be analyzed in a separate NEPA document. The temporary change from agricultural crops to a tree crop may result in a temporary change in resident animal and plant species, but after harvest and term ination of the proposed permits, it is reasonably foreseeable that the land will return to agriculture or be replanted to tree production or research. At the end of the field test, transgenic plant material will be removed from the test site and/or destro yed in accordance with supplemental permit conditions established for these permits. The effect of propagation,

cross

- breeding, and invasiveness on listed species or native ecosystems was not considered a potential impact because of the supplemental permi t conditions that prevents these events from occurring. Fire risk to TES was not considered a potential impact because the small size of the field trial combined with the unlikelihood of TES presence within the proposed test area. Likewise, the effect o n hydrology was not considered an impact because of the small size of the field trial combined with the distance from water sources that listed species rely upon. In addition, the small size of the field plots and the distance between test sites spread o ver thousands of miles indicate no cumulative effects would result from APHIS issuing the proposed field release permits. Therefore the only past, present, and reasonably foreseeable actions associated with the locations for the proposed releases under pe rmit are those related to agricultural or forest tree production. Based on the analysis provided in the EA, APHIS has determined that there are no past, present, or reasonably foreseeable actions that would aggregate with effects of the proposed action to create cumulative impacts or reduce the long

- term productivity or sustainability of any of the resources (soil, water, ecosystem quality, biodiversity, etc.) associated with the release sites or the ecosystem in which they are situated. No resources will be significantly impacted due to cumulative impacts resulting from the proposed action.Page 37 of 91 

### E. Risks to Threatened and Endangered Species

APHIS analyzed the potential for effects from the preferred alternative of this EA on federally listed threatened an d 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 t hat the proposed environmental release and interstate movement of GE Eucalyptus trees will have no effect on federally listed threatened or endangered species or species proposed for listing, an d no effect on designated critical habitat or habitat proposed for designation. Consequently, consultation with the United States Fish and Wildlife Service is not required for the action described in the preferred alternative of this EA . Appendix IV includes the BRS analysis of threatened and endangered species in the areas of the field releases.



### F. Impacts on Unique Characteristics of Protected Areas

Appendix V includes the BRS analysis of the release locations for their p roximity to State and Federal p rotected a reas. Based on this analysis

, i ssuance of the permit to allow flowering of these additional constructs i s not expected to impact unique characteristics of protected areas such as park lands, wetlands, wild and scenic areas, or ecologically critical areas.

None of the release sites are within protected areas. Common forestry practices that would be carried out in the cultivation of the engineered Eucalyptus are not expected to deviate from current practices. The trees will be grown on agricultural land currently suitable for the fi eld testing of forest trees. There are no proposed major ground disturbances; no new physical destruction or damage to property; no alterations of property, wildlife habitat, or landscapes; and no prescribed sale, lease, or transfer of ownership of any pr operty. This action is limited to issuance of the permit. This action would not convert land use to nonagricultural use. Standard forestry or agricultural practices for land preparation, planting, irrigation, and harvesting of plants would be used on th e permitted sites, including the use of EPA registered pesticides. Applicant’s adherence to EPA label use restrictions for all pesticides will mitigate potential impacts to the human environment.



### G . Other Considerations

Consideration of Executive Orders, Standards and Treaties 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

co nduct their programs, policies, and activities that substantially affect human health or the environment in a manner so as not to exclude persons and populations fromPage 38 of 91 participation in or benefiting from such programs. It also enforces existing statutes to prevent minority and low

- income communities from being subjected to disproportionately high and adverse human health or environmental effects.

EO 13045, “Protection of Children from Environmental Health Risks and Safety Risks ,” acknowledges that children may suffer disproportionately from environmental health and safety risks because of their developmental stage, greater metabolic activity levels, and behavior patterns, as compared to adults. The EO (to the extent permitted by law and consistent with the agency’s mission) requires each Federal agency to identify, assess, and address environmental health risks and safety risks that may disproportionately affect children.

Each alternative was analyzed with respect to the above EO 12898 and 13045. The hum an health and environmental impacts of the action alternatives are presented in Section V of this EA. No human health or environmental effects were identified for any of the action alternatives that would have a disproportionate adverse effect or that wou ld exclude a particular group of persons or populations, including minority and low

- income populations, or children, from expected benefits.

EO 13112, “Invasive Species” , states that federal agencies take action to prevent the introduction of invasive species and provide for their control and to minimize the economic, ecological, and human health impacts that invasive species cause. As presented in Section V of this EA, 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 impact 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, collect ing samples and eventual harvest of the treesPage 39 of 91 should have no adverse impact on migratory birds since they would not be expected to nest or permanently inhabit these types of field test sites

.
Consistency of Proposal with other Environmental Requirements:

The proposal is believed to be consistent with other environmental requirements. This environmental assessment was prepared in accordance with: (1) The National Environmental Policy Act of 1969 (NEPA), as amended (42 U.S.C 4321 et seq .); (2) regulations o f the Council on Environmental Quality for implementing the procedural provisions of NEPA (40 CFR parts 1500

- 1508); (3) USDA regulations implementing NEPA (7 CFR part 1b); and (4) APHIS’ NEPA Implementing Procedures (7 CFR part 372).



### H . Conclusion

As out lined under the Purpose and Need section s of this document, this EA was prepared by APHIS to evaluate the potential impacts to the human environment resulting from the inclusion of new constructs in field test releases of flowering Eucalyptus

, which could potentially lead to a lack of confinement of the field tests and other impacts to the environment. APHIS has evaluated the permit application to determine whether the environmental release and interstate movement , with appropriate conditions imposed by AP HIS , can be carried out while preventing the dissemination and establishment of plant pests. After preparing this EA

, APHIS has concluded that because there is no increase in the number of sites or acreage where trees will be allowed to reach maturity and flower,

o ver those already authorized by APHIS to flower under permits 08

- 011

- 106rm, 08

- 014

- 101rm, 10

- 112

- 101r and 11

- 201

- 103r

, there is no substantially greater risk of loss of confinement and risk to the environment. Based on the analysis and informati on provided in th is EA and supporting permit application , t he new genes that are engineered into the trees should also not pose any greater risk of loss of confinement and risk to the environment

. The addition of new genes to increase cold tolerance, alte r lignin and growth should not compromise the ability of these to remain confined field tests. Therefore, APHIS concludes that the releases will remain a s confined field test s and that the release and interstate movement of genetically engineered trees will not pose a significant plant pest risk. In addition, n o threatened and endangered species or critical habi t at w ould be impacted by allowing the trees reach maturity and flower.



## VI. Listing of Agencies and Persons Consul ted

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 Fi sh 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 OfficePage 40 of 91 Caroline Stahaller

- US Fish and Wildlife Service

- Panama City, Florida Field Office Brad Rick

- US Fish and Wildlife Service

- Vero Beach, Florida Field Office Candice Martino

- US Fish and Wildlife Service

- Jacksonville, F lorida Field OfficePage 41 of 91 

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- 570. Pino, M. T., J. S. Skinner, E. J. Pa rk, Z. Jeknić, H. P.M., M. F. Thomashow, and T. H. Chen. 2007. CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis . Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield. Plant Biotechnol J. 5 :591

- 604.Page 45 of 91 Potts, B. M., R. C. Barbour, A. B. Hingston, and V. R.E. 2003. Turner Review: the risk of genetic pollution of native Eucalyptus gene pools. Australian J. Botany 51 :1

- 25. Potts, B. M. and H. S. Dungey. 2004. Interspecific hybridization of Eucalyptus: Key issues for breeders and geneticists. New Forest 27 :115

- 138. Pound, L. M., M. A. B. Wallwork, B. M. Potts, and M. Sedgley. 2002. Self

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

- 372. Reigosa, M. J. and L. Gonzáles. 2006. Forest Ecosystems and Allelopathy. Pages 451

- 463 in R. Reigosa, N. Pedrol, and L. Gonzáles, editors. Allelopathy: A Physiological Process with Ecological Impli cations. Springer. Riechmann, J. L. and E. M. Meyerowitz. 1998. The AP2/EREBP family of plant transcription factors. . Biol Chem 379 :633

- 646. Richardson, A. D. 1998. Forestry Trees as Invasive Aliens. Conservation Biology 12 : 18

- 26. Rockwood, D. L., D. R. Carter, and J. A. Stricker. 2004. Commercial Tree Crops for Phosphate Mine Lands, Fourth Year Cumulative Progress Report

- Prepared for Florida Institute of Phosphate Research FIPR Project Number 99

- 03

- 141R. University of Florida. Sanginga, N. and M. J. Swift. 1992. Nutritional effects of Eucalyptus litter on the growth of maize ( Zea mays ). Agriculture, Ecosystems and Environment 41 :55

- 65. Sellers, C. H. (1910). Eucalyptus: its history, growth, and utilization , A.J. Johnston

- Original from Harvard Univer sity

. 93 pp. Skolmen, R. G. and F. T. Ledig. 1990 . Bluegum Eucalyptus. in R. M. Burns and B. H.
Honkala , editors. Silvics of North America: Volume 2. Hardwoods. Agriculture Handbook 654. U.S. Department of Agriculture, Forest Service, Washington, DC.

. Stricker, J., D. L. Rockwood, S. A. Segrest, G. R. Alker, R. M. Prine, and D. R. Carter. 2000. Short rotation woody crops for Florida. Pages 15

- 23 in Proc. 3rd Biennial Short Rotation Woody Crops Operations Working Group Conference, October 10

- 13, 2000, Sy racuse, NY. Sunder, S. 1995. T he Ecological, Economic and Social Effects of Eucalyptus

. in Proceedings of the Regional Expert Consultation on Eucalyptus . FAO Regional Office for Asia and the Pacific, Bangkok. Upton, A.

, J. A. Fraser

, S. E. Kidd

, C. Bretz

, K. H. Bartlett

, J. Heitman , and K. A. Marr

. 2007. First Contemporary Case of Human Infection with Cryptococcus gattii in Pugent Sound: Evidence for Spread of the Vancouver Island Outbreak. . Journal of Clinical Microbiology 45 : 3086

- 3088

. Volker, P. W. 199 5. Evaluation of Eucalyptus nitens x globulus for commercial forestry. Pages 222

- 225 in Eucalyptus Plantations: Improving Fiber Yield and Quality, Proc CRCTHF

- IUFRO Conference. CRC for Temperate Hardwood Forestry, Hobart. Australia. 19

- 24 Feb Wellington, A. B. 1989. Seedling regeneration and population dynamics of eucalyptus. Pages 155

- 167 in J. C. Noble and R. A. Bradstock, editors. Mediterranean Landscapes in Australia: Mallee Ecosystems and their Management. CSIRO, Melbourne.Page 46 of 91 White, K. J. 19 95. Silviculture of Eucalyptus Plantings

– Learning from the Region (Australia). in Proceedings of the Regional Expert Consultation on Eucalyptus

. FAO Regional Office for Asia and the Pacific, Bangkok. Wood, D., J. Setubal, R. Kaul, D. Monks, J. Kitajima, V . Okura, Y. Zhou, L. Chen, G. Wood, A. J. N., L. Woo, Y. Chen, I. Paulsen, J. Eisen, P. Karp, S. Bovee, D., P. Chapman, J. Clendenning, G. Deatherage, W. Gillet, C. Grant, T. Kutyavin, R. Levy, M. Li, E. McClelland, C. Saenphimmachak, Z. Wu, P. Romero, D. Gordon, S. Zhang, H. Yoo, Y. Tao, P. Biddle, M. Jung, W. Krespan, M. Perry, B. Gordon

Kamm, L. Liao, S. Kim, C. Hendrick, Z. Zhao, M. Dolan, F. Chumley, S. Tingey, J. Tomb, M. Gordon, M. Olson, and E. Nester. 2001. The Genome of the Natural Genetic Enginee r Agrobacterium tumefaciens C58. Science 294: 2317

- 2323. Xuan, T. D., S. Tawata, T. D. Khanh, and I. M. Chung. 2005. Decomposition of Allelopathic Plants in Soil. J. Agronomy and Crop Science 191 :162

- 171. Yamaguchi

- Shinozaki, K. and K. Shinozaki. 1993. Char acterization of the expression of a desiccation

- responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol. Gen. Genet. 23 :331

- 340. Yamaguchi

- Shinozaki, K. and K. Shinozaki. 1994. A novel cis

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- 30.Page 47 of 91 

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

The applicant has been authorized by APHIS to allow transgenic Eucalyptus trees flower under APHIS permits 06

- 325

- 111r (renewed as 10

- 112

- 101r ); 08

- 151

- 101r (now covered under 08

- 014

- 101rm); 08

- 011

- 106

- rm and 08

- 014

- 101rm. The following is a summary of information that has been collected by the applicant and provided to APHIS as part of their annual report ing requirements under the existing per mits that allow flowering.

All transgenic and non

- transgenic trees in field tests covered under Permit 06

- 325

- 111r (renewed as 10

- 112

- 101r ), spanning 6.2 acres in Baldwin County, Alabama produced flowers in the late summer months of years 2007 to 2010. The field test from Permit 08

- 151

- 101r (now covered under 08

- 014

- 101rm) spanning 1.4 acres in Highlands County, Florida produced mature flowers on all transgenic and non

- transgenic trees in the late summer months of 2008, to 2010. Three field tests flow ered in the late summer months of 2010 under Permit 08

- 011

- 106

- rm; the field test spanning 0.75 acre in Baldwin County, Alabama; the field test spanning 0.2 acres in Evans County, Georgia; and the field test spanning 0.7 acre in Jasper, County Texas. A fie ld test covered under permit 08

- 014

- 101rm in Gadsden County, Florida spanning 0.2 acres flowered in 2010 as well. Where flowering occurred in these tests, several hundred to a few thousand flowers were estimated on each tree. For the field tests i n Alabam a , Georgia, and Texas that flowered in summer of 2010; the transgenic trees produced more flowers per tree compared to the

non

- transgenic controls as a result of significant cold damage to non

- transgenic trees . In Florida, there was no difference in the nu mber of flowers produced by both transgenic and non

- transgenic control trees of the same parental genotype. O bservations from the replicated field study conducted in Alabama under permit 06

- 325

- 111r (renewed as 10

- 112

- 101r ) and Florida under permit 08

- 151

- 101r (now covered under 08

- 014

- 101rm) confirm ed that cold tolerant trans lines grown in these field test did not produce any pollen. The results to date have shown that the barnase gene cassette that has been engineered into these trees is effective at pre venting pollen formation.

Mature (but not yet opened) capsules have been collected from non

- transgenic and transgenic trees in the following field tests: Baldwin County, Alabama under Permit 06

- 325

- 111r (renewed as 10

- 112

- 101r ) in January 2008 and 2009; Highlands County, Florida under Permit 08

- 151

- 101r (now covered under 08

- 014

- 101rm) in January 2009 and 2010; Evans County, Georgia; and Jasper County, Texas under Permit 08

- 011

- 106rm in January 2011.

Replicate capsule samples were collected from transg enic trees and non

- transgenic controls in the field tests. For the January 2008 collection only from Baldwin County, AL (Permit 06

- 325

- 111r

- renewed as 10

- 112

- 101r ), capsules from a subset of transgenic trees plus non

- transgenic controls were dried in the laboratory and allowed to open to evaluate the presence of seed or seed like structures in the capsules. Approximately 100 capsules for each of the two replicate samples were analyzed. Microscopic examination of the material inside the capsules did not show any seed or seed like structures in capsules of either non

- transgenic or transgenic lines. Controlled germination studies of the material extracted from the capsules did not produce any germinating seeds.Page 48 of 91 Mature s eed capsules , prior to opening, w ere sampled from select trees in three field tests in Baldwin County, Alabama (Permit 06

- 325

- 111r

- renewed as 10

- 112

- 101r ); and trees from the single field test in Highlands County, Florida (Permit 08

- 151

- 101r

- now covered under 08

- 014

- 101rm) in early Ma rch 2009

. Samples consisted of approximately 70 to 100 capsules collected from different positions in the crown. T he capsules were returned to ArborGen’s greenhouse facility where they were dried and contents of the capsules were extracted and stored at 4°C. A controlled germination test was conducted using a pproximately 0.1 g of the extracted contents of each sample spread on moist filter paper in a standard Petri dish. Open pollinated seed of EH1 obtained from Brazil were germinated as control seedlings for comparison.

Of the samples collected and analyzed from trees in Baldwin County, AL, approximately 4% of the samples showed a low level of germination while approximately 83% of the samples collected from trees in the field trial in H ighlands County, FL, showed germination, including samples from both transgenic and non

- transgenic control trees. For both transgenic and non

- transgenic tree samples analyzed, the applicant observed 2

- 8 seedlings for each 0.1g sample plated for germinatio n . In the literature, for the same sample size tested for E. grandis 31 to 65 seedlings have been reported. The applicant indicates that, as expected from limited self

- pollination that may have occurred in these trees, the number of viable seeds produced in these tests is much lower than would be expected for open pollinated mixed stands of Eucalyptus

.

Mature s eed capsules , collected prior to opening, were sampled from the single field test in Highlands County, Florida in early March 2010 (Permit 08

- 151

- 1 01r

- now covered under 08

- 014

- 101rm)

. As expected from self

- fertilization, a very low number of viable seeds (less than 1) per capsule were observed for transgenic lines and non

- transgenic controls trees tested compared to the number of viable seeds ( ~2 5 per capsule) expected from an open pollinated mixed stands of Eucalyptus

.
The controlled seed germination studies with seed capsules collected over three years (2008 to 2010) from field trials allowed to flower have indicated that either no, or a very low number of viable seeds are produced in translines and control EH1 trees compared to what would be expected for an open

- pollinating mixed stand of Eucalyptus . These data are consistent with the hypothesis that limited self

- pollination can occur from vi able pollen produced by the non

- transformed control trees. The applicant has also not observed any volunteer seedlings in or around the test sites where trees have flowered and produced seed capsules. Volunteer monitoring and reporting the presence of vol unteers is required in the supplemental permit conditions.

Similar collections of seed capsules have also occurred during 2011 in field tests from Evans County, Georgia and Jasper County, Texas. The seed has not yet been analyzed.

Monthly field test monitoring observations have not identified any differences in diseases and insects or other non

- target organisms between the transgenic and non

- transgenic trees in the field test.Page 49 of 91 

## APPENDIX II: Hydrology considerations for planted Eucalyptus submitted by ArborGen LLC

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

August 12, 2008

Introduction

The relevant scientific literature and conclusions drawn by experts in the field of hydrology, ecology, and plantation management on the hydrology of Eucalyptus plantations are discussed in this document. Extensive research on hydrology has been conducted in countries where large plantations of Eucalyptus have been established for many years, including India, China, South Africa and Brazil, as well as in its native Australia. The FAO, in response to the criticisms and concerns expressed about Eucalyptus plantations, has developed several expert reviews on the ecological impacts of Eucalyptus (discussed below).

The main hydrological concerns voiced against Eucalyptus plantations are that they deplete water supplies. The authors of an early FAO report (Poore and Fries, 1985) noted that these same criticisms would apply equally to any other plantation tree species, and that society tends to judge more harshly forestry crops relative to agricultural crops. As stated in Poore and Fries, 1985, “... most crops in many parts of the world are of foreign ori gin (wheat, maize, rice, potatoes, manioc, rubber, oil palm, coconut and many others. No one is surprised either that the soil under agricultural crops becomes depleted if these are continuously cropped without adding fertiliser. But both of these features are considered grounds for criticism in forestry.” Ironically, Eucalyptus evolved to be water efficient as the Australian continent itself became more dry (Davidson, 1995). In fact Eucalyptus uses less water per unit weight of biomass produced than do other kinds of trees (Chaturvedi, 1987) and many agricultural crops (Davidson, 1995).

It is also important to take into account the breadth of the genus Eucalyptus , often referred to broadly as eucalypts, where different species have different characteris tics 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 savann ah, 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 pr oduction. 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 ju dged with reference to the pre

- existing conditions before the establishment of the planting of Eucalyptus . In the case of field trials of Eucalyptus requested in ArborGen’s permit applications, all sites are on land previously managed for forestry, agricul tural production or maintained as pasturePage 50 of 91 land.

The Water Cycle

In considering the potential hydrological impacts of Eucalyptus it is important to put these in the context of the water cycle. Poore and Fries (1985) provide a good overview of the water cycle (see Figure 1 in Poore and Fries, 1985, or see Figure 1

- 1 in NRC, 2008). One key variable is the amount of rain that is intercepted by the canopy and is then evaporated back into the atmosphere. As a result, such intercepted water does not contribute to water in the soil. Of the water that does reach the soil some of this is absorbed while some fraction runs off the surface or is evaporated. A certain amount of water is maintained in the soil layer against the forces of gravity (called the ‘fieldcapac ity’ and dependent on soil texture and organic content) while any excess drains to the water table, the level at which the soil is permanently saturated. Depending on the depth of the water table it may be accessed by deep rooting plants such as trees. Eve n without roots that reach to the water table, plants may be able to access water from deeper, wetter soil layers through capillary action, depending on the soil type, where there can actually be an upward movement of water.

Through normal transpiration p lant roots take up available water which is transported through the stem to the leaves, the majority of which is lost to the atmosphere. Evapotranspiration is the total water returned to the atmosphere through transpiration and evaporation from the ground, bodies of water, plus intercepted water in the canopy. The relative rates of evapotranspiration and precipitation are often compared in assessments of hydrological systems. Where precipitation exceeds evapotranspiration then there is a net water gain to g roundwater or downstream systems. Where evapotranspiration exceeds precipitation then the available water resources may be depleted. It is important to understand, as pointed out in Poore and Fries (1985), that water loss is a “price that plants must pay f or 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 qua ntities 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 o n 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 be low) where the hydrology of Eucalyptus has been studied in detail. FAO has also released two annotated bibliographies (FAO, 2002a, 2002b) that collate and summarize publications on environmental, social and economic impacts of Eucalypts, and which include many references to water use. While there are specific examples and geographic regions where Eucalyptus (and other trees) canPage 51 of 91 negatively impact hydrology, in general, the literature indicates that Eucalyptus can be grown in a sustainable manner and that it s associated water use is not a major ecological threat. Most of the reviews indicate that soil and water characteristics of the site should be taken into consideration when establishing and maintaining a Eucalyptus plantation, in the same way that would b e appropriate for plantings of agricultural crops.

The first FAO review was published in 1985 by Poore and Fries

. At that time the authors suggested that there were relatively few existing studies in several important areas including hydrology (Lima, 1984; Poore and Fries, 1985). Where comparative studies showed that for dry alpine conditions the water regime for Eucalyptus did not differ from adjacent grasslands (see Lima, 1984), this was attributed to Eucalyptus ’ ability to control the rate of transp iration, an evolutionary adaptation for survival of drought stress which is often typical of the rainfall regimens of their native habitats. For deep soils and higher rainfall Eucalyptus plantations might be expected to reduce streamflow or groundwater

rec harge 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 nea r 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

repo rt recognized the potential benefits of Eucalyptus and noted that many of the criticisms of the species were based on inappropriate government policies on afforestation or social concerns rather than the biology of the trees themselves (see also Casson, 19 97). Calder et al (2004) highlights that many early policies were based on public misconceptions about the impacts of forests on water. With regard to hydrological effects on intercropping with other species, the experts in the FAO report concluded that wh ile Eucalyptus can have negative effects in drier climates, in regions where rainfall is above 1,200 mm/year this is not expected to be a problem. The report suggests that for Eucalyptus plantings in those regions where water is scarce or demanded by other sectors, biomass production could be adjusted to match the amount of water available, for example by planting fewer trees per unit area or by thinning existing plantations.

As part of this expert consultation Sunder (1995) reported that the overall use o f 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 signi ficantly 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). Pa til (1995) reported data on water consumption at sites in India, which although high in Eucalyptus , was the most efficient in terms of water consumed per gram of biomass produced (see also Silva et al 2004). In fact, water use efficiency in Eucalyptus actu ally increases with greater water availability (Stape et al, 2004a, 2004b). Patil (1995) also noted that there were noPage 52 of 91 hydrological impacts of Eucalyptus on adjacent crops at these sites. White (1995) stated that large plantings of Eucalyptus may reduce water yield and lower water tables but this varies from one situation to another and most importantly can be mitigated through management practices such as changes in tree stocking regimes. The environmental considerations of Eucalyptus are the same as tho se for agricultural crops. Davidson (1995) noted that drawing water from shallow or deep wells to supply high water demanding crops such as rice or cotton can have a greater impact on drawing down water tables than fast growing tree plantations. He also co ncluded that many potential adverse effects are reversible, as noted earlier by Poore and Fries (1985).

A review of the environmental issues of Eucalyptus plantations in Brazil was published by Oak Ridge National Laboratory (Couto and Betters, 1995). This report summarized that the hydrology of Eucalyptus plantations was comparable to other tree plantations or natural forest cover and that any effects would largely depend on management practices. Numerous studies demonstrate that forest cover and any chang es 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 pl antations must address similar issues of sustainability as seen in agriculture. They refer to the many hundreds of trials that have been conducted in Brazil, with particular reference to a very large watershed project conducted in collaboration with Aracru z Cellulose Company (reported by Almeida et al 2007, described in more detail below). Binkley and Stape conclude that in semi

- arid environments afforestation with any species of trees may increase water use, lower ground water levels and reduce streamflow. Given appropriate silvicultural management however, wood production should face no barriers to sustainability.

Whitehead and Beadle (2004) provided a comprehensive review of the physiological regulation of water use in Eucalyptus . These species have evolved several mechanisms to allow them to cope with drought conditions in their native habitats. These include dynamic changes in leaf area index (LAI), arrangement of leaves, high stomatal sensitivity to air saturation deficit, osmotic manipulation to m aintain turgor in leaves, as well as an ability to form deep roots. Maximum potential rates of photosynthesis are high in Eucalyptus compared to other broad

- leaved trees, but actual rates are often much less because of water limitations. Some examples are noted where Eucalyptus plantings have led to reductions in yields of water catchments. Conversely, the high water usage by Eucalyptus may be valuable in purposefully lowering water tables to reduced potential salinity problems. It is therefore important to assess productivity and water use in relation to climate variables, nutrient supply and options for silvicultural management, and careful matching of species to sites where available water may be limited. One of the physiological responses of Eucalyptus t o limited water noted by Whitehead and Beadle is to reduce LAI, thus although Eucalyptus are evergreen species there can be large seasonal changes in LAI in response to dry seasons. Similar observations were made in reduced LAI along a gradient of water av ailability by Ares and Fownes (2000). The root systems of Eucalypts are dimorphic, with widely spreading lateral root systems below the surface plus a deep tap root system. In a plantation of 7 year old Eucalyptus trees in Brazil thePage 53 of 91 tap root extended to a depth of about 2.5m (Almeida and Soares, 2003) consistent with other observations (see Srivastava et al, 2003). When artificially stressed, by using plastic sheets on the soil surface to prevent rain entering the soil, young Eucalyptus developed roots 8 m or greater in depth. Under other conditions water was utilized from soil below the root zone by upward movement from wetter levels. In considering these physiological adaptations Whitehead and Beadle conclude that in the case of South Africa, where plante d Eucalyptus replaced native grasslands, the decreased water yields resulted from increased transpiration in the evergreen and deep rooted Eucalyptus during the dry season compared to the seasonally dormant grasses. It is well established that forests have greater evapotranspiration than grasslands (Zhang et al, 1999).

Specific Hydrology Issues for Planted Eucalyptus :

Eucalyptus Afforestation and Hydrology.

In those cases where Eucalyptus has been shown to have negative impacts on hydrology this has been associated with afforestation, most notably of lands where trees were previously absent. Typically, these are areas of low rainfall that are normally dominated by grasses. Under these conditions afforestation with different species of trees, including
Euc alyptus , has led to changes in the water balance including lowering of water tables and restricting stream flows. Calder and colleagues have published several reports on afforestation efforts in India including examples where deep

- rooted Eucalyptus were ab le 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 grassl ands have documented negative impacts of Eucalyptus on the water balance in South Africa (Lesch and Scott, 1997; Scott and Lesch, 1997; Scott et al, 1998) and Argentina (Jobbagy and Jackson, 2004; Engel et al, 2005; Nosetto, 2005). In many of these cases o ther introduced trees including pines had similar impacts and particularly in South Africa impacts on water balance result from a wide variety of introduced species (Le Maitre et al, 2000; 2002).

These examples contrast with the experience in Brazil where there has been extensive reforestation with Eucalyptus over many decades. Much of this literature is in Portuguese but often abstracts are published in English. Lima and colleagues have published a number of reports that analyzed potential impacts of both Eucalyptus and pine plantations on the cerrado grasslands in Brazil. Lima et al (1990) showed that in the region there was adequate rainfall to meet the evapotranspiration demands of Eucalyptus

. A comparison of 6

- year old Eucalyptus and pine plantings showed comparable levels of evapotranspiration during the dry season (May through September) as herbaceous vegetation (Lima and Freire, 1976). In these trials Eucalyptus actually showed greater interception than pine (Lima, 1976) and contrasts with referen ces above, but likely reflected the greater average height of the Eucalyptus at 13.4 m compared to an average of 6 m for pine.

Similarly, an examination of the water balance of Eucalyptus plantations in China were not considered to be deleterious for wate r supplies (Lane et al 2004). WhilePage 54 of 91 evapotranspiration exceeded precipitation in the dry season, water storages were replenished during the wet season.

Comparison of Water Use by Eucalyptus with other Tree Species.

In addition to the reports cited above many authors have concluded that the hydrological impacts of Eucalyptus are comparable to and should be viewed in the context of other tree species (see for example Myers et al, 1995; Wullschleger et al, 1998).

One of the largest studies comparing Eucalyp tus and native trees conducted to date has been a catchment area in Brazil of over 280 hectares (owned by Aracruz Cellulose S.A.) consisting of 190 ha of planted hybrid Eucalyptus and almost 90 ha of native Atlantic rainforest, that was analyzed over a per iod of six years. Average precipitation at this site was 1147 mm, which is similar or less than the sites listed in this permit (range from ~1160 mm in Glades County Florida to almost 1750 mm in Escambia County, Alabama). Mean high temperature at the Aracr uz site was 32.6 C (~91 F) for February (the summer season in the southern hemisphere), again, comparable to mean high temperatures in the summer for the sites in this permit. Data from the studies of this catchment area in Brazil indicated that evapotranspiration was strongly influenced by precipitation (Almeida et al, 2007). In an unusually dry year evapotranspiration was about half that compared to when water was readily available. In this dry year evapotranspiration exceeded precipitation but conversely in wetter years evapotranspiration was much less than precipitation. Over the length of the study evapotranspiration was ~95% of precipitation. This adjustment in response to varying conditions and water availability was indicative that these hy brid trees exert strong stomata control and utilize water according to its availability. In a series of studies in this same area conducted over a period of 8 years Almeida and Soares (2003, text in Portuguese with abstract and figure legends in English) e xamined 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 str ong stomatal control. Rainfall interception by the Eucalypts averaged ~11% compared to ~24% in the native forest and water availability (at a depth of 2.5m) is almost identical in the native forests and the Eucalyptus plantations during the wet summer mont hs 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 availa ble water in the shallower levels. Finally, the authors compared the ratio of evapotranspiration and precipitation (ET/P) of the planted Eucalyptus with the native forest. In years with normal precipitation ET/P was comparable for both the Eucalyptus and n ative forest. In years with less than normal precipitation the native forest had higher ET/P (that is, evapotranspiration was much greater than precipitation) compared to the Eucalyptus . Based on their data, the authors suggest that the native forest has a greater consumption of water relative to the growth/harvest cycle of Eucalyptus , since in the first few years after planting transpiration in the plantation is much less than the native forest.

Competition for Resources between Eucalyptus and Adjacent Cr opsPage 55 of 91 There has been speculation that water use by Eucalyptus could have a negative impact on water resources available for adjacent vegetation or agricultural crops. Such issues have been extensively researched in relation to widespread agroforestry systems (reviewed by Nuberg 1998, and Schroth, 1999). There are important tradeoffs between the positive effects from windbreaks and shelter belts versus potential competition for light, nutrients and water resources. Such effects typically occur within 1 to 2 tree

- heights (50 to 100 feet for a 50 foot tall tree, Nuberg, 1998) and can be attributed to direct competition by roots for available soil moisture. Often this can be managed by root pruning to reduce the area occupied by the tree roots. Impacts attr ibuted to Eucalyptus depend on specific site conditions, and as with other concerns, there are examples where no negative impact on adjacent agricultural crops were observed (e.g. Patil, 1995). At some sites this could be attributed to deeper rooted trees versus shallow rooting crops utilizing water from different soil profiles. Finally, tree planting has been proposed as a mitigation strategy where rising water levels increase salinity and reduce crop yields (Hatton and George, 2001).

Summary and Conclusi ons:

Many studies report that water use in Eucalyptus is comparable to other tree species. There are some cases where afforestation with Eucalyptus (or other tree species) has lead to reduced water run

- off and supply of streams or changes in water table l evels, especially in regions with limited rainfall. However, in many well documented cases Eucalyptus plantations do not have any significant negative impacts on hydrology. Where there have been purported negative impacts, these often reflect more complex issues such as socioeconomic and land ownership disputes rather than the physiology of Eucalyptus itself. A key finding of many experiments has been that Eucalyptus is highly effective in regulating its water consumption relative to available supplies and regulates its growth accordingly. Based on numerous comparisons that have been made between the potential hydrological impacts of Eucalyptus and other tree species, we do not expect that the Eucalyptus trials planted under these permits would be any more impactful on local hydrology than planting other fast growing trees species.

References:

Almeida, A.C. and Soares, J.V. (2003). Comparison of water use in Eucalyptus grandis plantations and Atlantic rainforest in eastern coast of Brazil. Revista Arvore 2 7(2): 159

- 170.

Almeida, A.C., Soares, J.V., Landsberg, J.J. and Rezende, G.D. (2007). Growth and water balance of Eucalyptus grandis hybrid plantations in Brazil during a rotation for pulp production. Forest Ecology and Management, 251:10

- 21

Ares, A. and Fownes, J.H. (2000) Productivity, nutrient and water

- use efficiency of Eucalyptus saligna and Toona ciliata in Hawaii. Forest Ecology and Management 139:227

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Binkley,D. and Stape, J. L. (2004). Sustainable Management of EucalyptusPage 56 of 91 Plantations in a Cha nging World. In: Borrelho, N., et al., Eucalyptus in a Changing World Proc. IUFRO Conference, Aviero, 11

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Bosch, J.M. and Hewlett, J.D. (1982). A review of catchment experiments to determine the effect of vegetation changes on water yield and evapotranspiration. Journal of Hydrology, 55:3

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Calder I.R., Rosier, P.T.W., Prasanna, K.T. and Parameswarappa, S. (1997). Eucalyptus water use greater than rainfall input

- a possible explanation from southern India. Hydrology and Earth System Sciences, 1:244

- 256.

Calder, I., Amezaga, J., Aylward, B., Bosch, J., Fuller, L., Gallop, K., Gosain, A., Hope, R., Jewitt, G., Miranda, M., Porras, I. and Wilson, V. (2004). Forest and Water Policies. The need to reconcile public and science perceptions. Geologica Acta, 2:157

- 166

Calder, I.R. (1994). Eucalyptus , Water and Sustainability: A Summary Report. ODA Forestry Series No. 6, United Kingdom Office of Development Assistance, London.

Casson, A. (1997). The controversy surrounding eucalypts in socia l forestry programs of Asia. National Center for development Studies. Working Paper 97/1

Chaturvedi, A.N. (1987). Silvicultural requirements of Eucalyptus for small farms. In Proc. International Workshop Multipurpose tree species for small farms. Winrock International Institute for Agricultural Development USA, International Development Research Centre of Canada.

Couto, L. and D. R. Betters. (1995). Short

- rotation Eucalypt Plantations in Brazil: Social and Environmental Issues, prepared for the Biofuels F eedstock Development Program, ORNL/TM

- 12846, Oak Ridge National Laboratory, Oak Ridge, Tennessee. Available on

- line at: http://bioenergy.ornl.gov/reports/euc

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Davidson, J., (1995). Ecological aspects of Eucalyptus plantations. In: Proceedings of the Regional Expert Consultation on Eucalyptus , 4

- 8 October, 1993, Vol. 1, FAO Regional Office for Asia and the Pacific, Bangkok. Available on

- line at: http://www.fao.org/docrep/005/ac777e/ac777e00.htm#Contents de Almeida, P.A. and Riekerk, H. (1990). Water balance of Eucalyptus globulus and Quercus suber forest stands in south Portugal. For. Ecol. Manage., 38: 55

- 64.

Engel V., Jobbagy, E.G., Stieglitz, M., Williams, M. and Jackson, R.B. (2005). Hydrological con sequences of Eucalyptus afforestation in the Argentine Pampas. Water Resour. Res., 41, W10409

FAO (2002a). Annotated bibliography on environmental, social and economic impacts of Eucalypts (Spanish Version), Forest Plantations Working Papers, Working Pape r 17 (S). Palmberg, C., Forest Resources Development Service, Forest Resources Division. FAO, Rome (unpublished).Page 57 of 91 FAO (2002b). Annotated bibliography on environmental, social and economic impacts of Eucalypts (Spanish Version), Forest Plantations Working Papers, Working Paper 16. Forest Resources Development Service, Forest Resources Division. FAO, Rome (unpublished).

Hatton,T. and George, R. (2001). The Role of Afforestation in Managing Dryland Salinity. In Plantations, Farm Forestry and water: Workshop Proceedings RIRDC Publication No. 01/20. Namibar, E.K.S. and Brown, A.G. (eds).

Jobbagy, E.G. and Jackson, R.B. (2004). Groundwater use and salinization with grassland afforestation. Global Change Biology 10:1299

– 1312

Lane, P.N.J., Morris, J., Zhang, N., Guangyi, Z., Guangyi, Z., Guoyi, Z. and Daping, X. (2004). Water balance of Eucalyptus plantations in southeastern China. Agricultural and Forest Meteorology, 124: 253

- 267

Le Maitre, D.C., Versfeld, D.B. and Chapman, R.A. (2000). The impact of invading alien plants on surface water resources in South Africa: A preliminary assessment. Water SA 26:397

- 408

Le Maitre, D.C., van Wilgen, B.W., Gelderblom, C.M., Bailey, C., Chapman, R.A. and Nel, J.A. (2002). Invasive alien trees and water resources in South A frica: case studies of the costs and benefits of management. Forest Ecology and Management 160:143

– 159

Lesch. W., and Scott, D.F. (1997). The response in water yield to the thinning of Pinus radiata

, Pinus patula and Eucalyptus grandis plantations. Forest Ecology and Management 99:295

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Lima, W. de P. (1976). Interceptação da Chuva em Povoamentos de Eucalipto e de Pinheiro. IPEF, n.13, p.75

- 90. (Portuguese with English abstract) Lima, W.P. (1984). The Hydrology of Eucalypts Forests in Australia

- A Revi ew. IPEF n.28, p.11

- 32

Lima, W. de P. and Freire, O. (1976). Evapotranspiração em Plantações de Eucalipto e de Pinheiro, e em Vegetação Natural. IPEF, n.12, p.103

- 117. (Potuguese with English abstract)

Lima, W.D. Zakia, M.J.B. Libardi, P.L. and Filho, A. (1990). Comparative evapotranspiration of Eucalyptus , pine and natural vegetation measured by the soil water balance method. IPEF International Piracicaba. 1:5

- 11.

Myers, B.J. Theiveyanathan, S. O’Brien, N.D. and Bond, W.J. (1995). Growth and

water use o f Eucalyptus grandis and Pinus radiata plantations irrigated with effluent. Tree Physiology, 16:211

- 219.Page 58 of 91 Nosetto, M.D., Jobbagy, E.G. and Paruelo, J.M. (2005). Land

- use change and water losses: the case of grassland afforestation across a soil textural gradient in central Argentina. Global Change Biology (2005) 11, 1101

– 1117

NRC (2008). Hydrologic Effects of a Changing Forest Landscape (Prepublication copy available on

- line at http://www.nap.edu/catalog/12223.html)

Nuberg, I.K. (1998). Effect of shelte r on temperate crops: a review to define research for Australian conditions. Agroforestry Systems 41:3

– 34.

Patil, V. (1995). Local Communities and Eucalyptus

- An Experience in India. In: Proceedings of the Regional Expert Consultation on Eucalyptus , 4

- 8 October, 1993, Vol. 1, FAO Regional Office for Asia and the Pacific, Bangkok. Available on

- line at: http://www.fao.org/docrep/005/ac777e/ac777e00.htm#Contents

Poore, M.E.D. and Fries, C. (1985). The Ecological Effects of Eucalyptus . FAO Forestry Paper No. 59

Sahin, V. and Hall, M.J. (1996). The effects of afforestation and deforestation on water yield. Journal of Hydrology 178:293

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Scott, D.F. and Lesch, W. (1997). Streamflow responses to afforestation with Eucalyptus grandis and Pinus patula and to felling in the Mokobulaan experimental catchments, South Africa. Journal of Hydrology 199:360

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Scott, D.F., Le Maitre, D.C. and Fairbanks D.H.K. (1998). Forestry and streamflow reductions in South Africa: A reference system for assessing extent and distribution. Water SA 24:187

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Silva, W., Sediyama, T., Silva, A., and Cardoso, A. (2004). Consump tion and Water Use Efficiency Index by E. citriodora and E. grandis plants cultivated in pots with three different water contents in the soil jointly with Brachiaria brisantha populations. Foresta, 32 (3): 325

- 335. (Potuguese with English abstract)

Srivas tava, R,J., Kumar, A. and Prasad, K. (2003). Studies on Soil Moisture Variations Under Eucalyptus Plantations. XII World Forestry Congress, 2003, Quebec City, Canada. Available on

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Stape, J.L. , Binkley, D. and Ryan, M.G. (2004a). Eucalyptus production and the supply, use and efficiency of use of water, light and nitrogen across a geographic gradient in Brazil. Forest Ecology and Management 193:17

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Stape, J.L. Binkley, D. Ryan, M.G. and Gomes , A.N. (2004b). Water use, waterPage 59 of 91 limitation, and water use efficiency in a Eucalyptus plantation. Bosque 25:35

– 41. Sunder, S. (1993). The Ecological, Economic and Social Effects of Eucalyptus . In: Proceedings of the Regional Expert Consultation on Eucalyptus , 4

- 8 October, 1993, Vol. 1, FAO Regional Office for Asia and the Pacific, Bangkok

White, K.J. (1995). Silviculture of Eucalyptus Plantings

– Learning from the Region (Australia). In: Proceedings of the Regional Expert Consultation on Eucalyptus , 4

- 8 October, 1993, Vol. 1, FAO Regional Office for Asia and the Pacific, Bangkok

White, K., Ball, J. and Kashio, M. (eds) (1995). Proceedings of the Regional Expert Consultation on Eucalyptus , 4

- 8 October, 1993, Vol. 1, FAO Regional Office for Asia and the Pacific, Bangkok

Whitehead, D. and Beadle, C.L. (2004). Physiological regulation of productivity and water use in Eucalyptus : A review. Forest Ecology and Management. 193:113

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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 U SDA APHIS BRS in support of consideration for approval of permits for field trials of Eucalyptus at multiple sites

Executive Summary

We reviewed the materials provided by ArborGen and synthesized the literature on water use by Eucalyptus and other veget ation in the southeastern US. Based on these materials and our best professional judgment, we provide the following assessments:

1. Water use efficiency (WUE) is not a good metric to evaluate impacts on hydrology From a hydrologic standpoint, total water use (transpiration + interception) is a more appropriate metric to assess hydrologic impacts. A species may have high WUE (defined by ArborGen as volume of wood produced per amount of water required), but still 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 Eucal yptus transpiration suggests that water use is at least 2

- fold greater than most other native forests in the southeastern US.

3. If Eucalyptus invades native forests, forest water use will increase Due to a combination of physiological and structural c haracteristics, Eucalyptus will use more water than most native species regardless of whether it is planted or invades native forests.

4. Afforestation from existing vegetation into Eucalyptus plantations reduces stream flow more so than afforestation to pine plantations Our review of the literature suggests that stream flow will be about 20% lower in Eucalyptus plantations vs. pine plantations.

5. Planting Eucalyptus hybrid plantations wil l 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 st ocking levels will be an acceptable management practice to reduce hydrologic impacts of Eucalyptus plantations. High biomass production requires fully stocked stands. Reducing stocking to minimize hydrologic impacts is likely to counter the benefits of planting fast growing Eucalyptus

.Page 61 of 91 

## Possible impacts of Eucalyptus hybrid plantations on southeastern US hydrology

Water use efficiency (WUE) is not a good metric to evaluate impacts on hydrology From a physiological standpoint, water use efficiency is defined as the ratio of the moles of carbon fixed to the moles of water lost. WUE is a leaf

- level metric . T he Hydro logy 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

m agnitude of evapotranspirational losses, nor do they integrate stand management effects (e.g., planting density, rotation age). Hence, WUE is a poor metric to evaluate the effects of Eucalyptus on water resources. For example, Eucalyptus can produce more b iomass per unit water consumed than native southeastern pines; however, their extremely rapid biomass production has proportionally higher transpirational costs and hence greater water use. Better metrics of evaluating the impacts of Eucalyptus hybrid pla ntations 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 southeast US will greatly exceed E t by other southeastern forest ty pes Previous studi es 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 contras t, a mature Eucalyptus plantation (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 comparison 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 62 of 91 Table 1

Vegetation type Mean transpiration

(mm yr - 1 ) Reference Longleaf pine savanna 244 (Ford et al. 2008)
Old field 250 (Stoy et al. 2006)
Oak

- pine

- hickory forest 278 (Oren and Pataki 2001) Upland oak forest 313 (Wullschleger et al. 2001) Mixed pine hardwood 355 (P hillips and Oren 2001) Mixed pine hardwood 442 (Stoy et al. 2006) Planted loblolly pine 490 (Stoy et al. 2006) Mixed pine hardwood 523 Schafer and others 2002 Slash pine flatwoods 563 (Powell et al. 2005) Eucalyptus hybrid plantation 882 Estimated for SW GA in average climate and rainfall year from model published in (Mielke et al. 1999)
Cotton (non

- irrigated, annual) 392 (Howell e t al. 2004) Strawberries (irrigated, 7

- month crop, 5

- month fallow) 1397 (Clark 1994, Allen et al. 1998)

Watermelon (irrigated, 3

- month crop, 9

- month fallow) 237 (Allen et al. 1998, Shukla et al. 2007)

Eucalyptus has much 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 ra tes that are roughly double the conductance rates of native southeastern pine species. This is one mechanism that confers a greater transpiration rate of the former compared to the latter.Page 63 of 91 Figure 1: Stomatal conductance response to VPD for pine and Eucalyptus (Oren et al. 1999)

If Eucalyptus invades native forests, forest water use will increase In general, species that evolved in arid climates have a more sensitive 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 greater compared to un

- invaded systems (Cavaleri and Sack 2008)

.
Afforestation from existing vegetation into Eucalyptus plantations reduces stream flow more so than afforestation to pine 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 flow , the reduction would be comparable to the reduction by pine plantations. Recent research suggests that Eucalyptus plantations would reduce stream flow more than pine plantations, and more importantly, Eucalyptus plantations could eliminate low flows. In a review of more than 20 catchment conversion studies, Farley and others (2005b) showed that converting existing vegetation to Eucalyptus plantations reduced stream flow by 20% more than converting it to a pine plantation. This review also showed that the loss of low flows were more complete for Eucalyptus plantations compared to pine plantations (100% vs. ~80% reduction of low flows).
In perennial streams throughout the southeast which have base flows sustained by subsurface flow from the water table (or unconfined aquifers), elimination of low flows VPD (kPa) 0 1 2 3 4 5 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 64 of 91 may have important ramifications for threatened and endangered aquatic species, such as th e 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 dep endent on subsurface flow patterns, local hillslope hydrology, and species

- specific rooting patterns. For example, in sites where the water table can be recharged laterally, if roots extend to the water table, then stomatal conductance and transpiration ca n 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 Pla in 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 root ing depths of pine plantation ( P. taeda and P. elliottii ) and grass species are 3 m and 2.6 m, respectively (Canadell et al. 1996b) . The average age of groundwater in the UFA is ~20 years (Happella et al. 2006) and groundwater is regularly recharged by surface water in this region (Opsahl et al. 2007) . Conversion to Eucalyptus on sites wit h 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 sp ecies 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 lowering water tables to reduced potential salinity problems. Eucalyptus has been used to aff orest areas and lower the saline groundwater in highly weathered landscapes (e.g., AUS). This application is not relevant to the southeastern US, as soils are not saline. Furthermore, receding groundwater levels in the UFA are being replaced in coastal are as by saltwater (i.e., saltwater intrusion) (Andersen et al. 2006) . Thus, lowering the water table, and the groundwater levels in the UFA would not reduce salinity problems (as stated in the Hydrology document), and may actually exacerbate them.

Key Point: The significance of the impact on groundwater and stream flow will depend greatly on the area exten t, size, and spatial distribution of the plantations. For example, a few small (i.e., < 10 ha) and well dispersed plantations may only have very localized impacts and negligible impacts at the watershed scale.

Management of Eucalyptus as coppice stands w ill affect water use of future rotations Management practices may create a perennial root stock in Eucalyptus plantations. If Eucalyptus plantations are managed as coppice stands, the remaining mature, deeply

-Page 65 of 91 penetrating root stock may be able to supply the second rotation stems with more water resources for use than similar sized stems in their first rotation (Swift and Swank 1981)

.
It is unlikely that lower stocking levels will be an acceptable management practice to reduce hydrologic impacts of Eucalyptus plantations Some of the reports cited in the A r borGen d ocument 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 descr ibe 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 lowe r 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 str eam flow is obtained. Large and sustained increases in stream flow typically require significant reductions in stocking because trees growing in more open conditions will increase transpiration rates in response to changes in micrometeorological condition s in the tree crown. From a practical standpoint, it is unlikely that lower stocking levels will be an acceptable management practice for Eucalyptus plantations because one of the primary objectives of growing Eucalyptus is to maximize biomass production

-- this requires fully stocked stands.

Monitoring Impacts on Hydrology Several options are available to monitor the impacts of Eucalyptus plantations on hydrology. Options include direct or indirect measurements of impacts, and vary in complexity and cost. The ArborGen document provided basic information on the hydrologic cycle and its components so these will not be repeated here.

Indirect Measurements : Transpiration & Interception Instrumentation required = rain gauges, throughfall collectors, sap flow sensors PROS: direct measure of change in water use component on hydrologic cycle CONS: does not directly measure impacts on 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 groundwater

Direct Measurements : Groundwater DepthPage 66 of 91 Instrumentation = access wells & pressure transducers (automated); access wells (manual) PROS: direct measure of impacts; reliable CONS: expensive

Stream flow Instrumentation = flumes & data loggers (automated); pressure transducer, rating curve, stream survey PROS: direct measure of impacts; reliable CONS: expensive, requires stream, expertise

Regardless of the monitoring approach chosen, the monitoring design will require a suitab le control for comparison. Ideally, the plantation site(s) and the control site(s) would be measured for at least 1 to 2 years prior to be being planted, and then both sites would be measured for the duration of the monitoring period. We recommend that t he monitoring period begin at plantation establishment and continue through canopy closure (approximately 5 years).

Additional analyses on groundwater dynamics and linkages with aquatic ecosystems are required We recommend that APHIS solicit input from experts on groundwater hydrology (e.g., from USGS) to assess the potential impacts on groundwater recharge and associated dynamics. In addition, our analysis suggests that stream flow will be reduced by at least 20% relative to pine plantations and perhaps even greater reductions will be observed relative to native ecosystems. We recommend that APHIS solicit input from aquatic ecologists to assess the potential impacts on aquatic ecosystems and associated species

.Page 67 of 91 References

Allen RG, Pereira LS, Raes D, Smith M. 1998. Crop evapotranspiration

- Guidelines for computing crop water requirements. FAO

- Food and Agriculture Organization of the United Nations. Paper 56. Andersen PF, Mercer JW, White Jr . HO. 2006. Numerical modeling of salt

- water intrusion at Hallandale, Florida. Ground Water 26: 619

– 630. Calder IR, Rosier PTW, Prasanna KT, Parameswarappa S. 1997. Eucalyptus water use greater than rainfall input

- a possible explanation from southern India . Hydrology and Earth System Sciences 1: 249

– 256. Canadell J, Jackson RB, Ehleringer JR, Mooney HA, Sala OE, Schulze ED. 1996. Maximum rooting depth of vegetation types at the global scale. Oecologia 108: 583

- 595. <Go to ISI>://A1996VX11000001 Cavaleri M, Sack L. 2008. Native vs. invasive plant water use at multiple scales: A global

meta

- analysis. Presented at the Ecological Society of America Annual Meeting in Milwaukee, WI. August 3

– 8. Clark GA. 1994. Water requirements for drip irrigated strawberries in south central Florida. Agricultural and Biological Engineering Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. AE253. Couch CA, McDowell RJ. 2006. Flint River Basin Regional Water Development and Conservation Plan. Georgia Dept. of Natural Resources, Environmental Protection Division. p. 242. Douglass JE, Swank WT. 1972. Streamflow modification through management of eastern forests. USDA Forest Service, Southeastern Forest Experimen t Station. Research Paper SE

- 94. Farley K, Jobbagy E, Jackson RB. 2005. Effects of afforestation on water yiel d : a global synthesis with implications for policy. Global Change Biology 11: 1565

– 1576. Ford CR, Goranson CE, Mitchell RJ, Will RE, Teskey RO. 20 04. Diurnal and seasonal variability in the radial distribution of sap flow: predicting total stem flow in Pinus taeda trees. Tree Physiol. 24: 951

- 960. Ford CR, Mitchell RJ, Teskey RO. 2008. Water table depth affects productivity, water use, and the respo nse to nitrogen addition in a savanna system. Can. J. For. Res. 38: 2118

– 2127. Golladay SW, Gagnon P, Kearns M, Battle JM, Hicks DW. 2004. Response of freshwater mussel assemblages (Bivalvia:Unionidae) to a record drought in the Gulf Coastal Plain of south western Georgia. Journal of the North American Benthological Society 23: 494

- 506. Happella JD, Opsahl S, Top Z, Chanton JP. 2006. Apparent CFC and 3 H/ 3 He age differences in water from Floridan Aquifer springs. J. Hydrol. 319: 410

– 426. Howell TA, Evett SR, Tolk JA, Schneider AD. 2004. Evapotranspiration of full

- , deficitirrigated, and dryland cotton on the northern Texas high plains. Journal of Irrigation an Drainage Engineering 130: 277

– 285. doi:10.1061/(ASCE)0733

- 9437(2004)130:4(277) McDowell N, Pockman WT, Allen CD, Breshears DD, Cobb N, Kolb T, Plaut J, Sperry J, West A, Williams DG, Yepez EA. 2008. Tansley review: Mechanisms of plantPage 68 of 91 survival and mortality during drought: why do some plants survive while others succumb to drought? New Phytol. 178: 719

– 739. Mielke MS, Oliva MA, deBarros NF, Penchel RM, Martinez CA, Almeida ACd. 1999. Stomatal control of transpiration in the canopy of a clonal Eucalyptus grandis plantation. Trees

- Structure and Function 13: 152

- 160. Opsahl SP, Chapal SE, Hicks DW, Wheeler CK. 2007. Evaluation of ground

- water and surface

- water exchanges using streamflow difference analyses. Journal of the American Water Resources Association 43: 1132

– 1141. Oren R, Pataki DE. 2001. Transpiration in response to variation in microclimate and s oil 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 d eficit. 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 e nergy 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 F ood and Agricultural Sciences (IFAS), University of Florida. Report No. WRP

- LY

- 0009 submitted to Southwest Florida Water Management District Stoy P, Katul G, Siqueira M, Juang J, Novick K, McCarthy HR, Oishi AC, Umbelherr J, Kim H, Oren R. 2006. Separating the effects of climate and vegetation on evapotranspiration along a successional chronosequence in the southeastern US. Global Change Biology 12: 2115

– 2135. Swift LW, Swank WT. 1981. Long term responses of streamflow following clearcutting and regrowth. Hydrological Sciences Bulletin 26: 245

– 256. Wullschleger SD, Hanson PJ, Todd DE. 2001. Transpiration from a multi

- species deciduous forest as estimated by xylem sap flow techniques. For. Ecol. Manage. 143: 205

- 213.Page 69 of 91 

## APPENDIX IV: Threatened and Endangered S pecies Analysis

Congress passed the Endangered Species Act (ESA ) of 1973, as amended, to prevent extinctions facing many species of fish, wildlife, and plants. The purpose of the ESA is to conserve endangered and threatened species and the ecosystems on which they depend as key components of America’s heritage. To implement the ESA, the U.S. Fish and Wildlife Service (USFWS ) works i n cooperation with the National Marine Fisheries Service (NMFS ); other Federal, State, and local agencies; Tribes; non

- governmental organizations; and private citizens. Before a plant or animal species can receive the protection provided by the ESA, it must first be added to the Federal list of threatened and endangered wildlife and plants.
A species is added to the list when it is determined by the USFWS/NMFS to be endangered or threaten ed because of any of the following factors:

• The present or threatened destruction, modification, or curtailment of its habitat or range;

• Overutilization for commercial, recreational, scientific, or educational purposes;

• Disease or predation;

• The inadequacy of existing regulatory mechanisms; and

• The natural or manmade factors affecting its survival.
Once an animal or plant is added to the list, in accordance with the ESA, protective measures apply to the species and its habitat. These measures include prote ction from adverse effects of Federal activities. Section 7 (a)(2) of the ESA requires that Federal agencies, in consultation with USFWS and/or the NMFS, ensure that any action they authorize, fund, or carry out is not likely to jeopardize the continued e xistence of a listed species or result in the destruction or adverse modification of designated critical habitat. It is the responsibility of the Federal agency taking the action to assess the effects of their action and to consult with the USFWS and NMFS if it is determined that the action “may affect” listed species or critical habitat.

The threatened and endangered species analysis was accomplished by considering the possibility of effects on all listed species including species proposed for listing and designated critical habitat including habitat proposed for designation within the cou nty where the test site is located. Normally consideration would be limited to only those species and critical habitat identified by the United States Fish and Wildlife Service and/or National Marine Fisheries Service to be likely found in the area of the field tests. However, because the locations are declared by the applicant to be confidential business information, APHIS instead obtained species lists and critical habitat information for the entire county where the field tests are to occur. Although it is very clear that there could be no effect on some species ( e.g. West Indian manatee ) all species on the list and all critical habitat in each county are included in the discussion below.

The following resources were used in the analysis: US Fish an d Wildlife Service Endangered Species Homepage: http://www.fws.gov/endangered US Fish and Wildlife Service Critical Habitat portal: http://criticalhabitat.fws.gov/crithab/ ArcGis Explorer with critical habitat metadata supplied by US Fish and Wildlife Service County s pecies lists from FWS field offices in Alabama, Mississippi, South Carolina and Florida obtained from http://www.fws.gov/offices/Page 70 of 91 S outh Carolina Heritage Trust Database Telephone contacts with USFWS Field Office personnel Discussions with property owners of field test sites

1. Baldwin County, AL

This location has been an agricultural research station for more than 20 years. The l ocat ion has been used for managed production of annual agricultural crops and forest trees. Approximately 3.55 acres of field trials of genetically modified Eucalyptus trees of some lines in the permit are being grown under issued permits 08

- 011

- 106rm (0.75 ac res) and 10

- 112

- 101r (2.8 acres) under which these trees are allowed to flower. The oldest of these trees were planted in August 2006. Site preparation will involve herbicide application, subsoiling, and planting of trees in flat beds. The

s urrounding area s of the test site consist of field plantings of agricultural crops, experimental forest trees and an abandoned pecan orchard. Under the new permit u p to 2.0 acres of field tests could be established at this location (at around 300

- 600 trees per acre) over the next three years.

Seventeen TES animals and one TES plant are listed in Baldwin County (list accessed from FWS website 8 / 30 /2011). The TES Animals include l oggerhead sea turtle ( Caretta caretta ), green sea turtle, ( Chelonia mydas ), Alabama red

- belly turtle ( Pseudemys alabamensis ), Kemp's ridley sea turtle ( Lepidochelys kempii ), Hawksbill sea turtle ( Eretmochelys imbricat a, Leatherback sea turtle (Dermochelys coriacea), Alabama beach deermouse ( Peromyscus polionotus ammobates ), Perdido Key beach deermouse ( Peromyscus polionotus trissyllepsis ), piping plover ( Charadrius melodus ), West Indian manatee ( Trichechus manatus ), wood stork ( Mycteria americana ), gulf sturgeon ( Acipenser oxyrinchus desotoi ), Alabama sturgeon ( Scaphirhynchus suttkusi ), southe rn clubshell ( Pleurobema decisum )

, Alabama inflated heelsplitter mussel ( Potamilus inflatus ), and eastern indigo snake ( Drymarchon corais couperi ) . The listed plant is American chaffseed ( Schwalbea
americana ).

The American chaffseed occurs on sandy peat, sandy loam, acidic, seasonally moist to dry soils. It is generally found in habitats described as open, moist pine flatwoods, fire

- maintained savannas, ecotonal areas between peaty wetlands and xeric sandy soils , and other open grass

- sedge systems. According to Jody Smithen (contacted February 20, 2008) of the Daphne Field Office USFWS, the only location this plant is known to be in the county is in the northeast corner, far from the release site. The plant has no critical habitat listed in the action area.

The six turtle species (loggerhead sea turtle, green sea turtle, Alabama redbelly turtle

, Kemp’s ridley sea turtle , Hawksbill sea turtle and the leatherback sea turtle ), the gulf and Alabama sturgeons, and the West Indian manatee occur in aquatic habitats, and their habitat systems (bays, lagoons, salt marshes, creeks, ship channels, and other saltwater and freshwater environments) do not overlap with the trial site. The inflated heelsplitter mussel occurs in the Tombigbee and Black Warrior rivers. The southern clubshell mussel occurs in major streams of the Mobile river basin. The closest stream where these mussels could possibly occur is at least a half a mile away from the release site so their habitat does not overlap with the trial site. The wood stork primarily inhabits wetland systems notably cypress or mangrove swamps and would not use the field test site. The

p iping plover uses sparsely vegetated dunes and coastal beaches in southern Baldwin Count y, also far away from the field site (about 60 miles). The two mouse species listed above ( Alabama beachPage 71 of 91 mouse and Perdido Key Beach deermouse ) are found only in coastal dune areas and Perdido Key Beach, respectively, where they feed on sea oats, bluestem s, and a variety of insects. Both habitats are located 65+ miles from the proposed field trial (distance estimated using ArcGis Explorer ). The Eastern indigo snake is known to inhabit a wide range of habitats (agriculture fields, pine flat

woods, wet dep ressions, stream bottom thickets and margins of swamps). It appears to be very rare in Baldwin County where a case has been reported in an unknown location (US Forest Service). According to Jody Smithen (contacted February 20, 2008) of the Daphne Field O ffice USFWS, the species has not been documented in the county for many years, but there are occasionally unsubstantiated reports. They do not feel there is any concern. Although it is highly unlikely that the species would be found at the site, the appl icant 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 , the Alabama beach mouse and the p iping plover

. There is no designated critical habitat in the immediate environment of the field test. The closest critical habitat (for the Alabama Beac h mouse, Perdido Key Beach d eermouse and the p iping p lover ) is about 26 miles away. The gulf sturgeon has proposed critical habitat but this does not occur in Baldwin County. ( http://crithab.fws.gov/ accessed 8/31 /2 011)

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 d oes 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 prop osed for designation. Therefore, the action will have no effect on listed species or species proposed for listing and would not affect designated critical habitat or habitat proposed for designation.

2. Escambia County, AL

This location ha d previ ously been used as an intensely managed pasture for more than 5 years. The test site is currently planted with grasses suitable for cattle grazing. Approximately 0.5 acres of field trials of genetically modified Eucalyptus of some lines in the permit are b eing grown under issued permit 08

- 011

- 106rm under which these trees are allowed to flower. The oldest of these trees were planted in July 2007. Site preparation will involve herbicide application to remove existing grasses, subsoiling, preparation for possible irrigation, and planting of the test trees in flat beds. The surrounding areas of the test site consist of greater than 30 years

- old slash pine, and a re

forested area with mixed stands of pine and hardwood species. Under the new permit u p to 2.0 acres of field tests could be established at this location (at around 300

- 600 trees per acre) over the next three years.

Five TES animals are listed for Escambia County (list accessed from FWS website 8/ 31 /2011)

. The animals are gulf sturgeon, ( Acipens er Oxyrinchus desotoi ), red

- cockaded woodpeckerPage 72 of 91 ( Picoides borealis )

, wood stork ( Mycteria Americana ) , gray bat ( Myotis grisescens ) and e astern i ndigo snake ( Drymarchon corais couperi )

. The listed plant is American chaffseed ( Schwalbea
americana ). The USFWS has recently proposed the listing of several mussel species ( Federal Register / Vol. 76, No. 192 / 10/0 4 / 11) , six of which occur in Escambia County. These are the Alabama pearlshell ( Margaritifera marrianae )

, r ound ebonyshell ( Fusconaia rotulata ) , southern kidneyshell ( ( Ptychobranchus jonesi) , Choctaw Bean ( Villosa Choctawensis) , Narrow Pigtoe ( Fusconaia Escambia) , and the Fuzzy Pigtoe ( Pleurobema Strodeanum)

. The gulf sturgeon occurs in the Gulf of Mexico and spawns in freshwater rivers. It w ill not be affected by the field test since the closest river is over 3.5 miles away (as measured via ArcGis Explorer ). T he red cockaded woodpecker inhabits old growth forests, primarily longleaf pine. It could visit the field test site but would not nes t there. The wood stork primarily inhabits wetland systems notably cypress or mangrove swamps and would not find the field test site hospitable. The gray bat might visit the field test location but would not nest there. The eastern indigo snake is highly unlikely to be found at the site, however, the applicant will provide all workers with identifying characteristics of the snake and instructions on what to do if the species is encountered. The American chaffseed occurs

on sandy peat, sandy loam, acidic, seasonally moist to dry soils. As noted above it is generally found in habitats described as open, moist pine flatwoods, fire

- maintained savannas, ecotonal areas between peaty wetlands and xeric sandy soils, and other open g rass

- sedge systems and wound not occur in the vicinity of the field test site. None of the proposed mussel species would be found in the release site. Mussels are found on the bottoms of streams and rivers. The closest known location of any of these spe cies is the Conecuh River, approximately 3.5 miles away.

Critical Habitat: The designated critical habitat for the gulf sturgeon includes the Escambia River System in Santa Rosa and Escambia counties, Florida and Escambia, Conecuh, and Covington

coun ties, Alabama. The establishment of the field test site would not impact this habitat. It is about 3.5 miles away from the Conecuh river ( http://crithab.fws.gov/ accessed 8 / 31 /2011)

. There is also proposed critical habitat for a number of freshwater mussel species in Escambia County ( Federal Register / Vol. 76, No. 192 / 10/0 4 / 11). Six of these habitats occur in Escambia County. These are Alabama pearlshell ( Margaritifera marrianae )

, r ound ebonyshell ( Fusconaia rotulata)

, southern kidneyshell ( ( Ptychobranchus jonesi) , Choctaw Bean ( Villosa Choctawensis) , Narrow Pigtoe ( Fusconaia Escambia) , and the Fuzzy Pigtoe ( Pleurobema Strodeanum) . The proposed critical habitat for these species in Escambia County occurs in Burnt Corn Creek, and in the lower Escambia and Conecuh rivers , and is limited to the creek and river channels up to the ordinary high

- water line . The closest CH to the release location is about 3.5 miles away in the Conecuh river.

Conclusion: No fede rally 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 constituen t 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. The refore, the action will have no effect on listed species or species proposed for listing and would not affect designated critical habitat or habitat proposed for designation.

3. Highlands County, FLPage 73 of 91 This location has been used for field trials of transgenic Eucalyptus trees for at least 5 years. Approximately 5

. 2 acres of field trials of genetically modified Eucalyptus trees are being grown under issued permit s 08

- 014

- 101rm , 09

- 070

- 101rm and 11

- 041

- 101rm at this location . The oldest of these trees were plant ed in July 2006. Current and future site preparation involves herbicide application, plowing, and planting of trees in flat beds. The test site is surrounded by a citrus production area and second

- growth pine and hardwood forests. Under the new permit u p to 4.0 acres of additional tests could be established under this permit.

Fifteen TES animals, nineteen TES plants, and one TES lichen are listed for Highland s County (list accessed from FWS website 9 / 1 /2011). . 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 ( Aphelocoma coerulescens ), Florida grasshopper s parrow ( Ammodramus savannarum flori danus ), 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

– similari ty of appearance), West Indian manatee ( Trichechus manatus ), Eastern i ndigo s nake ( Drymarchon corais couperi ), b luetail mole s kink ( Eumeces egregius 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 ( Crotalaria avonensis ), Garrett’s mint ( Dicerandra christmanii ), scrub m int ( Dicerandra frutescens ), scrub b uckwhea t ( 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 swamps and would not find the field test site hospitable. The crested caracara ( Caracara cheriway ) is associat ed with open country; dry prairie with scattered cabbage palms, wetter prairies, and to some extent also improved pastures and sometimes wooded areas having associated limited areas of open grassland. The center of range is the Kissimmee Prairie, an area of shallow ponds and sloughs with scattered hummocks of live oaks and cabbage palms. 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 sand, in open areas without a dense canopy associated with Palmetto, sand pine and rosemary. This includes scrub with no canopy, sandpine scrub, scrubby flatwoods, and coastal scrub. The Florida grasshopper sparrow ( Ammodramus

savannarum floridanus ) prefers dry prairie with stunted saw palmetto and dwarf oaks, bluestems and wiregrass and unimproved cattle pastures. Its habitat is maintained by periodic fires. The snailPage 74 of 91 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 States and Cuba and has declined to extinction or near extinction. It once occupied swampy forests, especially large bottomland river swamps of coastal plain and Mississippi Delta and cypress swamps of Florida, in areas with many dead and dying trees. It would not occur in an agricultural environment such as the field test site. Whooping crane ( Grus

a mericana ) pr efers freshwater marshes and wet prairies. It nests in dense emergent vegetation (sedge, bulrush) in shallow ponds, freshwater marshes, wet prairies, or along lake margins, within large expanses of undisturbed wilderness. The cranes listed in this count y are an experimental reintroduction of whooping cranes in Florida initiated in 1993 to establish a non

- migratory population. This is an experimental, non

- essential population

. Any of the bird species could potentially visit the field test site but woul d not nest there. The Florida panther ( Puma concolor coryi ) generally occurs in heavily forested areas in lowlands and swamps, also upland forests in some parts of range; areas with adequate deer or wild hog population. Habitats include tropical hammocks , pine flatwoods, cabbage palm forests, mixed swamp, cypress swamp, live oak hammocks, sawgrass marshes, and Brazilian pepper thickets. It depends on large contiguous blocks of wooded habitat, though interspersed fields and early successional habitats may be beneficial through their positive effect on prey populations. Its day

- use sites typically are dense patches of saw palmetto surrounded by swamp, pine flatwoods, or hammock. It would not occur in the trial area due to the openness and continued presen ce of humans in the area. The West Indian

manatee ( Trichechus manatus ) occurs in shallow coastal waters, rivers, bays and lakes; none of which are close to any of this release location. For the Eastern indigo snake ( Drymarchon cosair couperi )

– accordin g to Candice Martino (904

- 232

- 2580 ext. 129) Section 7 Endangered Species biologist from the Jacksonville, FL Field Office, (contacted February 25, 2008) the species is seldom seen but could be anywhere. However, the habitat at the release site would not be suitable. The historic and continuous use of the release site and the surrounding area as a citrus grove and Eucalyptus field trials makes it extremely unlikely that the species would be found in the area. Therefore, the appropriate determination woul d 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

measure s are a variation of standard protective measures the USFWS uses when they have reached a “may affect” determination for construction sites

.
The bluetail mole skink ( Eumeces egregius lividus ) inhabits sand pine

- rosemary scrub or, less frequently, longleaf pine

- turkey oak association sandhills. It occupies localized pockets of sufficient leaf litter and moisture to provide abundant food and nesting sites. The sand skink ( Neoseps reynoldsi ) occurs only on Florida's central ridges, at elevations of 27 m or more. It inhabits loose sands of sand pine

- rosemary scrub, less often longleaf pine

- turkey oak sandhills or turkey oak barrens adjacent to scrub, especially high pine

- scrub ecotones. It was determined that the release site is within a geographic area whe re these two skink species are found. According to Brad Rick, (contacted February 27 and 28, 2008) of the Vero Beach Field Office USFWS, sand skinks and bluetail mole skinks are found in scrub habitat with areas of open sand. The literature indicates that skinks are sometimes found in active and abandoned citrus groves and the applicant confirmed that sandy soils are predominant in the area. It was decided to have the applicant conduct a survey of the species using USFWS protocols to determine if the spec ies is present. The protocols were provided by the Vero Beach Field Office. The USFWS protocol recommends thatPage 75 of 91 surveys be conducted between March 1 and May 15 as this is an ideal time to observe evidence of the skinks. A coverboard survey was conducted over a one month period from March 18 to April 15 , 2008

. The coverboards were checked weekly on March 25, April 1, April 8 and April 15. No evidence indicating the presence of sand skinks or bluetail mole skinks was observed and they are presumed absent. Therefore, the appropriate determination would be “no effect.”

For the plants, the Florida bonamia ( Bonamia grandiflora ) grows in natural clearings of bare ground and invades disturbed areas of open sand. Although not common, it is often locally abunda nt where there is little or no shade from trees or shrubs. It is locally abundant on deep, white, dry sands of ancient dunes and sandy ridges in clearings or openings of scrub habitat on the Central Ridge of Florida. Pygmy f ringetree ( Chionanthus pygmaeu s ) is generally found in xeric, coarse white sand of scrub/oak scrub areas found at the southern end of the Central Florida Ridge. It is also found occasionally in longleaf pine

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

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

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

- oak scrub with scattered overstory of sand pine and scrub oak. Avon Park hare

- bells ( Crotalaria avonensis ) occurs in upland habitats (scrub and sandhill), often along tr ails. 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 frut escens ) 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 foun d in areas cleared for pasture, or areas in which wholesale site prepa ration has taken place. Scrub b uckwheat ( Eriogonum longifolium var. gnaphalifolium ) is long

- lived, slow growing and flowers and reproduces primarily after fires or other disturbances (e .g. logging, mowing) that increase light availability. It prefers dry pinelands, sandhills, and scrub (longleaf pine

- turkey oak, scrub oaks) and is more commonly found in transition habitats between scrub and high pine and in turkey oak barrens than in ei ther dense scrub or open high pine. S nakeroot ( Eryngium cuneifolium ) is generally found in areas of open sand, including blowouts and other highly disturbed soil surfaces, such as road shoulders. It occurs in exposed sunny openings; areas in scrub, espe cially rosemary scrub. Highlands s crub hypericum ( Hypericum cumulicola ) occurs in patches of open, nutrient

poor sand within oak and rosemary scrub. It is often associated with reindeer lichen ( Cladonia spp.) and snakeroot ( Eryngium cuneifolium ). Scrub blazingstar ( Liatris ohlingerae ) occurs in openings in oak

- rosemary scrub and sand pine scrub. Britton's b ear

- grass ( Nolina brittoniana ) occurs in deep, fine

- textured, well

- drained sands of sand pine

- evergreen oak scrub or longleaf pine

turkey oak sandhil ls. Nolina is entirely dependent on fire or some other mechanism to maintain an 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 w hite sand cl earings or blowouts. 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 pow erline 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.Page 76 of 91 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, excessively leache d, 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 ( Ziziphus celata ) is a scrub that occurs on gently rolling hills with vegetation dominated by Carya floridana and Quercus species. It prefers open, sunny areas. The Perforate Reindeer Lichen ( Cladonia perforate ) occurs in sandy openings in stabilized sand dunes with Flo rida 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 su rrounding 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 ( http://crithab.fws.gov/ accessed 9/1/2011).

Conclusion: No federally listed threatened or endangered species or specie s 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 w ill 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 specie s or species proposed for listing and would not affect designated critical habitat or habitat proposed for designation

4. Pearl River County, MS

This location has been an agricultural research station for more than 5 years. The location has been used for co nducting research experiments with agricultural crops and grasses. The test site has been used for experimental planting of grasses. Approximately 3 acres of field trials of genetically modified Eucalyptus trees of some lines in the permit are being grown under issued permit 08

- 011

- 106rm under which these trees are allowed to flower. The oldest of these trees were planted in October 2007. Current and future site preparation involves herbicide application to remove existing grasses, subsoiling, preparation f or possible irrigation installation, and planting of trees in flat beds. The surrounding areas of the test site consist of a grape research farm, mixed stands of hardwoods and pine, and a residential area. Under the new permit u p to 2.0 acres of field tests could be established at this location (at around 300

- 600 trees per acre) over the next three years.

There are six TES animals listed in this county and one TES plant (list accessed from FWS website 9/1 /2011) . The animals are the r inged map turtle ( Graptemys oculifera ), g opher t ortoise ( Gopherus polyphemus ), Louisiana black bear ( Ursus a. luteolus ), g ulf sturgeon ( Acipenser oxyrhynchus desotoi ) , i nflated heelsplitter ( Potamilus inflatus ) and r ed

- cockaded woodpecker

. The listed plant is Louisiana q uillwort ( Isoetes louisianensis ).Page 77 of 91 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 longl eaf pine forests, but its numbers have decreased with the replacement of longleaf pine fores ts with loblolly pine forests. This field test is locate d in an agricultural research station that would be an inhospitable environment for the gopher tortoise. Ac cording to James Harris (contacted February 6 and 7, 2008), Supervisory Wildlife Biologist with the USFWS in Lacombe, LA, the species is found in a large geographic area that includes the release site. However, the species is not found everywhere within t his geographic area. The species is not likely to be on the site because of its location on a facility used for many years as an agricultural research station. The research facility was contacted to determine if gopher tortoises have been observed at the facility. The farm manager has not observed the species at the facility, and no sightings have been reported to him during his nine years as manager. He is familiar with the species and has seen them at another location about 7

- 8 miles from the release site. Another employee contacted has worked at the site for over 35 years and has never seen a gopher tortoise at the facility but did observe one approximately ten years ago about 1⁄4 mile from the facility. The applicant surveyed the site for the

presenc e of gopher tortoise burrows on January 29, 2008 and none were found. Considering the use of the facility, testimony of the facility employees, and the negative result of the survey, it can be concluded that the species is not present now and would be unl ikely 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 rem ote areas with little or no human activity so it would not likely be found at the site. T he red cockaded woodpecker ( Picoides borealis ) inhabits old growth forests, primarily longleaf pine. It might visit the field test site but would not nest there. Th e gulf sturgeon, the inflated hellsplitter mussel, and the Louisiana quillwort occur in aquatic environments so would not be affected by the field test.

Critical habitat: The gulf Sturgeon has designated critical habitat in this county in the Pearl River system. The Pearl River is about 11 miles from the field test site. Tributaries of the Pearl River system are approximately 3.5 and 7.7 miles from the test site ( http://crithab.fws.gov/ accessed 9/1/2011).

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the release site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain constituent elements required by the species. Field activities will result in no changes to the habitat used by any listed species or species

propos ed 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 o r habitat proposed for designation.

5. Berkeley County, SC

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

- transgenic plants for more than 8 years. The 0.5 acre release site is located adjacent toPage 78 of 91 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. Tr ees will not be allowed to flower at this location.

Five threatened or endangered animals and three endangered plants are listed in Berkeley County (list accessed from FWS website 9/1 /2011) . The TES animal species are shortnose sturgeon ( Acipenser brevir ostrum ), frosted flatwoods salamander ( Ambystoma cingulatum ), red

- cockaded woodpecker ( Picoides borealis ), West Indian manatee ( Trichechus manatus ), wood stork ( Mycteria americana ). The TES Plants are pondberry ( Lindera melissifolia ), Canby's dropwort (Ox ypolis canbyi ) and American chaffseed ( Schwalbea Americana ).

Canby’s dropwort is an herbaceous perennial whose existing populations are maintained mainly through asexual reproduction. This species is strongly clonal, reproducing vegetatively by means

o f stoloniferous rhizomes. It has been found in a variety of habitats, including cypress ponds,

grass

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

pine swamps or sloughs. The largest and most vigorous populations re ported occur in open bays or ponds which are flooded throughout most of the year and which have little or no canopy cover. It grows in soils with a medium to high organic content, high water table, that are deep, poorly drained, and acidic. The pondberry occurs in similar locations, in wetland habitats such as bottomland and hardwoods in the interior areas, and the margins of sinks, ponds and other depressions in the more coastal sites. The plants generally grow in shaded areas but may also b e found in full sun. The American chaffseed occurs on sandy peat, sandy loam, acidic, seasonally moist to dry soils. It is generally found in habitats described as open, moist pine flatwoods, fire

maintained savannas, ecotonal areas between peaty wetland s and xeric sandy soils, and other open

grass

- sedge systems. The site in Berkeley County is a fenced research plot/holding area which would be very inhospitable to these species. Laura Zimmerman (843

- 727

- 4707 ext. 226) (contacted February 14 and 20, 2008 ) of the Charleston Field Office of the USFWS states that she does not believe the species would be likely to be in the area. According to the species’ recovery plan, most known occurrences are on US Forest Service land and the only two occurrences on pri vate land are not near the release site. A check of the SC Heritage Trust Database did not identify any occurrences in the area of the release.

The shortnose sturgeon occurs in rivers and estuaries. The West Indian manatee occur s in shallow coastal wat ers, rivers, bays and lakes; none of which are close to this release location. The red cockaded woodpecker inhabits old growth forests, primarily longleaf pine and might visit the field test site but would not nest there. The wood stork primarily inhabit s wetland systems notably cypress or mangrove swamps and would not find the field test site hospitable. The frosted flatwoods salamander inhabits longleaf or slash pine forests lying between drier land upslope and wetlands and seasonally inhabits wet pine flat

- woods with vernal pools. Discussions with Laura Zimmerman (843

- 727

- 4707 ext. 226) (contacted February 14 and 20, 2008) of the Charleston Field Office of the USFWS indicate that the species is not known to be in the area of the release. Known popula tions in the county are far from the release site in the Francis Marion National Forest. A check of the SC Heritage Trust Database did not identify any occurrences in the area of the release.Page 79 of 91 Critical Habitat: There is critical habitat for the frosted flatwoods salamander in Berkeley County. This is located in the Francis Marion National Forest which is about 19 miles away from the release location ( http://crithab.fws.gov/ accessed 9/1/2011).

Conclusion: No fed erally listed threatened or endangered species or species proposed for listing are likely to found at the release site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain constituent elements required by the species. Field activities will result in no changes to the habitat used by any listed species or species proposed for listing. The site is not within or near designated critical habitat or habitat proposed for designation. There fore, the action will have no effect on listed species or species proposed for listing and would not affect designated critical habitat or habitat proposed for designation.

6. Dorchester County, SC

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

transgenic plants. This location was previously cleared in 2010 for development as a light industrial park, prior to which it had been a managed pine plantation for more than 15 years. The surrounding area consists of man

- made ponds, hardwoods and pine plantations. This site is a secure fenced holding area where trees growing in containers are transferred from the greenhouse to th e out

- of

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

Five threatened or endangered species (TES) are listed in Dorchester County (TES list dated March 2010 obtained from FWS website 8 / 1 6/11). The TES animal species are shortnose sturgeon ( Acipenser brevirostrum ), red

- cockaded wo odpecker ( Picoides borealis ) and wood stork ( Mycteria americana ). The TES Plants are pondberry ( Lindera melissifolia ) and Canby's dropwort (Oxypolis canbyi ).

Canby’s dropwort is an herbaceous perennial whose existing populations are maintained mainly

t hrough asexual reproduction. This species is strongly clonal, reproducing vegetatively by means of stoloniferous rhizomes. It has been found in a variety of habitats, including cypress ponds,

grass

- sedge dominated Carolina bays, wet pine savannahs, shall ow 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 occurs in similar locations, in wetland habitats such as bottomland and hardwoods in the interior areas, and the margins of sinks, ponds and other depressions in the more coastal sites. The plants generally grow in shaded areas but may also be found in full sun. The site in Dorchester County is a fenced research plot/holding area which would be very inhospitable to these species. It is located in an industrial park that was established a year ago so no new land is being disturbed.

The shortnose sturgeon occurs in rivers and estuaries. The red cockaded woodpecker inhabits old growth forests, primarily longleaf pine and might visit the release site but would not nest there. The wood stork primarily inhabits wetland systems notably cypress or mangrove swamps and would not find the field test site hospitable.Page 80 of 91 Critical Habitat: There is no critical habitat or proposed critical habitat list for Dorchest er County ( http://criticalhabitat.fws.gov/crithab/ accessed 8 / 1 6/11)

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to found at the relea se site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain constituent elements required by the species. Field activities will result in no changes to the habitat used by any listed species or species proposed for listing. The site is not within or near designated critical habitat or habitat proposed for designation. Therefore, the action will have no effect on listed species or species proposed for listing and would not affect des ignated critical habitat or habitat proposed for designation.

7. Interstate Movement

APHIS considered the possible effects that interstate movement of GE Eucalyptus trees authorized by APHIS could potentially have on threatened or endangered species and th ei r critical habitat. APHIS could authorize the interstate movement of transgenic Eucalyptus trees in accordance with the regulatory requirements identified in 7 CFR 340.4, 340.7 and 340.8, including specific permit conditions ( see Appendices VI) assigned to this permit that would prevent the dissemination of the trees into the environment. As specified in the supplemental permit conditions, ArborGen would be moving trees in accordance with an APHIS approved variance that only allows trees to be moved in labeled containers within an enclosed vehicle. For the time period between 2005 to 2010, APHIS issued 218 permits and notifications that authorized the interstate movement of forest

trees. Of these 218 authorized movements, there was one compliance incid ent associated with a shipment of plants via mail. This compliance incident was associated with a movement that did not have similar APHIS approved variances as those that would be required for ArborGen to move GE Eucalyptus trees under this permit reques t. From 2005 to 2010, no compliance incidents of unauthorized release were reported to APHIS for moving GE Eucalyptus trees using similar APHIS approved variances that would be required to move trees under this permit request.

Conclusion: Considering the specific permit conditions that must be adhered to by the permit requirements identified in 7 CFR 340.4, 340.7 and 340.8, the conditions specified under the APHIS approved variance, the proposed supplemental permit conditions for interstate movement of the regulated article, and past compliance history of similar types of movements, APHIS has determined that the movement of GE Eucalyptus trees in accordance with APHIS approved supplemental permit conditions will have no effect on listed spec ies or species proposed for listing and would not affect designated critical habitat or habitat proposed for designation.

Overall Conclusions

The field test sites in th ese permit application s have been in agricultural or forest research, or in agricultur al production or forest tree plantations for from 5 to 1 5 years

. No federally listed threatened or endangered species or species proposed for listing are likely to be found at any of the release sites. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not c ontain 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 sitesPage 81 of 91 are not within or near designated critical habitat or habitat proposed for de signation. Potential effects from moving trees would be negligible to non

- existent due to t he applicants adherence to regulatory requirements identified in 7 CFR 340.4, 340.7 and 340.8, including specific permit conditions ( see Appendi x VI) assigned to th is permit that would prevent the dissemination of the trees into the environment. Therefore, the action will have no effect on listed species or species proposed for listing and would not affect designated critical habitat or habitat proposed for designat ion. Because of this no effect determination, consultation and/or the concurrence of the USFWS and/or the NMFS are not required.Page 82 of 91 

## APPENDIX V: Proximity of release sites to Protected Areas

The proposed release locations were accessed for their p roximit y to State and Federal p rotected

a reas and any p otential f oreseeable i mpacts to these areas . The analysis used the Protected Areas Database (PAD

- US 1.1) ( http://www.databasin.or g/protected

- center/features/PAD

- US

- CBI ) loaded into ArcGis Explorer. Proximity to the protected areas was determined using distance measuring tools in ArcGis Explorer.

Baldwin County, AL

Bon Secour National Wildlife Refuge is located in Baldwin County http://www.fws.gov/bonsecour/ . The Bon Secour National Wildlife Refuge comprises provides habitat for the endangered Alabama beach mouse and serves as a nesting site for loggerhead , and includes the Kemp ’s Ridley sea turtl e , the Perdido Key beach mouse and the piping plover. The Baldwin County release location is approximately 21 miles away from the Bon Secour National Wildlife Refuge. Another protected area located in Baldwin County is the Mobile

- Tensaw Delta Wildlife Management Area. http://www.dcnr.state.al.us/public

lands/stateLands/foreverwild/FWTracts/mobile

- tensawComplex/

. It is located a pproximately 16 miles from the field test location.

Escambia County, AL

Conecuh National Forest is located in Escambia County http://www.stateparks.com/conecuh.html

. Clear

- cut in the 1930s, the Cone cuh was reforested with slash pine that reduced the number of nesting trees for the endangered red

- cockaded woodpecker. The forest is currently undergoing a reforestation from slash pine to the native longleaf. In time, this should increase the number of r ed

cockaded woodpeckers as the trees mature. The Escambia County release location is approximately 8.5 miles from the western edge of the forest

. T he Perdido River Wildlife Management Area is approximately 35 miles away http://myfwc.com/viewing/recreation/wmas/cooperative/Perdido

- River . The northern border of Blackwater River State Forest in Florida is located about a mile away from the release location http://www.fl

- dof.com/state_forests/blackwater_river.html . Blackwater River State Forest is known for its longleaf pine/wiregrass ecosystem, which, in combination with the Conecuh National Forest to t he north and Eglin Air Force Base to the south, is the largest contiguous ecological community of this type in the world.

Highlands County, FL

Wales Ridge National Wildlife Refuge occurs in Highlands County http://www.fws.gov/lakewalesridge/ . The release location is about 7 miles from one section of the refuge. The refuge contains twenty

- two different threatened or endangered species of plants. Highlands Hammock State Park is also located in this count y and the field test site is adjacent to one border of the State park. http://floridastateparks.org/highlandshammock/

. Th is park is known for its old

- growth hammock, cypress swamps and offers a number of recreational activities.Page 83 of 91 Pearl River County, MS

Bogue Chitto National Wildlife Refuge occurs in Pearl River County http://www.fws.gov/boguechitto/ . The refuge’s bottomland forests contain sweetgum

- w ater oak stands interspersed with bald cypress

- tupelo brakes and overcup oak

- water hickory stands. It includes habitat for a number of endangered species. The release site is approximately 9 miles away from the eastern boundary of the refuge.

Berkeley County, South Carolina.

Berkeley County, Georgia contains parts of the Francis Marion National Forest which provides habitat for black bear, alligator, and the endangered red

- cockaded woodpecker http://www.fs.usda.gov/scnfs/ . T he field test site is located approximately 15 miles from Francis Marion National Forest.

Dorchester County, SC.

Dorchest er County contains Colonial Dorchester State Historic Site http://www.southcarolinaparks.com/park

- finder/state

- park/725.aspx and Givhans Ferry State Park http://www.southcarolinaparks.com/park

- finder/state

- park/1219.aspx . Both of these areas are protected recreational areas maintained by the State of South Carolina. The field test site is loca ted approximately 5 miles from Givhans Ferry State Park and approximately 7 miles from the Colonial Dorchester State Historic Site.Page 84 of 91 

## APPENDIX VI: Proposed Supplemental Permit Conditions for Movement

For Movement of Eucalyptus grandis x Eucalyptus urop hylla under permit 11

- 052

- 101rm

1. BRS should be notified in writing of any proposed changes to the permit application (or approved permit) including for example changes in movement protocols, additional transgenic lines or constructs, new destinations, or amount introduced. Changes usually require amendments to the permit and must be pre

- approved by BRS. Requests should be directed to Regulatory Permit Specialist, USDA APHIS BRS, Biotechnology Permit Services, 4700 River Road, Unit 147, Riverdale, Maryland 20737.

2. The regulated article is to be shipped in containers as specified in the Title 7 Code of Federal Regulation Section 340.8 (7 CFR 340.8) unless a variance request has been reviewed and approved by APHIS/BRS. BRS Notes that the trees would be moved under a previously approved Variance 07

- 014.

3. While in storage all regulated articles covered under this permit are to be kept in a locked storage facility with limited access to only authorized personnel. Storage containers must be identified as containi ng a genetically engineered regulated article. At least one sign stating "Authorized Personnel Only" must be posted in each area where the regulated article is stored.

4. Upon completion of research, all regulated articles (except those retained for future s tudies) should be rendered non

- viable by an appropriate method (e.g., heat or steam sterilization, bleach treatment, etc.).

5. There is to be no further distribution of this regulated article under this permit without prior approval from State (intrastate mo vement) and Federal regulatory officials (interstate movement).

6. APHIS/BRS and/or APHIS/PPQ personnel may conduct inspections of facilities and/or records at any time.

7. Reporting an Unauthorized or Accidental Release

1. According to the regulation in 7 C FR § 340.4(f)(10)(i), APHIS shall be notified orally immediately upon discovery and notified in writing within 24 hours in the event of any accidental or unauthorized release of the regulated article.

- For immediate verbal notification, contact APHIS BRS Com pliance Staff at (301) 734

- 5690 and ask to speak to a Compliance and Inspection staff member. Leave a verbal report on voicemail if the phone is not answered by a Compliance Officer.

- In addition, in the event of an emergency in which you need to spe ak immediately to APHIS personnel regarding the situation, you may call: The APHIS/BRS Regional Biotechnologist assigned in the region where the field test occurs:Page 85 of 91 For Western Region, contact the Western Region Biotechnologist at (970) 494

- 7513

or e

- mail: BRSWRBT@aphis.usda.gov For Eastern Region, contact the Eastern Region Biotechnologist at (919) 855

- 7622 or e

- mail: BRSERBT@aphis.usda.gov

Or

The APHIS State Plant Health Director for the state where the unauthorized release occurred. The list of APHIS State Plant Health Directors is available at: http://www.aphis.usda.gov/services/report_pest_disease/report_pest_disease.shtml.

or http://pest.ceris.purdue.edu/stateselect.html

2. Written notification should be sent by one of the following means:

By e

- ma il: BRSCompliance@aphis.usda.gov

By mail: Biotechnology Regulatory Services (BRS) Regulatory Operations Program USDA/APHIS 4700 River Rd. Unit 91 Riverdale, MD 20737

3. Additional instructions for reporting compliance incidents may be found at http://www .aphis.usda.gov/biotechnology/compliance_incident.shtml

8. No person shall move a regulated article interstate unless the number of the limited permit appears on the outside of the shipping container.Page 86 of 91 

## APPENDIX V I I : Proposed Supplemental Permit Conditions fo r Release

For Release of Eucalyptus grandis x Eucalyptus urop hylla under permit 11

- 052

- 101rm

1. The test sites and adjacent land within 100 meters shall be monitored for any volunteer Eucalyptus plants every 6 months during the field test (as indicated in the permit) and for one year after completion of the field test, during which time any volunteer plants will be destroyed before they flower. During the monitoring period following completion of the field test, the site will not be planted with Eucalyptus , so that any volunteer seedlings that emerge can be easily identified. If volunteers or stump sprouts are still emerging at the end of the first year, a second year will be added to the monitori ng period to ensure that no shoots are continuing to be produced. (2) Please note that transportation of all test and plant materials to and from the field test location must be done in accordance with APHIS/USDA regulations outlined in "Container requirements for the movement of regulated articles", 7CFR 340.8(b) unless a shipping container variance has been approved by APHIS

- BRS.

2. Any unusual flowering or seed set much be reported to APHIS as an unusual occurrence. If there is an overabundan ce of flowers or seeds produced this must be reported as soon as observed.

3. BRS should be notified in writing of any proposed changes to the permit application (or approved permit) including for example confinement protocols, transgenic lines or constructs, rel ease locations, acreage, etc. Changes usually require amendments to the permit and must be

pre

- approved by BRS. Requests should be directed to Regulatory Permit Specialist, USDA APHIS BRS, Biotechnology Permit Services, 4700 River Road, Unit 91, Riverdale, Maryland 20737.

4. Any regulated article introduced not in compliance with the requirements of 7 Code of Federal Regulation Part 340 or any standard or supplemental permit conditions, shall be subject to the immediate application of such remedial measures o r safeguards as an inspector determines necessary, to prevent the introduction of such plant pests. The responsible party may be subject to fines or penalties as authorized by the Plant Protection Act (7 U.S.C. 7701

- 7772).

5. This Permit does not eliminate t he 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.Page 87 of 91 7. Harvested plant material may not be used for food or animal feed unless it is first devitalized and approved for such use by the U.S. Food and Drug Administration; and for plant

incorporated prote ctants, a tolerance for the pesticide must first be established by the U.S. Environmental Protection Agency.

8. Reporting an Unauthorized or Accidental Releas e

1. According to the regulation in 7 CFR § 340.4(f)(10)(i), APHIS shall be notified orally immedia tely 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 Com pliance and Inspection staff member. Leave a verbal report on voicemail if the phone is not answered by a Compliance Officer.

- In addition, in the event of an emergency in which you need to speak immediately to APHIS personnel regarding the situation, you may call: The APHIS/BRS Regional Biotechnologist assigned in the region where the field test occurs: For Western Region, contact the Western Region Biotechnologist at (970) 494

- 7513

or e

- mail: BRSWRBT@aphis.usda.gov For Eastern Region, contact the Eastern Region Biotechnologist at (919) 855

- 7622 or e

- mail: BRSERBT@aphis.usda.gov Or The APHIS State Plant Health Director for the state where the unauthorized release occurred. The list of APHIS Stat e Plant Health Directors is available at: http://www.aphis.usda.gov/services/report_pest_disease/report_pest_disease.shtml

. o r http://pest.ceris.purdue.edu/st ateselect.html 2. Written notification should be sent by one of the following means: By e

- mail: BRSCompliance@aphis.usda.gov By mail: Biotechnology Regulatory Services (BRS) Regulatory Operations Program USDA/APHIS 4700 River Rd. Unit 91 Riverdale, MD 2073 7 3. Additional instructions for reporting compliance incidents may be found at http://www.aphis.usda.gov/biotechnology/compliance_incident.shtml

9. R eporting Unintended Effects:

According to the regulation in 7 CFR § 340.4(f)(10)(ii), APHIS shall be notified in writing as soon as possible but within 5 working days if the regulated article or associated host organism is found to have characteristics substantially differen t from those listed in the permit application or suffers any unusual occurrence (excessive mortality or morbidity, or unanticipated effect on

non

- target organisms). Written notification should be sent by one of the following means:Page 88 of 91 By e

- mail: BRSCompliance@aphis.usda.gov By mail: Biotechnology Regulatory Services (BRS) Regulatory Operations Program USDA/APHIS 4700 River Rd. Unit 91 Riverdale, MD 20737

10. Reports and Notices: Send notices and all reports (CBI and CBI

- deleted or non

- CBI copies) to BRS by e

- mail, mail, or fax. BRS E

- mail: BRSCompliance@aphis.usda.gov BRS Mail: Animal and Plant Health Inspection Service (APHIS) Biotechnology Regulatory Services (BRS) Regulatory Operations Program 4700 River Rd. Unit 91 Riverdale, MD 20737 BRS Fax: Reg ulatory Operations Program (301) 734

- 8910

a. Planting or Environmental Release Report

Planting reports must be submitted to BRS by the 15th of the month following the month in which the environmental release was started and must include the following dat a:

- Permit number

- Regulated article

- State

- County

- Location Name (Unique ID)

- GPS coordinates of the planting

- Planting Unique ID

- Planting Start Date

- Total acreage of the regulated article planted or otherwise released

- List of all constructs planted (This list is optional in the planting report but must be included in the Annual and Field Test Reports)

b. Annual Report

Within 30 days after the anniversary date (one

- year increments from the effective date listed on the permit) an Annual Repo rt must be submitted to APHIS. If the permit has been amended, the Annual Report is due 30 days from the anniversary date (one year increments from the effectivePage 89 of 91 date) of the ORIGINAL permit. FAILURE TO SUBMIT ANNUAL REPORTS MAY RESULT IN REVOCATION OF THE PERMIT. The Annual Report shall reflect the current status and observations to date for each location. It shall include:

- Permit number

- State

- County

- Location Name(s)

- Location Unique ID(s)

- Any plantings that occurred at each location

- GPS coor dinates for each planting

- Size of the plantings (in acres) at each location

- Phenotypic designations (all constructs that were planted)

- Include an accounting of the total acreage that remains in the ground

- Include a detailed map of the plantings

- Indicate if any of the planted material was destroyed before harvest If so provide the Pre

- Harvest destruction completion date and describe how the pre

harvested m aterial was destroyed

- Indicate if any of the planted material was harvested If so provide t he harvest completion date

- Describe how the harvested material was terminated

- If the material was terminated in the field and not removed from the field, provide the date the

f ield test was completely terminated and describe the method of termination

- If the material was removed from the field and terminated off site describe how it was disposed and provide the date of off site destruction.

- If material was removed from the field and placed in storage, provide the amount of material that was stored an d provide a description of the storage location

- Describe any other disposition methods that may be applicable

- Describe any deleterious effects on plants, non

- target organisms, or the environment

- Describe methods of observations and resulting data and analyses

- Indicate if you have submitted any of the following: 1. A report on the accidental or unauthorized release of the regulated article; 2. A report that characteristics of the permitted species are substantially different from those listed in the application; or 3. A report of any unusual occurrence Also include a ny reports of any overabundance of flowering or seed set that occur r ed and how th is was handled.

We encourage the inclusion of other types of data if the applicant anticipates submission of a petition for determination of non

- regulated status for their regulated article. APHIS considers these data reports as critical to our assessment of plant pest risk and development of regulatory policies based on the best scientific evidence. Failure by an applicant to provide data reports in a timely manner for a field trial may result in the withholding of permission by APHIS for future field trials.

c. Field Test ReportPage 90 of 91 Within six months after the expiration date of the permit, the permittee is req uired to submit a Field Test Report. Field Test Reports provide the final status and observations at each location and must include:

- Permit number

- State

- County

- Location Name(s)

- Location Unique ID(s)

- Any plantings that occurred at each location

- GPS coordinates for each planting

- Size of the plantings (in acres) at each location

- Phenotypic designations (all constructs that were planted)

- Indicate if any of the planted material was destroyed before harvest If so provide the Pre

- Harvest destru ction completion date and describe how the pre

- harvested material was destroyed

- Indicate if any of the planted material was harvested If so provide the harvest completion date

- Describe how the harvested material was terminated

- If the material was ter minated in the field and not removed from the field, provide the date the field test was completely terminated and describe the method of termination

- If material was removed from the field and terminated off site describe how it was disposed and provide the date of off site destruction.

- If material was removed from the field and placed in storage, provide the amount of material that was stored and provide a description of the storage location

- Describe any other disposition methods that may be applicab le

- Describe any deleterious effects on plants, non

- target organisms, or the environment

- Describe methods of observations and resulting data and analyses

- Indicate if you have submitted any of the following: 1. A report on the accidental or unauthorized release of the regulated article; 2. A report that characteristics of the permitted species are substantially different from those listed in the application; or 3. A report of any unusual occurrence Also include any reports of any overabundanc e of flowering or seed set that occurred and how this was handled.

We encourage the inclusion of other types of data if the applicant anticipates submission of a petition for determination of non

- regulated status for their regulated article. APHIS conside rs these data reports as critical to our assessment of plant pest risk and development of regulatory policies based on the best scientific evidence. Failure by an applicant to provide data reports in a timely manner for a field trial may result in the with holding of permission by APHIS for future field trials.

d. Volunteer Monitoring Report

T he report must include:Page 91 of 9 1 - Permit number

- State

- County

- Location Name(s)

- Location Unique ID(s)

- Dates when the field site and perimeter fallow zone were inspected for volunteers

- Number of volunteers observed

- Any actions taken to remove or destroy volunteers The final monitoring report is due no later than three months from the end of the volunteer monitoring period.