1

United States Department of Agriculture

Marketing and Regulatory Programs

Animal and

Plant Health Inspection
Service 

# Permit applications 08-011-106rm

and 08-014-101rm received from

ArborGen LLC

Field testing of genetically engineered Eucalyptus grandis X Eucalyptus urophylla

Revised Draft Environmental

Assessment December 17, 2009

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 require2

alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’S TARGET Center at (202) 720–2600 (voice and TDD).

To file a complaint of discriminatio n, write USDA, Director, Office of Civil Rights, Room 326–W, Whitten Building, 1400 Independence Avenue, SW, Washington, DC 20250–9410 or call (202) 720–5964 (voice and TDD). USDA is an equal opportunity provider and employer.

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

____________________________________ This publication reports research in volving 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 wildlif e—if they are not handled or applied properly. Use all pesticides selectiv ely and carefully. Follow recommended practices for the disposal of surplus pesticides and pesticide containers.3

TABLE OF CONTENTS

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

II. PURPOSE AND NEED.......................................................................................................... .... 5

A. P ROPOSED A CTION ................................................................................................................... 5

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

C. N EED FOR T HIS A CTION ........................................................................................................... 6

D. P URPOSE AND D ESCRIPTION OF THE R ESEARCH ................................................................... 7

III. AFFECTED ENVIRONMENT ............................................................................................... 7

IV. ALTERNATIVES.................................................................................................................... 15

A. N O A CTION

– D ENY THE P ERMIT .......................................................................................... 16

B. P REFERRED A LTERNATIVE

– I SSUE THE P ERMIT ................................................................. 16

V. ENVIRONMENTAL CONSEQUENCES .............................................................................. 17

A. N O A CTION .............................................................................................................................. 1 7

B. P REFERRED A LTERNATIVE .................................................................................................... 17

C. C UMULATIVE E FFECTS .......................................................................................................... 31

D. T HE D EGREE TO W HICH THE P OSSIBLE E FFECTS ARE H IGHLY U NCERTAIN OR I NVOLVE
U NIQUE OR U NKNOWN R ISKS ..................................................................................................... 32

E. R ISKS TO T HREATENED AND E NDANGERED S PECIES .......................................................... 36

F. O THER C ONSIDERATIONS ...................................................................................................... 36

G. C ONCLUSION ........................................................................................................................... 37

VI. LISTING OF AGENCIES AND PERSONS CONSULTED............................................... 38

VII. REFERENCES............................................................................................................... ........ 38

APPENDIX I: STATUS OF EXISTING FIELD TESTS ALLOWED TO FLOWER ............ 44

APPENDIX II: HYDROLOGY CONS IDERATIONS FOR PLANTED

EUCALYPTUS SUBMITTED BY ARBORGEN LLC................................................................ 46

APPENDIX III: USDA FORE ST SERVICE ASSESSME NT OF IMPACTS ON

HYDROLOGY...................................................................................................................... .......... 57

APPENDIX IV: THREATENED AND END ANGERED SPECIES ANALYSIS .................... 664

APPENDI X V: PROPOSED SUPPLEM ENTAL PERMIT CONDITIONS ............................ 935



## I. Summary

USDA/APHIS has prepared a draft environm ental assessment in response to permit applications (APHIS Number 08-011- 106rm and 08-014-101rm) received from ArborGen LLC (ArborGen), to cont inue research on transgenic Eucalyptus trees currently permitted, to be issued permits to plant additional trees, and to be issued permits for the environmental release of ge netically engineered (transgenic) Eucalyptus trees which will be allowed to flower. These plants are a clone 1 coded EH1 derived from a hybrid of Eucalyptus grandis X Eucalyptus urophylla. These have been genetically engineered with different c onstructs. The purpose of the environmental release is to assess the effectiveness of gene constructs which are intended to confer cold tolerance; to test the efficacy of a gene introduced to alte r lignin biosynthesis; a nd to test the efficacy of a gene designed to alter fertility. In a ddition the trees have b een engineered with a selectable marker gene.

ArborGen previously applied for a permit (06-325-111r) requesting that Eucalyptus trees
be allowed to flower in the field in Al abama. The permit was granted following the completion of an EA and reaching of a FONSI (http://www.aphis.usda.gov/br s/aphisdocs/06_325111r_ea.pdf ). The original permit allowed flowering on 1.1 acres. It was subsequently ame nded to allow flowering on 5.1

acres at the same location. In addition, the company was granted permit 08-151-101r to allow flowering on 1.4 acres at another site in Florida. As of the date of this EA, ArborGen had approximately 50 acres of the transgenic Eucalyptus hybrid planted and has permits that allow 7.6 acres of the trees to flower. ArborGen has now submitted two new permit applications for planting and gr owing trees on 29 sites and requesting that trees on 28 sites be allowed to flower; includ ing the locations that have already been authorized for flowering. These two comb ined permits would allow flowering on up to

330 acres across all 28 locations. The size of ea ch field test site ranges from 0.5 to 20

acres. All locations except one would be allowed to flower. The genes are the same as those in permits 06-325-111r and 08-151-101r, with the addition of a gene aimed at altering lignin biosynthesis that is being tested on some of th e sites. The status of the field tests permitted under permits 06-325-111r and 08-151-101r is included in Appendix

I.



## II. Purpose and Need

A. Proposed Action

The proposed action is for APHIS, Biotechnol ogy Regulatory Services (BRS) to issue two permits to allow the planting, field testing and flowering of a Eucalyptus hybrid clone engineered to express va rious genes. The genes are intended to confer increased tolerance to cold temperatures, alter fertility and alter lignin biosynt hesis. In addition there is a gene used as a selectable marker.

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.6



### B. Purpose of this Environmental Assessment

The purpose of this EA is to assess any poten tial adverse environm ental impacts of field research studies being conducted on research sites in Alabama, Florida, Georgia, Louisiana, Mississippi, South Carolina, a nd Texas. Two permit applications were received by APHIS–BRS in January 2008 from ArborGen, in order to continue research

on Eucalyptus hybrid trees originally planted und er various notifications, to plant additional trees under the permits , and to allow all the trees except the trees on one of the sites to flower. These are permit applic ation numbers 08-011-106rm and 08-014-101rm.

This environmental assessment (EA) wa s conducted pursuant to: (1) The National Environmental Policy Act of 1969 (NEP A), 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' NE PA Implementing Procedures (7 CFR part 372). Generally, issuance of a permit for field tr ials of regulated arti cles in categorically excluded from requirements for an environm ental assessment. However, when APHIS

determines that a confined field release of genetically engineered organisms has the potential to affect significantly the quality of the human envi ronment, as those terms are defined in 40 CFR 1508.27 and 1508.14, an envi ronmental assessment or environmental impact statement will be prepared, pursuant to 7 CFR § 372.5(d). This EA has been prepared because the permittee intends to allow the trees to grow under permit for a number of years and intends to let the trees reach maturity and flower. The actions described in the permit applications involve the release of transgenic Eucalyptus grandis

x Eucalyptus urophylla into the environment. The release of flowering Eucalyptus on many sites and in a number of new States a nd locations needs to be evaluated by APHIS to determine the potential for environmental impacts from such releases. APHIS has prepared this EA because there is a concern that the increased number of locations and

size of the releases that would be allowed to flower could po tentially result in the release of pollen and/or seed; which would lead to a lack of confinement of the field tests and impacts to the environment. Other impacts su ch as hydrology, allelopathy and fire are

also addressed.



### C. Need for This Action

The United States Department of Agricu lture (USDA), Animal and Plant Health Inspection Service (APHIS), Biotechnology Regulatory Services’ (BRS) mission is to protect America’s agriculture and the envi ronment using a dynamic and science-based regulatory framework that allows for the safe development and use of genetically engineered (GE) organisms. APHIS’ re gulations in 7 CFR part 340, which were promulgated pursuant to authority granted by the Plant Protection Act, as amended, (7 U.S.C. 7701–7772), regulates the introduction (importation, interstate movement, or release into the environment) of certain GE organisms and products. The Plant

Protection Act directs the USDA to facil itate imports and interstate commerce in agricultural products in ways that will reduce, to the exte nt practicable, the risk of dissemination of plant pests. Under APHIS regulations, the APHIS Administrator has authority to regulate any organism or product altered or pr oduced through genetic7

engineering that the Adm inistrator determines is a plant pest or has reason to believe is a plant pest. When APHIS receives an applica tion for a permit for e nvironmental release, the application is evaluated to determine whether the environmental release, with appropriate conditions imposed, can be carri ed out while preventing the dissemination and establishment of plant pe sts. The receipt of a permit application to introduce a genetically engineered organism require s a response from the Administrator:

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

The applicant has provided the information a ssociated with this request in the permit application. This information has been reviewed and analyzed in this EA.



### D. Purpose and Description of the Research

The purpose of the research is to assess the efficacy of the introduced cold tolerance genes and gene to alter lignin biosynthesis in Eucalyptus . According to the applicant, genetically engineered cold tolerant Eucalyptus would enable the production of this hardwood species for pulping and for biofuel applications in mana ged plantation forests in the southeastern U.S. In addition, the a pplicant is researching mechanisms for altered fertility. The release of the trees in differe nt areas of the southeast U.S. will allow the applicant to obtain data on pe rformance of the transgenic trees and the efficacy of the inserted genes in a wide variety of environments.



## III. Affected Environment

The field tests are taking place on land cont rolled by ArborGen or through contracts for field testing. The exact locations are clai med as CBI. Under the two permits, there are 29 sites where trees have been planted or w ill be planted, and on 28 sites the trees will be

allowed to flower. See below for the States and Counties in which these 29 sites are

located. An additional site in South Carolina is a holding area for plan ts in pots and trees. Trees will be held there for planti ng and will not be allowed to flower.

All the test sites listed in this permit are either on privately owned managed plantation forests and agricultural farm lands or e xperimental research stations managed by academic institutions and industry. The standard agricultural and silv icultural practices for land preparation, planting, irrigation, and ha rvesting of plants have been routinely

used on these sites. Sites that include ma naged pastures have had intense activity including the use of heavy machinery for general upkeep, irrigation, fertilization, controlled grazing and management of grasses. Standard silvicultura l practices will be

used at these sites for the duration of the fiel d tests. Surveys conducte d by the applicant at

each of these locations indicat e that there are not any old growth forests or undisturbed

natural areas in the immediate surroundings of the test sites. The trees will be planted8

from 0.5 up to 20 acres, depending on the location. In the case of these tests the planting density will be from 300 - 600 trees per acre 2 . An acre is about the size of a football

field.

Baldwin County Alabama Site:

This location has been an agricultural resear ch station for more than 20 years. The location has been used for managed producti on of annual agricultural crops and forest trees. Site preparation will involve herbic ide application, subsoiling, and planting of

trees in flat beds. The surr ounding areas of the test site c onsist of field plantings of agricultural crops, experimental forest trees and an abandoned pecan orchard. There are existing transgenic Eucalyptus field trials of the same clone (EH1) at this location that were planted on approximately 7 acres of test plots under Notifications and Permits 06- 325-111r, 08-039-102rm, 05-256-03n, 06- 135-01n, 06-150-02n, 07-093-113n and, 07- 159-103n. Trees at this location have been allowed to flower under permit 06-325-111r on approximately 6.2 acres. Up to 8,000 additional trees will be transf erred to this field test site and planted in fi eld plots of up to 10 acres (a t around 300 - 600 tr ees per acre) over the next three years.

Escambia County Alabama Site:

This location has been used as an intensely managed pasture for more than 5 years. The test site is currently planted with grasses suitable for cattle grazing. Site preparation has involved and will involve herbicide applicati on to remove existing grasses, subsoiling, preparation for possible irriga tion, and planting of the test trees in flat beds. The surrounding areas of the test site consist of approximately 30 year-old slash pine, and a re-forested area with less than 7 year-old mixed stands of pine and hardwood species. There are existing transgenic Eucalyptus field trials of the same clone (EH1) at this location that were planted on approximately 0.3 acres of test plot under Notification 07- 159-103n and are now covered under permit 08- 039-102r. Up to 8,000 additional trees will be transferred to this field test site and planted in field plots of up to 10 acres over the next three years.

Evans County Georgia Site:

This location has been a commercial nursery for forest seedling production for over 30 years. Site preparation has and will involve herbicide application, subsoiling and planting of trees in flat beds. The surrounding areas of the test site consist of nursery beds of forest tree seedlings, agricultu ral crops and mixed stands of hard wood and pine. A field trial of the same transgenic Eucalyptus clone (EH1) was planted on approximately 0.2 acres at this site under BRS permit # 08-039-102rm. An experimental test of non-transgenic E.

2 Planting density typically refers to the number of trees per acre. Planting densities can vary greatly depending upon the tree species and the environment, but densities of short rotation hardwood trees in the southeastern US are typically in the range of 300–800 trees per acre. Therefore sites ranging from 10 to 20 acres can have from 3000 to 16,000 total trees planted in the ground. Twenty acres, as defined by forest plantation standards in the southeast, is considered a small planting.9

macarthurii is planted within 100 m eters of the test plot location. Up to 4,000 additional

trees will be transferred to this field test si te and planted in field plots of up to 5 acres over the next three years.

Saint Landry’s Parrish Louisiana Site:

This location has been used as an experiment al agricultural farm for more than 25 years. The location has been used for conducting rese arch experiments with soybean, cotton and wheat. Site preparation has and will involve herbicide application, subsoiling and planting of trees in flat beds. The surrounding area s of the test site c onsist of agricultural fields of rice, sugarcane and millet. There are existing transgenic Eucalyptus field trials of the same clone (EH1) at this location that were planted on approximately 1.7 acres of test plots under Notifications 07-145- 102n and 07-159-103n; and these are now under Permit #08-039-102rm. Up to 8,000 additional trees will be transferred to this field test site and planted in field plots of up to 10 acres over the next three years.

Marshall County Mississippi Site:

This location has been an agricultural rese arch station for more than 50 years. The location has been used for conducting resear ch experiments with agricultural crops and grasses. The test site was used for experime ntal planting of grasses. Site preparation involved herbicide application, subsoiling a nd planting of trees in flat beds. The surrounding areas of the test site consist of agricultural fiel ds, and less than 5 year-old pine plantations. There are existing transgenic Eucalyptus field trials of the same clone (EH1) at this location that were planted on approximately 0.5 acres of test plot under

Notification 07-159-103n and these are now under permit 08-039-102rm. These plantings are under permit application 08-011-106rm. This trial is expected to continue

until at least 2011 and would be allowe d to flower under the submitted permit application. No additional plantings are anticipated at this site.

Pearl River County Mississippi Site:

This location has been an agricultural researchstation for more than 5 years. The location has been used for conducting research experi ments with agricultura l crops and grasses.

The test site has been used fo r experimental planting of grasse s. Site preparation has and will involve herbicide application to remove existing grasses, subsoiling, preparation for possible irrigation installation, and planting of trees in flat beds. The surrounding areas

of the test site consis t of a grape research farm, mixed stands of hardwoods and pine, and a residential area. There are existing transgenic Eucalyptus field trials of the same clone (EH1) at this location that were planted on approximately 2.0 acres of test plot under

Notification 07-159-103n and th ese are now included under permit 08-039-102rm. Up to 4,000 additional trees will be transferred to this fi eld test site and plan ted in field plots of up to 5 acres over the next three years.

Bamberg County South Carolina Site:10

This location has been a m anaged forest plan tation for more than 12 years. The location has been specifically used for short rotati on planting of hardwoods and softwood trees for forestry research. The standard silvicultural practices for site preparation, irrigation, fertilization, planting and harv esting have been used at th is location. Similar practices

will be used for the additional field tests to be established at this site. The surrounding areas of the test site consist of young pine plantations, mixed stands of hardwoods and pine, and agricultural fields. There are expe rimental test plots ofnon-transgenic coldhardy Eucalyptus species ( E. macarthurii, E. benthamii, E. viminalis, E. badjensis , and E. dorrigoensis ) planted at least 1000 meters from the te st plot location. There are existing transgenic Eucalyptus field trials of the same clone (EH1) at this location that were planted on approximately 3.3 acres of test plots under the Noti fications 07-093-113n, 07- 145-107n, 07-145-106n, 07-159-104n, 07-145-1 02n, 07-159-103n, and these are now under permit 08-039-102rm. Up to 4,000 additional trees will be transferred to this field test site and planted in fi eld plots of up to 5 acres over the next three years.

Berkeley County South Carolina Site 1:

This is an extension of a gr eenhouse facility that has been used for acclimatization of transgenic and non-transgenic pl ants for more than 5 years. The 0.5 acre release site is located adjacent to greenhouse facilities and is surrounded by hardwoods and pine plantations. This site is a secure fenced holding area where trees growing in containers are transferred from the greenhouse to the out -of-doors for acclimatiz ation prior to field planting. Trees will not be allowe d to flower at this location.

Berkeley County South Carolina Site 2:

This location has been a managed forest plan tation for more than 5 years. The location has been specifically used for short rotati on planting of cottonwood fo r forestry research. Site preparation has and will involve herbicide application, subsoiling, drip irrigation installation, and planting of trees in flat beds. The test site is located adjacent to greenhouse facilities and is surr ounded by pine plantations. Th ere are existing transgenic Eucalyptus field trials of the same clone (EH1) at this location that were planted on approximately 0.5 acres of test plot u nder the Notifications 07-145-102n and 07-159- 103n and are now under permit 08-039-102rm. Th ese would be allowed to flower under the new permit application. No additional plantings are requested for this site.

Charleston County South Carolina Site:

This location has been a managed forest plan tation for more than 10 years. The location has been specifically used for short-term planting of hardwoods and softwood trees for forestry research. The standard silvicultural practices for site preparation, irrigation, fertilization, planting and harv esting have been used at th is location. Similar practices

will be used for the additional field tests to be established at this site. The test plots adjacent to the field test site include young mi xed stands of hardwoods and pines. There are existing transgenic Eucalyptus field trials of the same clone (EH1) at this location that were planted on approximately 3.1 acres of test plots under Notifications 07-093-113n,11

07-145-107n, 07-145-106n, 07-145-102n, and 07- 159-103n and are now under permit 08- 039-102rm. These plantings are under perm it application 08-011- 106rm. These would be allowed to flower under the new permit application. No additional plantings are requested for this site.

Marlboro County South Carolina Site:

This location has been a commercial nursery for forest seedling production for over 30 years. Site preparation has and will involve herbicide application, subsoiling and planting of trees in flat beds. The surrounding areas of the test site consist of field plantings of agricultural crops, nursery beds of forest tree seedlings a nd less than 30 years-old mixed hardwood and pine plantations. There are existing transgenic Eucalyptus field trials of the same clone (EH1) at this location that we re planted on approximately 0.3 acres of test plot under Notification 07-159-103n and ar e now under permit 08-039-102rm. These would be allowed to flower under the new permit application. No additional plantings are requested for this site.

Hardin County Texas Site:

This location has been a managed forest plan tation for more than 30 years. The location consists of mixed hardwood tree plantations pl anted using standard silvicultural practices and was harvested by the owner in 2004. The test site is within the larger harvested area and has been re-bedded by the owner for planti ng. Site preparati on included herbicide application and sub-soiling. The surrounding area s of the test site consist of mixed hardwood stands and managed loblolly pine plantations. Up to 16,000 trees will be transferred to this field test site and planted in field test plots of up to 20 acres over three

years.

Jasper County Texas Site 1:

This location has been under managed pine pl antations for more than 25 years. Previous plantings were cultivated in beds using sta ndard silvicultural practices. The existing pine plantation at this site was harvested by the owner in 2007 an d re-bedded for planting. The test site is within the larger harvested and bedded site prepared by the site owner. Further site preparation has involved herbic ide application, subsoiling and planting of

trees in flat beds. The surr ounding areas of the test site consist of harvest age pine plantations. A field trial of appr oximately 0.7 acres of transgenic Eucalyptus was planted at this site under permit 08-039-102rm. Up to 16,000 trees will be transferred to this field test site and planted in fi eld test plots of up to 20 acres over three years.

Jasper County Texas Site 2:

This location has been under managed pine pl antations for more than 25 years. Previous plantings were cultivated in beds using standa rd silvicultural practices. The existing pine plantation at this site was harvested by the owner in 2007 an d re-bedded for planting. The test site is within the larger harvested and bedded site prepared by the site owner.12

Further s ite preparation has involved herbic ide application, subsoiling and planting of

trees in flat beds. The surr ounding areas of the test site c onsist of less th an 20 year-old managed hardwood and pine stands. Up to 8,000 tr ees will be transferred to this field test site and planted in fiel d test plots of up to 10 acres over three years.

Jefferson County Texas Site:

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

containerized trees will be transferred to this fi eld test site and plante d in field test plots of up to 10 acres over three years.

Newton County Texas Site 1:

This location has been a managed loblolly pi ne plantation for at least 30 years. The previous plantings were cultiv ated in beds using standard silvicultural practices and the area has recently been harvested. Additional site preparation will include herbicide application, plowing to remove stumps, and plan ting of trees in raised or flat beds. The surrounding areas of the test site consist of managed loblolly pine plantations and mixed hardwood stands. Up to 16,000 trees will be transf erred to this field test site and planted in field test plots of up to 20 acres over three years.

Newton County Texas Site 2:

This location has been a managed loblolly pi ne plantation for at least 30 years. The previous plantings were cultiv ated in beds using standard silvicultural practices and harvested by the owner in 2005 and the site was prepared for replanting. The test site is within the areas replanted in 2006. The surrounding areas of the test site consis t of managed loblolly pine plantations and mixed hardwood stands. Up to 16,000 trees will be transferred to this field test site and planted in field test plots of up to 20 acres over three years.

Newton County Texas Site 3:

This location has been a managed loblolly pi ne plantation for at least 30 years. The previous plantings were cultiv ated in beds using standard silvicultural practices and harvested by the owner in 2005. Site prep aration will include he rbicide application, plowing, and planting of trees in raised or flat beds. The surrounding areas of the test site consist of managed loblolly pine plantati ons and mixed hardwood stands. Up to 16,000

containerized trees will be transferred to this fi eld test site and plante d in field test plots of up to 20 acres over three years.

Newton County Texas Site 4:

This location has been a managed loblolly pi ne plantation for at least 30 years. The previous plantings were cultiv ated in beds using standard silvicultural practices and the13

area has recently been h arvested. Site pr eparation will include herbicide application, plowing, and planting of trees in raised or flat beds. The surrounding areas consist of a small cemetery on the property,

an area with a few mobile homes to the east a nd managed loblolly pine plantations to the west, south and north. Up to 16,000 trees will be transferred to this field test site and planted in field test plots of up to 20 acres over three years.

Bay County Florida Site:

This location has been used as an intensely managed pasture for more than 5 years. The test site is currently planted with grasses suitable for cattle grazing. Site preparation has and will involve herbicide application to re move existing grasses, subsoiling, preparation for possible irrigation installa tion, and planting of trees in flat beds. The surrounding areas of the test site consist of agricultur al crops and less than 25 years-old hardwoods and pine. There are existing transgenic Eucalyptus field trials of the same clone (EH1) at this location that were planted on approximate ly 1.0 acre of test pl ot under Notification 07-222-104n that are now covered under perm it 08-039-102rm. Up to 4,000 trees will be transferred to this site and planted in field te st plots of up to 5 acres over the next three

years.

Columbia County Florida Site:

This location has been a managed pine planta tions for more than 20 years. Previous plantings were cultivated in beds using standa rd silvicultural practices. This area of the tract was burned in a fire in May 2007. Af ter the fire, the area was raked and bedded by the site owner in preparation fo r re-planting. The test site is within the larger harvested and bedded area, which is surrounded by exis ting pine plantations and additional harvested tracts. Up to 16,000 trees will be transf erred to this site and planted in field test plots of up to 20 acres over the next three years.

Gadsden County Florida Site 1:

This location has been an agricultural resear ch station for more than 10 years. The location has been used for conducting resear ch experiments on agri cultural crops. Site preparation will involve herbicid e application, plowing, and planting of trees in flat beds. The surrounding areas of the test site cons ist of agricultural fi elds, plantings of horticultural crops and an experimental planting of Eucalyptus . There is an experimental plot of non-transgenic Eucalyptus species including E. grandis, E. amplifolia, and E.

camaldulensis at least 1000 meters away from the tran sgenic test location. A field trial of approximately 0.2 acres of transgenic Eucalyptus clone EH1 was planted at this site under BRS permit # 08-039-102rm. Up to 8,000 trees will be transferred to this site and planted in field test plots of up to 10 acres over the next three years.

Gadsden County Florida Site 2:14

This locatio n has been an agricultural research station for more than 10 years. The field has been fallow for approximately seven years. Standard silvicultural practices will be used for site preparation, in cluding herbicide application, pl owing and planting of trees in raised or flat beds. The surrounding areas of the test site consist of mixed pine-hardwood forests and pine plantations, as well as resear ch plantings of agricu ltural and horticultural crops. Up to 12,000 trees will be transferred to th is site and planted in field test plots of up to 15 acres over the next three years.

Glades County Florida Site:

This location has been used for hay or vege table production for at le ast 5 years. Site preparation has and will invol ve herbicide application to remove existing vegetation, subsoiling and planting of trees in flat beds. The surrounding areas of the test site consist of agricultural fields. There are existing transgenic Eucalyptus field trials of the same clone (EH1) at this location that were plante d on approximately 2.5 acres of test plot

under Notification 07-222-104n that are now under permit 08-039-102rm. Up to 8,000

trees will be transferred to this field site a nd planted in the field plots of up to 10 acres over the next three years.

Highlands County Florida Site:

This location was previously used for managed production of citrus for at least 15 years. The planting area at this location was previ ously used for field trials of transgenic Eucalyptus which were terminated over a year ago and monitored to confirm that all previous transgenic material was destroye d. Site preparation has and will involve herbicide application, plowing, and planting of trees in flat beds. The surrounding areas

of the test site consist of less than 5 year-old second-growth pine and hardwood with mixed grasses. The existing field trial of transgenic Eucalyptus clone EH1 at this location was planted on approximately 1.4 acres of test plot under Notification 07-145- 102n and has been allowed to flower under perm it 08-151-101r. It will be transferred to permit 08-014-101rm where it would be allowed to continue to flower. Up to 8,000 trees will be transferred to this site and planted in field test plots of up to 10 acres over the next three years.

Marion County Florida Site:

This location has been an agricultural research station for more than 5 years. The location has been used for conducting research e xperiments on agricultural crops. Site preparation has and will invol ve herbicide application, plow ing, and planting of trees in flat beds. The surrounding areas of the test site consist of agricultural fields, plantings of horticultural crops and an experimental Eucalyptus test plot. An e xperimental test of non-transgenic E. amplifolia is planted approximately 200 meters from the proposed test plot location. An approximately 0.5 acre of test plot of transgenic Eucalyptus clone EH1 was planted under Notification 07-222-104n and is now under permit 08-039-102rm at this location. Up to 8,000 trees will be transferred to this site and plan ted in field test plots of up to 9.5 acres over the next three years.15

Taylor County Florida Site 1:

This location has been a m anaged pine plan tation for over 50 years and contains third rotation plantings of pine. The previous plantings were cultiv ated in beds using standard silvicultural practices. The ex isting pine plantation at the site was harvested by the owner prior to 2007 and prepared fo r re-planting. Additional s ite preparation will involve herbicide application, plowing to remove stumps, and planting of trees in raised beds. The surrounding areas of the test site consist of less than 25 year-old third rotation pine plantings. A field trial of appr oximately 1.5 acres of transgenic Eucalyptus clone EH1 was planted at this site under permit 08-039-102rm. Up to 16, 000 trees will be transferred to this site and planted in fi eld test plots of up to 20 acres over the next three years.

Taylor County Florida Site 2:

This location has been a managed pine plan tation for over 50 years and contains third rotation plantings of pine. The previous plantings were cultiv ated in beds using standard silvicultural practices. The ex isting pine plantation at the site was harvested by the owner prior to 2007 and prepared fo r re-planting. Additional s ite preparation will involve herbicide application, plowing to remove stumps, and planting of trees in raised beds. The surrounding areas of the test site consist of less than 25 year-old third rotation pine plantings. A field tr ial on approximately 1.25 acres of transgenic Eucalyptus clone EH1 was planted at this site under BRS perm it # 08-039-102rm. Up to 16,000 trees will be transferred to this site and planted in field te st plots of up to 20 acres over the next three

years.

Taylor County Florida Site 3:

This location has been a managed pine plantatio n for more than 20 years. Previous pine plantings were cultivated in beds using standa rd silvicultural practices. The existing pine plantation at this site was harvested by the owner in 2008 an d prepared for re-planting. Additional site preparation will involve herbicide application, plowing and planting of

trees in raised beds. The test site is with in the larger area harves ted by the owner and is surrounded by managed stands of pine plantatio ns. Up to 16,000 trees will be transferred to this site and planted in fi eld test plots of up to 20 acres over the next three years.



## IV. Alternatives

This EA analyzes the potentia l environmental consequences of a proposal to issue two permits to allow existing transgenic Eucalyptus trees to flower and to plant additional

trees that would also be allo wed to flower in 28 locations in the southeast U.S. and to plant the trees on a 29 th site where trees will not be allo wed to flower. Two alternatives are considered in this EA: (1) deny the permit and (2) to issue the permit.16



### A. No Action – Deny the Permit

Under APHIS–BRS regulations, the Administrator must eith er grant or deny permits properly submitted under 7 CFR part 340. For the purposes of this Environmental Assessment, the No Action alte rnative would be the denial of permit applications 08-011-

106rm and 08-014-101rm.

Transgenic Eucalyptus trees have been previously a pproved for planting under various Notifications, and permits. Two field tests have been allowed to flower under permits 06-325-111r and 08-151-101r. An EA was prepar ed for the field test under 06-325-111r. The applicant has now submitted two new permit applications to add additional field sites where more trees will be planted and allowed to flower. Under the No Action Alternative, if these permits are denied, the transgenic Eucalyptus plants currently

released will not be allowed to flower. Th e trees could remain in the field, but the applicant will be required to ei ther remove developing flower s or remove the trees from the field test if removing fl owers becomes too difficult. Denying the permits would not allow the applicant 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.



### B. Preferred Altern ative – Issue the Permit

The APHIS-preferred alternative is to i ssue the permits with supplemental permit conditions for the requested three-year period. The permits will need to be renewed to allow the transgenic plants to remain in the ground beyond this time period. Under this alternative, APHIS would issue the permit to allow the research to proceed at the field

test sites in the different St ates and Counties, with supplemental permit conditions based on APHIS scientific analysis of the permit a pplication, input from the States, and public

comment from this environmental assessment. If warranted, based on the environmental risk of escape of the engineered organism, APHIS will require further mitigating measures and monitoring to prevent spread of the organism outsi de the field production area. If the permit is issued, the trees would remain in the fi eld and be allowed to flower where the applicant can gather data on pe rformance of the tran sgenic trees over a multiyear period and the efficacy of the genes in a wide variety of environments. This would allow the safe development and use of genetically engineered (GE) organisms

under the mission of BRS.

APHIS proposes to include the following require ments to allow the applicant to conduct

a confined field release and to ensure no significant harm to the environment. These are also the current requirements for those permits where trees have already been allowed to

flower.

a. Trees in the field test sites will be monitored for flowering and seed formation. Data will be provided to APHIS in an annual report documenting which trees produced flowers and which, if any, produced

viable seeds.17

b. The field test sites will be m onitored for volunteer seedlings. Any volunteers found will be sprayed with herbicide or physically removed. The presence and elimination of any volunteers will be reported to APHIS in an annual report. c. All non-engineered control Eucalyptus trees in the field test plot and any plant material removed from the field site will be treated as regulated articles, i.e., with respect to mon itoring for flowering and volunteers, maintaining the identity while in use, and devita lization when no longer in

use.



## V. Environmental Consequences

A. No Action

Under the no action alternative, the applicant would not be allowed to plant additional acres and let the trees on 28 sites produce fl owers. Since preventing flower formation would prove impossible over time, it would mean that the trees will have to be cut down prior to maturity. The Eucalyptus trees currently planted at the field test sites would remain in the ground but would be cut down with in 2 or 3 years since they would not be

allowed to flower. At each of the sites the locations could remain planted in short

rotation Eucalyptus field tests, or the s ites would be returned to forest tree production or agriculture/forestry research. Trees would be cut down and replanted with the same or

different trees. Some sites could be returned to pasture or other agri cultural activities. Intense activity includi ng the use of heavy machiner y for land preparation, general upkeep, irrigation, and fertilization for the management of tree plantings and grasses would continue. For forest tree plantings, stan dard silvicultural prac tices would continue

to be used at these sites



### B. Preferred Alternative

Under this alternative, the a pplicant would be allowed to le t the trees produce flowers and would continue to plant more tr ees at the release sites. Th is means that the trees would remain in the ground for at least 3 years and most likely longer sinc e the applicant plans to renew the permits. The trees could be a llowed to stay in the ground until maturity or when normally harvested (age 7-9). The standard silvicultural practices for land preparation, planting, irrigation, and harvesting of trees would continue to be routinely

used on these sites.



### Potential Environmental Impact of the Preferred Alternative

Biology of Eucalyptus and Status in the United States

The genus Eucalyptus belongs to family Myrtacea e (subfamily: Leptospermoideae) which includes over 700 species. Eucalyptus is native to Australia with the exception of some species that are native to the Timo r Islands (Groves 1994, Ladiges 1997). There18

are no wild relatives of Eucalyptus that occur naturally in th e United States. An overview of the biology of Eucalyptus grandis has been published by the US Forest Service (Meskim en 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.

There have been numerous attempts to grow Eucalyptus as a commercial forest tree in the southeastern United States, but due to its sens itivity to cold temperatures, these attempts have not met with success. It is only gr own in commercial plantations in central and southern Florida, where it normally survives freezing temperatures. Eucalyptus is adapted to live in the mild arid and semi-ari d climate of Australia. Severe freezing events

that can occur in the southern United States have limited its establishment as a

commercial forest tree. There are plantations of Eucalyptus grandis and E. amplifolia currently grown in south central Florida as short rotation energy crops and for mulch production (Stricker et al. 2000, Rockwood et al. 2004). These trees are generally planted in areas where severe freezing events are rare.

The species hybrid E. grandis x E. urophylla that ArborGen wishes to allow to mature and flower under this permit has not been cate gorized as invasive. However, one of the parents of the hybrid, E. grandis has been shown to be natura lized in the State of Florida ( http://www.florida .plantatlas.usf.edu ). In South Africa, E. grandis has been shown to be an invasive species (Forsyth et al., 2004). It is also na turalized in both New Zealand (New Zealand Plant Co nservation Network 2005: http://www.nzpcn.org.nz/exotic_plan t_life_and_weeds/ weed_list.asp .) and Ecuador ( http://i3n.iabin.net/participants/ecuador.html ). Daehler (1998) listed E. grandis as an invader of natural areas. These and other data were recently used in the University of

Florida’s IFAS Assessment of Non-native Pl ants in Florida’s Natural Areas. The

assessment conclusion for E. grandis is that it is now: "Pre dicted to be invasive: recommend only under specific management pr actices that have been approved by the IFAS Invasive Plant 2 Working Group" ( http://plants.ifas.ufl.edu/assessm ent/predictive_response_form s.html ). Specific management practices for f our specific cultivars of E. grandis have been approved by the Working Group ( http://plants.ifas.ufl.edu/assessment/ -- see Approved, Specified and Limited Uses link). The extent to which the hybrid between E. grandis and E. urophylla

is invasive has not been assessed.

Numerous species of Eucalyptus were introduced into Californ ia during that State’s early history (see Santos: http://wwwlibrary.csustan.edu/bsantos/euctoc.htm ), and some of these species have become es tablished. Two of these, E. globulus (Tasmanian blue gum)

and E. camaldulensis (Red gum) are now categorized as invasive by the California Invasive Plant Council ( http://www.cal-ipc.org/ip /inventory/weedlist.php ). Neither of these species is being proposed to be planted at the permitted field site.

Traits Engineered into Eucalyptus19

ArborGen LLC wishes to field test ge netically en gineer ed (transgenic) Eucalyptus trees during which time the trees may flower. Th ese plants are a clone coded EH1 derived from a hybrid of Eucalyptus grandis X Eucalyptus urophylla. These have been genetically engineered with diffe rent constructs. The purpose of the field trials is to test the effectiveness of the CBF gene which is inte nded to confer cold tolerance and to test the efficacy of the Barnase gene designed to al ter fertility. In a small set of experiments the CBF and Barnase genes are also being test ed in combination with genes introduced to alter lignin biosynthesis (claimed as CBI). In addition the trees have been engineered with a common selectable marker gene ( nptII ) which confers resistance to the antibiotic kanamycin.

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

Harbor Plant Pests

There has been no intentional genetic change in these plants to affect their susceptibility to disease or insect damage. All of th e genes have been previously tested in Eucalyptus in existing field tests and the permittee has observed no changes in the incidence of pests, beneficial insects or pathogens between the tr ansgenic and non-transge nic controls. None of the genes being e ngineered into the Eucalyptus plants are expected to alter the susceptibility of the transgenic Eucalyptus plants to disease or insect damage. There might be a concern that altered lignin could le ad to an increase in insect or disease susceptibility, but the results so fa r with this particular gene do not indicate that this is the case (see below). The prescribed periodic monitoring of the field plots in the permit will allow the detection of any unexpected infest ation by plant disease organisms or animal pests. The permittee is required to report a ny 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 pr opagated in sterile tissue cultu re and were free of pests upon importation into the U.S. prior to their in troduction. All materials were handled in accordance with the USDA–APHIS requirements for import and quarantine under a USDA–APHIS PPQ Post-entry quarantine permit.

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

Risk of the Gene Produc ts on the Environment

Gene used as selectable marker

The kanamycin resistance se lectable marker gene ( nptII ) engineered into the trees is generally accepted as being safe (Fuchs et al . 1993) and has been used in thousands of

field tests with no evidence that it has led to an increase in plant pest characteristics. This gene does not alter the expression of a gene pr oduct or change plant metabolism in such a way that it would be expected to cause risk to the environment. In a number of instances, plants transformed with this gene have been deregulated by APHIS (e.g. corn, petition 01-137-01p; rapeseed, petition 01-206-02p; cotton, petition 95-045-01p; and papaya, petition 96-051-01p). Consequently, APHIS has determined the presence of this gene will have no significant environmental impacts.20

Genes conferring cold tolerance

The C-Repeat Binding Factor ( CBF) genes are transcription fa ctors that belong to the AP2/EREBP family of DNA bi nding proteins (Riechmann and Meyerowitz 1998) and like other transcription factors act as control switches for the coordinated expression of other genes in defined metabolic pathways. CBF protein recognizes and binds to a coldand drought-responsive DNA regulatory seque nce designated as the C-repeat (CRT)/dehydration-responsive element (DRE ) (Baker et al. 1994, Yamaguchi-Shinozaki and Shinozaki 1994), which is found in the promoter regions of many cold-inducible genes (Maruyama et al. 2004).

A common observation across experiments in which CBF genes are overexpressed in transgenic plants is that constitutive expression of CBF negatively impacts a number of other traits (Hsieh et al. 2002). In pota to, for example, constitutive expression of Arabidopsis CBF genes using the CaMV35S promoter was associated with smaller leaves, stunted plants, delayed flowering, and reduction or lack of tuber production (Pino et al. 2007). In contrast, CBF genes under the control of a cold-induced promoter, rd29A (Yamaguchi-Shinozaki and Shinozaki 1993, Kas uga 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 pr oduction 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 obs erved after only a few hours of exposure to low but non-freezing temperatures. These results suggest that using a stress-inducible promoter to direct CBF transgene expression could significantly improve freeze tolerance without negatively impacting other agronomically important traits. In the case of these Eucalyptus trees, the CBF gene is under the contro l 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 a ny toxic substances and is not expected to

alter the characteristics of th e engineered plants other than imparting tolerance to cold temperatures. These genes do not alter the e xpression of a gene product or change plant metabolism in such a way that it would be expe cted to cause risk to the environment. These genes are in a number of previous a nd existing field tests and have not produced unanticipated phenotypes that would indica te there have been changes to plant metabolism leading to increase plant pest characteristics. Therefore APHIS has determined the presence of this gene will have no significant environmental impacts.

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 deregul ation 95-288-01p, 97-342-01p and 98-349-01p), rapeseed (petitions 98-278-01p and 01-206- 01p) and chicory (petition 97-148-01p) have been reviewed and granted non-regulated status by APHIS. There is no reason to believe21

that the function and expression of this gene will be any different from the plants in which it has been previously assessed. There were no toxicity or allergenicity issues found with this gene in previous FDA reviews (See BNF Nos. 31, 32, 45, 57 and 66 at: http://www.cfsan.fda.gov/~lrd/biocon.html ).). The presence of th is 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 envir onment. 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 s howed complete pollen ablation. Recent observations from the replicated field study being conducted in Alabama under the approved BRS permit (BRS # 06-3 25-111r-a1) confirm that cold tolerant trees grown at the site and allowed to flower did not produ ce any viable pollen (see also Appendix I). APHIS has therefore determined the presence of this gene will have no significant environmental impacts.

Gene for altered lignin

This gene has been engineered into other crops that have been prev iously released into

the environment under both notifications and permits. The gene engineered into the plants in these field tests has been previously tested in ArborGen field trials for more than two years. There might be a concern that altere d lignin could lead to an increase in insect or disease susceptibility sin ce lignin is often associated wi th resistance to insects and disease organisms (Pederson et al. 2005), but the results of field tests with this particular gene have shown no differences in plant pest susceptibility. Growth measurements have indicated that trees containing this gene had normal to a moderately reduced growth phenotype. The trees were also visually insp ected on a monthly basis for the presence of any insect and disease damage and these obs ervations found that there have been no differences in insect or diseases occurrence in the transgenic lines compared to the control trees. However, if duri ng the tests there is evidence of increase disease or insect susceptibility, the applicant is re quired to report this to APHIS. The permittee is required to report any such unanticipated effects (inc luding excessive mortality or morbidity) to APHIS under the terms of the permit - see 7 CFR 340.4(f)(10)(ii).

Non-coding sequences

The transgenic Eucalyptus also contains non-coding regulatory sequences 3 derived from plants and plant pathogens. The non-coding re gions of the plant pathogens will not result in the production of an infectious entity or cause plant disease symptoms. None of these sequences are expected to pose a plant pest risk.

3 A non-coding sequence is the stra nd of DNA that does not carry th e 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 thegene. Therefore promoters and terminators, by themselves, cannot result in the production of a diseasecausing entity.22

Method of transform ation

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 th e recipient plant (see review s by (Zambryski 1988, Klee and Rogers 1989.) A. tumefaciens is a bacterial plant pathoge n 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 firs t removed, and therefor e the recipient plant does not have crown gall disease. Following transformation, the bacteria are eliminated from the transformed plant tissue, and the DNA sequences introduced into the plant are maintained and inherited as any other genes of the plant cell.

Alteration in Weediness characteri stics – Potential of the Engineered Eucalyptus to

be Invasive.

The potential of the engineered Eucalyptus to be weedy and become invasive was covered in the previous EA and re sponse to comments for permit 06-325-111r ( http://www.aphis.usda.gov/br s/aphisdocs/06_325111r_ea.pdf ) and is herein incorporated by reference. Information on the invasiveness of E. grandis , received since the publication of the original draft EA on June 3, 2009, has also been included in the Biology section above.

As indicated above, the hybrid Eucalyptus EH1 used to produce the transgenic trees has not been shown to be weedy or invasive in the U.S., but no formal assessment has been conducted on its weediness or invasiveness poten tial. None of the genes introduced into Eucalyptus code for traits that would be expected to make the GE hybrids more weedy or invasive. The genes introduced to affect cold tolerance could make the engineered Eucalyptus more adapted to cold temperatures in the southern United States, but this trait alone would not impart invasive or weedine ss characteristics (Kolar and Lodge, 2001) to the engineered plants. The trees would be cons idered weedy or invasive if they were to produce many seedlings that were readily spread away from the field test sites Where the non-engineered hybrid Eucalyptus (EH1) has been grown in Brazil, on an estimated 400,000 acres planted over 15 years, there has been no indication that large numbers of seedlings are being produced and are b ecoming invasive from the commercial plantations.

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. The areas surrounding the field releas e sites would not be readily conducive to the establishment of seedlings because they are managed or unmanaged areas where other plant species are growing. So any seeds that attempted to germinate would face competition. The addition of the cold-tolerance genes are not expected to affect the reproductive biology such as s eed production or vegetative reproduction23

capabilities. The gene in trodu ced to alter lignin biosynthesis would also not be expected to affect seed production or vegetative reproduction capabilities. The selectable marker gene, when used previously, did not contribute to weediness or invasive properties of the genetically engineered plants. The gene fo r altered fertility shoul d not contribute to weediness or invasive properties and should reduce the ability of the trees to produce progeny. None of the traits in troduced into the transgenic Eucalyptus will compromise the ability to control these plants as weeds.

Possibility of Gene Flow within the Field Test

All of the trees in the test plots, including control non-transgenic trees, have the same parental genotype EH1. The high le vel of self incompatibility in Eucalyptus (Campinhos et al. 1998, Pound et al. 2002) is expected to significantl y reduce the potential for crossing 4 (gene flow) within the test plots. Seed set from any self pollination is expected to be poor, and the vigor of any selfed progeny is also expected to be greatly reduced. In experiments conducted in Brazil and Alabama, the control self-pollinated seed obtained from this genotype had abnormal morphol ogy 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 pr oduction. However, no seedlings have been

found established beneath the trees or in th e 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 consequent ly the dispersal of seed is very limited, generally being confined within a radius of twi ce 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 cons equence of the very small size of Eucalyptus seeds is that they have very limited reserv es and are intolerantof shade or weedy competition. In order to successfully germinate and establish, Eucalyptus seed needs contact with bare mineral soil and lack of competition either as a result of human intervention or naturally following a fire event (Meskimen and Francis 1990, Bell and Williams 1997). Eucalyptus plantations are typically esta blished using ro oted plantlets because of poor establishment using direct s eeding methods. Even for the rooted plants, competition control is recommended for seve ral months after planting to ensure good survival (Meskimen and Francis 1990). Ther efore there is limited possibility that volunteer seedlings could become established in any unmanaged areas that may be close to the site. However, if they were to a ppear, the conditions of the permits will require that all volunteers be reported, found and dest royed to prevent any sp read of trees from

the field release site.

4 When plants or trees “cross” the male pollen from one tree can pollinate (fer tilize) the female ovule (or egg) on the same tree or on another tree. Unlike an imals, some plants can fertilize themselves when the pollen and ovule are produced on the same tree. In th is 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 a nd reduced numbers of viable seed compared to fully sexually compatible Eucalyptus trees since they are genetically identical individuals.24

Eucalyptus seeds do not have any dorm ancy barrier s 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 are par ticularly adapted to a Mediterranean climate subject to summer fog, which is conducive to seed germination in those species ( http://ucce.ucdavis.edu/datastore/detailreport.cfm?usernumber=48&surveynumber=182 ) . This type of climate does not exist in the S outheastern U.S. In the event that any viable seeds are produced, these seeds would be exp ected to germinate w ithin a short period. The bordering fields within 100 meters from th e edge of the trials will be monitored every six months for germinating seedlings. This distance is twice the 50 meter distance that seeds would be expected to be disperse d from a tree at harvest age. If transgenic seedlings are observed they will be destr oyed either by uprooting or by spraying with herbicides (e.g., glyphosate or other herbicides to which these trees are susceptible) and

APHIS will be notified of their occurrence.

Possibility of Gene Flow Outside of the Field Test

Eucalyptus is adapted for insect pollination, with bees being the predominant vector (Pacheco et al. 1986, Pacheco 1987, House 1997). Under ideal conditions of humidity and temperature, viable Eucalyptus pollen can only be found within approximately 100 meters from the edge of nearest tree sta nd (Peters et al. 1990, Linacre and Ades 2004). Pacheco et al. (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) indi cated that the minimum distance to prevent undesirable pollen contamination of seed pr oducing areas is approximately 300 meters. Even if bees were to transpor t pollen farther distances from th e 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 routes of gene flow outside of the fiel d test. One could be with nearby transgenic Eucalyptus field test trees and the othe r could be with other nearby non-transgenic Eucalyptus species trials.

Transgenic trials, approved under BRS notif ications and permits, of the same hybrid Eucalyptus variety EH1 are planted adjacent to or at the proposed field test plot locations at the sites in Berkeley, Ch arleston, Marlboro Counties in South Carolina; Escambia and Baldwin Counties in Alabama; Saint Landr y’s Parish, Louisiana; Pearl River and

Marshall Counties in Mississippi; Jasper C ounty (Site 1) in Texas; and Bay, Glades, Highlands, Marion, Taylor (S ite 2) and Gadsden (Site 1) Counties in Florida. The applicant is not aware of any comm ercial plantings of compatible Eucalyptus species within 1000 meters of the test pl ot location at these sites. Observations by the applicant from self pollination experiments in Brazil show ed that the hybrid genotype used in these trials is unable to produce a ny viable seed from transgen ic or non-transgenic trees. However, in the two field trials in Alabama and Florida allowed to flower, the hybrids are producing low numbers of viable seeds, but no volunteer seedlings have occurred in the field trials to date. Any seed production as a result of crossing between trees will result25

from trees (including non-transg enic controls) crossing with each other within the test

site. There are no other commerc ial plantings of compatible Eucalyptus species within 1000 meters of these test plot locations. So the likelihood of gene flow from the new field tests is virtually nil.

At all test sites in Hardin, Jasper (except Site 1), Jefferson and Ne wton counties in Texas; Taylor (except Site 2), Columbia and Gadsden (except Site 1) Counties in Florida, there are no existing transgenic or non-transgenic Eucalyptus field trials. The applicant is not aware of any commercial plantings of compatible Eucalyptus species within 1000 meters of the test plot location at these sites.

In Bamberg County South Carolina, there are experimental test plots of non-transgenic cold-hardy Eucalyptus species ( E. macarthurii

, E. benthamii

, E. viminalis

, E. badjensis

, and E. dorrigoensis ) planted within 1000 meters of th e test plot location. As noted below, these cold hardy Eucalyptus species are highly unlikely to be compatible with the hybrid Eucalyptus variety because of distant phylogenetic relationship and asynchronous flowering. The applicant will monitor flower development in both the transgenic trial and the non-transgenic trial to determine if there is any overlap in the occurrence of mature flowers. If overlaps are foun d these will be reported to APHIS. In the event that pollen from the non transgenic Eucalyptus trees fertilizes the transgenic trees, seed dispersal, if any, is expected w ithin 100 m from the test plot. Should any hybridization and viable seed production o ccur, the monitoring for and removal of

volunteers within 100 m from th e edge of transgenic test plot would eliminate any offspring produced.

In Evans County Georgia, there are no existing tran sgenic field trials at this site. There are no commercial plantings of compatible Eucalyptus species within 10 00 meters of the proposed test plot location at this site. An experimental test of non-transgenic E.

macarthurii is planted within 100 meters of the test plot location. As i ndicated below, this cold-hardy Eucalyptus species is unlikely to be compatible with the hybrid Eucalyptus variety EH1 because of distant phylogenet ic relationship and asynchronous flowering.The applicant will monitor flower development in both the transgenic trial and the nontransgenic trial to determine if there is any overlap in the occurrence of mature flowers. In the event that pollen from the non transgenic Eucalyptus trees fertilizes the transgenic trees, seed dispersal, if any, is expected w ithin 100 m from the test plot. Should any hybridization and viable seed production o ccur, the monitoring for and removal of

volunteers within 100 m from th e edge of transgenic test plot would eliminate any offspring produced.

At the Marion County Florida site, an e xperimental test of non-transgenic E. amplifolia is planted approximately 200 meters from the proposed test plot location. T his Eucalyptus species is also unlikely to be compatible with the hybrid Eucalyptus variety EH1 because of its distant phylogenetic relationship a nd asynchronous flowering. As noted above, pollen dispersal is generally limited to 100 m w ith very low levels beyond that distance. All trees to be planted in this trial have b een shown in a previous trial to exhibit the reduced fertility trait. Results from these st udies have shown that the engineered trait is26

highly effective in preventing the form ation of mature pollen. As such they are not expected to fertilize the non transgenic E. amplifolia trees. Observations will be made on developing flowers in the test to verify th at these trees do have reduced fertility. Phenology of the Eucalyptus hybrid used in the transgenic trial (mature flowers in mid to late summer) and E. amplifolia (mature flowers in the spring) indicates that there would not be any overlap in flowering times. The applicant will monitor flower development in both the transgenic trial and the non-transgenic tr ial to determine if th ere is any overlap in

the occurrence of matu re flowers. In the unlikely event that pollen from the non transgenic E. amplifolia trees could fertilize the transgenic trees, seed dispersal, if any, is expected within 100 m from the test plot. Should any hybridization and viable seed production occur, the monitoring for and re moval of volunteers within 100m from the edge of transgenic test plot w ould eliminate any offspring produced.

At the Gadsden County Florida Site 1, there are experimental plantings of non-transgenic Eucalyptus species including E. grandis

, E. amplifolia , and E. camaldulensis which are at least 1000 meters away from th e transgenic test location.

The hybrid genotype used in these studies fo r transformation may be compatible with E. grandis trees grown in Florida. However, E. grandis produces mature flowers in the fall whereas the hybrid genotype used in these tests produces mature flowers in mid to late summer. Because of asynchronous flowering, hy bridization of transgenic trees with the

more common Eucalyptus species grown in Florida would be virtually negligible.

There are other spec ies 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 States where these field test occur. 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.

The transgenic hybrids are not lik ely to be sexually compatible with any of the cold hardy species listed above. It is well documente d that natural cross-compatibility between different species of eucalypts is limited to closel y related series within distinct sections of

the Eucalyptus genus. For example, E. grandis and E. urophylla , for which hybrids have been generated in directed breeding programs, are in the Salignae and Resiniferae series, respectively, of section Transversaria ( http://plantnet.rbgsyd.nsw.gov.au/cgibin/eucclass.pl?gn= Eucalyptus ). In contrast, E. cinerea , and other cold hardy species mentioned above are far removed genetically fr om 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 gene rally very low (Volker 1995). The published literature supports the fact that natural hybridization am ong distantly related species within genus Eucalyptus is rare and hybrid inviability incr eases with increasi ng taxonomic distance between parents (Potts and Dungey 2004). Wh ere hybridization is possible, it often requires significant human intervention in di rected breeding/crossing efforts. Potts and27

Dungey (2004) m ake reference to th e high degree of inviability in F 1 hybrids (offspring). Inviability of these offspring may be expresse d at germination, in the nursery and even after planting in the field. Slower germin ation 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 de veloping 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 potentia l crossing between the transg enic trees with ornamental E.

cinerea and other species is the expected differe nces in flowering times between species (Gore and Potts 1995, Potts et al. 2003). For example, E. cinerea flowers in spring, while the transgenic hybrid genotype us ed in this test initiates fl owers in early summer with expected maturation in mid to late summer. In the United States, ArborGen data indicate that flowering of the clone bei ng tested occurs in the summer.

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

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

flower have shown no mature pollen formation; 2) other sp ecies that are or could be grown in the area are unlikely to be compatib le; 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 e xpected to be of very poor vigor.

Possibility of Vegetative Propagation / Persistence Outside of the Field Test

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

via natural abscissions of bran ches, or cladoptosis. The as exual propagation of shoots via rooted cuttings requires specific environmenta l conditions such as a greenhouse or a high humidity environment (Hartney 1980), so it is highly unlikely that any sh oots that fall or

that are removed from the trees would propagate themselves in the wild.

Suckering (production of shoots from subte rranean roots) does not occur in this Eucalyptus hybrid. Regrowth of shoots from stum ps of felled trees is common and this practice, known as coppicing, is used to regrow trees in a plantation after harvest. This regrowth will be managed in this field test at termination by devita lizing any sprouts that form from the stumps of harvested trees using registered herbicide treatments.

Potential of the Eucalyptus in the Field Tests to Become an Invasive Species that

Threatens Native Plant and Animal Communities.

There could be a concern that adding the cold tolerance trait would make the engineered Eucalyptus more adaptive and invasive in the sout heastern U.S. It has been hypothesized28

that eng ineered traits such as cold tolera nce could significantly a ffect the engineered variety’s ability to propagate, surviv e, and impact native ecosystems.

As indicated above there is no evidence to date that the untran sformed clone of the Eucalyptus hybrid in these permits is weedy or inva sive in the U.S. None of the genes

introduced into Eucalyptus code for traits that would be expected to make the plants more weedy or invasive. The gene s introduced to affect cold tolerance could make the engineered Eucalyptus more adapted to cold temperatur es in the southern United States,

but this trait in and of itself would not impart invasive or w eediness characteristics to the engineered plants. There are multiple mechanisms in place that would prevent these Eucalyptus 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 (see details above and in Appendix 1). Monitoring of seed set and seedling viab ility will be required during the field tests so that the extent of seed production and seedling establishment can

be determined. In addition altered fertil ity leading to the l ack of viable pollen development has been engineered into the trees. The addition of the cold-tolerance genes are not expected to affect th e reproductive biology such as seed production or vegetative reproduction capabilities. The gene introduced to alter lignin biosynt hesis would also not be expected to affect seed production or vegetative reproduction capabilities. The selectable marker gene, when used previ ously, did not contribute to weediness or invasive properties of the ge netically engineered plants. The gene for altered fertility

should not contribute to weedin ess or invasive properties and should reduce the ability of the tree to produce progeny. In event that s eeds are formed and seedlings are produced,

none of the traits introdu ced into the transgenic Eucalyptus will compromise the ability to control these plants as weeds so spread of s eedlings and trees from the field test sites is highly unlikely. Volunteers can be re adily identified and controlled.

Impact on Existing Agricultural Practices

The establishment and growth of these sma ll field tests will not have any significant impact on existing agricultural practices because they are solely for research purposes. Current practices will essentially remain the sa me. The field sites that are being proposed under this permit have been used as forest tree plantations, as pastures , or for forestry and agriculture research and are specifically desi gned for field testing cr op plants or forest

trees.

Potential Impacts to Wildlife

Native floral communities

The field sites in the permit applications are located in Bamberg, Berkeley, Charleston, and Marlboro counties, South Carolina; Ev ans county, Georgia; Baldwin and Escambia counties, Alabama; St. Landry's Parish, L ouisiana; Marshall and Pearl River counties, Mississippi; Hardin Jasper, Jefferson and Ne wton counties, Texas; and Bay, Columbia, Gadsden, Glades, Highlands, Marion, and Tayl or counties, Florida. The sites are a mixture of pasture, crop lands a nd forested areas. If viable seed are formed, these areas29

are unsuitab le for the establishment of the Eucalyptus hybrid clone in this permit. Eucalyptus is intolerant of shade or weedy co mpetition. In order to successfully germinate and establish, Eucalyptus seed need contact with bare mineral soil and the removal of competing plants, either as a result of human intervention or naturally following a fire event. With the exception of the field test area, the agricultural areas surrounding the field sites are not co nducive to the es tablishment 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 plantations will be cultivated and weeds controlled by herbicides. All sites are intensively managed. The inhos pitable conditions for seed germination, in combination with the confinement conditions imposed by the permittee and APHIS, will successfully limit the establishment of any of these species in the surrounding area. Therefore APHIS

concludes there would be no significant effect on any native floral species.

Terrestrial animals

The most likely animals to encounter the transgenic Eucalyptus trees in this field experiment would be browsing mammals (e.g., deer), burrowing animals (such as rodents), and leaf consuming insects (cons idered 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 in terest do not produce a ny toxin or have any similarity to known toxins (see Section V - Risk of the Gene products on the Environment). Therefore APHIS concludes th ere would be no significant effect on any native vertebrate or invertebrate animal species.

Aquatic organisms

Eucalyptus normally grows in areas of bare minera l soils and would not be expected to establish in aquatic or riparian environments away from the field trial. Furthermore, as stated above, there is no expect ation of toxicological effect s on any organism due to the ingestion of the transgenic plant material in this study. For potential impacts due to Eucalyptus effects on hydrology, see section D. APHIS concludes there would be no significant effect on any aquatic species.

Potential Impacts by Fire

Most Eucalyptus communities in Australia have evolve d 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 re sistant or susceptible to fire damage (Gill 1997). Eucalyptus fires can be very hot and move rapi dly. The bark catches fire readily, and deciduous bark streamers tend to carry fi re 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 pendul ous branches, which encourages maximum updraft (Esser 1993, Gill 1997). In the U.S., ther e have been reports of significant fires30

in California and m any have been bl amed on the widespread planting of Eucalyptus

. Fuel buildup occurs very rapidly in unmanaged bluegum Eucalyptus stands in California which has lead to significant forest fires. The build up of litter and dead grass are primary responsible for the spread of these fires (see Santos: http://wwwlibrary.csusta n.edu/bsantos/euctoc.htm ) .) . The Forest Service indicates that fuel reduction programs and the establishment of firebreaks in Eucalyptus plantings can reduce wildfire hazard. (Esser 1993).

There is always a risk of forest fire in the southeastern U.S., however, the probability that

these field tests will increase the risk and severity of forest fires in their respective locations is very small. These plantings are small (none greater than 20 acres) and they will be highly managed to preven t litter buildup. They are al so physically isolated from nearby plantations. If they were to catch fire, the fires should be readily contained.

Potential Impacts to Human Health

During the comment period for the EA pr epared for permit 06-325-111r, there were concerns expressed that Eucalyptus field tests could be a source of Cryptococcus neoformans gattii. C. neoformans gattii is a fungal pathogen that is hosted on a variety of species of Eucalyptus as well as other tree spec ies. It causes systemic fungal infections in humans, leading to fungal meningitis and death. C. neoformans gattii has been found on a number of Eucalyptus hosts, some of which are being grown in commercial plantations and imported and exported for ornamental use. People have contracted and died from cryptococcosis in India, Africa, Ta iwan, South America and California. C. neoformans infections are found particular ly in AIDS patients due to their weak immune systems. Infections with this fungus are rare in thos e with fully functioning immune systems. For this reason, C. neoformans 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. The disease was previously only known to occur in tropical or semi-tropical climates. APHI S 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/br s/aphisdocs/06_325111r_ea.pdf )

It is unlikely that the trees that are the subject of the pr oposed field release can be a source that might introduce the pathogen into the U.S because the trees were derived from sterile tissue culture lines. The transgenic Eucalyptus started as a hybrid developed in Brazil. In Brazil, small pieces of the tissue derived from the hybrid were put into sterile

tissue culture and sent to New Zealand for transformation. The transformed lines were

sent to the U.S as sterile tissue culture lines that were inspected by APHIS Plant Protection and Quarantine inspectors prior to entry into the U.S. C. gattii spores readily germinate in culture. If C. gattii spores were present in the tissue culture, contamination, would be evident and the affected lines would be discarded pr ior to regeneration of trees

for introduction into the environment. Another reason it is unlikely th at spores could be or were ever present in the hybrid lines used in the field trial is that in the Eucalyptus species where C. gattii is associated, the pathogen is primarily found colonizing the bark31

or decay ing wood in hollows of older trees and the tissue culture was not derived from woody tissue nor was woody tissue generated du ring 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 negligib le 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 or animal heal th 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 beli eve that the genetic modification of the hybrids will a lter 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 cons iderations we have concluded that an increase of additional acreage planted to Eucalyptus would not impact the likelihood that these field trials should lead to a higher incidence of C. gattii in the U.S. and therefore should not pose an unnecessary risk to human or animal health.

Transfer of Genetic Information to Organ isms with which it Cannot Interbreed - Horizontal Gene Transfer to Other Organisms

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. HGT and expression of DNA from these plant speci es to bacteria is unlikely to occur. First, many genomes (or parts thereof) have b een 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 sequen ce data implied that horizontal gene transfer occurred, these even ts are inferred to occu r on an evolutionary time scale on the order of millions of year s (Koonin et al. 2001, Brown 2003). Third, transgene DNA promoters and coding sequences are optimized for plant expression, not prokaryotic (i.e., bacterial) ex pression. Thus even if horiz ontal gene transfer occurred, proteins corresponding to the transgenes are not likely to be produced. Fourth, many common transgenes used in plant biotechnol ogy are derived from bacteria commonly

found in the environment. The FDA has evalua ted horizontal gene tr ansfer from the use of selectable marker genes a nd concluded that the likelihood of transfer of such genes from plant genomes to microorganisms in the ga strointestinal tract of humans or animals, or in the environment, is remote ( http://vm.cfsan.fda.gov/~dms/opa-armg.html )

. Therefore APHIS concludes that horizontal gene transfer poses no significant

environmental risk.



### C. Cumulative Effects32

The f ield test sites in this permit application have been in agricultural or forest research, or in agricultural production or forest tree pl antations for from 5 to 50 years. Therefore the land has been in continuous agricultural or forest tree production for years prior to these releases and it is reasonably foreseeable that if the permit were not issued that the

sites would continue to be unde r agriculture or forestry prod uction. It is also reasonably foreseeable that the applicant may request to further extend the permit for this

environmental release for addi tional years to observe the growth of these trees to maturity. Moreover, APHIS has received a petition for the deregulation of these transgenic Eucalyptus trees, however, the environmental effects of that petition will be analyzed in a separate document. The temporar y change from agricultural crops to a tree crop may result in a temporary change in re sident animal and plant species, but after harvest it is reasonably foreseeable that the la nd 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 dest royed. Therefore the onl y past, present, and reasonably foreseeable actions associated with the locations for the proposed releases under permit are those related to agricultur al or forest tree production. APHIS has determined that there are no past, present, or reasonably foreseeable actions that would aggregate with effects of the proposed acti on to create cumulative impacts or reduce the long-term productivity or sustainability of a ny of the resources (soil, water, ecosystem quality, biodiversity, etc.) associ ated with the releas e sites or the ecosystem in which they are situated. No resources will be significantly impacted due to cumulative impacts resulting from the proposed action.



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

Potential Effects of Growing Eucalyptus on Soil Hydrology

Eucalyptus is recognized as having impacts on hydr ology and large widespread plantings could have potential impacts on hydrology in the southeastern United States (Farley et al. 2005). Since large plantings of Eucalyptus have not been grown in many parts of the southeastern US (other than southern Florid a) the potential impacts of such plantings on hydrology are unknown. APHIS requested additiona l information on potential impacts of hydrology from ArborGen and also consulted with the USDA Forest Service to assess the potential impacts of planting Eucalyptus on hydrology. The additional information supplied by ArborGen and the Forest Service ar e included as Appendices II and III. The document supplied by the Forest Service repr esents only their opi nion on the potential impacts of these field tests on hydrology and do es not represent the position of the USDA on the pros and cons of deploying Eucalyptus as a biofuel, bioenergy or fiber crop.

The Forest Service indicates that planting large-scale Eucalyptus plantations may potentially lower the water table, and aff ect groundwater recharge and stream flow dynamics. Eucalyptus is very efficient at using water. It can produce more biomass per

unit water consumed than native southeaste rn pines; however, their extremely rapid biomass production has proportiona lly higher transpirational co sts and therefore greater

water use. The Forest Service has estimated that a mature Eucalyptus plantation growing33

in southwest Georgia could potentially tran spire 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 prev ious 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 pl ants depending on the crop, the growing season, and the management practices.

Eucalyptus has a dimorphic rooting pattern which means that it has surface roots that draw water from the surface as well as deep roots which draw water from deep within the soil. The mean maximum rooting depth for Eucalyptus is 15 meters, which is a characteristic of a dimorphic rooting pattern. In contrast, mean maximum rooting depths of pine plantation ( P. taeda and P. elliottii ) and grass species are 3 meters and 2.6 meters, respectively (Canadell et al. 1996). Accord ing to the Forest Service, conversion to Eucalyptus on sites where the water tables are le ss than 10 meters will likely lower downslope water tables via direct means (i.e., dire ct use of ground water by deep roots), affect groundwater-aquifer dynamics, and result in evapotranspiration rates that exceed precipitation input, as have been reported for th is species in other lo cations (Calder et al. 1997).

Recent research suggests that Eucalyptus plantations would redu ce stream flow more than pine plantations, and could potentially e liminate low flows. In a review of more

than 20 catchment 5 conversion studies, Farley and others (Farley et al. 2005) showed that converting existing vegetation to Eucalyptus plantations reduced stream flow by 20% more than converting it to a pine plantation. Th is 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). E limination of low stream flows could have important ramifications for threatened and e ndangered aquatic species, such as the gulf strain striped bass, and species of endemi c freshwater mussels (Golladay et al. 2004, Couch and McDowell 2006).

Due to a lack of available data in the southeastern U.S. on planting Eucalyptus , it is difficult to determine the significance of the effects on hydrology if large acreage of Eucalyptus were to be planted. The Forest Servi ce has indicated that collection of data and modeling will be useful to determine the long-term impacts of planting large acreages of the genus. The Forest Service has al so pointed out that the significance of the impact on groundwater and stream flow will depend greatly on the area extent, size, and spatial distribution of the plantations. For example, a few small (less than 10 hectares, i.e. approximately 25 acres) and well-dispersed plantations may only have very localized impacts and negligible impacts at the watershed scale.

The field test sites requested under the two pe rmits are well dispersed and are limited in size (none are greater than 20 acr es) and it is anticipated that they are not likely to have

5 A catchment or drainage basin is an extent of land where water from rain or snow-melt drains downhill into a body of water, such as a river, lake, reserv oir, estuary, wetland, sea or ocean. The drainage basin includes both the streams and rivers that convey the water as well as the land surfaces from which water drains into those channels, and is separated from adjacent basins by a drainage divide.34

significant impacts on hydrology. At the request of APHIS, ArborGen has supplied data indicating th e maximum size of each of the plantings at each site, the individual watersheds where the plantings occur, th e area of the watershed, how much of the watershed will be occupied by the field tests, the location of the closest primary and secondary streams, and the location of any crit ical habitat for Federally listed threatened and endangered species w ithin the watershed.

Using the 8 digit HUC (Hydrologic Unit Code) as the Watershed to be analyzed, the data provided by ArborGen show that none of the sites occupy more than 0.03% of any given

watershed. The closest critical habitat for an aquatic species (such as a fish and mussel) is 6 kilometers at one location and ranges from 6 to 90 km for any of the sites having any proximity to habitats that could be imp acted. There are no nearby threatened or endangered plant species that could be im pacted by hydrological effects (see also Appendix IV). Any effects would be very localized on existing ne arby agricultural and forestry plantings. Therefore APHIS c oncludes that while the effects on hydrology, including the watershed and aquifers, are unkno wn and uncertain for very large plantings

of Eucalyptus , these small-scale field tests are unlik ely to have any significant negative impacts on hydrology and on native flora and fauna.

Potential Allelopathic Effects of Eucalyptus

Allelopathy refers to “any process involving secondary me tabolites produced by plants, microorganisms, viruses and fungi that in fluence the growth and development of agricultural and biol ogical systems” (S ee: International A llelopathy Foundation - http://www.allelopathy-jour nal.com/allelopathy.aspx ). Allelochemicals from plants are released into the environment by exudation fr om roots, leaching from stems and leaves, or decomposition of plant material. Alle lopathy 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 increased research activ ity in this area, one of which is taking advantage of plants that produce allelopath ic compounds in devel oping agroforestry and sustainable agriculture system s (Kohli et al. 2006, Narwal 2006)

. Allelopathy has been demonstrated in many commercially important tree species including Acacia

, Ailanthus Eucalyptus, Juglans, Quercus, Leuc aena, Pinus, Picea, Aibes, Populus and Acer ; and has been demonstrated in agronomic crops such as rye, wheat and alfalf a (Nandal et al. 1994, Ferguson and Rathinasabapathi 2003, Reigosa and Gonzáles 2006, Mallik 2008).

There have been extensive studies conducted on allelopathy in Eucalyptus and there are several comprehensive reports and review articles on this genus (Ong 1993, Sunder 1995) (Nandal et al. 1994, Davidson 1995, White 1995). Eucalyptus species are known to produce chemical compounds that are required by the plant for defense against herbivores and pathogens. There are several studies in the literature that dem onstrate the negative, positive and neutral allelopathic interaction of Eucalyptus species and their hybrids with other crop plants (Sanginga and Swift 1992) (Khan et al. 2004) (Espinosa-Garcia et al. 2008). These interactions vary greatly de pending upon the crop sp ecies and conditions under which they are grown. There is inconclu sive data as to whether these compounds produced by Eucalyptus are exclusively responsible for allelopathic influence on35

understory vegetation in Eucalyptus plantations. Most alle lo pathic studies in Eucalyptus species have involved laboratory experiments with extracts obtained from different plant parts or leaf litter to investigate allelopath ic effects on seed germ ination and growth in potted plants. These laboratory bioassays and pot culture studies may or may not be applicable to field conditions. The perceived allelopathic effects obs erved in the field on growth of understory or adjacent intercr opped food crops could also result from competition for water, nutrients and light.

Allelopathy tends to be an inexact scie nce and many studies in allelopathy are inconclusive and difficult to interpret due to potential interactions with other aspects of the environment. For example in a recent study, Nandal and Dhillon (2005) tested the allelopathic effects of poplar ( Populus deltoides ) leaf extracts on ge rmination and growth

of ten wheat varieties under la boratory conditions. They report ed that lower concentration of leaf extracts from poplar had stimulatory effects on root length in all wheat varieties whereas higher concentrations adversely aff ected germination and seedling growth of

some of the wheat varieties tested. In a field experiment, the performance of all ten

wheat varieties was also evaluated under four different poplar spacings in an agrisilviculture system. Although the grain yield of wheat varieties wa s significantly lower under all spacings of poplar compared to cont rols, yields increased significantly with increased spacing of poplar, possibly due to reduced competition for light and nutrients. However, no correlation was found between th e laboratory bioassay us ing leaf extracts

and the field studies.

In a recent study, the allel opathic 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 investigat ed (Espinosa-Garcia et al. 2008). Soil samples were collected from different soil horizons and at varying distances from Eucalyptus trees growing at the plantation edge and used for growth studies in pots. The dried soil samples used for growth studies we re 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 sl ightly inhibitory, to a stimulatory effect on germination and radicle 6 growth of test crops. Among the three Eucalyptus species tested, the soil samples from E. grandis x urophylla plantations had an inhibitory effect on germination of maize, bean and watermel on but had a stimulatory effect on squash.

The soil from E. grandis plantations had an inhibitory e ffect on squash. The total soluble phenolics varied in different soil samples but did not explain the di fferential 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 demonstr ate allelopathic properties, the presence of any allelochem icals is not going to make the Eucalyptus planted under these permits more invasive or present a plan t pest risk. Since a ll these field tests are

6 The radicle is the first part of a seedling (a growin g plant embryo) to emerge from the seed during the process of germination. It is an embryonic root.36

conf ined and limited in size, a ny allelopathic effects should be small. As a standard silvicultural practice, herbicides will also be used within the field test sites and any of their effects on understory vegetation will be as severe or more severe than any allelopathic effects. In the future, should any negative allelopathic or other competitive

interactions be observed under field conditions outside of the immediate field tests sites, these could be mitigated by adjusting th e tree spacing, irrigation and fertilization practices or by planting the fiel d 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 conditions of the permit; including any indications of allelopathic

effects.



### E. Risks to Threatened and Endangered Species

APHIS has reached a determination that the proposed environmental release will have no effect on federally listed thre atened or endangered species or species proposed for listing, and no effect on designated cr itical habitat or habitat proposed for designation in the action area. Consequently, consultation under Section 7 of the Endangered Species Act

with the United States Fish and Wildlife Serv ice 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 area of the field release.



### F. Other Considerations

Consideration of Executive Orders, Standards and Treaties Relating to Environmental Impacts.

Executive Order (EO) 12898, "Federal Actions to Address Environmental Justice in Minority Populations and Low -Income Populations," requir es Federal agencies to conduct their programs, policies, and activities that substantia lly affect human health or the environment in a manner so as not to exclude persons and populations from participation in or benefiting from such program s. It also enforces existing statutes to prevent minority and low-income communities from being subjected to disproportionately high and adverse huma n health or environmental effects.

EO 13045, “Protection of Children from Environmental Health Risks and Safety Risks,” acknowledges that children may suffer dispr oportionately from envi ronmental 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) require s each Federal agency to identify, assess,

and address environmental health risks and safe ty risks that may disproportionately affect

children. Each alternative was analyzed with respect to the above EO 12898 and 13045. The human health and environmental impacts of the action alternatives are presented in

Section V of this EA. No human health or environmental effects were identified for any

of the action alternatives that would have a disproportionate adverse effect or that would37

exclude a particular group of persons or populations, includ ing m inority and low-income populations, or children, from expected benefits.

EO 13112, “Invasive Species”, states that fede ral agencies take action to prevent the introduction of invasive speci es and provide for their co ntrol and to minimize the economic, ecological, and human health impact s that invasive species cause. The hybrid species of Eucalyptus being grown is not considered an invasive species and does not

establish itself without human intervention (as described abov e). Based on historical experience with the Eucalyptus in these field tests, the engine ered plant is not expected to have an increased invasive potential.

Executive Order 12114, “Environmental Effect s Abroad of Major Federal Actions” requires Federal officials to take into cons ideration any potential environmental effects outside the U.S., its territories and possessi ons that result from actions being taken. APHIS has given this due consideration and does not expect an environmental impact

outside the United States shoul d APHIS choose any of the two alternatives. These field tests are being conducted in th e continental U.S. and would not be expected on have

environmental effects outside of the U.S.

Migratory Bird Treaty Act, 1918 as am ended and Executive Order 13186. Migratory

birds include all nativ e wild birds found in the United St ates except the house sparrow, starling, feral pigeon, and resident game birds such as pheasant, grouse, quail, and wild turkeys. A reference list of migratory game birds is found in Title 50, Code of Federal Regulations, Part 10. The Mi gratory Bird Treaty Act ma kes it unlawful for anyone to kill, capture, collect, possess, buy, sell, trade, ship, import, or export any migratory bird, including feathers, parts, ne sts, or eggs. Executive Or der 13186 “Responsibilities of Federal Agencies to Protect Mi gratory Birds” requires Federa l officials to consider the impacts of planned actions on migratory bird populations and habitats for all planning

activities. APHIS has determined that it is reasonable to assume that the activities at the field test sites such as planting, collecting samples and eventual harvest of the trees should have no impact on migrat ory birds since they would not be expected to inhabit

these sorts of field tests.

Consistency of Proposal with ot her Environmental Requirements:

The proposal is believed to be consistent w ith other environmental requirements. This environmental assessment was prepared in accordance with: (1) The National Environmental Policy Act of 1969 (NEPA), as amended (42 U.S.C 4321 et seq .); (2) regulations of the Council on Environmental Quality for implementing the procedural provisions of NEPA (40 CFR parts 1500- 1508); (3) USDA regulations implementing NEPA (7 CFR part 1b); and (4) APHIS’ NE PA Implementing Procedures (7 CFR part 372).



### G. Conclusion38

As outlined under the Pu rpose and Need sections of this document, this EA was prepared because it was necessary for APHIS to eval uate the potential environmental impacts resulting from the increased number of locati ons and size of the releases of flowering Eucalyptus , which could potentially lead to a lack of confinement of the field tests and and other impacts to the environment. APHI S has evaluated the permit applications to determine whether the environmental release, with appropriate c onditions imposed, can be carried out while preventing the disseminatio n and establishment of plant pests. After preparing this draft EA, APHIS has concluded th at even though there is an increase in the

number of sites where trees will be allowe d to reach maturity and flower, over those already allowed to flower under permits 06-325-111r and 08-151-101r, there is no substantially greater ri sk of loss of confinement and ri sk to the environment. APHIS

concludes that the releases will remain as c onfined field tests and that the genetically engineered trees will not pose a significant pl ant pest risk. In a ddition, APHIS concludes that granting permits will not significantly a ffect the quality of the human environment. No threatened and endangered species or critic al habitat should be impacted by letting the trees reach maturity and flower at the increased number of locations.



## VI. Listing of Agencies and Persons Consulted

James M. Vose - USDA-Forest Service Co weeta Hydrologic Labor atory, Otto, NC Chelcy R. Ford - USDA-Forest Service Coweeta Hydrologic La boratory, Otto, NC Jody Smithen – US Fish and Wildlife Service – Daphne, Alabama Field Office Kathy Chapman - US Fish and Wildlife Service - Coastal Georgia Field Office

James Harris – US Fish and Wildlife Serv ice – Lacombe, Louisiana Field Office

Laura Zimmerman - US Fish and Wildlife Se rvice – Charleston, South Carolina Field

Office Caroline Stahaller - US Fish and Wildlife Service - Panama City, Florida Field Office

Brad Rick - US Fish and Wildlife Serv ice - Vero Beach, Florida Field Office

Candice Martino - US Fish and Wildlife Serv ice - Jacksonville, Florida Field Office



## VII. References

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## APPENDIX I: Status of Existing Field Tests Allowed to Flower

The applicant has been allowed to let transgenic Eucalyptus trees flower under permits

06-325-111r-a2 and 08-151-101r. These trees had the same constructs as those in these two permit applications. All transgenic and non-transgenic trees in these approved tests spanning 5.1 acres in Alabama a nd 1.4 acres in Florida, produ ced mature flowers in late August-early September of 2007 and/or 2008. On the 25-30 foot tall tr ees in these tests

there were estimated to be several thousand flowers on each tree. No differences were noted in flower formation in transgenic trees compared to non-tran sgenic controls (the same parental genotype).

In January 2008, mature (but not yet ope ned) capsules were collected from nontransgenic and transgenic trees under pe rmit 06-325-111r. Replicate samples were collected from transgenic trees and non-tran sgenic controls in two separate blocks. Capsules from a subset of transgenic trees pl us non-transgenic contro ls were dried in the laboratory and allowed to open to evaluate the pr esence of seed or seed like structures in the capsules. Approximately 100 capsules fo r each of the two replicate samples were analyzed. Microscopic examination of the ma terial inside the capsules did not show any seed or seed like structures in capsules of e ither non-transgenic or transgenic lines. Controlled germination studies of the mate rial extracted from the capsules did not produce any germinating seeds. Observati ons from the replicated field study being conducted in Alabama under permit No. 06-325- 111r confirm that cold tolerant lines grown in this field test also did not produce any viable pollen. The results to date have shown that the barnase gene that has been e ngineered into these trees is effective at preventing viable pollen formation.

After the initial draf t EA was published, the applicant submitted annual reports for permits (06-325-111r-a2 and 08-151-101r) as required under the supplemental conditions. The transgenic and non-transgen ic trees planted unde r these permits in Baldwin County, Alabama and Highlands Count y, Florida were prev iously allowed to

flower.

In Alabama, the transgenic trees produced several hundred to a few thousand flowers per tree whereas the non-transgenic controls had mu ch fewer flowers as a result of significant cold damage. In Florida, there was no diffe rence in the number of flowers produced by both transgenic and non-transgenic trees. Matu re seed capsules, pr ior to opening, were sampled from select trees in three field te sts in Baldwin County, Alabama and trees from the single field test in Highlands Co unty, Florida in early March 2009. Samples consisted of approximately 70 to 100 capsules collected from different positions in the crown. The capsules were returned to Arbor Gen’s greenhouse facility where they were dried and contents of the capsules were extr acted and stored at 4°C. A controlled germination test was conducted using approxi mately 0.1 g of the extracted contents of each sample spread on moist filter paper in a st andard Petri dish. Open pollinated seed of EH1 obtained from Brazil were germinated as control seedlings for comparison.45

Of the sa mples collected and analyzed from trees in Baldwin County, AL, approximately

4% of the seeds showed a low level of germination while approximately 83% of the samples collected from trees in the fiel d trial in Highlands County, FL, showed germination, including sample s from both transgenic and non- transgenic control trees. For both transgenic and non-transgenic tree sa mples analyzed, the applicant observed 2-8 seedlings for each 0.1g sample plated for germ ination. 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 seedlings observed in these tests is much lower than would be expected for open pollinated seeds.

The controlled germination tests from samples collected in spring 2009 from field trials at two sites indicate that a very low level of seedlings can be produced from these trees. The number of seedlings produced is significan tly less than what would be expected for an open-pollinating mixed stand of Eucalyptus . So far the data are consistent with the hypothesis that limited self pollination can o ccur from viable pollen produced by the nontransformed control trees. Th e applicant has also not obser ved any volunteer seedlings in

or around the test sites. Volunteer monito ring and reporting the pr esence of volunteers is required in the supplemental permit conditions.

Monthly field test monitoring obs ervations have not identified any differences in diseases and insects or other non-target organisms betw een the transgenic and non transgenic trees

in the field test.46



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

Submitted by ArborGen. LLC to USDA APHI S 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 c onclusions drawn by experts in the field of hydrology, ecology, and plantation ma nagement on the hydrology of Eucalyptus plantations are discussed in this document. Extensive re search on hydrology has been

conducted in countries wher e 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 fore stry crops relative to agricultural crops. As stated in Poore and Fries, 1985, “... most crops in many parts of the world are of foreign origin (wheat, maize, rice, potatoes, manioc, rubber, oil palm, coconut and many others. No one is surprised either that the soil unde r agricultural crops becomes depleted if these are continuously cropped wit hout 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 itse lf became more dry (Davidson, 1995). In fact Eucalyptus uses less water per unit weight of bi omass 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 eucalypt s, where different species ha ve different characteristics which may prove detrimental or beneficial und er 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 na tural vegetation such as savannah, scrub or grassland; on degraded or waste land either as a potential crop or to assist in the control of erosion or salinity; within agricultural land as shelter belts or as components of agroforestry systems, or as intensivel y managed crops for wood production. It is important to understand the particular appli cation in order to evaluate the potential ecological impact of Eucalyptus in a scientific manner. For example, the effect on soil moisture content and the water table can only satisfactorily be judged with reference to47

the pre-existing conditions before th e establishm ent of the planting of Eucalyptus . In the

case of field trials of Eucalyptus requested in ArborGen’s permit applications, all sites are on land previously managed for forestry, agricultural production or maintained as pasture

land.

The Water Cycle

In considering the potentia l 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 Figur e 1 in Poore and Fries, 1985, or see Figure 1-1 in NRC, 2008). One key variable is the amount of ra in that is intercep ted 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 su rface or is evaporated. A certain amount of water is maintained in the soil layer ag ainst the forces of gravity (called the ‘fieldcapacity’ and dependant on soil textur e and organic content) while any excess drains to the water table, the level at which the soil is permanently saturated. Depending on the depth of the water table it may be accessed by deep rooting plants such as trees.

Even without roots that reach to the water tabl e, plants may be able to access water from deeper, wetter soil layers through capillary action, depending on the soil type, where there can actually be an upward movement of water.

Through normal transpiration plant roots take up available water which is transported through the stem to the leaves , the majority of which is lost to the atmosphere. Evapotranspiration is the total water returned to the atmosphere th rough transpiration and evaporation from the ground, bodies of water, plus intercepted water in the canopy. The relative rates of evapotranspi ration and precipitation are of ten compared in assessments of hydrological systems. Where precipitation ex ceeds evapotranspiration then there is a net water gain to groundwater or downst ream systems. Where evapotranspiration exceeds precipitation then the available water resources may be depleted. It is important to understand, as pointed out in Poore and Fries (1985), that water loss is a “price that plants must pay for growth”. When stomata in plant leaves close th en photosynthesis and growth both cease. In general te rms, the rate of growth or biomass production in a plant is proportional to its water use. Consequent ly fast growing trees, of any species, use large quantities of water. As described below, Eucalyptus is actually more efficient in terms of water used per gram of biom ass produced than many other tree species.

Hydrology of Eucalyptus – Key Literature Reviews

There has been extensive literat ure published on the hydrology of Eucalyptus . Google Scholar for example lists over 60,000 hits for the keywords ‘ Eucalyptus ’ and ‘water’. It is therefore not possible to pr ovide summaries of the entire breadth of the literature.

Several review articles are available including reviews s ponsored by FAO that assessed the ecological impacts of Eucalyptus plantations, including anal ysis of the impact of Eucalyptus on hydrology. We provide here a summary of these reviews together with

data from some specific reports (see below) where the hydrology of Eucalyptus has been48

studied in detail. FAO has also released two annotated bibliographies (FAO, 2002a, 2002b) that collate and summ ar ize publications on environmen tal, social and economic impacts of Eucalypts, and which include many references to water use. While there are specific examples and geographic regions where Eucalyptus (and other trees) can negatively impact hydrology, in general, the literature indicates that Eucalyptus can be grown in a sustainable manner and that its a ssociated water use is not a major ecological

threat. Most of the reviews indicate that soil and water char acteristics of the site should

be taken into consideration when establishing and maintaining a Eucalyptus plantation, in the same way that would be appropriate for plantings of agricultural crops.

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

. At that time the authors suggested that there were relatively few ex isting studies in several important areas including hydrology (Lima, 1984; Poore and Fries, 1985). Where comparative studies showed that for dry alpine c onditions the water regime for Eucalyptus did not differ from adjacent grasslands (see Lima, 1984), this was attributed to Eucalyptus ’ ability to control the rate of transpiration, an evolutionary adaptation for survival of drought stress which is often typical of the rainfall regimens of thei r native habitats. For deep soils and higher

rainfall Eucalyptus plantations might be expected to reduce streamflow or groundwater recharge but that this is comparable to these same effects in pine plantations. In contrast, the water intercepted and re-evaporated by the foliage, and therefore not available to the soil, is less for Eucalyptus when compared to pines, due in part to the near vertical

orientation of leaves in Eucalyptus (Whitehead and Beadle, 2004) . It was concluded that the conditions of a particular site need to be taken into account as well as balancing local demands for forest products and water.

By 1993, at an FAO sponsored regional Expert Consultation on Eucalyptus (White et al, 1995) more information was available about hydrology and Eucalyptus plantations. These experts presented their experiences with Eucalyptus plantations from Asia. The report recognized the potential benefits of Eucalyptus and noted that many of the criticisms of the species were base d on inappropriate government policies on

afforestation or social concerns rather than the biology of the trees themselves (see also Casson, 1997). Calder et al (2004) highlight s that many early policies were based on public misconceptions about the impacts of fore sts on water. With regard to hydrological effects on intercropping with ot her species, the experts in th e FAO report concluded that

while Eucalyptus can have negative effects in drier cl imates, 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 wa ter 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 ex isting plantations.

As part of this expert consultation Sunder (1995) reported that the overall use of water by Eucalyptus is lim ited 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 di ffer significantly from other trees. As an example, monthl y evapotranspiration of an E. globulus plantation in Portugal was the same as that of a natural open stand of cork oak ( Quercus suber ) with a49

developing understory of shrubs (de Al meid a and Riekerk, 1990). Patil (1995) reported data on water consumption at site s in India, which although high in Eucalyptus , was the

most efficient in terms of water consumed per gram of biomass produced (see also Silva et al 2004). In fact, water use efficiency in Eucalyptus actually increases with greater water availability (Stape et al, 2004a, 2004b). Patil (1995) also noted that there were no hydrological impacts of Eucalyptus on adjacent crops at these sites. White (1995) stated that large plantings of Eucalyptus may reduce water yield and lower water tables but this

varies from one situation to another and most importantly can be mitigated through management practices such as changes in tree stocking regimes. The environmental

considerations of Eucalyptus are the same as those for agricultural crops. Davidson (1995) noted that drawing water from sha llow or deep wells to supply high water demanding crops such as rice or cotton can ha ve a greater impact on drawing down water tables than fast growing tree plantations. He also concluded that many potential adverse effects are reversible, as noted earlier by Poore and Fries (1985).

A review of the environmental issues of Eucalyptus plantations in Brazil was published by Oak Ridge National Laboratory (Couto and Betters, 1995). This report summarized that the hydrology of Eucalyptus plantations was comparable to other tree plantations or natural forest cover and that any effects w ould largely depend on management practices.

Numerous studies demonstrate that forest cove r and any changes in this alter water yield: reducing forest cover typically increases wate r yield and vice versa (Bosch and Hewlett, 1982; Sahin and Hall, 1996).

More recent reviews support the points made above. Binkley and Stape (2004) contend that very large tree plantations must address similar issues of sust ainability 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 conduc ted in collaboration with Aracruz Cellulose Company (reported by Almeida et al 2007, described in more detail below). Binkley and Stape conclude th at in semi-arid environments afforestation with any species of trees may increase water use, lower ground wate r levels and reduce streamflow. Given appropriate silvicu ltural management however, wood production should face no barriers to sustainability.

Whitehead and Beadle (2004) provided a co mprehensive 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 ma nipulation to maintain turgor in leaves, as well as an ability to form deep roots. Ma ximum 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 ca tchments. Conversely, the high water usage by Eucalyptus may be valuable in purposefully lowe ring water tables to reduced potential salinity problems. It is therefore important to assess productivity and water use in

relation to clim ate variables, nutrient supply and options fo r silvicultural management, and careful matching of species to sites where available water may be limited. One of the50

physiological responses of Eucalyptus to lim ited water noted by Whitehead and Beadle is to reduce LAI, thus although Eucalyptus are evergreen species ther e can be large seasonal changes in LAI in response to dry seasons. Similar observations were made in reduced LAI along a gradient of water availability by Ares and Fownes (2000). The root systems of Eucalypts are dimorphic, with widely spre ading lateral root systems below the surface plus a deep tap root system. In a plantation of 7 year old Eucalyptus trees in Brazil the tap root extended to a depth of about 2.5m (Almeida and Soares, 2003) consistent with other observations (see Srivas tava et al, 2003). When arti ficially 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. Unde r other conditions wate r was utilized from soil below the root zone by upward movement from wetter levels. In considering these physiological adaptations Whitehead and Bead le conclude that in the case of South Africa, where planted Eucalyptus replaced native grasslands, th e decreased water yields resulted from increased transpiration in the evergreen and deep rooted Eucalyptus during the dry season compared to the seasonally dorm ant grasses. It is well established that forests have greater evapot ranspiration than grassla nds (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 afforestati on, 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 afforestati on with different specie s of trees, including Eucalyptus , has lead to changes in the water bala nce including lowering of water tables and restricting stream flows. Calder and colleagues have published several reports on afforestation efforts in India including examples where deep-rooted Eucalyptus were able to tap into water resources not previously util ized by short-rooted sp ecies (Calder et al , 1997), but also describes cas es where water use by Eucalyptus was comparable to indigenous forests at some sites (Calder, 1994) . Similar studies of na tive grasslands have documented negative impacts of Eucalyptus on the water balance in South Africa (Lesch and Scott, 1997; Scott and Lesch, 1997; Sc ott et al, 1998) and Argentina (Jobbagy and Jackson, 2004; Engel et al, 2005; Nosett o, 2005). In many of these cases other introduced trees including pines had similar impacts and particular ly in South Africa impacts on water balance result from a wide va riety of introduced species (Le Maitre et al, 2000; 2002).

These examples contrast with the experien ce in Brazil where there has been extensive

reforestation with Eucalyptus over many decades. Much of this literature is in Portuguese but often abstracts are publishe d in English. Lima and colleagues have published a number of reports that analyzed potential im pacts 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 herbaceous51

vegetation (Lim a and Freire , 1976). In these trials Eucalyptus actually showed greater interception than pine (Lima, 1976) and c ontrasts with references above, but likely reflected the greater average height of the Eucalyptus at 13.4 m compared to an average of 6 m for pine.

Similarly, an examination of the water balance of Eucalyptus plantations in China were not considered to be deleterious for water supplies (Lan e et al 2004). While 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 au thors have concluded that the hydrological impacts of Eucalyptus are comparable to and should be viewed in the context of other tree species (see for example Myers et al, 1995; Wullschleger et al, 1998).

One of the largest studies comparing Eucalyptus and native trees conducted to date has

been a catchment area in Brazil of over 280 hectares (owned by Aracruz Cellulose S.A.) consisting of 190 ha of planted hybrid Eucalyptus and almost 90 ha of native Atlantic rainforest, that was analyzed over a period of si x years. Average precip itation at this site was 1147 mm, which is similar or less than th e sites listed in this permit (range from ~1160 mm in Glades County Florida to al most 1750 mm in Escambia County, Alabama). Mean high temperature at the Aracruz site was 32.6 C (~91 F) for February (the summer season in the southern hemisphere), again, co mparable 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 evapotranspirati on was strongly influenced by precipitation (Almeida et al, 2007). In an unusually dry year evapotranspiratio n was about half that compared to when water was readily available. In this dry year evapotranspiration exceeded precipitation but conversely in wetter year s evapotranspiration was much less than precipitation. Over the length of the study evapot ranspiration was ~95% of preci pitation. This adjustment in response to varying conditions and water ava ilability was indicativ e that these hybrid trees exert strong stomata control and utili ze water according to its availability. In a

series of studies in this same area conducte d over a period of 8 years Almeida and Soares (2003, text in Portuguese with abstract and fi gure legends in English) examined a number of other hydrological parameters. Stomatal conductance was steady over several months with adequate water and then dropped significantly as available water dropped and the predawn leaf water potential ( Ψ ) increased, again demonstrating strong stomatal control. Rainfall interception by the Eucalypts averaged ~11% compared to ~24% in the native forest and water availability (at a depth of 2.5 m) is almost identical in the native forests

and the Eucalyptus plantations during the we t summer months but is less in the area with Eucalyptus during the drier winter. The authors attribute this to the deeper roots systems (>5m) of the native trees accessing wa ter at deeper levels, while the Eucalyptus (with roots only to ~2.5m) are limite d to th e available water in the shallower levels. Finally, the authors compared the ratio of evapotra nspiration and precipitation (ET/P) of the planted Eucalyptus with the native forest. In years with normal precipitation ET/P was comparable for both the Eucalyptus and native forest. In years with less than normal52

precipitation the native forest had higher ET/P (that is, evapo transpiration was much greater than precipitation) compared to the Eucalyptus . Based on their data, the authors suggest that the native forest has a great er consumption of water relative to the growth/harvest cycle of Eucalyptus , since in the first fe w years after planting transpiration in the plantation is much less than the native forest.

Competition for Resources between Eucalyptus and Adjacent Crops

There has been speculation that water use by Eucalyptus could have a negative impact on water resources available for adjacent vegeta tion 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 o ccur within 1 to 2 tr ee-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 r oot pruning to reduce the area occupied by the tree r oots. Impacts attributed to Eucalyptus depend on specific site conditions, and as with other concerns , there are examples where no negative impact on adjacent agricultural crops were observed (e.g. Patil, 1995). At some sites this could be attributed to deeper rooted trees versus shallow rooting crops utilizing water from different soil profiles. Finally, tree planting has been proposed as a mitigation strategy where rising water levels increase salinity and reduce crop yields (Hatton and George, 2001).

Summary and Conclusions:

Many studies report that water use in Eucalyptus is comparable to other tree species.

There are some cases where afforestation with Eucalyptus (or other tree species) has lead to reduced water run-off and supply of st reams or changes in water table levels, especially in regions with limited rainfa ll. However, in many well documented cases Eucalyptus plantations do not have any signi ficant negative impacts on hydrology. Where there have been purported negative impacts, these often reflect more complex

issues such as socioeconomi c 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 co mparisons 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 othe r fast growing trees species.

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## APPENDIX III: USDA Forest S ervice assessment of impacts on hydrology

This document was prepared by C. R. Ford a nd J. M. Vose in response to the document titled “Hydrology considerations for planted Eucalyptus ” submitted by ArborGen LLC to USDA APHIS BRS in support of consideration fo r approval of permits for field trials of Eucalyptus at multiple sites

Executive Summary

We reviewed the materials provided by Arbor Gen and synthesized the literature on water use by Eucalyptus and other vegetation in the southeas tern US. Based on these materials and our best professional judgment, we provide the following assessments:

1. Water use efficiency (WUE) is not a good metr ic to evaluate impacts on hydrology From a hydrologic standpoint, total water use (transpiration + interception) is a more appropriate metric to assess hydrologic impacts. A species may have high WUE (defined by ArborGen as volume of wood produced per amount of water required), but still transpire and inter cept 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 liter ature and estimate of Eucalyptus transpiration suggests that water use is at least 2-fold greater than most other native forests in the

southeastern US.

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

invades native forests.

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

5. Planting Eucalyptus hybrid plantations will lower the water table, and affect groundwater recharge and stream flow dynamics The combination of shallow and deep roots typical of Eucalyptus species has the potential to impact both surface and groundwater hydrology.

6. It is unlikely that lower stocking levels will be an acceptable management practice to reduce hydrologic impacts of Eucalyptus plantations.58

High biom ass production requires fully st ocked stands. Reducing stocking to minimize hydrologic impacts is likely to counter the benefits of planting fast growing Eucalyptus.



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

Water use efficiency (WUE) is not a good me tric to evaluate impacts on hydrology From a physiological standpoint, water use effici ency is defined as the ratio of the moles

of carbon fixed to the moles of water lost. WUE is a leaf-level metric. The Hydrology document prepared by ArborGen provided a ratio of liters of water consumed to grams of biomass produced. While these ratios pr ovide good information regarding the transpirational cost of biomass production, th ey do not incorporat e information on the magnitude of evapotranspirational lo sses, nor do they integrate stand management effects (e.g., planting density, rotation ag e). Hence, WUE is a poor metr ic to evaluate the effects

of Eucalyptus on water resources. For example, Eucalyptus can produce more biomass per unit water consumed than native southeas tern pines; however, their extremely rapid biomass production has proportionally higher tr anspirational costs and hence greater water use. Better metrics of evaluating the impacts of Eucalyptus hybrid plantations on hydrology exist. In order of scale, th ese are evapotrans piration (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 la ndscape scale using precipitation input minus stream flow output on paired-catchments ( P

- R
o ). The net effects of greater evapotranspiration losses are reduced so il moisture, reduced groundwater depth and recharge, and reduced stream flow. These pa rameters 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 types Previous studies have quantified annual E t from various southeastern US forested and crop lands (Table 1). Native pine plantations consume nearly twice the water consumed by longleaf pine savannas, but only margina lly more than mature upland hardwood forests. In contrast, 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. The Hydrology document prepared by ArborGen states that the propos ed sites are on land previously managed for forestry, agricultural production or main tained as pasture land. In these cases, we may expect Eucalyptus E t to exceed that of previous pine plan tations by a factor of 1.6, and previous pasture land by a factor of 3.5. The comparison wi th 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.59

Table 1

Vegetation type Mean transpiration

(mm yr -1 ) Reference Longleaf pine savanna 244 (Ford and others 2008)

Old field 250 (Stoy and others 2006)
Oak-pine-hickory forest 278 (Oren and Pataki 2001)
Upland oak forest 313 (Wullschleger and others 2001)
Mixed pine hardwood 355 (Phillips and Oren 2001)
Mixed pine hardwood 442 (Stoy and others 2006)
Planted loblolly pine 490 (Stoy and others 2006)
Mixed pine hardwood 523 Sc hafer and others 2002
Slash pine flatwoods 563 (Powell and others 2005)
Eucalyptus hybrid plantation 882 Estimated for SW GA in average climate and rainfall year from model published in (Mielke and others 1999)

Cotton (non-irrigated, annual) 392 (Howell and others 2004)
Strawberries (irrigated, 7- month crop, 5-month fallow) 1397 (Clark 1994; Allen and others 1998)

Watermelon (irrigated, 3- month crop, 9-month fallow) 237 (Allen and others 1998; Shukla and others 2007)

Eucalyptus has much higher 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 va por 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 variat ion in leaf water potential. The benefit of having a high  , is having a high conductance in humid environments ( g s ref). The southeastern US is a relatively humid envi ronment, with average daily VPD values around 1.5 kPa (Ford and others 2004). In this humid environment, we can expect that Eucalyptus hybrid plantations will have stomat al conductance rates that are roughly

double the conductance rates of na tive southeastern pine species. This is one mechanism that confers a greater transpiration rate of the former compared to the latter.60

Figure 1: Stom atal conductance response to VPD for pine and Eucalyptus (Oren and others 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 and others 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 leve l transpiration was significantly greater compared to un-invaded systems (Cavaleri and Sack 2008). VPD (kPa) 0123456gs(mmol m-2s-1)

0 100 200 300 400 500 600 700 800

Eucalyptus

 = -194

g
s ref = 328 Pine

 = -81

g
s ref = 144

Afforesta tion from existing vegetation into Eucalyptus plantations reduces stream flow more so than afforestation to pine plantations The Hydrology document prepared by ArborGen st ates that afforestation with any tree species may reduce stream flow; and that while Eucalyptus plantations might reduce stream flow, the reduction would be compar able to the reduction by pine plantations. Recent research suggests that Eucalyptus plantations would redu ce stream flow more than pine plantations, and more importantly, Eucalyptus plantations could eliminate low

flows. In a review of more than 20 catchm ent conversion studies, Farley and others (2005) showed that conver ting 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).61

In perennial streams thro ughout the southeast which have base flows sustained by subsurface flow from the water table (or unconf ined aquifers), elimination of low flows may have important ramifications for threaten ed and endangered aquatic species, such as the gulf strain striped bass, and thee species of endemic freshwater mussels (Golladay and others 2004; Couch and McDowell 2006).

Planting Eucalyptus hybrid plantations will lower the water table, and affect groundwater recharge and stream flow dynamics The Hydrology document prepared by ArborGen st ates that afforestation with any tree species may lower ground water levels. This is highly dependent on subsurface flow patterns, local hillslope hydrol ogy, and species-specific rooting patterns. For example, in sites where the water table can be recharged la terally, if roots extend to the water table, then stomatal conductance and transpiration can be maintained even when water in the upper soil layers is insufficient to maintain tr anspiration. If Eucalyptus hybrid plantations mine water from the saturated zone (i.e., wa ter table), groundwater recharge could be

reduced. The southeastern Coastal Plain is characterized in many places by karst geology in which groundwater from the semi-confin ed Upper Floridan Aquifer (UFA) is hydraulically connected to the water table (surface water) (Opsahl and others 2007). Mean water table depths typically range 3–8 m (Ford and others 2008). The mean maximum rooting depth for Eucalyptus is 15 m, characteristic of its dimorphic rooting pattern; in contrast, mean maximum r ooting depths of pine plantation ( P. taeda and P.

elliottii ) and grass species are 3 m and 2.6 m, re spectively (Canadell and others 1996). The average age of groundwater in the UFA is ~20 years (Happella and others 2006) and groundwater is regularly recharged by surface water in this region (Opsahl and others 2007). Conversion to Eucalyptus on sites with water tables <10 m will likely lower down-slope water tables via di rect means (i.e., direct use of ground water by deep roots), affect groundwater-aquifer dynamics, and result in ET rates that exceed precipitation input, as have been reported for this species in other locations (C alder and others 1997). The Hydrology document prepared by ArborGe n states that the high water usage by Eucalyptus may be valuable in purposefully lo wering water tables to reduced potential salinity problems. Eucalyptus has been used to afforest areas and lower the saline groundwater in highly weathered landscapes (e.g ., AUS). This application is not relevant to the southeastern US, as soils are not sali ne. Furthermore, receding groundwater levels in the UFA are being replaced in costal areas by saltwater (i.e., saltwater intrusion) (Andersen and others 2006). Thus, lowering th e water table, and the groundwater levels in the UFA would not reduce salinity problem s (as stated in the Hydrology document), and may actually exacerbate them.

Key Point: The significance of the impact on groundwater and stream flow will depend greatly on the area extent, size, and spa tial 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.62

Management of Eucalyptus as cop pice stands will affect water use of future rotations Management practices may create a perennial root stock in Eucalyptus plantations. If Eucalyptus plantations are managed as coppice st ands, the remaining mature, deeplypenetrating root stock may be able to supply the second ro tation stems with more water

resources for use than similar sized stems in thei r first rotation (Swift and Swank 1981).

It is unlikely that lower stocking levels w ill be an acceptable management practice to reduce hydrologic impacts of Eucalyptus plantations Some of the reports cited in the ArborGen document discuss the potential for altering management practices to minimize th e impacts of intensively managed Eucalyptus plantations on hydrology. The most viable op tion for reducing hydrologic impacts is to manage stocking (“stocking” is a term to desc ribe the how much of the site is occupied by the species of interest; stand basal area expressed in m 2 stem area hectare -1 is often used as a measure of stocking). Water use is highly regulated by stand leaf area and reducing basal area will result in lower stand leaf area. Empirical research (Douglass and Swank 1972) at Coweeta has shown that stand basal area needs to be reduced by at least 15 % before any impact on stream flow is obtained. Large and su stained increases in stream flow typically require significant redu ctions in stocking because trees growing in more open conditions will increase transpir ation rates in response to changes in micrometeorological conditions in the tree crown. From a practical standpoint, it is unlikely that lower stocking levels will be an acceptable management practice for Eucalyptus plantations because one of the primary objectives of growing Eucalyptus is to maximize biomass produc tion -- 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 imp acts on stream flow or groundwater; expensive, high maintenance

Soil Moisture63

Instrumentation = TDR probes and data loggers (automated); soil probe for gravimetric (manual) PROS: easy to implement, relatively inexpensive CONS: does not directly measure imp acts on stream flow or groundwater

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

Stream flow Instrumentation = flumes & data loggers (automated); pressure transducer, rating

curve, stream survey PROS: direct measure of impacts; reliable CONS: expensive, requires stream, expertise

Regardless of the monitoring approach c hosen, the monitoring design will require a suitable control for compar ison. Ideally, the plantation si te(s) and the control site(s)

would be measured for at leas t 1 to 2 years prior to be be ing planted, and then both sites

would be measured for the duration of the monitoring period. We recommend that the monitoring period begin at plan tation establishment and co ntinue through canopy closure (approximately 5 years).

Additional analyses on gro undwater dynamics and linkage s with aquatic ecosystems are required We recommend that APHIS solicit input from experts on groundwater hydrology (e.g., from USGS) to assess the potential impact s on groundwater recharge and associated dynamics. In addition, our analysis suggests th at stream flow will be reduced by at least 20% relative to pine plantations and perhap s 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 aq uatic ecosystems and associated species.References Allen RG, Pereira LS, Raes D, Smith M. 1998. Crop evapotranspiration - Guid elines for computing crop water requirements. FAO - Food and Agri culture Organization of the United Nations. Paper 56. Andersen PF, Mercer JW, White Jr. HO. 2006. Numerical modeling of salt-w ater intrusion at Hallandale, Florida. Ground Water 26: 619–630. Calder IR, Rosier PTW, Prasanna KT, Parameswar appa S. 1997. Eucalyptus water use greater than rainfall input-a possible explanation from southern India. Hydr ology 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. Oecologi a 108: 583-595. <Go to

ISI>://A1996VX11000001 Cavaleri M, Sack L. 2008. Native vs. invasive plan t water use at multiple scales: A global metaanalysis. Presented at the Ecological Society of America Annual Meeting in Milwaukee, WI. August 3–8. Clark GA. 1994. Water requirements for drip irriga ted strawberries in south central Florida. Agricultural and Biological Engineering Department, Florida Cooperative Extension Service, Institute of Food and Agricultura l Sciences, University of Florida. AE253. Couch CA, McDowell RJ. 2006. Flint River Basin Re gional Water Development and Conservation Plan. Georgia Dept. of Natural Resources , Environmental Prot ection Division. p. 242. Douglass JE, Swank WT. 1972. Stream flow modification through manage ment of eastern forests. USDA Forest Service, Southeastern Forest Experiment Station. Research Paper SE-94. Farley K, Jobbagy E, Jackson RB. 2005. Effects of afforestation on water yiel: a global synthesis with implications for policy. Global Change Biology 11: 1565–1576. Ford CR, Goranson CE, Mitchell RJ, Will RE, Teskey RO. 2004. Diurnal and seasonal variability

in the radial distributi on of sap flow: predicti ng total stem flow in Pinus taeda trees. Tree Physiol. 24: 951-960. Ford CR, Mitchell RJ, Teskey RO. 2008. Water tabl e depth affects productivity, water use, and the response to nitrogen addition in a sava nna 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 reco rd drought in the Gu lf Coastal Plain of southwestern Georgia. Journal of the North American Benthological Society 23: 494-506. Happella JD, Opsahl S, Top Z, Chanton JP. 2006. Apparent CFC and 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. Ev apotranspiration of full- , deficit-irrigated, 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, C obb N, Kolb T, Plaut J, Sperry J, West A, Williams DG, Yepez EA. 2008. Tansley review: Mechanisms of plant 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. TreesStructure and Function 13: 152-160.

Page 64 of 97Opsahl SP, Chapal SE, Hicks DW , Wheeler CK. 2007. Evaluation of ground-water and surfacewater exchanges using streamflow difference analyses. Journal of the American Water

Resources Association 43: 1132–1141. Oren R, Pataki DE. 2001. Transpiration in response to variation in microclimate and soil moisture

in southeastern deciduous fo rests. Oecologia 127: 549-559. Oren R, Sperry JS, Katul GG, Pataki DE, Ew ers BE, Phillips N, Schafer KVR. 1999. Survey and synthesis of intra- and interspe cific variation in stomatal se nsitivity to vapour pressure

deficit. Plant Cell Environ. 22: 1515-1526. Phillips N, Oren R. 2001. Intra- and inter-annual vari ation in transpiration of a pine forest. Ecol. Appl. 11: 385–396. Powell TL, Starr G, Clark KL, Martin TA, Gholz HL. 2005. Ecosystem and understory water and energy exchange for a mature, naturally regenera ted pine flatwoods forest in north Florida.

Can. J. For. Res. 35: 1568-1580. Shukla S, Jaber F, Srivastava S, Knowles J. 2007 . Water use and crop coefficient for watermelon in

southwest Florida. Institute of Food and Agricultural Scie nces (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 clim ate and vegetation on evapotranspiration along a successional chronosequence in the sout heastern US. Global Change Biology 12: 2115–

2135. Swift LW, Swank WT. 1981. Long term responses of streamfl ow following clearcutting and regrowth. Hydrological Scie nces Bulletin 26: 245–256. Wullschleger SD, Hanson PJ, Todd DE. 2001. Transpir ation from a multi-species deciduous forest as estimated by xylem sap flow techni ques. For. Ecol. Manage. 143: 205-213.

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## APPENDIX IV: Threatened and Endangered Species Analysis

The following analysis was conducte d using the following resources: US Fish and Wildlife Service ECOS system: http://ecos.fws.gov/ ecos_public/index.do NatureServe Explorer: http://www.natureserve.org/explorer/index.htm Google Earth with critical habi tat metadata supplied by US Fish and Wildlife Service Regional Species lists for FWS offices in Alab ama, Georgia, Louisiana, Mississippi, South Carolina, Texas and Florida. Telephone contact with Field Office personnel with USFWS.

1. Baldwin County, AL (AL-BAL-01)

This location has been an agricu ltural research station for more than 20 years. The location has been used for managed production of annual agricu ltural crops and forest trees. Site preparation will involve herbicide applicati on, subsoiling, and planting of trees in flat beds. The surrounding

areas of the test site consist of field plantings of agricultural crops, experimental forest trees and an abandoned pecan orchard.

Seventeen TES animals and one TES plant are listed in Baldwin County. The TES Animals include loggerhead sea turtle ( Caretta caretta ), green sea turtle, ( Chelonia mydas ), Alabama redbelly turtle ( Pseudemys alabamensis ), Eastern indigo snake (Dry marchon corais couperi), redcockaded woodpecker ( Picoides borealis ), Alabama beach deermouse ( Peromyscus polionotus

ammobates ), Perdido Key beach deermouse ( Peromyscus polionotus trissyllepsis ), piping plover ( Charadrius melodus ), Least Tern ( Sternula antillarum ), West Indian manatee ( Trichechus

manatus ), wood stork ( Mycteria americana ), Kemp's ridley sea turtle ( Lepidochelys kempii ), gulf sturgeon ( Acipenser oxyrinchus desotoi ), Alabama sturgeon ( Scaphirhynchus suttkusi ), heavy pigtoe mussel ( Pleurobema taitianum ), inflated heelsplitter mussel ( Potamilus inflatus ), and reticulated flatwoods salamander ( Ambystoma bishopi ). The listed plant is American chaffseed ( Schwalbea americana ).

The American chaffseed occurs on sandy peat, sandy lo am, 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 wetlan ds and xeric sandy soils, and othe r 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 north east corner, far from the release site. The plant has no critical habitat listed in the action area.

The four turtle species (loggerhead sea turtle, green sea turtle, Al abama redbelly turtle and Kemp’s ridley sea turtle), the gulf and Alabama sturgeons, and the West Indian manatee occur in aquatic habitats, and their habitat systems (bays, lagoons, salt marshes, creeks, ship channels, and other saltwater and freshwater environments) do not ov erlap with the trial site. The red-cockaded woodpecker could potentially visit the field tests but this species prefers mature pine stands as habitat for nesting. The wood stork primarily inhabits wetland systems notably cypress or mangrove swamps and would not use the field test site. The reticulated flatwoods salamander uses

Page 66 of 97the wet pine flat-woods associated w ith ephemera l wetlands. They typically breed in the low wetlands where they lay their eggs . After hatching, they spend 11 to 18 weeks as larvae before metamorphosing into adults and l eaving the wetland for higher ground to burrow into the soil. It is important that the area through whic h the species moves is vegetate d with grasses. Reticulated flatwood salamanders may move as far as 450 me ters from their breeding sites. The area surrounding the release site out to beyond this distance has be en intensively managed for over 20 years as an agricultural research station, making it unlikely that the species would find this habitat useable. The two mouse species listed above (Alabama beach mouse and Perdido Key Beach deermouse) are found only in coastal dune areas and Perdido Key Beach, respectively, where they feed on sea oats, bluestems, and a variety of insect s. Both habitats are located approximately 65+ miles from the proposed field trial. The piping plover uses sparsely vegetated dunes and coastal beaches in southern Baldwin County, also far away from the field site (about 60 miles). The Least Tern breeds on seacoasts, beaches, bays, estuarie s, lagoons, lakes, and rivers and rests on sandy beaches, mudflats, and salt-pond dikes which are far away from the field test site. The Eastern indigo snake is known to inhabit a wide range of habitats (agricul ture fields, pine flat-woods, wet depressions, stream bottom thickets and margins of swamps). It appe ars to be very rare in Baldwin County where a case has been reported in an unk nown location (US Forest Service). According to Jody Smithen (contacted February 20, 2008) of th e Daphne Field Office USFWS, the species has not been documented in the county for many year s, but there are occa sionally unsubstantiated reports. They do not feel there is any concer n. Although it is highly unl ikely that the species would be found at the site, the appl icant will provide all workers w ith identifying char acteristics of

the snake and instructions on what to do if the species is encountered. These measures are a variation of standard protective measures the USFW S uses when they have reached a “may affect”

determination for construction sites.

Critical Habitat: Most of the TES animals within the county area use inshore or wetland systems most of which are concentrated essentially in th e southern and southeastern coastal beaches of Baldwin County. There is criti cal habitat listed for the Perd ido Key beach deermouse and the piping plover. Notice of designa tion of critical habita t for the reticulated flatwood salamander (final rule) was published in the Federal Register on February 10, 2009 ( http://edocket.acce ss.gpo.gov/2009/pdf/E9-2403.pdf ). There is no designated critical habitat for this species in Alabama. The closest critical ha bitat (for the Perdido Key Beach deermouse and the piping plover) is about 65 miles away. The gulf sturgeon has propos ed critical habitat in Alabama but this does not occur in Baldwin County.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

2. Escambia County, AL (AL-ESC-01)

Page 67 of 97This location has been used as an intensely m anaged pasture for more than 5 years. The test site is currently planted with grasses suitable for cattle grazing. Site preparati on will involve herbicide application to remove existing grasses, subsoili ng, preparation for possibl e irrigation, and planting of the test trees in flat beds. The surrounding areas of the test site consis t of ~ 30 year-old slash pine, and a re-forested area with less than 7 year -old mixed stands of pine and hardwood species.

Three TES animals are listed for Escambia County. The animals are gulf sturgeon, ( Acipenser Oxyrinchus desotoi ), red-cockaded woodpecker ( Picoides borealis ) and wood stork ( Mycteria

Americana ). The gulf sturgeon occurs in the Gulf of Mexico and spawns in freshwater rivers. It will not be affected by the field test since the closest river is over 3.5 miles away. The red cockaded woodpecker inhabits old growth forests, primarily longleaf pine. It could visit the field

test site but would not nest th ere. The wood stork primarily i nhabits wetland systems notably cypress or mangrove swamps and would not find the field test site hospitable.

Critical Habitat: The designated critical habitat for the gulf sturgeon includes the Escambia River System in Santa Rosa and Escambia counties, Florida and Escambia, Conecuh, and Covington counties, Alabama. The establishmen t of the field test site would not impact this habitat. It is about 3.5 miles away from the Conecuh river.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

3. Evans County, GA (GA-EVA-01)

This location has been a commercial nursery for fo rest seedling production for over 30 years. Site preparation will involve herbicide application, subsoiling and planting of trees in flat beds. The surrounding areas of the test site c onsist of nursery beds of forest tree seedlings, agricultural crops and mixed stands of hardwood and pine.

There are four TES animals listed for Evans County. There are no listed plants. The animals are: Eastern Indigo Snake ( Drymarchon couperi ), Wood Stork ( Mycteria Americana ), Red-cockaded Woodpecker ( Picoides borealis ), and frosted flatwoods salamander ( Ambystoma cingulatum ).

The listed birds (red-cockaded woodp ecker and wood stork) could all potentially visit the field test

site but would not nest there. The red-cockaded woodpecker nests in old growth longleaf pine.

The wood stork nests in marshe s, floodplain lakes, and swam ps. The flatwoods salamander ( Ambystoma cingulatum ) was a species that required further analysis because it typically inhabits longleaf or slash pine forests lying between drier land upslope and wetlands a nd seasonally inhabits wet pine flat-woods with vernal pools. Originally it was associat ed with a unique community of longleaf pine/wire grass, but m uch of this hab itat is now replaced by sl ash pine plantations. According to Kathy Chapman (contacted February 22 and 26, 2008) of the Coastal Georgia Field

Page 68 of 97Office, there should not be an effect on the species because there would be no chang e to the habitat. However, she suggested verifying th at there is no suitable breeding habitat near the release site. She provided information on life history and suitable ha bitat for the species. Viewing the site using Google Earth does not readily identif y any suitable breeding habitat. To ensure that there is no suitable habitat in the area, BRS provided the species information to the applicant and in early March 2008, the applicant conducted a breeding habitat survey of th e area within 450 meters (1476 feet) of the release site. No suitable habitat was found. For the Eastern Indigo Snake ( Drymarchon couperi ) according to Kathy Chapman (contacted Februa ry 22 and 26, 2008) of the Coastal Georgia Field Office, the species is asso ciated with the gopher tortoise in sandhill areas with wetlands. However, land use is a determining factor on the likelihood of either specie s being present. The site has been used as a commercial nursery for forest seedling production for over 30 years. She

indicated that she was not concerned with the site because there would be no change to the habitat. Although it is highly unlikely that the species woul d be found at the site, the applicant will provide all workers with identifying characteristics of the snak e and instructions on what to do if the species

is encountered. These measures are a variation of standard protective measures the USFWS uses when they have reached a “may affect ” determination for construction sites.

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

county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

4. St. Landry’s Parish, LA (LA-SLP-01)

This location has been used as an experimental agricultural farm for more than 25 years. The location has been used for conducting research experiments with soybea n, cotton and wheat. Site preparation will involve herbicide application, subsoiling and planting of trees in flat beds. The surrounding areas of the test site consist of agri cultural fields of rice, sugarcane and millet.

There is one TES animal listed for St. Landry’s Parish; the pallid sturgeon ( Scaphirhynchus albus ). There are no listed plants. The pallid sturgeon occurs in larger channels of the MississippiMissouri river system. The field test site is approximately 24 miles aw ay from the Mississippi river, so would not be imp acted by the field test.

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

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent elements required by the

Page 69 of 97species. F ield 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

5. Marshall County, MS (MS-MAR-01)

This location has been an agricultural research station for more than 50 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. Site preparation will involve herbicide application, subsoiling and planting of trees in flat beds. The surrounding areas of the test site consist of agricultural fields, and less than 5 year-old pine plantations.

There are no listings for TES anim als or plants in the county. Critical Habitat: There is no designated critical habitat or habita t proposed for designation in the

county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. 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 designati on. Therefore, the acti on will have no effect on listed species or species proposed for listing and w ould not affect designated critical habitat or habitat proposed for designation.

6. Pearl River County, MS (MS-PRC-01)

This location has been an agricultu ral research station for more than 5 years. The location has been used for conducting research experime nts with agricultural crops and gr asses. The test site has been used for experimental planting of grasses. Site preparation will involve herbicide application to remove existing grasses, subsoili ng, preparation for possible irriga tion 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 pi ne, and a residential area.

There are five TES animals listed in this county and one TES plant. There is no critical habitat listed for this county. The animals are the ringed map turtle ( Graptemys oculifera ), gopher tortoise ( Gopherus polyphemus ), Louisiana black bear ( Ursus a. luteolus ), gulf sturgeon ( Acipenser oxyrhynchus desotoi ) and inflated heelsplitter ( Potamilus inflatus ). The listed plant is Louisiana quillwort ( Isoetes louisianensis ). There is critical ha bitat for the gulf sturgeon and proposed critical

habitat for the Louisiana bl ack bear in Mississippi.

The ringed map turtle ( Graptemys oculifera ) inhabits wide rivers with strong currents, adjacent white sand beaches, and an abundance of basking site s in the form of brush, logs, and debris. The field test will not impact this aquatic speci es which occurs in the Pearl River system – approximately 11 miles from the fiel d test site. The gopher tortoise ( Gopherus polyphemus ) inhabits dry sand ridges do minated by pine and areas maintained by fire. It is common in longleaf pine forests, but its numbers have decreased with the replacement of longleaf pine forests with

Page 70 of 97loblolly pine forests. This field test is located in a n agricultural research station that would be an inhospitable environment for the gopher tortoise. According to James Harris (contacted February 6 and 7, 2008), Supervisory Wildlife Biologist with the USFWS in Lacombe, LA, the species is found in a large geographic area that includes the release site. However, the species is not found everywhere within this geographic ar ea. 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 ha ve been observed at the facility. The farm manager has not observed the species at the facili ty, 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. A nother employee contacted has worked at the site for over 35 years and has never seen a gopher tort oise at the facility but did observe one approximately ten years ago about 1⁄4 mile from the f acility. The applicant surveyed the site for the presence of gopher tortoise burrows on January 29, 2008 and none were found. Considering the use of the facility, testimony of th e facility employees, and the negative result of the survey, it can be concluded that the species is not present now and would be un likely to use the site while it operates as an agricultural research station. The Louisiana black bear ( Ursus a. luteolus ) prefers bottomland forests with diverse food resources, in cluding a variety of hard-mast-producing species.

Its habitat includes remote areas w ith little or no human activity so it would not likely be found at the site. The gulf sturgeon, the inflated hellspli tter mussel, and the Louisiana quillwort occur in aquatic environments so would not be affected by the field test.

Critical habitat: The critical habi tat proposed for the Louisiana black bear is an area lying south of Washington County and west of the main channel of the Mississippi River. The proposed areas are the Tensas River Basin, Atchafalaya River Floodwa y, Lower Iberia and St. Mary Parishes all of which are well over a hundred miles away. The critic al habitat for the gulf Sturgeon occurs in this county in the Pearl River system. The Pearl River is a bout 11 miles from th e field test site.

Tributaries of the Pearl River system are approximately 3.5 and 7.7 miles from the test site.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

7. Bamberg County, SC (SC-BAM-01)

This location has been a managed forest plantati on for more than 12 years. The location has been specifically used for short rotati on planting of hardwoods and softwood trees for forestry research. The standard silvicultural practices for site preparation, irrigation, fe rtilization, planting and harvesting have been used at this location. Simila r practices will be used for the additional field

tests to be established at this site. The surr ounding areas of the test si te consist of young pine plantations, mixed stands of hardwoods and pine, and agricultural fields.

Three TES are listed in Bamber g County. The TES species listed are a plant, Canby’s dropwort

Page 71 of 97( Oxypolis canbyi ), and two anim als, the wood stork ( Mycteria americana) and the red cockaded woodpecker ( Picoides borealis )

.

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

of stoloniferous rhizomes. It has been found in a variety of habitats, including cypress ponds, grass-sedge dominated Carolina bays, wet pine savannahs, shallow pi neland ponds and cypresspine swamps or sloughs. The largest and most vigor ous 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 co ntent, high water table, that are deep, poorly drained, and acidic. The Lisa Matthews Memorial Bay is a 52 acre site in Bamberg County which was given to the South Carolina Native Plants Society by the Nature C onservancy for the purpose of preserving Canby's Dropwort ( Oxypolis canbyi ). This depression wetland is apparently a remnant Carolina Bay which is being restored and expanded to protect this endangered species. The location of the field test under this permit is a managed forest research area and does not provide the proper habitat for the species.

The wood stork inhabits riparian areas with lagoons and shallow wa ter. It lives in freshwater situations: marshes, swamps, lagoons, ponds, flooded fi elds and brackish wetlands. It nests mostly in the upper parts of cypress trees, mangroves, or dead hardwoods over water or on islands along streams or adjacent to shallow lakes and feed s in freshwater marshes, swamps, lagoons, ponds, flooded pastures and flooded ditches, and in depre ssions in marshes. The field trial location is

unsuitable habitat for the wood stork so it will be very unlikely to occur in this location. The red cockaded woodpecker inhabits old growth forests, primarily longleaf pine a nd might visit the field

test site but would not nest there.

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

county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

8 & 9. Berkeley County, SC (SC-BER-01 and SC-BER-02)

There are two release site locations in Berkeley County. One site is an extension of a greenhouse facility that has been used for acclimatization of transgenic and non-transgenic plants for more than 5 years. The release site is located adjacent to greenhouse facilities and is surrounded by hardwoods and pine plantations. The trees will only be held here on a tem porary basis until planted in the field. The other location has been under managed forest plantation for more than 5 years. The location has been specifically us ed for short rotation planting of cottonwood for forestry research. Site preparation will invo lve he rbicide application, subsoiling, drip irrigation installation, and

Page 72 of 97planting of trees in flat beds. The test site is located adjacent to gr eenhouse facilities and is surrounded by pine plantations.

Six threaten ed or endangered species (TES) animals and three plants are listed in Berkeley County. The TES animal species are shortnose sturgeon ( Acipenser brevirostrum ), frosted flatwoods salamander ( Ambystoma cingulatum ), red-cockaded woodpecker ( Picoides borealis ), West Indian manatee ( Trichechus manatus ), wood stork ( Mycteria americana ), and loggerhead sea turtle ( Caretta caretta ). The TES Plants are pondberry ( Lindera melissifolia ), Canby's dropwort (Oxypolis canbyi ) and American chaffseed ( Schwalbea americana ).

The shortnose sturgeon ( Acipenser brevirostrum ) occurs in rivers and es tuaries. The West Indian manatee (Trichechus manatus), and loggerhead sea turtle ( Caretta caretta ) occur in shallow coastal waters, rivers, bays and lakes; none of which are close to any of these re lease locations. The red cockaded woodpecker ( Picoides borealis ) inhabits old growth forests, primarily longleaf pine and might visit the field test site but w ould not nest there. The wood stork ( Mycteria americana ) primarily inhabits wetland systems notably cypr ess or mangrove swamps and would not find the field test site hospitable. The flatwoods salamander ( Ambystoma cingulatum ) 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 Zimmerma n (contacted February 14 and 20, 2008) of the Charleston Field Office of the USFWS i ndicate that the species is not known to be in

the area of the release. Known populations in the county are in the Francis Marion National Forest,

far from the release site. A check of the SC Heritage Trust Database did not identify any

occurrences in the area of the release. One s ite in Berkeley County is a fenced research plot/holding area so it is highly unl ikely that these animal species w ould occur at this location. The other site is a managed forest plantation for short rotation pl anting of cottonwood for forestry research. The bird species might visit the location but would not nest there.

Canby’s dropwort (Oxypolis canbyi ) is an herbaceous perennia l whose existing populations are maintained mainly through asexual reproduction. This species is strongly clonal, reproducing vegetatively by means of stoloniferous rhizomes. It has been found in a variety of habitats, including cypress ponds, grass-sedge dominated Carolina bays, wet pine savannahs, shallow pineland ponds and cypress-pine swamps or sl oughs. 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 dr ained, and acidic. The pondberry ( Lindera melissifolia ) occurs in similar locations, in wetland habitats such as botto m land 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 chaffseed ( Schwalbea americana ) occurs on sandy peat, sandy loam, acid ic, seasonally moist to dry soil s. It is generally found in habitats described as open, moist pine flatwoods , fire-maintained savannas, ecotonal areas between peaty wetlands and xeric sandy soil s, and other open grass-sedge sy stems. The site in Berkeley County is a fenced research plot/holding area whic h would be very inhospitable to these species. Laura Zimmerman (contacted February 14 and 20, 2008) of the Charleston Field Office of the USFWS states that she does not believe the species would be likely to be in the area. According to the species’ recovery plan, most known occurren ces are on US Forest Service land and the only

Page 73 of 97two occurrences on priv ate land are not near the re lease site. A check of the SC Heritage Trust Database did not identify any occurren ces in the area of the release.

Critical Habitat: Notice of designation of critical habitat for th e frosted flatwood salamander (final rule) was published in the Federal Register on February 10, 2009 ( http://edocket.acce ss.gpo.gov/2009/pdf/E9-2403.pdf ). Unit FFS-6 is within Berkeley County.

This is located in the Francis Marion National Forest which is about 19-20 miles away from the release location. There is proposed critical habi tat listed for the West I ndian manatee in South

Carolina but it does not occur in this county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

10. Charleston County, SC (SC-CHA-01)

This location has been a managed forest plantati on for more than 10 years. The location has been specifically used for short-term planting of hardw oods and softwood trees for forestry research. The standard silvicultural practices for site preparatio n, irrigation, fertilization, planting and harvesting have been used at this location. Similar practices will be used for the additional field tests to be

established at this site. The test plots adjacent to the field test site include young mixed stands of hardwoods and pines.

Eleven TES animals and 4 TES plants are listed for Charleston County. The animal species are: shortnose sturgeon ( Acipenser brevirostrum ), West Indian manatee ( Trichechus manatus ), Kemp's ridley sea turtle ( Lepidochelys kempii ), leatherback sea turtle ( Dermochelys coriacea ) and green sea turtle, ( Chelonia mydas ), loggerhead sea turtle ( Caretta caretta ), wood stork ( Mycteria

Americana ), Bachman's warbler ( Vermivora bachmanii ), red-cockaded woodpecker ( Picoides

borealis ), piping plover ( Charadrius melodus ), and flatwoods salamander ( Ambystoma cingulatum ). The plant species are: seabeach amaranth ( Amaranthus pumilus ), Canby's dropwort ( Oxypolis canbyi ), pondberry ( Lindera melissifolia ) and American chaffseed ( Schwalbea

americana ).

The shortnose Sturgeon, West Indi an manatee, Kemp's ridley sea turtle, leatherback sea turtle, green sea turtle, loggerhead sea tu rtle, and West Indian manatee ar e all aquatic animals and would not be affected by the field test. The birds (Bachman’s warbler, piping plover, red-cockaded woodpecker and wood stork) could all potentially visit the field test si te but would not nest there. The piping plover nest along the coast. The red-cockaded woodpecker nests in old growth longleaf pine. The wood stork nests in marshes, floodplain lakes, and swamps. Bachman’s warbler nests in bushes, blackberry vines, or canes, on swamp palme tto leaf, in densely vegetated watery swamps.

The flatwoods salamander inhabits longleaf or slash pine forests lying between drier land upslope and wetlands and seasonally inhabi ts wet pine flat-woods with vernal pools. Discussions with

Page 74 of 97Laura Zimmerm an (contacted February 14 and 2 0, 2008) of the Charleston Field Office of the USFWS indicate that the species is not known to be in the area of the rele ase. Known populations in the county are far from the release site in the Santee Coastal Reserve. A check of the SC Heritage Trust Database did not identify any occurrences in the area of the release.

For the plants, the seabeach amaranth occurs in areas just above the high tide line on accreting shorelines; those where the beach is building up or expanding. Canby's dropwort occurs in a variety of coastal plain communities, including p ond cypress savannahs, the shallows and edges of cypress and pond pine ponds, sloughs, and wet pine savannas. Pondberry is found in swamp and pond margins, sandy sinks, swampy depr essions or wet flats that are subject to drying but the roots are submerged at times. American chaffseed is found in various sandy soil areas on the coastal plain; plants are usually found on margins of savannas and cypre ss ponds that are seasonally wet; best managed by prescribed fire. The location of the field test, which has been a managed forest plantation for more than 10 years, would not be hospitable habitat for any of these species.

Critical Habitat: Notice of designation of critical habitat for th e frosted flatwood salamander (final rule) was published in the Federal Register on February 10, 2009 ( http://edocket.acce ss.gpo.gov/2009/pdf/E9-2403.pdf ). Unit FFS-7 is within Charleston County.

This is located in the Santee Coastal Reserve wh ich is about 43 miles away from the Berkeley county sites and about 57 miles away from the Charleston County site. The proposed critical

habitat listed for the West Indian manatee does not occur in this county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not adversely modify designate d critical habitat or habitat proposed for designation.

11. Marlboro County, SC

This location has been a commercial nursery for fo rest seedling production for over 30 years. Site preparation will involve herbicide application, subsoiling and planting of trees in flat beds. The surrounding areas of the test site consist of field plantings of ag ricultural crops, nursery beds of forest tree seedlings and less than 30 yea r-old mixed hardwood and pine plantations.

There are two TES animals listed for Marlboro County; the red-co ckaded woodpecker ( Picoides

borealis ) and the shortnose sturgeon ( Scaphirhynchus albus ). The listed plant is Canby's Dropwort ( Oxypolis canbyi ). The red-cockaded woodp ecker nests in old growth longleaf pine not found at

the location of the field test . The shortnose sturgeon ( Acipenser brevirostrum ) occurs in rivers and

estuaries. Little is known about the status of a ny of the populations in Sout h Carolina. The closest body of water where the sturgeon could possibly live is about 5 miles away. Canby's dropwort occurs in a variety of coastal plain communitie s, including pond cypress savannahs, the shallows and edges of cypress and pond pine ponds, sloughs, and wet pine savannas. The location of the

Page 75 of 97field test, which has been a m anaged nursery for more than 30 years, would not be hospitable habitat for any of these species.

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

county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

12. Hardin County, TX (TX-HAR-01)

This location has been a managed forest plantation fo r more than 30 years. The location consists of mixed hardwood tree plantations planted using sta ndard silvicultural prac tices and was harvested by the owner in 2004. The test site is within the larger harvested area and has been re-bedded by the owner for planting. Site preparation incl uded herbicide application and sub-soiling. The surrounding areas of the test site consist of mi xed hardwood stands and managed loblolly pine plantations.

One TES animal and one TES plant are listed for Ha rdin County. The animal s is the red-cockaded woodpecker ( Picoides borealis ), and the plant is th e Texas trailing phlox ( Phlox nivalis ssp. texensis). The red-cockaded woodpecker might vis it the field test site bu t would not nest there since it prefers old growth pine forests, partic ularly longleaf pine. Th e Texas trailing phlox is endemic to the Pineywoods of the west gulf coastal plain of east Texas. It occurs in deep sandy soils in fire-maintained openings in upland longleaf pine sava nnas or post oak-bluejack oak woodlands. Since this site has been under managed forest plantations for more than 30 years this species would not find the field te st site a suitable habitat.

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

county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

Page 76 of 9713 & 14. Jasper County, TX (TX-JAS-01 and TX-JAS-02)

There are tw o locations in Jasper County Texas. One location has been under managed pine plantations for more than 25 years. Previous pl antings were cultivated in beds using standard silvicultural practices. The existi ng pine plantation at this site was harvested by the owner in 2007 and re-bedded for planting. The test site is within the la rger harvested and bedded site prepared by the site owner. Further site preparation will involve herbicide application, subsoiling and planting of trees in flat beds. The surrounding areas of the test site consist of harvest age pine plantations.

The other location has been under managed pine pl antations for more than 25 years. Previous plantings were cultivated in beds using standard silvicultural practi ces. The existing pine plantation at this site was harvested by the owner in 2007 and re-bedded for planting. Th e test site is within the larger harvested and bedded site prepared by the site owner. Further site preparation will involve herbicide application, subs oiling and planting of trees in fl at beds. The surrounding areas

of the test site consist of less than 20 years-old manage d hardwood and pine stands.

Two threatened or endangered species (TES) animal s and one plant are listed in Jasper County. The animals are the red-cockaded woodpecker ( Picoides borealis ) and the Louisiana black bear ( Ursus americanus luteolus ). The plant is Navasota ladies'-tresses ( Spiranthes parksii ).

The red-cockaded woodpecker ( Picoides borealis ) inhabits old growth forests, primarily longleaf pine and might visit the field te st sites but would not nest ther e. The Louisiana black bear ( Ursus

americanus luteolus ) depends on diverse, productive bottomla nd forest with diverse food resources, including a variety of hard-mast- producing species. High quality habitat includes remote areas with little or no human activity so it would not likely occur at either site. Navasota ladies'-tresses ( Spiranthes parksii ) occurs at the margins of post oak ( Quercus stellata ) woodlands in sandy loams along intermittent tributaries of rivers. The fiel d test sites have none of these characteristics.

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

county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

15. Jefferson County, TX (TA-JEF-01)

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

Page 77 of 97There are six TES animals listed for Jefferson Count y. There are no plants listed. T he animals are green turtle ( Chelonia mydas ), hawksbill turtle ( Eretmochelys imbricata imbricate ), Kemp's ridley sea turtle ( Lepidochelys kempii ), leatherback turtle ( Dermochelys coriacea ), loggerhead sea turtle ( Caretta caretta ) and piping plover ( Charadrius melodus ). All of the turt les occur in the open

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

Critical Habitat: There is critical habitat listed for the Piping Plover but it does not occur in this county. The closest critical ha bitat is in neighbori ng Galveston County, approximately 40 miles

from the field test site.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

16-19. Newton County, TX (TX-NEW-01, TX-NEW-02, TX-NEW-03, TX-NEW-04)

There are four locations in Newton County. All lo cations have been under managed loblolly pine plantations for at least 30 years. The previous plantings were cu ltivated in beds using standard silvicultural practices and the areas have recently been harvested. Site preparation has been or will include herbicide application, plow ing, and planting of trees in rais ed or flat beds. The surrounding areas of the test site consist of managed loblo lly pine plantations and/or mixed hardwood stands.

Two TES animals are listed for Newton County. The animals are the red-cockaded woodpecker ( Picoides borealis ), and Louisiana black bear ( Ursus americanus luteolus ). There are no plants listed for this county.

The red-cockaded woodpecker might visit the field te st site but would not ne st there since it prefers old growth pine forests, pa rticularly longleaf pine. The Louisiana black bear ( Ursus a. luteolus ) prefers bottomland forests with diverse food resources, including a variety of hard-mast-producing species. According to the Black Bear Recovery Plan ( http://www.bbcc.org/web/images/stories/inform ation/pdf/FinalRestorationPlanwithF igures.pdf ) in east Texas, potentially occupied habitat may occur in at least four Counties: Cass, Shelby, Panola and Angelina Counties. All of th ese locations are from fifty to over a hundred miles away from

these test sites. Louisiana black bear habitat includes remote areas with little or no human activity so it is unlikely to be found at the site.

Critical Habitat: The proposed crit ical habitat is locat ed in Avoyelles, East Carroll, Catahoula, Concordia, Franklin, Iberia, Iber ville, Madison, Pointe Coupee, Ri chland, St. Martin, St. Mary,

Page 78 of 97Tensas, W est Carroll, and West Feliciana Parishes , Louisiana. All of th ese locations are over a hundred miles away from these test sites.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

20. Bay County, FL (FL-BAY-01)

This location has been used as an intensely managed pasture for more than 15 y ears. The test site is currently planted with grasses suitable for cattl e grazing. Site preparati on will involve herbicide application to remove existing grasses, subsoili ng, preparation for possible irrigation installation, and planting of trees in flat beds . The surrounding areas of the test si te consist of agricultural crops and less than 25 year-old hardwoods and pine.

Sixteen TES animals and 7 TES plants are li sted for Bay County. The animals are gulf moccasinshell ( Medionidus penicillatus ), oval pigtoe ( Pleurobema pyriforme ), gulf sturgeon ( Acipenser oxyrinchus ) loggerhead turtle ( Caretta caretta ), eastern indigo snake ( Drymarchon couperi ), piping plover ( Charadrius melodus ), red-cockaded woodpecker ( Picoides borealis ), West Indian manatee ( Trichechus manatus ), Choctawhatchee beach mouse ( Peromyscus polionotus allophrys ), St. Andrews beach mouse ( Peromyscus polionotus peninsularis ), reticulated flatwoods salamander ( Ambystoma bishopi ), green turtle ( Chelonia mydas ), leatherback turtle ( Dermochelys

coriacea ), hawksbill turtle ( Eretmochelys imbricata imbricate ), Kemp's ridley turtle ( Lepidochelys kempii ), and wood stork, ( Mycteria Americana ). The plants are telephus spurge ( Euphorbia telephioides ), white birds-in-a-nest ( Macbridea alba ), paper-like whitlow-wort ( Paronychia

chartacea ), violet-flowered butterwort ( Pinguicula ionantha ), Florida skullcap ( Scutellaria floridana ), Harper's beauty, ( Harperocallis flava ) and Crystal Lake nailwort, ( Paronychia

chartacea minima ).

The gulf moccasinshell, oval pigtoe, gulf sturge on, West Indian manatee, and the turtles (loggerhead, green, leatherback, hawksbill and Kemp's ridley) are all aquatic animals and would not be affected by the field test. The birds (piping plover, red-cockaded woodpecker and wood stork) could all potentially visit th e field test site but would not nest there. The piping plover nests along the coast which is about 20 m iles away from the release site . The red-cockaded woodpecker nests in old growth longleaf pine . T he wood stork nests in marshes, floodplain lakes, and swamps. The two mouse species live along the coast in beach areas so would not be impacted by the field release. For the eastern indigo snake, accordi ng to Caroline Stahaller (contacted February 20, 2008) of the Panama City Field Office USFWS, the species has not been pos itively identified in the county or anywhere else on the Florida panhandle in 15-20 years. Consideri ng the area is currently managed as a pasture, a habitat where the species is unlikely to occur, th ere is no concern for the species at this site. Although it is highly unlikely that the species woul d be found at the site, the applicant will provide all workers with identifying characteristics of the snake and instructions on

Page 79 of 97what to do if the species is encountered. These m easures are a variation of standard protective measures the USFWS uses when they have reache d a “may affect” determ ination for construction

sites. The reticulated flatwoods salamander inhabits longleaf or slash pine forests lying between drier land upslope and wetlands and seasonally i nhabits wet pine flat-woods with ephemeral wetlands. They typically breed in the low wetlands where they lay their eggs. After hatching, they spend 11 to 18 weeks as larvae before metamorp hosing into adults and leaving the wetland for higher ground to burrow into the soil. It is impor tant that the area through which the species moves is vegetated with grasses. Management of the ar ea with controlled burning is generally needed to provide quality flatwoods salamander habitat. Re ticulated flatwoods salamanders may move as far as 450 meters from their breeding sites. Althoug h the release site is not an area with known populations, and is at least 10 miles from the criti cal habitat, Ms. Stahalle r of USFWS suggested a

closer look at the site to determ ine if the habitat would be suitab le for the species. The property owner was forwarded survey protocol informati on provided by USFWS. In late March 2008, the owner, (who is 64 and has lived at the site his entire life) walked the site within 450 meters of the release. The owner was contacted on April 7, 200 8 to discuss his observations. The owner looked for adult salamanders but did not find any, and does not recall ever seeing them at the site. There were four naturally wet areas that were found. One wa s in the pasture itself. It was a small site of about 8 ft. by 10 ft. and is surrounded by an area that has been in continuous agricultural use for at least 60 years. The other two sites were heav ily wooded areas surrounded by pasture and remain constantly wet in most years. They are approxim ately 250 yards from the release site. The fourth site is approximately 400 to 500 yards from the rele ase site and is a 5 acre wetland that shares the same elevation with an adjacent creek and remains constantly wet, but does dry around the perimeter. Vegetation in the site is longleaf pine with a wiregrass groundc over – habitat the species finds most suitable. Vegetation betw een this potential breeding site and the release site is pasture. It is unlikely that the flatwoods salamander would be using the releas e site for a number of reasons. First, the only possible breeding s ites within 450 meters of the rel ease remain constantly wet during most years, a condition that woul d not serve as breeding habitat. The five acre site where the perimeter dries could perhaps allow for breeding in some years but it is about at the farthest possible distance from the release s ite that the adults are known to tr avel from their breeding area. Second, the sites are either surrounde d by land that has been used fo r agricultural purposes for at least 60 years or is separated from the release site by such land. Activities associated with agricultural production, disking, plow ing, application of pesticides etc. would make the habitat unsuitable for the species. In addition, none of this area is managed by controlled burning, a management practice that is generally used in ar eas where the species has viable populations. The lack of suitable breeding habita t, combined with the ongoing and hi storic agricultura l activities at the site, point to a conclusion that the reticula ted flatwood salamander will not be present in the area of the release, nor will they be likely to enter the site during the field trial. None of the seven listed plant species would be expected to grow in the existing pasture. They grow in wiregrass dominated longleaf pine savannahs, in the mesi c flatwoods, on low sand ri dges of pine-scrub oak near the Gulf of Mexico or in wet prairies and seepage bogs.

This location is being converted fr om a pasture to a forest tree re search plot. Based on the above evaluation, APHIS has determined that disturbance of the field site for the release (e.g. plowing,

rem oving vegetation, burning etc.) will not directly or indirectly affect a fede rally listed threatened or endangered species or species proposed for listing, or affect the habitat for these species.

Page 80 of 97Critical Habitat: There is designa ted critical habitat for the Gulf sturgeon along the entire coast of Bay County. Critical habitat for the Choctawhatchee beach mouse, St. Andrew’s beach m ouse, and the piping plover is along the coast in the Panama C ity area. The field test site is approximately 18 miles away from the coast so would not impact thes e critical habitats. No tice of designation of

critical habitat for the reticulated flatwood salamander (final rule) was published in the Federal Register on February 10, 2009 ( http://edocket.acc ess.gpo.gov/2009/pdf/E9-2403.pdf ). Unit RFS-6, subunit B is just beyond the northern border of Bay County in neighbori ng Washington County. The field test site is approximately 28 miles aw ay from this location. Unit RFS-9 subunit A is located in neighboring Calhoun County approximatel y 10 miles from the site. There is proposed

critical habitat listed for th e West Indian manatee but it does not occur in this county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

21. Columbia County, FL (FL-COL-01)

This location has been under managed pine plantations for more th an 20 years. Previous plantings were cultivated in beds using standard silvicultura l practices. This area of the tract was burned in a fire in May 2007. After the fire, the area was raked and bedded by the site owner in preparation for re-planting. The test site is within the larger ha rvested and bedded area, which is surrounded by existing pine plantations and additional harvested tracts.

Five TES animals are listed for Columbia County. The animals are oval pigtoe ( Pleurobema pyriforme ), gulf sturgeon ( Acipenser oxyrinchus ), eastern indigo snake ( Drymarchon couperi ), redcockaded woodpecker ( Picoides borealis ), and wood stork ( Mycteria Americana ). There are no plants listed for this county.

The oval pigtoe and gulf sturgeon ar e aquatic animals and would not be affected by the field test. The birds (red-cockaded woodpecker and wood stork) could all potentially vis it the field test site but would not nest there. The red-cockaded woodp ecker nests in old growth longleaf pine. The wood stork nests in marshes, floodplain lakes, and swamps. For the eastern indigo snake, according to Caroline Stahaller (contacted Februa ry 20, 2008) of the Panama City Field Office USFWS, the species has not been positively identified in the Florida panhandle in 15-20 years. Considering the area has been managed as a pine plantation for many years, it is unlikely to be found at this site. Although it is highly unlikely that the specie s would be found at the site, the applicant will provide all workers with identifying characteristics of the snake and instructions on what to do if the species is encountered. These measures are a variation of standard protective measures the USFWS uses when they have reache d a “may affect” determ ination for construction

sites.

Page 81 of 97Critical Hab itat: There is designated critical habitat for the Gulf sturgeon but it is not listed in this county. There is no proposed critical habitat listed for this county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

22 & 23. Gadsden County, FL (FL-GAD-01, FL-GAD-02)

There are two planting sites at this location. One location has been an agri cultural research station for more than 10 years and has been used fo r conducting research expe riments on agricultural crops. Standard silvicultural practices will be used for site preparation, including herbicide application, plowing and planting of trees in raised or flat beds. The surrounding areas of the test site consist of mixed pine-hardwood forests and pine plantations, as well as research plantings of agricultural and horticultural crops.

This other location has also been an agricultural re search station for more than 10 years. The field has been fallow for approximately seven years. Sta ndard silvicultural practices will be used for site preparation, including herb icide application, plowing and planting of trees in raised or flat beds. The surrounding areas of the test site consist of mixed pine-hardwood forest s and pine plantations, as well as research plantings of ag ricultural and horticultural crops.

Ten TES animals and six TES plants are listed for Gadsden County. The animals are: fat threeridge mussel ( Amblema neislerii ), purple bankclimber mussel ( Elliptoideus sloatianus ), shinyrayed pocketbook ( Hamiota subangulata ), ochlockonee moccasinshell ( Medionidus simpsonianus ), oval pigtoe ( Pleurobema pyriforme ), chipola slabshell ( Elliptio chipolaensis), gulf sturgeon ( Acipenser oxyrinchus desotoi ), wood stork ( Mycteria americana ), red-cockaded woodpecker, ( Picoides borealis ), Eastern indigo snake ( Drymarchon couperi ). The plants are: Florida torreya ( Torreya taxifolia ), Chapman's rhododendron ( Rhododendron chapmanii ), American chaffseed ( Schwalbea americana ), fringed campion ( Silene polypetala ), Miccosukee gooseberry ( Ribes echinellum ) and gentian pinkroot ( Spigelia gentianoides ).

The six mussel species (fat threeridge mussel, purple bankclimber mussel, shinyrayed pocketbook, Ochlockonee moccasinshell, oval pigtoe and ch ipola slabshell), and the gulf sturgeon ( Acipenser oxyrinchus desotoi ) are all aquatic anim als and would not be affected by the field test. (See CH info below on the gulf sturgeon). The red cockaded woodpecker ( Picoides borealis ) inhabits old growth forests, primarily longleaf pi ne. It could visit the field test site but w ould not nest there. The wood stork ( Mycteria americana ) primarily inhabits wetland systems notably cypress or mangrove swamps and would not find the field test site hospitable. For the Eastern indigo snake ( Drymarchon couperi ), according to Caroline Stahaller, (contacted February 20, 2008) of the Panama City Field Office USFWS, the species has not been seen anywhere on the Florida panhandle in 15-20 years. She had no concern that the action could affect the species and agreed

Page 82 of 97the action w ould have “no effect.” Although it is highly unlikely that the species would be found at the site, the applicant will pr ovide all workers with identifying characteristics of the snake and instructions on what to do if the species is encoun tered. These measures are a variation of standard protective measures the USFWS us es when they have reached a “may affect” determination for

construction sites.

None of the six listed plant species would be expected to grow at the field site/research station. For American chaffseed ( Schwalbea Americana ), according to the species recovery plan, the only known extant population in the county is on pr ivate land. A site visit in 1994 found the area changed to a residential developm ent. It is assumed that the population may be extirpated. For Chapman’s rhododendron ( Rhododendron chapanii ), according to the Federal Register notice listing the species, there is onl y one population in Gadsden C ounty. The population is on land owned by a paper company near the Gadsden – Libe rty county line, and is many miles from the release site. The Florida torreya ( Torreya taxifolia ) occurs on hardwood hammock slopes, ravines, and bluffs of the Apalachicola River region, usua lly in steephead ravines (deep cuts made by erosion into coastal plain sedime nts). The ravines are much cooler and more moist than the land surface above and harbor remnants of the more temp erate flora that existed in the region during the Tertiary ice ages. The fringed campion ( Silene polypetala ) prefers well-drained, sandy-loam soils of deciduous woods, usually hillside s. The Miccosukee gooseberry ( Ribes echinellum ) is only known to exist on sites along east ba nk of Lake Miccosukee which is about 36 miles away from the field test site. The gentian pinkroot ( Spigelia gentianoides ) is also listed in Gadsden County and specimens were once collected in the county, but presently the only population currently known to

exist is at the Three Rivers State Recrea tion Area, Lake Seminole, Jackson County, FL. ( http://www.centerforplantconservation.org/ ). None of these species wo uld find the field test site

as suitable habitat.

Critical Habitat: The designated critical habitat for the gulf sturgeon includes the Apalachicola River mainstream, beginning from the Jim W oodruff Lock and Dam, Gadsden and Jackson Counties, Florida, downstream to its discharge at East Bay or Apalachicola Bay, Franklin County,

Florida. The establishment of the field test site would not impact this habi tat. The field site is about 19 miles away from the Apalachicola rive r mainstream below the Jim Woodruff Lock and

Dam.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to found at the release s ite. If they were to enter the site, their pres ence would be fleeting

as the habitat is either not suitable or does not co ntain constituent elements required by the species. Field activities will result in no changes to th e habitat used by any listed species or species proposed for listing. The site is not within or near designated critical habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

24. Glades County, FL (FL-GLA-01)

This location has been used for hay or vegetable production for at least 5 y ears. Site preparation will involve herbicide application to remove existi ng vegetation, subsoiling and planting of trees in flat beds. The surrounding areas of the test site cons ist of agricultural fields.

Page 83 of 97Eleven TES anim als and two TES pl ants are listed for Glades Count y. The animals are: Eastern indigo snake ( Drymarchon couperi ), American crocodile ( Crocodylus acutus ), wood stork ( Mycteria Americana ), snail kite ( Rostrhamus sociabilis plumbeus ), crested caracara ( Caracara cheriway ), Florida scrub-jay ( Aphelocoma coerulescens ), Florida grasshopper sparrow ( Ammodramus savannarum floridanus ), West Indian manatee ( Trichechus manatus ), Florida panther ( Puma concolor coryi ), Ivory-billed woodpecker ( Campephilus principalis ), and Redcockaded woodpecker ( Picoides borealis ). The plants are Carter’s mustard ( Warea carteri ) and Okeechobee gourd ( Cucurbita okeechobeensis ssp. Okeechobeensis ).

The snail kite ( Rostrhamus sociabilis plumbeus ) prefers large, open freshwater marshes and lakes with shallow open waters. The crested caracara ( Caracara cheriway ) is associated with open country; dry prairie with scattered cabbage palms, wetter prairies, a nd to some extent also improved pastures and sometimes even rather wooded ar eas having associated limited areas of open grassland. The center of its ra nge is the Kissimmee Prairie, an area of shallow ponds and sloughs

with scattered hummocks of live oaks and ca bbage palms. The red cockaded woodpecker ( Picoides borealis ) inhabits old growth forests, primarily longleaf pine. The Florida scrub-jay ( Aphelocoma coerulescens ) prefers oak scrub on white, drained sand, in open areas without a dense canopy associated with Palmetto, sand pine and rosemary. This includes scrub with no canopy, sandpine scrub, scrubby flatwoods, and coasta l scrub. The Florid a grasshopper sparrow ( Ammodramus savannarum floridanus ) prefers dry prairie with st unted saw palmetto and dwarf oaks, bluestems and wiregrass and unimproved cattle pa stures. Its habitat is maintained by periodic fires. The ivory-billed woodpecker ( Campephilus principalis ) formerly occurred in the

southeastern United States and Cuba and has declin ed to extinction or near extinction. It once occupied swampy forests, especially large bo ttomland river swamps of coastal plain and Mississippi Delta and cypress swam ps of Florida, in areas with many dead and dying trees. It would not occur in an agricultural environment such as the field test site. The wood stork ( Mycteria Americana ) primarily inhabits wetland systems notably cypress or mangrove swamps

and would not find the field test s ite hospitable. Any of the six bi rd species could potentially visit

the field test site but would not ne st there. The Florida panther ( Puma concolor coryi ) generally occurs in heavily forested areas in lowlands a nd 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 thic kets. It depends on large conti guous blocks of wooded habitat, though interspersed fields and early successional ha bitats may be beneficial through their positive effect on prey populations. Day- use sites typically are dense pa tches of saw palmetto surrounded by swamp, pine flatwoods, or hammock. It woul d shy away from areas with a lot of human activity. The site is surrounded by large areas of land under agricultural pr oduction. The American crocodile ( Crocodylus acutus ) inhabits coastal ma ngrove swamps, brackish and salt water bays, lagoons, marshes, tidal rivers, brackish creeks, abandoned coastal canals and borrow pits. It occupies mostly nonsaline waters in nonbreeding season, moving to saline waters when breeding. The field test site is miles away from any area that the crocodile might inhabit. The same would be true for the West Indian manatee ( Trichechus manatus ) which inhabits an aquatic environment. For the Eastern indigo snake ( Drymarchon couperi ) – According to Brad Rick, (contacted February 27, 2008) of the Vero Beach Field Office USFWS, the species is quite rare, but could be found anywhere. He agreed that the historic and continuous use of th e release site and the surrounding

Page 84 of 97area for hay and vegetable production m akes it extr emely unlikely that the species would be found in the area, and the appropriate determination w ould be “no effect”. The change from hay and vegetable production to a tree planting could actua lly benefit the Eastern indigo snake because the

current habitat is unsuitable. Given the unlike lihood of the species being in the surrounding area,

USFWS does not see a need to cons ult on this potentially beneficial 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 instruc tions 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.

For the plants, Carter’s mustard ( Warea carteri ) occurs in sandy clearings in sand scrub and sandhills; scattered overstory of sand; longleaf or slash pine and scrub oaks. The Okeechobee gourd ( Cucurbita okeechobeensis ssp. Okeechobeensis ) was originally found in swampy forests and hammocks on muck soils. Today, this species is restri cted to disturbed areas that are not cultivated,

such as ditch banks and wet road shoulders. It curr ently persists at a few s ites on the shore of Lake

Okeechobee in south Florida. It has also been co llected in Glades County on an island in Lake

Okeechobee and in Broward and Dade counties where it was apparently ephemeral. According to the recovery plans ( http://www.fws.gov/verobeach /im ages/pdfLibrary/waca.PDF

, ( http://www.fws.gov/verobeach/images/pdflibrary/cuok.PDF ) for both of these species, neither is

found in the area of the field release.

Critical Habitat: The American crocodile has designate d critical habitat but this occurs in the lower parts of Dade County and the Florida keys. There is no critical habitat fo r this species in this county. There is proposed critical ha bitat listed for the West Indian manatee but it does not occur in this county. The snail kite has critical habita t in Glades and Hendry C ounties, along the western

shore of Like Okeechobee which is at le ast 14 miles from the field test site.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to found at the release s ite. If they were to enter the site, their pres ence would be fleeting

as the habitat is either not suitable or does not co ntain constituent elements required by the species. Field activities will result in no adverse changes to the habitat used by any listed species or species proposed for listing. The site is not within or near designated critical habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

25. Highlands County, FL (FL-HIG-01)

This location was previously used for managed production of citrus for at least 15 years. The planting area at this location was previous ly used for field trials of transgenic Eucalyptus which were terminated more than 12 months ago and mon itored to confirm that all previous transgenic material was destroyed. Site preparation will i nvolve herbicide application, plowing, and planting of trees in flat beds. The surrounding areas of the test site consist of le ss than 5 year-old secondgrowth pine and hardwood with mixed grasses.

Fourteen TES animals, nineteen TES plants, and one TES lichen are listed for Highlands County. The animals are: American Alligator ( Alligator mississippiensis ), Wood Stork ( Mycteria

Page 85 of 97americana ), Crested Caracara ( Cara cara cheriway ), Red-cockaded Woodpecker ( Picoides

borealis ), Florida Scrub-jay ( Aphelocoma coerulescens ), Florida Grasshopper Sparrow ( Ammodramus savannarum floridanus ), Everglade snail kite ( Rostrhamus sociabilis plumbeus ), Ivory-billed woodpecker ( Campephilus principalis ), Whooping crane ( Grus Americana ), Florida Panther ( Puma concolor coryi ), West Indian manatee ( Trichechus manatus ), Eastern Indigo Snake ( Drymarchon couperi ), Bluetail Mole Skink ( Eumeces egregius lividus ) and Sand Skink ( Neoseps reynoldsi ).

The plants are: Large-flowered Bonamia ( Bonamia grandiflora ), Pygmy Fringetree ( Chionanthus

pygmaeus ), Pigeon Wings ( Clitoria fragrans ), Short-leaved Rosemary ( Conradina brevifolia ), Avon Park Rabbit-bells ( Crotalaria avonensis ), Yellow Scrub Balm ( Dicerandra christmanii ), Scrub Mint ( Dicerandra frutescens ), Scrub Buckwheat ( Eriogonum longifolium var. gnaphalifolium ), Wedge-leaved Button-snakeroot ( Eryngium cuneifolium ), Highlands Scrub St. John's-wort ( Hypericum cumulicola ), Florida Gayfeather ( Liatris ohlingerae ), Britton's Bear-grass ( Nolina brittoniana ), Paper-like Whitlow-wort ( Paronychia chartacea ssp. Chartacea ), Lewton's Polygala ( Polygala lewtonii ), Wireweed ( Polygonella basiramia ), Small's Jointweed ( Polygonella myriophylla ), Scrub Plum ( Prunus geniculata ), Carter's Mustard ( Warea carteri ), and Scrub Ziziphus ( Ziziphus celata ).

The lichen is: Perforate Reindeer Lichen ( Cladonia perforate ).

The American alligator ( Alligator mississippiensis ) is no longer biologi cally endangered or threatened; however, it is liste d by USFWS as Threatened thr oughout 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 hosp itable. The crested caracara ( Caracara cheriway ) is associated with open country; dry prairie with scattered cabbage pa lm s, wetter prairies, and to some extent also improved pastures and sometimes wooded areas having associated lim ited 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 pa lms. The red cockaded woodpecker ( Picoides borealis ) inhabits old growth forests, primarily longleaf pine. The Florida scrub-jay ( Aphelocoma

coerulescens ) prefers oak scrub on white, drained sa nd, in open areas without a dense canopy associated with Palmetto, sand pine and rosemar y. This includes scrub with no canopy, sandpine scrub, scrubby flatwoods, and coastal sc rub. The Florida grasshopper sparrow ( Ammodramus savannarum floridanus ) prefers dry prairie with stunted sa w palmetto and dwarf oaks, bluestems and wiregrass and unimproved cattle pastures. Its habi tat is maintained by periodic fires. The snail kite ( Rostrhamus sociabilis plumbeus ) prefers large, open freshwater marshes and lakes with shallow open waters. The ivory-billed woodpecker ( Campephilus principalis ) formerly occurred in

the southeastern United States a nd Cuba and has declined to extinc tion or near extinction. It once occupied swampy forests, especially large bo ttomland river swamps of coastal plain and Mississippi Delta and cypress swam ps of Florida, in areas with many dead and dying trees. It would not occur in an agricultural environment such as the field test site. Whooping crane ( Grus

Americana ) prefers freshwater marshes and wet prairies . It nests in dens e emergent vegetation (sedge, bulrush) in shallow ponds, freshwater marshes, wet prairies, or along lake margins, within large expanses of undisturbed wilderness. The cran es listed in this county are an experimental reintroduction of whooping cranes in Florida initiated in 1993 to establish a non-migratory

Page 86 of 97population. This is an experim e ntal, non-essential popula tion. Any of the bird species could potentially visit the field test site but would not nest th ere. 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. Hab itats include tropical hammocks, pine flatwoods, cabbage palm fo rests, mixed swamp, cypress swamp, live oak hammocks, sawgrass marshes, and Brazilian pepper thickets. It depends on large contiguous blocks of wooded habitat, though interspersed fi elds and early successional habitats may be beneficial through their positive effect on prey popul ations. Its day-use sites typically are dense patches of saw palmetto surrounded by swamp, pine fl atwoods, or hammock. It would not occur in the trial area due to the openness and continued pres ence 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 couperi ) – according to Candice Martino (904-232-2580 ext. 129) Section 7 Endangered Species biologist from the Jacksonville, FL Field Office, (contacted February 25, 2008) the species is seldom seen but could be anywhere. However, the habitat at the release site would not be suitable. The historic

and continuous use of the re lease site and the surrounding ar ea as a citrus grove and Eucalyptus field trials makes it extremely unlikely that the sp ecies would be found in th e area. Therefore, the appropriate determination would be “no effect.” Although it is highly unl ikely that the species would be found at the site, the appl icant will provide all workers w ith identifying char acteristics of

the snake and instructions on what to do if the species is encountered. These measures are a variation of standard protective measures the USFW S 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 sandhill s. It occupies localized pockets of

sufficient leaf litter and moisture to provide abundant food and ne sting 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 scr ub, less often longleaf pine-turkey oak sandhills or turkey oak barrens adjacent to scrub, especially high pine-scrub ecotones. It was determined that

the release site is within a geographic area where these tw o 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 scru b habitat with areas of open sand. The literature

indicates that skinks are sometimes found in ac tive 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 protocol s to determine if the sp ecies is present. The protocols were provided by the Vero Beach Fiel d Office. The USFWS protocol recommends that surveys be conducted between March 1 and May 15 as this is an ideal time to observe evidence of the skinks. A coverboard survey was conducted over a one month period from March 18 to April 15. The coverboards were checked weekly on March 25, April 1, April 8 and April 15. No evidence indicating the presence of sand skinks or bluetail m ole skinks was observed and they are presumed absent. Therefore, the appropria te determination would be “no effect.”

For the plants, the large-flowered bonamia ( Bonamia grandiflora ) grows in natural clearings of bare ground and invades disturbe d areas of open sand. Although not common, it is often locally

abundant where there is little or no shade from tr ees 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

Page 87 of 97Central Ridge of Florida. Pygm y Fringetree ( Chionanthus pygmaeus ) is generally found in xeric,

coarse white sand of scrub/oak sc rub areas found at the southern e nd of the Central Florida Ridge. It is also found occasionally in longleaf pine-t urkey oak vegetation, high pineland, dry hammocks, and transitional habitats. Pigeon Wings ( Clitoria fragrans ) is widely scattered in undisturbed clearings of xeric sandhill and sc rub communities on well-drained upl and soils. It is typically found in undisturbed clearings in sc rub areas but also occurs in very open scrub as well. Shortleaved Rosemary ( Conradina brevifolia ), is found in white sands of sand pine-oak scrub with scattered overstory of sand pine a nd scrub oak. Park Rabbit-bells ( Crotalaria avonensis ) occurs in upland habitats (scrub and sandhill ), often along trails. It grow s in full sun or partial shade provided by characteristic scrub shrubs or sand pine. Yellow Scrub Balm ( Dicerandra christmanii ) occurs in openings in sand pine-oak scrub on yellow soils of the Central Flor ida Ridge. Scrub Mint ( Dicerandra frutescens ) occurs in well-drained soils of scr ub or sandhill vegetation. It is locally abundant in and around the sand pine-evergreen oa k scrub, where it may occur in the low shrub layer or in open stands, clearings, or adjacent sandy places. It is not found in areas cleared for pasture, or areas in which wholesale site preparation has taken place. Scrub Buckwheat ( Eriogonum longifolium var. gnaphalifolium ) is long-lived, slow gr owing and flowers and reproduces primarily after fires or other disturbances (e.g. loggi ng, mowing) that increase light availability. It prefers dry pi nelands, sandhills, and scrub (lon gleaf pine-turkey oak, scrub oaks) and is more commonly found in transition habitats between scrub and high pine and in turkey oak barrens than in either dense scrub or open high pine. Wedge-leaved Button-snakeroot ( Eryngium cuneifolium ) is generally found in areas of open sand, including blowouts and other highly disturbed soil surfaces, such as road shoulders. It occurs in exposed sunny openings; areas in scrub, especially rosemary scrub. Highlands Scrub St. John's-wort ( Hypericum cumulicola ) occurs in patches of open, nutrient-poor sand within oak a nd rosemary scrub. It is often associated with reindeer lichen ( Cladonia spp.) and the rare wedge-leaved button snakeroot ( Eryngium cuneifolium ). Florida Gayfeather ( Liatris ohlingerae ) occurs in openings in oak-rosemary scrub and sand pine scrub. Britton's Bear-grass ( Nolina brittoniana ) occurs in deep, fine-textured, welldrained sands of sand pine-evergreen oak sc rub or longleaf pine-t urkey oak sandhills. Nolina is entirely dependent on fire or some other mech anism to maintain an open successional stage in scrub or sandhills. Paper-like Whitlow-wort ( Paronychia chartacea ssp. Chartacea ) is a sand

scrub that occurs on ancient dunes in the lake region, in white sand clearings or blowouts. Lewton's Polygala ( Polygala lewtonii ) occurs in sandhills characte rized by longleaf pine and low scrub oaks, including low turkey oak woods, and tr ansitional sandhill/scrub habitats. This species occasionally inhabits powerline cleari ngs or new roadsides. Wireweed ( Polygonella basiramia ) is

restric ted to bare patches within sand pine-evergreen oak scrub vege tation. It grows on areas of bare sand within sand pine ( Pinus clausa ) and Florida rosemary ( Ceratiola ericoides ). Small's Jointweed ( Polygonella myriophylla ) occurs in areas of sand pine scrub and ancient sand dunes. Scrub Plum ( Prunus geniculata ) has a very narrow range and small widely scattered populations. It frequently forms small colonies of several plants but may grow as solitary individuals. It grows in deep, yellow sands of longleaf pine-turkey oa k sandhill and white, excessively leached, winddeposited soils of evergreen scrub oak- sand pine scrub. Carter's Mustard ( Warea carteri ) occurs in sandy clearings in sand scrub and sandhills; scattered overstory of sand; longleaf or slash pine and scrub oaks. Scrub Ziziphus ( Ziziphus celata ) is a scrub that occurs on gently rolling hills with vegetation dominated by Carya floridana and Quercus species. It prefers open, sunny areas. The Perforate Reindeer Lichen ( Cladonia perforate ) occurs in sandy openings in stabilized sand dunes with Florida scrub vegetation. It is often associated with Ceratiola . None of the plants and the

Page 88 of 97lichen listed above would find the fiel d test site as suitable habita t and w ould not be present given

the historic and continuous use of the release s ite and the surrounding area as a citrus grove and research area used for growing Eucalyptus

.

Critical Habitat: There is proposed critical habi tat listed for the West Indian manatee and the Everglade snail kite but none of the proposed habitat occurs in this county. The whooping crane has designated critical habitat but it does not occur in this county. Th e population in this county is a non-essential experimental population a nd does not have critical habitat.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

26. Marion County, FL (FL-MAR-01)

This location has been an agricultu ral research station for more than 5 years. The location has been used for conducting research experiments on agri cultural crops. Site preparation will involve herbicide application, plowing, and planting of trees in flat beds. The surrounding areas of the test site consist of agricultural fields, plantings of horticultura l crops and an experimental Eucalyptus test plot.

Nine TES animals and five TES plants are listed for Marion County. The an imals are: sand skink ( Neoseps reynoldsi ), Eastern indigo snake ( Drymarchon couperi ), American alligator ( Alligator mississippiensis ), wood stork ( Mycteria Americana ), snail kite ( Rostrhamus sociabilis plumbeus ), red-cockaded ( Woodpecker Picoides borealis ), Florida scrub-jay ( Aphelocoma coerulescens ), West Indian manatee ( Trichechus manatus ), hoary bat ( Lasiurus cinereus ). The plants are: largeflowered bonamia ( Bonamia grandiflora ), longspurred mint ( Dicerandra cornutissima ), scrub buckwheat ( Eriogonum longifolium var. gnaphalifolium ), Britton's bear-grass ( Nolina brittoniana ), Lewton's polygala ( Polygala lewtonii ).

The American alligator ( Alligator mississippiensis ) is no longer biologi cally endangered or threatened; however, it is liste d by USFWS as Threatened thr oughout its entire range due to similarity of appearance to other endangered or threatened crocodilians. The snail kite ( Rostrhamus sociabilis plumbeus ) prefers larg e, open freshwater marshes and lakes with shallow open waters. The red cockaded woodpecker ( Picoides borealis ) inhabits old growth forests, primarily longleaf pine. The Florida scrub-jay ( Aphelocoma coerulescens ) prefers oak scrub on white, drained sand, in open areas without a dens e canopy associated with Palmetto, sand pine and rosemary. This includes scrub with no ca nopy, sandpine scrub, scrubby flatwoods, and coastal scrub. The wood stork ( Mycteria Americana ) primarily inhabits wetland systems notably cypress or mangrove swamps and would not find the field test site hospitable. Any of the four bird species could potentially visit the field test site but would not nest there. The West Indian manatee occurs in shallow coastal waters, rivers, bays and lakes; none of which are close to any of this release

Page 89 of 97location. The hoary bat pr efers deciduous and coniferous forests and woodlands. It roosts usually in tree foliage 3-5 m above ground, with dense folia ge above and open flying room below, often at the edge of a clearing and commonl y in hedgerow trees. It sometimes roosts in rock crevices and rarely uses caves in most of its range. It would be unlikely to find the rele ase site hospitable habitat since there are no trees in the plot itself. Once the trees are esta blished and growing, if the trees were to reach maturity, it would not likely find the trees suitable habitat since they do not have dense foliage and the plot is in the middle of an agricultural research ar ea, not in a deciduous or coniferous forest. For the Eastern indigo snake ( Drymarchon couperi ) – according to Candice Martino, Section 7 Endangered Specie s biologist from the Jacksonville, FL Field Office, (contacted February 25, 2008) the species is seldom seen but could be anywhere. Howe ver, the habitat at the

release site would not be suitable . The historic and continuous use of the release site and the surrounding area as an agricultural research stati on makes it extremely unlikely that the species would be found in the area. Therefore, the appr opriate determination would be “no effect”. Although it is highly unlikely that the species woul d be found at the site, the applicant will provide all workers with identifying characteristics of the snak e 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” determin ation for construction sites. Also for the Sand Skink ( Neoseps renoldsi ) – according to Candice Martino (contacted February 25, 2008), although the release site may be within a greater geograp hic area where the species may be found, the habitat

of the release site would not be suitable for the species. The hi storic and continuous use of the release site and the surrounding area as an agricultural research station makes it extremely unlikely that the species would be found in the area. Ther efore, the appropriate determination would be “no

effect”.

For the plants, the large-flowered bonamia ( Bonamia grandiflora ) grows in natural clearings of bare ground and invades disturbe d areas of open sand. Although not common, it is often locally

abundant where there is little or no shade from tr ees or shrubs. It is locally abundant on deep, white, dry sands of ancient dunes and sandy ridges in clearings or openings of scrub habitat on the Central Ridge of Florida. The longspurred mint ( Dicerandra cornutissima ) is scattered in openings (natural or artificial) in longl eaf pine-turkey oak scrub/sandhill or on low rises in slash pinepalmetto scrub. The scrub buckwheat ( Eriogonum longifolium var. gnaphalifolium ) is an herbaceous perennial that occurs in dry pineland s, sandhills, and scrub (longleaf pine-turkey oak, scrub oaks). It is more comm only found in transition habitats be tween scrub and high pine and in turkey oak barrens than in either dense scr ub or open high pine. Britton's bear-grass ( Nolina

brittoniana ), occurs in deep, fine-textu red, well-drained sands of sand pine-evergreen oak scrub or longleaf pine-turkey oak sandhill. Nolina is entirely dependent on fire or some other mechanism to maintain an open successional stage in the scrub or sandhill. Lewton's polygala ( Polygala

lewtonii ), oc curs in sandhills characterized by longleaf pine and low scrub oaks, including low turkey oak woods, and transitional sandhill/scrub habitats. This species occasionally inhabits powerline clearings or new roadsides. None of the six plant species listed above would find an agricultural research area as suitable habitat and would not be present given the historic and

continuous use of the release site and the surrou nding area as an agricultural research station.

Critical Habitat: There is no designated critical habitat or habita t proposed for designation in the county. There is proposed critical ha bitat listed for the West Indian manatee but it does not occur in this county.

Page 90 of 97Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to found at the release s ite. If they were to enter the site, their pres ence would be fleeting

as the hab itat is either not suitable or does not co ntain constituent elements required by the species. Field activities will result in no changes to th e habitat used by any listed species or species proposed for listing. The site is not within or near designated critical habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

27 - 29. Taylor County, FL (FL-TAY-01, FL-TAY-02 and FL-TAY-03)

There are three release site lo cations in Taylor County.

Two of the locations have been under managed pine plantations for over 50 years and contain third rotation plantings of pine. The previous plantings were cultiv ated in beds using standard silvicultural practices. The existi ng pine plantation at this site was harvested by the owner prior to 2007 and prepared for re-planting. Additional site preparation will involve herbicide application, plowing to remove stumps, and pl anting of trees in raised beds. The surrounding areas of the test site consist of less than 25 year-o ld third rotation pine plantings.

The third location has been under managed pine plan tation for more than 20 years. Previous pine plantings were cultivated in beds using standard silvicultural practices. The existing pine plantation at this site was harvested by the owner in 2008 and prepared for re-pla nting. Additional site preparation will involve herbicide ap plication, plowing and planting of trees in raised beds. The test

site is within th e larger area harvested by the owner and is surrounded by managed stands of pine plantations.

There are eleven TES animals listed for Taylor County. There are no liste d plants. The animals are: Eastern indigo snake ( Drymarchon couperi ), American alligator ( Alligator mississippiensis ), wood stork ( Mycteria Americana ), piping plover ( Charadrius melodus ), red-cockaded woodpecker ( Picoides borealis ), West Indian manatee ( Trichechus manatus ), Gulf sturgeon ( Acipenser oxyrinchus desotoi ), green turtle ( Chelonia mydas ), leatherback turtle ( Dermochelys coriacea ), Kemp's ridley sea turtle ( Lepidochelys kempii ) and loggerhead sea turtle ( Caretta caretta ).

The American alligator ( Alligator mississippiensis ) is no longer biologi cally endangered or threatened; however, it is liste d by USFWS as Threatened thr oughout its entire range due to similarity of appearance to other endangered or threatened crocodilians. According to Caroline Stahaller, (contacted February 20, 2008) of the Panama City Fi eld Office USFWS, the Eastern indigo snake ( Drymarchon couperi ) has not been seen anywhere on the Florida panh andle in 15-20 years. She had no concern that the action could a ffect the species. Although it is highly unlikely that the species would be found at the site, the applicant will pr ovide all workers with identifying

characteristics of the snake and instructions on wh at to do if the species is encountered. These

measures are a variation of sta ndard protective measures the USFW S uses when they have reached a “may affect” determination for construction s ites. The birds (piping plover, red-cockaded woodpecker and wood stork) could all potentially visit the field test si te but would not nest there. The piping plover nests along the coast which is seven or more miles away from the field test sites. The red-cockaded woodpecker nests in old growth longleaf pine. The wood stork nests in marshes,

Page 91 of 97floodplain lakes, and swamps. The rem aining species: West Indian manatee ( Trichechus manatus ), Gulf sturgeon ( Acipenser oxyrinchus desotoi ), green turtle ( Chelonia mydas ), leatherback turtle ( Dermochelys coriacea ), Kemp's ridley sea turtle ( Lepidochelys kempii ) and loggerhead sea turtle ( Caretta caretta ) are all aquatic species and would not occur anywhere clos e to the field test site. The closest body of water is the Ec onfina river which is about 2.5 miles away from one of the test

sites.

Critical Habitat: There is designated critical habita t listed for the piping plove r in Taylor county. It is located along the coast in the southwest part of the county. It is approximately 15, 27 and 32 miles away from the three test sites. There is pr oposed critical habitat lis ted for the West Indian

manatee but it does not occur in this county.

Conclusion: No federally listed threatened or endangered species or species proposed for listing are likely to be found at the releas e site. If they were to enter the site, their presence would be fleeting as the habitat is either not suitable or does not contain cons tituent 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 habita t or habitat proposed for designation. Therefore, the acti on will have no effect on listed sp ecies or species proposed for listing and would not affect desi gnated critical habitat or ha bitat proposed for designation.

Overall Conclusions

The field test sites in this permit application have been in agricultural or forest research, or in agricultural production or forest tr ee plantations for from 5 to 50 years. No federally listed threatened or endangered species or species proposed for listing are li kely to be found at any of the release sites. If they were to enter the site, thei r presence would be fleeting as the habitat is either

not suitable or does not contain constituent elements required by the species. Field activities will result in no changes to habitat used by any listed species or species proposed for listing. The sites are not within or near designated critical habitat or habitat proposed for designation. Therefore, the action will have no effect on listed species or sp ecies proposed for listing and would not affect designated critical habitat or habitat proposed for designation.

Page 92 of 97

## APPENDIX V: Proposed Supplemental Permit Conditions

For Release of Eucalyptus grandis x Eucalyptus urop hylla under permits 08-011-106rm and

08-014-101rm

(1) The test sites and adjacent land within 100 meters shall be monitored for any 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 co mpletion of the field test, the site will not be planted with Eucalyptus , so that any volunteer se edlings that emerge can be easily identified. If volunteers or stump sprouts are st ill emerging at the end of the fi rst year, a second year will be added to the monitoring period to ensure that no shoots ar e 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 re gulations outlined in "Container requirements for the movement of regulated articles", 7CFR 340.8(b) unless a shippi ng container variance has been approved by APHIS-BRS.

(3) BRS should be notified in writing of any proposed changes to the permit application (or approved permit) including for example confinemen t protocols, transgenic lines or constructs, release locations, acreage, etc. Changes usually re quire amendments to the permit and must be preapproved by BRS. Requests shou ld be directed to Regulator y Permit Specialist, USDA APHIS BRS, Biotechnology Permit Services, 4700 Rive r Road, Unit 91, Riverdale, Maryland 20737.

(4) Any regulated article introduced not in complia nce with the requirements of 7 CFR part 340 or any standard or supplemental permit conditions, sha ll be subject to the immediate application of such remedial measures or safeguards as an inspector determines necessary, to prevent the introduction of such plant pests. The responsible party may be subject to fines or penalties as authorized by the Plant Protection Act (7 U.S.C. 7701-7772).

(5) This Permit does not eliminate the permittee's legal responsibility to obtain all necessary Federal and State approvals, incl uding for the use of: (1) any non-ge netically 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 unreg istered 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 re cords at any time.

(7) Harvested plant material may not be used for food or animal feed unless it is first devitalized and approved for such use by the U.S. Food and Dr ug Administration; and fo r plant-incorporated protectants, a tolerance for the pesticide must first be establ ished by the U.S. Environmental Protection Agency.

Page 93 of 97(8) The perm ittee shall provide all workers with identifying characteristics of the threatened Eastern indigo snake and in structions on what to do if the species is encountered. This shall be done for release sites within the known range of the snake which include the following counties: Baldwin AL, Evans GA, Glades FL, Bay FL, Hi ghlands FL, Marion FL, Gadsden FL, Columbia, FL and Taylor FL.

(9) Reporting an Unauthorized or Accidental Release 1. According to the regulation in 7 CFR 340.4(f)(10), APHIS shall be notified orally immediately upon discovery and notified in writing within 24 hours in the event of any accidental or

unauthorized release of the regulated article.

- For immediate verbal notification, contact AP HIS BRS Compliance Sta ff at (301) 734-5690 and ask to speak to a Compliance and Inspection staff member. Leave a verbal report on voicemail if the phone is not answered by a Compliance Officer. - In addition, in the event of an emergency in which you need to speak immediately to APHIS personnel regarding the situation, you may call:

The APHIS/BRS Regional Biotechno logist assigned in the region where the field test occurs: For Western Region, contact the Wester n Region Biotechnolog ist 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 st ate where the unauthorized release occurred. The

list of APHIS State Plant Health Directors is available at: http://www.aphis.usda.gov/se rvices/report_pest_disease/ report_pest_disease.shtml. or http://pest.ceris.purdue.edu/stateselect.html

2. Written notification should be sent by one of the following means: By e-mail: BRSCompliance@aphis.usda.gov By mail: Biotechnology Regulatory Services (BRS) Compliance and Inspection Branch

USDA/APHIS

4700 River Rd. Unit 91 Riverdale, MD 20737

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

(10) Reporting Unintended Effects: According to the regulation in 7 CFR 340.4(f)(10)(ii) , APHIS shall be notified in writing as soon as possible but within 5 working days if the regulated article or associ ated host organism is found to

have characteristics substa ntially different from those listed in the permit application or suffers any

Page 94 of 97unusual occurrence (excessive m ortality or morb idity, or unanticipated effect on non-target organisms).

Written notification should be sent by one of the following means: By e-mail: BRSCompliance@aphis.usda.gov By mail: Biotechnology Regulatory Services (BRS) Compliance and Inspection Branch

USDA/APHIS

4700 River Rd. Unit 147 Riverdale, MD 20737

(11) 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) Compliance and Inspection Branch

4700 River Rd. Unit 91 Riverdale, MD 20737

BRS Fax: Compliance and Inspection Branch (301) 734-8669

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

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

RESULT IN REVOCATION OF THE PERMIT. The A nnual Report shall reflect the current status

Page 95 of 97and observations to date for each location. It shal l include the inform ation submitted in the Planting Report, plus the following: i. An accounting of the acreage or number of plants per line (event) for each construct that remain in the ground; ii. A detailed map of the plantings; iii. Total remaining acreage (include acreage of border rows if appropriate); iv. The methods of observation; v. The resulting data and analysis regarding all de leterious effects on plants, non-target organisms, or the environment. This should include, but not be limited to, data on insect damage, disease susceptibility, gross morphology and any indications of weediness; vi. If any material was harvested, removed, or terminated or otherwise destroyed, a disposition table with the following information for each line (event) released should be provided: date(s) of harvest, removal, and/ or termination; a formal record of how the regulated material was removed from the environment; what material an d how much was harvested or removed and where it was transported, stored and further processed up to the time it is or was to be taken to a contained facility; and what was done to devitalize residua l and/or harvested mate rial at the location.

In this report also provide data documenting wh ich trees produced flowers and which if any produced viable seeds. Also document seed ling volunteer monitoring, including any volunteers

found and the method of devitalization.

c. Field Test Report Within 6 months after the expira tion date of the permit, the permittee is required to submit a Field Test Report. NOTE: If a new application is approved to continue the field test past its scheduled expiration date, an annual report should continue to be submitted un til the final expiration date, at which point the Field Test Report will be due after 6 months. Fi eld Test Reports provid e the final status and

observations at each location and shall include: i. List of all constructs and specifi c transformed lines (event) planted; ii. Planting date(s), and harvest date s if any material was harvested; iii. Total acreage of regulated article planted; iv. Total acreage of any border rows planted; vi. The methods of observation; vii. The resulting data and analysis regarding all deleterious effect s on plants, non-target organisms, or the environment. This should include, but not be limited to, data on insect damage, disease susceptibility, gross morphology and any indications of weediness. viii. A disposition table with the following information: Site name (or GPS), crop, harvest date(s), and dis position of harvested material. Date(s) of harvest, removal, and/or termination; a formal record of how the regulated material was removed from the environment; what material and how much was harv ested or removed and wh ere it was transported, stored and further processed up to the time it was taken to a contained fac ility; and what was done

to devitalize residual and/or harv ested material at the location.

We encourage the inclusion of ot her types of data if the applic ant anticipates submission of a petition for determination of non-re gulated status for their regulate d article. APHIS considers these data reports as critical to our assessment of plant pest risk and development of regulatory policies

Page 96 of 97Page 97 of 97 based on the best scientific evidence. Failure by an applicant to provide da ta reports in a timely manner for a field trial may result in the withholdi ng of permission by APHIS fo r future field trials.

d. Flowering monitoring report In the locations where there are other species of eu calyptus within 1000 meters of the test plots, if there is any overlap in flowering between the transgenic trial and th e non-transgenic trees, this must be reported to APHIS.

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