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United States Department of Agriculture

Agricultural Research Service

Beltsville Area Beltsville Agricultural Research Center

Environ. Chem. Lab Bldg. 007, BARC-W Beltsville, MD 20705

Email= RCHANEY@ASRR.ARSUSDA.GOV Phone= 301-504-8324; Fax= 301-504-5048

CONFIDENTIAL CRADA DOCUMENT September 8, 1997 Dan Hilburn Oregon Department of Agriculture 635 Capitol St., NE Salem, OR 97310 Dear Mr. Hilburn: I am sending you the information which we discussed by telephone today for use in your official capacity with the Oregon Department of Agriculture. Please treat this information as confidential business information from the cooperators in a Cooperative Research and Development Agreement. The cooperators are the USDA-Agricultural Research Service, the University of Maryland, the University of Sheffield, UK, and the commercial cooperator, NKT Inc. We are conducting research to develop a commercial agricultural phytoextraction technology for Ni+Co from naturally mineralized or contaminated soils. Dr. J.S. Angle is the scientific cooperator at the University of Maryland, and Dr. A.J.M. Baker at the University of Sheffield. Further, we are cooperating with Dr. V. Van Volk at Oregon State University-Corvallis, and Dr. Richard Roseberg of the Medford Agricultural Experiment Station, and will cooperate with a local farmer. 1. The species we want to use in 1997 test plantings include Alyssum murale WALDST. & KIT, Alyssum corsicum, Alyssum pterocarpum, Alyssum falacinium, Alyssum caricum, and Alyssum tenium. These species have been identified as nickel hyperaccumulators based on analysis of herbarium specimens and field collected samples by Brooks, Reeves, Baker et al. [Brooks, et al., 1979; Brooks and Radford, 1978; Morrison, et al., 1980; Reeves, et al., 1983). A detailed description of the genus Alyssum is reported in Dudley (1965). Full citations and abstracts for the noted references are provided in an enclosure to this letter. 2. These species occur on serpentine soils from Albania, Italy, Corsica and Greece through Bulgaria and across Turkey, and related species in Spain and Portugal. These Alyssum species are serpentine endemic species; that is, they are only found on serpentine soils. Such plants are able to tolerate the unfavorable low Ca:Mg ratio and other low fertility aspects of serpentine soils, and reproduce, while normal plants and crops do very poorly on such soils (on the other hand, crops and normal weeds compete very strongly on normal soils, and the Alyssum serpentinophyte species are unable to persist on non-serpentine soils). From the literature, and the observations of Reeves and Baker in the field, A. murale and other endemic serpentine Alyssum species do not

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colonize non-serpentine soils. Serpentine soils naturally contain elevated Ni, Co and Cr compared to most soils, and are usually nearly neutral pH until all of the Mg silicate minerals are leached from the soil. The Ni levels are commonly 2000-3000 mg Ni/kg soil. Non-accumulator plant species contain about 5-20 mg Ni/kg, while the shoots of A. murale plants from which seed was collected in Bulgaria in 1996 contained about 1.5% Ni on a dry weight basis. This strain or genotype of A. murale has grown very well at our test garden on serpentine soil near Baltimore, MD. Several of these species have performed very well in experiments we have conducted. Yields near 20 t dry matter/ha were obtained in the UK. Our own studies indicate that the genotypes we selected for testing, based on the size in the field, and their Ni+Co concentrations in these fields, are very promising for development as an alternative crop. These Alyssum species occur across a wide range (Dudley, 1965), within the Mediterranean climate such that they are dormant in the summer after flowering and setting seed in the late spring. Based on their adaptation, we believe these species will grow well on Oregon serpentine soils if fertilized and managed as a crop. Some of these species have been grown in North America for many years (Dudley, 1966). The natural populations are extensive; we searched thousands of hectares to find the most promising genotypes. In order to collect genetic diversity which might be used to breed improved cultivars for commercial use, we have been collecting seed of individual plants in different niches within the serpentine soil areas where the plants occur. Although the evidence is not absolute at this time, much evidence indicates that this species is "selfing". Insects do not appear to be required for its self-fertilization, although bees and some other insects do visit the flowers. Some other Ni hyperaccumulator species do require specific insects for pollen transfer, and this trait was considered during the process of selecting the most promising species for development. Clearly, our collection methods do not threaten any of these species. We have been searching for test fields in Josephine Co, Oregon, in which to conduct germplasm evaluation, and identification of soil fertility and agronomic management practices which allow maximum value in the crop. We have several objectives in the field test. These include: Can this species survive the winter and have their usual perennial growth habit in southwestern Oregon? Does this species hyperaccumulate Ni and Co from the mineralized soils which occur in that area. Does soil pH affect Ni hyperaccumulation from these mineralized Oregon soils? [Because lower soil pH usually increases Ni uptake by plants, and our pot studies confirmed this model for A. murale and three other species, we would lower soil pH in specific test plots by addition of sulfur so that higher accumulation might be achieved in the test. All other soil amendments in these tests are common fertilizers and gypsum used to attain higher yields of crop species on these infertile soils.] What is the seasonal pattern of shoot Ni concentration and shoot biomass yield during the year? Can these species be mechanically harvested at low cost? We plan to conduct a field test in which pelleted seed are sown in early fall, 1997. We will vary soil fertility factors in controlled experiments, establish the phenology of the species in Oregon, and evaluate many genotypes under controlled soil conditions. Previous information about these species were from many different locations, with unfavorable conditions for maximum accumulation of metals, and severe fertility and climatic

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limitations. The seeded test would evaluate our present highly promising genotypes of A. murale, other genotypes of A. murale, seed of which were collected, with the parents being unusually large plants with high Ni levels, with consideration for winter temperatures, etc. From our more extensive germplasm collection work in summer 1997, we will include several other Alyssum hyperaccumulators in this seeded test (e.g., A. pterocarpum; A. corsicum; A. falacinium; A. tenium and/or A. caricum). Each genotype or species and each treatment would be sampled over the growing year for a two year period. These species will be evaluated in our phytoextraction development program and a standard breeding program as noted in this letter; information about the related species is in the cited papers. This species is not a "weed" except on serpentine soils where other plants do not compete with A. murale on disturbed serpentine soils. Our cooperators Dr. Alan Baker (University of Sheffield, UK) and Roger Reeves (Massey University, New Zealand) noted that they have observed serpentine soils used as low value pastures in Greece and other countries and that sheep and goats did not eat the A. murale. The seeds are small and do not attract wildlife. Because large animals have wider ranges, we do not expect large mammals to be threatened by this crop. It is possible that field mice and other small herbivore mammals with a small range could suffer toxicity if they consume a large dietary fraction of A. murale or related hyperaccumulator species. It is well established, however, that Ni is not biomagnified in environmental food-chains; instead it is "biominified" because so little Ni accumulates in the first trophic level which consumes the plant material. Meadow voles fed soybeans containing nearly 30 mg Ni/kg DW as a large fraction of their diet suffered no toxicity, and had little or no increase in tissue Ni compared to Ni-salt fed voles (Alexander, et al., 1979). Further, during the test period, the plots would be enclosed with an electric fence to keep out wildlife which could interfere with the experiment by eating parts of plants, uprooting plants, etc. Ni in the plants is expected to prevent feeding by chewing insects. To illustrate the low risk these plants comprise in North America, when we brought Alyssum species seeds into the US, the USDA Animal and Plant Health Inspection Service evaluated the situation and allowed us to bring the seeds directly to Beltsville. No Alyssum species occurs in continental United States (one does occur in Alaska), and no closely related species is grown as a crop. Brassica crop species are quite distant from the Alyssum family regarding cross-fertilization. If we acidify the soil to favor Ni and Co uptake, we expect Ni phytotoxicity to limit weed competition with the A. murale. We are evaluating common herbicides used on canola for use to control grassy weeds which might grow under these conditions, and will seek an appropriate experimental use permit before using the herbicide approved for canola on the Alyssum species. In our telephone conversation, you noted that you believe that such species may be grown as new crops without specific pre-approval. Thus we will be working to install the field test plots in Josephine Co. in late September or early October, 1997. We believe

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that phytoextraction of metals from such mineralized soils and mine wastes present in Oregon, with biomass energy production and recycling of the biomass ash as a high grade ore, is a very promising agricultural technology which is much less disruptive than traditional mining practices. And that soil materials which cannot be used for economic mining and smelting can be cropped with these species which use biological specificity and energy to become an economic crop. Several locations in Oregon could benefit if we can develop the new crop and the crop and soil management practices needed to make the system effective. We will be conducting plant breeding to improve the annual Ni phytoextraction (yield times shoot Ni concentration) ability of Alyssum species using standard procedures of crossing after characterizing the genotypes we have collected. No transgenic plant materials are involved in this project or expected to be required to make a successful technology. Please consider the information in this letter, and with appropriate confidentiality restrictions, discuss this planned research and development with other Oregon officials who you believe should be aware that such a test is planned. Because this alternative, non-food crop is expected to be produced on infertile soils with low productivity, the farmers who own such land should have new opportunities not otherwise available. It is our intent to cooperate with the Oregon Department of Agriculture to provide information about our research and about these species which would help you in your official capacity. Because we will conduct a seed increase on part of the approximately 10 acres to be planted in 1997, it is possible to begin commercial production in the fall of 1998 if the technology is found to be economically promising considering the investments in fertilizers and soil amendments to improve yields of even the serpentine adapted species to this land. Sincerely yours, Rufus L. Chaney Research Agronomist Environmental Chemistry Laboratory Enc. Cited references with abstracts. cc: Jay Nelkin, New York, NY. J. Scott Angle, University of Maryland, College Park. Alan J.M. Baker, University of Sheffield, Sheffield, UK. Darrell F. Cole, Beltsville Area Technology Transfer Manager.

V. Van Volk, Oregon State University, Corvallis.

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Cited references and abstracts: Alexander, J., R. Koshut, R. Keefer, R. Singh, D.J. Horvath and R.L. Chaney. 1979. Movement of nickel from sewage sludge into soil, soybeans, and voles. Trace Subst. Environ. Health 12:377-388.

[Sewage Sludge: USDA-Chaney et al.] BIOSOLIDS. FOOD-CHAIN. "Fertilizing a Wheeling sandy loam with sewage sludge resulted in up to 4-fold greater nickel concentrations in soybean seeds. Nickel from serpentinite chips in composted sludge had a similar effect in acid soil. In keeping with reports of other workers, plant Ni concentration appeared to be inversely related to pH of the amended soil. Feeding voles (Microtus pennsylvanicus) diets containing beans with elevated Ni caused small but sometimes significant increases in carcass Ni with no short term (under 3 months) adverse effects. Animals fed diets containing Ni salts providing approximately 2000 mg Ni/kg diet grew poorly and had 70% mortality. When carcasses were analyzed for Ni and other elements, possible genetic differences in tissue concentrations of Cu and P were observed. Barring evidence of carcinogenicity of plant forms of Ni, Ni in the food chain is not likely to be hazardous. The data do not, however, justify indiscriminate use of sludge as a soil amendment."

Brooks, R.R., R.S. Morrison, R.D. Reeves, T.R. Dudley and Y. Akman. 1979. Hyperaccumulation of nickel by Alyssum Linnaeus (Cruciferae). Proc. Roy. Soc. Lond. B203:387-403.

[Hyperaccumulators: Brooks et al.] "Herbarium specimens of all except one of the 168 recognized species of Alyssum Linnaeus have been analyzed for their nickel content in order to identify hyperaccumulators (> 1000 Fg/g dry mass) of nickel. A further 31 hyperaccumulators (all in section Odontarrhena) were discovered in addition to the 14 European species reported earlier. Pot trials on the non-accumulator A. serpyllifolium Desfontaines and the hyperaccumulator A. pintodasilvae Dudley in ed. involving addition of nickel to the medium in which the plants were growing, showed that not all species of section Odontarrhena were able to act as hyperaccumulators of nickel. Hyperaccumulation occurred almost exclusively in the eastern Mediterranean area and Turkey. there appeared to be a definite correlation between species diversity, proliferation and endemism on the one hand, and extremely high nickel concentrations (> 1%) on the other. The data have been used to assess the evidence for promoting section Odontarrhena to generic rank."

Brooks, R.R., and C.C. Radford. 1978. Nickel accumulation by European species of the genus Alyssum. Proc. Roy. Soc. Lond. B200:217-224.

[Hyperaccumulators: Brooks et al.] "Herbarium specimens (over 400) of all the 64 species of Alyssum listed in the Flora Europaea were analyzed for their Ni and Co content in order to identify hyperaccumulators (> 1000 Fg/g dry mass) of either element. In addition to the three previously recorded hyperaccumulators of nickel (A. bertolonii, A. murale, and A. serpyllifolium subsp. lusitanicum), a further 11 were identified. With the exception of A. fischerianum, all hyperaccumulating species are from Section Odontarrhena of the genus, and include A. alpestre, A. argenteum, A. corsicum, A. euboenum, A. fallacinum, A. heldreichii, A. markgrafi, A. robertianum, A. smolikanum and A. tenium. Nickel contents exceeding 10,000 Fg/g (1%) were recorded in A. bertolonii, A. corsicum, A. heldreichii, A. markgrafi, and A. robertianum. The occurrence of 14 hyperaccumulators in a single genus is the highest density so far recorded and poses interesting questions concerning the plant chemistry of these species."

Chaney, R.L. 1983. Plant uptake of inorganic waste constituents. pp 50-76. In J.F. Parr, P.B. Marsh and J.M. Kla (eds.) Land Treatment of Hazardous Wastes. Noyes Data Corp., Park Ridge, NJ.

[Hyperaccumulators: Chaney et al.] REVIEW. Summarizes information on metal uptake and tolerance by plants in relation to soil metals concentrations and other soil properties such as pH and OC. Reviewed metal hyperaccumulator crops, as reported by Brooks et al. in 1977. First published description of using hyperaccumulator plants to phyto-remediate metal contaminated soils, considering Ni removal by corn vs. Alyssum.

Dudley, T.R. 1965. Alyssum. pp. 362-409. In P.H. Davis (ed.) Flora of Turkey and the

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East Aegean Islands. Vol. 1. Edinburgh University Press, Edinburgh. [Hyperaccumulators: Dudley et al.] "Annuals, biennials or perennials. Indumentum of stellate hairs, often lepidote or sublepidote; simple setae occasionally present. Leaves simple, entire, without persistent, swollen bases. Inflorescence racemose, corymbose, paniculate or subumbellate. Sepals erect, free, monomorphic or dimorphic and appearing connate, never saccate nor forming a cup-shaped calyx. Petals yellow or sometimes whitish. Long filaments unilaterally or bilaterally winged, or wingless. Nectaries one at each side of the short filaments. Siliculae dehiscent or indehisent, loculi 1-8-ovulate with nearly apical, or distinctly lateral placentation; valves compressed or inflated. Seeds often mucilaginous. The informal groups used in the key to the species correspond in large part to the Sections used in the enumeration." 75. A. murale Waldst. & Kit., Pl. Rar. Hung. 1:5, t. 6 (1799) subsp. murale. Perennial up to 60 cm. Leaves of sterile shoots oblanceolate or obovate. Cauline leaves increasing in size upwards, lanceolate or oblanceolate, acute, ofter bracteate. Indumentum of stellate hairs with equal or unequal and branched rays, rarely strigose. Pedicels rigid, ascending to patent or horizontal, or slender and subflexuous, 3-5 mm. Petals obovate, entire or rarely retuse, 2-3.5 (-4.5) x (0.5-)1-1.5 mm, glabrous or with sparse indumentum. Fruits orbicular, ovate or broadly elliptic, obtuse or truncate or emarginate, 2.5-5 x 1-4 mm, with a ± dense indumentum; valves compressed, usually undulate, wingless. Styles (0.5-)102 mm, slender, glabrous or with sparce indumentum. Seed wings 0.2-1 mm wide. I. Fruiting stems arcuate-ascending, (15-)30-60 cm; corymbs usually up to 20 cm; wings of seeds 0.4-1 mm wide; 1.2 Leaves of sterile shoots (2-)5-10 x 1-3 mm, rarely larger = var murale. 1.2 Leaves of sterile shoots 10-15 x 3-5 mm = var haradjianii. 1. Fruiting stems dwarf, lax, sprawling, not more than 20 cm; corymbs dense, (2-)5-8 cm; wings of seeds 0.2-0.3 mm wide = var. alpinum. Figure 17, p. 371. Synonyms A. trochocarpum and A. mesopotamicum Fenzl. A. chalcidicum; etc. Widespread. Central and Southern Europe and South West Asia.

Dudley, T.R. 1966. Ornamental madworts (Alyssum) and the correct name of the goldentuft alyssum. Arnoldia 26:33-45.

[Hyperaccumulators: Dudley et al.] "No Abstract." "INTRO: Many of the standard horticultural reference works list the "Madworts" as a group of annuals, biennials, perennials, or subshrubs in the family Cruciferae, which with the exception of a few species, including the goldentuft madwort, are not widely cultivated. The purposes of this article are twofold. First, to inform interested gardeners, horticulturists, and plantsmen that this exception, with a number of cultivars, does not belong to the genus Alyssum, but because of certain critical and technical characters, should be placed in the genus Aurinia of the same family. The second goal is to emphasize that many species of the 'true' Alyssum are notable ornamentals and merit greater popularity and cultivation." A summary of the popular or ornamental use of Alyssum species, or mis-use of the botanic name. He stresses that many of the commercially available seed called Alyssum saxitale should be correctly called Aurinia saxitale. Genus name from Linnaeus in 1758 based on Alyssum montanum. But this species or others were used as medicinal plants by Greeks, especially for bites of mad dogs. The 'sweet Alyssum' flower species were transferred to another genus, making it Lobularia maritima. He also discusses the incorrect naming of many commercial Alyssum seed; Alyssum argenteum was commonly used for the real Alyssum murale. Finally Graf von Waldstein and P. Kitaibel described a plant, originally collected in Transylvania, and called it A. murale. The A. argenteum had already been used to name the short species from Italy, which is in large difference from A. murale. Dudley then did a trial planting at the Arnold Arboretum (Harvard) with 100 different seed lots in 1964. Tested the hardiness of the plants, finding that 85-90% survived the hard winter of 1964-1965 after planting in the Spring of 1963. Evaluated the ornamental value of these species; and compared the plants with those of known species of wild origin. The perennials were the most interesting horticulturally, and some species (e.g. A. scardicum) planted in June, actually started to flower sporadically in September of the same year. The blooming season of these plants was long compared to many other ornamental plants. And flower color, dominantly yellow, ranged from pale to deep orange yellow, to white and even purple. He felt they preferred neutral or basic soils (no mention of ultrabasic soils). Most of the species grew into a desirable winter rosette phase that remained green during the cold dormant season; then shot up to 3-4 feet during flowering. "None of the plants grown at the Case Estates during the past two years was apparently susceptible to insect or disease infestations." He lists several commercial sources, and the American Rock Garden Society. His description of Alyssum murale: "Subshrub with crowded and erect flowering stems up to two feet tall, subtended by dense and spreading rosettes of evergreen sterile shoots; oblong-spathulate to linear-oblanceolate leaves, greenish above and silvery or white below; flowers deep golden yellow in widely spreading, strongly branched flattish corymbs; fruits orbicular and flat, often undulated, indumentum variable.

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June-September. Very variable. Southeastern and central Europe, and throughout the Levant. (Widely cultivated as A. argenteum, and occasionally as A. chalcidicum). A very attractive, long flowering and long-lived, species suitable for permanent perennial plantings or as a ground cover." A few more of the hyperaccumulator Alyssum species are listed as interesting for horticulture.

Morrison, R.S., R.R. Brooks and R.D. Reeves. 1980. Nickel uptake by Alyssum species. Plant Sci. Lett. 17:451-457.

[Hyperaccumulators: Brooks et al.] "Experiments were carried out on 11 species of Alyssum of which 9 had the capacity to accumulate very high concentrations of nickel. These hyperaccumulators were grown in substrates containing 30-10,000 Fg/g Ni. Their nickel content after 6 weeks was a function of the nickel content of the soil and reached a limiting value of approximately 10,000 Fg/g in dried leaves of the 9 species. The tolerance of 4 of the species towards Ni was determined by measuring the length of new roots on excised seedlings suspended in aerated solutions containing 0-100 Fg/mL nickel. Four hyperaccumulating taxa tolerated Ni concentrations in the range 15-60 Fg/mL. In rate-of-uptake experiments on A. euboeum, it was observed that Ni uptake was complete in approx. 6 days after addition of Ni to the substrate."

Reeves, R.D., R.R. Brooks and T.R. Dudley. 1983. Uptake of nickel by species of Alyssum, Bornmuellera and other genera of Old World Tribus Alysseae. Taxon 32:184-192.

[Hyperaccumulators: Brooks et al. SNi] ALYSSUM. "It has been shown previously that 45 species of Alyssum (Cruciferae), found on serpentine soils, can accumulate nickel to levels above 1000 Fg/g (on a dry mass basis). An investigation of most species of the Old World genera of Tribus Alysseae has now demonstrated that this remarkable nickel accumulation is exhibited by five additional serpentine taxa from Turkey and the Balkans: Alyssum peltarioides subsp. virgatiforme, Bornmuellera glabrescens, B. baldaccii, B. tymphaea, and the hybrid B. X petri. All of these taxa had maximum nickel concentrations in the range 1.0-3.1%. Additional data are presented for Alyssum species, including some for taxa not previously reported. Although adaptation by plant species to unfavorable edaphic factors may take place in a relatively short time, it is noteworthy that all species so far recognized as nickel accumulators are from regions that were beyond the maximum advance of the Pleistocene ice-caps. These plants may therefore represent a type of behavior which started to evolve prior to the most recent glacial episodes. Possible reasons for the endemism of many of the nickel accumulators are outlined."

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Dear Colleagues: This afternoon I called the USDA Animal and Plant Health Inspection Service to ask if there were any limitations or permits required for culture of the plants used in the CRADA R&D. I explained about the review at Customs, and not competing on normal soils. After considering this question, the APHIS representative advised that the only agencies who may have an interest in the R&D tests would the the State Department of Agriculture for any states where we would have plots or production fields. She gave me the telephone number of the Plant Protection contact in Maryland and in Oregon. I called Oregon and talked with Mr. Dan Hilburn of the Oregon Department of Agriculture in Salem. I first requested that the informtion I would be disclosing be given the confidentiality appropriate for such commerical information. We discussed different aspects of the tests and the plan to produce a new economic crop. Mr. Hilburn advised that while that such field testing activities we plan are not required to obtain specific approvals from ORDA, it would be appropriate for me to write him an information letter about what we planned. If he had any concerns, he would bring them to our attention. I revised the letter we prepared for Terry McIntyre to the situation in Oregon, reviewed it carefully, and sent it by fax on Sept. 8 afternoon. His phone number is 503-986-4663, and fax number is 503-986-4786. So far, so good. I guess I am not surprised. And I gave him information about the questions which are normally raised in such reviews.