Plant Variation in Cd, Pb, Zn and as Accumulation and Bioavailability and Methods to Limit Risk

APPROACH: <OL> <LI> Conduct animal feeding studies on the effect of dietary iron, zinc and calcium
supply, and crop Zn level, on absorption of Cd in lettuce, polished rice and other
crops for which Cd is important in understanding of human Cd risks from foods (durum
wheat; bread wheat; etc.) <LI> Grow commercial and garden carrot varieties with a wide
range of properties on contaminated orchard soils rich in Pb and As; include tests of
soil amendments expected to reduce uptake of Pb or As. Measure in vitro
bioaccessibility and if needed bioavailability of crop Pb or As to animals. Examine
metal residues in peel layer vs. internal storage root tissue. <LI>Complete testing of
inheritance of grain Cd concentration in sunflower hybrids, flax genotypes shown to
differ in grain Cd accumulation, and durum wheat breeding lines; assist plant
breeders develop germplasm releases with lower Cd than present commercial types.
Examine physiology of genetic differences in Cd accumulation in relation to soil
properties where crops are grown. <LI>Test methods for rapid direct analysis, or
preparation or extraction of Cd, As, and/or Pb in foods of plant or animal origin for
spectrometric analysis at lower cost than present usual methods of analysis and
verify the application of the methods developed for commercial food samples.</OL>

PROGRESS: 2005/10 TO 2006/09
<BR>Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? <BR>Excessive levels of some trace elements in crops [cadmium (Cd), lead (Pb), arsenic (As)] may cause risk to consumers when crops are grown on mineralized or contaminated land. International sales of nonoilseed sunflower kernels, flax, durum wheat and Romaine lettuce have been limited by high natural crop Cd levels. We have obtained evidence that the bioavailability of Cd in some foods (especially rice) is much greater than most foods, because rice diets cause deficiency of Fe and Zn in rice subsistence consumers. International limits on Cd in crops which ignore real differences in food Cd bioavailability will increase costs for export of US crops significantly. One solution to such Cd limits is searching for germplasm and breeding genotypes which industry can use for production of lower Cd commercial cultivars. Another solution is demonstration that bioavailability is different for different crops and limits should be based on bioavailable Cd rather than total Cd in crops. Also, marketing of carrots grown on land which had been used for apple orchards (where lead arsenate was used as a pesticide for decades) has been limited by higher than background levels of Pb in carrots roots. Previous research identified differences between crops in the bioavailability of Cd or Pb in the crops, and this may affect either the need for regulation of grain Cd and carrot Pb or the nature of such regulations. Whether growing carrots on old orchard soils should be prohibited depends on whether the carrots absorbed the soil Pb, if soil amendments reduce uptake, if Pb contamination occurred during mechanical peeling by food processors, and if the crop Pb has sufficient bioavailability to comprise concern. Experiments can clarify the mechanism by which soil Pb was present in peeled carrots and whether soil testing can identify whether Pb in a particular soil comprises sufficient risk to prohibit use of the soil in the production of vegetable crops. <BR><BR>2. List by year the currently approved milestones (indicators of research progress) <BR>Objective 1. Characterize the influence of zinc and iron concentrations in edible crop tissues on bioavailability of crop cadmium to animals; the influence of crop Zn deficiency on Cd accumulation in edible plant tissues; and whether dietary Zn deficiency increases Cd retention, and to what extent higher crop Zn affects Cd retention by animals. <BR>FY2006 Prepare plan to grow lettuce with needed levels of Cd and Zn to conduct test with rats on the role of food Zn on absorption of food Cd. Prepare manuscript on effect of Zn deficiency stress on uptake of Cd by lettuce. Cooperate in production of nonoilseed sunflower kernels to test effect of genetic reduction of kernel Cd on bioavailability of kernel Cd to rats. <BR>FY2007 Cooperate in feeding test of the effect of lettuce Zn on bioavailability of crop Cd. Cooperate in feeding test of sunflower hybrids with varied Cd. Cooperate in preparation of manuscripts on Cd bioavailability tests. Assist Thai researchers in evaluation of food chain Cd risks from contaminated Thai soils. <BR>FY2008 Identify other crops which contain high enough Cd to threaten sales due to natural Cd accumulation. Plan tests of plant uptake and bioavailability of crop Cd. <BR>FY2009 Prepare manuscripts on relationships of soil properties to Cd accumulation in crops grown on U.S. soils. Depending on finding new animal nutritionist collaborator, conduct additional feeding tests to clarify issues of crop Cd risks and needs for limits on crop Cd levels. <BR>FY2010 Conduct studies of the fundamental plant processes which affect bioavailability of crop Cd to consumers. <BR><BR>Objective 2. Characterize potential transfer of soil lead, arsenic, and copper historically applied to orchards and other crops on potential transfer of these elements into vegetable crops grown on the contaminated soils, and the potential for application of Fe and phosphate rich soil amendments to reduce uptake or bioavailability of crop Pb and As. <BR>FY-2006 Identify methods of analysis of trace levels of Pb and As in food crops using ICP-MS for Pb and arsine evolution atomic absorption spectrophotometry method for As. Test role of soil particle contamination of peel layer in Pb contamination. <BR>FY-2007 Identify, and collect soils, iron, P and organic rich byproducts. Measure Pb, As, Cu, P, Fe & organic carbon in soils and byproducts. Initiate field test for promising soil amendments to reduce Pb and As accumulation in crops grown on orchard soils. <BR>FY-2008 Evaluate relative contribution of P, Fe organic matter in limiting metal uptake or toxicity in lead arsenate contaminated soils. Analyze plant samples from field tests at the first test location. If results are promising for growers, initiate additional field demonstration tests on orchard soils. <BR>FY-2009 Report relative importance of Fe, P and organic matter on Pb, As and Cu availability to plants. <BR>FY-2010 Complete publication of risk assessments for elements in lead arsenate amended soils.<BR><BR> Objective 3: Characterize inheritance of grain Cd concentration to reduce cadmium concentrations in flax; test whether reducing grain Cd of nonoilseed sunflower or flax reduces absorption of Cd by animals to verify that breeding lower Cd cultivars can reduce crop Cd risks to consumers. <BR>FY2006 Analyze durum wheat samples from present breeding program of a researcher to aid in selecting lines to be entered into regional yield trials. Make first crosses of flax genotypes with varied Cd levels. <BR>FY2007 Prepare manuscript to report genetic diversity of grain Cd in flax. Analyze durum wheat samples from regional yield tests to compare with other commercial lines in the field. <BR>FY2008 Conduct plant analyses needed to assist cooperator in breeding lower grain Cd phenotypes in adapted flax cultivars for ND. <BR>FY2009 Cooperate in identifying durum lines for release. Continue to support plant breeding work by analyzing samples for Cd. Prepare manuscripts on inheritance of Cd in flax grain. <BR>FY2010 Support breeding work needed to combine other commercially important properties with low Cd accumulation in flax grain. <BR><BR>4a List the single most significant research accomplishment during FY 2006. <BR>Orchard soil Pb and As studies: Identifying the source of laboratory lead contamination in processed plants tissue. Several laboratory methods and procedures were examined in identifying the source of the lead contamination which was caused by a drying oven that is used by many scientists at Beltsville. Identifying the source of Pb contamination prevent future Pb contamination in harvested plant samples and will save time and money. <BR><BR>4b List other significant research accomplishment(s), if any. <BR>Food Cd bioavailability studies: Using methods developed, grew the Romaine lettuce and sunflower kernels needed for feeding studies. Cooperatively characterized unusual Cd accumulation in lettuce and spinach grown in Lockwood soils, mineralized in Cd from marine shale parent rocks. Lettuce grown on these soils would violate CODEX limits for leafy vegetables if these are established. These tests show the need for a technology to either phytoextract Cd from such soils or to treat the soils with amendments which can substantially reduce Cd accumulation by lettuce and spinach. Orchard soil Pb studies: Used the modified microwave method developed for preparation of food samples with low levels of Pb and As to digest carrot (roots, peel and leaves), lettuce and Irish potatoes tissue from growth experiments with several orchard soils and soils amended with phosphate or iron oxide plus phosphate to restrict uptake of lead and arsenic. Difficulty with variation among replications had required additional study; multiple samples run at one time gave unequal results while if run one sample at a time, the results were much more reproducible. Thus digestion methods were now available to analyze the carrots, lettuce and potato from study of orchard soils with Pb and As contamination from long use of Pb arsenate pesticide. Digested samples with low food levels of Pb and As were analyzed using Inductively Coupled Plasma-Optical Emission Spectrometry, ICP-Mass Spectrometry and Atomic Absorption-Graphite furnace to determine which method gave the most reliable results considering recovery of added Pb, replication of sample results, and analysis of Standard Reference Materials. The ICP-Mass Spectrometry approach would provide results more quickly than the other methods, and an ICP-MS was obtained and installed during the FY. With proper quality assurance, the other methods can be reliable in Pb and As analysis in food samples. Results are being analyzed and a manuscript is being prepared. Durum wheat breeding lines lower in Cd: Analysis of grain from early testing of breeding lines allowed cooperator to select lower Cd lines for testing in Regional Variety Testing program. Lower Cd durum wheat cultivars will aid growers meet CODEX limits on exported grain Cd. 5. Describe the major accomplishments to date and their predicted or actual impact. Research identified soil series which could reliably produce low Cd sunflower kernels, flax and durum wheat, and then identified soil properties which appear to have caused higher grain Cd on some soil series (poorly drained soils with substantial chloride in the soil; certain soils developed from mineralized parent materials rich in Cd). This knowledge gave U.S. sunflower marketing companies the ability to purchase and market lower Cd nonoilseed sunflower kernels which protected a market for $30 million of kernels per year in Europe. Genetic testing identified sources of lower grain Cd levels for sunflower kernels and flax, and methods to use these genes to develop low Cd nonoilseed sunflower hybrids. With the release of lower Cd inbreds and restorer lines needed to produce lower Cd nonoilseed sunflower hybrids, we have provided the industry improved ability to meet international kernel Cd limits. Further, a greater number of growers (with soil series which previously caused their crop to exceed Cd market limits) will be able to choose to contract to grow nonoilseed sunflower rather than only oilseed types. The earlier information protected companies and the industry but limited farmers with the soil series which produce higher Cd kernels from obtaining the profits from this crop. International Cd concern is based on total Cd in foods, considering all Cd in food to have equivalent risk, and ignores many observations that concurrent Zn enrichment in most foods whenever Cd was enriched reduces Cd absorption or bioavailability, or that phytate and fiber strongly affect Cd bioavailbility. Research identified the unique nature of rice (low bioavailable Fe and Zn in polished rice cause subsistence rice consumers to absorb a 10-20 fold higher fraction of dietary Cd than other diets) in promoting Cd risk by characterizing the overall diet factors which affect Cd absorption from foods. Previous Cd research was mostly of the toxicological type in which Cd salts were added to basal diets. By demonstrating that Cd risk from different foods were significantly different, and not mimicked by addition of Cd salts alone, we brought attention to the role of rice diets in Cd risk, and the lack of adverse effects on populations which obtained increased Cd from other foods. Present international Cd limits assume that Cd in all foods is equal and some proposed limits (based on that assumption) will limit sale of a few US crops (durum wheat, peanut). Because current Cd regulations are limited to major foods, limits on Cd in flax or sunflower kernels are not included in current Codex proposed limits, although limits for these crops have been established in the EU and some other nations. The demonstration that rice promotes risk from soil Cd compared to all other crops, and the characterization of the role of low Fe, Zn and Ca in polished rice in causing higher bioavailability of rice Cd, has shown that previous approaches to regulation of crop Cd levels were technically flawed. This finding was confirmed in the kinetic study of intestinal absorption and excretion of 109Cd after a test meal was fed to marginal or adequate Fe-Zn-Ca rats. And additional evidence that previous models of dietary Cd risk related to metallothionein binding of Cd are also flawed should improve understanding that testing must use diets and Cd concentrations relevant to food Cd risk assessment rather than traditional toxicological methods. Further, we strongly illustrated that rice differs from other crops by excluding Zn from grain when the crop is grown on highly Zn-contaminated soils. Thus, the approach for regulation of crop Cd in Codex should be changed to account for bioavailability of Cd in a crop and presence of Zn with Cd, which we believe would alleviate concern about Cd in all U.S. crops. These results have application to soil and food Cd risk assessment and limit setting, and to Cd levels allowed in P fertilizers, biosolids, and livestock manures applied on agricultural land. For example, application of manure increases soil Cd because imported feeds carried some of the feed Cd into manure. But field tests have shown that when manure causes increase in soil Cd, it actually causes reduction in grain Cd due to changes in soil Cd phytoavailability. This further helps industry and farmers protect their markets from technically flawed claims about risks from Cd. We predicted that clarification of the relationship of soil properties, contaminant source properties, and crop properties would strongly affect the potential for risk from soil Cd, and in the absence of consideration of the science about potential for risk from foodchain transfer of cadmium, would limit sales of otherwise valuable foods. Potential food Cd limits are presently still under consideration at Codex; and failure to consider the bioavailability of Cd from different foods or diets will yield unnecessary limits and public fear of Cd. Information is provided to FDA and commodity groups to help them in the international discussion of how any Cd limits should be structured. Conducted survey of several orchards in Maryland, Michigan, New York, Virginia Washington and West Virginia with history of lead arsenate use in order to identify soils with high levels of lead and arsenic. Soils used for apple and some other tree fruits before 1950 are commonly high in Pb and As. Although apple fruits are not increased in either Pb or As when grown on such soils, concern about using these soils to produce vegetable crops has been raised. Soils were collected from six orchards and analyzed for lead and arsenic using hot HNO3 digestion. Plant grown tests were conducted in growth chambers with six Pb and As contaminated orchard soils to characterize Pb and As uptake by four cultivars of carrots, five cultivars of Irish potatoes and one cultivar of lettuce. Soil particles contaminate washed potato peels which increases crop Pb. A greenhouse experiment tested the ability of eastern gamagrass to phytoextract soil Pb and As to remediate lead arsenate contaminated orchard soils. Developed improved carrot As analysis methods and showed that low levels of As in vegetable crops can be reliably measured using microwave digestion and ICP-OES hydride generation for measurement. Determined solubility and potential leaching of Pb and As for two orchard soils contaminated with lead arsenate and treated with phosphate to reduce Pb uptake. <BR><BR>6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? <BR>Limits on Cd in foods has limited sale of U.S. nonoilseed sunflower kernels, flax, durum wheat and peanuts. Market constraints must be met by companies which sell U.S. agricultural products, and they have to decrease purchase of higher Cd crops they cannot be assured they can market. The strong influence of soil series on Cd in several crops has been transferred to Extension advisors and to food processors to assure that they can market the crops produced. This protects consumers, agribusiness, and farmers. Developing lower Cd sunflower kernels, and identification of lower Cd cultivars of other crops allows production of lower Cd crops for these markets. For flax, progress on understanding the inheritance of high seed Cd and breeding of low Cd cultivars with other market demanded properties must be achieved before more farmers can produce flax for human consumption. For durum wheat, the absence of market enforcement of expected Codex Cd limits in the U.S. has allowed agribusiness to ignore the possible benefit of lower Cd cultivars. New limits at Codex are causing breeders to seek to develop lower Cd cultivars using known genetic methods. But the Codex process does not obtain technically valid consideration of the difference among crops in Cd bioavailability and risk, international markets which presently are not considering grain Cd may demand that only grain meeting those limits may be shipped. The costs of monitoring and purchasing only lower Cd grains would reduce the competitiveness of U.S. durum wheat significantly. Unless industry and government understand this possible effect of Codex limits and sales of U.S. crop, it is difficult to gain the support for the research and technology transfer regarding food-chain Cd risks. Our findings about food Cd bioavailability have been transferred to regulatory agencies including U.S.-FDA; we believe that if the role of concurrent Zn accumulation in foods other than rice were taken into account, Cd limits for other crops would need to be set higher than those for rice. Ongoing feeding studies focus on clarification of this aspect of Cd bioavailability. <BR><BR>7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). <BR>Codling presented poster on Pb and As contamination of orchard soils in the U.S., and implications for Pb and As transfer to foods. World Congress of Soil Science, Philadelphia, PA, July 9-14, 2006. Chaney presented invited lecture Risk Assessment for Soil Cd Should Consider Soil and Crop Variation and Cd Bioavailability at the PROLAND OECD Conference Fate and Impact of Persistent Pollutants in Agroecosystems. Pulawy, Poland, Mar. 8-13, 2005.