Poly-D- (Chitosan) Crops 1 2 Identification of Petitioned Substance

3 Chemical Names: CAS Numbers: 4 poly-D-glucosamine 9012-76-4 5 6 Other Name: 7 Chitosan Other Codes: 8 128930 (EPA/OPP Chemical Code) 9 Trade Names: 10 Chito-stik 11 12 Characterization of Petitioned Substance 13 14 Composition of the Substance: 15 Chitosan (poly-D-glucosamine) is a polymer1 of glucosamine , specifically glucosamine and N-acetyl- 16 glucosamine (Hadwiger 2004). Its structure and composition are similar to both (i.e., the primary 17 structural component of plant fiber) and . Like chitin, chitosan is found naturally in the shells of all 18 and , as well as certain other organisms such as many fungi, algae, and yeast. Chitosan is one 19 of the most common polymers found in nature (EPA 2003). 20 21 Properties of the Substance: 22 Chitosan is a chemically stable, white to pale yellow powder or flake (Polysciences 2003). Chitosan has a 23 strong positive charge, which is the basis of its use as a “sticking” agent (i.e., an adhesive adjuvant). The 24 positively charged molecules adhere to negatively charged pesticides and plant surfaces. 25 26 Chitosan is not soluble in water. It can be made soluble in water, however, by treating it with an acid to 27 form soluble chitosan ions (Rabea et al. 2003). See Evaluation Question #1 form more information the 28 production of chitosan. 29 30 Specific Uses of the Substance: 31 The petitioned use of the substance is as an adhesive adjuvant for use in organic crop production 32 (Hadwiger 2004). As an adhesive adjuvant, the substance would be used to make a pesticide or fungicide 33 stick to plant surfaces. Specifically, the petitioner seeks approval to test chitosan as a sticking agent for the 34 fungicide copper sulfate pentahydrate for the control of potato late blight. 35 36 Approved Legal Uses of the Substance: 37 Chitosan is a registered pesticide (OPP No. 128930) that is used in crop production as a plant growth 38 enhancer and plant defense booster (EPA 2003). In these uses, chitosan is applied to treat field crops, 39 ornamentals, turf, home gardens, and nurseries. Target pests include early and late blight, downy and 40 powdery mildew, and gray mold. Proposed application rates for the petitioned use as a sticking agent are 41 much lower than the application rates for use as a pesticide/fungicide. Chitosan is exempt from the 42 requirement for a pesticide tolerance (EPA 1995). See Evaluation Question #6 for more information on 43 chitosan application rates and Evaluation Question #8 for more information on the modes of action for 44 approved legal uses as a biopesticide. 45 46 According to the petition, chitosan is listed as an animal feed component in the Official Publication of the 47 Association of American Feed Control Officials (Hadwiger 2004).

1 A polymer is a large molecule that is a chain of linked, identical or similar molecular units called monomers. ______February, 2004 Technical Evaluation Report Page 1 of 8 Compiled by (ICF Consulting) for the USDA National Organic Program Technical Evaluation Report Poly-D-Glucosamine (Chitosan) Crops

48 Chitosan is used as a human dietary supplement for weight loss and reduction (Rabea et al. 49 2004). 50 51 Chitosan is also used as a flocculating (i.e., settling) agent in wastewater treatment systems, a hydrating 52 agent in cosmetics, a pharmaceutical agent in , and an antimicrobial food wrap (Rabea et al. 53 2003). The State of Oregon has approved the use of chitosan in unrestricted amounts as a soil amendment 54 (fertilizer). This use is not regulated by EPA under the Federal Insecticide, Fungicide, and Rodenticide Act 55 (EPA 1995). 56 57 Status 58 59 Action of the Substance: 60 61 Chitosan has a positive chemical charge, which causes it to attract negatively charged materials. This 62 property is the mode of action for the petitioned use as an adhesive adjuvant. Specifically, chitosan would 63 be used to adhere to negatively charged copper sulfate particles and plant surfaces. 64 65 International 66 67 Chitosan is not specifically listed for the petitioned use or other uses in the following international organic 68 standards: 69 70 • Canadian General Standards Board 71 • CODEX Alimentarius Commission 72 • European Economic Community (EEC) Council Regulation 2092/91 73 • International Federation of Organic Agriculture Movements 74 • Japan Agricultural Standard for Organic Production 75 76 Evaluation Questions for Substances to be used in Organic Crop or Livestock Production 77 78 Evaluation Question #1: Is the petitioned substance formulated or manufactured by a chemical process? 79 (From 7 U.S.C. § 6502 (21)) 80 81 According to the petition, the raw material for chitosan is shell waste byproduct (Hadwiger 2004). 82 Other potential raw material sources for commercially produced chitosan include shells (e.g., FDA 83 2002), shells (EPA 1995), and cultured fungi (Rabea et al. 2003). 84 85 The process used to formulate chitosan is shown in Figure 1. The process begins with chitin obtained from 86 seafood byproducts. Non-chitinous components of the seafood byproduct are stripped with a hydrochloric 87 acid (not shown in Figure 1). Next, (NaOH), which is a base, and heat are used to 88 remove residual meat attached to the shell material. Next, a stronger sodium hydroxide solution is used 89 (Step 1 in Figure 1), in a step called deacetalation, to convert some N-acetyl glucosamine (the primary 90 component of chitin) to glucosamine (the primary component of chitosan) (Rabea et al. 2003). 91 92 Following deacetalation, the chitosan is rinsed with water to remove remaining sodium hydroxide and 93 impurities (Step 2 in Figure 1). A mild organic acid, such as lactic or acetic acid, is then applied (Step 3 in 94 Figure 1) to adjust the pH of the chitosan below neutral (i.e. from basic to acidic). This step is required to 95 make the chitosan soluble in water (Rabea et al. 2003). In the last manufacturing step, the chitosan is dried.

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96

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98 Evaluation Question #2: Is the petitioned substance formulated or manufactured by a process that 99 chemically changes the substance extracted from naturally occurring plant, animal, or mineral sources? 100 (From 7 U.S.C. § 6502 (21).) 101 102 In the chitosan manufacturing process, the primary component of chitin (i.e., N-acetyl glucosamine) is 103 chemically changed to the primary component of chitosan (glucosamine). Both the starting and ending 104 chemicals in this process are natural components of both chitin and chitosan and are present in the natural 105 animal byproduct source (e.g., crab shells). The proportion of the two chemicals determines whether a 106 mixture is chitin or chitosan. According to the petitioner, chitosan is approximately 80 percent 107 glucosamine and 20 percent N-acetyl glucosamine (Hadwiger 2004). There are no precise definitions, 108 however, of chitin and chitosan based on the percentage composition of glucosamine and N-acetyl 109 glucosamine (Rabea et al. 2003). In both chitin and chitosan, these two chemicals are linked together in 110 chains, called polymers, of as many as 5,000 glucosamine and N-acetyl glucosamine molecules (i.e., 111 monomers). 112 113 Although N-acetyl glucosamine is converted to glucosamine in nature, the conversion does not occur by 114 the controlled process used for commercial production (Figure 1). 115 116 Evaluation Question #3: Is the petitioned substance created by naturally occurring biological 117 processes? (From 7 U.S.C. § 6502 (21).) 118 119 In nature, N-acetyl glucosamine may be deacetylated to glucosamine. The natural deacetalation process, 120 however, does not occur as a result of the specific process (i.e., application of NaOH) used for commercial 121 manufacturing. 122 123 Evaluation Question #4: Is there environmental contamination during the petitioned substance’s 124 manufacture, use, misuse, or disposal? (From 7 U.S.C. § 6518 (m) (3).) 125 126 There is no information available from EPA or FDA to suggest that environmental contamination results 127 from the manufacture, use, misuse, or disposal. Chitosan is a registered pesticide, which implies a 128 potential for misuse or improper disposal. It is a naturally occurring and biodegradable chemical, 129 however, and EPA exempted it from the requirement for a tolerance limit when used as a pesticide in the 130 production of any raw agricultural commodity (EPA 1995). In exempting chitosan from the requirement 131 for a tolerance limit, EPA cited the following considerations: 132 133 “Chitosan (1) is not toxic, as demonstrated in acute toxicity studies in mice, rats, and 134 rabbits; (2) is naturally occurring in the environment in large concentrations; (3) has been 135 exempted from the requirement of a tolerance in or on barley, beans, oats, peas, and 136 wheat (40 CFR 180.1072) when used as a seed treatment at an application rate of 4 137 oz./100 lbs. seed; (4) has been approved by the State of Oregon for use in unrestricted 138 amounts as a soil amendment (fertilizer), a use not regulated by EPA under the Federal 139 Insecticide, Fungicide, and Rodenticide Act.” (EPA 1995) 140 141 In addition, according to EPA’s pesticide fact sheet for chitosan, it is not expected to harm people, pets, 142 wildlife, or the environment when used according to label directions due to its low potential for toxicity 143 and abundance in the natural environment (EPA 2003). 144 145 The petitioner notes that manufacturing chitosan from crab shell waste reduces the potential for 146 environmental contamination associated with disposal of the wastes (Hadwiger 2004). 147 148 Evaluation Question #5: Is the petitioned substance harmful to the environment? (From 7 U.S.C. § 6517 149 (c) (1) (A) (i) and 7 U.S.C. § 6517 (c) (2) (A) (i).) 150 151 Chitosan is a naturally occurring chemical and is one of the most common polymers found in nature (EPA 152 2003). EPA exempted chitosan from the requirement for a tolerance limit due to its low potential for 153 toxicity and abundance in the environment. EPA concluded that chitosan is not expected to harm people, February, 2004 Page 4 of 8 Technical Evaluation Report Poly-D-Glucosamine (Chitosan) Crops

154 pets, wildlife, or the natural environment, in part because chitosan was found to be nontoxic in acute 155 toxicity studies in mice, rats, and rabbits (EPA 1995). 156 157 Evaluation Question #6: Is there potential for the petitioned substance to cause detrimental chemical 158 interaction with other substances used in organic crop or livestock production? (From 7 U.S.C. § 6518 159 (m) (1).) 160 161 The adhesive property of chitosan that is the basis of its petitioned use could cause negatively charged 162 particles other than the co-applied organic pesticide to stick to plant surfaces. Potential examples of other 163 particles that may be attracted to the chitosan adhesive adjuvant include other agricultural products or fine 164 soil particles. No information sources reviewed for this report described or evaluated potential adverse 165 impacts of this nature. 166 167 Biochemically, however, chitosan is unlikely to cause detrimental chemical interaction with other 168 substances used in organic crop or livestock production. As a component of the shells of insects and 169 crustaceans, as well as certain other organisms such as many fungi, algae, and yeast (EPA 2003), chitosan is 170 naturally present in agroecosystems. In addition, plants and microbes (e.g., in soil) have enzymes called 171 chitinases and chitosanases that can break chitosan down to utilizable (Hadwiger 2004; 172 Brzezinski and Neugebauer 2004). 173 174 Evaluation Question #7: Are there adverse biological or chemical interactions in the 175 agro-ecosystem by using the petitioned substance? (From 7 U.S.C. § 6518 (m) (5).) 176 177 For the petitioned use, chitosan is unlikely to cause adverse biological or chemical interactions in the 178 agroecosystem. Chitosan is found naturally in agroecosystems, and it may be broken down and utilized by 179 plants and microbes (Hadwiger 2004; Brzezinski and Neugebauer 2004). 180 181 Although chitosan attracts positively charged particles, it is not highly reactive and it is not known to be 182 toxic (e.g., EPA 1995). EPA has approved the use of chitosan as a pesticide and plant growth promoter at 183 much higher application rates than proposed for its use as an adhesive adjuvant (see Evaluation Question 184 #6). 185 186 Evaluation Question #8: Are there detrimental physiological effects on soil organisms, crops, or 187 livestock by using the petitioned substance? (From 7 U.S.C. § 6518 (m) (5).) 188 189 Chitosan has documented physiological effects on plants and soil organisms, including plant growth 190 enhancement, and antimicrobial ability. These effects, which are regarded as beneficial to crop production, 191 are not involved in the petitioned use of chitosan as an adhesive adjuvant. The rate at which chitosan 192 would be applied for the petitioned use (i.e., 5 to 10 grams per acre) is well below the recommended 193 application rates for these other uses (e.g., 180 to 1,080 grams per acre [EPA 2001]). The known 194 physiological effects of chitosan are described further below. 195 196 Chitosan has been shown to have antimicrobial, antifungal, and antiviral effects, and it is also known to be 197 a plant growth enhancer (Rabea et al. 2003). The antimicrobial and antifungal effects are influenced by the 198 length and composition of the chitosan polymers, environmental conditions, and other factors (Rabea et al. 199 2003). For example, very short chitosan polymers have the strongest antimicrobial and antifungal effects. 200 Although Rabea et al. (2003) summarized several hypotheses about chitosan’s mode of antimicrobial 201 action, the exact mode of action is still unknown. 202 203 As a plant growth enhancer, the mode of action is believed to be that chitosan is taken up by plant cells 204 where it enters the cell nucleus and stimulates messenger RNA and enzyme production. This action 205 stimulates the plant to produce more in the stems, resulting in stronger stems (EPA 1995, Rabea et al. 206 2003). 207

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208 At the proposed application rates, chitosan is unlikely to create unacceptable changes in soil temperature, 209 water availability, pH levels, nutrient availability, or salt concentration. 210 211 Evaluation Question #9: Is there a toxic or other adverse action of the petitioned substance or its 212 breakdown products? (From 7 U.S.C. § 6518 (m) (2).) 213 214 Breakdown products of chitosan include smaller chitin and chitosan polymers (i.e., shorter chains of 215 glucosamine and N-acetyl-glucosamine monomers), unlinked monomers, and other -related 216 molecules. These breakdown products are all nontoxic. Glucose is a that can be utilized by many 217 organisms. 218 219 Evaluation Question #10: Is there undesirable persistence or concentration of the petitioned substance 220 or its breakdown products in the environment? (From 7 U.S.C. § 6518 (m) (2).) 221 222 Chitosan and its breakdown products are not persistent in the environment, and significant environmental 223 accumulation of chitosan and its breakdown products would not result from repeated use of chitosan at the 224 proposed application rate. Chitosan is biodegradable. For example, plants and microbes (e.g., in soil) have 225 enzymes called chitinases and chitosanases that can break chitosan down to utilizable carbohydrates 226 (Hardwiger 2004). 227 228 Evaluation Question #11: Is there any harmful effect on human health by using the petitioned 229 substance? (From 7 U.S.C. § 6517 (c) (1) (A) (i), 7 U.S.C. § 6517 (c) (2) (A) (i)) and 7 U.S.C. § 6518 (m) (4).) 230 231 Chitosan is not known to be toxic to humans. Chitosan is marketed as a human dietary supplement for 232 control of obesity and high cholesterol. The scientific evidence of these benefits is questionable, however, 233 and FDA sent a warning letter concerning unsubstantiated claims to the maker of one chitosan supplement 234 (FDA 2004). 235 236 In 2001, Primex Ingredients, ASA, submitted a Generally Regarded as Safe (GRAS) notification to FDA for 237 chitosan produced from shrimp. Primex subsequently withdrew the GRAS notification (FDA 2002), and 238 chitosan is not currently GRAS. 239 240 Evaluation Question #12: Is there a wholly natural product that could be substituted for the petitioned 241 substance? (From 7 U.S.C. § 6517 (c) (1) (A) (ii).) 242 243 The availability of alternative products was investigated by consulting organic industry resources, 244 researching sources cited by the poly-D-glucosamine petition, and conducting Internet searches. This 245 investigation identified one adhesive adjuvant product formulated with the functional agents , 246 , and casein. These ingredients are recognized as natural by organic industry sources (e.g., OMRI, 247 2005). The investigation also identified two similar adjuvant products formulated with pine-based 248 functional agents (i.e., di-1-P-menthene, poly-1-P-menthene). However, it is unknown whether these 249 products are synthetic or not. 250 251 ATTRA (Kuepper and Sullivan 2004) published a guide to organic alternatives for late blight control in 252 potatoes. Although this source does not discuss adhesive adjuvants, it does describe alternative late blight 253 control practices (e.g., application of compost tea) that do not involve fungicides and thus would not 254 require an adhesive adjuvant. 255 256 Evaluation Question #13: Are there other already allowed substances that could be substituted for the 257 petitioned substance? (From 7 U.S.C. § 6518 (m) (6).) 258 259 Based on a review of organic industry resources, there are at least three products marketed as organic 260 adhesive adjuvants. As described in Evaluation Question #12, one of the products contains the functional 261 agents bentonite, lactose, and casein. The two other products are pine-based. However, it is unknown 262 whether the pine-based functional agents of these closely-related products are synthetic. No information

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263 about the manufacturing process for these products was found and National List petitions have not been 264 submitted for any uses of the functional agents. 265 266 Evaluation Question #14: Are there alternative practices that would make the use of the petitioned 267 substance unnecessary? (From 7 U.S.C. § 6518 (m) (6).) 268 269 The petition proposes the use of chitosan as an adhesive adjuvant for use with an approved organic 270 fungicide, such as copper sulfate (7 CFR 205.601(i)(2)). The petitioned use would enhance the ability of the 271 fungicide to stick to plant surfaces, thereby improving effectiveness and reducing fungicide application 272 rates. Potential alterative practices include application of the organic fungicide without the adhesive 273 adjuvant or use of an alternative organic adhesive adjuvant (see Evaluation Questions #12 and #13). 274 275 Although the petitioner states that there is no effective control currently available for potato late blight, a 276 publication by ATTRA (Kuepper and Sullivan 2004) provides guidance on organic control of late blight in 277 potatoes. The ATTRA guidelines include cultural controls, such as: 278 279 • Field scouting and inspection of tubers going into storage to catch outbreaks in their earliest 280 stages; 281 • Avoiding piling and leaving culls; 282 • Using certified seed and mixing seed lots; 283 • Using an AireCup® planter; 284 • Carefully monitoring seed planting depth and hilling operations; 285 • Using organic contact herbicides to kill infected plants; 286 • Managing irrigation to regulate leaf wetness; 287 • Destroying green vines; 288 • Harvesting tubers two weeks after destroying green vines; and 289 • Minimizing damage to tubers and keeping regulated air flow through storage piles. 290 291 In addition, ATTRA identified foliar feeding (e.g., with products made from kelp or horsetail) and 292 application of compost tea as a method of enhancing disease resistance (Kuepper and Sullivan 2004). 293 294 References 295 296 Brzezinski and Neugebauer 2004. “The Chitosanase Web Page.” Prepared by Ryszard Brzezinski and 297 Andrzej Neugebauer. Contact: Ryszard Brzezinski, Département de Biologie, Faculté des Sciences, 298 Université de Sherbrooke, Sherbrooke, PQ, Canada. Updated October 25, 2004. 299 http://www.callisto.si.usherb.ca/~rbrzezin/ 300 301 EPA 2003. “Chitosan; Poly-D-Glucosamine (128930) Fact Sheet,” Biopesticide Active Ingredient Fact 302 Sheets, U.S. Environmental Protection Agency, 303 www.epa.gov/pesticides/biopesticides/ingredients/factsheets/factsheet_128930.htm Issued June 2003. 304 305 EPA 2001. EPA Pesticide Registration Number 70464-3 (ELEXA 4), Issued February 15, 2001. 306 307 EPA 1995. “Poly-D-Glucosamine (Chitosan); Exemption from the Requirement of a Tolerance.” U.S. 308 Environmental Protection Agency, Final Rule. Federal Register, Volume 60, Number 75, Pages 19523-19524, 309 April 19, 1995. 310 311 FDA 2004. Warning Letter for Weight Loss Products “Super Chitosan.” Letter to Genesis Nutrition from 312 Joseph Baca, Director, Office of Compliance, Center for Food Safety and Applied Nutrition, U.S. Food and 313 Drug Administration. March 26, 2004. http://www.cfsan.fda.gov/~dms/wl-ltr4.html 314

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315 FDA 2002. Agency Response Letter, GRAS Notice No. GRN 000073. Letter to Lee B. Dexter, Lee B. Dexter 316 and Associates, from Linda Kahl, Division of Biotech and GRAS Notice Review, Office of Food Additive 317 Safety, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration. February 2, 318 2002. http://www.cfsan.fda.gov/~rdb/opa-g073.html 319 320 Hadwiger 2004. Petition to add chitosan (poly-D-glucosamine) to the National List of materials approved 321 for use by organic growers. Submitted by Dr. Lee A. Hadwiger, Department of Plant Pathology, 322 Washington State University, Pullman, WA, Received January 26, 2004. 323 324 Kuepper, G. and P. Sullivan. 2004 “Organic Alternatives for Late Blight Control in Potatoes.” Appropriate 325 Technology Transfer to Rural Areas (ATTRA), April 2004. www.attra.ncat.org/attra- 326 pub/PDF/lateblight.pdf 327 328 OMRI 2005. OMRI Brand Name Product List. Organic Materials Review Institute. Eugene, Oregon. 329 January 2005. 330 331 Polysciences 2003. Material Safety Data Sheet for Chitosan Powder Purified. Prepared by Polysciences, 332 Inc., Issue date 10/21/2003. Obtained from www.MSDSOnline.com 333 334 Rabea, E., M. Badawy, C. Stevens, G. Smagghe, W. Steubaut. “Chitosan as Antimichrobial Agent: 335 Applications and Mode of Action.” Biomacromolecules. 2003 Nov-Dec;4(6):1457-65.

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