February 2006

Issue: Cumulative Impacts to Amphibians Species.

Atrazine: Atrazine is now known to cause hermaphroditism among frogs both in laboratory settings and in the wild, at exposure rates as low as 0.1 ppb (parts per billion dilution rate). (Hayes 2002, 2003, 2005). Male frogs develop multiple ovaries and lose sexual characteristics needed to find mates and reproduce. This level of exposure is easily encountered by wildlife for weeks or months after spraying (ibid). The types of internal changes seen by Dr. Hayes requires endocrinology expertise for the species at issue, and has been missed during the types of laboratory studies currently required by the EPA for chemical registration. The issue of endocrine disrupting chemical impacts was addressed by Congress in 1996 with the passage of the Food Quality Protection Act, but to date, the EPA has yet to require standardized testing for the endocrine disrupting potential of any of the chemicals it has registered. Thus the product labels that SPI points to as being protective for wildlife if followed properly are not adequate to be protective. Atrazine exposure in the range of 0.1 ppb or even at 0.1 ppm could easily be encountered by frogs or other amphibian species potentially for months after spraying due to run off and or drift. Even small amounts of atrazine found in ephemeral pools and drainages where amphibians congregate is sufficient to cause mutations. Exposure to atrazine can seriously impair the ability of amphibians to reproduce, and may be one of the causal factors in amphibian declines. (Ibid).

SPI mischaracterizes its use of atrazine. SPI claims that it uses atrazine “normally” only once in the 80-year average rotation age of trees on SPI lands. County Agricultural Commissioner records of herbicide use by SPI in Tuolumne County showed applications occurring up to five times in one month in one watershed, amounting to hundreds of pounds of atrazine (2004 data).

SPI also fails to disclose herbicide impacts of the full formulation of products as they are actually applied in the field-as mixtures. A team of scientists from University of California, Berkeley, found highly significant increases in adverse effects to tadpoles exposed to mixtures of pesticides at low concentrations. The frogs developed an array of health problems including meningitis, because the chemicals suppressed their immune systems. They also took longer to complete the transformation from tadpole to frog, which reduces their chances of survival (Hayes et al. 2006).

In the paper, published in January, 2006 in the journal Environmental Health Perspectives, the authors conclude: "the current study revealed that estimating ecological risk and the impact of pesticides on amphibians using studies that examine single pesticides at high concentrations, only, may lead to gross underestimations of the role of pesticides in amphibian decline.”(emphasis added).

SPI is misleading and provides false information in regards to its use of herbicides.

Hexazinone: According to Dr. Hayes (pers. comm.), hexazinone may also cause similar mutations in frog organs as atrazine. Dr. Hayes is currently conducting laboratory studies on the effects of hexazinone on frogs. Hexazinone is chemically related to atrazine; they are both triazine chemicals and their herbicidal action is through the same mechanism. Like atrazine, EPA did not require testing of hexazinone for its endocrine disrupting effects on amphibians or other organisms during registration, and it has not been tested as as a full mixture the way that it is applied in the field.

Triclopyr: SPI does not acknowledge the known toxicity of triclopyr to frogs and to fish. Berrill et al. (1993) tested the formulations of triclopyr to determine their impacts on frogs and concluded, “Ranid tadpoles are likely to be paralyzed or killed by residues of the ester formulation [BEE or Garlon 4] of triclopyr that could occur in small ponds as a result of forest management spraying programs. Paralysis is likely to render tadpoles more vulnerable to predation, and when it is associated with slower growth it could also reduce later reproductive fitness.” Triclopyr products are among those under a court ordered restriction for use in listed salmonid habitat; however, that restrictive information is not found on the labels. Currently, triclopyr cannot be applied within 20 yards of salmonid bearing streams. Similar impacts can be expected to non-listed species. SPI's statement that following the labels is sufficient to protect wildlife is not factual.

Glyphosate: SPI uses Roundup more than any other glyphosate formulation on its ownership (Source: DPR 2004 Pesticide Use Report). Roundup is a glyphosate herbicide formulation that contains the surfactant POEA and is now known to be highly toxic to amphibians. Relyea (2005a, 2005b, 2005c) found high mortality among three different species of tadpole and juvenile (terrestrial) amphibians at a single exposure to Roundup that was designed to simulate an exposure encountered in the wild. Across all species, 96-100 percent of the tadpoles and 68-86 percent of the juveniles were killed from one exposure to Roundup.

Roundup was also recently found to interfere with a vital step in cell mitosis, transcription, at regular label-approved exposure rates after one spray application, in experimental sea urchin embryos. (Marc et al. 2002; 2005). The mechanism involved in cell transcription is universally conserved among all organisms, therefore virtually any other organism could have the same response (ibid).

Richard et al. (2005) found additional evidence that Roundup is a hormone mimic chemical, in experiments with human placental cells. The cells developed mutations when exposed to Roundup at environmentally relevant low concentrations, but these changes were not observed with exposure to glyphosate by itself. Physician Sawada (1988) hypothesized that the acute toxicity and even lethality in humans seen from Roundup poisoning was due to the surfactant POEA. Glyphosate by itself is not highly toxic but Roundup is a mixture-it is not simply glyphosate.

It cannot be said that the chemical herbicides proposed for use by SPI will not have significant adverse impacts on public trust resources, especially for rare species such as amphibians and salmonids.
A summary of responses to the paper (Richard et al. 2005) relative to chemical mixtures and specific to glyphosate follows. (Source: Environmental Health Perspectives, October 2005):
Environmental Health Perspectives, Vol. 113, No. 10 October 2005: Toxicity Tests: "Inert" and Active Ingredients
The findings of Richard et al. (2005) are an important addition to our understanding that the health and environmental effects of formulated pesticide products are not fully reflected in tests conducted on the active ingredient(s) alone. It has been long known that the adjuvants (commonly and misleadingly called "inert" ingredients) may be toxic and may enhance or supplement the toxic effects of the active pesticidal ingredient.
In the case of glyphosate-containing products, this phenomenon was well demonstrated in the data submitted to the (EPA) by the registrant (Monsanto), and summarized by the U.S. EPA in the Reregistration Eligibility Document (RED) for glyphosate (U.S. EPA 1993). For example, based on the registrant's own tests of acute toxicity to freshwater fish, the U.S. EPA classified technical grade glyphosate as "slightly toxic" to "practically non-toxic" and formulated products ranged from "moderately toxic" to "practically non-toxic." Tested alone, the surfactant adjuvant (identified as "inert") was "highly toxic" to "slightly toxic." Similar differences were reported in tests of acute toxicity to freshwater invertebrates.
Based in part on the data in the glyphosate RED (U.S. EPA 1993), the New York State Attorney General's office successfully pursued an action against Monsanto in 1996 (Attorney General of the State of New York 1996). At that time, Monsanto was making advertising claims about the toxicity of the Roundup products based on data from tests on the active ingredient alone. Such claims are scientifically unfounded and inherently deceptive. The Attorney General's action was facilitated by the availability of at least some limited information about the inert ingredients and their toxicity. That same sort of information enabled Richard et al. (2005) to conduct their study.
Unfortunately, that is not always the case, and for many pesticide products, little or no information about the identity of inert ingredients is publicly available. Registrants are generally required to conduct acute toxicity tests on formulated products, but they traditionally conduct chronic toxicity tests on the active ingredient alone. Even when formulated products are tested, the identity of inert ingredients is rarely revealed in the open literature, publicly available regulatory documents, or product labels. Therefore, independent research is stymied, and the public is ill-informed in the marketplace.
The author is the chief scientist in the New York State Attorney General's Environmental Protection Bureau and was actively involved in the 1996 action against Monsanto.
Michael H. Surgan
Environmental Protection Bureau
New York State Attorney General's Office
New York, New York
E-mail: michael.surgan@oag.state.ny.us
References
Attorney General of the State of New York. 1996. In the Matter of Monsanto Company, Respondent. Assurance of Discontinuance Pursuant to Executive Law § 63(15). New York:Attorney General of the State of New York, Consumer Frauds and Protection Bureau, Environmental Protection Bureau.

Richard S, Moslemi S, Sipahutar H, Benachour N, Seralini G-E, 2005. Differential effects of glyphosate and Roundup on human placental cells. Environ Health Perspect 113:716-720.

U.S. EPA. 1993. Reregistration Eligibility Decision (RED). Glyphosate. EPA-738-R-93-014. Washington, DC: U.S. Environmental Protection Agency. Available: http://cfpub.epa.gov/oppref/rereg/status.cfm?show=rereg [accessed 1 September 2005].
"Inert" and Active Ingredients: Séralini Responds:
Surgan raises interesting points in his analysis. This interest has been confirmed by reactions of agriculture authorities all over the world after publication of the article by Richard et al. (2005).
Indeed, scientific problems do exist in the registration of pesticides today, when chronic toxicity tests are conducted with the active ingredient alone--which is generally the case. First of all, chemists from companies may work hard for several years to find the right formulation that best amplifies the effects of the active ingredient. his formulation will allow penetration and stability and/or bioaccumulation of the active ingredient within plant, fungi, or insect cells, for instance, to reach the best toxicity. If there are any side effects in other animal or human cells, these will be also amplified by adjuvants, and thus not measured in chronic toxicity tests with the active ingredient alone. The active compound absorption by skin is generally calculated in the presence of formulated adjuvants, but this is clearly a short-term study and not sufficient to detect, for example, endocrine disruption or carcinogenesis, possibly promoted in vivo by the described synergy. This should even necessitate further care in case of the use of formulated products such as glyphosate-based herbicides on tolerant, edible plants.
As a matter of fact, most genetically modified crops have been modified and selected only to tolerate high-formulated herbicide absorption, but the plants are not submitted for registration requiring chronic toxicity studies involving long-term feeding of animals. Moreover, in the case of environmental pollution, active pesticide ingredients may encounter detergents or other lipohilic xenobiotics with comparable effects other than those of their own adjuvants, for instance, forming microvesicles to penetrate the cells. These combined effects should also be taken into account in authorized thresholds of pollution in order to avoid effects on wildlife or humans.
The author declares he has no competing financial interests.
Gilles-Eric Séralini
Laboratoire de Biochimie et Biologie Moleculaire
Université de Caen
Caen, France
E-mail: criigen@ibfa.unicaen.fr
Reference
Richard S, Moslemi S, Sipahutar H, Benachour N, Seralini G-E, 2005. Differential effects of glyphosate and Roundup on human placental cells. Environ Health Perspect 113:716-720.



References
Berrill, M. et al. 1993. Effects of low concentrations of forest-use pesticides on frog embryos and tadpoles. Env. Tox. Chem. 13(4):657-664.

Hayes, T.B., A. Collins, M. Lee, M. Mendoza, N. Noriega, A.A. Stuart, and A. Vonk. 2002. Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proc. National Academy of Sciences 99(8):5476-5480.

Hayes, T.B., K. Haston, M. Tsui, A. Hoang, c. Haeffele, and A. Vonk. 2003. Atrazine-induced hermaphroditism at 0.1 ppb in American Leopard frogs (Rana pipens): laboratory and field evidence. Env. Health Persp. 111(4):568-575.

Hayes, T.B. 2005. There is no denying this: defusing the confusion about atrazine. BioScience 54(12):1139-1149.

Hayes, T.B. P. Case, S. Chui, D. Chung, C. Haefele, K. Haston, M. Lee, V. Pheng Mai, Y. Marjuoa, J. Parker, and M. Tsui. 2006 (in press). Pesticide mixtures, endocrine disruption, and amphibian declines: Are we underestimating the impact? Env. Health Persp. Online 24 January 2006.

Lajmanovich, R.C., M.T. Sandoval, P.M. Peltzer. 2003. Induction of mortality and malformation in Scinax nasicus tadpoles exposed to glyphosate formulations. Bull. Environ. Contam. Toxicol. 70:612-618.

Marc, J., O. Mulner-Lorillon, S. Boulben, D. Hureau, G. Durand, and R. Belle. 2002. Pesticide Roundup provokes cell division dysfunction at the level of CDK1/Cyclin B activation. Chem. Research Toxicol. 15, 326-331.

Marc, J., M. Le Breton, P. Cormier, J. Morales, R. Belle´, O. Mulner-Lorillon.
2005. A glyphosate-based herbicide pesticide impinges on transcription. Toxicology and Applied Pharmacology 203, 1-8.

Relyea, R.A. 2005a. The impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities. Ecol. App. 15(2):618-627.

Relyea, R.A. 2005b. The lethal impacts of Roundup and predatory stress on six species of North American tadpoles. Arch. Environ. Contam. Toxicol. 48, 351-357.

Relyea, R.A. 2005c. The lethal impact of Roundup on aquatic and terrestrial amphibians. Ecol. App. 15(4):1118-1124.

Relyea, R.A., N.M. Schoeppner, and J.T. Hoverman. 2005. Pesticides and amphibians: the importance of community context. Ecol. App. 15(4):1125-1134.

Richard, S., S. Moslemi, H. Sipahutar, N. Benachour, and G. Seralini. 2005. Differential effects of glyphosate and Roundup on human placental cells. Env. Health Persp. 113:716-720.

Saenz, M.E., W.D. DiMarzio, J.L. Alberdi, M. del Carmen Tortorelli. 1997. Effects of technical grade and a commercial formulation of glyphosate on algal population growth. Bull. Environ. Contam. Toxicol. 59:638-644.

Sawada, Y. et al. 1988. Probable toxicity of surface-active agent in commercial herbicide containing glyphosate. Lancet Feb. 6, 1988:299.

Storrs, S.I. and J.M. Kiesecker. 2004. Survivorship patterns of larval amphibians exposed to low concentrations of atrazine. Environ Health Perspect. 112:1054-1057.

Tsui, Martin T.K. and L.M. Chu. 2003. Aquatic toxicity of glyphosate-based formulations: comparison between different organisms and the effects of environmental factors. Chemosphere 52, 1189-1197.