The Fate and Effect of Glyphosate

on Amphibians

Evan Hallas

Matt Berg

Kate Johannesen

Final Report

ENSC 202

May 11, 2007

Executive Summary:

Glyphosate, the world’s most widely used herbicide, has been considered environmentally inert until recently. Amphibians, long recognized as important indicator species, show susceptibility to this herbicide. The largest manufacturer is the Monsanto Company, which invented it, and it is often found under the brand name Roundup (as well as others). It is a non-selective, post-emergent, systemic herbicide.

Because animals lack the catabolic pathway that glyphosate inhibits, as well as its strong adsorption to soil, and non-volatile nature, the pesticide registration and subsequent reregistration required by the Environmental Protection Agency (EPA) pursuant to the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) have found glyphosate to be “practically non-toxic”.

However, it has been fourteen years since registration, suggesting that more modern test methods and updated equipment should be used to double check the chemical due to its widespread application. Also, patent law protects the manufacturer from listing the inert ingredients (meaning inactive, not ecologically harmless) on the pesticide label. This includes surfactants, the amphibian toxicity of which recent studies have called into question.

Recent research on amphibian toxicity of glyphosate was compiled and compared with background information compiled by the EPA during registration. It was concluded that the chemical glyphosate itself is about as environmentally inert as Monsanto claims. However, at very high doses tadpoles respond negatively to glyphosate. More importantly, the un-labeled surfactants have been found to be toxic. It is the recommendation of this team that patent law be revised to insist on the listing of all pesticide ingredients such that they will fall under the jurisdiction of FIFRA and the EPA.

Problem Statement

Despite research on the effects of glyphosate on the environment, there is still much argument over what the true effects of glyphosate are.

Background

Glyphosate (C3H8NO5P) is the world’s top selling herbicide (USGS, 2007). This post-emergent systemic herbicide was developed by Dr. John Franz under the Monsanto Company in 1970 and was ready for retail under the brand name Roundup in 1974 (Monsanto, 2007). In 1990 Dr. Franz was awarded the Perkin Medal, the highest honor in industrial chemistry, for glyphosate’s massive contribution to modern agriculture(Society of Chemical Industry, 2007). Its usage has tripled since 1997 (USGS, 2007). Until recently Monsanto held the patent on glyphosate, and is still its largest producer; largely in union with their line of Roundup Ready crops, which are resistant to the herbicide, allowing for its widespread application without damage to crop yield (Monsanto, 2007). There are three salt formulations of glyphosate, the most prevalent of which is Monsanto’s isopropylamine salt (EPA, 1993).

In its pure form, it is a white, crystalline organophosphate salt, but for retail it is dissolved in water and a surfactant solution (USGS, 2007). Surfactant simply means “surface active agent” and these chemicals allow for the transport of glyphosate through the leaves of the plant (thus post-emergent as the plant has emerged) throughout the body of the plant down to the roots (thus the term systemic) (Alibhai et Al., 2001). An organophosphate is a chemical containing phosphorus and carbon. Glyphosate imitates glycine, the simplest amino acid, and acts as a plant growth inhibitor (Alibhai et Al., 2001).

As mentioned before glyphosate is a non-selective, post-emergent, systemic herbicide. It is non-selective in that it kills all plants by inhibiting an important plant catabolism pathway. Post-emergent indicates that the plants have already germinated at the time of application. It is systemic because chemicals in solution with glyphosate (known as surfactants) help the herbicide absorb into the leaves and travel throughout the plant body.

The mode of action for glyphosate is now nearly completely understood. Succinctly, it inhibits the shikimate enzyme pathway. This series of steps allows plants to synthesize aromatic amino acids, which are basic building blocks for peptide bonds and other necessary metabolites. Fortunately for humans, the shikimate pathway doesn’t exist in animals, as they obtain aromatic amino acids via a heterotrophic diet (Alibhai et Al., 2001).

There is a world of controversy surrounding the wide-spread use of glyphosate. The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) requires that any new pesticide be licensed for use by the EPA after registration. Registration calls for over one hundred different laboratory tests to ensure that the pesticide’s degradents do not linger in or harm the environment and maximum pesticide residue levels are established for pesticides to be applied to food crops (EPA, 2007). The 1988 amendments to FIFRA required the re-registration of pesticides registered before November 1, 1984 to ensure that they complied with more modern safety standards (EPA, 2006). This doesnot appear to be a set system for how often pesticides should be reregistered. Since pesticides meant for use on food crops were slated for the earliest phase of reregistration, the process has been complete for glyphosate since September 1993 (EPA, 1993).

The reregistration eligibility decision (RED) fact sheet for glyphosate (EPA, 1993) states that glyphosate bonds to soil so effectively as to become practically inert and is not expected to move farther down into the soil than six inches. It has been placed in Toxicity Category III (I being most toxic), meaning low oral and dermal acute toxicity (though it is in Category I as an eye irritant). These studies were conducted with rats and rabbits, and the RED explicitly states that in reproductive studies “glyphosate does not cause mutations”. Although when used aquatically EPA states that it may kill non-target aquatic plants, for birds it is considered “no more than slightly toxic” and “practically non-toxic” to aquatic insects, fish, honeybees, and mammals (EPA, 1993).

Given that glyphosate is a food pesticide and therefore was a priority for reregistration, the RED is now fourteen years old and possibly out of date in the face of more current studies with better equipment. Also, reregistration was a one-shot-deal and will not occur again without another FIFRA amendment. The RED did also state that an inert ingredient in some formulations of glyphosate is toxic to fish. As it turns out, patent law protects the manufacturer from listing inert ingredients. Only the percent by weight of the active ingredient must be listed on the label (EPA, 2007), and the majority of any glyphosate formulation is inert ingredients. However, “inert” only refers to the ingredient’s non-participation in actually killing plants (glyphosate is the active ingredient that actually kills the plant). Inert does not mean harmless. There is growing concern that the surfactant system used may actually be far more toxic to non-intended organisms than glyphosate itself.

Another cause for concern over the RED is that it waived inhalation studies given the highly non-volatile nature of glyphosate and its affinity for soil bonding (EPA, 1993). However, when the crops are harvested soil dust is sent into the air, making it likely that inhalation of the soil particles with the bonded glyphosate might occur.

In recent years the world has seen amphibian populations steadily decreasing. A number of factors may be causing this trend, one of which is the presence of pollutants. As glyphosate is one of the most widely used herbicides in the world it is important to examine the effects it has on amphibian growth and mortality rates. Amphibians act as indicator species for ecosystems and therefore can help lend insight to the potential effects of specific pollutants (The World Conservation Union, 2007).

Purpose statement

This review intends to analyze previous research on glyphosate and come to a conclusion about its ecological impacts, specifically towards growth and survival of amphibians.

Objectives

  1. Research how exactly glyphosate travels through the environment and ultimately affects amphibians.
  2. Identifyscientifically valid, non-biased studies concerning response of amphibians, particularly during larval stages, to glyphosate and glyphosate containing products.
  3. Comparethe findings of these studies to EPA classifications of the substance, as well as claims made by manufacturers such as Monsanto.
  4. Make recommendations for any necessary changes in toxicity ratings or for further research.

Approach

When beginning the research portion of this project it was evident that there was a plethora of information on the subject, so the topic was pared down to amphibians. Studies were broken down into different subcategories such as location of the study, source of study (such as Monsanto or independent), and whether the study examined glyphosate or Roundup®.

One of the major components of the evaluation of studies examined was the validity of the data. There have been many different studies conducted including many different variables. Acute versus chronic exposure was a major influence in the findings of various studies. The majority of studies were conducted within laboratory settings, although some did take place in actual or simulated ecosystems. Of these studies, only those that used concentrations of glyphosate that would realistically be found within a natural body of water were included.

As more research was conducted, it became apparent that there is a large difference between the stress that glyphosate causes the organisms versus glyphosate products that also contain surfactants. Studies were examined that looked at glyphosate itself versus products containing glyphosate as well as surfactants. Studies that were able to specifically outline the way in which glyphosate and other substances within products affected amphibian development and mortality were examined for greater understanding of the subject.

Findings

It is unlikely for glyphosate to leach into groundwater of runoff due to the fact that it is strongly absorbed by soils. In field studies the immobility of glyphosate in soils has been demonstrated by the lack of detectable concentrations of glyphosate in runoff waters. However there are still a number of ways that the chemical can contaminate bodies of water. In aerial application there is a large risk of drift and overspray. In addition large areas are usually sprayed at once and there are often times small wetlands located with the spray boundaries (Giesy et. Al., 2000).

Exposure levels have been predicted following direct application to water at a number of rates. Some examples of surface water concentration levels in mg Active Ingredient (AI)/L are listed in Table 1. The actual concentrations observed are dependent on several factors including application rate, interception by target vegetation, water depth, amount of suspended solids, and type of water body (Giesy et. Al., 2000).

Table 1. (Giesy et. Al., 2000)

A number of studies have been completed focusing directly on specific concentrations of glyphosate herbicides and how it affects mortality rates of tadpoles. Once research into the subject was conducted, it became evident that the most accurate studies completedwere the ones that analyzed the toxicity of the surfactant POEA as well as glyphosate.

Dr. Rick Relyea of the University of Pittsburgh has completed a number of these experiments over the last few years. One of his experiments tested the effects of Roundup®on six species of larval amphibians from North Americarepresentinga diverse set of species that span a large geographic range. The six species of tadpoles were exposed to six different levels of glyphosate concentrations; 20, 10, 5, 1, 0.1 and 0 mg AI/L. Each day the number of surviving tadpoles was counted and any dead tadpoles were removed until 16 days had passed (Figure 1). From the results,LC5016-d were estimated for all the species ranging between 0.55 and 2.07 mg AI/L (Table 2 and Figure 2). Previous work has concluded that Roundup® is only slightly to moderately toxic to larval amphibians but this study shows that Roundup is moderately toxic (1 to 10mg AI/L) to highly toxic (0.1 to 1 mg AI/L) (Relyea, 2005a).

Table 2. (Relyea, 2005a)

Figure 1. (Relyea, 2005a)Figure 2. (Relyea 2005a)

In another experiment conducted byDr. Relyea, the maximum amount of glyphosate that is likely to occur in a natural wetland was applied to tanks with naturally coexisting tadpoles. This was done by simulating a direct overspray, (crop dusting.) The overspray of Roundup® created a glyphosate concentration of 3.8 mg AI/L (Relyea, 2005b) representing levels of a worst case scenario that could be found in a natural setting (Giesy et. Al., 2000). The results of this study showed that Roundup® caused a large reduction in the survival rates of all three tadpole species when compared with the control groups. Roundup® reduced tree frog survival from 75% to 2%, toad tadpole survival from 97% to 0%, and leopard frog tadpole survival from 98% to 4%(Figure 3). This study did not isolate glyphosate from the added surfactant, POEA, so no determination can be made as to which component of Roundup® caused the mortality (Relyea, 2005b).

Figure 3. (Relyea, 2005b)

A study done by K. Cauble and R. S. Wagner investigated low dose chronic exposure to Roundup® affected amphibian growth and metamorphosis. This study was conducted in a laboratory setting using 0 ppm, 1 ppm, and 2 ppm concentrations of Roundup®. Significant impacts on R. Casadae were found; including survivability, rate of metamorphosis, and post-metamorphosis size (Cauble and Wagner 2004).

Decreased size due to toxic exposure can have two major effects on amphibian species. The first concern is that it is believed that species must reach a certain size or they will not undergo metamorphosis. The second is that they are more vulnerable to predators. However predation is not much of a concern if the organism is not going to metamorphose.

Research has also been done with Vision®, a glyphosate based herbicide similar to Roundup® also containing POEA surfactant. Researchers in Canada found no significant impact on larval leopard frogs and green frogs when exposed to concentrations of 1.43 mg AI/L, an expected environmental concentration based on forest wetlands sampled. Substantial mortality was only found to occur when concentrations were much higher. This study was interesting in that it inoculated a section of a natural wetland; other natural factors may have influenced mortality (Wojtaszek et al., 2004).

Further experimentation has alsoprovided some more interesting insight on the validity of laboratory testing. An experiment completed by the University of Guelph in Ontario compared the effects of pH levels and Vision® concentrations on early life stages of four amphibian species. The results showed a significant interaction between pH and Vision® concentration such that the toxicity of Vision® was elevated when pH increases. These types of interaction are reasons why more research should be completed especially focusing on field studies (Edginton et. Al., 2004).

Some of the studies reviewed examined multiple sources of glyphosate. All of these studies included roundup. A study on responses by North American frog species carried out by Howe, et. Al. in August, 2004 exposed larval frogs to five different substances. This study exposed amphibians to multiple sources of glyphosate as well as the isolated surfactant found within Roundup®, polyethoxylated tallowamine (POEA). The study found POEA to be the most toxic followed by Roundup Original®. The largest effects of these two substances were on metamorphosis, believed to be caused by disruption of hormone signaling (Howe et. Al., 2004). Many of the other herbicides used in the study had negligible impacts because they are specifically designed for application in aquatic areas.

The toxicity of glyphosate and several other glyphosate formulations was tested on the larval and adult stages of four species of southwestern Australian frogs. Acute toxicity of technical grade glyphosate acid, glyphosate isopropylamine, and three glyphosate formulations was determined. As expected the glyphosate formulas that contained a surfactant had the lowest LC50 after both 24 and 48 hours. The LC50 for 48 hours for Roundup® ranged from 2.9 mg AI/L to 11.6 mg AI/L. However as glyphosate isopropylamine LC50 concentrations were significantly larger and levels that would never occur in a natural setting raging from 373 mg AI/L to 466 mg AI/L for 48 hours. The rest of the data can be viewed below in Table 3 (Mann and Bidwell, 1999).

Table 3. (Mann and Bidwell, 1999)

A similar study conducted by Perkins et. Al. in April, 2000 compared Roundup Original® and POEA to a salt form of glyphosate, isopropylamine (ipa) found in the product Rodeo®, which does not contain a surfactant (Perkins et. Al., 2000). The study found that POEA was three times more toxic than Roundup Original® (Perkins et. Al., 2000). Additionally, Rodeo® was found to have an LC50 of 5,407 mg acid equivalent (AE)/L (Perkins et. Al., 2000), while Roundup Original® had an LC50 as high as 9.4 mg AE/L (Perkins et. Al., 2000). This shows how drastically toxic POEA is on its own. A similar study was conducted by Tsui and Chu where glyphosate and its surfactant were studied separately. However this study examined the effect on algae, bacteria, protazoa, and crustaceans. There is some validity to looking at this study because although glyphosate is meant to be toxic to photosynthetic organisms, POEA is not supposed to be. This study found that POEA was toxic to all organisms present (Tsui and Chu, 2003).

Conclusion/Recommendations

The Perkins et al study on the comparison of glyphosate from different sources on amphibians serves as an expedient model for the logic of this team’s conclusions. Monsanto has expended much energy in public relations assuring consumers that their pesticide glyphosate is environmentally sound. This group concludes the same, but only within the chemical parameters of the glyphosate molecule itself. Although the research outlined above does indicate that glyphosate has some adverse affects on amphibian development, when one considers an LC50 of 5,407 mg AI/L for the glyphosate product Rodeo® it seems unlikely that such an exorbitant amount of the chemical would leach into surface waters. However, this is one of the common white crystalline salts of glyphosate described in the background section, and Rodeo® doesn’t contain a surfactant system. The product Roundup Original®, which contains the surfactant POEA, is far more deadly (Mann and Bidwell, 1999).