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The Environmental Fate and Effects Division has developed an interim approach for determining data requirements for non-racemic mixtures of stereoisomeric pesticides. These data are needed in order to assess the risk posed to ecosystems and drinking water sources by these mixtures. This policy considers enantiomers or "optical isomers" only. Enantiomers exhibit the same physical and chemical properties, except for the sign of rotation of plane-polarized light and chemical behavior in the presence of chiral chemical environments. The approach assumes that EFED already has an acceptable and full data set for the racemic mixture prior to the receipt of an application for registration of enriched mixtures of enantiomers. EFED's objective in its assessment of these mixtures is to determine, in the most cost-effective manner possible, whether the enriched mixture(s) are of greater concern environmentally and from a human health perspective than the (already registered) racemic mixture.

Because enantiomers may exhibit selective biological effects (stereoselectivity) towards inherently chiral-rich organisms, some data are needed on the enriched mixture in order to assess the relative behavior of the enriched mixture versus the racemic compound. A minimal data set is needed in order to make the determination of whether the enriched mixture MAY present a greater risk than the already registered compound. Additional data, above the minimal data set, to more fully understand the fate, transport and ecological risk picture may also be necessary depending upon the results of the initial tier of testing (i.e., the minimal data set). Over time, as EFED learns more about the behavior of non-racemic mixtures versus racemic mixtures, the minimal data set for enriched mixtures may change. This is why EFED has designated this policy as an interim policy.

Minimal Data Set for Enantiomer-enriched Active Ingredients

When a full complement of ecotoxicity and environmental fate data for a racemic mixture has been submitted and reviewed and a registrant submits a new, enhanced enantiomer purity active ingredient and/or formulation, OPP will require the following minimal data set using species and conditions (including testing laboratories) that are identical to those for the data submitted for the previous racemic mixture.

Environmental Fate

Analytical chemistry methods capable of identifying and quantifying each separate enantiomer and chiral transformation products in soil, water, and fish tissue are needed. The ability to differentiate enantiomers in environmental media is important in distinguishing biotransformation, accumulation or preferential sorption of enantiomers. These analytical methods are required to assess the potential for stereoselectivity. Regardless of the enantiomeric purity, chemical and physical characterization would be required for all formulations containing a single enantiomer, racemic mixtures, or enantiomerically-enriched mixtures.

1 Lewis, Garrison, et al. Nature, Vol 401, Oct.18, 1999, pp. 898-901 and Garrison, et al. Environmental Science and Technology, Vol 30 (8), 1996, pp.2449-2455)

The following chemical information is required and should be included with all environmental fate and ecotoxicity studies:

  1. Enantiomeric purity (enantiomeric excess),

  2. Ratio of geometrical isomers

  3. Pesticide activity of each stereoisomer, when applicable

  4. Mode of pesticide action

  5. Chemical names for each enantiomer (CAS and IUPAC nomenclatures)

  6. Chemical Abstracts Service Registry Number for each isomer, if applicable

  7. Chemical structure(s)

  8. Chemical family

  9. Physical and chemical properties (solubility in water, vapor pressure, octanol-water partition coefficient, Henry's Law constant, pKa, if applicable)

Fate Data

Because enantiomers may exhibit selective biological effects, OPP believes that biotic processes (e.g., soil and aquatic metabolism) may cause preferential degradation when compared to abiotic processes (e.g., hydrolysis and direct photolysis in water). Based on this premise, an aerobic soil metabolism study (GLN 162-1/835.3300) is required as part of the minimal data set for enantiomeric enriched mixtures. If the biotransformation rates and degradation products are substantially similar for the racemic and the enantiomeric enriched mixture, then no additional environmental fate data are needed. If on the other hand, the biotransformation rates and degradation products for the racemic and enantiomeric enriched mixture differ in a manner that suggests greater potential risk, then additional environmental fate data may be required on a case-by-case basis.

Ecotoxicity Data

OPP will require the following minimal data set using species and conditions (including testing laboratories) that are identical to those for the data submitted for the previous racemic mixture. If the toxicity values are substantially similar for the racemic and enantiomeric enriched mixture and do not result in significantly greater potential risk, then no additional ecotoxicity data are needed. If, however, the toxicity data for the racemic and enantiomeric enriched mixture differ significantly and suggests that the enriched mixture poses a significantly greater risk, then additional ecotoxicity data may be required on a case-by-case basis.

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Acute Avian Oral (GLN 71-1/850.2100) - use same test species

Avian Reproduction (GLN 71-4/850.2300) if birds are more sensitive than mammals


Acute Fish Toxicity (GLN 72-1/850.1075) or Acute Invertebrate Toxicity (GLN 72-2/850.1010) -test most sensitive species

Fish Early Life Stage (GLN 72-4a/850.1400) or Invertebrate Life Cycle (GLN 72-4b/850.1399) - test most sensitive species

Nontarget Plant

  1. Seedling Emergence (GLN 122-1a/850.4100) or Vegetative Vigor (GLN 122-1b/850.4150) - test most sensitive monocotyledon and dicotyledon

  2. Aquatic Plant Growth, algae (GLN 122-2a/850.5400) - test most sensitive of the tested algae

  3. Aquatic Plant Growth, lemna (GLN 122-2a/850.4400)

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APPENDIX I - Stereoisomers and Pesticide Activity

Biological stereospecificity (bioactivity), including pesticide activity, originates when a single isomer interacts with a "receptor" in a "key-complementing-its-lock" manner and triggers a specific response in a specific organism. The interaction of a chiral chemical with bioreceptors that are made up of chiral molecules, such as amino acids, carbohydrates, and triglycerides is a typical example of sterereospecificity. The following pesticide activity cases are possible for stereoisomeric pesticides:

  1. Both of the stereoisomers exhibit comparable pesticide activity

  2. Only one of the stereoisomers exhibits pesticide activity. The other stereoisomer is inactive.

  3. Both stereoisomers exhibit pesticide activity, but one is more active than the other.

Interactions of stereoisomeric pesticides with target and/or non-target organisms have the possibility that:

  1. The pesticide activity towards a target pest does not imply that the pesticide-active stereoisomer will be necessarily toxic to non-target organisms.

  2. The pesticide-inactive stereoisomer may or may not be toxic to non-target organisms.

  3. The pesticide active and inactive stereoisomer may have the same toxicological mode of action.

  4. The pesticide-active and inactive stereoisomer may have different toxicological modes of action.

Risk Assessment Uncertainties with the Current Environmental Fate and Ecotoxicity Data on Stereoisomeric Pesticides

Environmental fate data are necessary to identify the stereoisomers and/or their transformation products to which non-target organisms might be actually exposed. The current environmental fate and ecotoxicity data with those pesticides that are chiral, or geometric isomers, or both leave the following uncertainties:

  1. If the pesticide-inactive stereoisomer may be toxic to non-target organisms.

  2. If transformation (including isomer interconversion), preferential adsorption, and/or bioaccumulation could increase the amount of the active stereoisomer and, therefore, its exposure in the environment

  3. If the transformation products of the active (or inactive) stereoisomer(s) are also chiral, geometric, or both and how exposure to these products may contribute to the overall ecotoxicity of the pesticide.

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APPENDIX II - Stereoisomers: Background

Stereoisomeric molecules have identical bonding, positioning of atoms or substituent groups, but differ in the spatial, three-dimensional arrangements of atoms or substituent groups. It is the different orientation of atoms and substituent groups that differentiate a pair of stereoisomers. Two major classes of stereoisomerism will be considered in this summary, as they are the most relevant for many bioactive compounds, including pesticides. One class of stereoisomers originates from the presence of an atom containing four different substituents (that is, the presence of an asymmetric atom, mostly referred to as a "chiral atom"). The other class of stereoisomers arises from the presence of a carbon-to-carbon double bond (-C=C-) and are known as geometric isomers. Conformational isomers are not being considered.

Selective Bioactivity

Biological stereospecificity originates when a single isomer interacts with a receptor in a "key-complementing-its-lock" manner that triggers a specific response. Biological stereospecificity of enantiomers connotes selective interaction of a single enantiomer with receptors that are made up of chiral molecules, such as amino acids, carbohydrates, and triglycerides. Below are some examples showing how different stereoisomers can produce different, and many times opposite, biological responses. Thus, from a point of view of biological activity, each stereoisomer should be envisioned as different substances.

Biological Responses Produced from Different Stereoisomers
Chemical R- S-
Butarbital Narcotic Anticonvulsant
1-Chloro-2,3-propanediol Poison Medicinal drug
Asparagine Sweet Bitter

Monosodium glutamate (+) Used as a meat flavoring agent (-) Insipid

S- Metolachlor a R-Inactive S-Active a 80% enrichment of S- isomer

Dimethomorph Z-Active E-Inactive

Chiral chemicals (Enantiomers; Optical Isomers)

If a molecule containing an atom with four different substituents (or isotopes) is non-superimposable to its mirror image, that molecule is chiral. If the mirror image is superimposable, that molecule is said to be achiral. A pair of non-superimposable molecules are called enantiomers. In most molecules, chirality is associated with a tetrahedral carbon atom. However, chirality is not restricted to tetrahedral carbon. Other tetrahedral atoms have the potential to be chiral, for example Si, N in quaternary salts (brucine, cinchonine), phosphate P. Examples of chirality at the phosphate-P atom are found among some organophosphate pesticides and their transformation products. More than one chiral atom in a molecule may not lead to formation of enantiomers because they may have superimposable mirror images. These stereoisomers are known as diastereoisomers.

Enantiomers have exactly the same physical and thermodynamic properties, that is, the same melting/boiling point, index of refraction, vapor pressure, free energy, enthalpy, entropy, etc. Enantiomers differ only in the left- and right- handness of their spatial orientation. Optical activity, that is, rotation of plane polarized light is the result of this different spatial orientation. Each enantiomer rotates plane polarized light in opposite directions, but by equal amounts. If plane polarized light is rotated clockwise, that enantiomer is said to be dextro-rotatory and is designated (+). If rotation is counterclockwise, that enantiomer is said to be levo-rotatory and designated (-).

Enantiomers react differently in the presence of a chiral environment (i.e, they can react at different rates), but in achiral environments (i.e, they can react at the same rate). A mixture of two enantiomers present in equimolar amounts of equal but opposite rotations are defined as racemic mixtures or racemates. Racemic mixtures are optically inactive because the equal (+) and (-) rotations cancel out. Separation of each optical isomer in a racemic mixture is known as resolution.

Absolute Configuration

Absolute configuration is the actual positioning of substituent atoms or groups around the chiral atom. Absolute configurations were assigned in the past using glyceraldehyde as the standard to assign a D- or L- absolute configuration to a molecule. The D- and L- notation is used to assign absolute configuration. The notation d- and l- is no longer recommended as it is sometimes used ambiguously for rotation and configuration.

The D- and L- system has been retained for amino acids or compounds containing an amino acid moiety, such as alanine in metalaxyl. The method presently utilized for assigning absolute configurations is based on the chemical nature of the four different atoms/substituent groups bonded to the chiral carbon (or other atom) using a set of rules. This ranking is made according to a set of sequence and priority rules. This is known as the Cahn-Ingold -Prelog (CIP) system, and it is unambiguous because it does not depend on correlations with glyceraldehyde.

The ranking rules in the CPI system are as follows:

R- (rectus) and S- (sinester) configurations are determined by the order of ranking. When the sequence goes clockwise from the highest ranking to the lowest, the R- configuration is assigned, but if the highest ranking to the lowest is counterclockwise, then the S- configuration is assigned. The R- and S- absolute configuration do not necessarily indicate the sign of rotation, (+), (-). For example, there are chemicals that are R- (-) or S- (+). In other words, the clock- or counterclockwise sequence of groups/atoms bonded to the chiral atom has nothing to do with the sign of rotation of plane-polarized light.

Confirmation of absolute configuration assignments may be done via chemical and biochemical reactions, nmr, or X-ray structural determinations.

Resolution of Racemic Mixtures

Among the procedures utilized for separating enantiomers in a racemic mixture are:

  1. Mechanical separation of crystalline racemic mixtures (Pasteur's work with tartaric acid).

  2. Using a chiral compound. For example, using the quaternary amines brucine or cinchonine to resolved a racemic mixture of an anion.

  3. Using biological substrates, such as those provided by microorganisms, where one of the enantiomers show preferential activity.

  4. Using purified enzymes.

  5. Chiral chromatography, mostly HPLC.

Attempts to resolve a racemic mixture do not always result in 100% separation of the two enantiomers. To assess the success of a racemic mixture resolution, some of the following approaches are commonly taken:

  1. measurement of optical rotation;

  2. chromatographic determination of ratio of each enantiomer; and

  3. nmr spectroscopy.

The enantiomeric purity is a criteria for the success of the resolution,

enantiomeric purity = enantiomeric excess (e.e.)= |% R - % S|

For example, if S- = 90% and R- = 10%, the enantiomeric purity (enantiomeric excess) is equal to |10 - 90| = |-80| = 80% enantiomeric excess of the S- enantiomer.

Industrial-scale Preparation of Optically Pure Chiral Chemicals

Naturally occurring, chiral compounds such as amino acids, carbohydrates and terpinoids constitute the "chiral pool" that can be used for transferring chirality to reagents. The chiral reagents are then used to synthesize the desired enantiomer. This is in contrast with "conventional" syntheses that result in racemic mixtures and not in an specific enantiomer. An example of a pesticide that uses a terpinoid chiral pool is S- Metolachlor, which results in a 80% enrichment of the most herbicide active S- enantiomer.

Use of chiral solvents is sometimes used as the reaction media for non-chiral synthesis. In many cases, chiral chromatography (mostly HPLC) may be used for separation or for analysis of enantiomer ratios. Microbial fermentation and use of purified enzymes are commonly used in industrial-scale operations. Metal complexes containing chiral ligands have been used to produce the insect growth regulators S- 7-methoxy-citronellal and S- 3,7-dimethyl-1-octanol.

Geometric Isomers

When a molecule has a carbon-to-carbon double bond (-C=C-), a substituent group (or atom) can be positioned at the same side of a double bond or at opposite sites. The isomer with the two groups positioned at the same site are known as cis- isomers. If the two groups are positioned at opposite sides, the isomers are known as trans-.

The use of cis- and trans- is currently acceptable for all disubstituted alkenes. However, when there are three or four groups attached to each carbon of the double bond, a different system of nomenclature is used. The nomenclature is based on Cahn, Ingold and Prelog (CIP) of ranking substituents, as presented for assigning absolute configurations of enantiomers. In the case of three or more atoms attached to the double bond stereoisomers, the E- and Z- nomenclature is then used. If the two highest ranking are on the same side, this isomer is designated as the Z- isomer (from the German word zusammen, "together"). If the two highest ranking groups are opposite to each other, the isomer is designated as the E- isomer (from the German word entgegen, "opposite").

Unlike enantiomers, geometrical stereoisomers exhibit different physical and chemical properties and can be separated.

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References for Appendices

General Bibliography

  1. Juaristi, E. Introduction to Stereochemistry and Conformational Analysis, Wiley Interscience, New York, N.Y., 1991.

  2. Eliel, E.L. Stereochemistry of Organic Compounds, Wiley Interscience, New York, N.Y., 1994.

  3. Rahman, A-U and Shah, Z. Stereoselective Synthesis in Organic Chemistry, Springer Verlag, New York, N.Y., 1994.

  4. Mazey, P.G. (Editor). New Developments in Molecular Chirality, Kluwer Academic Publishers, Drdrecht, The Netherlands, 1991.

  5. König, W.A. The Practice of Enantiomer Separation by Capillary Gas Chromatography, Dr. Alfred Hüthig Verlag, New York, N.Y., 1988.

  6. Ager, D.J. (Editor). Handbook of Chiral Compounds, Marcel Dekker, Inc., New York, N.Y., 1999.

  7. March, J. Advanced Organic Chemistry, 3rd. Edition, John Wiley & Sons, New York, N.Y., 1988.

  8. Moss, J.P. Basic Terminology of Stereochemistry, IUPAC Recommendations 1996, Pure and Applied Chemistry, Vol. 68, 1996, pp. 2193- 2222.

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