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field methods are not available for several of the suspected endocrine disrupters currently under
review by the environmental scientific community. An understanding of the modes of
lexicological action, measurements of the concentrations of these substances in the various
environmental media, and improved means of modeling their movement and transformation among
environmental media are fundamental to conducting human and ecosystem risk assessments.
Improved methods and models are needed to measure and to predict the exposure to these
substances. A modicum of understanding of exposure is also needed to design studies related to
endocrine disruption; e.g., studies of biological mechanisms and effects should address chemical
species of endocrine disruptors that are most prevalent in the environment. The consensus
emerging from the scientific debate surrounding endocrine disruptors is that there are insufficient
data to resolve objectively the relative ecological and human health risks associated with these
environmental contaminants.
The suspected endocrine disruptors that have been studied are predominantly organic
compounds or organic forms of heavy metals that are persistent, can bioaccumulate, and can
biomagnify in the food chain. Subtle variations in chemical form and physicochemical
characteristics (e.g., planarity, isomerization, equilibria, and sorption affinities), may manifest
themselves in numerous ways that may affect exposure (e.g., differences in transport and routes
of exposure, increased or decreased bioavailability, changes in exposure pathways, potential for
atmospheric and hydrological transformation, and fate). Most.polychlorinated biphenyls, for
example, would be expected to have more affinity for the sediment than for the water, since they
are relatively hydrophobic. Risk analysts and exposure researchers must understand complex
exposure patterns, rather than net annual exposure estimates. Developmental biology dictates that
certain exposure windows of vulnerability can be expected to follow temporal and seasonal
patterns of endocrine functions.
A RISK ASSESSMENT PERSPECTIVE
A national endocrine disrupter research program should follow EPA's risk assessment
framework, and explore methods and models to estimate and to predict exposure to these
substances. The exposure component of this research plan should follow the steps shown in
Figure 1. Calculations of exposure are a function of dose. The U.S. Environmental Protection
Agency (1992) defines four types of dose: potential dose (D ); applied dose (D ); internal dose
(Dp; delivered dose (DD); and biologically effective dose (Dfi ). The exposure pathways begins
with an organism's first contact with a substance (D ) to its intake, absorption, and metabolism
(D , D and D ) to its effect on the target organ (D ).
AID . BE
Measurements of D can often provide a reasonable estimate of exposure; i.e., the
concentration of a contaminant around an organism. For airborne contaminants, D is a function of
concentration, time, and ventilation. It is difficult or impossible to measure D directly, so D , D
and D are most often expressed by bioraarkers, i.e., "indicators of changes or events in human
biological systems" (1991). Biomarkers may either be the contaminant itself or metabolites
indicating exposure to the contaminant; e.g., increased concentration of cotinine (a metabolite of
nicotine) in blood resulting from exposure to tobacco smoke. Similarly, biomarkers in ecosystems
are "biochemical, physiological, or nistological indicators of either exposure to or effects of
xenobiotic chemicals at the suborganismal or organismal level" (Huggett, et al., 1992). Biomarkers
can also apply to ecological exposure, although they are not often classified as measures of dose
(ecologists may apply the terms, "biotic and abiotic accumulation"). For example, Hunsaker et al.
(1990) have suggested measuring chclinesterase levels and porphyrin accumulation to indicate the
level of ecosystem exposure..
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Hazard Identification")
Exposure Assessment
*
~~ i** ~- fm, """
Risk
Characterization
Effects Assessment
Figure 1 Simplified Exposure Assessment Paradigm. A substance is released to the environment, is
transported, may be transformed chemically and physically, and can move through various pathways;
e.g., water, air, soil and sediment. After a substance reaches an environmental pathway; i.e., media
change, this can be tantamount to being a new source, in essence starting the process again from
source characterization. The fate is then determined iteratively via mass balance.
Methods for estimating ecosystem exposure can be similar to those for human exposure
assessment, as indicated in Figure 2, but the methods may differ in important ways. Both
ecosystem and human exposure assessments are often concerned with sensitive subpopulations,
many pollutants are both human and ecological stressors, and ambient measurements for some
pollutants can be indicators of both human and ecosystem exposure (e.g., ozone). Human risk
assessments provide an expression of the likelihood that an adverse outcome will result from a
given hazard; e.g., 10~6 chance of cervical cancer in a population exposed to a particular pollutant.
Ecological risk assessments are also expressions of the likelihood of an adverse outcome, but the
expression depends upon the "environmental value" of concern; e.g., biological diversity,
sustainability, and aesthetics (Environmental Monitoring and Assessment Program, 1993). A
major difference between human and ecological exposure paradigms is the level of biological
organization at which contaminants are typically studied; i.e., human epidemiology considers
population exposure for one species (human) and medical research considers responses at various
doses and exposures for an individual human being. Ecological exposure assessments often
attempt to address substances that affect the whole ecosystem. These may include exposures to a
community (several species), as evidenced by contaminant concentrations in certain indicator
organisms or "sentinel" species. Ecosystem exposure also considers population exposures for a
target species (e.g., top predator tissue concentrations of a contaminant suspected of reducing
fecundity). Ecosystem exposures can even be extrapolated from measurements of abiotic media;
e.g., an estimate of fish community exposure extrapolated from water column concentrations of a
contaminant.
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Human
« Source Identification, Characterization
ft Apportionment
Transport/Transformation/
Interaction/Fate
Environmental Concentration
Exposure Measurements (Potential Dose)
t
Actual Dose
Applied Dose
Internal Dose
Delivered Dose
Biologically Effective Dose
1
Biomarkers
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Ecological
Source Identification, Characterization
tf Apportionment
Transport/Transformation/
Interaction/Fate
Deposition
Physical/Chemical Measurements of
Srressor In Ecosystem
JL Phys/Chem/Bio
^f Degradation
Accumulation Into Abiotic and Biotic
Components of Ecosystem
t
Biomarkers
Figure 2 Exposure components of risk paradigms are similar for humans and ecosystems (Vallero, 1996a).
Human exposure can be expressed as the lifetime average daily dcse (LADD). Each route of
exposure must be considered; i.e., ingestion (water, food, and soil), inhalation of gases and
particles, and derma) exposures. Based .upon Derelanko's (1995) expressions of LADD, total
LADD may be calculated as the sum of all LADD values via all routes:
LADDT =LADD
where:
« 1 U
LADDT = lifetime average daily dose (mg/kg/d) via all routes
LADDA = lifetime average daily dose (mg/kg/d) via inhalation
LADDj = lifetime average daily dose (mg/kg/d) via ingestion
LADDD = lifetime average daily dose (mg/kg/d) via dermal r
(1)
routes.
Further, each route can be further, subdivided. For example, LADDT = LADDg + LADDp
LADD =
where:
(BW)(TL)
LADDg = lifetime average daily dose (mg/kg/d) from inhaling vapors;
C = concentration in air (mg/m^);
IR = inhalation rate (m^ /h);
EL = exposure length (h/d);
AF = uptake or absorption factor (dimensionless, fraction of inhaled C absorbed);
ED = duration of exposure (d);
BW = body wt (kg);
TL = typical lifetime (d)
(2)
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LADDD =
(3)
(BW)(TL)
where: LADD = lifetime average daily dose (mg/kg/d) from inhaling particle matter (liquid
and solid);
Cp = concentration of contaminant sorbed on or in particle (mg/m^);
PC = particle concentration in air (mg/m^);
10"6 = converts kg to mg.
Equations 2 and 3 also indicate that exposure models should incorporate physicochemical
properties associated with transport, transformation and fate in air, soil, water, and sediment
transport capabilities of existing compartmental models. For example, Figure 3 illustrates three
idealized bimodal distributions for particles. Such distributions can provide weight-of-evidence for
i i
IDEALIZED MASS/SIZE DISTRIBUTION FOR
URBAN AEROSOLS:VARIES BY CITY FOR
MASS, SIZE DISTRIBUTION, AND CHEMICAL
COMPOSITION.-from Hidy (1975).
PRIMARILY NATURAL
OR QUASI-NATURAL
'E
t
O
I I
Combustion
dominated
ambient aerosol;
e.g. East Coast
such as
Philadelphia, and
Washington, DC,
Also Los Angeles
when wind is from
[offshore.
LU '
O
Z
O
O
CO 0.01 0.1 1.0 10
W AERODYNAMIC DIA. (urn)
< I I I
10C
O
Q.
Soil/dust dominated
ambient aerosol.; e.g.
desert, farming
Industrial grinding
andmining. Also, Los
Angeles when wind is
from tho nearby
eastern desert.
0.5 ~0.7 1.0-2.5-3.0
AERODYNAMIC DIAMETER (urn)
10
0.01 0.1 1.0 10
AERODYNAMIC DIA. (um)
100
Figure 3 Particles often display a bimodal distribution by mass, originate from multiple sources, show dynamic
growth and reactivity, and are carriers of other pollutants (Hidy, 1975). The upper right mass
distribution is typical for an area dominated by anthropogenic (combustion) sources, while the bottom
distribution is typical for areas where particles are generated from noncombustion sources (e.g., re-
entrained soil and mining activities).
anthropogenic or natural sources of contaminants. Karickhoff and Long (1996) have developed the
SPARC model which characterizes the potential environmental fate of substances based upon
vapor pressure, lipophih'city (e.g., Kow), activity coefficients, water solubility, phase partitioning
-------
(i.e., Henry's Constant) and ionization potential (pKa). Therefore, physical and chemical
characteristics, such as phase distribution and a substance's affinity to accumulate in various
environmental compartments, can profoundly affect the estimates of exposure to humans and
wildlife.
Exposure assessments should be conducted at appropriate spatial and temporal scales,
depending upon the hypothesis or research question being investigated. Timing is complicated by
triggering and response mechanisms in the endocrine system at certain stages of development in
humans and wildlife. These windows of exposure, where the organism is particularly vulnerable
to hormonal dysfunction, must be addressed in any exposure calculations. The author
recommends:
CWDDeDc = (LADDT + CW)(SF)(MT) (4)
where: OV/DD^ = Total critical window endocrine disrupter exposure (mg/kg/d);
CW = Additive dose during critical windows of vulnerability (mg/kg/d);
SF = sensitivity factors; e.g., demographics for human populations, species
sensitivities for wildlife (dimensionless);
MT = maternal transfer and transgenerational multiplier (dimensionless).
POTENTIAL ENDOCRINE DISRUPTOR EXPOSURE RESEARCH AREAS
The National Academy of Sciences (1991) has recommend approaches for assessing
human exposure to airborne pollutants (Figure 4), emphasizing the need for data from direct
measurements (personal and biomarker monitoring) and from indirect approaches (especially to
gain knowledge about activities). At the outset, however, exposure research for endocrine
disrupters should emphasize the physicochemical characterization of known or highly suspect
endocrine disrupters. As indicated, even slight differences in physicochemical properties can
greatly affect environmental fate; therefore exposure estimates must begin with an understanding of
how these substances can be expected to behave in the various media and their fate. The
development and adaptation of compartmental transport and fate models can be a major focus of
this research.
Characterizing Chemodynamic Fate
Chemical substances reside and move through environmental "compartments;" i.e., air,
soil, water, sediment, and biota, being transported and transformed before reaching the sites of
their ultimate fate. Such compartments can be simulated in mathematical models to predict the
persistence, bioaccumulation, bioconcentration, and biomagnification of these substances within
each medium, according to physicochemical properties such as vapor pressure, water/lipid
solubility, bioaccumulation factors, and chemical half-life. Modeling can apply to both human and
ecosystem exposures; such as food chain models, however, compartmental exposure models have
principally been used to address outcomes other than endocrine disruption. A number of
researchers, including Cohen and Clay (1994), have developed models to simulate the movement
and change of chemical substances in the environment, as a function of their physicochemical
characteristics. These properties dictate the potential sorption, transformation, transfer, and fate in
soil, sediment, water and air and uptake by biota; the ability of substances to enter the food chain;
and the magnification of chemical concentrations at higher trophic levels for those substances that
accumulate.
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Exposure Analysis
Approaches
Environmental
Monitoring
Models
Questionnaires
'"1
Diaries
Pharmacokinetic &
Phartnacodynamic Models
\
Mitigation
Measures
Figure 4 Possible approaches for analysis of air contaminant (From: National Academy of Sciences, 1991).
The dashed line between pharmacokinetic and pharmacodynamic models and exposure models have
been added by author to show that exposure models can be derived from direct measurement data, from
routines from other models, and from combinations of measured and derived data.
In sediments, for example, transformation pathways and kinetics are determined by a
complex interaction of microbial, chemical, and physical processes. The interplay of these
processes should be an overarching theme for any study of chemical fate in the environment.
Many of the suspect endocrine disrupting chemical substances identified to date are low solubility,
neutral organic compounds that are highly sorbed on the organic carbon phases of sediments.
Currently available predictive tools are based on hydrophobic solution theory, and are reliable for
estimating the magnitude of sorption of such compounds on sediments. Comparable tools for
estimating the kinetics of the sorption and desorption processes are lacking. Work is also needed
to develop models for predicting the sorption of endocrine disrupters to particles in sediment, soil,
water, and air, under varying environmental conditions (e.g., pH, moisture, organic matter types
and concentrations, ionic strength, and concentrations, shapes and sizes of particles).
Providing Improved Exposure Data
Reliable and standardized data bases are vital in testing effects/exposure hypotheses, and in
evaluating exposure and effects models. A strategic approach for using or modifying existing
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monitoring programs to assess current and historical effects of endocrine disrupters should be
developed. Federal and other data bases need to be reviewed for reliability (meta-data, quality
assurance, documentation, frequency, and methods), and an assessment be given to the scientific
community regarding the data quality and the means for accessing these data (electronically and
manually). Several existing monitoring programs that collect data could be used to help in
problem formulations for risk assessments, or to support exposure or effect characterizations in
retrospective risk assessments. Examples in the U.S. include the Environmental Monitoring and
Assessment Program (EMAP) of the EPA, the National Status and Trends Program (BEST) of the
Department of the Interior's National Biological Service, the National Water Quality Assessment
Program (NAWQA) administered by the U.S. Geological Survey, and a variety of state and joint
international monitoring programs.
Identifying Major Knowledge Gaps and Uncertainties
The ranges of uncertainty must be identified and incorporated into exposure models.
Improved toxicokinetic and structure activity models need to be linked with the physicochemical
characteristics of suspect endocrine disrupters, especially at critical and sensitive early life-stages.
Compartmental models and laboratory studies must be linked to field research by developing
mechanism-based dose response models. Exposure levels observed in the field will be used as a
basis for identifying realistic dose ranges in laboratory experiments.
Exposure scientists will need specific and sensitive biofnarkers. Effects and exposure
biomarkers must be calibrated to adverse individual- and population-level effects. Biomarkers of
exposure are an essential adjunct to environmental measurements in developing and verifying
human and ecosystem exposure models. They are also needed to screen ecosystems for exposures
and to improve exposure estimates in future epidemiological studies. Field evaluations of these
markers should establish which are most predictive of population-level effects (i.e., which are
most useful for establishing cause and effect relationships). This necessitates the evaluation of
"normal" values and the uncertainty associated with their measurement.
Research at Appropriate Spatial, Biologic, and Temporal Scales
Endocrine disrupter research will take place at spatial scales ranging from subcellular
exposure to regional. Methods for assessing exposure for an individual organism (e.g., one
human being) differ from methods used to assess population exposure. Likewise, estimating
exposures for a single ecosystem component; e.g., a lake or wetland, will be different from a
large-scale exposure assessment of region or biome. Geographic scale also plays a crucial role in
model selection. There may be a need for predictive capability on the micro-scale (e.g.,
occupational, residential), field-scale (e.g., production plant emissions impacting adjacent
ecosystems or human populations), regional scale (e.g., farm applications and resulting human and
ecosystem exposures in an entire watershed) and global-scale (e.g., long-range transport and
exposure at remote sites).
At lower levels of biological organization, a researcher may be able to determine signals of
exposure for a wide array of contaminants, and provide detailed and specific information about a
subject's activity patterns. Often, however, scientists are asked to estimate exposure of entire
populations or target groups, wherein gathering detailed and specific information about the
exposure of each individual in a population is scientifically and economically infeasible.
Moreover, in the case of ecosystems, detailed information about individuals may have less
importance than the interrelationships and diversity of a larger ecological community; true to the
adage, "not seeing the forest for the trees." The hypothesis or study objective determines the scale
of an exposure assessment.
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The temporal scale can range from very short duration, single event to random episodic
events to long-term, discrete exposures (e.g. annual or seasonal) to continuous exposures. The
temporality of the exposure must be considered in the study design. For example, an episodic
event may require monitoring until the chemical and biological indicators regain equilibrium, and
long-term, continuous exposure studies may call for seasonal and annual time series and status and
trend assessments. Even episodic exposures may require lengthy follow-up studies, however,
since the exposures may be transgenerational, and successive generations become exposed via
maternal transfer.
Exposure Hypothesis Testing
Comprehensive investigations of a small number of experimental sites/systems with
problems that are known or strongly-suspected to be related to endocrine disrupters could yield
valuable information about human and wildlife exposures. Such an integrated study, conducted at
multiple levels of biological organization, with both laboratory and field components, may provide
insights into the identification of sensitive measurement endpoints and species, and extrapolation
among endpoints, species and chemicals.
Both biological and exposure measurements need to be collected for areas expected to have
elevated concentrations of suspected endocrine disrupters in various environmental media.
Biomarker researchers can test screening tools, in situ results can be compared to in vivo and in
vitro findings, and biologically plausible hypotheses linking exposure and effects can be tested.
At the outset, using professional judgment, scientists could select pilot study sites based upon
weight-of-evidence that populations have been affected by exposure to endocrine disrupters. Such
evidence could be derived from ecological epidemiology, exposure screens, historical data
suggesting a likelihood that endocrine disrupters are present in one or more environmental media,
or suggestions from source or fate models of a "hot spot." For ecosystems, pilot studies should
address direct and indirect effects of endocrine disruption in multiple phylogenetic groups and
trophic levels.
An emerging area of concern is the impact of endocrine disrupters on mammalian immune
systems. Some agricultural chemicals, such as DDT and its metabolites, can act as both endocrine
disrupters and immunosuppressants. The initial mode of action is to suppress adrenal secretions
which, in turn, directly and indirectly decreases the immune response to bacterial infection. A
possible exposure study may include measurements of mammalian serum antibody tilers. Some
veterinary Pharmaceuticals and antibiotics are also administered to promote growth in livestock,
which may select out the more resistant strains of enteric bacteria, the so-called "super bugs." The
fate and transport in the various environmental media of these multi-active compounds should be
examined. Various species of bacteria at contaminated sites should be analyzed for increased
resistance to selected antibiotics. An important exposure research question is whether the bacteria
are being transported among environmental compartments, thus spreading resistance to unexposed
bacterial populations. Identifiable bacteria strains may also prove useful as exposure biomarkers
for specific endocrine disrupting agricultural Pharmaceuticals.
Studies will be needed to test and confirm results from predictive, integrated models that
incorporate structure-activity relationships, toxicokinetics, bioenergetics, environmental chemistry,
and population ecology. They can provide a means for testing effects and exposure screening
tools, and would provide multimedia samples for analytical methods development Although these
pilots would likely focus upon ecosystem level effects and endocrine disrupter concentrations in
environmental media, they may also present the opportunity to conduct human residential studies
compare expected exposures from human exposure models to actual exposures under actual
environmental conditions where weight-of-evidence suggests a human biological response; e.g.
concentrations in carpet, food, indoor and outdoor ah, and drinking water at a small number of
sites around a former facility where a suspect endocrine disrupting substance was manufactured.
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In addition to measurements of'endocrine disrupters in highly contaminated areas, some
estimate of variability of contamination in different regions may be obtained via monitoring sites
where air, water, soil, sediment, and vegetation samples can be gathered. These samples can
provide valuable information about areas other than the highly contaminated areas that will be
addressed in the pilot studies. The National Exposure Research Laboratory, for example, is
establishing such a site near each of its laboratory facilities in Georgia, Nevada, North Carolina,
and Ohio. At a minimum, these "near-laboratory" sites will provide a means for developing and
testing measurement protocols for a wide range of pollutants, including suspected endocrine
disrupters.
FUNGICIDES IN SOIL: A POTENTIAL AREA OF ENDOCRINE DISRUPTOR
CHEMODYNAMICS RESEARCH
Agricultural operations have long used many neurologically active pesticides that have
subsequently been shown to effect the endocrine systems of animals. Several suspect endocrine
disrupters presently under review by the U.S. EPA (1996) are agricultural chemicals or their decay
products; e.g., DDT and DDE, that are relatively lipophilic and tend to bioaccumulate in the
environment. Recently, in response to concerns about groundwater contamination, pesticide
manufacturers have reformulated pesticides to dissipate upwardly to the atmosphere to prevent
downward migration. These changes in chemodynamics accentuate the importance of advancing
the understanding of soil-to-air fluxes.
Several fungicides are form dated with active organochlorine and organometaUic functional
groups (Meister, 1996), often making them persistent and semi-volatile substances (vapor pressure
=. 10'2 to 10'8 kilopascals). Semi volatile compounds do not readily dissipate and can remain active
for longer periods of time than volatile compounds (Lewis and Gordon, 1996). Unlike
nonvolatiles, after being incorporated into the soil, semivolatiles may later be transported from soil
to air. This flux rate is diffusion controlled, and is proportional to vapor pressure. In the
atmosphere, they may remain as gaseous pollutants or may be sprbed to particles that can travel
long distances and later concentrate in various environmental media and biotic tissue.
Within soil and sediment, sorption and degradation processes exert the largest controls over
the fate of agricultural chemicals in the environment. Adsorption to soil particles decreases the
vapor pressures of pesticides, and is dependent upon soil conditions. The rate of evaporation from
the soil column also affects xenobiotic flux. Plant uptake rates of dissolved chemicals and
transport to the ground and surface waters are controlled by the chemicals' physical and chemical
properties and the conditions of the soil (Rao, et al. 1993). Spencer and Cliath (1990) identified
soil water content, physical and chemical properties, concentration of the compound, and soil
properties, especially sou organic matter, as the most important factors controlling adsorption rates
(Nash and Hill, 1990). Fungicide half-life in the soil and sediment, degradation product
formation, and kinetics in the soil matrix and air column and in the sediment and. water column
must be characterized properly before reliable endocrine risk assessments are possible.
Characteristics of the compounds affect its potential for transport and transformation, such as water
solubility, lipophilicity, dissociation, and molecular weight. Characteristics of the environmental
media also determine transport and transformation, such as soil and solution pH and redox
conditions, soil moisture, soil texture and structure, and type and amount of soil organic matter.
The modes of toxic actions of these substances are determined by the physicochemical
characteristics. These same characteristics may influence a compound's ability to persist and to
bioaccumulate, and to elicit longer term effects in humans and wildlife. Slight variations in
physicochemical characteristics, such as planarity and isomerization, may drastically change soil
fungicide exposure and effects.
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Movement and transformation in the environment can be illustrated by two fungicide
classes associated with endocrine effects in humans and wildlife; i.e., dicarboximides and
organotins.
Vinclozolin
Vinclozolin is a dicarboximide fungicide whose structure is shown in Figure 5. Since it has
a dichlorobenzene group, Vinclozolin may be classified as an organochlorine compound, the group
presently most often associated with endocrine disruption. It may also be classified as a carbamate
pesticide, since the right side of the structure is derived from carbamic acid. Two principal
degradation products result from opening the carbamate ring: a buteonic acid (2-[[(3,5-
Dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid), referred to as "Ml"; and an enanilide
(3',5'-Dichloro-2-hydroxy-2-methylbut-3-enanilide), called "M2". Both degradation products
have been isolated in plants and soils (Kelce, et al., 1994). Ml is a reversible reaction, where the
carbamate ring closes and returns to the Vinclozolin structure, whereas M2 is a non-reversible
degradation product.
Vinclozolin is registered in the United States and Europe as a fungicide for grapes,
strawberries, sunflowers, rape seed, soft fruits, hops, ornamental plants. It has been shown to
alter mammalian sex differentiation by inhibiting androgen receptor activity (Kelce et al, 1994).
Developing fetuses are extremely sensitive to vinclozohn exposure; exposures to rat fetuses has
been associated with infertility, deformed genitalia, and reduced sperm counts (Wong et al., 1995;
Gray, et al., 1994). Kelce, et al. (1996) have found that it induces antiandrogenic developmental
effects in vivo and that it inhibits androgen receptor (AR) binding and AR gene expression in vitro.
Vln clozolln
Figure 5 Structural formulae and degradation pathways of Vinclozolin and its principal degradation products
(from Szeto, et al., 1989a).
Physical and chemical processes determine the amount and form of Vinclozolin that may
reach the soil. Application rates and methods affect Vinclozolin degradation and persistence,
since it may applied be to foliage and above-ground plant parts and migrate downward to soils,
or it may be incorporated directly into soil; e.g., it is used to prevent onion white rot and other
fungi in bulb crops (Meister, 1996).
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Szeto, et al. (1989b) found that the pH of the soil, sediment, and water column is a
principal factor in vinclozolin degradation rates. The degradation is quite rapid at higher pH, and
much slower at low pH. At 35 ° C, the half-life at pH 8.3 is less than one hour, and at pH 4.5 is
530 hours. This difference can be explained in part to vinclozolin's increased resistance to
hydrolysis at lower pH. The pH also determines the principal degradation pathway that
vinclozolin will take, with higher pH yielding more Ml and lower pH yielding more M2. A third
degradation product, 3,5-dichloroaniline, has been detected after considerable time (672.3 h at pH
6.5, 1537 h at pH 5.5, and 505.8 h at pH 4.5). This points to important considerations for
estimating the fate of vinclozolin. Not only does increased soil and solution acidity increase
vinclozolin's persistence, but acidity also influences the degradation pathways and the appearance
of secondary degradation products.
The type of application solvent also influences vinclozolin degradation and bioavailability
of vinclozolin. Szeto, et al (1989c) compared the persistence of vinclozolin by applying the
fungicide in water and acetone solutions to young pea plants and analyzing the concentration of
vinclozolin in leaflets. The acetone-vinclozolin solution was significantly more persistent than the
water solution. This is likely the result of the dichorophenyl group's influence on the lipophilicity
of vinclozolin. The persistence observed in the field was lower than in the laboratory studies,
likely to do increased photodegradation and greater moisture gradients. However, these findings
indicate the importance of the original application solutions and suspensions when estimating the
ultimate fate of vinclozolin. They also indicate the importance of available moisture in the soil
column, which could serve to dilute the lipophilic application solvents over time.
The amount and degradation pathways in the atmosphere and fluxes from soil to air are
potentially important research areas. Since vinclozolin's vapor pressure is 1.6 xlO'5 kilopascals
(kPa) at 20° C, which is considered semivolatile, the atmospheric transport could be an important
exposure pathway. The volatility of its degradation products should also be considered in these
studies.
Organotin Fungicides
Organometals are an important group of endocrine disrupters. These are compounds with
covalent bonds between a carbon (C) and metal atom (Pelletier, 1995). Organotins have been
associated with endocrine dysfunction (Briischweiler, etai, 1996; Ochlmann, et al., 1993). Their
structures are shown in Figure 6.
Triphenyltin Acetate Triphenyltin Hydroxide
Figure 6 Structural formulae of triphenyltin acetate and triphenyltin hydroxide.
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Agricultural fungicide and scab biocide applications comprise the principal source of the
triphenyltins. Triphenyltin hydroxide is directly applied to soil and plant tissue as a fungicide and is
used to treat seeds, root, and tubers. Triphenyltin hydroxide is also the degradation product of
triphenyltin acetate, a fungicide for potatoes, rice, and sugar beets, and scab biocides for peanuts
(Tnayer, 1974). Other tin (Sn) compounds are used as fungicides and antifouling agents, most
notably tributyltin; but their usage has been greatly restricted in the past decade.
The mechanisms of toxic action for organotins are more diverse than those of vinclozolin.
They include cytotoxicity in the liver, disturbance of calcium homeostasis and induction of
apoptosis in thympcytes, inhibition of ATP-synthesis and mitochondrial oxidative
phosphorylation, inhibition and uncoupling of chloroplasts, ion pump inhibition and cell
membrane damage, Cytochrome P450 inhibition, and intracellular enzyme inhibition (Pent, 1996).
The principal endocrine endpoint observed to date is increased imposexual response in gastropods.
Mud snails and other gastropods exposed to tributyltin compounds exhibit an increased incidence
of pseudohermaphroditism (Bryan, et al., 1989 and Smith, 1981). Like mercury, Sn is neurotpxic.
The endocrine response may be the indirect result of Sn activity in neurological system, which
induces a chain of endogenous responses that ultimately elicit an endocrine response.
Most Sn-related research has focused on the aquatic environment, but fate and transport in
soils and the atmosphere must also be better explained. Figure 7 shows the principal degradation
pathway from the less stable acetate compounds to alkylated tin species mediated by microbes,
which theoretically will ultimately degrade the fungicides and metabolites to elemental Sn (Keijzer
and Loch, 1995). The process would likely move in the opposite direction under aerobic,
microbially mediated conditions (from oxides and elemental tins to alkylated species).
The amount of Sn species fluxing to the atmosphere is an area in need of research. The
volatility varies by species. The alkyltins are more volatile than the aryltins. Methyltins, for
example, are significantly more volatile than the phenyltins. Within the phenyltins, triphenyltin
acetate's vapor pressure is 1.9 xlO"6 kPa at 20° C, which is considered semivolatile, but
triphenyltin hydroxide is a salt that is nonvolatile. The volatility of each degradation product should
also be studied.
Sn_OH
SnO
MkrobM 2
Figure 7 Degradation pathway expected for tin fungicides under aerobic soil conditions (from Keijzer and Loch,
1995). ' '
Even the less stable organotin species can remain undegraded in soil under certain redox
and acidity conditions, and if soil organic matter is plentiful. For example, triphenyltin acetate has
been shown to accumulate in organic-rich upper soil horizons as a result of heavy application rates
in the Netherlands (Keijzer and Loch, 1995). This phenomenon can have profound effects on the
ability of certain tin compounds to migrate, after deposition under certain atmospheric and aquatic
conditions, but to remain in place for much longer time periods under other conditions.
Environmental acidity and redox conditions can also be affected by type of organic matter present
in soils and sediment. Kuballa, et al. (1995) found that humic substances act as methylating agents
for Sn in sediment in reduced soil profiles. Therefore, anaerobic soil conditions; e.g., in wetlands
and rice paddies, can play a major role in Sn methylation. Little is known about the persistence of
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the triphenyliin fungicides, but the persistence appears to be enhanced in reduced environments
(Pent, 1996). Speciation of organotins is also pH dependent. Within normal natural ranges,
higher pH values, where the hydroxides dominate, generally increase acute toxicity and uptake by
organisms. However, this is mediated by the presence of organic material. Soil moisture will
affect speciation of the tin compounds. For example, triphenyltin acetate is relatively hydrophilic
(9 mg/1 at 20° C), and is not lipophilic. Application rates will also affect soil-air fluxes by kinetic
and cheraicd processes; e.g., mass action.
In aquatic systems, increasing dissolved organic carbon reduces bioavaUability by the
creation of Sn-organic complexes. Therefore, organic matter in soils likely will play similar roles,
especially under saturated conditions.
Accurate means of extracting, separating, and detecting the various organotin species is
necessary to generate reliable estimates of exposures and risks. Analytical techniques for
speciating tins are improving. Barshick, et alr (1996) recently found new chromatographic
methods that show promise in improving the ability to speciate inorganic and organic forms of Sn
from a single soil. Future research applying th procedures to different soil types and specific Sn-
related research should advance analytical capability even further.
CONCLUSIONS
The challenge of addressing endocrine disrupter risks and exposures is daunting; however,
is has been shown that EPA's risk paradigm relates well to both human and ecosystem exposure
assessment. Presently, some strong weight-of-evidence in isolated studies of invertebrates, fish,
reptiles, birds and mammals has provided compelling reasons for linking exposures to a number of
chemical compounds to endocrine disruption mechanisms in populations. These ecosystem
observations suggest that similar human weight-of-evidence data bases, could be valuable in
directing future human endocrine disrupter exposure research.
The chemical classes represented by agricultural fungicides are sufficiently different in
chemical structure and mechanisms of endocrine action to provide insights into the chemodynamic
factors that are likely to influence human and ecosystem exposures. The two fungicides reviewed
in the present study represent chemical groups that have been associated with in vitro, in vivo, and
in situ endocrine effects. .
In soils and sediments, transformation pathways and kinetics are determined by a complex
interaction of processes that affect the amount and the degree of speciation of substances in the
soil, and the potential for their release and uptake by the atmosphere, surface water, groundwater,
and biota. Transport and transformation are function of characteristics of the compounds and
characteristics of the environmental media. Slight variations in physicochemical characteristics may
drastically change the potential for exposure and effects.
The cheraodynamic behavior of fungicides in soil is basic to predicting future exposures
and the efficacy of agricultural endocrine disruptor exposure prevention strategies. Improved flux
measurement methods to screen and to model exposures to endocrine disrupting pesticides in
various media nee1.' to be developed, validated, and incorporated into test guidelines, especially
those required unc~: Toxic Substances and Control Act and Federal Insecticide, Fungicide and
Rodenticide Act.
ACKNOWLEDGMENTS ,
I thank Dr. Ellen Cooler, Dr. Nancy Wilson, and Joe Bumgarner of the National Exposure
Research Laboratory for critical reading of the manuscript . Dr. Cooler's insights regarding
modeling and deposition were very helpful. Dr. Wilson's advice regarding chemical structures and
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Mr. Bumgamer's insights about nontraditonal pollutants improved the quality of the paper. I am
grateful to Dr. William Kelce of the .National Health and Environmental Effects Laboratory for
recommendations concerning toxicity and fate of vinclozolin. Finally, I acknowledge the valuable
advice of Dr. J. Jeffrey Peirce of Duke University and Dr. Viney P. Aneja of North Carolina State
University regarding contaminant fluxes from soil to the atmosphere.
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DISCLAIMER
This paper has been reviewed in accordance with the United States Environmental
Protection Agency's peer and administrative review policies and approved for presentation and
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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