Use and Role of Invertebrate Models in Endocrine Disruptor Research and Testing
Peter L. deFur
Abstract
Historically, invertebrates have been excellent models for
studying endocrine systems and for testing toxic chemicals.
Some invertebrate endocrine systems are well suited for
testing chemicals and environmental media because of the
ease of using certain species, their sensitivity to toxic
chemicals, and the broad choice of models from which to
choose. Such assays will be useful in identifying endocrine
disruptors to protect invertebrate populations and as screening systems for vertebrates. Hormone systems are found in
all animal phyla, although the most simple animals may
have only rudimentary endocrine systems. Invertebrate endocrine systems use a variety of types of hormones, including steroids, peptides, simple amides, and terpenes. The
most well-studied hormone systems are the molting and
juvenile hormones in insects, the molting hormones in crustaceans, and several of the neurohormones in molluscs and
arthropods. These groups offer several options for assays
that may be useful for predicting endocrine disruption in
invertebrates. A few invertebrate phyla offer predictive capabilities for understanding vertebrate endocrine-disrupting
chemicals. The echinoderms, and to a lesser extent molluscs, have closer evolutionary relationships with the vertebrates than the arthropods and these phyla. The recently
identified estrogen receptor structure within the genome of
the marine gastropod, Aplysia, indicates that the estrogens,
and probably the basic steroid receptor, are quite old evolutionarily. This review of the recent literature confirms the
effects of some endocrine-disrupting chemicals on invertebrates—tributyltin on snails, pesticides on insects and crustaceans, and industrial compounds on marine animals.
Key Words: ecdysone; endocrine disruptors; invertebrates;
juvenile hormone; methyl farnesoate; molting; testing
Introduction
Colborn and Clement (1992) addressed the problem of environmental chemicals interfering with endogenous hormone systems in a publication that assembled information
from scientists conducting research on experimental animals, wildlife, and human health. The contributing scientists and editors focused on the toxicological and
endocrinological similarities among responses of humans
and wildlife, specifically vertebrate wildlife. Since that
1992 publication, public interest has focused on human
health-related aspects of endocrine-disrupting chemical
(EDC1) exposures (Colborn et al. 1996; NRC 1999), although research scientists have investigated well-known
cases in fish and wildlife, especially those with obvious
endocrine effects (Kendall et al.1998; NRC 1999). Few reports (deFur et al. 1999) have considered the invertebrates
despite the abundance, importance, and utility of invertebrate systems in toxicology, the importance of invertebrates,
and the knowledge of insect endocrine systems (Fingerman
1997; Nijhout 1994). Authors of at least two recent reviews
(Hutchinson et al. 2000; Oberdorster and Cheek 2001) and
one research needs summary (Ankley et al. 1998) evaluated
the use of invertebrate assays in assessing EDC activity,
and concluded that invertebrate assays are an important part
of a comprehensive program that seeks to protect natural
systems.
There are excellent reasons to include invertebrate assays as necessary components in the research program and
efforts to control or regulate EDCs (Guillette and Crain
2000). First, invertebrate animals are essential elements of
the planet Earth’s ecosystems, necessitating protection from
the harmful effects of EDCs. Invertebrates provide food for
other animals, transfer and store metabolic energy in trophic
systems, and decompose plant and animal material. The
effects of EDCs on invertebrates, however, may well go
un-noticed for some time, as was the case with marine gastropods (reviewed in deFur et al. 1999), with far-reaching
and even devastating consequences.
Second, invertebrate EDC assays may be useful in predicting or indicating potential EDC responses in vertebrates,
in part because some invertebrates can be more readily manipulated than vertebrate systems. In this capacity, invertebrate assays may serve either as sentinels of potential effects
from exposure to conditions or chemicals, or as actual predictors of effects that have a counterpart in other or many
1
Peter L. deFur, Ph.D., is an Affiliate Associate Professor at the Center for
Environmental Studies, Virginia Commonwealth University, Richmond,
Virginia.
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Abbreviations used in this article: ASTM, American Society for Testing
and Materials; Ecy, ecdysone; ED, endocrine-disrupting; EDC, endocrinedisrupting chemical; EPA, Environmental Protection Agency; JH, juvenile
hormone; MF, methyl farnesoate; MIF, molt-inhibiting hormone; TBT,
tributyltin.
ILAR Journal
species. Invertebrates have been used for decades as the
primary research model in such fields as neurobiology,
muscle physiology, and structure and function of hemoglobin, among other areas (Mangum and Hochachka 1998).
Advantages of select invertebrates systems include the ease
of manipulation, diversity of animals for test systems, short
generation times, ease of culture of some species, and fewer
legal/regulatory issues.
This is not to say that all species are useful or usable for
all purposes, but that among the hundreds of thousands of
species of invertebrates, a large number have great value
and potential in EDC assays. Two notable challenges using
invertebrate assays as predictors for vertebrate systems are
(1) identifying the functional relationships between invertebrate and vertebrate responses, and (2) the inability of
invertebrate systems to duplicate whole animal vertebrate
systems. Luciferase and its gene comprise one notable example of invertebrate material that has been used with great
success in various molecular and cellular assays. In addition, some invertebrates may contain genes or other biological components that are sufficiently similar to vertebrates to
offer predictive powers. Thornton and colleagues (2003)
recently demonstrated that invertebrates also contain the
DNA sequences (and thus, the putative genes) for vertebrate
hormonal systems, even when the invertebrate gene is a
homolog or analog.
The current federal effort of the Environmental Protection Agency (EPA1) to screen and test chemicals for endocrine activity emphasizes the effects of EDCs on humans.
The Endocrine Disruptor Screening and Testing Advisory
Committee (EDSTAC1) recommended the examination of
three specific vertebrate hormonal systems—estrogen, androgen, and thyroid (EDSTAC 1998). Although the screening and testing program now focuses on vertebrates, both
the congressional authorization (the Safe Drinking Water
Act of 1996) and EPA directives include other hormones
and other animals. The battery of assays that EDSTAC recommended does include one arthropod, the Mysid shrimp
assay. The basis for including the Mysid assay is that the
assay is sensitive, is known to respond toxicologically, and
has been used for decades in aquatic toxicology (ASTM
1993). This assay should be able to detect at least some of
the invertebrate endpoints, at least for arthropods. However,
incorporation of additional invertebrate assays will offer
greater species diversity and improve the likelihood of preventing harm to ecological and human systems.
In this article, the role of invertebrates in the suite of
tools for detecting EDCs and protecting environmental resources against harm from these compounds is discussed.
The text provides a context for EDCs in invertebrates, a
brief summary of invertebrate endocrine systems, and several examples of known EDC effects with assays that have
proven useful in evaluating chemicals. Exhaustive reviews
of invertebrate endocrinology, toxicology, or the literature
on endocrine disruptors are beyond the scope of this article,
and the reader is referred to other sources for those reviews
(e.g., deFur et al. 1999; Fingerman 1997).
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2004
Historical Consideration of EDCs in Insects
Synthetic chemicals were first intentionally developed and
used as endocrine disruptors in the late 1970s as agents to
control invertebrates, specifically insects (summarized in
deFur et al. 1999). These insecticides were the modern generation of chemicals intended to target a specific group of
pests by disrupting the hormone system that controlled
growth or molting or both, and they were first called insect
growth regulators. Chemical companies focused on the
molting system and on juvenile hormones (JHs1) that control maturation or development at the molt in insects. The
molting hormone itself did not prove to be highly specific
and practicable, but the JHs could be mimicked or blocked,
and with some specificity. Interestingly, enzyme systems
that regulate chitin synthesis (chitin synthetase) are also
sensitive to chemical alteration by pesticides (e.g., diflubenzuron), even though chitin synthesis is not functionally part
of an endocrine pathway (deFur et al. 1999).
The discovery of the JH structure led to the synthesis of
JH agonists for use as insect control agents that would disrupt the normal molting and metamorphosis processes (LeBlanc et al. 1999). The JH agonists proved to be stable,
specific, readily manufactured and effective on target species with little effect on nontarget organisms. The ecdysteroids, developed to disrupt the molting system, had not
demonstrated all of these features; they were more difficult
to synthesize, proved less stable, and affected nontarget organisms. Since their discovery, several insecticides have
been synthesized commercially and have mainly targeted
the JH system, although the newer pesticide tebufenozide is
an agonist of 20-OH ecdysone (Ecy1) (reviewed in LeBlanc
et al.1999).
Insect endocrine-disrupting growth regulators may target either the JH or Ecy system in several ways. First, the
agent may act as an antagonist or agonist, thereby directly
interfering with the target tissues in the organism. Second,
the agent may target the organ that synthesizes JH, the
corpora allata in insects. Third, the agent may affect one of
the other hormones or organs involved in the process, such
as uptake or release of calcium or cholesterol on which the
molting hormone system depends (reviewed in LeBlanc et
al. 1999).
Summary of Invertebrate Endocrinology
The invertebrate phyla include an incredibly diverse array
of animal forms, from simple sponges to insects, hemichordates (acorn worms), and urochordates (sea squirts). Arthropods constitute the largest and most abundant group of
invertebrates, in no small part from the number of insect
species and the sheer abundance of microscopic plankton in
the sea. Molluscs are often considered the most diverse
phylum because of the body forms and life histories represented by bivalves, which comprise seven diverse classes,
including the bivalves, gastropods, and cephalopods, plus
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four minor groups. A number of invertebrate groups (annelids, nematodes, and arthropods) include important parasites
(endo or ecto) on humans, livestock, or domestic animals,
making these animals important in the work of veterinary
(as well as human) medicine.
Invertebrates use a wide variety of hormones to regulate
growth, development, metabolism, and other physiological
processes in the same manner as vertebrates. Invertebrate
hormones include, at a minimum, steroids, proteins, terpenoids, and amides, with the great likelihood that other molecules will be identified as research progresses (reviewed in
deFur et al. 1999). The most well-known and -studied hormone systems are those of the insects because of the economic and ecological importance of this class of animals.
The crustacean endocrine systems, similar to insects, are not
as well known but appear equally as complex, relying on
steroid, terpenoid, and protein hormones. Other invertebrates have not been as well studied, with the result that the
distribution of hormonal types is poorly known. Most invertebrates, however, appear to rely on nonsteroidal hormones (and receptors) to regulate biological functions,
although not to the complete exclusion of steroid hormones.
Invertebrate endocrine systems are composed primarily
of neuroendocrine components, with fewer true glands than
are present in the vertebrates. The arthropods, notably the
insects and crustaceans, are the only group with true endocrine glands derived from epithelial tissue and functioning
the way vertebrate glands function. The arthropods, especially insects, have been studied more extensively than other
invertebrate phyla. Other arthropods, such as the horseshoe
crabs, spiders, and mites, possess true glands but the systems are not as well described.
The most well-studied hormonally controlled system in
arthropods is molting. The process of development and/or
maturation is critical in a specific molt during the life of
every arthropod. In most if not all arthropods, there is a
specific molt at which the reproductively viable form develops from the juvenile. The maturation and molting processes are under endocrine control from hormones other
than the molting hormone.
Molting hormone, or Ecy, is a steroid hormone that acts
in arthropods much as the vertebrate steroid hormones act,
through a nuclear receptor system that binds to DNA. Ecy
occurs in arthropods in several forms (Figure 1), depending
on species and metabolism. The active form in most arthropods is 20-OH Ecy.
The molting hormone in insects is chemically identical
to the molecule found in other arthropods, including crabs,
shrimp, crayfish, lobster, spiders, mites, and horseshoe
crabs. One of the more interesting properties of the Ecy
system is the structural similarity between the arthropod
nuclear protein receptor for Ecy and the vertebrate retinoic
acid receptor. The Ecy nuclear receptor is a dimer, of which
one unit, termed ultraspiracle, is homologous to the retinoid
X receptor. This characteristic places Ecy in the retinoic
acid group, rather than the estrogen steroid family.
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Crustacean molting hormone systems are not as well
known as insect molting hormone systems and have several
important differences. The molting hormone Ecy is the
identical molecule in both groups, and is active as the 20OH form of the compound. Ecy and 20-OH Ecy are not the
only active compounds; rather, several different ecdysteroids occur in the various arthropods, although 20-Ecy is the
most common (Figure 1). In both groups, Ecy released into
the hemolymph prepares metabolic processes before the actual molt. To this point, all arthropods are similar. The
differences reside in the control over release synthesis and
of ecdysteroids. In insects, Ecy synthesis and release from
the prothoracic gland is under positive control exerted by
the prothoracotropic hormone, which is secreted by neurosecretory cells in the brain (reviewed in deFur et al., 1999,
LeBlanc et al. 1999; Nijhout 1994). In crustaceans, Ecy
synthesis and release are under negative control from moltinhibiting hormone (MIH1). MIH secretion declines in the
premolt period, permitting Ecy titers to increase in preparation for molting.
Insect molting and maturation are also affected by the
hormone that maintains the juvenile form, JH (Nijhout
1994). The presence of this hormone at the appropriate time
preceding the molt will result in a juvenile form; the withdrawal of JH will cause the molting insect to develop mature features and characteristics when other factors such as
body mass are sufficient. Thus, JH exerts a negative or
inhibitory action on the maturation process at the molt in
insects. Changes in normal JH titer in the premolt period can
alter the normal molt cycle and pattern in insects.
The crustacean counterpart to insect JH differs in several functional aspects from the insect system. After a scientific search of many years for the JH counterpart in
crustaceans, Laufer and colleagues (1987) finally identified
methyl farnesoate (MF1) (Figure 2) as the homologous compound in crustaceans. MF is structurally similar to one form
of JH, JH III. In crustaceans, MF has juvenoid activity. It
also stimulates reproductive organ growth and maturation in
several species. The presence of MF in premolt animals
appears to stimulate vitellogenesis and organ maturation in
both male and female aquatic crustaceans. Research by LeBlanc (1999) indicates that MF plays an important role in
reproductive maturation and development in the microcrustacean Daphnia, and the conservative nature of crustacean
endocrine systems suggests that similar patterns exist in
other species.
Invertebrate Endocrine Disruptors
Several compounds or groups of compounds are known to
disrupt specific invertebrate endocrine systems, including
development and reproductive function (Table 1). These
substances currently serve as the reference standards for the
scientific and regulatory communities to use in developing
screening and testing assays, model systems, and investigative approaches to detect EDCs and increase knowledge of
ILAR Journal
Figure 1 Ecdysteroid hormones of insects and crustaceans.
the phenomenon. These cases address disruption of insect
molting and development, molluscan sexual development,
and crustacean development.
JH analogs constitute the class of modern insecticides
manufactured successfully to target specific insect groups or
species in an attempt to avoid mortalities in nontarget populations. As described above and summarized in several reviews (deFur et al. 1999; Hutchinson et al. 2000;
Oberdorster and Cheek 2001), these compounds act as JH
agonists or antagonists and thereby interfere with normal
metamorphosis at the molt, resulting in death, lethal deformities, or sterility.
Tributyltin (TBT1), one of the best known invertebrate
EDCs, is an organo-tin compound that was formulated and
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2004
used as an antifouling compound in the paint of ship hulls
for many years. One of several organotin compounds, TBT
was the most commonly used antifouling agent and the one
that has received the most attention. In the 1980s, researchers discovered that TBT leaches out of paint into marine
waters and alters reproductive development of gastropod
snails at low levels (as low as a few parts per billion [see
Bender and Huggett 1987 for discussion of early investigations]). TBT stimulates penis growth in female snails, imposing one organ on the other and preventing reproduction.
The resulting conditions, termed imposex or intersex, result
in reproductive failure sterility that has caused population
declines in snails in countries around the world. The mechanism involves alteration of normal testosterone activity but
487
Figure 2 Juvenoid hormones of insects (JH I, II, and II) and crustaceans (methyl farnesoate).
may include other mechanisms as well (Gooding and LeBlanc 2002).
Chlordecone is an organo-chlorine pesticide that was
manufactured in Hopewell, Virginia, under the trade name
Kepone, until the mid 1970s, when a series of events raised
the alarm that the James River was contaminated with the
pesticide. Since that time, chlordecone has been found to be
estrogenic in in vitro cell assays (Soto et al. 1992), although
it was originally shown to disrupt several hormonally regulated biological processes such as shell formation and molting in blue crabs and insects (Roberts and Leggett 1980;
Schimmel and Wilson 1977). The manufacture and sale of
Kepone was subsequently prohibited; however, the contamination was so extensive that cleanup was not consid-
Table 1 Recent examples of invertebrate endocrine disruption
Compound
Animal
Effect
Reference (see text)
Bisphenol A
Snails
Maris cornuarietis
Nucella lapillus
Snails
Maris cornuarietis
Nucella lapillus
Oyster
Crassostrea gigas
Daphnia magna
Abalone
Haliotis gigantea
Grass shrimp
Palaemonetes pugio
Grass shrimp
Palaemonetes pugio
Copepod
Amphiascus tenuiremis
Impaired/enhanced reproductive organ growth
Oehlmann et al. 2000
Impaired/enhanced reproductive organ growth
Oehlmann et al. 2000
Feminization; altered sex ratios
Nice et al. 2003
LeBlanc et al. 2000
Octylphenol
Nonylphenol
Tributyltin
Endosulfan
Methoprene
Fipronil
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Impairs development
Masculinization of females
Horiguchi et al. 2002
Acute mortality; delayed embryo hatch
Wirth et al. 2001
Acute mortality; delayed embryo hatch;
altered growth
Male infertility
Wirth et al. 2001
McKenney and Celestial 1993
Chandler et al. 2004
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ered practical and “natural attenuation” was used to deal
with the problem.
Several modern pesticides and industrial chemicals are
known to disrupt invertebrate endocrine systems, or exert
toxic effects that are consistent with endocrine disruption
(Table 1; Figure 3). As with vertebrates, endocrine disruption is more difficult to document in practice because of the
need to know the functional interaction of the chemical and
the endocrine system (EDSTAC 1998). Inherent in the characteristics of these chemicals are two important types of
evidence regarding screening and testing for endocrine
disruption. First is the fact that invertebrates are equally as
susceptible to the effects of chemicals that have been
described in vertebrates (Colborn and Clement 1992; Kendall et al. 1998; NRC 1999) and attributed as endocrinedisrupting (ED 1 ). Second is the promise of using
invertebrates in additional or replacement screening and
testing assays for EDCs. This suggestion was made by a
group of experts on the subject (deFur et al. 1999), and
included lists of assays, endpoints, species (Table 2), and
fruitful areas for further investigation in laboratory and field
work.
Pesticides are not the only chemicals that have been
identified as EDCs in invertebrates; several industrial
compounds have been found to alter arthropod hormone
systems. The phenolic compounds bisphenol A, nonylphenol, and octylphenol all have been identified as estrogenic
in in vitro assays (Soto et al 1992) and in some animal
assays (Colborn et al. 1993). The similarities in structure
among the ecdysone and estrogenic steroids suggest crossreactivity among the systems, despite the fact that the
receptors are not considered in the same molecular families.
Endosulfan is a pesticide commonly applied in the
southeastern United States to various crops, including tomatoes (reviewed in Wirth et al. 2001). Endosulfan exists in
three chemical forms, as alpha or beta forms, plus the sulfate salt; all are toxic. Endosulfan has estrogenic activity in
vitro (Soto et al. 1992) and is highly toxic to aquatic animals, notably invertebrates. Recent research indicates that
endosulfan interferes with molting and development in both
freshwater (Foersom et al. 2001) and marine (Wirth et al.
2001) crustaceans at exposures of parts per trillion. The
mechanism of action of endosulfan has not been elucidated
in invertebrates.
Fipronil is a recently licensed pesticide used for insect
control. One use is as an internal pesticide used for domestic
animals (Chandler et al. 2004). Fipronil is an intentional endocrine disruptor that targets the JH system in insects and is
Table 2 Examples of existing assays that could be used for EDC determination in invertebratesa
Group
Assays
Endpoints
Insects
Expose early instar larvae: precocious
metamorphosis
Expose normal Manduca sexta: If it becomes
black = JHb antagonist
In vitro Manduca sexta epidermal (wound) assay
Galleria wax wound assay
Black Manduca sexta larval test
Tenebrio: morphological assay
Locust adult: destroy CA;b agonist to
vitrellogenin production
Cockroach: accessory gland development assay
Mosquito larvae: prevent emergence of adults,
egg hatch
Inverse of Calliphora pupariation assay
Molting: premature metamorphosis (small adults)
Inhibition of vitellogenin synthesis in some
insects
Insects
Insects/Crustaceans
Insects/Crustaceans
Calliphora pupariation assay
Ecdysteroid-responsive cell lines
Ecdysone receptor binding assay
Insects/ Crustaceans
Various types
Mysid shrimp and grass shrimp assays–96 hr to
multiple generations
Molting: extra larval instars, or deformed
intermediates formed, or absence of adults
Fecundity
Egg mortality
Inhibition of molting and metamorphosis
Inhibtion of egg production in Diptera
Inhibition of feeding
Molting: premature incomplete molt with double
head capsule in Lepidoptera, followed by
lethality (deformed and molt susceptible to
predation)
Vitellogenin synthesis in Diptera and some
crustaceans
Mating behavior (sex attractants)
Disruption of behavior
Reproduction–egg production, viability
development—sex ratio, survival
a
Summarized from deFur PL, Crane M, Ingersoll C, Tattersfield L, eds. 1999. Endocrine Disruption in Invertebrates: Endocrinology, Testing, and
Assessment. Pensacola FL: Society of Environmental Toxicology and Chemistry.
b
JH, juvenile hormone; CA, corpora allata.
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nontoxic to vertebrates. Hence, it is possible to use this
compound internally without harming vertebrates.
Methoprene is manufactured as an insecticidal endocrine disruptor that targets the juvenile hormone system of
juvenile (larval) insects as a JH agonist. This compound has
been used effectively to control mosquitoes and other
aquatic insects. The crustacean hormone methyl farnesoate
is remarkably similar in structure to insect JH (variant III),
therefore there is reason to suspect that methoprene and/or
its breakdown products will be active in crustaceans. Methoprene is toxic to several aquatic crustaceans, notably the
grass shrimp Palaemonetes pugio, at parts per billion levels
(Wirth et al. 2001).
Invertebrate Testing Models
EPA and the American Society for Testing and Materials
(ASTM1) have developed a range of assay systems for testing chemicals under different regulatory programs and applications. Each organization has prepared a list of the
assays that use invertebrates for toxicity assessment; the
descriptions include specific procedures and methods as
well as endpoints. These assays often use aquatic animals
because of the practical ease of administering dose. Test
systems that were recommended by experts in the field
(deFur et al. 1999) include clams, worms, microcrustaceans,
grass shrimp, fruit flies, midges, snails, and echinoderms
(sea urchins and star fish), among others. ASTM (1993) has
procedures for these animals or groups and for the types of
endpoints of interest in ED evaluations. What is not certain
is the ED connection—whether the effect on a hormonally
activated system is evidence of endocrine disruption, and
whether the converse is also the case. An exhaustive review
of all invertebrate assays of possible use in EDC screening
is not practical in this review, but some important developments are discussed below.
Researchers have assigned great importance to the potential results and possibilities of using aquatic crustaceans
in assays for EDCs. Indeed, of the three different species
used in assays, each offers excellent options for EDC
screening: benthic copepods (Amphiascus tenuiremis)
(Chandler and Green 2001), grass shrimp (Palaemonetes
pugio) (McKenney 1998), and water fleas (Daphnia magna)
(LeBlanc et al. 2000). All ASTM methods and EPA procedures support each assay, and larvae of other species (e.g.,
crayfish) could be used in these assays, once they have
adapted and appropriately validated.
Staton and colleagues (2002) described a method that
uses modern approaches for assaying chemicals in saline
waters using a benthic copepod. The assay uses a 96-well
plate, making the procedure and equipment straightforward
and simple for replicating treatments. The problems of the
microscopic size of the copepod are offset by the advantages of short life cycle, ease of culture, and capability of
replicating and isolating individuals. Because many chemical measurements can now be made on microscale samples,
490
the size of the biological material for sampling is not a
problem for the most part.
Grass shrimp and mysid shrimp (Mysidopsis spp. and
Neomysis spp.) have been used in aquatic toxicity testing for
a number of years. EPA has published and approved methods for these species (EPA 2002), as has ASTM (ASTM
1993). The procedures have been described and the effectiveness evaluated by McKenny (1998) and McKenny and
Celestial (1993). The procedures call for collecting wild
stock to establish clean cultures that are free from infections, or for using stocks from other laboratories (EPA,
university, or commercial laboratories). Animals held in
flow-through conditions are exposed to test chemicals for
various periods, and reproductive and developmental outcomes are assessed via gross observation and microscopic
examination of the eggs and offspring. Egg production and
offspring viability are two of the important endpoints, but
other endpoints can be added as appropriate. Grass shrimp
have alternating generations in summer and winter, and the
production of seasonally appropriate eggs is an important
endpoint that will have significant effects on the populations
in the field (deFur et al. 1999).
Molluscs, especially snails, oysters, and clams, will continue to be a significant component of the invertebrate EDC
assay system because TBT-induced imposex remains the
best case of field-demonstrated ED effects. Recent reports
of effects of EDCs on bivalves (Table 1) demonstrate the
importance of continuing research in such other animal systems. Bivalves are particularly important owing to the economic and ecological significance of oysters (and clams),
their worldwide distribution, and their availability in areas
where bivalves are cultured.
Sea urchins have been used historically as an experimental model for embryology and developmental biology
because of the similarity to vertebrates in the early developmental processes. Evolutionarily, the phylum is on the
branch with vertebrates (and molluscs), giving support to
the biological basis for this group. ASTM methods exist for
urchins, and the vertebrate steroids have been reported in
the echinoderms (Botticelli et al. 1960).
Future Research
EPA is currently developing and validating screening assays
to identify EDCs and to estimate the effects of EDCs on fish
(Ankley and Johnson 2004), wildlife (vertebrates and Mysid
shrimp,) and humans (see http://www.epa.gov/scipoly/
oscpendo/index.htm for program status). As noted above,
both on EPA’s web site and in the EDSTAC report (EDSTAC 1998), the assays under development by EPA focus
on human health, with some consideration for lower vertebrates; however, the EPA battery of assays includes only
one invertebrate assay. In the opinion of this author, the
program will fall short of its potential to estimate the effects
of EDCs if a representative range of invertebrate species are
not assessed. Invertebrate assays are necessary to provide
ILAR Journal
comprehensive environmental protection, to predict harmful
effects, and to use evolutionary conservatism among animals as a means of assaying more efficiently for effects of
chemicals on a wide array of species. EPA should develop
and validate additional assays to screen chemicals (both
industrial and pesticides) effectively for ED activity and
effects. This task will be challenging because of the diversity of forms, life histories, and biology in the invertebrate
phyla, and the nearly exclusionary focus on mammalian
experimental models in the past few decades. Fortunately,
several options exist now for EPA and the other federal
agencies to select and validate invertebrate assays.
The National Science Foundation continues to support
basic research in comparative endocrinology, and to provide
a scientific base for the protection of animals from EDCs.
The National Institutes for Environmental Health Sciences,
and EPA (in the STAR program (http://es.epa.gov/ncer/
grants/), support applied research on EDCs, especially related to human health. ASTM and EPA have developed and
published numerous laboratory assays using invertebrates
for evaluating toxicity of chemicals. These assay systems
include animals that range from Daphnia to earthworms to
crayfish (Table 2). The most popular assays use microcrustaceans, owing to their ease of culture, short life cycles, and
replicability in a small space (see LeBlanc 1999). At least
two other arthropods, mysids and harpactacoid copepods,
will hopefully receive increased attention, and for quite different reasons.
The east coast of the United States is home to one of
four living species of horseshoe crabs, Limulus polyphemus,
that occurs from Maine to Florida, with spawning throughout the range. Horseshoe crabs were once abundant in MidAtlantic coastal waters and on the beaches during the spring
spawns (May-June), but commercial harvesting for bait and
blood (hemolymph) has greatly reduced the population.
This arthropod uses the same 20-OH Ecy as the molt hormone, but juvenoid hormones have not been identified (Jengla 1982). This species is of critical economic and
ecological importance to the coastal region, and the eggs are
the primary food item of migratory shore birds. Thus, determining the basic biology of this species and further elucidating the cause of population declines and spawning
limits have great importance. It is especially important to
understand whether EDCs may be contributing to this population decline.
The evolutionary relation among the animal phyla
places molluscs and echinoderms in a more central position
in relation to vertebrates and invertebrates. For this reason,
it is not surprising that many molecules found in vertebrates
appear in the lower taxa (reviewed in Mangum and Hochachka 1998). Nor should it be surprising that an estrogen
receptor ortholog was recently reported in the marine gastropod Aplysia californica (Thornton et al. 2003). Thornton
and colleagues (2003) note that the gene sequence identified
in Aplysia is not a functional estrogen receptor that participates in the neuroendocrine system, but is instead an evolutionary vestige lost from the arthropod lineage and not
hormonally active. The significance of this result is twofold.
First, the finding supports the hypothesized evolutionary
age of the estrogen family of steroids and receptors, not-
Figure 3 Examples of pesticides that have juvenile hormone activity and may also affect crustaceans.
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2004
491
withstanding existing data on the presence of estrogens and
androgens in the molluscs and echinoderms. Second, the
presence of these genes in Aplysia offers the possibility of
using more simple systems to study and identify EDCs in a
range of phyla if the Aplysia gene is found to be functional
in other groups.
Conclusions
Invertebrate species have been used effectively in screening
and testing assays for many years, and such applications
should now be used in screening and testing programs for
EDCs. This application of basic biological research builds
on decades of laboratory investigations demonstrating the
conservative nature of basic biological processes, structures,
and functions. Basic research has elucidated a number of
characteristics of invertebrate endocrine systems that are
both common to vertebrates, as well as unique. Both types
of characteristics can and should be exploited in using invertebrates in EDC screening and testing. At present, the
endocrine systems of insects, crustaceans, echinoderms, and
molluscs offer real opportunities for identifying EDCs that
may act on higher vertebrates or on the ecologically and
economically important invertebrate resources of the earth.
These groups, as well as others, also deserve further investigation in research and development laboratories as EPA
continues to develop a comprehensive screening and testing
program for EDCs.
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