STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C.
Requesting Phototoxicity Study Protocol for Light-Dependent
Request for Phototoxicity Study Protocol for Light-Dependent
Elizabeth Leovey, Acting Director Environmental Fate and
Effects Division Office of Pesticide Programs
Norman B. Birchfield, Ph.D. Thomas M. Steeger, Ph.D. Brian
Montague Aquatic Biology Tech Team
light-dependent peroxidizing herbicides (LDPHs) are a growing
class of weed control chemicals (see partial listing attached).
They act in plants by inhibiting the enzyme protoporphyrinogen
oxidase (protox), which is the last common enzyme in the
heme and chlorophyll biosynthetic pathways.1 Protox exists
in both plants and animals and the enzyme from both sources
has been shown to be highly sensitive to many LDPHs.2.
LDPH protox inhibition in plants results in a rapid accumulation
of protoporphyrin IX, a phototoxic heme and chlorophyll
precursor. In the presence of light, protoporphyrin IX is
a powerful generator of singlet oxygen which in plants causes
lipid membrane peroxidation leading to a rapid loss of turgidity
and foliar burns. LDPH exposure in mammals has been shown
to result in excretion of porphyrins in urine (porphynuria)
and feces, increased liver weight, elevated blood porphyrin
levels, developmental abnormalities, and cancer. Humans
with a hereditary protox disorder (variegate porphyria)
which results in lowered protox activity exhibit many symptoms
similar to LDPH exposure in addition to photosensitivity.
However, photosensitivity is not a commonly reported symptom
of LDPH exposure in animals.
LDPH-induced occurrence of phototoxicity in rats 3 and increased
cytotoxicity to human skin cells grown in culture in the
presence of light and an LDPH 4 have been reported but many
other LDPH toxicity studies make no mention of phototoxicity
in animals. The scarcity of phototoxicity data in animals
could result from physiological or biochemical distinctions
from plants. For instance, animals exposed to LDPHs may
not normally accumulate protoporphyrin IX in their epidermis.
However, phototoxicity may not be reported in many LDPH
toxicity tests because of relatively low light conditions
in laboratories and/or protection afforded by the animals'
fur or feathers. Animals without fur or feathers existing
in sunny environments would be expected to be at highest
risk for potential phototoxic effects.
Aquatic Biology Tech Team (ABTT) recommends that phototoxicity
studies be conducted on herbicides with this mode of action
to determine if animals exposed to LDPHs and intense light
(similar to sunlight) show increased toxicity relative to
controls exposed to LDPHs and low intensity light. The
results of these studies will help to determine if animals
that are exposed to sunlight in LDPH use areas are at higher
risk than guideline toxicity studies suggest.
ABTT is requesting that a LDPH phototoxicity protocol be
submitted for review and agreement by EFED and the registrant
prior to study initiation. Protocols for standard toxicity
tests have also been published.5 In nature, fish and other
aquatic organisms are expected to be exposed to LDPHs through
run-off and spray drift. Aquatic organisms inhabiting small,
shallow water bodies, exposed to high levels of solar radiation
would be expected to be at greatest risk for potential phototoxic
effects. Therefore, the ABTT is requesting a small fish
species be used in a phototoxicity assay to assess the potential
of light to increase LDPH toxicity.
ABTT requests that the study adequately address the following
issues and suggests the paper, "Photoenhanced Toxicity of
a Carbamate Insecticide to Early Life Stage Anuran Amphibians",5
and other studies in the peer-reviewed scientific literature
serve as sources of additional guidance:
The fathead minnow may be an appropriate test species because
of existing toxicity protocols which may be adapted for
A subchronic exposure duration would be adequate for proof
of principle. A single exposure may not allow adequate time
for porphyrin accumulation, however, a life-cycle is not
necessary to identify a phototoxic effect.
A range finding study should be conducted under defined
low light conditions to identify an LC50 value and lower
dose levels expected to be similar to controls. Doses used
in the phototoxicity study should not be expected to result
in significant mortality in low light controls. Dissolved
concentrations of the test chemical should be confirmed
by an appropriate analytical method.
Behavioral observations should be made in addition to measurements
of mortality, growth, weight, morphology, and appearance.
Ideally, measurements of protoporphyrin and heme concentrations
in the blood and protox activity in the liver of each test
organisms should be made.
Artificial light may be preferred to natural light that
will vary in different regions and seasons as well as with
weather. If artificial light is used, the light should resemble
full, natural sunlight as closely as possible, particularly
around 400 nm. The most important wavelength for porphyrin
induced phototoxicity in ~400 nm. No matter what the light
source, the duration and intensity of UV and visible light
should be reported at all wavelengths (200-800 nm). At this
point EFED does not have a specific recommendation for an
artificial light source.
light, and positive controls
As this study is intended to identify potential effects
of light on LDPH toxicity, an appropriate study protocol
should include a dark, or low light, control group. Another
group not exposed to chemicals but exposed to full light
should be included (a full light control). In addition to
the dark and light controls, a positive control group using
protoporphyrin IX may be useful.
chambers and light filters
Light intensity should be measured inside test chambers
if glass or any other material is placed between the light
source and the test animals. Any filters should be cured
under the study light for 72-hours prior to study initiation
to ensure consistent transmittance.
Matringe, M., J.-M. Camadro, P. Labbe, and R. Scalla. 1989.
Protoporphyrinogen oxidase as a molecular target for diphenyl
ether herbicides. Biochem. J. 260: 231-235.
Birchfield, N.B., and J.E. Casida. 1997. Protoporphyrinogen
oxidase of mouse and maize: Target site selectivity and
thiol effects on peroxidizing herbicide action. Pesticide
Biochemistry and Physiology 57, 36-43.
Halling, B.P., D.A. Yuhas, V.F. Fingar, and J.W. Winkleman.
1994. "Protoporphyrinogen oxidase inhibitors for tumor therapy"
in Porphyric Pesticides: Chemistry, Toxicology, and Pharmaceutical
Applications, (S.O. Duke and C.A. Rebeiz, Eds.) pp. 280-290,
American Chemical Society Symposium Series 559, Am. Chem.
Soc., Washington, D.C., 1994.
Birchfield, N.B. Protoporphyrinogen Oxidase as a Herbicide
Target: Characterization of the [ 3 H]Desmethylflumipropyn
Binding Site. Dissertation. University of California, Berkeley.
American Society for Testing and Materials. 1994. Standard
guide for conducting the frog embryo teratogenesis assay-Xenopus.
E 1439-91. In Annual Book of ASTM Standards, Vol 11.5, pp.
825-835. Philadelphia, PA.
Ref: Dec 11, 2001 - US EPA. Revised Environmental
Fate and Effects Division Preliminary Risk Assessment for the Oxyfluorfen Reregistration
Eligibility Decision Document.