Environmental Toxicology and Chemistry, Vol. 23, No. 6, pp.

1441–1451, 2004
q 2004 SETAC
Printed in the USA
0730-7268/04 $12.00 1 .00

Environmental Toxicology
DEVELOPMENT OF FRESHWATER WATER-QUALITY CRITERIA FOR PERCHLORATE

KIRK E. DEAN,*† RANDY M. PALACHEK,† JILL M. NOEL,† RYAN WARBRITTON,‡ JOHN AUFDERHEIDE,‡ and
JODY WIREMAN§
†Parsons, 8000 Centre Park Drive, Suite 200, Austin, Texas 78754, USA
‡ABC Laboratories, Chemical Development Group, 7200 E. ABC Lane, Columbia, Missouri 65202, USA
§Air Force Institute for Operational Health, AFIOH/RSRE, 2513 Kennedy Circle, Brooks AFB, Texas 78235, USA

( Received 2 January 2003; Accepted 10 October 2003)

Abstract—The anion perchlorate (ClO24 ) is an oxidizing component commonly used in solid propellants for rockets and missiles;
in explosives, flares, fireworks, chemical processes, and automobile air-bag inflators; and for other assorted uses. With recent
advances in analytical detection capability, perchlorate has been found in a variety of ground and surface waters throughout the
United States. Because perchlorate has been associated with thyroid problems in humans and may have similar effects on wildlife,
it is desirable to develop a water-quality criterion to assist in identifying concentrations of perchlorate in water likely to pose an
ecological health risk. In the present study, we compiled all available data regarding the effects of perchlorate to aquatic organisms,
and we performed additional toxicity and bioconcentration tests as required by the U.S. Environmental Protection Agency (U.S.
EPA) for the development of water-quality criteria for aquatic life. A criterion maximum concentration of 20 mg/L and a criterion
continuous concentration of 9.3 mg/L were calculated based on the test results. Although these are not formal Clean Water Act
Section 304(a) criteria, which must be published by the U.S. EPA, these criteria may be useful in the determination of remedial
action levels for contaminated sites, for National Pollutant Discharge Elimination System permit limits, and other water-quality
management practices.

Keywords—Perchlorate Water-quality criteria

INTRODUCTION ample, the aqueous solubility of sodium perchlorate is almost

In the 1990s, the perchlorate ion emerged as a contaminant
of concern in ground- and surface waters [1]. Even at low
doses, perchlorate (ClO24 ) is reported to modify mammalian
thyroid function by competitive inhibition of iodide uptake,
resulting in reduced thyroid hormone production as well as in
thyroid tumors [2–6]. Thyroid hormone deficiencies can affect
normal metabolism, growth, and development. Because of the
mobility and stability of perchlorate in the aqueous environ-
ment, aquatic organisms are predicted to receive the highest
exposure [7]. The objective of the present study was to develop
acute and chronic water-quality criteria for perchlorate to pro-
tect aquatic life in freshwater.
Perchlorate is an oxidizing component used in solid pro-
pellants for rockets and missiles; in explosives, flares, fire-
works, chemical processes, and automobile air-bag inflators;
and for other assorted items. Perchlorate also occurs naturally,
notably in some Chilean caliche ores, which are used as a
nitrate fertilizer in the United States [8].
Environmental perchlorate contamination has resulted from
discharges to ground and surface waters from manufacturing
and production operations, waste handling and washing op-
erations, research and testing, and use of perchlorate in in-
dustrial, military, and agricultural practices [9]. In particular,
the military, space program, and supporting industries, which
use a high percentage of all manufactured perchlorate, have
been identified with perchlorate releases to the environment.
Perchlorate has been used or manufactured in at least 40 states
[9].
Perchlorate salts tend to be highly water-soluble; for ex-
* To whom correspondence may be addressed
(kirk.dean@parsons.com).
1441
8 M [10]. Perchlorate is a strong oxidizing agent, but its re-
duction is so kinetically limited that it is practically unreactive
in dilute aqueous solution at environmental temperatures and
at pH .1 [10]. The perchlorate ion is also a weakly complexing
anion, and its adsorption to minerals tends to be weak and
reversible [8]. For these reasons, perchlorate is exceedingly
mobile and can persist for many decades under typical ground-
and surface water conditions [9].
Perchlorate was not observed in natural waters, except at
a few Superfund sites, until approximately 1997, when a new
analytical method was developed with a much lower detection
limit (;0.004 mg/L). To date, few measurements of perchlo-
rate concentrations in natural waters have been obtained. Most
of the ambient water measurements have been collected from
shallow groundwater near contaminated sites. Perchlorate con-
centrations in groundwater reached 3,700 mg/L near a man-
ufacturing facility in Henderson (NV, USA), 640 mg/L at a
rocket manufacturing facility in Rancho Cordova (CA, USA),
and 169 mg/L at the Longhorn Army Ammunition Plant in
Karnack (TX, USA) [9]. Reports of perchlorate measurements
in surface waters are even rarer. In East Camden (AR, USA),
a perchlorate concentration of 480 mg/L was observed in sur-
face water near a rocket manufacturing facility [9]. A per-
chlorate concentration of 130 mg/L was observed in the Las
Vegas Wash (USA), a stream flowing into Lake Mead (USA)
that is influenced by perchlorate-contaminated groundwater
[11]. At Allegany Ballistics Laboratory (Rocket Center, WV,
USA), perchlorate was detected in 21 of 23 samples, with
average and maximum measured concentrations of 0.212 and
0.280 mg/L, respectively [11]. At Indian Head Naval Surface
Warfare Center (MD, USA), perchlorate was detected in 27
of 36 surface water samples, with average and maximum mea-
1442 Environ. Toxicol. Chem. 23, 2004 K.E. Dean et al.

Table 1. Maximum perchlorate concentrations observed in various environmental media at six U.S. locationsa

Measured perchlorate concentration

(mg/L [liquid] or mg/kg [solid])

Media n % Detectsb Average Maximum Site of maximum concentration

Soil 113 38 57.7 1,470 Lake Mead area (NV, USA)

Terrestrial vegetation 177 50 34.7 428 Lake Mead area
Aquatic vegetation 50 24 38.8 176 Lake Mead area
Surface water 147 57 10.5 130 Lake Mead area
Pore water 10 50 19.6 98.0 Lake Mead area
Sediment 93 23 12.8 56.0 Lake Mead area
Terrestrial mammal 88 18 13.4 53.0 Lake Mead area
Fish 107 3 16.4 44.3 Lake Mead area
Terrestrial insect 33 12 12.6 6.2 Allegany Ballistics Laboratory (WV, USA)
Terrestrial bird 42 12 1.5 4.2 Lake Mead area
Amphibian 22 12 1.1 1.7 Longhorn Army Ammunition Plant (TX, USA)
Aquatic insect 4 75 0.74 1.0 Longhorn Army Ammunition Plant
a Data from Parsons [11].
b The percentage of samples in which perchlorate was determined to be present.

sured concentrations of 0.018 and 0.025 mg/L, respectively
[11].
In surface waters not in the immediate vicinity of contam-
inated sites, it appears that perchlorate concentrations are typ-
ically less than 0.1 mg/L. Low concentrations of perchlorate
have been found in Lake Mead (,0.10 mg/L) and in the Col-
orado River downstream of Lake Mead (,0.01 mg/L) [9].
Further downstream, in the vicinity of Yuma (AZ, USA), per-
chlorate was detected in 15 of 24 Colorado River water sam-
ples, but the maximum concentration was 0.0056 mg/L [11].
In Goose Prairie Creek near Karnack (TX, USA), three per-
chlorate measurements ranged from 0.044 to 0.085 mg/L [7].
The U.S. Environmental Protection Agency (U.S. EPA) and
several states have initiated research and monitoring efforts to
protect human health from excessive doses of perchlorate in
drinking water. Perchlorate was added to the Contaminant Can-
didate List of the federal Safe Drinking Water Act on March
2, 1998, where it was identified as a contaminant needing
additional research in the areas of health effects, treatment
technologies, analytical methods, and more complete occur-
rence data before regulations could be developed [12]. In 1999,
the U.S. EPA added a perchlorate monitoring requirement for
large public water systems nationwide under the Unregulated
Contaminants Monitoring Rule of the Safe Drinking Water Act
[13]. Some states, including California, Nevada, Arizona, and
Texas, have already established provisional action levels for
perchlorate in drinking water or remedial action thresholds at
concentrations ranging from 0.004 to 0.013 mg/L.
Perchlorate has been reported in tissues of aquatic plants
and animals in the vicinity of contaminated sites. Parsons [11]
performed a survey of perchlorate concentrations in various
biota and media at six different perchlorate-contaminated sites
across the United States. The 965 samples collected included
surface water, groundwater, soil, sediment, sediment pore wa-
ter, aquatic and terrestrial birds, fish, aquatic and terrestrial
reptiles, amphibians, terrestrial mammals, aquatic and terres-
trial insects, other aquatic and terrestrial invertebrates, and
aquatic and terrestrial plants to cover a wide range of potential
sources, pathways, and receptors. Average measured perchlo-
rate concentrations (not corrected for nondetected values)
ranged from 0.7 to 58 ppm for all media (Table 1). Of all the
media sampled, soil and terrestrial vegetation typically exhib-
ited the highest perchlorate concentrations and frequency of
detection, followed by aquatic vegetation, surface water, sed-
iment pore water, and sediments. Perchlorate was only occa-
sionally present at detectable concentrations in organisms at
the higher trophic levels.
Smith et al. [7] also reported perchlorate measurements in
aquatic organisms in perchlorate-contaminated streams and
ponds near the Longhorn Army Ammunition Plant. Perchlorate
concentrations ranged from 0.811 to 2.038 mg/kg in aquatic
insects, below detection (BD) to 0.207 mg/kg in fish, BD to
0.58 mg/kg in frogs, BD to 2.328 mg/kg in mammals, and
0.555 to 5,557 mg/kg in vegetation. As in the Parsons study
[11], many of the highest perchlorate concentrations were mea-
sured in terrestrial plants, followed by water, sediment, and
aquatic plants. Concentrations of perchlorate in aquatic biota
were lower than or similar to those in water at most sites,
indicating a general lack of bioconcentration.
Clearly, aquatic organisms are exposed to perchlorate in
contaminated areas; however, little information exists regard-
ing the ecological effects of perchlorate or any of its salts. The
effects of perchlorate on thyroid function observed in humans
and laboratory rats may occur in fish and other aquatic ver-
tebrates, because thyroid hormones also play important roles
in their development [14].
Existing data suggest that at concentrations ranging from
10 to 1,000 mg/L, effects on aquatic animals include changes
in thyroid hormone production, alterations in metamorphosis,
changes in development, and population growth. Potassium
perchlorate concentrations of 100 mg/L (;72 mg/L perchlorate
ion) induced metamorphosis and reduced lipid storage in larval
sea lamprey (Petromyzon marinus) and American brook lam-
prey (Lampetra appendix) [15,16]. Potassium perchlorate con-
centrations of 340 mg/L (;244 mg/L perchlorate ion) halted
normal metamorphosis of Argentine toad (Bufo arenarum)
tadpoles [17]. A recent report indicates that ammonium per-
chlorate inhibited forelimb emergence and/or tail resorption
in African clawed frog (Xenopus laevis) tadpoles even at con-
centrations as low as 0.005 mg/L [18]. Perchlorate is also
reported to inhibit seed germination and growth of agricultural
crops. Soil potassium perchlorate concentrations as low as 107,
8.6, and 18 mg/kg reduced biomass growth of oats, lettuce,
and tomato, respectively, by 50% [19].
Section 304(a)(1) of the federal Water Pollution Control
Act [20], also known as the Clean Water Act, requires the U.S.
EPA Administrator to develop and publish, and from time to
time to revise, water-quality criteria
Freshwater water-quality criteria for perchlorate
. . . accurately reflecting the latest scientific knowledge (A) on the
kind and extent of all identifiable effects on health and welfare
including, but not limited to, plankton, fish, shellfish, wildlife,
plant life, shorelines, beaches, esthetics, and recreation which may
be expected from the presence of pollutants in any body of water,
including ground water; (B) on the concentration and dispersal of
pollutants, or their byproducts, through biological, physical, and
chemical processes; and (C) on the effects of pollutants on bio-
logical community diversity, productivity, and stability, including
information on the factors affecting rates of eutrophication and
rates of organic and inorganic sedimentation for varying types of
receiving waters.
These National Ambient Water Quality Criteria are generally
applicable to waters of the United States. The U.S. EPA expects
states and tribes to adopt either these recommended criteria,
modified criteria reflecting site-specific conditions, or other
scientifically defensible criteria in their water-quality standards
to protect aquatic life, human health, and other designated uses
of water bodies. These standards, and the criteria on which
they are based, serve as the basis for implementing a large
number of environmental regulations, including limiting pol-
lutant discharges or releases, and, often, in setting remedial
goals for contaminated sites [21].
Because of concern over potential ecological effects of per-
chlorate and the need for scientifically derived, risk-based
cleanup levels for a number of contaminated sites in different
states, a group of scientists within the U.S. Department of
Defense concluded that an ambient water-quality criterion for
perchlorate was needed. A review of existing toxicity infor-
mation regarding the toxicity of perchlorate, however, indi-
cated that insufficient data were available to develop a crite-
rion.
The objective of this effort was to develop acute and chronic
water-quality criteria for perchlorate to protect aquatic life in
freshwater in accordance with U.S. EPA procedures. A cri-
terion for perchlorate in salt water was not within the scope
of this effort. The project included assessment of existing data
regarding ecological effects of perchlorate and performance
of additional laboratory toxicity and bioconcentration tests
needed to provide the remaining required data for development
of water-quality criteria. These criteria should not be construed
as Clean Water Act Section 304(a) water-quality criteria, which
can only be issued by the U.S. EPA. Also, the U.S. EPA has
not formally reviewed or approved these criteria. However,
these criteria were developed in strict accordance with U.S.
EPA requirements. The U.S. Department of Defense, U.S.
EPA, and state regulators may utilize them in determination
of remedial action levels, in evaluation of ambient water-qual-
ity data, and, potentially, for National Pollutant Discharge
Elimination System permit limits and other water-quality man-
agement activities.
Current U.S. EPA guidelines for development of water-
quality criteria are described by Stephan et al. [22] as well as
in the U.S. EPA water-quality guidance for the Great Lakes
system [23]. The calculation of National Ambient Water Qual-
ity Criteria requires chemical-specific aquatic toxicity data
from both acute and chronic exposures. Acute toxicity test
results are required to develop a final acute value (FAV),
whereas chronic toxicity test results, or ratios of acute to chron-
ic toxicity, are required to develop a final chronic value (FCV).
Tests of a number of different taxa are required to ensure
protection of at least 95% of the species. Toxicity tests with
one or more aquatic vascular plants or algae are required to
develop a final plant value. If plants are among the aquatic
organisms most sensitive to the material in question, results
Environ. Toxicol. Chem. 23, 2004 1443
of a test with a plant in another phylum (division) should also
be available. Finally, bioconcentration tests are required to
develop a final residue value from a U.S. Food and Drug
Administration (U.S. FDA) Action Level or a long-term wild-
life feeding study, if either is available.
The minimum taxonomic requirements to establish a FAV
must include acceptable acute toxicity data from at least one
species in eight different families, including each of the fol-
lowing: The family Salmonidae in the class Osteichthyes, a
second family in the class Osteichthyes (preferably a com-
mercially or recreationally important, warm-water species,
e.g., the bluegill sunfish or channel catfish), a third family in
the phylum Chordata, a planktonic crustacean, a benthic crus-
tacean, an insect, a family in a phylum other than Arthropoda
or Chordata, and a family in any order of insect or any phylum
not already represented.
The minimum taxonomic requirements for the FCV are the
same as those for the FAV, except that the U.S. EPA also allows
derivation of the FCV based on acute to chronic ratios (ACRs)
with at least three aquatic species in at least three different
families, provided that the three species include at least one
fish, at least one invertebrate, and at least one acutely sensitive
freshwater species.
In addition to taxonomic requirements, U.S. EPA guidelines
include requirements for the test methodologies. As a general
rule, acceptable tests include flow-through exposures (for most
taxa) of an acceptable duration in which the concentration of
the test substance and species-appropriate endpoints are mea-
sured [22,23]. For acute tests, these endpoints include severe
effects, such as loss of equilibrium and immobilization, in
addition to mortality. Thus, a median effect concentration
(EC50) is preferred to a median lethal concentration (LC50),
which is based on mortality alone.
A review of existing literature identified several studies that
met U.S. EPA requirements for test methodologies used in
determining water-quality criteria. EA Engineering, Science,
and Technology (EA) measured an acute perchlorate LC50 of
66 mg/L and a 7-d chronic value of 18.2 mg/L for the cla-
doceran crustacean Ceriodaphnia dubia [24]. The chronic val-
ue is the geometric mean of the lowest-observable-effect con-
centration (LOEC) and the no-observable-effect concentration
(NOEC). EA also measured an acute perchlorate LC50 of 490
mg/L for the cladoceran crustacean Daphnia magna [24], an
acute LC50 exceeding 1,000 mg/L (the highest tested con-
centration) for the amphipod Hyalella azteca [25], and an
acute LC50 of 1,655 mg/L [24] and chronic value exceeding
490 mg/L [25] with the fathead minnow (Pimephales pro-
melas) (Table 2). Block Environmental Services [26] inde-
pendently measured an acute perchlorate LC50 of 77.8 mg/L
and a chronic value of 15.2 mg/L for the cladoceran crustacean
Ceriodaphnia dubia in acceptable tests. To our knowledge,
no reports of acceptable bioconcentration factors (BCFs) have
been published. An acceptable test by EA [27] generated a
96-h plant value of 775 mg/L perchlorate to the aquatic alga
Raphidocelis subcapitata. The plant value is calculated iden-
tically to the chronic value for animals.
A number of studies containing acute and chronic toxicity
data for the species Daphnia magna [28], P. promelas [24–
26], the coelenterate Hydra attenuata [29], the Argentine toad
B. arenarum [17], the African clawed frog X. laevis [18,30],
the lampreys L. appendix [16], and Petromyzon marinus [15]
did not meet the U.S. EPA’s minimum requirements for use in
developing water-quality criteria because of the use of non-
1444 Environ. Toxicol. Chem. 23, 2004 K.E. Dean et al.

Table 2. Acute median effective concentrations of perchlorate to various freshwater animal species

Species
Category mean
(exposure duration Acute median effect acute value
and type) Common name Scientific name concn. (mg/L) (mg/L) Rank Source

Invertebrate Cladoceran crustacean Ceriodaphnia dubia 66 (40–144)a 71.7 1 [24]

(48-h static) 77.8 (not given) [26]
Invertebrate Cladoceran crustacean Daphnia magna 490 (406–591) 490 2 [24]
(48-h static)
Invertebrate Amphipod Hyalella azteca .1,000 .1,000 3 [25]
(96-h static)
Fish—centrarchid Bluegill Lepomis macrochirus 1,470 (1,270–1,710) 1,470 4 Present study
(96-h flow-through)
Fish—cyprinid Fathead minnow Pimephales promelas 1,655 (1,507–1,817) 1,655 5 [25]
(96-h static)
Fish—salmonid Rainbow trout Oncorhynchus mykiss 2,010 (1,810–2,220) 2,010 6 Present study
96-h flow-through)
Invertebrate Oligochaete Lumbriculus variegatus 3,710 (3,550–3,880) 3,710 7 Present study
(96-h flow-through)
Amphibian Green frog Rana clamitans 5,100 (4,380–5,990) 5,100 8 Present study
(96-h flow-through)
Invertebrate Asiatic clam Corbicula fluminea 6,680 (5,300–8,400) 6,680 9 Present study
(96-h flow-through)
Invertebrate Midge Chironomus tentans 8,140 (6,600–10,000) 8,140 10 Present study
(48-h flow-through)
a Values in parentheses represent the 95% confidence interval.

native species, unacceptable dilution waters, differences in the
length of exposure, excessive mortality among the control pop-
ulation, and probable toxicity from the ammonium, sodium,
or potassium perchlorate counterion. Also, plant tests with the
algae Chlorococcales [31], Scenedesmus quadricauda [32],
and Microcystis aeruginosa [32] did not meet requirements
for use in criteria development, because the tests did not in-
clude a control, exposures were not of sufficient duration, and
supporting information was not available. The acute LC50s
from the rejected studies tended to agree with the acceptable
values, with none falling outside the range of 269 to 670 mg/
L. However, in one study, a chronic effect on amphibian de-
velopment was noted at 0.005 mg/L [18], a concentration three
orders of magnitude less than that reported for any of the
acceptable data. Also, the rejected plant data included esti-
mated plant values at 79 and 360 mg/L [32], well below the
single acceptable plant value. A complete listing and evalu-
ation of all rejected aquatic studies of perchlorate has been
prepared and reported elsewhere [33].
To fill the remaining data gaps for criteria development, a
series of acute, chronic, and bioconcentration tests were per-
formed. These tests were performed according to requirements
in the U.S. EPA guidelines for development of water-quality
criteria [22], including flow-through exposures with measured
toxicant concentrations and use of appropriate controls, ex-
posure type, duration, and sensitive life stages of the test or-
ganisms.
glass beaker and refrigerated, then shipped on ice to APPL
Laboratories (Fresno, CA, USA) for analysis. Perchlorate con-
centrations in water and tissue were measured by ion chro-
matography according to U.S. EPA Method 314.0 [34]. Ho-
mogenized tissues (10 g) were shaken in 100 ml of deionized
water for 1 h on an orbital shaker, then filtered through What-
man (Clinton, NJ, USA) GF/F and 0.45-mm filters. Equipment
included the Dionex (Sunnyvale, CA, USA) DX500 ion chro-
matography system equipped with a GP40 pump, an AS40
autosampler, AMMS-III 4-mm ion suppressor, IonPac AG16
guard column (4 3 50 mm), IonPac AS16 analytical column
(4 3 250 mm), and CD20 detector. Conditions were as follows:
Injection volume, 1.0 ml; eluent, 50 mM NaOH; eluent flow
rate, 1.5 ml/min; regenerant, 50 mN H2SO4; regenerant flow
rate, 8 to 15 ml/min. The system was calibrated at least weekly
with a laboratory reagent blank and six calibration standards
that ranged in concentration from 1 to 100 mg/L perchlorate
ion. Each analysis batch included an instrument performance
check standard, a laboratory reagent blank, an initial calibra-
tion check standard, a laboratory fortified blank, unknown
samples with one or more duplicates, a continuing calibration
check standard (when the batch included .10 samples), an
end-calibration check standard, and a laboratory fortified ma-
trix sample. Samples were diluted with laboratory reagent wa-
ter to the calibration range, as required. Additional details of
specific testing protocols and methods are available elsewhere
[33].

MATERIALS AND METHODS General test conditions

Test material
Sodium perchlorate monohydrate (CAS 7791-07-3) with a
purity of greater than 99.9% was purchased from Aldrich
Chemical (Milwaukee, WI, USA) and used as received.
Perchlorate analytical procedures
Water samples (125 ml) for perchlorate determination were
collected directly from the middle of test chambers using a
All tests were performed at ABC Laboratories (Columbia,
MO, USA) under flow-through test conditions with a propor-
tional diluter system continuously delivering the test solutions
at a rate of at least two complete test-system volume replace-
ments per day. Diluters were volumetrically calibrated before
each test, and the perchlorate concentrations in the resulting
solutions were quantified to ensure the diluters were function-
ing properly before introduction of the test organisms. Fluo-
Freshwater water-quality criteria for perchlorate
rescent lighting was maintained at an intensity of 492 6 44
lux on a 16:8-h light:dark photoperiod with 30-min simulated
dawn and dusk periods. The dilution water in all tests was
prepared by blending naturally hard well water with well water
that had been demineralized by reverse osmosis to achieve a
moderately hard water, as recommended in U.S. EPA guidance
[22]. Hardness, alkalinity, and specific conductance of the di-
lution water ranged from 140 to 160 mg/L as CaCO3, 150 to
162 mg/L as CaCO3, and 303 to 333 mS/cm, respectively, for
all tests. Test chambers were immersed in a circulating water
bath adjusted to maintain the water temperature at 22 6 28C,
except where otherwise noted. Temperature, dissolved oxygen,
and pH were measured in test chambers on a daily basis for
short-term acute toxicity tests and at least weekly for longer-
term chronic and bioconcentration tests. Temperature and pH
were measured with a Denver Instrument (Arvada, CO, USA)
pH meter, which was calibrated before use each day with pH
4, 7, and 10 National Institute of Standards and Technology
(Gaithersburg, MD, USA)–traceable buffer solutions (Fisher
Scientific, Pittsburgh, PA, USA). Dissolved oxygen was mea-
sured with either a YSI (Yellow Springs, OH, USA) Model
95 or a WTW (Ft. Myers, FL, USA) Oxi 330 dissolved oxygen
meter, which was calibrated before use each day, according to
the manufacturer’s instructions, to ambient atmospheric oxy-
gen levels adjusted for barometric pressure and altitude. Tem-
perature was also monitored and recorded continuously with
an electronic data-logging system. Hardness, alkalinity, and
specific conductance in the exposure chambers were measured
at test initiation and at least weekly thereafter. Biological ob-
servations and test monitoring were performed on a daily basis
at minimum. Perchlorate concentrations in 48- or 96-h acute
toxicity tests were measured at initiation and termination.
Each toxicity test consisted of one or more preliminary
range-finding tests, performed to identify the appropriate con-
centration range for quantifying toxicity endpoints, and a de-
finitive test to quantify those endpoints. The range-finding tests
consisted of static exposures to a series of five widely spaced
test concentrations, typically an order of magnitude different
from each other (e.g., 1, 10, 100, 1,000, and 10,000 mg/L).
The definitive tests were flow-though exposures consisting of
a series of five or more perchlorate treatment concentrations,
each approximately half the next-higher concentration, as well
as a perchlorate-free control. In bioconcentration tests, the two
perchlorate exposure concentrations were set at 1% and 0.1%
of the measured perchlorate 96-h LC50 for these species. To
account for potential toxicity from the sodium cation of the
sodium perchlorate, sodium chloride controls were also in-
cluded with the preliminary and/or definitive toxicity tests.
The sodium chloride controls were additional exposures with
sodium chloride added to dilution water at the same sodium
concentration as that in the one or two highest sodium per-
chlorate treatment concentrations. Animals were not fed during
96-h acute exposures.
For each test, the measured perchlorate EC50 and its 95%
confidence limits were calculated by the trimmed Spearman–
Karber method in TOXCALC (Ver 5; Tidepool Scientific Soft-
ware, McKinleyville, CA, USA). Fisher’s exact test was used
to calculate the NOEC and LOEC.
Rainbow trout acute toxicity test
Rainbow trout (Oncorhynchus mykiss) embryos were ob-
tained from Trout Lodge (Sumner, WA, USA). Twenty rainbow
trout fry per treatment level were exposed to sodium perchlo-
Environ. Toxicol. Chem. 23, 2004 1445
rate in 15-L glass aquaria for 96 h. The mean wet weight of
the fish was 0.104 g. The temperature of the test chambers
was maintained at 14.9 to 15.98C. The test procedures were
designed to meet U.S. EPA Office of Prevention, Pesticides,
and Toxic Substances (OPPTS) protocol 850.1075 [35] and
American Society for Testing and Materials (ASTM) E729-96
Guidelines [36]. Nominal (i.e., target) exposure concentrations
in the definitive test were 0 (control), 188, 375, 750, 1,500,
and 3,000 mg/L perchlorate ion.
Bluegill sunfish acute toxicity test
Juvenile bluegill sunfish (Lepomis macrochirus) were ob-
tained from Osage Catfisheries (Osage Beach, MO, USA).
Twenty bluegill per treatment level were exposed to sodium
perchlorate in 15-L glass aquaria for 96 h. The mean wet
weight of the fish was 0.355 g. The temperature of the test
chambers was maintained at 21.2 to 23.78C. The test proce-
dures were designed to meet U.S. EPA OPPTS 850.1075 [35]
and ASTM E729-96 Guidelines [36]. Nominal exposure con-
centrations in the definitive test were 0 (control), 625, 1,250,
2,500, 5,000, and 10,000 mg/L perchlorate ion.
Lumbriculus variegatus acute toxicity test
Adult oligochaete annelids of the species Lumbriculus var-
iegatus were obtained from in-house cultures at ABC Labo-
ratories. Twenty organisms per treatment level, in two repli-
cates of 10 each, were exposed to sodium perchlorate in 500-
ml glass jars with a screen collar for 96 h. The test procedures
were designed to meet U.S. EPA OPPTS 850.1075 [35] and
ASTM E729-96 Guidelines [36]. Nominal exposure concen-
trations in the definitive test were 0 (control), 625, 1,250,
2,500, 5,000, and 10,000 mg/L perchlorate ion.
Green frog acute toxicity test
Green frog tadpoles (Rana clamitans) were obtained from
Amphibians of North America (Nashville, TN, USA). The
tadpoles were approximately seven to eight months old at test-
ing, were in a late stage of development, but had no apparent
limb-bud development. The temperature of the test chambers
was maintained at 18.4 to 20.18C. Twenty tadpoles per treat-
ment level were exposed to sodium perchlorate in 15-L glass
aquaria for 96 h. The test procedures were designed to meet
U.S. EPA OPPTS 850.1075 [35] and ASTM E729-96 Guide-
lines [36]. Nominal exposure concentrations in the definitive
test were 0 (control), 625, 1,250, 2,500, 5,000, and 10,000
mg/L perchlorate ion.
Clam acute toxicity test
Adult Asiatic clams (Corbicula fluminea) were collected
from Little Dixie Lake near Columbia (MO, USA). Twenty
clams per treatment level were exposed to sodium perchlorate
in 15-L glass aquaria for 96 h. The test procedures were de-
signed to meet U.S. EPA OPPTS 850.1075 [35] and ASTM
E729-96 Guidelines [36]. Nominal exposure concentrations in
the definitive test were 0 (control), 625, 1,250, 2,500, 5,000,
and 10,000 mg/L perchlorate ion.
Midge acute toxicity test
Midges of the species Chironomus tentans were obtained
from in-house cultures at ABC Laboratories. The midges uti-
lized were approximately 11-d posthatch at study initiation
and were in the third instar. Twenty organisms per treatment
level, in two replicates of 10 each, were exposed to sodium
1446 Environ. Toxicol. Chem. 23, 2004
perchlorate in 500-ml glass jars with a screen collar for 48 h.
A thin layer of silica sand coated the bottom of the test cham-
bers to provide a substrate for the midges. The test procedures
were designed to meet U.S. EPA OPPTS 850.1075 [35] and
ASTM E729-96 Guidelines [36]. Nominal exposure concen-
trations in the definitive test were 0 (control), 625, 1,250,
2,500, 5,000, and 10,000 mg/L perchlorate ion.
Midge life-cycle chronic toxicity test
Midges of the species C. tentans were obtained from egg
masses produced by adult midges in cultures at ABC Labo-
ratories. The midges utilized were approximately 1-d posthatch
at study initiation. The test was initiated with the addition of
12 midge larvae into 12 replicate test chambers, for a total of
144 midges per treatment. The test chambers were glass jars
holding a solution volume of approximately 500 ml and ap-
proximately 100 g of hydrated artificial sediment. The artificial
sediment consisted of 76% fine silica sand, 20% kaolinite clay,
and 4% fine peat moss (passed through a 500-mm mesh sieve).
The sediment was hydrated with water of approximately 43%
of its dry weight, and calcium carbonate was added to the
sediment to adjust its pH to between 6.5 and 7.0 before use.
Four additional replicate test chambers were initiated with the
addition of 12 midge larvae on day 10 to provide additional
male midges during the reproduction evaluation portion of the
study. An emergent trap covered each of the test replicates.
The emergent traps consisted of a plastic cylinder with a screen
top. The jars were placed in a temperature-controlled water
bath set to maintain test temperature at 23 6 28C.
An intermittent feeding system was used to deliver con-
centrated algae (i.e., a mixture of Selenastrum capricornutum
and Ankistrodesmus falcatus) to each test chamber during each
diluter cycle. This feeder was used for 14 d before testing to
coat the sediment with algae and for 19 d during the early
development of the midge larvae. The larvae were fed 3.0 to
4.5 ml of a 4 g/L flake food suspension for the first 6 d of
testing. The feeding rate was reduced to 1.5 ml/d on day 7
and then to 0.5 ml/d on day 35 to prevent dissolved oxygen
depletion.
Nominal perchlorate exposure concentrations in the defin-
itive test were 0 (controls), 62.5, 125, 250, 500, 1,000, and
2,000 mg/L perchlorate ion. The sodium concentration in the
sodium chloride control was equivalent to that in a 4,000 mg/
L sodium perchlorate solution. The proportional diluter system
delivered approximately 125 ml of dilution water or test so-
lution to each test chamber once every 3 h (two test-chamber
volumes per day) for the first 7 d of testing. From this point
until termination, the diluter rate was increased to replace the
test solution every 90 min (16 test-chamber volumes per day)
to maintain adequate dissolved oxygen within the test cham-
bers.
Four replicate test chambers for each treatment were ter-
minated after 20 d of exposure to the perchlorate ion to record
mortality and sublethal observations. The surviving larvae
were composited into a tared, preashed aluminum weigh boat
and dried at a temperature range of 63 to 828C for approxi-
mately 24 h. The dried larvae were weighed and then ashed
at a temperature of 5508C for approximately 3.5 h, cooled, and
then reweighed. The replicate mean individual ash-free dry
weight (i.e., tissue wt) was determined by subtracting the post-
ashed weight from the preashed weight and then dividing by
the number of larvae present.
Emergent adults were gently aspirated into clean flasks,
K.E. Dean et al.
which contained a minimal amount of water for egg deposition.
The numbers of emergent male, female, and unknown adults
were recorded daily. Adults that were not observed in the
emergence trap but left a pupal skin were recorded as unknown.
The unknown adults were included in the total number of
emergent adults. Available females were paired with at least
one or more males, if available. The flasks containing the adult
pairs were capped with a gas-permeable, foam plug and placed
at a slight angle in a sand matrix within a glass tray. The adult
pairs were observed daily for the production of an egg mass.
The number of successful and unsuccessful (i.e., the female
died before depositing an egg mass) pairings were recorded.
Where possible, the number of eggs/egg mass (i.e., per female)
was estimated by multiplying the number of egg rings within
the mass by the average number of eggs per egg ring. If the
egg mass was malformed, then a total egg count was per-
formed. All egg counts were made with a dissecting micro-
scope. After 42 d of exposure, the numbers of surviving larvae
and pupae within the test chambers were recorded. The total
number of emergent adults and surviving midge were added
together and reported as the 42-d survival.
Temperature, dissolved oxygen concentration, and pH were
measured in all replicate solutions at test initiation, then week-
ly, and again at test termination. No aeration was provided to
any control or test chamber during the test. The test procedures
were designed to meet U.S. EPA Method 100.5 [37] and ASTM
E1706-00 Guidelines [38].
Bluegill sunfish bioconcentration test
Juvenile bluegill sunfish (Lepomis macrochirus) were ob-
tained from Osage Catfisheries (Osage Beach, MO, USA). At
study initiation, the fish weighed 6.3 6 2.4 g (wet wt), were
62 6 7.7 mm in standard length, and were 76 6 9.8 mm in
total length. Sixty bluegill per treatment level were exposed
to sodium perchlorate in 70-L glass aquaria for 28 d. The
nominal treatment levels were 0 (control), 1.5, and 15 mg/L
perchlorate. The proportional diluter system delivered six vol-
ume replacements per day. Fish were fed daily 2% of their
body weight with commercial fish food (Rangen, Buhl, ID,
USA). Any food remaining after 1 h was siphoned from the
aquaria. Observations for mortality and sublethal responses
were made twice daily. The concentrations of perchlorate in
water and tissue were measured in all treatment solutions on
days 0, 1, 2, 3, 7, 14, 21, and 28. Six fish were collected on
each sampling date, and whole fish were homogenized using
a Polytron (Elkhart, IN, USA) homogenizer. Tissue samples
were shipped to APPL Laboratories on dry ice for analysis.
These test procedures were designed to meet U.S. EPA Guide-
line 165-4 [39] and Organisation for Economic Cooperation
and Development Guideline 305 [40].
Clam bioconcentration test
Adult Asiatic clams (Corbicula fluminea) were collected
from Little Dixie Lake near Columbia (MO, USA). For each
treatment level, 200 active clams (diameter, $2 cm) were ex-
posed to sodium perchlorate in 70-L glass aquaria for 28 d.
The nominal treatment levels were 0 (control), 6.5, and 65
mg/L perchlorate. The proportional diluter system delivered
six volume replacements per day. Clams were fed a mixture
of the algal species Chaetoceros and Isochrysis throughout
the exposure period. The concentrations of perchlorate in water
and tissue were measured in all treatment solutions on days
0, 1, 2, 3, 7, 14, 21, and 28. An appropriate number of clams
Freshwater water-quality criteria for perchlorate
was collected on each sampling date, shucked, and homoge-
nized using a Polytron homogenizer to obtain 23 to 33 g of
tissue for analysis. Tissue samples were shipped to APPL Lab-
oratories on dry ice for analysis. These test procedures were
designed to meet U.S. EPA OPPTS 850.1710 [41] and ASTM
E1022-94 [42].
Additional details of specific testing protocols and methods
are available elsewhere [33].
RESULTS AND DISCUSSION
Rainbow trout acute toxicity test
Mean measured perchlorate concentrations ranged from 140
to 2,760 mg/L (74–91% of the nominal concentrations). No
sublethal effects were observed during the test; all observa-
tions were either of mortality or normal behavior. No mortality
was observed in the control, the sodium control, or the 140
or 442 mg/L treatments at 96 h. One fish (5%) died in the 738
mg/L treatment, 3 (15%) in the 1,460 mg/L treatment, and 17
(85%) in the 2,760 mg/L treatment. The 96-h NOEC for O.
mykiss was 1,460 mg/L, and the LOEC was 2,760 mg/L. The
96-h EC50 was calculated to be 2,010 mg/L based on mean
measured perchlorate concentrations, with a 95% confidence
interval of 1,810 to 2,220 mg/L.
Bluegill sunfish acute toxicity test
Mean measured perchlorate concentrations ranged from 547
to 9,715 mg/L (88–104% of the nominal concentrations). No
sublethal effects were observed during the test; all observa-
tions were either of mortality or normal behavior. No mortality
was observed in the control, the 547 mg/L treatment, or the
level 4 (5,220 mg/L equivalent) sodium control, but one fish
(5%) died in the level 5 (9,715 mg/L equivalent) sodium con-
trol. Five fish (25%) died in the 1,260 mg/L treatment, and all
20 (100%) died in the 2,530, 5,220, and 9,715 mg/L treatments.
The 96-h NOEC for L. macrochirus was 547 mg/L, and the
LOEC was 1,260 mg/L. The 96-h EC50 was calculated to be
1,470 mg/L based on mean measured perchlorate concentra-
tions, with a 95% confidence interval of 1,270 to 1,710 mg/L.
Lumbriculus variegatus acute toxicity test
Mean measured perchlorate concentrations ranged from 470
to 10,700 mg/L (75–115% of the nominal concentrations).
Sublethal effects included discoloration and annular constric-
tions in the body walls, forming sausage-like links, at exposure
concentrations of 470 mg/L and higher. Neither of these effects
was immobilizing or appeared to lead to death, and the worms
recovered from these effects in many cases, possibly by ex-
cising the segments posterior to the kink. No mortality was
observed in the control or the sodium controls. One organism
(5%) died in each of the 470, 1,240, and 2,500 mg/L treat-
ments. All 20 (100%) organisms died in the 5,760 and 10,700
mg/L treatments. The U.S. EPA Guidance [22] indicates that
the sublethal effects to consider in calculating an EC50 for an
acute test include severe effects leading to death, such as im-
mobilization or loss of equilibrium. Based on mortality, the
96-h NOEC for L. variegatus was 2,500 mg/L, the LOEC was
5,760 mg/L, and the EC50 was 3,710 mg/L, with a 95% con-
fidence interval of 3,550 to 3,880 mg/L.
Green frog acute toxicity test
Mean measured perchlorate concentrations ranged from 680
to 9,800 mg/L (96–116% of the nominal concentrations). No
mortality was observed in the control, the sodium controls, or
Environ. Toxicol. Chem. 23, 2004 1447
the 680, 1,200, or 2,400 mg/L treatments at 96 h. Nine tadpoles
(45%) died in the 5,800 mg/L treatment, and all 20 (100%)
died in the 9,800 mg/L treatment. Two additional tadpoles in
the 5,800 mg/L treatment exhibited loss of equilibrium at 96
h. The 96-h NOEC for R. clamitans was 2,400 mg/L, and the
LOEC was 5,800 mg/L. The 96-h LC50 was calculated to be
5,500 mg/L based on mortality, with a 95% confidence interval
of 4,700 to 6,420 mg/L. The 96-h EC50 was calculated to be
5,100 mg/L based on mortality plus loss of equilibrium, a
severe sublethal effect, with a 95% confidence interval of 4,380
to 5,990 mg/L.
Corbicula fluminea acute toxicity test
Mean measured perchlorate concentrations ranged from 713
to 10,800 mg/L (93–114% of the nominal concentrations). No
sublethal effects were observed during the test; all observa-
tions were either of mortality or normal behavior. A single
clam (5%) died in the control. No mortality was observed in
the level 4 (4,850 mg/L equivalent) sodium control, but all 20
clams died in the level 5 (10,800 mg/L equivalent) sodium
control at 96 h. In the perchlorate treatments, mortality was
0, 5, 25, 20, and 85% in the 713, 1,400, 2,710, 4,850, and
10,800 mg/L treatments, respectively. The 96-h NOEC for C.
fluminea was 4,850 mg/L, and the LOEC was 10,800 mg/L.
The 96-h LC50 was calculated to be 6,680 mg/L based on
mortality, with a 95% confidence interval of 5,300 to 8,400
mg/L. Because of mortality in the sodium control, we cannot
conclude that the sodium ion did not contribute to the observed
toxicity attributed to perchlorate. However, we can conclude
that the 96-h LC50 exceeded 4,850 mg/L.
Chironomus tentans acute toxicity test
Mean measured perchlorate concentrations ranged from 772
to 12,700 mg/L (113–127% of the nominal concentrations).
One midge (5%) was missing and considered to be dead in
the control and in the 1,000 mg/L equivalent sodium control,
but no mortality was observed in the level 5 (10,000 mg/L
equivalent) sodium control. In the perchlorate treatments, mor-
tality (missing midge were considered to be dead) was 5, 20,
15, 15, and 90% in the 772, 1,545, 3,045, 5,640, and 12,700
mg/L treatments, respectively. The 48-h NOEC for C. tentans
was 5,640 mg/L, and the LOEC was 12,700 mg/L. The 48-h
EC50 was calculated to be 8,100 mg/L, with a 95% confidence
interval of 6,600 to 10,000 mg/L.
Chironomus tentans life-cycle chronic toxicity test
Mean measured perchlorate concentrations ranged from
58.5 to 2,080 mg/L (93–104% of the nominal concentrations).
Survival in the control and the sodium control over the 42-d
exposure was 90 and 81%, respectively. In perchlorate treat-
ments, survival was 87, 62, 24, 21, 4, and 0% in the 58.5,
118, 233, 489, 1,020, and 2,080 mg/L exposures. Most of the
surviving larvae from exposure concentrations greater than
233 mg/L were discolored. However, this sublethal effect was
not considered in the calculation of the EC50. The NOEC,
LOEC, and EC50 for survival were 58.5, 118, and 208 mg/L,
respectively, with a 95% confidence interval of 184 to 236
mg/L for the EC50.
Adult midge emergence was first recorded after 21 d of
exposure. Emergence totaled 89 and 28% in the control and
sodium control, respectively, through day 42, when the ex-
posure was terminated. Emergence in the perchlorate treat-
ments ranged from 0 to 85%. The NOEC, LOEC, and EC50
1448 Environ. Toxicol. Chem. 23, 2004
for emergence were 58.5, 118, and 146 mg/L, respectively,
with a 95% confidence interval of 134 to 159 mg/L for the
EC50.
The mean individual ash-free dry weight of surviving lar-
vae at day 20 was 1.6 and 0.55 mg/L in the control and sodium
control, respectively, and 1.4, 1.4, 0.93, 0.48, and 0.40 in the
58.5, 118, 233, 489, and 1,020 mg/L exposures, respectively.
The NOEC, LOEC, and EC50 for growth were 118, 233, and
363 mg/L, respectively, with a 95% confidence interval of 158
to 834 mg/L for the EC50.
The control and sodium control females produced egg mas-
ses with an average of 936 and 1,792 eggs per egg mass,
respectively. The mean number of eggs per egg mass was 918,
900, and 393 in the 58.5, 118, and 233 mg/L exposures, re-
spectively. A single female emerged in the 1,080 mg/L treat-
ment but could not be paired. The NOEC, LOEC, and EC50
for reproductive output were 118, 233, and 211 mg/L, re-
spectively, with a 95% confidence interval of 131 to 338 mg/
L for the EC50.
The adult emergence was the most sensitive biological end-
point measured during the midge life-cycle exposure to per-
chlorate. Midges in the sodium chloride control group appar-
ently were developmentally inhibited. This developmental in-
hibition affected the growth and emergence rates, but it did
not lower the survival rate or the individual reproductive out-
put of the emergent females. Because the 28% emergence rate
in the sodium control occurred at a sodium concentration
equivalent to 4,000 mg/L perchlorate, whereas an emergence
of 11% was observed in the 233 mg/L treatment, the observed
effect likely was caused by the perchlorate ion rather than by
the sodium cation. The chronic value for C. tentans was 83.1
mg/L, calculated as the geometric mean of the LOEC and
NOEC for adult emergence.
Bluegill sunfish bioconcentration test
Mean measured perchlorate concentrations were 0, 1.4, and
11.9 mg/L in water in the control and the low- and high-level
exposures, respectively. All fish were observed to be normal
and healthy for the 28-d duration of the test. Concentrations
of perchlorate in fish tissue in the low treatment were not
detected until day 21 but reached 1.1 mg/kg on day 21 and
1.5 mg/kg on day 28. In the high-level treatment, perchlorate
concentrations in bluegill increased throughout the test, reach-
ing 6.8 mg/kg on day 28. According to U.S. EPA Guidance
[22] for water-quality criteria development, if steady-state con-
ditions are not reached, the BCF should be taken as the highest
BCF. Thus, the BCF value for the low treatment was 0. 73 L/
kg and the BCF for the high treatment 0.68 L/kg for whole
bluegill. The geometric mean BCF value for perchlorate in
bluegill is 0.70 L/kg.
Clam bioconcentration test
Mean measured perchlorate concentrations were less than
0.026, 7.05, and 51.6 mg/L in water in the control and the
low- and high-level perchlorate exposures, respectively. All
clams were observed to be normal and healthy for the 28-d
duration of the test. It was not determined if steady-state con-
ditions were reached. According to U.S. EPA Guidance [22],
if steady-state conditions are not reached, the BCF should be
taken as the highest BCF. Thus, the BCF value for the low
treatment was 3.1 L/kg (from day 21), and the BCF for the
high treatment was 1.1 L/kg (from day 28). The geometric
mean BCF value for perchlorate in the clam is 1.85 L/kg.
K.E. Dean et al.
Calculation of the FAV
The FAV is an estimate of the concentration of perchlorate
corresponding to a cumulative probability of 0.05 of the acute
toxicity values [22]. That is, the FAV is a calculated estimate
of the concentration of perchlorate so that 95% of the genera
tested will have a higher genus mean acute value (GMAV)
than FAV. Acceptable acute toxicity data were available for
10 species meeting the taxonomic requirements for water-qual-
ity criteria development [22]. For each species used to develop
the criteria, the species mean acute value (SMAV) was cal-
culated. The SMAVs were either a single measured EC50 value
for mortality or other severe effects, such as immobilization
or loss of equilibrium, or were calculated as the geometric
mean of the EC50s when two or more acceptable acute tests
were available. The U.S. EPA [22] recommends the geometric
mean instead of the arithmetic mean, because the distributions
of the sensitivities of individual organisms in toxicity tests on
most materials are more likely to be log-normal than normal.
The SMAVs for perchlorate ranged from 71.7 to 8,140 mg/L
(Table 2). The most sensitive species to acute perchlorate tox-
icity was the cladoceran crustacean Ceriodaphnia dubia, with
LC50 values of 66 mg/L [24] and 77.8 mg/L [26], yielding a
SMAV of 71.7 mg/L. The two most perchlorate-tolerant in-
vertebrate species in acute exposures were Corbicula fluminea
and Chironomus tentans, with SMAVs of 6,680 and 8,140 mg/
L, respectively. The bluegill was the most sensitive vertebrate
species, with a SMAV of 1,470 mg/L, but the fathead minnow
and rainbow trout were similarly sensitive, with SMAVs of
1,655 and 2,010 mg/L, respectively. The green frog R. clam-
itans was the most tolerant vertebrate species tested, with a
SMAV of 5,120 mg/L. In the present study, the GMAVs were
all equivalent to the SMAVs.
Calculation of the FAV was performed according to U.S.
EPA Guidance [22]. The GMAVs were ordered from highest
to lowest and assigned a rank, r, from r 5 1 for the lowest
GMAV (Ceriodaphnia dubia) to r 5 10 for the highest GMAV
(Chironomus tentans) (Table 2). The cumulative probability,
P, for each GMAV was calculated as P 5 r/(n 1 1), where n
is the number of GMAVs. The four GMAV values with cu-
mulative probabilities closest to 0.05 were selected, and the
FAV for perchlorate was calculated to be 39.9 mg/L using the
following equations:
O [(ln GMAV ) ] 2 O 4
3
(ln GMAV )4
2
2
O (P) 2 3 O (ÏP )4
S2 5
2
4
L5
O (ln GMAV ) 2 S 3 O (ÏP )4
4
A 5 S (Ï0.05) 1 L FAV 5 e A
The values that are reported as greater-than-values (e.g., Hy-
alella azteca) were included in the calculation of the FAV,
because not having including these values would unnecessarily
lower the FAV by eliminating values for resistant species. The
three most sensitive species to perchlorate were invertebrates
(Ceriodaphnia dubia, D. magna, and H. azteca), and all
SMAVs were higher than the FAV.
Calculation of the FCV
The FCV is a calculated estimate of the concentration of
perchlorate such that 95% of the genera tested will have a
Freshwater water-quality criteria for perchlorate
Table 3. Chronic median effective concentrations (EC50s) and acute to chronic EC50 ratios for perchlorate in three freshwater animals
Acute EC50 Chronic EC50 Acute to
Common name Scientific name (mg/L)
Cladoceran crustacean Ceriodaphnia dubia 66
77.8
Midge Chironomus tentans 8,140
Fathead minnow Pimephales promelas 1,655
genus mean chronic value higher than the FCV. The FCV can
be calculated in the same way as the FAV, using chronic values
for eight or more families, or it can be calculated from the
FAV divided by the appropriate ACR. The ACR may be cal-
culated from acute and chronic toxicity test data from three
or more families, provided that at least one species is a fish,
at least one is an invertebrate, and at least one is an acutely
sensitive freshwater species [22]. Acute and chronic toxicity
data were available for Ceriodaphia dubia, Chironomus ten-
tans, and Pimephales promelas. Ceriodaphia dubia was the
most acutely sensitive. Thus, these ACRs met the requirements
for calculation of the ACR. Chronic values ranged from 15.3
to greater than 490 mg/L. The chronic value for a 7-d chronic
exposure of C. dubia was 18.2 mg/L [24], whereas that for
an independent 6-d chronic test was 15.3 mg/L [26] (Table 3).
Although results of both tests for C. dubia were similar, they
were not from the same testing facility, so U.S. EPA Guidelines
[22] suggest that ACRs should be calculated for each test and
the geometric mean of the ACRs calculated. Therefore, ACRs
of 3.63 (66.0/18.2 mg/L) and of 5.12 (77.8/15.2 mg/L) were
calculated, and the geometric mean of these two ACR values
gives a species mean ACR of 4.31 for C. dubia.
The chronic value for the second most sensitive species,
Chironomus tentans, was 83.1 mg/L. The corresponding acute
value for C. tentans was 8,140 mg/L, yielding an ACR of 98.0.
No significant effects of perchlorate were observed even at
the highest concentrations used in a 35-d, early life-stage
chronic test [25] with P. promelas, and the chronic value was
greater than 490 mg/L. The corresponding acute value for the
fathead minnow was 1,655 mg/L, yielding an ACR of less than
3.38.
The species mean ACRs are similar for Ceriodaphia dubia
and P. promelas (4.31 and ,3.38, respectively), but the spe-
cies mean ACR for Chironomus tentans (98.0) is more than
a factor of 10 higher. The U.S. EPA Guidance [22] states that
if the species mean ACR seems to increase or decrease as the
SMAV increases, the final ACR (FACR) should be calculated
as the geometric mean of the ACR for those species with
SMAV near the FAV. In this case, the species with the highest
SMAV (C. tentans) does have the highest ACR (Table 3). Only
Ceriodaphnia dubia has a SMAV (71.7 mg/L) near the FAV
(39.9 mg/L). Thus, the FACR is 4.31, which is the species
mean ACR for C. dubia.
The FCV is then calculated to be 9.26 mg/L, as the FAV
(39.9 mg/L) divided by the FACR (4.31). The most sensitive
species to chronic perchlorate toxicity was C. dubia, and all
the SMCVs were higher than the FCV.
Calculation of the final plant value
Acceptable data regarding the effects of perchlorate on
aquatic plants are available for one plant species. The toxicity
of perchlorate to the freshwater green alga Raphidocelis sub-
capitata was measured in a 96-h exposure test [27]. The 25%
Environ. Toxicol. Chem. 23, 2004 1449
Species mean
acute to
(mg/L) chronic ratio chronic ratio Rank Source
18.2 3.63 4.31 1 [24]
15.2 5.12 [26]
83.1 98.0 98.0 2 Present study
.490 ,3.38 ,3.38 3 [24,25]
inhibition concentration (i.e., a point estimate of the toxicant
concentration that caused a 25% reduction in growth) was
calculated to be 615 mg/L. Because plants did not appear to
be more sensitive to perchlorate than animals, no other plant
taxa data were used, and the final plant value was equal to
615 mg/L. Additional toxicity testing using acceptable pro-
tocols with other species may be warranted to increase con-
fidence in the final plant value and to confirm that plants are
less sensitive than aquatic animals to perchlorate.
BCFs and the final residue value
A final residue value is designed to prevent concentrations
of contaminants in commercially or recreationally important
aquatic species from affecting marketability because of ex-
ceedance of a U.S. FDA Action Level and to prevent health
effects in wildlife that eat aquatic species [22]. Limiting tissue
concentrations of contaminants are derived from U.S. FDA
Action Levels or a long-term wildlife feeding study and then
linked to water concentrations by a BCF, which is the ratio of
contaminant concentration in tissue to that in water. To date,
a U.S. FDA Action Level for perchlorate has not been estab-
lished, nor has a long-term wildlife feeding study, to our
knowledge, been performed. Thus, it is not possible to develop
a final residue value at this time.
Acceptable data regarding bioconcentration (using whole-
body tissue analysis) of perchlorate in aquatic organisms is
available for an aquatic invertebrate, the Asiatic clam Cor-
bicula fluminea and the bluegill Lepomis macrochirus (present
study). The geometric mean BCF value for perchlorate in
clams, based on the highest measured BCFs in each of two
exposure concentrations, was 1.85 L/kg for C. fluminea and
0.70 L/kg for L. macrochirus. These BCFs indicate that these
animals take up perchlorate at concentrations less than or
slightly exceeding those in the exposure media. This conclu-
sion is supported by field measurements of BCFs that are
typically less than one [11].
Statement of water-quality criteria
National ambient water-quality criteria consist of two val-
ues, a criterion maximum concentration (CMC) and a criterion
continuous concentration (CCC). The CMC for perchlorate is
calculated to be 20 mg/L, or half the FAV (39.9 mg/L) rounded
to two significant digits. The CMC represents an estimate of
the concentration in water to which an aquatic community can
be exposed briefly without unacceptable effects [43].
The CCC is equal to the lowest of the FCV (9.26 mg/L),
the final plant value (615 mg/L), and the final residue value,
rounded to two significant digits. Because a final residue value
cannot be calculated in the absence of a U.S. FDA Action
Level or a long-term wildlife feeding study, the CCC for fresh-
water was equal to the FCV, which was 9.3 mg/L. The CCC
is an estimate of the highest concentration to which an aquatic
1450 Environ. Toxicol. Chem. 23, 2004
community can be exposed indefinitely without unacceptable
effects [44].
Our proposed freshwater perchlorate criteria can be stated
according to the instructions in the U.S. EPA Guideline [22]
as follows:
The procedures described in the Guidelines for Deriving Nu-
merical National Water-Quality Criteria for the Protection of
Aquatic Organisms and Their Uses indicate that, except pos-
sibly where a locally important species is very sensitive, fresh-
water aquatic organisms and their uses should not be affected
unacceptably if the 4-d average concentration of perchlorate
ion does not exceed 9.3 mg/L more than once every three
years on the average and if the 1-h average concentration does
not exceed 20 mg/L more than once every three years on the
average.
As the toxicity of perchlorate to additional plants and an-
imals is measured, these criteria should be recalculated to in-
corporate these data. Amphibians are thought to be very sen-
sitive environmental indicator species, and additional studies
using native amphibians in long-term chronic toxicity tests are
planned.
Acknowledgement—This work was performed for the Department of
the Air Force under delivery order 0063 of Contract F41624-95-D-
9018.
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