1

1 Running head: Freshwater water quality criteria for perchlorate

2 Corresponding Author: Kirk Dean

3 Parsons

4 8000 Centre Park Drive, Suite 200

5 Austin, TX 78754

6 (512) 719-6000

7 Fax: (512) 719-6099

8 e-mail: Kirk.Dean@Parsons.com

10 Word count: Text: 8,169

11 References: 1,083

12 Tables: 334

13 Figure legends:0
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1 Title: Development of freshwater water quality criteria for perchlorate

2 Authors: Kirk E. Dean†, Randy M. Palachek†, Jill M. Noel †,║, Ryan Warbritton‡,

3 John Aufderheide‡, and Jody Wireman#

4
†
5 Parsons

6 8000 Centre Park Drive, Suite 200

7 Austin, TX 78754

8
‡
9 ABC Laboratories

10 Chemical Development Group

11 7200 E. ABC Lane

12 Columbia, MO 65202

13
#
14 Air Force Institute for Environment, Safety, and Occupational Health Risk Analysis

15 AFIERA/RSRE

16 2513 Kennedy Circle

17 Brooks AFB, TX 78235

18
║
19 Current Address: University of Texas at Austin

20 Department of Plant Biology

21 Mail code: A6700

22 Austin, TX 78713
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1 Corresponding author e-mail: kirk.dean@parsons.com

2 Disclaimer:
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1 ABSTRACT

2 The anion perchlorate (ClO4-) is an oxidizing component commonly used in solid

3 propellants for rockets and missiles, and in explosives, flares, fireworks, chemical

4 processes, automobile air bag inflators, and other assorted uses. With recent advances in

5 analytical detection capability, perchlorate has been found in a variety of ground and

6 surface waters throughout the United States. Because perchlorate has been associated

7 with thyroid problems in humans and may have similar effects on wildlife, it is desirable

8 to develop a water quality criterion to assist in identifying levels of perchlorate in water

9 likely to pose an ecological health risk.

10 In this study, we compiled all available data on the effects of perchlorate to

11 aquatic organisms, and performed additional toxicity and bioconcentration tests required

12 by the USEPA for the development of water quality criteria for aquatic life. A Criterion

13 Maximum Concentration (CMC) of 20 mg/L and a Criterion Continuous Concentration

14 (CCC) of 9.3 mg/L were calculated based on the test results. Though these are not formal

15 Clean Water Act Section 304(a) criteria, which must be published by the USEPA, these

16 criteria may be useful in determination of remedial action levels for contaminated sites,

17 for NPDES permit limits, and other water quality management practices.

18

19 Keywords: Perchlorate, water quality criteria

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1 INTRODUCTION

2 In the 1990s, the perchlorate ion emerged as a contaminant of concern in ground

3 and surface waters [1]. Even at low doses, perchlorate is reported to modify mammalian

4 thyroid function by competitive inhibition of iodide uptake, resulting in reduced thyroid

5 hormone production as well as thyroid tumors [2,3,4,5,6]. Thyroid hormone deficiencies

6 can affect normal metabolism, growth, and development. Due to its mobility and stability

7 in the aqueous environment, aquatic organisms are predicted to receive the highest

8 exposure to perchlorate [7]. The objective of this study was to develop acute and chronic

9 water quality criteria for perchlorate for the protection of aquatic life in freshwater.

10 Perchlorate (ClO4-) is an oxidizing component used in solid propellants for

11 rockets and missiles, and in explosives, flares, fireworks, chemical processes, automobile

12 air bag inflators, and other assorted items. Perchlorate also occurs naturally, notably in

13 some Chilean caliche ores, which are used as a nitrate fertilizer in the United States [8].

14 Environmental perchlorate contamination has resulted from discharges to surface

15 water and ground water from manufacturing and production operations, waste handling

16 and washing operations, research and testing, and the use of perchlorate in industrial,

17 military, and agricultural practices [9]. In particular, the military, space program, and

18 supporting industries, which use a high percentage of all manufactured perchlorate, have

19 been identified with perchlorate releases to the environment. Perchlorate has been used

20 or manufactured in at least 40 states [9].

21 Perchlorate salts tend to be highly water-soluble; for example, the aqueous

22 solubility of sodium perchlorate is almost 8 M [10]. While perchlorate is a strong

23 oxidizing agent, its reduction is so kinetically limited that it is practically unreactive in

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1 dilute aqueous solution at environmental temperatures and at pH >1 [10]. The

2 perchlorate ion is also a weakly complexing anion, and its adsorption to minerals tends to

3 be weak and reversible [8]. For these reasons, perchlorate is exceedingly mobile and can

4 persist for many decades under typical ground water and surface water conditions [9].

5 Perchlorate was not observed in natural waters, except at a few Superfund sites,

6 until approximately 1997, when a new analytical method was developed with a much

7 lower detection limit (~0.004 mg/L). To date, there have been few measurements of the

8 levels of perchlorate in natural waters. Most of the ambient water measurements were

9 collected from shallow ground water near contaminated sites. Perchlorate concentrations

10 in groundwater reached 3,700 mg/L near a manufacturing facility in Henderson, Nevada,

11 640 mg/L at a rocket manufacturing facility in Rancho Cordova, California, and 169

12 mg/L at the Longhorn Army Ammunition Depot in Karnack, Texas [9]. Reports of

13 perchlorate measurements in surface waters are even rarer. In East Camden, Arkansas, a

14 perchlorate concentration of 480 mg/L was observed in surface water near a rocket

15 manufacturing facility [9]. A perchlorate concentration of 130 mg/L was observed in the

16 Las Vegas Wash, a stream flowing into Lake Mead that is influenced by perchlorate-

17 contaminated ground water [11]. At Allegany Ballistics Laboratory in West Virginia,

18 perchlorate was detected in 21 of 23 samples, with average and maximum measured

19 concentrations of 0.212 and 0.280 mg/L, respectively [11]. At Indian Head Naval Surface

20 Warfare Center, Maryland, perchlorate was detected in 27 of 36 surface water samples,

21 with average and maximum measured concentrations of 0.018 and 0.025 mg/L,

22 respectively [11].
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1 In surface waters not in the immediate vicinity of contaminated sites, it appears

2 that perchlorate concentrations are typically less than 0.1 mg/L. Low levels of perchlorate

3 have been found in Lake Mead, Nevada (<0.10 mg/L) and the Colorado River

4 downstream of Lake Mead (<0.01 mg/L) [9]. Further downstream, in the vicinity of

5 Yuma, Arizona, perchlorate was detected in 15 of 24 Colorado River water samples, but

6 the maximum concentration was 0.0056 mg/L [11]. In Goose Prairie Creek near

7 Karnack, Texas, three perchlorate measurements ranged from 0.044 to 0.085 mg/L [7].

8 The United States Environmental Protection Agency (USEPA) and several States

9 have initiated research and monitoring efforts to protect human health from excessive

10 doses of perchlorate in drinking water. Perchlorate was added to the Contaminant

11 Candidate List (CCL) of the federal Safe Drinking Water Act (SDWA) on March 2,

12 1998, where it was identified as a contaminant needing additional research in the areas of

13 health effects, treatment technologies, analytical methods, and more complete occurrence

14 data before regulations could be developed. In 1999, the USEPA added a perchlorate

15 monitoring requirement for large public water systems nationwide under the Unregulated

16 Contaminants Monitoring Rule of the SDWA. Water utilities began to report perchlorate

17 measurements in drinking water to the USEPA in August 2002. Some states, including

18 California, Nevada, Arizona, and Texas, have already established provisional action

19 levels for perchlorate in drinking water or as remedial actions thresholds at levels ranging

20 from 0.004 to 0.013 mg/L.

21 Perchlorate has been reported in tissues of aquatic plants and animals in the

22 vicinity of contaminated sites. Parsons [11] performed a survey of perchlorate

23 concentrations in various biota and media at six different perchlorate-contaminated sites

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1 across the United States. The 965 samples collected included surface water, ground

2 water, soil, sediment, sediment pore water, aquatic and terrestrial birds, fish, aquatic and

3 terrestrial reptiles, amphibians, terrestrial mammals, aquatic and terrestrial insects, other

4 aquatic and terrestrial invertebrates, and aquatic and terrestrial plants to cover a wide

5 range of potential sources, pathways, and receptors. Average measured perchlorate

6 concentrations (not corrected for non-detected values) ranged from 0.7 to 58 ppm for all

7 media (Table 1). Soil and terrestrial vegetation typically exhibited the highest

8 perchlorate concentrations and frequency of detection of all the media sampled, followed

9 by aquatic vegetation, surface water, sediment pore water, and sediments. Perchlorate

10 was only occasionally present at detectable levels in higher trophic level organisms.

11 Smith et al. [7] also reported perchlorate measurements in aquatic organisms in

12 perchlorate-contaminated streams and ponds near the Longhorn Army Ammunitions

13 Plant near Karnack, Texas. Perchlorate concentrations ranged from 0.811 to 2.038 mg/kg

14 in aquatic insects, below detection (BD) to 0.207 mg/kg in fish, BD to 0.58 mg/kg in

15 frogs, BD to 2.328 mg/kg in mammals, and 0.555 to 5557 mg/kg in vegetation. As in the

16 Parsons study, many of the highest perchlorate concentrations were measured in

17 terrestrial plants, followed by water, sediment, and aquatic plants. Concentrations of

18 perchlorate in aquatic biota were lower than or similar to those in water at most sites,

19 indicating a general lack of bioconcentration.

20 Clearly, aquatic organisms are exposed to perchlorate in contaminated areas;

21 however, little information exists on ecological effects of perchlorate or any of its salts.

22 The effects of perchlorate on thyroid function observed in humans and laboratory rats
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1 may occur in fish and other aquatic vertebrates because thyroid hormones also play

2 important roles in their development [12].

3 Existing data suggest that at concentrations ranging from 10 to 1000 mg/L, effects

4 on aquatic animals include changes in thyroid-hormone production, alterations in

5 metamorphosis, changes in development, and population growth. Potassium perchlorate

6 concentrations of 100 mg/L (~72 mg/L perchlorate ion) induced metamorphosis and

7 reduced lipid storage in larval Sea Lamprey (Petromyzon marinus), and American Brook

8 Lamprey (Lampetra appendix) [13, 14]. Potassium perchlorate concentrations of 340

9 mg/L (~244 mg/L perchlorate ion) halted normal metamorphosis of tadpoles of the

10 Argentine Toad (Bufo arenarum) [15]. A recent report indicates that ammonium

11 perchlorate inhibited forelimb emergence and/or tail resorption in tadpoles of the African

12 Clawed Frog (Xenopus laevis) even at concentrations as low as 0.005 mg/L perchlorate

13 [16]. Perchlorate is also reported to inhibit seed germination and growth of agricultural

14 crops. Soil potassium perchlorate concentrations as low as 107, 8.6, and 18 mg/kg

15 reduced biomass growth of oats, lettuce, and tomato, respectively, by 50% [17].

16 Section 304(a)(1) of the Clean Water Act of 1977 requires the USEPA

17 Administrator to develop and publish, and from time to time revise, water quality criteria

18 “…accurately reflecting the latest scientific knowledge (A) on the kind and extent of all

19 identifiable effects on health and welfare including, but not limited to plankton, fish,

20 shellfish, wildlife, plant life, shorelines, beaches, aesthetics, and recreation which may be

21 expected from the presence of pollutants in any body of water, including ground water;

22 (B) on the concentration and dispersal of pollutants, or their byproducts, through

23 biological, physical, and chemical processes; and (C) on the effects of pollutants on
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1 biological community diversity, productivity, and stability, including information on the

2 factors affecting rates of eutrophication and rates of organic and inorganic sedimentation

3 for varying types of receiving waters.” These national ambient water quality criteria

4 (NAWQC) are generally applicable to waters of the United States. USEPA expects

5 States and Tribes to adopt either these recommended NAWQC, modified criteria

6 reflecting site-specific conditions, or other scientifically defensible criteria in their water

7 quality standards to protect aquatic life, human health, and other designated uses of water

8 bodies. These standards, and the criteria on which they are based, serve as the basis for

9 implementing a large number of environmental regulations, including limiting pollutant

10 discharges or releases, and often in setting remedial goals for contaminated sites [18].

11 Due to concern over potential ecological effects of perchlorate, and the need for

12 scientifically-derived risk-based cleanup levels for a number of contaminated sites in

13 different states, a group of scientists within the Department of Defense (DOD) concluded

14 that an ambient water quality criterion for perchlorate was needed. A review of existing

15 toxicity information on the toxicity of perchlorate indicated that insufficient data were

16 available to develop a criterion.

17 The objective of this effort was to develop acute and chronic water quality criteria

18 for perchlorate for the protection of aquatic life in freshwater in accordance with USEPA

19 procedures. A criterion for perchlorate in saltwater was not within the scope of this effort.

20 The project included assessment of existing data on ecological effects of perchlorate, and

21 performance of additional laboratory toxicity and bioconcentration tests needed to

22 provide the remaining required data for water quality criteria development. These criteria

23 should not be construed as Clean Water Act Section 304(a) water quality criteria, which
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1 can only be issued by the USEPA. Also, the USEPA has not formally reviewed or

2 approved these criteria. However, the criteria were developed in strict accordance with

3 USEPA requirements for criteria development. The DOD, USEPA, and state regulators

4 may utilize them in determination of remedial action levels, in evaluation of ambient

5 water quality data, and potentially for NPDES permit limits and other water quality

6 management activities.

7 Current USEPA guidelines for development of water quality criteria are found in

8 Stephan et al. [19] as well as USEPA water quality guidance for the Great Lakes system

9 [20]. The calculation of NAWQC requires chemical-specific aquatic toxicity data from

10 both acute and chronic exposures. Acute toxicity test results are required to develop a

11 Final Acute Value (FAV), whereas chronic toxicity test results, or ratios of acute to

12 chronic toxicity, are required to develop a Final Chronic Value (FCV). Tests of a number

13 of different taxa are required to ensure protection of at least 95% of the species. Toxicity

14 tests with one or more aquatic vascular plant(s) or algae are required to develop a final

15 plant value. If plants are among the aquatic organisms most sensitive to the material,

16 results of a test with a plant in another phylum (division) should also be available.

17 Finally, bioconcentration tests are required to develop a final residue value from a Food

18 and Drug Administration (FDA) Action Level or a long-term wildlife feeding study, if

19 either is available.

20 The minimum taxonomic requirements to establish a final acute value must

21 include acceptable acute toxicity data from at least one species in eight different families,

22 including each of the following: (i) the family Salmonidae in the class Osteichthyes, (ii) a

23 second family in the class Osteichthyes (preferably a commercially or recreationally

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1 important warm water species such as the bluegill sunfish or channel catfish), (iii) a third

2 family in the phylum Chordata, (iv) a planktonic crustacean, (v) a benthic crustacean, (vi)

3 an insect, (vii) a family in a phylum other than Arthropoda or Chordata, and (viii) a

4 family in any order of insect or any phylum not already represented.

5 The minimum taxonomic requirements for the final chronic value are the same as

6 those for the final acute value, except that the USEPA also allows derivation of the final

7 chronic value based on acute:chronic ratios with at least three aquatic species in at least

8 three different families, provided the three species include: (i) at least one fish, (ii) at

9 least one invertebrate, and (iii) at least one acutely sensitive freshwater species.

10 In addition to taxonomic requirements, USEPA guidelines include requirements

11 on the test methodologies. As a general rule, acceptable tests include flow-through

12 exposures (for most taxa) of an acceptable duration in which the concentration of the test

13 substance was measured and species-appropriate endpoints are measured [19, 20]. For

14 acute tests, these endpoints include severe affects such as loss of equilibrium and

15 immobilization, in addition to mortality. Thus, a median effects concentration (EC50) is

16 preferred to a median lethal concentration (LC50), which is based on mortality alone.

17 A review of existing literature identified several studies that met USEPA

18 requirements for test methodologies used in determining water quality criteria. EA

19 Engineering, Science, and Technology (EA) measured an acute perchlorate median lethal

20 concentration (LC50) of 66 mg/L and a 7 d chronic value of 18.2 mg/L for the cladoceran

21 crustacean Ceriodaphnia dubia [21]. The chronic value is the geometric mean of the

22 lowest observable effects concentration (LOEC) and the no observable effect

23 concentration (NOEC). EA also measured an acute perchlorate LC50 of 490 mg/L for the
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1 cladoceran crustacean Daphnia magna [21], an acute LC50 exceeding 1000 mg/L (the

2 highest tested concentration) for the amphipod Hyalella azteca [22], and an acute LC50 of

3 1655 mg/L [21] and chronic value exceeding 490 mg/L [22] with the fathead minnow

4 Pimephales promelas (Table 2). Block Environmental Services [23] independently

5 measured an acute perchlorate LC50 of 77.8 mg/L and a chronic value of 15.2 mg/L for

6 the cladoceran crustacean Ceriodaphnia dubia in acceptable tests. No reports of

7 acceptable bioconcentration factors were discovered. An acceptable test by EA [24]

8 generated a 96 h plant value of 775 mg/L perchlorate to the aquatic alga Raphidocelis

9 subcapitata. The plant value is calculated identically to the chronic value for animals.

10 A number of studies containing acute and chronic toxicity data for the species

11 Daphnia magna [25], P. promelas [21,22,23], the coelenterate Hydra attenuata [26], the

12 Argentine toad Bufo arenarum [15], the African Clawed Frog Xenopus laevis [16,27], the

13 lampreys Lampetra appendix [14] and Petromyzon marinus [13] did not meet the

14 USEPA’s minimum requirements for use in developing water quality criteria. Also, plant

15 tests with the alga Chlorococcales [28], Scenedesmus quadricauda [29], and Microcystis

16 aeruginosa [29] did not meet requirements for use in criteria development. The acute

17 LC50s from the rejected studies tended to agree with the acceptable values, with none

18 falling outside the range of 269 to 670 mg/L perchlorate. However, in one study a chronic

19 effect on amphibian development was noted at 0.005 mg/L perchlorate [16], a level three

20 orders of magnitude below that reported for any of the acceptable data. Also, the rejected

21 plant data included estimated plant values at 79 and 360 mg/L perchlorate [29], well

22 below the single acceptable plant value. A complete listing and evaluation of all rejected

23 aquatic studies of perchlorate has been prepared and reported elsewhere [30].
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1 To fill the remaining data gaps for criteria development, a series of acute, chronic,

2 and bioconcentration tests were performed. These tests were performed according to

3 requirements in the USEPA guidelines for water quality criteria development [19],

4 including flow-through exposures with measured toxicant concentrations, use of

5 appropriate controls and exposure type and duration, and sensitive life stages of the test

6 organisms.

7 MATERIALS AND METHODS

8 Test Material

9 Sodium perchlorate monohydrate (CAS 7791-07-3) with a purity of greater than

10 99.9% was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA) and used as

11 received.

12 Perchlorate Analytical Procedures

13 Water samples (125 mL) for perchlorate determination were collected directly

14 from the middle of test chambers using a glass beaker and refrigerated, then shipped on

15 ice to APPL Laboratories (Fresno, CA) for analysis. Perchlorate concentrations in water

16 and tissue were measured by ion chromatography according to USEPA Method 314.0

17 [31]. Homogenized tissues (10 g) were shaken in 100 ml of deionized water for 1 h on an

18 orbital shaker, then filtered through Whatman (Clinton, NJ) GF/F and 0.45 µm filters.

19 Equipment included the Dionex (Sunnyvale, CA) DX500 ion chromatography system

20 equipped with a GP40 pump, an AS40 autosampler, AMMS-III 4 mm ion suppressor,

21 IonPac AG16 guard column (4x50mm), IonPac AS16 analytical column (4x250mm), and

22 CD20 detector. Conditions were as follows: injection volume: 1.0 mL; eluent: 50mM

23 NaOH; eluent flow rate: 1.5 mL/min; regenerant: 50mN H2SO4; regenerant flow rate: 8 to
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1 15 mL/min. Additional details of specific testing protocols and methods are available in

2 Parsons [30].

3 General Test Conditions

4 All tests were performed at ABC Laboratories (Columbia, MO) under flow-

5 through test conditions, with a proportional diluter system continuously delivering the

6 test solutions at a rate of at least two complete test-system volume replacements per day.

7 Diluters were volumetrically calibrated before each test, and the perchlorate

8 concentrations in the resulting solutions were quantified to ensure the diluters were

9 functioning properly before introduction of the test organisms. Fluorescent lighting was

10 maintained at an intensity of 492 ± 44 lux on a 16 h daylight photoperiod, with 30 min

11 simulated dawn and dusk periods. The dilution water in all tests was prepared by

12 blending naturally hard well water with well water that had been demineralized by

13 reverse osmosis to achieve a moderately hard water, as recommended in USEPA

14 guidance [19]. Hardness, alkalinity, and specific conductance of the dilution water ranged

15 from 140 to 160 mg/L as CaCO3, 150 to 162 mg/L as CaCO3, and 303 to 333 µS/cm,

16 respectively, for all tests. Test chambers were immersed in a circulating water bath

17 adjusted to maintain the water temperature at 22 ºC (± 2 ºC), except where otherwise

18 noted. Temperature and pH were measured with a Denver Instruments pH meter.

19 Dissolved oxygen was measured with a YSI Model 95 or WTW Oxi 330 dissolved

20 oxygen meter. Temperature, dissolved oxygen, and pH were measured in test chambers

21 on a daily basis for short-term acute toxicity tests, and at least weekly for longer term

22 chronic and bioconcentration tests. Temperature and pH were measured with a Denver

23 Instrument Company (Arvada, CO) pH meter, while dissolved oxygen was measured
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1 with either a YSI Model 95 (Yellow Springs, OH) or a WTW Oxi 330 (Ft. Myers, FL)

2 dissolved oxygen meter. Temperature was also monitored and recorded continuously

3 with an electronic data logging system. Hardness, alkalinity, and specific conductance in

4 the exposure chambers were measured at test initiation and at least weekly thereafter.

5 Biological observations and test monitoring were performed on a daily basis at a

6 minimum. Perchlorate concentrations in 48 h or 96 h acute toxicity tests were measured

7 at initiation and termination.

8 Each toxicity test consisted of one or more preliminary range-finding tests and a

9 definitive test. The range-finding tests consisted of static exposures to a series of five

10 widely spaced test concentrations, typically an order of magnitude different from each

11 other (e.g., 1, 10, 100, 1000, and 10000 mg/L). The definitive tests were flow-though

12 exposures consisting of a series of five or more perchlorate treatment concentrations,

13 each approximately half of the next higher concentration, as well as a perchlorate-free

14 control. In bioconcentration tests, the two perchlorate exposure concentrations were set at

15 1% and 0.1% of the measured perchlorate 96 h LC50 for these species. To account for

16 potential toxicity from the sodium cation of the sodium perchlorate, sodium chloride

17 controls were also included with the preliminary and/or definitive toxicity tests. The

18 sodium chloride controls were additional exposures with sodium chloride added to

19 dilution water at the same sodium concentration as that in the highest one or two sodium

20 perchlorate treatment concentrations. Animals were not fed in 96 h acute exposures.

21 For each test, the measured perchlorate EC50 and its 95% confidence limits were

22 calculated by the Trimmed Spearman-Karber method in TOXCALC version 5 (Tidepool

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1 Scientific Software, McKinleyville, CA). Fisher’s Exact Test was used to calculate the

2 NOEC and LOEC.

3 Rainbow trout acute toxicity test

4 Rainbow trout (Oncorhynchus mykiss) embryos were obtained from Trout Lodge

5 (Sumner, WA). Twenty rainbow trout fry per treatment level were exposed to sodium

6 perchlorate in 15 L glass aquaria for 96 h. The mean weight of the fish was 0.104 g. The

7 temperature of the test chambers was maintained at 14.9 ºC to 15.9 ºC. The test

8 procedures were designed to meet USEPA OPPTS 850.1075 [32] and ASTM E729-96

9 guidelines [33]. Nominal (target) exposure concentrations in the definitive test were 0

10 (control) 188, 375, 750, 1500, and 3000 mg/L perchlorate ion.

11 Bluegill Sunfish acute toxicity test

12 Juvenile Bluegill Sunfish (Lepomis macrochirus) were obtained from Osage

13 Catfisheries (Osage Beach, MO). Twenty bluegill per treatment level were exposed to

14 sodium perchlorate in 15 L glass aquaria for 96 h. The mean weight of the fish was

15 0.355 g. The test procedures were designed to meet USEPA OPPTS 850.1075 [32] and

16 ASTM E729-96 guidelines [33]. Nominal (target) exposure concentrations in the

17 definitive test were 0 (control) 625, 1250, 2500, 5000, and 10000 mg/L perchlorate ion.

18 Lumbriculus variegatus acute toxicity test

19 Adult oligochaete annelids of the species Lumbriculus variegatus were obtained

20 from in-house cultures at ABC Laboratories. Twenty organisms per treatment level, in

21 two replicates of ten each, were exposed to sodium perchlorate in 500 mL glass jars with

22 a screen collar, for 96 h. The test procedures were designed to meet USEPA OPPTS

23 850.1075 [34] and ASTM E729-96 guidelines [33]. Nominal (target) exposure
 18

1 concentrations in the definitive test were 0 (control) 625, 1250, 2500, 5000, and 10000

2 mg/L perchlorate ion.

3 Green frog acute toxicity test

4 Green frog tadpoles (Rana clamitans) were obtained from Amphibians of North

5 America (Nashville, TN). The tadpoles were approximately seven to eight months old at

6 testing, and were in a late stage of development, but had no apparent limb bud

7 development. The temperature of the test chambers was maintained at 18.4 ºC to 20.1 ºC.

8 Twenty tadpoles per treatment level were exposed to sodium perchlorate in 5 L glass

9 aquaria for 96 h. The test procedures were designed to meet USEPA OPPTS 850.1075

10 [32] and ASTM E729-96 guidelines [33]. Nominal (target) exposure concentrations in the

11 definitive test were 0 (control) 625, 1250, 2500, 5000, and 10000 mg/L perchlorate ion.

12 Clam acute toxicity test

13 Adult Asiatic clams (Corbicula fluminea) were collected from Little Dixie Lake

14 near Columbia, MO. Twenty clams per treatment level were exposed to sodium

15 perchlorate in 15 L glass aquaria for 96 h. The test procedures were designed to meet

16 USEPA OPPTS 850.1075 [34] and ASTM E729-96 guidelines [33]. Nominal (target)

17 exposure concentrations in the definitive test were 0 (control) 625, 1250, 2500, 5000, and

18 10000 mg/L perchlorate ion.

19 Midge acute toxicity test

20 Midges of the species Chironomus tentans were obtained from in-house cultures

21 at ABC Laboratories. The midges utilized were approximately 11 d post hatch at study

22 initiation, in the third instar. Twenty organisms per treatment level, in two replicates of

23 ten each, were exposed to sodium perchlorate in 500 mL glass jars with a screen collar,
 19

1 for 48 h. A thin layer of silica sand coated the bottom of the test chambers to provide a

2 substrate for the midges. The test procedures were designed to meet USEPA OPPTS

3 850.1075 [34] and ASTM E729-96 guidelines [33]. Nominal (target) exposure

4 concentrations in the definitive test were 0 (control) 625, 1250, 2500, 5000, and 10000

5 mg/L perchlorate ion.

6 Midge life-cycle chronic toxicity test

7 Midges of the species Chironomus tentans were obtained from egg masses

8 produced by adult midges in cultures at ABC Laboratories. The midges utilized were

9 approximately 1 d post hatch at study initiation. The test was initiated with the addition

10 of 12 midge larvae into 12 replicate test chambers for a total of 144 midges per treatment.

11 The test chambers were glass jars holding a solution volume of ~500 mL and

12 approximately 100 g of hydrated artificial sediment. The artificial sediment consisted of

13 76% fine silica sand, 20% kaolinite clay, 4% fine peat moss (passed through 500 μm

14 mesh sieve). The sediment was hydrated with water of approximately 43% of its dry

15 weight, and calcium carbonate was added to the sediment to adjust its pH to 6.5 to 7.0

16 before use. Four additional replicate test chambers were initiated with the addition of 12

17 midge larvae on day 10 to provide additional male midge during the reproduction

18 evaluation portion of the study. An emergent trap covered each of the test replicates.

19 The emergent traps consisted of a plastic cylinder with a screen top. The jars were placed

20 in a temperature-controlled water bath set to maintain test temperature at 23 ºC (± 2 ºC).

21 An intermittent feeding system was used to deliver concentrated algae (i.e., a

22 mixture of Selenastrum capricornutum and Ankistrodesmus falcatus) to each test

23 chamber during each diluter cycle. This feeder was used for 14 d prior to testing to coat
 20

1 the sediment with algae and for 19 d during the early development of the midge larvae.

2 The larvae were fed 3.0 to 4.5 mL of a 4 g/L flake food suspension for the first 6 d of

3 testing. The feeding rate was reduced to 1.5 mL/d on day 7 and then to 0.5 mL/d on day

4 35 to prevent dissolved oxygen depletion.

5 Nominal (target) perchlorate exposure concentrations in the definitive test were 0

6 (controls) 62.5, 125, 250, 500, 1000, and 2000 mg/L perchlorate ion. The sodium

7 concentration in the sodium chloride control was equivalent to that in a 4000 mg/L

8 sodium perchlorate solution. The proportional diluter system delivered approximately

9 125 mL of dilution water or test solution to each test chamber once every 3 h (2 test-

10 chamber volumes/d) for the first 7 d of testing. From this point until termination, the

11 diluter rate was increased to replace the test solution every 90 min (16 test-chamber

12 volumes/d) to maintain adequate dissolved oxygen levels within the test chambers.

13 Four replicate test chambers for each treatment were terminated after 20 d of

14 exposure to the perchlorate ion to record mortality and sublethal observations. The

15 surviving larvae were composited into a tared pre-ashed aluminum weigh boat and dried

16 at a temperature range of 63 to 82 °C for approximately 24 h. The dried larvae were

17 weighed and then ashed at a temperature of 550 °C for approximately 3.5 h, cooled, and

18 then weighed. The replicate mean individual ash-free dry weight (i.e., tissue weight) was

19 determined by subtracting the post-ashed weight from the pre-ashed weight and dividing

20 by the number of larvae present.

21 Emergent adults were gently aspirated into clean flasks, which contained a

22 minimal amount of water for egg deposition. The numbers of emergent male, female,

23 and unknown adults were recorded daily. Adults that were not observed in the
 21

1 emergence trap, but left a pupal skin were recorded as unknown. The unknown adults

2 were included in the total number of emergent adults. Available females were paired with

3 at least one or more males, if available. The flasks containing the adult pairs were capped

4 with a gas-permeable foam plug and placed at a slight angle in a sand matrix within a

5 glass tray. The adult pairs were observed daily for the production of an egg mass. The

6 number of successful and unsuccessful (i.e., the female died prior to depositing an egg

7 mass) pairings were recorded. Where possible the number of eggs/egg mass (i.e., per

8 female) was estimated by multiplying the number of egg rings within the mass by the

9 average number of eggs per egg ring. If the egg mass was malformed, then a total egg

10 count was performed. All egg counts were made with a dissecting microscope. After 42

11 d of exposure the numbers of surviving larvae and pupae within the test chambers were

12 recorded. The total number of emergent adults and surviving midge were added together

13 and reported as the 42 d survival.

14 Temperature, dissolved oxygen concentration, and pH were measured in all

15 replicate solutions at test initiation, weekly, and at test termination. No aeration was

16 provided to any control or test chamber during the test. The test procedures were

17 designed to meet USEPA Method 100.5 [35] and ASTM E1706-00 guidelines [36].

18 Bluegill sunfish bioconcentration test

19 Juvenile Bluegill Sunfish (Lepomis macrochirus) were obtained from Osage

20 Catfisheries (Osage Beach, MO). At study initiation the fish weighed 6.3 ± 2.4 g (wet

21 weight), were 62 ± 7.7 mm in standard length, and 76 ± 9.8 mm in total length. Sixty

22 bluegill per treatment level were exposed to sodium perchlorate in 70 L glass aquaria for

23 28 d. The nominal treatment levels were 0 (control), 1.5 mg/L and 15 mg/L perchlorate.
 22

1 The proportional diluter system delivered six volume replacements per day. Fish were

2 fed daily 2% of their body weight with commercial fish food. Any food remaining after

3 one hour was siphoned from the aquaria. Observations for mortality and sublethal

4 responses were made twice daily. The concentrations of perchlorate in water and tissue

5 were measured in all treatment solutions on days 0, 1, 2, 3, 7, 14, 21, and 28. Six fish

6 were collected on each sampling date and the whole fish were homogenized using a

7 Polytron (Elkhart, IN) homogenizer. Tissue samples were shipped to APPL Laboratories

8 (Fresno, CA) on dry ice for analysis. These test procedures were designed to meet

9 USEPA Guideline 165-4 [37] and OECD guideline 305 [38].

10 Clam bioconcentration test

11 Adult Asiatic clams (Corbicula fluminea) were collected from Little Dixie Lake

12 near Columbia, MO. For each treatment level, 200 active clams (at least 2 cm in

13 diameter) were exposed to sodium perchlorate in 70 L glass aquaria for 28 d. The

14 nominal treatment levels were 0 (control), 6.5 mg/L and 65 mg/L perchlorate. The

15 proportional diluter system delivered six volume replacements per day. Clams were fed a

16 mixture of the algal species Chaetoceros sp. and Isochrysis sp. throughout the exposure

17 period. The concentrations of perchlorate in water and tissue were measured in all

18 treatment solutions on days 0, 1, 2, 3, 7, 14, 21, and 28. An appropriate number of clams

19 were collected on each sampling date, shucked, and homogenized using a Polytron

20 homogenizer to obtain 23 to 33 g of tissue for analysis. Tissue samples were shipped to

21 APPL Laboratories (Fresno, CA) on dry ice for analysis. These test procedures were

22 designed to meet USEPA Guideline OPPTS 850.1710 [39] and ASTM E1022-94 [40].
 23

1 Additional details of specific testing protocols and methods are available in

2 Parsons [30].

3 RESULTS AND DISCUSSION

4 Rainbow trout acute toxicity test

5 Mean measured perchlorate concentrations ranged from 140 to 2760 mg/L (74 to

6 91% of the nominal concentrations). No sub-lethal effects were observed during the test;

7 all observations were either mortality or normal behavior. There was no mortality in the

8 control, the sodium control, or in the 140 or 442 mg/L treatments at 96 h. One fish (5%)

9 died in the 738 mg/L treatment, three (15%) in the 1460 mg/L treatment, and 17 (85%) in

10 the 2760 mg/L treatment. The 96 h NOEC for O. mykiss was 1460 mg/L and the LOEC

11 was 2760 mg/L. The 96 h EC50 was calculated to be 2010 mg/L based on mean measured

12 perchlorate concentrations.

13 Bluegill sunfish acute toxicity test

14 Mean measured perchlorate concentrations ranged from 547 to 9715 mg/L (88 to

15 104% of the nominal concentrations). No sub-lethal effects were observed during the test;

16 all observations were either mortality or normal behavior. There was no mortality in the

17 control, the 547 mg/L treatment, or in the level 4 (5220 mg/L equivalent) sodium control,

18 but one fish (5%) died in the level 5 (9715 mg/L equivalent) sodium control. Five fish

19 (25%) died in the 1260 mg/L perchlorate treatment, and all 20 (100%) died in the 2530,

20 5220, and 9715 mg/L perchlorate treatments. The 96 h NOEC for L. macrochirus was

21 547 mg/L perchlorate and the LOEC was 1260 mg/L. The 96 h EC50 was calculated to be

22 1470 mg/L based on mean measured perchlorate concentrations.

 24

1 Lumbriculus variegatus acute toxicity test

2 Mean measured perchlorate concentrations ranged from 470 to 10700 mg/L (75 to

3 115% of the nominal concentrations). Sub-lethal effects included discoloration and

4 sausage-like kinks in body walls, in exposure concentrations of 470 mg/L perchlorate and

5 higher. Neither of these effects was immobilizing or appeared to lead to death, and the

6 worms recovered from these effects in many cases, possibly by excising the segments

7 posterior to the kink. There was no mortality in the control or the sodium controls. One

8 organism (5%) died in each of the 470, 1240, and 2500 mg/L perchlorate treatments. All

9 20 (100%) organisms died in the 5760 and 10700 mg/L perchlorate treatments. USEPA

10 guidance [19] indicates that sublethal effects to consider in calculating an EC50 for an

11 acute test include severe effects leading to death, such as immobilization or loss of

12 equilibrium. Based on mortality, the 96 h NOEC for L. variegatus was 2500 mg/L, the

13 LOEC was 5760 mg/L, and the EC50 was 3710 mg/L perchlorate.

14 Green frog acute toxicity test

15 Mean measured perchlorate concentrations ranged from 680 to 9800 mg/L (96 to

16 116% of the nominal concentrations). There was no mortality in the control, the sodium

17 controls, or in the 680, 1200, or 2400 mg/L perchlorate treatments at 96 h. Nine tadpoles

18 (45%) died in the 5800 mg/L treatment, and all 20 (100%) died in the 9800 mg/L

19 treatment. Two additional tadpoles in the 5800 mg/L treatment exhibited loss of

20 equilibrium at 96 h. The 96 h NOEC for R. clamitans was 2400 mg/L and the LOEC was

21 5800 mg/L. The 96 h LC50 was calculated to be 5500 mg/L perchlorate based on

22 mortality. The 96 h EC50 was calculated to be 5100 mg/L perchlorate based on mortality

23 plus loss of equilibrium, a severe sub-lethal effect.

 25

1 Corbicula fluminea acute toxicity test

2 Mean measured perchlorate concentrations ranged from 713 to 10800 mg/L (93 to

3 114% of the nominal concentrations). No sub-lethal effects were observed during the

4 test; all observations were either mortality or normal behavior. A single clam (5%) died

5 in the control. There was no mortality in the level 4 (4850 mg/L equivalent) sodium

6 control, but all 20 clams died in the level 5 (10800 mg/L equivalent) sodium control at 96

7 h. In the perchlorate treatments, mortality was 0%, 5%, 25%, 20%, and 85% in the 713

8 mg/L, 1400 mg/L, 2710 mg/L, 4850 mg/L, and 10800 mg/L treatments, respectively. The

9 96 h NOEC for C. fluminea was 4850 mg/L and the LOEC was 10800 mg/L. The 96 h

10 LC50 was calculated to be 6680 mg/L perchlorate based on mortality. Due to mortality in

11 the sodium control, we could not conclude that the sodium ion did not contribute to the

12 observed toxicity attributed to perchlorate. However, we can conclude that the 96 h LC50

13 exceeded 4850 mg/L perchlorate.

14 Chironomus tentans acute toxicity test

15 Mean measured perchlorate concentrations ranged from 772 to 12700 mg/L (113

16 to 127% of the nominal concentrations). One midge (5%) was missing and considered

17 dead in the control and in the 1,000 mg/L equivalent sodium control, but there was no

18 mortality in the level 5 (10,000 mg/L equivalent) sodium control. In the perchlorate

19 treatments, mortality (missing midge were considered dead) was 5%, 20%, 15%, 15%,

20 and 90% in the 772 mg/L, 1545 mg/L, 3045 mg/L, 5640 mg/L, and 12700 mg/L

21 treatments, respectively. The 48 h NOEC for C. tentans was 5640 mg/L and the LOEC

22 was 12700 mg/L. The 48 h EC50 was calculated to be 8100 mg/L perchlorate.
 26

1 Midge life-cycle chronic toxicity test

2 Mean measured perchlorate concentrations ranged from 58.5 to 2080 mg/L (93 to

3 104% of the nominal concentrations). Survival in the control and the sodium control over

4 the 42 d exposure was 90% and 81%, respectively. In perchlorate treatments, survival

5 was 87%, 62%, 24%, 21%, 4%, and 0% in the 58.5, 118, 233, 489, 1020, and 2080 mg/L

6 exposures. Most of the surviving larvae from exposure concentrations greater than 233

7 mg/L were discolored. However, this sublethal effect was not considered in the

8 calculation of the EC50. The NOEC, LOEC, and EC50 for survival were 58.5, 118, and

9 208 mg/L, respectively.

10 Adult midge emergence was first recorded after 21 d of exposure. Emergence

11 totaled 89% and 28% in the control and sodium control through day 42, when the

12 exposure was terminated. Emergence in the perchlorate treatments ranged from 85% to

13 0%. The NOEC, LOEC, and EC50 for emergence were 58.5, 118, and 146, respectively.

14 The mean individual ash-free dry weight of surviving larvae at day 20 was 1.6

15 and 0.55 mg/L in the control and sodium control, and 1.4, 1.4, 0.93, 0.48, and 0.40 in the

16 58.5, 118, 233, 489, and 1020 mg/L exposures. The NOEC, LOEC, and EC50 for growth

17 were 118, 233, and 211, respectively.

18 The control and sodium control females produced egg masses with an average of

19 936 and 1792 eggs per egg mass, respectively. The mean number of eggs per egg mass was

20 918, 900, and 393 in the 58.5, 118, and 233 mg/L exposures. A single female emerged in

21 the 1080 mg/L treatment level but could not be paired. The NOEC, LOEC, and EC50 for

22 reproductive output were 118, 233, and 211 mg/L, respectively.

 27

1 The adult emergence was the most sensitive biological endpoint measured during the

2 midge life cycle exposure to perchlorate. Midges in the sodium chloride control group

3 were apparently developmentally inhibited. This developmental inhibition affected the

4 growth and emergence rates, but did not lower the survival rate or the individual

5 reproductive output of the emergent females. Because the 28% emergence rate in the

6 sodium control occurred at a sodium concentration equivalent to 4,000 mg/L perchlorate,

7 whereas an emergence of 11% was observed in the 233 mg/L perchlorate treatment, it is

8 considered likely that the observed effect was due to the perchlorate ion rather than the

9 sodium cation. The chronic value for C. tentans was 83.1 mg/L, calculated as the geometric

10 mean of the LOEC and NOEC for adult emergence.

11 Bluegill sunfish bioconcentration test

12 Mean measured perchlorate concentrations were 0, 1.4, and 11.9 mg/L in water in

13 the control and low and high level perchlorate exposures. All fish were observed to be

14 normal and healthy for the 28 d duration of the test. Concentrations of perchlorate in fish

15 tissue in the low treatment were not detected until day 21, but reached 1.1 mg/kg on day

16 21 and 1.5 mg/kg on day 28. In the high level treatment, perchlorate concentrations in

17 Bluegill increased throughout the test, reaching 6.8 mg/kg on day 28. According to

18 USEPA guidance (19) for water quality criteria development, if steady-state conditions

19 are not reached then the BCF should be taken as the highest BCF. Thus the BCF value

20 for the low treatment was 0.73 L/kg and the BCF for the high treatment was 0.68 L/kg for

21 whole Bluegill. The geometric mean BCF value for perchlorate in Bluegill is 0.70 L/kg.
 28

1 Clam bioconcentration test

2 Mean measured perchlorate concentrations were <0.026, 7.05, and 51.6 mg/L in

3 water in the control and low and high level perchlorate exposures. All clams were

4 observed to be normal and healthy for the 28 d duration of the test. It was not determined

5 if steady-state conditions were reached. According to USEPA guidance (19), if steady-

6 state conditions are not reached then the BCF should be taken as the highest BCF. Thus

7 the BCF value for the low treatment was 3.1 L/kg (from day 21) and the BCF for the high

8 treatment was 1.1 L/kg (from day 28). The geometric mean BCF value for perchlorate in

9 the clam C. fluminea is 1.85 L/kg.

10 Calculation of the Final Acute Value

11 The Final Acute Value (FAV) is an estimate of the concentration of perchlorate

12 corresponding to a cumulative probability of 0.05 of the acute toxicity values [19]. That

13 is, the FAV is a calculated estimate of the concentration of perchlorate so that 95% of the

14 genera tested will have a higher Genus Mean Acute Value (GMAV) than the FAV.

15 Acceptable acute toxicity data were available for ten species meeting the taxonomic

16 requirements for water quality criteria development [19]. For each species used to

17 develop the criteria, the species mean acute value (SMAV) was calculated. SMAVs were

18 either a single measured EC50 value, or were calculated as the geometric mean of the

19 EC50s when two or more acceptable acute tests were available. The USEPA [19]

20 recommends the geometric mean instead of the arithmetic mean because the distributions

21 of the sensitivities of individual organisms in toxicity tests on most materials are more

22 likely to be lognormal than normal. SMAVs for perchlorate ranged from 71.7 mg/L to

23 8140 mg/L (Table 2). The most sensitive species to acute perchlorate toxicity was the
 29

1 Cladoceran crustacean C. dubia, with LC50 values of 66 mg/L [21] and 77.8 mg/L

2 perchlorate [23], yielding a SMAV of 71.7 mg/L. The two most perchlorate tolerant

3 invertebrate species in acute exposures were C. fluminea and C. tentans with SMAVs of

4 6680 mg/L and 8140 mg/L perchlorate, respectively. The Bluegill was the most sensitive

5 vertebrate species, with a SMAV of 1470 mg/L, though the fathead minnow and rainbow

6 trout were similarly sensitive, with SMAVs of 1655 and 2010 mg/L, respectively. The

7 green frog R. clamitans was the most tolerant vertebrate species tested, with a SMAV of

8 5120 mg/L. In this study, the GMAVs were all equivalent to the SMAVs.

9 The calculation of the FAV was performed according to USEPA guidance (19).

10 The GMAVs were ordered from highest to lowest and assigned a rank, R, from R=1 for

11 the lowest GMAV (Ceriodaphnia) to R=10 for the highest GMAV (Chironomus) (Table

12 2). The cumulative probability, P, for each GMAV was calculated as P=R/(N+1). The

13 four GMAV values with cumulative probabilities closest to 0.05 were selected, and the

14 FAV for perchlorate was calculated to be 39.9 mg/L using the following equations:

(∑ (ln GMAV )) 2

∑ ((ln GMAV ) ) −
2

15 S2 = 4
(∑ ( P ))
2

∑ (P ) − 4

16 L=
∑ (ln GMAV ) − S (∑ ( P ))
4

17 A=S ( )
0.05 + L

18 FAV = e A

19 The values that are reported as “greater than” values (e.g., H. azteca) were

20 included in the calculation of the FAV because not having including these values would
 30

1 unnecessarily lower the FAV by eliminating values for resistant species. The three most

2 sensitive species to perchlorate were invertebrates (C. dubia, Daphnia magna, and H.

3 azteca), and all of the SMAVs are higher than the FAV.

4 Calculation of the Final Chronic Value

5 The Final Chronic Value (FCV) is a calculated estimate of the concentration of

6 perchlorate such that 95% of the genera tested will have a higher Genus Mean Chronic

7 Value (GMCV) than the FCV. The FCV can be calculated in the same way as the FAV,

8 using chronic values for eight or more families, or can be calculated from the FAV

9 divided by the appropriate acute-chronic ratio (ACR). The ACR may be calculated from

10 acute and chronic toxicity test data from three or more families, provided that at least one

11 species is a fish, at least one is an invertebrate, and at least one is an acutely sensitive

12 freshwater species (19). Acute and chronic toxicity data were available for C. dubia, C.

13 tentans, and P. promelas. C. dubia was the most acutely sensitive. Thus, these ACRs met

14 the requirements for calculation of the ACR. Chronic values ranged from 15.3 mg/L to

15 greater than 490 mg/L perchlorate. The chronic value for a 7 d chronic exposure of C.

16 dubia was 18.2 mg/L perchlorate [21], while that for an independent 6 d chronic test was

17 15.3 mg/L perchlorate [23]. (Table 3). Although results of both tests for C. dubia were

18 similar, because they were not the same testing facility, USEPA guidelines [19] suggest

19 that acute-chronic ratios (ACR) should be calculated for each test, and the geometric

20 mean of the ACRs calculated. Therefore, ACRs of 3.63 (66.0/18.2 mg/L) and 5.12

21 (77.8/15.2 mg/L) were calculated, and the geometric mean of these two ACR values

22 gives a species mean ACR of 4.31 for C. dubia.

 31

1 The chronic value for the second most sensitive species, C. tentans, was 83.1

2 mg/L. The corresponding acute value for C. tentans was 8140 mg/L perchlorate, yielding

3 an ACR of 98.0.

4 No significant effects of perchlorate were observed even at the highest

5 concentrations used in a 35 d early life-stage chronic test [22] with P. promelas, and the

6 chronic value is greater than 490 mg/L. The corresponding acute value for the Fathead

7 Minnow was 1655 mg/L, yielding an ACR of less than 3.38.

8 The species mean ACRs are similar for C. dubia and P. promelas (4.31 and

9 <3.38, respectively), but the species mean ACR for C. tentans (98.0) is more than a factor

10 of 10 higher. USEPA guidance (19) states that if the species mean ACR seems to

11 increase or decrease as the SMAV increases, the final acute-chronic ratio (FACR) should

12 be calculated as the geometric mean of the ACR for those species with SMAV near the

13 FAV. In this case the species with the highest SMAV (C. tentans) does have the highest

14 ACR (Table 3). Only C. dubia has a SMAV (71.7 mg/L) near the FAV (39.9 mg/L).

15 Thus, the FACR is 4.31, which is the species mean ACR for C. dubia.

16 The final chronic value (FCV) is then calculated to be 9.26 mg/L perchlorate, as

17 the FAV (39.9 mg/L) divided by the FACR (4.31). The most sensitive species to chronic

18 perchlorate toxicity was C. dubia, and all of the SMCVs are higher than the FCV.

19 Calculation of the Final Plant Value

20 Acceptable data on the effects of perchlorate on aquatic plants are available for

21 one plant species. The toxicity of perchlorate to the freshwater green alga Raphidocelis

22 subcapitata, was measured in a 96 h exposure test [24]. The IC25, a point estimate of the

23 toxicant concentration that caused a 25% reduction in growth, was calculated to be 615
 32

1 mg/L. Because plants did not appear to be more sensitive to perchlorate than animals, no

2 other plant taxa data were used and the Final Plant Value (FPV) is equal to 615 mg/L.

3 Additional toxicity testing using acceptable protocols with other species may be

4 warranted to increase confidence in the FPV and confirm that plants are less sensitive to

5 perchlorate than aquatic animals.

6 Calculation of the Final Residue Value

7 A Final Residue Value (FRV) is designed to prevent concentrations of

8 contaminants in commercially or recreationally important aquatic species from affecting

9 marketability due to exceedance of an FDA Action Level, and to prevent health effects in

10 wildlife that eat aquatic species [19]. Limiting tissue concentrations of contaminants are

11 derived from FDA Action Levels or a long-term wildlife feeding study, and linked to

12 water concentrations by a bioconcentration factor, which is the ratio of contaminant

13 concentration in tissue to that in water. To date, an FDA Action Level for perchlorate has

14 not been established, nor has a long-term wildlife feeding study been performed. Thus, it

15 is not possible to develop a FRV at this time.

16 Acceptable data on bioconcentration (using whole body tissue analysis) of

17 perchlorate in aquatic organisms is available for an aquatic invertebrate, the Asiatic clam

18 C. fluminea and the Bluegill L. macrochirus [this study]. The geometric mean

19 bioconcentration factor (BCF) value for perchlorate in clams, based on the highest

20 measured BCFs in each of two exposure concentrations, was 1.85 L/kg for C. fluminea

21 and 0.70 L/kg for L. macrochirus. These BCFs indicate that these animals take up

22 perchlorate at concentrations less than or slightly exceeding those in the exposure media.

23 This conclusion is supported by field measurements of BCFs typically less than one [11].
 33

1 Statement of Water Quality Criteria

2 National ambient water quality criteria consist of two values, a Criterion

3 Maximum Concentration (CMC) and a Criterion Continuous Concentration (CCC). The

4 CMC for perchlorate is calculated to be 20 mg/L, half of the Final Acute Value (39.9

5 mg/L), rounded to two significant digits. The CMC represents an estimate of the

6 concentration in water to which an aquatic community can be exposed briefly without

7 unacceptable effects.

8 The CCC is equal to the lowest of the FCV (9.26 mg/L), the FPV (615 mg/L), and

9 the FRV, rounded to two significant digits. Because an FRV cannot be calculated in the

10 absence of a FDA Action Level or a long-term wildlife feeding study, the CCC for

11 freshwater was equal to the FCV, which was 9.3 mg/L. The CCC is an estimate of the

12 highest concentration to which an aquatic community can be exposed indefinitely without

13 unacceptable effects.

14 The freshwater perchlorate criteria can be stated according to the instructions in

15 the USEPA Guideline [19] as follows:

16 The procedures described in the “Guidelines for Deriving Numerical National

17 Water Quality Criteria for the Protection of Aquatic Organisms and Their Uses”

18 indicate that, except possibly where a locally important species is very sensitive,

19 freshwater aquatic organisms and their uses should not be affected unacceptably if

20 the 4 d average concentration of perchlorate ion does not exceed 9.3 mg/L more

21 than once every 3 year on the average and if the 1 h average concentration does

22 not exceed 20 mg/L more than once every 3 years on the average.
 34

1 As the toxicity of perchlorate to additional plants and animals is measured, these

2 criteria should be re-calculated to incorporate this data. Amphibians are thought to be

3 very sensitive environmental indicator species, and additional studies using native

4 amphibians in long-term chronic toxicity tests are planned.

5 ACKNOWLEDGEMENTS

6 This work was performed for the Department of the Air Force under delivery order 0063

7 of contract F41624-95-D-9018.

8 REFERENCES

9 [1] U.S. Environmental Protection Agency. 1997. Announcement of the draft drinking

10 water contaminant candidate list; notice. 62 FR No. 193 52194-52219, October 6.

11 [2] Brechner RJ, Parkhurst GD, Humble WO, Brown MB, Herman WH. 2000.

12 Ammonium perchlorate contamination of Colorado River drinking water is

13 associated with abnormal thyroid function in newborns in Arizona. J. Occup.

14 Environ. Med. 42: 777-782.

15 [3] Lawrence JE, Lamm SH, Pino K, Richman K, Braverman LE. 2000. The effect of

16 short-term, low-dose perchlorate on various aspects of thyroid function. Thyroid

17 10:659-663.

18 [4] Lawrence JE, Lamm SH, Braverman LE. 2001. Low-dose perchlorate (3 mg daily)

19 and thyroid function. Thyroid 11: 295.

 35

1 [5] Greer MA, Goodman G, Pleus RC, Greer SE. 2000. Does environmental perchlorate

2 exposure alter human thyroid function? Determination of the dose-response for

3 inhibition of radioiodine uptake. Endocr. J. 47 (suppl): 146.

4 [6] Schwartz J. 2001. Gestational exposure to perchlorate is associated with measures of

5 decreased thyroid function in a population of California neonates. Thesis.

6 University of California, Berkeley, CA.

7 [7] Smith PN, Theodorakis CW, Anderson TA, Kendall RJ. 2001. Preliminary

8 assessment of perchlorate in ecological receptors at the Longhorn Army

9 Ammunition Plan (LHAAP), Karnack, TX. Ecotoxicology 10: 305-313.

10 [8] Urbansky ET, Collette TW, Robarge WP, Hall WL, Skillen JM, Kane PF. 2001.

11 Survey of fertilizers and related materials for perchlorate (ClO4-). USEPA/600/R-

12 01/049. Final Report. U.S. Environmental Protection Agency Office of Research

13 and Development, Cincinnati, OH.

14 [9] U.S. Environmental Protection Agency. 2002. Perchlorate environmental

15 contamination: toxicological review and risk characterization. External review

16 draft. NCEA-1-0503, Office of Research and Development. Washington, DC.

17 [10] Urbansky ET. 1998. Perchlorate chemistry: implications for analysis and

18 remediation. Biorem. J. 2: 81-95.

19 [11] Parsons. 2001. Scientific and technical report for perchlorate biotransport

20 investigation: a study of perchlorate occurrence in selected ecosystems. Interim

 36

1 Final Report for the Air Force Institute for Environment, Safety and Occupational

2 Health Risk Analysis, Brooks AFB, TX.

3 [12] Kendall RJ, Dickerson RL, Giesy JP, and Suk WP. 1998. Principles and processes

4 for evaluating endocrine disruption in wildlife. SETAC Press, Pensacola, FL.

5 [13] Kao Y, Manzon RG, Sheridan MA, Youson JH. 1999. Study of the relationship

6 between thyroid hormones and lipid metabolism during KClO4-induced

7 metamorphosis of landlocked lamprey, Petromyzon marinus. Comp. Biochem.

8 Physiol. (C) 122: 363-373.

9 [14] Holmes JA, Chu H, Khanam SA, Manzon RG, Youson JH. 1999. Spontaneous and

10 induced metamorphosis in the American brook lamprey, Lampetra appendix.

11 Can. J. Zool. 77: 959-971.

12 [15] Miranda LA, Paz DA, Dezi RE, Pisano A. 1995. Immunocytochemical and

13 Morphometric Study of TSh, PRL, GH, and ACTH Cells in Bufo arenarum

14 Larvae with Inhibited Thyroid Functions. Gen. Comp. Endocrinol. 98(2): 166-

15 176.

16 [16] Goleman WL, Urquidi LJ, Anderson TA, Smith EE, Kendall RJ, Carr JA. 2002.

17 Environmentally relevant concentrations of ammonium perchlorate inhibit

18 development and metamorphosis in Xenopus laevis. Environ. Toxicol. Chem. 21:

19 424-430.
 37

1 [17] Adema DMM, Henzen L. 1989. A comparison of plant toxicities of some industrial

2 chemicals in soil culture and soilless culture. Ecotoxicol. Environ. Saf. 18: 219-

3 229.

4 [18] Suter GW, Efroymson RA, Sample BE, Jones DE. 2000. Ecological risk assessment

5 for contaminated sites. Lewis Publishers, Boca Raton, FL.

6 [19] Stephan CE, Mount DI, Hansen DJ, Gentile JH, Chapman GA, Brungs WA. 1985.

7 Guidelines for deriving numerical national water quality criteria for the protection

8 of aquatic organisms and their uses. USEPA 822/R-85/100. National Technical

9 Information Service, Springfield, VA.

10 [20] U.S. Environmental Protection Agency. 1995. Final water quality guidance for the

11 Great Lakes system; final rule. March 23, 1995. Fed. Regist. 60(56): 15365-

12 15425.

13 [21] EA Engineering, Science, and Technology. 1998. Results of acute and chronic

14 toxicity testing with sodium perchlorate. Report Number 2900. Brooks Air Force

15 Base, TX.

16 [22] EA Engineering, Science, and Technology. 2000. Results of chronic toxicity testing

17 with sodium perchlorate using Hyalella azteca and Pimephales promelas. Report

18 Number 3505. Brooks Air Force Base, TX.

19 [23] Block Environmental Services. 1998. LC50 aquatic toxicity test results for

20 ammonium perchlorate - a two species chronic definitive bioassay. Final Report.

21 Santa Clara Valley Water District. San Jose, CA.

 38

1 [24] EA Engineering, Science, and Technology. 1999. Results of algal toxicity testing

2 with sodium perchlorate. Report Number 3188. Brooks Air Force Base, TX.

3 [25] Bringmann G, Kuhn R. 1977. The effects of water pollutants on Daphnia magna. Z.

4 Wasser-Abwasser-Forsch. 10: 161-166.

5 [26] Confer PD, Wolfe RE, Kinkead ER. 1996. Developmental toxicity screen of

6 ammonium perchlorate using Hydra attenuata. AL/OE-TR-1996-0162.

7 Armstrong Laboratory. Wright-Patterson Air Force Base, OH.

8 [27] Dumont JN, Bantle JA. 1998. FETAX analysis of ammonium perchlorate.

9 Oklahoma State University. Stillwater, OK.

10 [28] Krebs F. 1991. Bestimmung der biologischen schadwirkung wassergefahrdender

11 stoffe im assimilations-zehrungs-test (a-z-test). Deutsche Gewasserkundliche

12 Mitteilungen 35: 161-170.

13 [29] Bringmann G, Kuhn R. 1978. The effect of water pollutants on blue-green algae

14 (Microcystis aeruginosa) and green algae (Scenedesmus quadricauda) in the cell

15 multiplication inhibition test. Vom Wasser. 50: 45-60.

16 [30] Parsons. 2002. Scientific and technical report for development of freshwater water

17 quality criteria for perchlorate. Final Report for the Air Force Institute for

18 Environment, Safety and Occupational Health Risk Analysis, Brooks AFB, TX.

19 [31] Hautman DP, Munch DJ, Eaton AD, Haghani AW. 1999. Method 314.0:

20 Determination of perchlorate in drinking water using ion chromatography. U.S.

 39

1 Environmental Protection Agency National Exposure Research Laboratory,

2 Cincinnati, OH.

3 [32] U.S. Environmental Protection Agency. 1996. Ecological effects test guidelines.

4 OPPTS 850.1075. Fish acute toxicity test, freshwater and marine. USEPA 712-C-

5 96-118. Office of Prevention, Pesticides, and Toxic Substances, Washington, DC.

6 [33] American Society for Testing and Materials. 1996. Standard guide for conducting

7 acute toxicity tests on test materials with fishes, macroinvertebrates, and

8 amphibians. ASTM Designation E729-96. Barr Harbor, PA.

9 [34] U.S. Environmental Protection Agency. 1996. Ecological effects test guidelines.

10 OPPTS 850.1010. Aquatic invertebrate acute toxicity test. USEPA 712-C-96-114.

11 Office of Prevention, Pesticides, and Toxic Substances, Washington, DC.

12 [35] U.S. Environmental Protection Agency. 2000. Methods for measuring the toxicity

13 and bioaccumulation of sediment-associated contaminants with freshwater

14 invertebrates. Second Edition. USEPA 600-R-99-064. Washington, DC.

15 [36] American Society for Testing and Materials. 2000. Test method for measuring the

16 toxicity of sediment-associated contaminants with freshwater invertebrates.

17 ASTM Designation E1706-00. Barr Harbor, PA.

18 [37] U.S. Environmental Protection Agency. 1982. Pesticide assessment guidelines,

19 subdivision N, chemistry: environmental fate. USEPA 540/09-82-031. Office of

20 Pesticide Programs, Washington, DC.

 40

1 [38] Organization for Economic Cooperation and Development. 1996. OECD guidelines

2 for testing of chemicals “Proposed guideline 305, bioconcentration: flow-through

3 fish test”. Paris.

4 [39] U.S. Environmental Protection Agency. 1996. Ecological effects test guidelines.

5 OPPTS 850.1710. Oyster BCF. USEPA 712-C-96-127. Office of Prevention,

6 Pesticides, and Toxic Substances, Washington, DC.

7 [40] American Society for Testing and Materials. 1994. Standard guide for conducting

8 bioconcentration tests with fishes and saltwater bivalve mollusks. ASTM

9 Designation E1022-94. Barr Harbor, PA.

 41

1 Table 1. Maximum perchlorate concentrations at six U.S. locations [11].

Media Measured perchlorate concentration Site of Maximum

(parts per million) Concentration

N % Detects Average Max

Soil 113 38% 57.7 1,470 Lake Mead Area

Terrestrial 177 50% 34.7 428 Lake Mead Area

Vegetation

Aquatic 50 24% 38.8 176 Lake Mead Area

Vegetation

Surface 147 57% 10.5 130 Lake Mead Area

Water

Pore Water 10 50% 19.6 98.0 Lake Mead Area

Sediment 93 23% 12.8 56.0 Lake Mead Area

Terrestrial 88 18% 13.4 53.0 Lake Mead Area

Mammal

Fish 107 3% 16.4 44.3 Lake Mead Area

Terrestrial 33 12% 12.6 6.2 Allegany Ballistics

Insect Laboratory
 42

Measured perchlorate concentration

(parts per million)

Terrestrial 42 12% 1.5 4.2 Lake Mead Area

Bird

Amphibian 22 12% 1.1 1.7 Longhorn Army

Ammunition Plant

Aquatic 4 75% 0.74 1.0 Longhorn Army

Insect Ammunition Plant

1
 43

1 Table 2. Acute toxicity of perchlorate to freshwater animals.

C S

a c

t i

e e

g n

o t

r i

y f

i
[
c
E

x
N
p
a
o
m
s
e
u

a
 44

Invertebrate Cladoceran Ceriodaphnia dubia 66 71.7 1 [28]

crustacean
[48h static] 77.8 [29]

Invertebrate Cladoceran Daphnia magna 490 490 2 [28]

crustacean
[48h static]

Invertebrate Amphipod Hyalella azteca >1,000 >1,000 3 [27]

[96h]

Fish – centrarchid Bluegill Lepomis macrochirus 1,470 1,470 4 This study

[96h flow-through]

Fish – cyprinid Fathead Minnow Pimephales promelas 1,655 1,655 5 [28]

[96h static]
 45

Fish – salmonid Rainbow Trout Oncorhynchus mykiss 2,010 2,010 6 This study

[96h flow-through]

Invertebrate Oligochaete Lumbriculus 3,710 3,710 7 This study

variegatus
[96h flow-through]

Amphibian Green Frog Rana clamitans 5,100 5,100 8 This study

[96h flow-through]

Invertebrate Asiatic Clam Corbicula fluminea 6,680 6,680 9 This study

[96h flow-through]

Invertebrate Midge Chironomus tentans 8,140 8,140 10 This study

[48h flow-through]

1
 46

1 Table 3. Chronic toxicity of perchlorate and acute-chronic ratios to freshwater

2 animals.

Common Scientific Acute Chronic ACR SMACR Rank Source

Name Name LC50 Value

(mg/L) (mg/L)

Cladoceran Ceriodaphnia 66 18.2 3.63 4.31 1 [28]

Crustacean dubia
77.8 15.2 5.12 [29]

Midge Chironomus 8,140 83.1 98.0 98.0 2 [26]

tentans

Fathead Pimephales 1,655 >490 <3.38 <3.38 3 [28, 27]

minnow promelas