Achieving Stable pH Without Buffers in Aquatic Animal Model Exposure Systems
IAAAM 2016
Scott Willens1*; Christine E. Baer2; Linda M. Brennan1; Ronald Miller Jr.2; David M. Kumsher2; William S. Eck3; Jonathan D. Stallings1
1United States Army Center for Environmental Health Research (USACHER), Fort Detrick, MD, USA; 2Excet, Inc., Fort Detrick, MD, USA; 3Army Public Health Center, Aberdeen Proving Ground, Aberdeen, MD, USA


Absorption of toxic industrial chemicals and materials by aquatic species is dependent on physicochemical properties of the toxicant, as well as physiological and environmental factors. Perchlorate - a common oxidizer with broad military and industrial explosive, pyrotechnic, and propellant applications - inhibits iodine uptake by the thyroid gland, causing developmental abnormalities in aquatic organisms, and potentially thyroid and neurological disease in humans and other mammals.1-7 Sodium (meta) periodate may provide a safer alternative, but the effects of periodate exposure are unknown.8

In a USACEHR study in progress, Xenopus laevis tadpoles will be exposed to different concentrations each of sodium perchlorate or sodium periodate to assess the effects of periodate on thyroid function. Concentrations will be determined through guidance from the Amphibian Metamorphosis Assay Guidelines and information generated from in-house range-finding studies.9 Presence of inorganic anions was found to have a profound effect on the solubility of the test substance. Sodium periodate is highly soluble in Milli-Q (Millipore, Billerica, MA) water, which was used to prepare stock solutions to be introduced at the target test concentrations using a flow-through system. However, periodate precipitates upon introduction to well water with a pH ≥ 7.0 due to formation of insoluble calcium periodate. Therefore, pH control is necessary to maintain target concentrations of test chemical without the necessity of a buffer control arm, significantly reducing the number of animals required.

For our preliminary diluter study, four test tanks with 20 stage 51 tadpoles were maintained within a pH range of 6.5–7.0. Acidified reverse osmosis water was pumped from a holding carboy into a mixing tank, which was continuously supplied with laboratory-processed well water and then into the diluter system for the test. The carboy contained 20 L of 0.116N HCl which was pumped into the mixing tank using an AA9 microprocessor dosing pump (LMI, Ivyland, PA). The normality of the acid in the carboy was selected based on trial runs with the dosing pump to obtain a reliable volume (> 1 mL/stroke). A sc200 Universal Controller (Hach, Loveland, CO) regulated output of the dosing pump to maintain the desired pH range in the mixing tank, which was confirmed with continuous pH readings from the PC1R1A probe (Hach). The sc200 pH set point was adjusted slightly below the desired pH ranges in the tanks. The difference between the set point pH and the desired pH increased as the study progressed due to the addition of air stones, increased biomass from maturing tadpoles, and resultant increased algae feedings.

At the conclusion of the 33d test, sc200 output was set at 6.42, and mean pH value across the four study tanks was 6.8 ± 0.08. Mean temperature was 22.3 ± 0.28°C, mean dissolved oxygen was 7.3 ± 0.54 mg/mL, and mean conductivity was 0.657 ± 0.006 uS/cm. All parameters were measured with an HQ40D Multimeter (Hach). Tadpoles exhibited normal growth and behavior without increased morbidity or mortality. Results suggest that this system is an effective model for environmental and developmental toxicology studies, pharmacology studies, and enhanced bioavailability of pharmaceuticals in aquaculture, husbandry, and clinical settings.9

Acknowledgements & Disclaimers

The authors thank Dr. Jason Koontz, Mr. Mark Widder, and Ms. Jordan McNairn for their assistance in the mechanical, electrical, and plumbing set-up and calibration of the diluter. Research was conducted in compliance with the Animal Welfare Act and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.

* Presenting author

Literature Cited

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2.  Dean KE, Palachek RM, Noel JM, Warbritton R, Aufderheide J, Wireman J. Development of freshwater water-quality criteria for perchlorate. Environ Tox Chem. 2004;23(6):1441–1451.

3.  [ATSDR] Agency for Toxic Substances and Disease Registry. Toxicological Profile for Perchlorates. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service; 2008.

4.  U.S. Environmental Protection Agency. Perchlorate Environmental Contamination: Toxicological Review and Risk Characterization. External Review Draft. NCEA-1-0503. Washington, DC: Office of Research and Development; 2002.

5.  Bernhardt RR, von Hippel FA, Creska WA. Perchlorate induces hermaphroditism in threespine sticklebacks. Environ Tox Chem. 2006;25(8):2087–2096.

6.  Goleman WL, Carr JA. Contribution of ammonium ions to the lethality and antimetamorphic effects of ammonium perchlorate. Environ Tox Chem. 2006;25(4):1060–1067.

7.  Goleman WL, Urquidi LJ, Anderson TA, Smith EE, Kendall RJ, Carr JA. Environmentally relevant concentrations of ammonium perchlorate inhibit development and metamorphosis in Xenopus laevis. Environ Tox Chem. 2002;21(2):424–430.

8.  Mainiero C. Picatinny to remove tons of toxins from lethal rounds. News Archives. 2013.

9.  [OECD] Organisation for Economic Cooperation and Development. OECD Guideline for the Testing of Chemicals: Amphibian Metamorphosis Assay. No. 231. Paris, France. 2009.


Speaker Information
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Scott Willens, DVM, PhD, DACVPM
United States Army Center for Environmental Health Research (USACHER)
Fort Detrick, MD, USA

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