U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2012–5008

Prepared in cooperation with Montana Fish, Wildlife, and Parks, U.S. Bureau of Land 

Management, and the U.S. Environmental Protection Agency

The Potential Effects of Sodium Bicarbonate, a Major 

Constituent of Produced Waters from Coalbed Natural 

Gas Production, on Aquatic Life
Cover Photograph Index

Photogragh showing Beaver Creek 

inflow to Powder River, Montana.

Photograph showing the greening of 

vegetation as a result of water inputs 

in the arid region (Lower Beaver Creek, 

Wyoming).
The Potential Effects of Sodium 

Bicarbonate, a Major Constituent of 

Produced Waters from Coalbed Natural 

Gas Production, on Aquatic Life

Edited by Aïda M. Farag and David D. Harper

Prepared in cooperation with Montana Fish, Wildlife, and Parks, U.S. Bureau of 

Land Management, and the U.S. Environmental Protection Agency

Scientific Investigations Report 2012–5008 

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2012

For more information on the USGS—the Federal source for science about the Earth, its natural and living 

resources, natural hazards, and the environment, visit http://www.usgs.gov or call 1–888–ASK–USGS.

For an overview of USGS information products, including maps, imagery, and publications, 

visit http://www.usgs.gov/pubprod

Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the 

U.S. Government.

Although this report is in the public domain, permission must be secured from the individual copyright owners to 

reproduce any copyrighted materials contained within this report.

Suggested citation:

Farag, A.M., and Harper, D.D., eds, 2012, The potential effects of sodium bicarbonate, a major constituent from coalbed natural gas production, on aquatic life: U.S. Geological Survey, Scientific Investigations Report 2012–5008, 101 p. 
iii

Acknowledgments

The research described in this report was a collaborative effort between the U.S. Geological 

Survey and Montana Fish, Wildlife, and Parks. Thanks to the many others from within and outside of these agencies that participated in the effort.

Connie Boese, University of Wyoming, and staff at the Montana Department of Public Health 

and Human Services performed chemical measurements. Review comments from Dr. Will Clements, Colorado State University, and J. Russell Hockett, U.S. Environmental Protection Agency, 

improved the report. Cathy Eakin, Montana Fish, Wildlife, and Parks, provided technical assistance in the field and with laboratory experiments performed in Montana. Elizabeth MacConnell, U.S. Fish and Wildlife Service provided histology analyses. Dr. Joseph Meyer, University of 

Wyoming, provided input and consultation during initial study designs. Brady Morris, University 

of Wyoming provided technical assistance with the fathead minnow laboratory experiments and 

with the presence/absence field work. The crew of the U.S. Fish and Wildlife Service, Jackson 

National Fish Hatchery, assisted with maintaining cultured organisms in Jackson, Wyoming. 

Montana, Fish, Wildlife and Parks staff provided sturgeon for experiments. Montana Department of Environmental Quality provided review comments on the draft report. Collaboration 

with Dave Zaft and additional staff from the Wyoming Game and Fish, Fisheries Research Unit, 

was instrumental in completion of the presence/absence surveys. The field experiments could 

not have been conducted without the assistance of the Wyoming Game and Fish Administration 

and Permitting Office who worked beyond traditional business hours to complete the approval 

process. 

Private landowners were gracious in allowing access to their property for water collection and 

performing in situ experiments. Thanks especially to John Iberlin and Tom Harriet who allowed 

access repeatedly over the years. Fidelity Exploration and Production Company allowed access 

to their property during sample collection from their outflow.

This project would not have been possible without funds provided by the U.S. Bureau of Land 

Management and the U.S. Environmental Protection Agency. The collaborative process with all 

of the members of Aquatic Task Group, formed under the Powder River Interagency Working 

Group, was instrumental as they provided suggestions for study design and focus throughout the 

project.

Mixing zone studies could not have been completed without Tom Cleasby, Colleague, U.S. Geological Survey who provided assistance in the field and with data interpretations. Brad Mueller, 

U.S, Geological Survey, provided technical assistance during the fathead minnow experiments. 

Darren Rhea, U.S. Geological Survey assisted with the presence/absence data collection and 

compilation. Luke May, U.S. Geological Survey provided assistance with data quality assurance 

and table construction. The suggestions provided by Kevin Buhl, U.S. Geological Survey were 

very valuable for the completion of this report. 

v

Volume Contents

1. Introduction: The Potential Effects of Sodium Bicarbonate, a Major Constituent of 

Produced Waters from Coalbed Natural Gas Production, on Aquatic Life ........................1

2. The Acute Toxicity of Sodium Bicarbonate Defined Under Laboratory Conditions ..........9

3. The Chronic Toxicity of Sodium Bicarbonate Defined Under Laboratory Conditions .....39

4. The Potential Toxicity Defined In Situ and Streamside in the Powder River and Tongue 

River Basins.................................................................................................................................61

5. Presence/Absence of Native and Non-native Species in the Powder and Tongue 

River Watersheds .......................................................................................................................79

6. Summary and Sample Calculations for Water-Quality Criteria...........................................93
vi

Conversion Factors, Abbreviations, and Datums

SI to Inch/Pound

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

millimeter (mm) 0.03937 inch (in.)

meter (m) 3.281 foot (ft) 

kilometer (km) 0.6214 mile (mi)

kilometer (km) 0.5400 mile, nautical (nmi) 

meter (m) 1.094 yard (yd) 

Volume

liter (L) 33.82 ounce, fluid (fl. oz)

liter (L) 2.113 pint (pt)

liter (L) 1.057 quart (qt)

liter (L) 0.2642 gallon (gal)

microliter (μL) 3.382 x 10-5 ounce, fluid (fl. oz)

2.113 x 10-5 pint (pt)

1.057 x 10-5 quart (qt)

0.2642 x 10-5 gallon (gal)

liter (L) 61.02 cubic inch (in3)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F=(1.8×°C)+32

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows:

°C=(°F-32)/1.8

Vertical coordinate information is referenced to the North American Vertical Datum of 1988 

(NAVD 88).

Horizontal coordinate information is referenced to the North American Datum of 1983 

(NAD 83).

Altitude, as used in this report, refers to distance above the vertical datum.

Specific conductance is given in microsiemens per centimeter at 25 degrees Celsius 

(μS/cm at 25°C).

Concentrations of chemical constituents in water are given either in milligrams per liter (mg/L) 

or micrograms per liter (μg/L).

NOTE TO USGS USERS: Use of hectare (ha) as an alternative name for square hectometer 

(hm2) is restricted to the measurement of small land or water areas. Use of liter (L) as a 

special name for cubic decimeter (dm3) is restricted to the measurement of liquids and gases. 

No prefix other than milli should be used with liter. Metric ton (t) as a name for megagram 

(Mg) should be restricted to commercial usage, and no prefixes should be used with it.
Introduction: The Potential Effects of 

Sodium Bicarbonate, a Major Constituent 

of Produced Waters from Coalbed Natural 

Gas Production, on Aquatic Life

By Aïda M. Farag, Don Skaar, and David D. Harper

Chapter 1 of

The Potential Effects of Sodium Bicarbonate, a Major Constituent of 

Produced Waters from Coalbed Natural Gas Production, on Aquatic 

Life

Edited by Aïda M. Farag and David D. Harper

Prepared in cooperation with Montana Fish, Wildlife, and Parks, U.S. Bureau of 

Land Management, and the U.S. Environmental Protection Agency

Scientific Investigations Report 2012–5008

U.S. Department of the Interior

U.S. Geological Survey
Contents

History .............................................................................................................................................................3

Data Gaps........................................................................................................................................................4

General Study Design....................................................................................................................................5

References Cited............................................................................................................................................7

Figures

1–1. Diagram showing approaches needed to define the potential effects of NaHCO3

(sodium bicarbonate) at both the individual and population levels, and to move the 

understanding from the laboratory to the field........................................................................4

1–2. Map showing the locations of the Tongue and Powder River Basins in the Tongue 

River/Powder River watershed of eastern Wyoming and Montana ....................................6

Table

1–1. Experiments completed to achieve minimum requirements for the establishment of a 

water-quality criterion for sodium bicarbonate.......................................................................5
History

A substantial expansion in coalbed natural gas production 

has taken place in the western United States. The production 

water from coalbed natural gas extraction contains many constituents. For some of these constituents, such as aluminum, 

ammonia, copper, lead, cadmium and zinc, the U.S. Environmental Protection Agency has established aquatic life criteria, 

and it is therefore possible to evaluate their risk to aquatic life. 

However, of the major ions associated with produced waters 

(sodium[Na+], calcium [Ca2+], magnesium [Mg2+], potassium 

[K+], chloride [Cl-], bicarbonate [HCO3

-

], sulfate [SO4

2-]), 

chloride is the only one with an established aquatic life criterion (U.S. Environmental Protection Agency, 1988). Previous 

research determined that different ions (and salts they form) 

induce varying degrees of toxicity to aquatic life (for example, 

Young, 1923; Mossier, 1971; Nelson, 1968; Held and Peterka, 

1974; Rawson and Moore, 1944). Mount and others (1997) 

completed more than a thousand acute experiments and developed a multiple regression model that described the toxicity 

of common ions in various combinations to zooplankton and 

fathead minnows (Pimephales promelas, FHM). One of the 

most pivotal findings of Mount and others (1997) was that all 

major ions have a lethal concentration, and the toxicity of a 

mixture of salts is generally equivalent to the additive toxicity 

of the individual salts.

During a toxicity identification evaluation, Boelter and 

others (1992) defined salinity as the cause of toxicity in an oil 

field discharge collected in northeastern Wyoming. The Mount 

and others (1997) study defined the salt most likely to be toxic 

to aquatic life that comes from coalbed natural gas production 

water as sodium bicarbonate (NaHCO3). A lethal concentration 

of NaHCO3 in three 96-hour experiments caused a 50-percent mortality (LC50) of less than 850 milligrams per liter 

(mg/L) for this salt, which is below the mean concentration of 

NaHCO3 discharged from some production sites, for example 

1,335 mg NaHCO3/L at the CX Ranch production field near 

Decker, Montana, not shown (Bauder, 2002). Few other published studies have investigated the acute toxicity of this salt. 

Beatty (1959) experimented with this salt on rainbow trout 

and determined that 65 percent of the fish were killed within 

a week at a NaHCO3 concentration of 1,000 mg/L. However, 

Beatty (1959) used reconstituted water (distilled water plus 

NaHCO3) in the experiments, without the addition of other 

salts that are necessary for normal physiological functioning; 

the results, therefore, are not a reliable predictor of effects of 

this salt in natural waters. 

The problem of interpreting the scant NaHCO3 acute toxicity data available is exaggerated by the absence of adequate 

laboratory studies on the chronic effects of NaHCO3. A few 

field studies exist that fall short of clearly demonstrating the 

effects of NaHCO3. For example, McCarraher and Thomas 

(1968) studied the tolerance of FHM in saline lakes in the sand 

hills of Nebraska where sodium bicarbonate was the dominant salt. Using in situ bioassays, they determined that lakes 

in which the minnows were able to survive for 6 months or 

more had bicarbonate + carbonate concentrations that averaged 1,061 mg/L and ranged from 336 to 1,979 mg/L. Lakes 

in which the minnows did not survive 6 months contained 

bicarbonate and carbonate concentrations that averaged 

4,282 mg/L and ranged from 1,410 to 20,760 mg/L. Minnows 

in the lake with 1,410 mg/L bicarbonate and carbonate began 

to die within 60 days and incurred total mortality within 

80 days. Total alkalinities of 1,800 and 2,000 mg/L were 

defined as thresholds for successful reproduction in two lakes 

in which more in depth studies were completed by McCarraher and Thomas (1968). Galat and others (1985) studied the 

Lahonton cutthroat trout (Oncorhynchus clarkii henshawi) in 

saline lakes in Nevada and Oregon, not shown. This species is 

well known for its ability to tolerate saline conditions, an ability perhaps developed throughout the millennia from living in 

Pleistocene Lake Lahonton, not shown. Histological changes 

in the tissues of these fish were studied, and in lakes with a 

range of alkalinity from 60 to 3,500 mg calcium carbonate 

(CaCO3)/L, there was a correlation between HCO3

-

, carbon 

dioxide (CO2-), Cl- concentrations and gill cell hyperplasia.

Neither the McCarraher and Thomas (1968) nor the Galat 

and others (1985) field studies reported in the literature are 

Introduction: The Potential Effects of Sodium Bicarbonate, 

a Major Constituent of Produced Waters from Coalbed 

Natural Gas Production, on Aquatic Life

By Aïda M. Farag1, Don Skaar2, and David D. Harper1

1

USGS, Columbia Environmental Research Center, Jackson Field Research 

Station, Jackson, Wyoming.

2

Montana Fish, Wildlife, and Parks, Helena, Montana.
4 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

suitable to develop chronic threshold concentrations for the 

effects of NaHCO3. The development of chronic threshold 

concentrations for NaHCO3 effects requires experimenting 

with a salt intolerant species and the measurement of sublethal 

effects on the most sensitive lifestage. Moreover, no other 

regulatory tools currently are available to assure long-term 

protection of aquatic life exposed to NaHCO3. Even whole 

effluent testing (WET) of coalbed natural gas well discharge is 

focused only on the measurement of and protection from acute 

toxicity, and WET testing results will certainly not be adequate 

for establishing Total Maximum Daily Loads (TMDLs) for 

drainages with coalbed natural gas production.

Data Gaps

Multiple approaches are needed to more completely 

define the potential toxicity of sodium bicarbonate on aquatic 

life. Approaches that define the potential effects at levels that 

range from the individual to the population and that work to 

explain implications as research moves from the laboratory 

to the field, can assist in better understanding the overall 

potential for NaHCO3 to adversely affect aquatic resources 

(fig. 1–1).

It is the intersection of information gathered as we move 

from the individual organism to effects at the population level, 

and as we move from the laboratory to the field, that guide 

an understanding and ability to predict the overall effects of 

NaHCO3 in real world conditions. This wide range of understanding also would give resource managers a science-based 

confidence upon which to employ adaptive management 

decisions.

To fine tune the general concept provided above, several 

types of experiments could be employed. First, laboratory 

experiments using water to mimic field conditions will define 

the toxicity attributable to NaHCO3. A series of acute and 

chronic laboratory experiments with multiple aquatic species, 

especially those present in areas of concern, would provide 

needed input for no-effect level (NOEL) determinations for 

NaHCO3. Second and concurrently, a field survey to define the 

presence/absence of aquatic species would be necessary and 

could define the transferability of laboratory data to the field. 

Third, in situ bioassays could further ground-truth laboratory 

data and refine information about the lifestage sensitivity in 

field conditions. Fourth, experiments with mixing zone waters 

could define the extent of potential toxicity in the areas of concern. These data could assist managers as they decide on the 

extent of the potential for effect concentrations to be reached 

in a watershed.

Additionally, 60-day studies that begin with fertilization will span the egg, hatch, sac-fry and swim-up stages of 

selected fish species. Therefore, experiments of this length will 

provide a chronic exposure of fish during their sensitive early 

life stages. In addition to survival, sublethal effects that can be 

measured to define chronic thresholds may include effects on 

growth, histology, whole-body or plasma ion concentrations 

and sodium-potassium adenosine triphosphatase (Na/K ATP–

ase) in early lifestage fish that have been exposed to NaHCO3

under chronic conditions. All of these parameters studied 

together may provide a pattern of events to explain changes 

in growth. For example, upsets in enzyme levels may result in 

ionic imbalance with histological changes at the gill to follow. 

These physiological changes, ultimately, may be manifested as 

growth reductions in the laboratory. As a result, not only will 

chronic no-effect levels be defined, attempts will be made to 

explain the mechanisms of toxicity that are involved. 

A TMDL dataset that includes NaHCO3 would not be 

complete without field validation of the laboratory assays. 

The presence/absence of native and game species of fish 

could ground truth the predicted no-effect levels established 

with laboratory studies. Furthermore, Clearwater and others (2002) suggest that an increase in salinity from product 

waters of coalbed methane production might increase salinity 

of the upper reaches of the Tongue River. The authors suggest increased salinity might affect the habitat quality of these 

reaches for fishes. Therefore, adequate baseline data of species 

distribution are needed in this watershed. The baseline data 

also would document the presence/absence of the species 

chosen for use during laboratory experiments.

Field toxicity experiments are important for multiple reasons. First, when supplemented with the proper water-quality 

information, in situ bioassays can further ground truth the 

laboratory investigations. Assessments can be made to define 

whether or not the no-effect levels of NaHCO3 determined 

in the laboratory are similar to those found at sites of critical importance. The water quality at various field sites may 

Laboratory

Acute/Chronic

Field

In situ

Population

Survival

Individual

Na/K Atpase

Histology

Figure 1–1. Approaches needed to define the potential effects 

of NaHCO3 (sodium bicarbonate) at both the individual and 

population levels, and to move the understanding from the 

laboratory to the field.
General Study Design 5

have additional constituents, so it is important that multiple 

constituents in the water be monitored during any in situ study. 

Second, in situ bioassays can be used to monitor changes 

in future survivability at sites in question. Therefore, in situ 

bioassays could alert managers to issues of degraded water 

quality or highlight areas where sites have been remediated 

and are more hospitable to fish survival.

General Study Design

The approaches described in Data Gaps above were 

used to determine the potential effects of NaHCO3, a major 

constituent of coalbed natural gas production water on aquatic 

life. Water qualities in the Tongue and Powder Rivers in 

Montana and Wyoming (fig. 1–2) were simulated and used in 

laboratory studies, and field studies were completed at sites 

along tributaries and the main stem of these rivers. The series 

of acute and chronic studies completed in the laboratory 

(table 1–1) span multiple species and phyla, and addresses 

requirements defined by the U.S. Environmental Protection 

Agency (Stephan and others, 1995) for establishment of waterquality criteria. 

This effort was completed to expand upon the limited 2002 knowledge base related to the potential effects of 

NaHCO3. To achieve balance between beneficial use of scarce 

water resources and the protection of aquatic life, managers 

need a broad range of fundamental science on which to base 

their far reaching decisions. The information gathered and the 

interpretations provided in the following chapters should provide managers with some needed information as they implement adaptive management practices. 

The focus of this research was NaHCO3, a compound that 

is a major constituent of coalbed natural gas produced waters 

in the Tongue and Powder River Basins. This compound was 

added during controlled experiments, and for this reason, we 

chose to formulate sample criteria and discussions of potential toxicity with concentrations of the compound NaHCO3

. 

However, criteria often are established for single elements or 

ions, in this case most likely HCO3

-

 as the toxic fraction of 

the compound NaHCO3 (Mount and others, 1997). Therefore, 

HCO3

-

 information has been provided for use if derivations 

with this single element are preferred. The sample criteria 

could also be calculated as alkalinity because it is an easily 

measured water chemistry property that is expressed as mg 

CaCO3/L, but defines the amount of HCO3

-

 in a sample with a 

pH less than 8.3 (American Public Health Association, 1975). 

In the Standard Method (American Public Health Association, 

1975) for alkalinity measurement, a sample with pH greater 

than 8.3 is titrated to pH = 8.3 with sulfuric acid. The amount 

of titrant used translates into mg CaCO3/L in the sample. The 

titration is continued until pH = 4.5 and the additional milliliters (mL) of titrant used translates into the amount of HCO3

-

 in 

the sample. The total amount of titrant used expresses the total 

alkalinity. Data has been provided for total alkalinity, HCO3

-

and CO3 measurements. With this information, managers can 

use this dataset to interpret the potential toxicity expressed in 

Table 1–1. Experiments completed to achieve minimum requirements for the establishment of a water-quality criterion 

for sodium bicarbonate. U.S. Environmental Protection Agency requirement/guidance taken from Stephen and others 

(1995).

U.S. Environmental Protection Agency 

requirement/guidance acute criteria Experiments completed during this project

One plant species 1

Selenastrum capricornutum.

Eight animal species

-Three families of fish from Osteichthyes, including one 

from Salmonidae, and one from a warmwater species

Salmonidae-cutthroat trout.

Percidae-walleye.

Catastomidae-white sucker.

Escoidae-northern pike.

Cyprinidae-fathead minnow (warmwater).

Centrarchidae-largemouth bass (warmwater).

Acipenseridae-pallid and shovelnose sturgeon (warmwater).

Planctonic crustacean Ceriodaphnia (literature exists for Ceriodaphnia and Daphnia).

Benthic crustacean Gammarus or Hyalella.

Insect Chironomus.

Non-Insect phylum Annelid-Tubifex.

Insect or any other phylum not already represented African clawed frog-Xenopus laevis.

One fish species White sucker.

Fathead minnows.

One invertebrate species Ceriodaphnia

One acutely sensitive freshwater species Freshwater mussel.

1

Results not available at time of publication. Also completed: (1) 96-hour in situ experiments during two different years at six different sites 

with pallid sturgeon and fathead minnow; (2) 96-hour laboratory experiments with pallid sturgeon exposed to water collected from several of 

the field sites; (3) 96-hour laboratory experiments with fathead minnow exposed to site water from coal bed natural gas mixing zones.
6 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Goose Creek

Beaver Creek

Prairie

Dog Creek

Clear Creek

Hanging

Woman Creek

Burger Draw

Bighorn River

Little Powder River

Crazy

Woman Creek

Big Porcupine

Creek

O'fallon Creek

Mizpah Creek

Rosebud Creek

Tongue River

Powder River

Yellowstone

River

Forsyth

Miles City

Broadus

Busby

Birney Day School

Ashland

Locate

Buffalo

106°

46°

44°

MONTANA

Index map

Montana

Wyoming

Map produced by

Montana Fish, Wildlife & Parks

Data Services Section

January 24, 2012

WYOMING

107° 105°

45°

Base map data from ESRI and Montana State Library

Lambert Conformal Conic projection

North American Datum, 1983 (NAD 83)

0 10 20 MILES

0 10 20 KILOMETERS

Figure 1–2. The locations of the Tongue and Powder River Basins in the Tongue River/Powder River watershed of 

eastern Wyoming and Montana.
References Cited 7

the manner that best suits their needs. Conductivity is another 

measurement that increases as concentrations of NaHCO3

increase in the water column. Measurements of conductivity also have been provided, especially for field experiments, 

should managers want to express toxicity in the context of 

conductivity measured as microsiemens per centimeter (μS/

cm).

References Cited

American Public Health Association Standard Methods, 1975, 

Alkalinity titration method for water and wastewater (14th 

ed.): Washington, D.C., American Public Health Association, 14th edition, p. 278. 

Bauder, J.W., 2002, Quality and characteristics of saline and 

sodic water affect irrigation suitability: Montana State 

Univeristy, accessed April 10, 2010, at http://waterquality.

montana.edu/, 7 p.

Beatty, D.D., 1959, An experimental study of the toxicity of 

sodium bicarbonate, sodium chloride, and sodium sulfate to 

rainbow trout: Laramie, University of Wyoming, Master’s 

Thesis, 69 p.

Boelter, A.M., Lamming, F.N., Farag, A.M., and Bergman, 

H.L., 1992, Environmental effects of saline oil-field discharges on surface waters: Environmental Toxicology and 

Chemistry, v. 11, p. 1,187–1,195.

Clearwater, S.J., Morris, B.A., and Meyer, J.S., 2002, A comparison of coalbed methane product water quality versus 

surface water quality in the Powder River Basin of Wyoming, and an assessment of the use of standard aquatic testing organisms for evaluating the potential effects of coalbed 

methane product waters: Laramie, University of Wyoming, 

Department of Zoology and Physiology, Report to the Wyoming Department of Environmental Quality, 131 p.

Galat, D.L., Post, G., Keefe, T.J., and Bouck, G.R., 1985, 

Histological changes in the gill, kidney and liver of Lahonton cutthroat trout, Salmo clarki henshawi, living in lakes of 

different salinity-alkalinity: Journal of Fish Biology, v. 27, 

p. 533–552. 

Held, J.W., and Peterka, J.J., 1974, Age, growth, and food habits of the fathead minnow, Pimephales promelas, in North 

Dakota saline lakes: Transactions of the American Fisheries 

Society., v. 103, p. 743–756.

McCarraher, D.B., and Thomas, R., 1968, Some ecological 

observations on the fathead minnow, Pimephales promelas, in the alkaline waters of Nebraska: Transactions of the 

American Fisheries Society, v. 97, p. 52–55.

Mount, D.R., Gulley, D.D., Hockett, J.R., Garrison, T.D., and 

Evans, J.M., 1997, Statistical models to predict the toxicity 

of major ions to Ceriodaphnia dubia, Daphnia magna, and 

Pimephales promelas (fathead minnows): Environmental 

Toxicology and Chemistry, v. 16, p. 2,009–2,019. 

Mossier, J.N., 1971, The effect of salinity on the eggs and sac 

fry of the fathead minnow (Pimephales promelas promelas), 

northern pike (Esox lucius) and walleye (Stizostedion vitreum vitreum): Fargo, North Dakota State University, Ph.D. 

dissertation, 47 p.

Nelson, J.S., 1968, Salinity tolerance of brook sticklebacks, 

Culaea inconstans, freshwater ninespine sticklebacks, 

Pungitius pungitius, and freshwater fourspine sticklebacks, 

Apeltes quadracus: Canadian Journal of Zoology, v. 46, 

p. 663–667.

Rawson, D.S., and Moore, J.E., 1944, The saline lakes of Saskatchewan: Canadian Journal of Research, v. 22, 

p. 141–201.

U.S. Environmental Protection Agency, 1988, Ambient water 

quality criteria for chloride: Cincinnati, Environmental 

Monitoring Systems Laboratory Office of Research and 

Development, U.S. Environmental Protection Agency EPA 

440/5–88–001, 39 p.

Young, R.T, 1923, Resistance of fish to salts and alkalinity: 

American Journal of Physiology, v. 63, p. 373–388.

The Acute Toxicity of Sodium Bicarbonate 

Defined Under Laboratory Conditions

By David D. Harper, Aïda M. Farag, Don Skaar, and Trevor Selch

Chapter 2 of

The Potential Effects of Sodium Bicarbonate, a Major Constituent of 

Produced Waters from Coalbed Natural Gas Production, on Aquatic 

Life

Edited by Aïda M. Farag, and David D. Harper

Prepared in cooperation with Montana Fish, Wildlife, and Parks, U.S. Bureau of 

Land Management, and the U.S. Environmental Protection Agency

Scientific Investigations Report 2012–5008

U.S. Department of the Interior

U.S. Geological Survey
Contents

Introduction...................................................................................................................................................13

Methods.........................................................................................................................................................14

Pallid Sturgeon....................................................................................................................................14

Fathead Minnow .................................................................................................................................14

White Sucker.......................................................................................................................................15

Newly Hatched Fry ....................................................................................................................15

Older Fry ......................................................................................................................................15

African Clawed Frog...........................................................................................................................15

Freshwater Mussels...........................................................................................................................15

Rainbow Trout......................................................................................................................................16

Ceriodaphnia........................................................................................................................................16

Chironomids.........................................................................................................................................16

Tubifex ..................................................................................................................................................16

Amphipods ...........................................................................................................................................16

Shovelnose Sturgeon.........................................................................................................................16

Walleye.................................................................................................................................................16

Northern Pike.......................................................................................................................................16

Results ...........................................................................................................................................................17

Pallid Sturgeon....................................................................................................................................17

Fathead Minnow .................................................................................................................................21

White Sucker.......................................................................................................................................23

Newly Hatched Fry ....................................................................................................................23

Older Fry ......................................................................................................................................23

African Clawed Frog...........................................................................................................................25

Freshwater Mussels...........................................................................................................................26

Rainbow Trout......................................................................................................................................27

Ceriodaphnia........................................................................................................................................28

Chironomids.........................................................................................................................................29

Tubifex ..................................................................................................................................................30

Amphipods ...........................................................................................................................................31

Shovelnose Sturgeon.........................................................................................................................32

Walleye.................................................................................................................................................33

Northern Pike.......................................................................................................................................34

Discussion.....................................................................................................................................................35

References Cited..........................................................................................................................................36

Table

2–1. Median 50-percent lethal concentrations of sodium bicarbonate for species used 

in experiments in the laboratory with simulated water from the Tongue and Powder 

Rivers, and water collected from the Yellowstone River.....................................................17

2–2. Median 50-percent lethal concentrations of alkalinity and bicarbonate for species used 

in 96-hour experiments with water reconstituted in the laboratory to simulate water from 

the Tongue, Powder and Yellowstone Rivers.................................................................................18
2–3. Water quality measured during acute 96-hour exposures to sodium bicarbonate. Dilution waters were reconstituted to simulate the Tongue River, Powder River, or YellowstoneRiver....................................................................................................................................18

2–4. Exposure concentrations of sodium bicarbonate during a 96-hour acute toxicity 

experiment with 4-day-post-hatch pallid sturgeon (Scaphirhynchus albus)...................19

2–5. Number alive and percent survival of 4-day-post-hatch pallid sturgeon (Scaphirhynchus 

albus) exposed to sodium bicarbonate in a 96-hour acute toxicity experiment completed in reconstituted Powder River water..........................................................................19

2–6. Water chemistry measured during a 96-hour acute toxicity experiment with 4-daypost-hatch pallid sturgeon (Scaphirhynchus albus).............................................................20

2–7. Number alive and percent survival of 4-day-post-hatch pallid sturgeon (Scaphirhynchus 

albus) exposed to sodium bicarbonate in a 96-hour acute toxicity experiment completed in reconstituted Tongue River water...........................................................................20

2–8. Water chemistry measured during a 96-hour acute toxicity experiment with 4-daypost-hatch fathead minnows (Pimephales promelas)..........................................................21

2–9. Number alive and percent survival of 4-day-post-hatch fathead minnows (Pimephales 

promelas) exposed to sodium bicarbonate in a 96-hour acute toxicity experiment completed in reconstituted Powder River water..........................................................................21

2–10. Water chemistry measured during a 96-hour acute toxicity experiment with 2-day-posthatch fathead minnows (Pimephales promelas)...................................................................22

2–11. Number alive and percent survival of 4-day-post-hatch fathead minnows (Pimephales 

promelas) exposed to sodium bicarbonate in a 96-hour acute toxicity experiment completed in reconstituted Tongue River water...........................................................................22

2–12. Water chemistry measured during a 96-hour acute toxicity experiment with 22-daypost-hatch white sucker (Catostomus commersoni)............................................................23

2–13. Number alive and percent survival of 22-day-post-hatch white suckers (Catostomus 

commersoni) exposed to sodium bicarbonate in a 96-hour acute toxicity experiment 

completed in reconstituted Tongue River water...................................................................23

2–14. Water chemistry measured during a 96-hour acute toxicity experiment with 22-daypost-hatch white sucker (Catostomus commersoni)............................................................24

2–15. Number alive and percent survival of 22-day-post-hatch white suckers (Catostomus 

commersoni) exposed to sodium bicarbonate in a 96-hour acute toxicity experiment 

completed in reconstituted Powder River water ..................................................................24

2–16. Water chemistry measured during a 96-hour acute toxicity experiment with less than 

24-hour-post-fertilization African clawed frog (Xenopus laevis)........................................25

2–17. Number alive and percent survival of African clawed frog (Xenopus laevis) in a 96-hour 

acute toxicity experiment completed in reconstituted Tongue River water.....................25

2–18. Water chemistry measured during a 96-hour acute toxicity experiment with newly 

transformed fatmucket mussels (Lampsilis siliquoidea)......................................................26

2–19. Number alive and percent survival of newly transformed fatmucket mussels (Lampsilis 

siliquoidea) in a 96-hour acute toxicity experiment completed in reconstituted Tongue 

River water...................................................................................................................................26

2–20. Water chemistry measured during a 96-hour acute toxicity experiment with experiment 

with rainbow trout (Oncorhynchus mykiss) completed in reconstituted Tongue River 

water.............................................................................................................................................27

2–21. Number alive and percent survival of juvenile rainbow trout (Oncorhynchus mykiss) 

exposed to sodium bicarbonate in a 96-hour acute toxicity experiment completed in 

reconstituted Powder River water...........................................................................................27
2–22. Water chemistry measured during a 96-hour acute toxicity experiment with adult 

(Ceriodaphnia dubia) completed in moderately hard reconstituted water, reconstituted 

Tongue River water, and reconstituted Powder River water ..............................................28

2–23. Number of live organisms at 0, 24, and 48 hours and percent survival at 48 hours for 

adult Ceriodaphnia dubia completed in moderately hard reconstituted water, reconstituted Tongue River water, and reconstituted Powder River water....................................28

2–24. Water chemistry measured in concentrated stock solutions and reconstituted (control) 

Tongue and Powder River waters used to make exposure concentrations for a 48-hour 

acute toxicity experiments with Chironomid larvae (Chironomus dilutus) .......................29

2–25. Number alive and percent survival of Chironomid larvae (Chironomus dilutus) in a 

48-hour acute toxicity experiment completed in reconstituted (control) Tongue and 

Powder River waters..................................................................................................................29

2–26. Water chemistry measured in concentrated stock solutions of 5,000 milligrams sodium 

bicarbonate per liter and reconstituted (control) Tongue and Powder River waters 

used to make exposure concentrations for 96-hour acute toxicity experiments with 

Tubifex worms (Tubifex tubifex)................................................................................................30

2–27. Number alive and percent survival (% in parentheses) of Tubifex worms (Tubifex tubifex) in 96-hour acute toxicity experiments completed in reconstituted (control) Tongue 

and Powder River waters..........................................................................................................30

2–28. Water chemistry measured in concentrated stock solutions of sodium bicarbonate 

and reconstituted (control) Tongue and Powder River waters used to make exposure 

concentrations (also presented) for 96-hour acute toxicity experiments with adult 

amphipods (Hyalella azteca).....................................................................................................31

2–29. Number alive and percent survival of adult amphipods (Hyalella azteca) in 96-hour 

acute toxicity experiments completed in reconstituted (control) Tongue and Powder 

River waters.................................................................................................................................31

2–30. Water chemistry measured during a 96-hour acute toxicity experiment with shovelnose sturgeon (Scaphirhynchus platorynchus) completed in Yellowstone River water 

(control)........................................................................................................................................32

2–31. Number alive and percent survival of 1-day-post-hatch shovelnose sturgeon 

(Scaphirhynchus platorynchus) completed in Yellowstone River water (control)..........32

2–32. Water chemistry measured during a 96-hour acute toxicity experiment with 

1-day-post-hatch walleye (Sander vitreus) completed in Yellowstone River 

water (control).............................................................................................................................33

2–33. Number alive and percent survival of 1-day-post-hatch walleye (Sander vitreus) 

completed in Yellowstone River water (control) ...................................................................33

2–34. Water chemistry measured during a 96-hour acute toxicity experiment with 2-daypost-hatch northern pike (Esox lucius) completed in Yellowstone River water ..............34

2–35. Number alive and percent survival of 2-day-post-hatch northern pike (Esox lucius) 

completed in Yellowstone River water (control) ...................................................................34
Introduction

Sodium bicarbonate (NaHCO3) is a major constituent of 

water produced during coalbed natural gas (CBNG) extraction 

in the Powder River Basin (ALL Consulting, 2003). In small 

ephemeral tributary streams, CBNG produced water may be a 

substantial part or all of the streamflow during late summer or 

drought periods. Little is known about the effects of NaHCO3

on aquatic organisms, and acute toxicity from exposure to 

NaHCO3 has not been investigated extensively. Mount and 

Gulley (1992) developed a model to predict the relation 

between salinity and toxicity in freshwater that evaluated the 

toxicity of a series of salts in freshwater. Mount and others 

(1997) completed the most diverse series of experiments to 

describe the toxicity of various salts and salt mixtures to assess 

the effects of salt exposure associated with oil field discharge; 

however, these experiments were limited to two species of 

daphnia, Ceriodaphnia dubia and Daphnia magna and one 

fish species, fathead minnows (Pimephales promelas). Compared to other compounds, relatively large concentrations of 

NaHCO3 are needed to induce acute toxicity (Dwyer and others, 2005), but among salts found in oilfield produced waters, 

NaHCO3 had some of the smallest lethal concentrations for 

fathead minnows and Ceriodaphnia (Mount and others, 1997). 

The toxicity of salts to aquatic organisms can be variable 

(Weber-Scannell and Duffy, 2007). Some fish, invertebrates 

and plant species have adapted to seasonal or periodic changes 

in salt concentrations because of evaporation or have evolved 

from marine environments and are more tolerant of salinity 

and high alkalinity compared to other freshwater biota (Wurts, 

1998; Nielsen and others, 2003). Therefore, it is critical that a 

wide range of aquatic organisms be used to assess the toxicity 

of NaHCO3. A dataset that includes multiple species is recommended for the establishment of water-quality criteria derived 

to protect 95 percent of species present (Stephan and others, 

1985), and the dataset would provide information about the 

range of potential toxicity of NaHCO3 to aquatic organisms as 

well.

One objective of the present study was to define the 

toxicity of NaHCO3 to multiple species of aquatic organisms. 

Then, regulatory entities could use information from the most 

sensitive species to calculate an acute water-quality criteria 

if one is desired (see chapter 6 of this report). To accomplish 

this objective, acute toxicity experiments were completed in 

the laboratory with a suite of organisms, including 7 species 

of fish, 5 species of invertebrates, and 1 amphibian species. 

The acute experiments with white sucker and fathead minnow 

included both newly hatched and older fry to assess the effects 

of age on survival. Acute toxicity experiments were performed 

at the Montana Fish, Wildlife and Parks laboratory in Helena, 

Montana and the USGS Columbia Environmental Research 

Center (CERC), Jackson Field Research Station, Jackson, 

Wyoming (hereafter referred to as Jackson Field Research 

Station).

Ideally, experimenting with a combination of species 

native to the region of concern and species that routinely are 

cultured in laboratories can provide a well-rounded assessment 

during toxicological investigations. Using species associated 

with the region of concern provides region-specific sensitivity information, whereas the routinely cultured species often 

defined as disease free, can serve as surrogate species, and 

the data obtained can be compared to data generated in other 

laboratories throughout the country or internationally. In the 

present study, the white sucker (Catostomus commersoni), 

pallid sturgeon (Scaphirhynchus albus), Shovelnose sturgeon 

(Scaphirhynchus platorynchus), fathead minnows (Pimephales 

promelas), chironomids (Chironomus dilutus), tubifex worms 

(Tubifex tubifex), amphipods (Hyalella azteca), freshwater 

mussels (fatmucket, Lampsilis siliquoidea), and Ceriodaphnia 

dubia species were used and are all native to the Tongue and 

Powder River Basins; most of the species were available commercially or from educational, State, or Federal facilities. The 

Northern Leopard frog (Rana pipiens), an amphibian native to 

the Powder and Tongue River Basins was unavailable because 

of logistical problems with obtaining eggs and adults at the 

proper lifestage for experiments, and the lack of an approved 

disease-free source of experimental animals. For these reasons, the African Clawed frog (Xenopus laevis) was selected 

as a surrogate species. Walleye (Sander vitreus), northern pike 

(Esox lucius), and rainbow trout (Oncorhynchus mykiss) also 

were used. Although these species are not native to the area of 

The Acute Toxicity of Sodium Bicarbonate Defined Under 

Laboratory Conditions

By David D. Harper1, Aïda M. Farag1, Don Skaar2, and Trevor Selch2

1

USGS, Columbia Environmental Research Center, Jackson Field Research 

Station, Jackson, Wyoming.

2

Montana Fish, Wildlife, and Parks, Helena, Montana.
14 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

concern, they are popular introduced sport fish species present 

in the Tongue and Powder River Basins. Additionally, various 

lifestages of disease-free walleye, northern pike, and rainbow 

trout are readily available from various sources and frequently 

are used as experimental organisms with established test 

protocols. 

Acute toxicity often is defined as the concentration of a 

chemical or substance that is lethal to 50 percent of the test 

population (median lethal concentration or LC50) within a 

specified time period, or as an effect concentration resulting in 

severe, measurable effects such as immobilization or developmental abnormalities (EC50; Sprague, 1973). Acute toxicity to aquatic organisms typically occurs within less than 96 

hours (Rand, 1995). Typically, after 96 hours (hereafter stated 

as 96-h) of exposure, the most sensitive organisms will have 

experienced acute toxic effects, and no significant increases 

in mortality occur with increased exposure time (Rand, 1995). 

Organisms generally are exposed for 96-h in acute aquatic toxicity experiments. Small organisms with a short lifespan, such 

as Ceriodaphnia spp., are typically exposed for 48-h. 

The acute toxicity to aquatic organisms is most efficiently 

determined in acute toxicity experiments that use the most 

sensitive species during their most sensitive lifestages (Rand, 

1995). For many organisms, the early life stages are the most 

sensitive to most toxicants (U.S. Environmental Protection 

Agency, 2002); however, research into the effects of age 

within lifestages is limited. For this reason, a second research 

goal was to determine the effects of age on the toxicity of 

NaHCO3 to early lifestage fish and other aquatic organisms.

Methods

The experiments were performed in reconstituted waters 

that simulated the Tongue River or Powder River or both, 

except for shovelnose sturgeon, walleye and northern pike, 

which were exposed to water collected from the Yellowstone 

River near Miles City, Montana, not shown. Reconstituted 

waters were prepared using metal-grade salts. Reconstituted 

waters were prepared by adding salts to well water from the 

Jackson Field Research Station, not shown. Well water was 

used to minimize the volume of salts necessary to mimic 

the Tongue and Powder Rivers during low-flow conditions. 

Reconstituted waters were mixed in 18L HDPE carboys, and 

were measured for conductivity, dissolved oxygen (DO), and 

temperature with YSI 85TM handheld meters (Yellow Springs 

Industries, Yellow Springs, Ohio). Exposure waters, including 

control waters, were sampled for cations and anions by the 

Montana Department of Health laboratory in Helena, Montana. All samples were filtered and refrigerated, and cation 

samples were acidified to pH-2 with nitric acid (HNO3

-

).

A series of increasing amounts of NaHCO3 were added to 

the reconstituted waters to obtain multiple exposure concentrations for all experiments. Unless otherwise noted, experiments 

to determine LC50s and EC50s were completed in accordance 

with procedures described in the protocol outlined by the 

U.S. Environmental Protection Agency (2002), test organisms were not fed during exposures, and the photo period was 

approximately 16-h daylight and 8-h darkness/day. All acute 

experiments lasted 96-h, except for C. dubia and Chironomid 

exposures, which were 48-h. Conductivity, DO, and temperature were measured at least once per day during all experiments with YSI 85TM handheld meters. 

 The Toxicity Relationship Analysis Program (TRAP; 

U.S. Environmental Protection Agency, 2010) was used to calculate LC50s and EC50s. Mean measured NaHCO3

 concentrations were 97 percent of the nominal values; however all LC50 

and EC50 calculations were completed with measured sodium 

bicarbonate, bicarbonate and alkalinity concentrations.

The methods used for each species are outlined in the 

following sections. 

Pallid Sturgeon

Newly hatched pallid sturgeon were obtained from the 

Miles City State Fish Hatchery, Miles City, Montana, not 

shown, where the eggs had been incubated and hatched in 

Yellowstone River water. Four-days-post-hatch (dph) sturgeon 

were exposed to nominal concentrations of 518, 864, 1,440, 

2,400, and 4,000 milligrams sodium bicarbonate per liter (mg 

NaHCO3/L) in reconstituted Tongue or Powder River water. 

Three replicates of each NaHCO3 concentration were used 

with 10 fish per replicate. 

Fathead Minnow 

Newly hatched fathead minnows (FHM, Pimephales 

promelas) were obtained from the Jackson Field Research 

Station where they had been incubated, hatched, and held in 

water that was reconstituted to simulate Tongue River water 

quality. Four-days-post-hatch (4-dph) FHM were exposed to 

nominal concentrations of 518, 864, 1,440, 2,400, and 4,000 

mg NaHCO3/L in reconstituted Tongue or Powder River water. 

Ten fish were exposed to three replicates of each experimental 

concentration in the reconstituted Tongue River water and 

five fish per replicate were exposed in the reconstituted 

Powder River water. Mortality was not sufficient to calculate 

an LC50 for FHM exposed in the reconstituted Tongue River 

water quality with only 23 percent mortality occurring after 

96-h of exposure to the largest concentration tested (4,000 

mg/L). Thus, the experiment was repeated with 2-dph FHM 

fry. The 2-dph fry were exposed to nominal NaHCO3 concentrations of 500, 800, 1,100, 1400, 2,000, and 3,000 mg 

NaHCO3/L in reconstituted Tongue River water and a control 

treatment of reconstituted water with no NaHCO3 added. 
Methods 15

White Sucker 

Newly Hatched Fry 

White sucker fry were obtained from the Jackson 

Field Research Station where the eggs had been incubated 

and hatched and the fry were held in water reconstituted to 

simulate Tongue River water quality. After the white sucker 

fry began to feed exogenously, they were transferred from 

Jackson, Wyoming to Helena, Montana, not shown, where 

they were held in water of a similar quality. Then, 22-dph fry 

were exposed to nominal concentrations of 864, 1,440, 2,400, 

4,000 and, 6,666 mg NaHCO3/L in glass Mason jars that 

contained 200 milliliters (mL) of exposure water for 96-h. The 

control treatment contained a nominal concentration of 267 as 

opposed to175 mg NaHCO3/L (to simulate Tongue River conditions) or 323 as opposed to 194 mg NaHCO3

/L (to simulate 

Powder River conditions). For the Tongue River experiment, 

10 fish were exposed to 3 replicates of each exposure concentration. For the Powder River experiment, eight fish were 

exposed to two replicates of each exposure concentration. 

Water temperature was maintained at 20 plus or minus 

(±) 2 degrees Celsius (°C) and natural lighting supplemented 

with fluorescent lights maintain at a light: dark photoperiod of 

16:8 hour (h). The fish were exposed under static-renewal conditions for 96-h and the fish were transferred to fresh solutions 

after 24-h, 48-h and 72-h. The fish were fed commercial Rangen Trout Starter (Rangen Inc., Buhl, Idaho) during the 2 days 

before the experiment was started, and 1 h before they were 

transferred to fresh exposure solutions during the experiment.

Older Fry 

Additional fish were transported from the Jackson Field 

Research Station to Helena, Montana to evaluate the toxicity 

of NaHCO3 to older fry. The experiment was started 

with 69-dph fish that had a mean length and weight of 

19.3 millimeters (mm) and 0.038 grams (g), respectively. The 

reconstituted Tongue River water (as described in the Newly 

Hatched Fry section) was used to expose the fish to nominal 

concentrations of 4,000 and 6,666 mg NaHCO3/L. Ten fish 

were exposed in each of two replicates of each concentration 

in a 96-h static renewal experiment. The fish were transferred 

to fresh experimental solutions after 24, 48 and 72 h. The fish 

were fed commercial Rangen Trout Starter during the 2 days 

before the experiment was started and 1 h before they were 

transferred to fresh solutions during the experiment. 

African Clawed Frog 

Acute toxicity experiments with African clawed frogs 

were performed in accordance with the guidelines outlined in 

the standard guide for Conducting Frog Embryo Teratogenesis Assay-Xenopus (FETAX; American Society for Testing 

and Materials International, 1998). Adult African clawed 

frogs were obtained from Xenopus 1, Dexter, Michigan, 

USA. Adults were held in well water from the Jackson Field 

Research Station in 100 L glass aquaria maintained at 23°C 

(±3°C). The frogs were fed beef liver to satiation, 2 to 3 times 

per day, 5 days/week, and held under a light:dark photoperiod 

of 11:13 h. On the day before egg collection, adult frogs were 

injected with human chorionic gonadotropin at approximately 

1800 h and pairs of male and female were placed together in 

darkened 4-L covered plastic food-grade containers to initiate amphiplexus, fertilization and egg deposition. Eggs were 

deposited on a 4-mm mesh high density polyethylene (HDPE) 

screen that allowed fertilized eggs to pass through the mesh 

into a water bath containing reconstituted Tongue River water. 

Fertilized eggs were collected the following morning between 

0900 h and 1200 h, sorted by developmental stage (Bantle 

and others, 1991), and 25 embryos were randomly placed in 

10-mL glass Petri dishes that contained 5 mL exposure waters, 

with 2 replicates per exposure. Following American Society 

of Testing and Materials International guidelines, 3 replicate 

experiments were completed.

The embryos were exposed to 750, 1,000, 1,500, 2,500, 

3,500, and 4,500 mg NaHCO3/L and a control of reconstituted 

Tongue River water. The embryos were exposed in a 96-h 

static-renewal acute toxicity experiment and the exposure 

water was renewed every 24 h. Embryo mortality at 96-h and 

embryonic abnormalities were observed and recorded during 

the experiments as described in Bantle and others (1991). 

Freshwater Mussels

Fatmucket mussels are native to the Tongue River 

drainage in both Wyoming and Montana (A.M. Cvancara, 

written commun., 2004; Montana Fish Wildlife and Parks, 

2010). Newly transformed juvenile fatmucket mussels were 

obtained from Missouri State University, Springfield, Missouri. Juvenile mussels were held in a water bath in well water 

from the Jackson Field Research Station maintained at 20 ± 

2°C. Mussels were fed a mixture of commercially prepared 

algae and shellfish diet during a 48-h acclimation period 

before the initiation of the exposure. Acute static-renewal 

toxicity experiments with mussels exposed to NaHCO3 were 

completed according to the protocol developed by Wang and 

others (2007), and American Society for Testing and Materials International (2006). The mussels were exposed for 96-h 

to nominal concentrations of 500, 1,000, 1,500, 2,000, and 

2,500 mg NaHCO3/L, and a control treatment of reconstituted 

Tongue River water. Five mussels were exposed to 4 replicates 

of each exposure concentration in 30-mL beakers. Renewal of 

experimental waters was performed after 48 h. At the end of 

the 96-h experiments foot movement and the lack of a visible 

foot movement was recorded during a 5-minute observation 

period for the exposure effect. 
16 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Rainbow Trout

Juvenile rainbow trout were obtained from the U.S. 

Fish and Wildlife Service, Ennis National Fish Hatchery, 

Ennis, Montana as 2-dph sac fry. These fish were exposed to 

a series of concentrations that ranged from 4,000–12,000 mg 

NaHCO3/L with reconstituted Tongue River water as 

the control and dilution water. Three replicates of 10 fish 

for each concentration were each held in 4-L glass aquaria. 

Experiments were static renewal with daily replacement of test 

waters. 

Ceriodaphnia

C. dubia were obtained as adults from Aquatic Biosystems Inc., Fort Collins, Colorado. Neonates produced by these 

adults were tested following the procedures described by 

the U.S. Environmental Protection Agency (2002). C. dubia

were exposed to a series of concentrations that ranged from 

900–3,000 mg NaHCO3/L with reconstituted moderately hard 

(as defined in U.S. Environmental Protection Agency, 2002), 

reconstituted Tongue and Powder River waters as controls and 

dilution waters. Ten replicates with one C. dubia per replicate 

for each exposure concentration were exposed in 30-mL plastic cups containing 15-mL exposure water. Experiments were 

static renewal with daily replacement of test waters.

Chironomids

Chironomids were obtained from the Columbia Environmental Research Center, Columbia, Missouri as egg masses 

and tested as 1–2 week old larvae. Experiments to determine 

the 48-h LC50 were completed following the protocol outlined 

in U.S. Environmental Protection Agency (2002). Pupae 

were exposed to a series of concentrations that ranged from 

4,000–16,000 mg NaHCO3/L with reconstituted Tongue and 

Powder River waters as the control and dilution waters. Four 

replicates with 10 Chironomids per replicate for each exposure 

concentration were exposed in 1-L glass aquaria. Experiments 

were static renewal with daily replacement of experimental 

waters.

Tubifex

The oligochaete annelid, Tubifex tubifex, were obtained 

from rearing ponds located at the U.S. Fish and Wildlife 

Service, Bozeman Fisheries Technology Center, Bozeman, 

Montana. Experiments to determine the 96-h LC50 were completed following the protocol outlined in U.S. Environmental 

Protection Agency (2002). Tubifex were exposed to a series of 

concentrations that ranged from 1,000–5,000 mg NaHCO3/L 

with reconstituted Tongue and Powder River waters as the 

control and dilution waters. Four replicates of 10 Tubifex per 

replicate for each exposure concentration were tested in 1-L 

glass aquaria. Experiments were static renewal with daily 

replacement of experimental waters.

Amphipods 

 Adult amphipods were obtained from a private farm 

pond in Lewis and Clark County, Montana. Experiments to 

determine the 96-h LC50 were completed following the protocol outlined in U.S. Environmental Protection Agency (2002). 

Amphipods were exposed to a series of concentrations that 

ranged from 1,000–2,000 mg NaHCO3/L with reconstituted 

Tongue River and Powder River waters as the control and 

dilution water. Four replicates of ten Amphipods per replicate for each exposure concentration were tested in 1-L glass 

aquaria. Experiments were static renewal with daily replacement of test waters.

Shovelnose Sturgeon

Newly hatched shovelnose sturgeon were obtained from 

the Miles City State Fish Hatchery where eggs had been incubated and hatched in Yellowstone River water. Experiments 

to determine the 96-h LC50 were completed with yolk-sac 

fry following the protocol outlined in U.S. Environmental 

Protection Agency (2002). Shovelnose sturgeon were exposed 

to a series of concentrations that ranged from 625–10,000 mg 

NaHCO3/L with Yellowstone River water as the control and 

dilution water. Four replicates of 5 sturgeon per replicate for 

each exposure concentration were tested in 1-L glass aquaria. 

Experiments were static renewal with daily replacement of 

experimental waters.

Walleye

Newly hatched walleye were obtained from the Miles 

City State Fish Hatchery where the eggs had been incubated 

and hatched in Yellowstone River water. Experiments to 

determine the 96-h LC50 were completed with 1-dph fry following the protocol outlined in U.S. Environmental Protection 

Agency (2002). Walleye were exposed to a series of concentrations that ranged from 625–5,000 mg NaHCO3

/L with Yellowstone River water as the control and dilution water. Four 

replicates of 5 walleye per replicate for each exposure concentration were tested in 1-L glass aquaria. Experiments were 

static renewal with daily replacement of experimental waters.

 Northern Pike

Northern pike were obtained from the Miles City State 

Fish Hatchery where the eggs had been incubated and hatched 

in Yellowstone River water. Experiments to determine the 96-h 

LC50 were completed with 1-dph fry following the protocol 

outlined in U.S. Environmental Protection Agency (2002). 

Northern pike were exposed to a series of concentrations that 
Results 17

ranged from 500–8,000 mg NaHCO3/L with Yellowstone 

River water as the control and dilution water. Four replicates 

of 10 pike per replicate for each exposure concentration were 

tested in 1-L glass aquaria. Experiments were static renewal 

with daily replacement of experimental waters.

Results

The freshwater mussel, C. dubia, pallid sturgeon, 

shovelnose sturgeon, and FHM were the most sensitive 

organisms exposed to NaHCO3, alkalinity and bicarbonate 

(HCO3

-

) (tables 2–1 and 2–2). Exposure conditions (DO, pH, 

and temperature) did not deviate outside of acceptable values 

during acute exposures and the mean and range for each 

experiment is presented in table 2–3. Results by species are 

presented in applicable tables.

Pallid Sturgeon

The 96-h LC50s for 4-dph fish were calculated as 1,356 

mg/L NaHCO3 with 95-percent Confidence Interval (CI) 

of 1,206–1,507 mg NaHCO3/L for the Powder River water 

and 1,295 mg/L NaHCO3 95-percent CI of 927–1,662 mg 

NaHCO3/L for Tongue River water. Water chemistry and survival data are presented in tables 2–4, 2–5, 2–6, 2–7 and 2–8. 

Table 2–1. Median 50-percent lethal concentrations of sodium bicarbonate for species used in experiments in the laboratory with 

simulated water from the Tongue and Powder Rivers, and water collected from the Yellowstone River. Exposure duration was 96 hours 

for all species except Ceriodaphnia and Chironomids, which was 48 hours. 

[LC50, median 50-percent lethal; NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; >, concentrations greater than the expressed value; --, experiments 

were not conducted in the specified water]

Species Tongue River

LC50, (mg NaHCO3/L)

Powder River

LC50, (mg NaHCO3/L)

Yellowstone River 

LC50, (mg NaHCO3/L)

Mean

LC50, (mg NaHCO3/L)

Pallid sturgeon (Scaphirhynchus albus) 1,295 1,356 1

1,249 1,326

Freshwater mussel (Lampsilis siliquoidea) 1,120 -- -- 1,120

White Sucker (Catostomus commersoni) 4,494 1

5,648 -- 4,494

Rainbow trout (Oncorhynchus mykiss) 8,070 -- -- 8,070

Fathead minnow (Pimphales promelas) 1,749 2,078 -- 1,914

Walleye (Sander vitreus) -- -- 3,249 3,249

Shovelnose sturgeon (Scaphirhynchus 

platorynchus)

-- -- 1,430 11,430

Northern pike (Esox lucious) >8,000 -- -- >8,000

African clawed frog (Xenopus laevis) 1,940 -- -- 1,940

Tubifex (Tubifex tubifex) 3,297 3,367 3,332

Amphipod (Hyalella azteca) 1.419 1

3,851 -- 2,635

Chironomid (Chironomus dilutus) 4,947 8,014 -- 6,481

Ceriodaphnia (Ceriodaphnia dubia) 989 1,355 2

1,288 1,211

1

Unreliable confidence intervals.

2

Experiment completed in moderately hard reconstituted water.
18 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Table 2–2. Median 50-percent lethal concentrations of alkalinity and bicarbonate for species used in 96-hour experiments with water 

reconstituted in the laboratory to simulate water from the Tongue and Powder Rivers. Yellowstone River water was collected directly 

from that river for experimental use. Exposure duration for Ceriodaphnia and Chironomids was 48 hours.

[LC50, median 50-percent lethal; CaCO3, calcium carbonate; mg/L, milligrams per liter; --, experiments were not conducted in the specified water; >, 

concentrations greater than the expressed value]

Species

 Tongue River Powder River Yellowstone River

Alkalinity 

(CaCO3, in mg/L) 

Bicarbonate 

(in mg/L)

Alkalinity 

(CaCO3, in mg/L) 

Bicarbonate

 (in mg/L)

Alkalinity 

(CaCO3, in mg/L) 

Bicarbonate 

(in mg/L)

Pallid sturgeon (Scaphirhynchus 

albus)

846 939 608 831 -- --

Freshwater mussel (Lampsilis 

siliquoidea)

670 844 -- -- -- --

White sucker (Catostomus commersoni)

2,668 3,208 3,852 4,276 -- --

Rainbow trout (Oncorhynchus 

mykiss)

5,134 5,699 -- -- -- --

Fathead minnow (Pimphales 

promelas)

1,139 1,254 1,089 1,418 -- --

Walleye (Sander vitreus) -- -- -- -- 2,022 2,352

Shovelenose sturgeon (Scaphirhynchus platorynchus)

-- -- -- -- 880 1,038

Northern pike (Esox lucius) -- -- -- -- >4,181 >8,000

African clawed frog 856 914 -- -- -- --

Tubifex (Tubifex tubifex) 2,119 2,395 2,427 2,532 -- --

Amphipod (Hyalella azteca) 966 1,031 -- -- -- --

Chironomid (Chironomus dilutus) 2,903 3,593 3,874 5,820 -- --

Ceriodaphnia (Ceriodaphnia 

dubia)

734 699 883 769 1846 1842

1

Experiment completed in moderately hard reconstituted water.

Table 2–3. Water quality measured during acute 96-hour exposures to sodium bicarbonate. Dilution waters were reconstituted to 

simulate the Tongue and Powder Rivers. Yellowstone River water was collected directly from that river for experimental use. Exposure 

duration for Ceriodaphnia and Chironomids was 48 hours.

[mg/L, milligrams per liter; DO, dissolved oxygen; °C, degrees Celcius]

Species Dilution water Exposure range 

(mg/L )

DO range

(mg/L) pH range Temperature range (°C)

Fathead minnow (Pimephales promelas) Tongue River 500–3,000 6.2–7.5 8.2–8.8 23–26

Powder River 518–4,000 6.3–8.5 8.05–8.69 24–26

White sucker (Catostomus commersoni) Tongue River 864–6,666 4.2–8.9 7.97–8.74 16–17

Powder River 864–6,666 6.2–8.7 8.06–8.68 16–17

Rainbow trout (Oncorhynchus mykiss) Tongue River 4,000–12,000 5.6–7.7 7.93–8.38 10–11

Pallid sturgeon (Scaphirhynchus albus) Tongue River 518–4,000 4.7–7.4 8.16–8.95 19

Powder River 518–4,000 4.3–7.7 8.06–8.76 19

Walleye (Sander vitreus) Yellowstone River 625–5,000 7.9–8.9 8.08–9.19 14–15

Shovelnose sturgeon (Scaphirhynchus platorynchus)

Yellowstone River 625–10,000 6.6–7.4 7.79–9.28 15–17

Northern Pike (Esox lucius) Yellowstone River 500–8,000 7.8–9.6 7.78–9.04 10–11

Freshwater mussel (Lampsilis siliquoidea) Tongue River 500–2,500 7.2 8.3–9.2 19–22

African clawed frog (Xenopus laevis) Tongue River 750–4,500 7.8 8.2–9.1 22–24

Tubifex (Tubifex tubifex) Tongue River 1,000 6.1–8.4 7.3–9.2 17–19

Powder River 5,000 6.2–8.1 8.2–9.3 17–19

Amphipod (Hyalella azteca) Tongue River 1,000–2,000 6.6–7.3 8.6–8.8 23

Powder River 1,000–2,000 6.5–7.2 8.1–9.0 23

Chironomid (Chironomus dilutus) Tongue River 4,000–16,000 5.1–6.5 8.1–9.5 21–23

Powder River 4,000–16,000 5.1–6.8 8.1–9.3 21–23

Ceriodaphnia (Ceriodaphnia dubia) Tongue River 900–3,000 7.3–7.6 8.4–9.2 24–27

Powder River 900–3,000 7.5–7.7 8.1–9.3 24–27
Results 19

Table 2–4. Exposure concentrations of sodium bicarbonate during a 96-hour acute toxicity experiment with 4-day-post-hatch pallid 

sturgeon (Scaphirhynchus albus). Experiments were completed in reconstituted Powder River water. Exposures presented are 

nominal concentrations of sodium bicarbonate. 

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium, Mg2+, magnesium; Na+, sodium; K, potassium, Cl-, 

chloride; SO4

2-, sulfate; ( ), range in parentheses; --, one sample was measured, no range provided. Concentrations are means unless no range is provided]

Exposure 

(mg NaHCO3/L )

Alkalinity 

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– SO4

2–

Control 207 0 252 68.9 87.9 324 16.8 98.6 856

(198–220) (5.3) (242–268) -- -- -- -- -- --

518 305 5.3 366 65.0 87.7 378 16.8 98.7 855

(300–312) (0–8.0) (361–381) -- -- -- -- -- --

864 465 26.7 535 61.5 87.5 429 16.8 98.8 855

(444–480) (8.0–48.0) (517–556) -- -- -- -- -- --

1,440 747 20 887 54.0 87.1 533 16.7 98.9 853

(720–768) (0–48.0) (859–927) -- -- -- -- -- --

2,400 1,240 40 1,470 39.1 86.4 742 16.7 99.3 851

4,000 2,020 208 2,200 9.4 84.9 1,160 16.5 100 846

Table 2–5. Number alive and percent survival of 4-day-post-hatch pallid sturgeon (Scaphirhynchus albus) exposed to sodium 

bicarbonate in a 96-hour acute toxicity experiment completed in reconstituted Powder River water. Exposure concentrations 

presented are nominal concentrations of sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, millgrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L ) 0 hour 24 hours 48 hours 72 hours 96 hours

Control 27 (100) 27 (100) 26 (96) 26 (96) 26 (96)

518 28 (100) 28 (100) 27 (96) 27 (96) 26 (93)

864 20 (100) 20 (100) 20 (100) 20 (100) 20 (100)

1,440 30 (100) 19 (63) 13 (43) 3 (10) 2 (6)

2,400 30 (100) 0 (0) 0 (0) 0 (0) 0 (0)

4,000 30 (100) 0 (0) 0 (0) 0 (0) 0 (0)
20 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Table 2–6. Water chemistry measured during a 96-hour acute toxicity experiment with 4-day-post-hatch pallid sturgeon 

(Scaphirhynchus albus). Experiments were completed in reconstituted Tongue River water. Exposures presented are nominal values 

for sodium bicarbonate. 

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, 

chloride; SO4

2-, sulfate; ( ), range in parentheses; --, one sample was measured, no range provided. Concentrations are means unless no range is provided]

Exposure 

(mg NaHCO3

/L )

Alkalinity

(CaCO3

, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– SO4

2–

Control 172 0 210 42.3 20.6 75.9 7.41 7.98 186

(168–178) (0–0) (205–217) -- -- -- -- -- --

518 302 4.0 363 40.4 20.5 134 7.39 7.87 186

(292–308) (0–8.0) (346–346) -- -- -- -- -- --

864 467 22.0 543 38.5 20.5 193 7.37 7.77 186

(448–492) (16–24) (517–571) -- -- -- -- -- --

1,440 753 261.3 844 34.6 20.35 309 7.33 7.55 186

(728–796) (56–72) (656–902) -- -- -- -- -- --

2,400 1,239 124 1,360 26.9 20.1 543 7.25 7.13 185

(1,216–1,270) (112–136) (1,336–1,400) -- -- -- -- -- --

4,000 2,188 216 2,404 11.5 19.6 1,110 7.10 6.27 185

(2,116–2,260) (200–232) (2,336–2,470) -- -- -- -- -- --

Table 2–7. Number alive and percent survival of 4-day-post-hatch pallid sturgeon (Scaphirhynchus albus) exposed to sodium 

bicarbonate in a 96-hour acute toxicity experiment completed in reconstituted Tongue River water. Exposures presented are nominal 

values for sodium bicarbonate. 

[NaHCO3, sodium bicarbonate; mg/L, millgrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3

/L) 0 hour 24 hours 48 hours 72 hours 96 hours

Control 30 (100) 30 (100) 30 (100) 30 (100) 30 (100)

518 30 (100) 30 (100) 30 (100) 29 (97) 27 (90)

864 30 (100) 29 (97) 29 (97) 28 (94) 28 (94)

1,440 20 (100) 20 (100) 20 (100) 20 (100) 20 (100)

2,400 20 (100) 0 (0) 0 (0) 0 (0) 0 (0)

4,000 30 (100) 1 (3) 0 (0) 0 (0) 0 (0)
Results 21

Fathead Minnow 

The 96-h LC50 for 4-dph FHM exposed in reconstituted 

Powder River water was 2,078 mg NaHCO3/L with 95-percent 

CI of 769–3,388 mg/L (table 2–9). With 2-dph FHM exposed 

in reconstituted Tongue River water, the LC50 was 1,749 mg 

NaHCO3/L with 95-percent CI of 1,604–1,895 mg NaHCO3/L, 

respectively. Water chemistry and survival data are presented 

in tables 2–10 and 2–11.

Table 2–8. Water chemistry measured during a 96-hour acute toxicity experiment with 4-day-post-hatch fathead minnows 

(Pimephales promelas). Experiments were completed in reconstituted Powder River water. Exposures presented are nominal values 

for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, 

chloride; SO4

2-, sulfate; ( ), range in parentheses; --, one sample was measured, no range provided. Concentrations are means unless no range is provided]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– SO4

2–

Control 201 8 193 78.3 92.3 285 11.8 101 929

(196–204) (8–8) (229–239) -- -- -- -- -- --

518 320.5 17 303.5 76.3 92.0 332 12.0 100.4 923

(312–332) (16–20) (361–380) -- -- -- -- -- --

864 491 21 470 72.8 91.6 417 12.4 99.2 911

(482–500) (20–24) (558–585) -- -- -- -- -- --

1,440 772 21 751 66.9 90.8 557 13.1 97.4 892

(762–786) (20–24) (905–929) -- -- -- -- -- --

2,400 1,285 62 1,223 57.2 89.5 791 14.2 94.3 861

(1,270–1,300) (56–68) (1,480–1,500) -- -- -- -- -- --

4,000 2,215 152 2,063 40.9 87.3 1,180 16.1 89.1 808

(2,170–2,250) (136–160) (2,450–2,550) -- -- -- -- -- --

Table 2–9. Number alive and percent survival of 4-day-post-hatch fathead minnows (Pimephales promelas) exposed to sodium 

bicarbonate in a 96-hour acute toxicity experiment completed in reconstituted Powder River water. Exposures presented are nominal 

values for sodium bicarbonate. 

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L) 0 hour 24 hours 48 hours 72 hours 96 hours

Control 15 (100) 15 (100) 15 (100) 15 (100) 15 (100)

518 15 (100) 15 (100) 15 (100) 14 (93) 12 (80)

864 15 (100) 15 (100) 15 (100) 14 (93) 12 (80)

1,440 15 (100) 14 (93) 13 (87) 9 (60) 9 (60)

2,400 15 (100) 11 (73) 11 (73) 8 (53) 8 (53)

4,000 15 (100) 7 (47) 4 (27) 2 (13) 1 (7)
22 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Table 2–10. Water chemistry measured during a 96-hour acute toxicity experiment with 2-day-post-hatch fathead minnows 

(Pimephales promelas). Experiments were completed in reconstituted Tongue River water. Exposures presented are nominal values for 

sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl, chloride; SO4

2

, sulfate; ( ), standard error in parentheses; --, one sample was measured, no range provided. Concentrations are means unless no range is provided]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– NO3 SO4

2–

Control 210 11 243 47 34 45 2.5 2.2 0.3 140

(11) (6) (18) (2.6) (.35) (.78) (.03) (.2) (.01) (2.5)

500 374 15 438 47 34 134 2.4 2.3 .3 142

(5.8) (7) (15) (.1) (.37) (1.9) (.07) (.11) (.01) (1.5)

800 532 114 627 37 34 216 2.4 2.2 .3 142

(13) (102) (27) (3.3) (.6) (4.5) (.04) (.03) (.01) (1.3)

1,100 704 60 766 27 34 293 2.5 2.2 .3 144

(18) -- -- -- -- -- -- -- -- --

1,400 899 62 1,020 22 34 390 2.6 2.4 .3 146

(3.5)( (34) (44) (8.2) (.94) (13) (.11) (.06) (.01) (3.5)

2,000 1,220 126 133 16 33 524 2.8 4 .3 143

(26) (21) (51) (4.3) (.33) (5.5) (.03) (.16) (.01) (2.1)

3,000 1,880 265 1,970 12 34 812 3.4 5.1 .3 148

(72) (79) (26) (5.1) (.79) (24) (.54) (.4) (.01) (3.2)

Table 2–11. Number alive and percent survival of 2-day-post-hatch fathead minnows (Pimephales promelas) exposed to sodium 

bicarbonate in a 96-hour acute toxicity experiment completed in reconstituted Tongue River water. Exposures presented are nominal 

values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L) 0 hour 24 hours 48 hours 72 hours 96 hours

Control 30 (100) 30 (100) 30 (100) 30 (100) 30 (100)

500 30 (100) 29 (97) 29 (97) 29 (97) 29 (97)

800 30 (100) 30 (100) 29 (97) 29 (97) 29 (97)

1,100 30 (100) 30 (100) 30 (100) 30 (100) 25 (83)

1,400 30 (100) 30 (100) 30 (100) 28 (93) 22 (73)

2,000 30 (100) 30 (100) 29 (97) 27 (90) 15 (50)

3,000 30 (100) 30 (100) 29 (97) 14 (47) 0 (0)
Results 23

White Sucker 

Newly Hatched Fry

There was greater mortality among fish exposed in the 

reconstituted Tongue River water compared with fish exposed 

in reconstituted Powder River water. The LC50 for white 

suckers exposed to reconstituted Tongue River water was 

4,494 mg NaHCO3/L with 95-percent CI of 2,891–6,097 mg 

NaHCO3/L. Water chemistry and survival data are presented 

in tables 2–12 and 2–13 In reconstituted Powder River water, 

the calculated 96-h LC50 was 5,648 mg NaHCO3/L. Reliable 

95-percent CI could not be calculated. Water chemistry and 

survival data are presented in tables 2–14 and 2–15.

Older Fry

The limited number of exposure levels did not allow the 

LC50 to be calculated for older white suckers, but mortality 

rates were 9.5 percent, 13.6 percent, and 50 percent for the 

control, 4,000 and 6,666 mg NaHCO3/L (table 2–13.), respectively. These results suggest that the older fry were somewhat 

more tolerant of the NaHCO3 than the younger fish.

Table 2–12. Water chemistry measured during a 96-hour acute toxicity experiment with 22-day-post-hatch white sucker 

(Catostomus commersoni). Experiments were completed in reconstituted Tongue River water. Exposures presented are nominal values 

for sodium bicarbonate. 

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, 

chloride; SO4

2-, sulfate; ( ), range; --, one sample was measured, no range provided. Concentrations are means unless no range is provided]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– SO4

2–

Control 177.5 0 217 37.6 19.1 70.9 5.90 5.01 172

(168–196) (0–0) (205–239) -- -- -- -- -- --

864 529.5 22.0 619 38.3 19.8 219 6.30 5.09 164

(518–542) (0–8.0) (346–346) -- -- -- -- -- --

1,440 829 49.0 951 20.6 19.1 381 6.50 4.74 168

(804–850) (48–52) (922–978) -- -- -- -- -- --

2,400 1,396 93.3 1,300 12.9 18.8 597 8.0 5.50 164

(1,370–1,410) (88–104) (1,565–1,600) -- -- -- -- -- --

4,000 2,330 227 2,610 10.9 19.5 1,030 8.40 7.31 167

(2,200–2,380) (216–240) (2,600–2,620) -- -- -- -- -- --

6,666 3,929 365 4,360 11.0 19.6 1,166 6.03 167 166

(3,900–4,010) (360–376) (4,220–4,400) -- -- -- -- -- --

Table 2–13. Number alive and percent survival of 22-day-post-hatch white suckers (Catostomus commersoni) exposed to sodium 

bicarbonate in a 96-hour acute toxicity experiment completed in reconstituted Tongue River water. Exposures presented are nominal 

values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L) 0 hour 24 hours 48 hours 72 hours 96 hours

Control 26 (100) 26 (100) 24 (92) 24 (92) 24 (92)

864 31 (100) 28 (90) 28 (90) 28 (90) 27 (87)

1,440 30 (100) 30 (100) 30 (100) 30 (100) 28 (93)

2,400 30 (100) 28 (93) 28 (93) 27 (90) 26 (87)

4,000 30 (100) 21 (70) 19 (63) 17 (57) 15 (50)

6,666 30 (100) 19 (63) 11 (37) 10 (30) 10 (30)
24 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Table 2–14. Water chemistry measured during a 96-hour acute toxicity experiment with 22-day-post-hatch white sucker (Catostomus 

commersoni). Experiments were completed in reconstituted Powder River water. Exposures presented are nominal values for sodium 

bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, 

chloride; SO4

2-, sulfate; ( ), range; --, one sample was measured, no range provided. Concentrations are means unless no range is provided]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– SO4

2–

Control 234 8 274 61.9 84.5 291 11.5 86.2 820

(216–270) (0–32) (254–300) -- -- -- -- -- --

864 552.5 33 633 61.8 88.7 491 12.4 85.7 820

(546–558) (24–44) (624–651) -- -- -- -- -- --

1,440 825.5 37 962 42.3 88.4 670 13.3 85.5 822

(820–836) (24–44) (941–980) -- -- -- -- -- --

2,400 1,372 37 1,580 23.2 87 793 14 85.2 811

(1,360–1,380) (64–96) (1,560–1,600) -- -- -- -- -- --

4,000 2,304 162 2,610 13.7 86.4 1190 15.5 86.4 812

(2,260–2,360) (120–192) (1,520–2,660) -- -- -- -- -- --

6,666 3,873 298 4,360 9.9 86.3 1,840 14.7 86.4 798

(3,830–3,900) (192–392) (4,240–4,530) -- -- -- -- -- --

Table 2–15. Number alive and percent survival of 22-day-post-hatch white suckers (Catostomus commersoni) exposed to sodium 

bicarbonate in a 96-hour acute toxicity experiment completed in reconstituted Powder River water. Exposures presented are nominal 

values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L) 0 hour 24 hours 48 hours 72 hours 96 hours

Control 16 (100) 15 (94) 15 (94) 15 (94) 15 (94)

864 16 (100) 16 (100) 16 (100) 15 (94) 15 (94)

1,440 14 (100) 14 (100) 13 (93) 13 (93) 13 (93)

2,400 16 (100) 16 (100) 16 (100) 16 (100) 15 (94)

4,000 16 (100) 16 (100) 15 (94) 15 (94) 15 (94)

6,666 16 (100) 5 (31) 5 (31) 5 (31) 5 (31)
Results 25

African Clawed Frog 

The mean LC50 for four acute toxicity experiments with 

the African clawed frog was 1,940 mg NaHCO3/L with mean 

95-percent CI of 1,482–2,890 mg NaHCO3/L. Non-Lethal 

abnormalities occurred with increasing NaHCO3 concentrations, primarily as mild stunting and mild dorsal and lateral 

flexure of the tail. In some individuals that ultimately were 

counted as mortalities, severe stunting and flexure of the tail 

was observed before the end of the 96-h exposure. Mild flexure of the tail was observed in some survivors after the 96-h 

exposure. The EC50 ranged from 955 to 1,307 mg NaHCO3/L, 

with a mean of 1,108 mg NaHCO3/L. Water chemistry and 

survival data are presented in tables 2–16 and 2–17.

Table 2–16. Water chemistry measured during a 96-hour acute toxicity experiment with African clawed frog (Xenopus laevis). 

Experiments were completed in reconstituted Tongue River water. Exposures presented are nominal values for sodium bicarbonate. 

Frog embryos were less than 24-hour-post-fertilization at the start of the exposure.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, 

chloride; NO3-, nitrate; SO4

2-, sulfate; ( ), standard error in parentheses. Concentrations are means]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Mean

Ca2+

Mean

Mg2+

Mean

Na+

Mean

K

Mean

Cl– NO3

– Mean

SO4

2–

Control 192 12.3 220 45 33 45 2.4 2 0.3 139

(4.3) (1.1) (3.5) (1.6) (.8) (.3) (.10) (.1) (.01) (.4)

500 306 39 325 33 30 131 2.3 2.2 .2 143

(4.4) (5.6) (11) (1.4) (.3) (.6) (.1) (.2) (.01) (.5)

750 464 46 508 18 30 193 4 3.2 .3 132

(6.3) (1.7) (9.8) (2.8) (.4) (1) (1.6) (1.3) (.01) (3.5)

1,000 600 77 637 12 30 261 2.6 2 .2 130

(8.5) (11) (22) (3.9) (.6) (2.9) (.1) (.1) (.02) (3.2)

1,500 859 120 901 13 31 390 2.7 2.7 .3 136

(6.7) (20) (32) (7.1) (.3) (2.2) (.1) (.3) (.01) (0)

2,500 1,500 177 1,730 16 31 628 2.7 3.5 .2 136

(10.8) (75) (37) (3.5) (.3) (1.5) (.1) (.05) (.01) (.4)

3,000 1,877 265 1,965 12 34 812 3.4 5.1 .3 148

(72) (79) (26) (5.1) (.79) (24) (.54) (.4) (.01) (3.2)

3,500 2,070 159 2,330 4.3 31 901 2.8 4.9 .3 135

(18) (5.2) (19.5) (.5) (.2) (8.9) (.1) (.1) (.01) (.4)

4,500 2,640 408 2,730 3.6 31 1,093 2.8 5.4 .3 135

(18) (50) (39) (1.0) (1.0) (4.8) (.1) (.2) (.01) (.8)

Table 2–17. Number alive and percent survival of African clawed frog (Xenopus laevis) in a 96-hour acute toxicity experiment 

completed in reconstituted Tongue River water. Exposures presented are nominal concentrations sodium bicarbonate. Frog embryos 

were less than 24-hour-post-fertilization at the start of the exposure. 

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival; --, not applicable]

Experiment 1 Experiment 2 Experiment 3 Experiment 4

Exposure

(mg NaHCO3/L)

96 hour 

survival

Exposure

(mg NaHCO3/L)

96 hour 

survival

Exposure

(mg NaHCO3/L)

96 hour 

survival

Exposure

(mg NaHCO3/L)

96 hour 

survival

Control 45 (90) Control 46 (92) Control 46 (92) Control 46 (92)

500 45 (90) 750 36 (72) 750 18 (36) 750 39 (78)

1,000 46 (92) 1,000 38 (76) 1,000 16 (32) 1,000 38 (76)

1,500 14 (28) 1,500 32 (64) 1,500 0 (0) 1,500 34 (68)

3,000 10 (20) 2,500 10 (20) 2,500 0 (0) 2,500 31 (61)

4,500 0 (0) 3,500 0 (0) 3,500 0 (0) 3,500 0 (0)

-- -- 4,500 0 (0) 4,500 0 (0) 4,500 0 (0)
26 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Freshwater Mussels

The EC50 for newly transformed juvenile fatmucket 

mussels exposed to reconstituted Tongue River water was 

1,120 mg NaHCO3/L, with 95-percent CI of 1,099–1,497 

mg NaHCO3/L. Although the lack of foot movement was 

the ultimate measure of toxicity, mussels that did not display 

foot movement within the 5-minute observation period were 

all observed as mortalities, and under closer examination as 

empty shells at the end of the experiment. Water chemistry and 

survival data are presented in tables 2–18 and 2–19.

Table 2–18. Water chemistry measured during a 96-hour acute toxicity experiment with newly transformed fatmucket mussels 

(Lampsilis siliquoidea). Experiments were completed in reconstituted Tongue River water. Exposures presented are nominal values for 

sodium bicarbonate. Sample size of two for each.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, chloride; SO4

2-, sulfate]

Experiment 1

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– NO3- SO4

2–

Control 182 10 211 39 33 45 2.4 2.2 0.3 142

500 344 24 390 29 33 131 2.3 2.2 .2 143

1,000 634 6.3 760 15 33 249 2.3 1.2 .5 146

1,500 939 22 1,120 17 32 365 2.2 1.3 .5 143

2,000 1,230 20 1,480 8.8 32 511 2.5 1.3 .5 144

2,500 1,550 20 1,870 14 31 618 2.4 1.2 .5 143

Experiments 2 and 3 

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– SO4

2–

Control 204 13.1 235 47.5 33.7 46.1 2.17 2.01 150

500 367 32.1 415 37.1 33.9 129 2.23 2.04 148

750 511 1.22 623 22.4 33.2 189 2.16 1.35 152

1,000 672 9.51 810 20.5 33.4 255 2.17 1.05 151

1,250 810 37.7 951 21.9 32.8 318 2.16 1.18 152

1,500 934 32.8 1,100 16.4 32.6 367 2.11 1.17 150

2,000 1,280 35.6 1,490 8.84 33.4 519 242 1.34 149

Table 2–19. Number alive and percent survival of newly transformed fatmucket mussels (Lampsilis 

siliquoidea) in a 96-hour acute toxicity experiment completed in reconstituted Tongue River water. 

Exposures presented are nominal values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival; --, not applicable]

Experiment 1 Experiment 2 Experiment 3

Exposure

(mg NaHCO3/L)

96-hour 

survival

Exposure

(mg NaHCO3/L)

96-hour 

survival

Exposure

(mg NaHCO3/L)

96-hour 

survival

Control 20 (100) Control 20 (100) Control 20 (100)

500 19 (95) 500 20 (100) 500 20 (100)

1,000 11 (55) 750 19 (95) 750 19 (95)

1,500 0 (0) 1,000 16 (80) 1,000 14 (70)

2,000 0 (0) 1,250 9 (45) 1,250 4 (25)

2,500 0 (0) 1,500 2 (10) 1,500 0 (0)

-- -- 2,000 0 (0) 2,000 0 (0)
Results 27

Rainbow Trout

The calculated LC50 for 2-dph rainbow trout exposed to 

reconstituted Tongue River water was 8,070 mg NaHCO3/L, 

with 95-percent CI of 7,493–8,647 mg NaHCO3/L. Water 

chemistry and survival data are presented in tables 2–20 and 

2–21. 

Table 2–20. Water chemistry measured during a 96-hour acute toxicity experiment with rainbow trout (Oncorhynchus mykiss) 

completed in reconstituted Tongue River water. Exposures presented are nominal concentrations of milligrams sodium bicarbonate. 

Sample size equals 1 for each.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, 

chloride; SO4

2-, sulfate]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– SO4

2–

Control 208 5.06 248 3.18 24.9 139 6.27 5.21 216

4,000 4,020 512 4,280 2.38 15.8 1,680 5.2 9.62 141

6,000 5,930 745 6,320 3.31 22.4 2,420 8.31 12.1 206

8,000 6,000 570 6,560 2.79 15.8 2,580 5.66 12.2 147

10,000 7,040 730 7,700 3.48 18.8 2,960 7.14 15 174

12,000 8,770 375 10,200 3.62 22.1 3,620 8.08 18.3 197

Table 2–21. Number alive and percent survival of juvenile rainbow trout (Oncorhynchus mykiss) completed in reconstituted Tongue 

River water. Exposures presented are nominal values for sodium bicarbonate. Sample size equals 1 for each.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L) 0 hour 24 hours 48 hours 72 hours 96 hours

Control 30 (100) 30 (100) 30 (100) 30 (100) 29 (97)

4,000 30 (100) 30 (100) 29 (97) 29 (97) 29 (97)

6,000 30 (100) 21 (100) 9 (30) 8 (27) 7(23)

8,000 30 (100) 15 (50) 10 (33) 3 (10) 1 (3)

10,000 30 (100) 3 (10) 0 (0) 0 (0) 0 (0)

12,000 30 (100) 0 (0) 0 (0) 0 (0) 0 (0)
28 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Ceriodaphnia

The calculated LC50s for C. dubia were; 1,288 mg 

NaHCO3/L, with a 95-percent CI of 1,171–1,406 mg 

NaHCO3/L in moderately hard reconstituted water; 989 mg 

NaHCO3/L with 95-percent CI of 910–1,069-mg NaHCO3/L 

in reconstituted Tongue River water; and 1,355 mg NaHCO3/L 

with 95-percent CI of 1,257–1,453 mg NaHCO3/L in reconstituted Powder River water. Water chemistry and survival data 

are presented in tables 2–22 and 2–23.

Table 2–22. Water chemistry measured during a 48-hour acute toxicity experiment with adult (Ceriodaphnia dubia) completed in 

moderately hard reconstituted water, reconstituted Tongue River water, and reconstituted Powder River water. Exposures presented 

are nominal values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, 

chloride; SO4

2-, sulfate; ( ), standard error in parentheses; Mod hard, moderately hard water]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– SO4

2–

Mod hard 

control

76 0 93 15 9.0 21 7.9 1.4 96

900 546 71 579 14 13 216 2.7 1.8 99

1,300 736 140 727 8.0 13 339 2.4 1.0 93

1,600 918 144 944 5.0 12 418 2.2 1.0 98

2,000 1,136 288 1,034 4.0 12 476 2.2 1.2 96

3,000 2,040 640 1,707 5.4 13 768 6.9 3.0 113

Tongue River 

control

204 1 249 47 34 43 2.4 1.7 157

900 604 104 610 44 34 185 2.0 2.3 164

1,300 746 180 715 38 33 303 2.7 2.0 157

1,600 900 236 810 30 33 390 2.7 2.2 162

2,000 1,100 280 1,000 4.6 33 476 3.3 2.8 158

3,000 1,880 680 1,460 5 32 765 4.1 3.9 171

Powder River 

control

212 16 239 13 180 283 13 11 1,320

900 534 120 505 11 174 438 15 11 1,430

1,300 710 168 661 12 177 488 15 11 1,410

1,600 912 276 776 12 176 583 16 11 1,410

2,000 1,100 324 944 6.0 176 740 16 10 1,330

3,000 1,860 720 1,370 6.0 176 931 17 12 1,530

Table 2–23. Number of live organisms at 0, 24, and 48 hours and percent survival at 48 hours for adult Ceriodaphnia dubia completed 

in moderately hard reconstituted water, reconstituted Tongue River water, and reconstituted Powder River water. Exposures 

presented are nominal concentrations of sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter]

Exposure 

(mg NaHCO3/L)

Moderately hard reconstituted Reconstituted Tongue River Reconstituted Powder River

Survival Survival Survival

0 hour 24 hour 48 hour Percent 0 hour 24 hour 48 hour Percent 0 hour 24 hour 48 hour Percent

Control 10 10 9 90 10 10 10 100 10 10 10 100

900 10 10 10 100 10 9 9 90 10 10 10 100

1,300 10 9 9 90 10 9 5 50 10 7 7 70

1,600 10 5 1 10 10 2 0 0 10 7 70

2,000 10 0 0 0 10 1 0 0 10 2 0 0

3,000 10 0 0 0 10 0 0 0 10 0 0 0
Results 29

Chironomids 

The calculated LC50 for 1–2 week post hatch chironomid larvae exposed to reconstituted Tongue River water was 

4,947 mg NaHCO3/L, with a 95-percent CI of 4,674–5,221 

mg NaHCO3/L, and 8,014 mg NaHCO3/L in reconstituted 

Powder River water, with a 95-percent CI of 7,513–8,514 mg 

NaHCO3/L. Water chemistry and survival data are presented in 

tables 2–24 and 2–25. 

Table 2–24. Water chemistry measured in concentrated stock solutions and reconstituted (control) Tongue River and Powder River 

waters used to make exposure concentrations for a 48-hour acute toxicity experiments with chironomid larvae (Chironomus dilutus). 

Sample size equals one for each. Exposures presented are nominal concentrations of sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium,; Cl-, 

chloride; SO4

2-, sulfate; <. less than 1]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– SO4

2–

Tongue River 

control

156 <1.00 190 4.23 20.7 105 6.76 6.96 178

Tongue River 

Stock

8,520 <1.00 10,400 2.48 19.4 3,730 9.22 8.51 169

Powder River 

control

320 112 254 67.4 108 363 20.8 111 1,010

Powder River 

Stock 

12,000 2,480 11,700 8.63 87.1 4,210 29.4 105 892

Table 2–25. Number alive and percent survival of chironomid larvae (Chironomus 

dilutus) in a 48-hour acute toxicity experiment completed in reconstituted (control) 

Tongue and Powder River waters. Exposures presented are nominal concentrations of 

sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L) 0 hour 24 hours 48 hours

Tongue River control 40 (100) 39 (98) 39 (98)

4,000 40 (100) 38 (95) 38 (95)

6,000 40 (100) 38 (95) 33 (83)

8,000 40 (100) 13 (33) 0 (0)

12,000 40 (100) 0 (0) 0 (0)

16,000 40 (100) 0 (0) 0 (0)

Powder River control 40 (100) 40 (100) 38 (95)

4,000 40 (100) 39 (98) 39 (98)

6,000 40 (100) 38 (95) 36 (90)

8,000 40 (100) 33 (83) 18 (45)

12,000 40 (100) 0 (100) 0 (97)

16,000 40 (100) 0 (100) 0 (97)
30 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Tubifex

The calculated LC50 for Tubifex exposed to reconstituted 

Tongue River water was 3,297 mg NaHCO3/L, with upper 

and lower 95-percent CI of 3,121–3,473 mg NaHCO3/L, and 

3,367 mg/L NaHCO3/L, with upper and lower 95-percent CI 

of 3,188 and 3,547 mg NaHCO3/L with reconstituted Powder 

River water. Water chemistry and survival data are presented 

in tables 2–26 and 2–27.

Table 2–26. Water chemistry measured in concentrated stock solutions of 5,000 milligrams sodium bicarbonate per liter and 

reconstituted (control) Tongue and Powder River waters used to make exposure concentrations for 96-hour acute toxicity experiments 

with Tubifex worms (Tubifex tubifex). Exposures presented are nominal values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, 

chloride; NO3-, nitrate; SO4

2-, sulfate]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– NO3- SO4

2–

Tongue River 

control

176 55.1 207 1 20.1 112 11.6 1 20.1 1

Tongue River 

5,000

3,030 104.3 3,560 1.96 18.8 1,220 5.64 1.96 18.8 1.96

Powder River 

control

192 3.23 229 53.5 60.1 291 11.5 53.5 60.1 53.5

Powder River 

5,000

2,910 66.5 3,460 72.2 87.5 1,420 12.7 72.2 87.5 72.2

Table 2–27. Number alive and percent survival of Tubifex worms (Tubifex tubifex) in 96-hour acute toxicity experiments completed in 

reconstituted (control) Tongue and Powder River waters. Exposures presented are nominal values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L) 0 hour 24 hours 48 hours 72 hours 96 hours

Tongue River control 40 (100) 40 (100) 40 (100) 40 (100) 40 (100)

1,000 40 (100) 40 (100) 40 (100) 40 (100) 40 (100)

2,000 40 (100) 40 (100) 40 (100) 40 (100) 39 (98)

3,000 40 (100) 40 (100) 40 (100) 37 (93) 33 (83)

4,000 40 (100) 40 (100) 34 (95) 14 (35) 7 (18)

5,000 40 (100) 40 (100) 14 (100) 1 (3) 0 (0)

Powder River control 40 (100) 40 (100) 40 (100) 40 (100) 38 (95)

1,000 40 (100) 40 (100) 40 (100) 40 (100) 40 (100)

2,000 40 (100) 40 (100) 40 (100) 39 (98) 40 (100)

3,000 40 (100) 40 (100) 40 (100) 40 (100) 39 (98)

4,000 40 (100) 40 (100) 40 (100) 30 (75) 29 (73)

5,000 40 (100) 40 (100) 34(85) 58 (98) 0 (0)
Results 31

Amphipods 

The calculated LC50 for Hyalella azteca exposed to 

reconstituted Tongue River water was 1,419 mg/L NaHCO3/L, 

with 95-percent CI of 1,340–1,4982 mg NaHCO3/L. It should 

be noted that this LC50 may be affected by low chloride (Cl-) 

concentrations in the reconstituted Tongue River water. An 

LC50 was calculated in Powder River reconstituted water as 

3,851 mg NaHCO3/L. However, the low mortality in the largest concentration (2,000 mg/L) and the resulting large CI of 

-3,642–9,450 mg/L, make this number suspect. Water chemistry and survival data are presented in tables 2–28 and 2–29.

Table 2–28. Water chemistry measured in concentrated stock solutions of sodium bicarbonate and reconstituted (control) 

Tongue and Powder River waters used to make exposure concentrations (also presented) for 96-hour acute toxicity experiments 

with adult amphipods (Hyalella azteca). Exposures presented are nominal values for sodium bicarbonate. Sample size equals 

one for each.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium, Mg2+; magnesium; Na+, sodium; K, potassium; 

Cl-, chloride; SO4

2-, sulfate]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– SO4

2

–

Tongue River control 156 8.98 147 2 19.6 108 6.03 3.32 177

Tongue River stock 1,210 90 1,120 2.53 18.9 553 7.06 3.7 174

Powder River control 199 6.15 192 55.4 90.1 288 13 97.5 829

Powder River stock 1,110 33.7 1,070 53.9 88.9 724 13.8 103 878

Tongue River 

nominal

(mg NaHCO3/L)

Measured 

(mg NaHCO3/L)

Tongue River

nominal

(mg NaHCO3/L)

Measured

 (mg NaHCO3/L)

Control 264 Control 486

1,000 879 1,000 923

1,250 1,100 1,250 1,150

1,500 1,320 1,500 1,380

1,750 1,540 1,750 1,620

2,000 1,760 2,000 1,850

Table 2–29. Number alive and percent survival of adult amphipods (Hyalella azteca) in 96-hour acute toxicity experiments completed 

in reconstituted (control) Tongue and Powder River waters. Exposures presented are nominal values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L) 0 hour 24 hours 48 hours 72 hours 96 hours

Tongue River control 40 (100) 40 (100) 40 (100) 40 (100) 40 (100)

1,000 40 (100) 40 (100) 40 (100) 36 (90) 35 (88)

1,250 40 (100) 40 (100) 40 (100) 34 (85) 29 (73)

1,500 40 (100) 40 (100) 40 (100) 31 (78) 22 (55)

1,750 40 (100) 40 (100) 38 (95) 25 () 19 (48)

2,000 40 (100) 40 (100) 38 (0) 29 (0) 9 (23)

Powder River control 40 (100) 40 (100) 40 (100) 40 (100) 40 (100)

1,000 40 (100) 40 (100) 40 (100) 40 (100) 40 (100)

1,250 40 (100) 40 (100) 39 (98) 39 (98) 38 (95)

1,500 40 (100) 40 (100) 40 (100) 40 (100) 39 (98)

1,750 40 (100) 39 (98) 39 (98) 39 (98) 39 (98)

2,000 40 (100) 40 (100) 39 (98) 39 (98) 37 (93)
32 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Shovelnose Sturgeon

The calculated LC50 for yolk-sac fry shovelnose sturgeon 

exposed to Yellowstone River water was 1,038 mg NaHCO3/L. 

A reliable 95-percent CI could not be calculated. Water chemistry and survival data are presented in tables 2–30 and 2–31.

Table 2–30. Water chemistry measured during a 96-hour acute toxicity experiment with shovelnose sturgeon 

(Scaphirhynchus platorynchus) completed in Yellowstone River water (control). Exposures presented are nominal values for 

sodium bicarbonate. Sample size equals one for each. 

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, 

potassium; Cl-, chloride; SO4

2-, sulfate; <, less than 1]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– SO4

2

–

Control 94.5 <1 115 3.75 10.75 65.95 3.8 3.2 109.5

625 469 6 565 3.6 10.7 225 4.2 3 109.5

1,250 843 11 1,020 3.4 10.6 384 4.5 2.9 109.6

2,500 1,591 20 1,920 3 10.5 710 5.3 2.5 109.6

5,000 3,087 47 3,720 1.9 10.4 1,338 6.7 1.9 109.75

10,000 6,080 80 7,320 .5 10.1 2,610 9.7 .5 110

Table 2–31. Number alive and percent survival of 1-day-post-hatch shovelnose sturgeon (Scaphirhynchus platorynchus) completed 

in Yellowstone River water (control). Exposures presented are nominal values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L) 0 hour 24 hours 48 hours 72 hours 96 hours

Control 20 (100) 20 (100) 20 (100) 20 (100) 20 (100)

625 20 (100) 20 (100) 20 (100) 20 (100) 20 (100)

1,250 20 (100) 20 (100) 20 (100) 12 (60) 11 (55)

2,500 20 (100) 20 (100) 20 (100) 0 (0) 0 (0)

5,000 20 (100) 20 (100) 20 (100) 0 (0) 0 (0)

10,000 20 (100) 20 (100) 7 (35) 0 (0) 0 (0)
Results 33

Walleye

The calculated LC50 for 1-dph walleye exposed to 

Yellowstone River water was 3,249 mg NaHCO3/L, with 

95-percent CI of 2,850–3,648 mg NaHCO3/L. Water chemistry 

and survival data are presented in tables 2–32 and 2–33.

Table 2–32. Water chemistry measured during a 96-hour acute toxicity experiment with 1-day-post-hatch walleye (Sander vitreus) 

completed in Yellowstone River water (control). Exposures presented are nominal values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L;, milligrams per liter,; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, 

chloride; SO4

2-, sulfate]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl– NO3- SO4

2

–

Control 172 1 209 44.5 20.5 142 6.1 17.3 217 44.5

625 519 5.9 626 41.7 19.2 289 5.7 16.2 203 41.7

1,250 866 10.9 1,010 38.9 17.9 435 5.3 15.1 190 38.9

2,500 1,560 20.7 1,880 33.4 15.4 729 4.6 13.0 163 33.4

5,000 2,950 40.5 3,540 22.5 10.3 1,320 3.0 8.65 109 22.5

Table 2–33. Number alive and percent survival of 1-day-post-hatch walleye (Sander vitreus) completed in Yellowstone River water 

(control). Exposures presented are nominal values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L) 0 hour 24 hours 48 hours 72 hours 96 hours

Control 20 (100) 20 (100) 20 (100) 20 (100) 20 (100)

625 20 (100) 20 (100) 20 (100) 20 (100) 20 (100)

1,250 20 (100) 20 (100) 20 (100) 20 (100) 19 (95)

2,500 20 (100) 20 (100) 20 (100) 20 (100) 19 (95)

5,000 20 (100) 20 (100) 17 (85) 8 (40) 0 (0)
34 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Northern Pike

The calculated LC50 for 1-dph northern pike exposed 

to Yellowstone River water was >8,000 mg NaHCO3/L, no 

confidence interval could be calculated. Water chemistry and 

survival data are presented in tables 2–34 and 2–35.

Table 2–34. Water chemistry measured during a 96-hour acute toxicity experiment with 2-day-post-hatch northern 

pike (Esox lucius) completed in Yellowstone River water (control). Exposures presented are nominal values for sodium 

bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; Cl-, 

chloride; SO4

2-, sulfate; ( ), range; --, one sample was measured, no range provided. Concentrations are means unless no range is provided]

Exposure 

(mg NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate 

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ Cl– SO4

2

–

Control 89.3

(82–102)

0

(0.0–0.0)

108.9

(100–124)

4 18.8 48.2 7.59 121

500 357

(348–364)

10.7

(0–32)

423

(385–444)

-- -- -- -- --

1,000 627

(604–640)

18.7

(0–56)

741

(668–780)

3.87 18.6 173 7.8 121

2,000 1,178

(1160–1210)

56

(0–128)

1,368

(1,260–1,430)

-- -- -- -- --

4,000 2,468

(2300–2560)

211

(120–240)

2,752

(2,610–2,830)

3.75 18.5 297 8 121

8,000 4,181

(4,120–4,220)

336

(120–464)

4,690

(3,650–4,100)

-- -- -- -- --

Table 2–35. Number alive and percent survival of 2-day-post-hatch northern pike (Esox lucius) completed in Yellowstone River water 

(control). Exposures presented are nominal values for sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), percent survival]

Exposure 

(mg NaHCO3/L) 0 hour 24 hours 48 hours 72 hours 96 hours

Control 40 (100) 40 (100) 40 (100) 40 (100) 40 (100)

500 40 (100) 39 (97) 39 (97) 39 (97) 27 (67)

1,000 40 (100) 39 (97) 39 (97) 39 (97) 38 (95)

2,000 40 (100) 40 (100) 40 (100) 40 (100) 40 (100)

4,000 30 (100) 30 (100) 30 (100) 30 (100) 30 (100)

8,000 30 (100) 28 (94) 28 (94) 28 (94) 26 (87)
Discussion 35

Discussion

Sodium bicarbonate generally has been considered a 

relatively harmless substance, and there has been very little 

research into its toxicity to aquatic organisms. This is likely 

because of the large concentrations required to induce toxicity 

when compared to other potential toxicants such as heavy metals, ammonia and pesticides. Because of the lack of research 

into the toxicity of salts, total dissolved solids (TDS) and 

conductivity have been used to predict toxicity; however, the 

toxicity of different salts can be quite variable and TDS and 

conductivity are not always good predictors of toxicity (Burnham and Peterka, 1975; Dickerson and others, 1996; WeberScannell and Duffy, 2007). 

In a series of experiments to predict the toxicity of salts 

to aquatic organisms, Mount and others (1997) calculated 

a 48-h LC50 of 1,020 mg NaHCO3/L for C. dubia exposed 

to moderately hard reconstituted water. Experiments in this 

study with C. dubia resulted in an LC50 of 989 mg NaHCO3/L 

in simulated Tongue River water, and 1,355 mg NaHCO3/L 

in simulated Powder River water. Mount and others (1997) 

also exposed FHM to NaHCO3 and determined them to be 

more sensitive than C. dubia, with a 96-h LC50 of <850 mg 

NaHCO3/L. In the present study experiments, FHM were not 

more sensitive than C. dubia, and the LC50 concentrations 

were 1,749 mg NaHCO3/L in simulated Tongue River water 

and 2,079 mg NaHCO3/L in simulated Powder River water. 

Overall, results of the present study demonstrate that NaHCO3 

is acutely toxic to aquatic organisms with LC50 or EC50 

values that range from 989–>8,000 mg/L. Further, the LC50 

values for 6 of the 13 species utilized were less than 2,000 mg 

NaHCO3/L. These results are comparable to those obtained by 

Mount and others (1997).

The Salinity Toxicity Relationship (STR) model, developed by Mount and Gulley (1992) and validated by Tietge 

and others (1997), predicts LC50 values for combinations of 

salts. Using the measured water chemistry from the present 

study of fathead minnow experiments in reconstituted Tongue 

River water, the STR model predicted 96-h LC50 of 1,590 

mg NaHCO3/L for FHM, which is comparable to our measured 96-h LC50 of 1,749 mg NaHCO3

/L. The STR model 

predicted 48-h LC50 of 1,571 mg NaHCO3/L for C. dubia in 

moderately hard reconstituted water; 1,776 mg NaHCO3/L in 

reconstituted Tongue River water; and 1,575 mg NaHCO3/L in 

reconstituted Powder River water. Predicted values in the STR 

model were all greater than the LC50 values calculated with 

the TRAP model in the present study of 1,288 mg NaHCO3/L 

in moderately hard reconstituted water; 989 mg NaHCO3/L in 

reconstituted Tongue River water; and 1,355 mg NaHCO3/L 

in reconstituted Powder River water. The STR model was 

conservative for FHM in reconstituted Tongue River water; 

however, the STR model under-predicted toxicity to C. dubia

in all reconstituted waters.

Dwyer and others (2005) observed that no single species was the most sensitive to a series of chemicals. Common experimental organisms such as rainbow trout are more 

tolerant of salinity, but are sensitive to dissolved metals or 

pesticides, whereas another common experimental organism, the FHM is less tolerant of salinity but more tolerant of 

dissolved metals. The relative tolerance to salinity of different 

fish species has been offered as a predictor of fish assemblages 

in prairie streams (Ostrand and Wilde, 2004). Although the 

concentrations of NaHCO3 required to induce toxicity appear 

larger than other commonly tested compounds, concentrations of NaHCO3

 can exceed 3,000 mg/L in untreated CBNG 

produced waters (Patz and others, 2004). This concentration is 

greater than the LC50 values reported for the majority of species exposed to NaHCO3

 in the present study.

Mount and others (1997) demonstrated that the toxicity 

of sodium and calcium salts was caused by the co-occurring 

anions (specifically Cl-, sulfate, and HCO3

-

). In the Tongue 

and Powder River waters that were simulated in the present 

experiments, HCO3

-

 was the predominant co-occurring anion. 

Therefore, it is likely that the primary source of toxicity of 

NaHCO3 can be attributed to HCO3

-

. 

Toxicity from NaHCO3 likely is caused by disruption of 

the mechanisms responsible for ionic regulation. Ionoregulation in aquatic organisms is controlled, in part, by chloride 

cells, also known as ionocytes or mitochondria rich cells. 

Located within the gills and the epithelium of fish, invertebrates and amphibians, ionocytes are densely packed with 

mitochondria and use energy to “pump” ions across the cell 

membrane (Hobe and others, 1984; Perry, 1997; Wilson and 

Laurent, 2002). An enzyme involved in this process, sodiumpotassium adenosine triphosphatase, (Na/K+

 ATPase), also 

known as the Na/K+ pump, likely is affected by exposure to 

NaHCO3 (see chapter 3 in this report). In addition to, or in 

conjunction with ionoregulation, chloride cells also play a 

role in maintaining acid/base balance (Sullivan and others, 

1995; Perry, 1997; Gilmour and Perry, 2009). Chloride cells 

excrete HCO3

-

 in exchange for Cl- and expand in size when 

blood alkalosis occurs, and the rapid exchange of HCO3

-

 and 

Cl- acts to reduce blood alkalinity (Wilson and Laurent, 2002). 

In fish, the acid/base balance is controlled exclusively through 

the exchange of H+ and HCO3

-

 with Na+ and Cl- (Gilmour and 

Perry, 2009). 

Bicarbonate also is involved in respiration. Carbon dioxide (CO2

) produced during respiration is converted into HCO3

-

, 

which allows for the removal of CO2 and the maintenance of 

pH balance in the blood and hemolymph (Haswell and others, 

1980; Burnett and McMahon, 1985; Randall and Tsui, 2006). 

Because HCO3

-

 may be excreted across the gill membrane 

for respiration and as part of the acid/base regulatory mechanism, it is possible that the inability to excrete HCO3

-

 because 

of elevated concentrations in the exposure water during the 

experiments in this study, ultimately, caused acute mortality. 

Hoke and others (1992) completed experiments to determine 

the toxicity of alkalinity to C. dubia, and concluded that 

HCO3

-

 was toxic to aquatic organisms when concentrations 

were sufficiently large enough to interfere with this exchange 

of Cl-.
36 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

The function of chloride cells for ionoregulation and to 

maintain acid/base balance appears to be interrelated (Wilson 

and Laurent, 2002). Exposure to brackish water resulted in an 

increase in the number of chloride cells, indicating a response 

to maintain ionic balance similar to alkalosis and acidosis 

(Wilson and Laurent, 2002). The response to increased salinity was an increase in the size and number of chloride cells, 

which are actively driving the transport of ions across the gill 

membrane to maintain ionic balance. The mortality associated 

with exposure to NaHCO3, in the present study, likely resulted 

from the inability of the organisms to rapidly compensate for 

increased alkalinity and the subsequent disruption of ionoregulation and acid/base balance, which has been observed 

in previous studies (Wilson and Laurent 2002; Gilmour and 

Perry, 2009).

The variability in the sensitivity to NaHCO3 among 

organisms has not been well researched; however, it has 

been hypothesized that differences in salinity tolerance are a 

result of evolutionary history of the organism (Wurts, 1998; 

Nielson and others, 2003). For example, salmonid fishes that 

evolved in environments with highly variable salinity, such as 

estuaries, likely have been selected for adaptations to changing salinity (Wurts, 1998). Likewise, prairie species such 

as the plains killifish and some copepod species exposed to 

drought conditions and the resulting alkaline environment also 

have adapted as a response, and may be more fit to survive 

increased alkalinity and conductivity (Nielson and others, 

2003; Ostrand and Wilde, 2004). 

Young organisms often are more sensitive than adults; 

however, the effects of toxicants on different species and 

lifestages of fish are variable (Lasier and others, 1997; U.S. 

Environmental Protection Agency, 2002; J.M. Besser, I.E. 

Greer, J.L. Kunz, C.G. Ingersoll, C.G., and N. Wang, written commun., 2004; Besser and others, 2005). In the present 

study, 2-dph and 4-dph FHM, and 22-dph and 69-dph white 

suckers were exposed to a range of NaHCO3 concentrations. 

In both experiments, the LC50s for younger fish and older 

fish, respectively, were 1,643 and approximately 4,000 mg 

NaHCO3/L for FHM, and 5,121 and approximately 6,600 

mg/L for white suckers. U.S. Environmental Protection 

Agency (2002) guidelines require the use of the most sensitive lifestage for toxicity testing, and FHM must be 1-dph to 

14-dph. However, because FHM more than 4-dph were less 

sensitive than 2-dph, experiments that use even slightly older 

fish may miss the most sensitive period of development for 

these fish. Therefore, it appears that with NaHCO3 toxicity, 

fish must be exposed immediately post hatch to insure that 

testing occurs during the most sensitive developmental period. 

Also, the effect of age on sensitivity was observed during field 

experiments in the present study completed in the Powder 

River Basin. In these experiments, a statistically significant 

difference in survival was observed between 2-dph and 6-dph 

fish, which further supports observations that very early lifestage (2-dph to 4-dph) are more sensitive than fish even just a 

few days older (see chapter 4).

In summary, the present study indicates that NaHCO3 is 

acutely toxic to freshwater organisms, including mussels, fish 

and C. dubia. The acute toxicity occurred within the range 

of concentrations defined by Mount and Gulley (1992) and 

Mount and others (1997), and are below concentrations measured in untreated CBNG produced water in the Powder River 

Basin (ALL Consulting, 2003; Patz and others, 2004). Additionally, the age at which fish are exposed to NaHCO3

 appears 

to be inversely related to the severity of toxic responses. 

Therefore, assessments of NaHCO3 toxicity to fish should be 

initiated immediately after hatching to ensure that the most 

sensitive lifestage is used.

References Cited

ALL Consulting, 2003, Handbook on coalbed methane produced water—Management and beneficial use alternatives: 

coalbed natural gas resources and produced water management, Tulsa: ALL Consulting, Report to the U.S. Department of Energy, accessed April 2010 at http://www.all-llc.

com/CBNG/BU/index.htm, 44 p.

American Society of Testing and Materials International, 

1998, Standard guide for conducting the frog embryo teratagenesis assay–Xenopus (FETAX): West Conshohocken, Pa., 

American Society of Testing and Materials International, 

E1439–98, 16 p.

American Society of Testing and Materials International, 

2006, Standard guide for conducting laboratory toxicity 

tests with freshwater mussels: West Conshohocken, Pa., 

American Society of Testing and Materials International, 

E 2455–06, 52 p.

Bantle, J.A., Dumont, J.N., Finch, R.A., and Linder, G., 1991, 

Atlas of abnormalities—a guide for the performance of 

FETAX: Stillwater, Oklahoma State University Publications, 63 p.

Besser, J.M., Wang, N., Dywer, F.J., Mayer, F.L., and Ingersoll, C.G., 2005, Assessing contaminant sensitivity of 

endangered and threatened aquatic species—Part I. Acute 

Toxicity of five chemicals chronic toxicity of copper and 

entachlorophenol to two endangered species and two surrogate species: Archives of Environmental Contamination 

and Toxicology, v. 48, p. 155–165.

Burnett, L.E., and McMahon, B.R., 1985, Facilitation of CO2

excretion by carbonic anhydrase located on the surface of 

the basal membrane of crab gill epithelium: Respiratory 

Physiology, v. 62, p. 341–348.

Burnham, B.L., and Peterka, J.J., 1975, Effects of saline water 

from North Dakota lakes on the survival of fathead minnows Pimephales promelas embryos and sac fry: Journal of 

Fisheries Research Board of Canada, v. 32, p. 809–812.
References Cited 37

Dickerson, K.K., Hubert, W.A., and Bergman, H.L., 1996, 

Toxicity assessment of water from lakes and wetlands 

receiving irrigation drain water: Environmental Toxicology 

and Chemistry, v. 15, no. 7, p. 1,097–1,101.

Dwyer, F.J., Mayer, F.L., Sappington, L.C., Buckler, D.R., 

Bridges, C.M., Greer, I.E., Hardesty, D.K., Henke, C.E., 

Ingersoll, C.G.,Kunz, J. L., Whites, D.W., Augspurger, 

T., Mount, D.R., Hattala, K., and Neuderfer, G.N., 2005, 

Assessing contaminant sensitivity of endangered and threatened aquatic species: Part I. acute toxicity of five chemicals: 

Archives of Environmental Contamination and Toxicology, 

v. 48, p. 143–154.

Gilmour, K.M., and Perry, S.F., 2009, Carbonic anhydrase and 

acid-base regulation in fish: Journal of Experimental Biology, v. 212, p. 1,647–1,661. 

Haswell, M.S., Randall, D.J., and Perry, S.F., 1980, Fish gill 

carbonic anhydrase: acid base regulation or salt transport?: 

American Journal of Physiology, v. 238, p. 240–245.

Hobe, H., Wood, C.M., and McMahon, B., 1984, Mechanisms 

of acid/base and ionoregulation in white suckers Catostomus commersoni in natural soft water: Journal of Comparative Physiology, v. 154, p. 35–46.

Hoke, R.A., Gala, W.R., Drake, J.B., Giesy, J.P., and Flegler, 

S., 1992, Bicarbonate as a potential confounding factor in 

cladoceran toxicity assessments of pore water from contaminated sediments: Canadian Journal of Fisheries and Aquatic 

Sciences, v. 49, p. 1,633–1,640.

Lasier, P.J., Winger, P.V., and Reinert, R.E., 1997, Toxicity 

of alkalinity to Hyalella azteca: Bulletin of Environmental 

Contamination and Toxicology, v. 59, p. 807–814.

Montana Department of Fish Wildlife and Parks, 2010, 

Fatmucket—Lampsilis siliquoidea, Montana Field Guide: 

Montana Natural Heritage Program, accessed February 16, 

2010, 2 p. at http://fieldguide.mt.gov/detail_IMBIV21280.

aspx.

Mount, D.R., and Gulley, D.D., 1992, Development of a salinity toxicity relationship to predict acute toxicity of saline 

waters to freshwater organisms: Chicago, Gas Research 

Institute, GRI–92/0301, 10 p.

Mount, D.R., Gulley, D.D., Hockett, J.R., Garrison, T.D., and 

Evans, J.M., 1997, Statistical models to predict the toxicity 

of major ions to C. dubia, Daphnia magna and Pimephales 

promelas (fathead minnows): Evironmnetal Toxicology and 

Chemistry, v. 16, p., 2009–2019. 

Nielsen, D.L., Brock, M.A., Rees, G.N., and Baldwin, D.S., 

2003, Effects of increasing salinity on freshwater ecosystems in Australia: Australian Journal of Botany, v. 51, 

p. 655–665.

Ostrand, K.G., and Wilde, G.R., 2004, Changes in prairie 

stream fish assemblages restricted to isolated streambed 

pools: Transactions of the American Fisheries Society, 

v. 133, p. 1,329–1,338.

Patz, M.J., Reddy, K.J., and Skinner, Q.D., 2004, Chemistry of 

coalbed methane discharge water interacting with semi-arid 

ephemeral stream channels: Journal of American Water 

Resources Association, v. 40, no. 5, p. 1,247–1,255. 

Perry, S.F., 1997, The chloride cell: structure and function in 

the gills of freshwater fishes: Annual Review of Physiology, 

v. 59, p. 325–347.

Rand, G.M., 1995, Fundamentals of Toxicology, 1: New York, 

Taylor and Francis, 125 p.

Randall, D J., and Tsui, T.J.N., 2006, Tribute to R.G. Boutilier—Acid-base transfer across fish gills: The Journal of 

Experimental Biology, v. 209, no. 1, p. 1179–1,184.

Sprague, J.B., 1973, The abc’s of pollutant bioassays using 

fish in Cairns, J., and Dickson, J.L., eds., Biological 

methods for the assessment of water quality: Philadelphia, 

American Society of Testing and Materials International, 

p. 6–30.

Stephan, C.E., Mount, D.I., Hansen, D.J., Gentile, J.R., Chapman, G.A., and Brungs, W.A., 1985, Guidelines for deriving 

numerical national water quality criteria for the protection 

of aquatic organisms and their uses: Duluth, U.S. Environmental Protection Agency, Office of Research and Development, EPA–PB85–227049, 106 p.

Sullivan, G. V., Fryer, J.N., and Perry, S.F., 1995, Immunolocalization of proton pumps (H+-ATPase) in pavement cells 

of rainbow trout: Journal of Experimental Biology, v. 198, 

no. 2, p. 2,619–2,629.

Tietge, J.E., Hockett, J.R., and Evans, J.M., 1997, Major ion 

toxicity of six produced waters to three freshwater species: 

Application of ion toxicity models and TIE procedures. 

Environmental Toxicology and Chemistry, v. 16, no. 10, 

p. 2,002–2,008.

U.S. Environmental Protection Agency, 2002, Methods for 

measuring the acute toxicity of effluents and receiving 

waters to freshwater and marine organisms, 5th edition: 

Washington, D. C., U.S. Environmental Protection Agency, 

Office of Water (4303T), EPA–821–R–02–012, 266 p.

U.S. Environmental Protection Agency, 2010, The Toxicity 

Relationship Analysis Program: Duluth, U.S. Environmental Protection Agency, Mid-Continent Ecology Division, 

21 p. accessed April 25, 2011, at http://www.epa.gov/med/

Prods_Pubs/trap.htm. 
38 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Wang, N., Ingersoll, C.G., Hardesty, D.K., Ivey, C.D., Kunz, 

J.L., May, T.W., Dwyer, F.J., Roberts, A.D., Augspurger, 

T. ,Kane, C.M., Neves, R.J., and Barnhart, M.C., 2007, 

Acute toxicity of copper, ammonia, chloride and chlorine to 

glochidea and juveniles of freshwater mussels (Unionidae): 

Evironmental Toxicology and Chemistry, v. 26, no. 2, 

p. 2,036–2,047.

Weber-Scannell, P.K., and Duffy, L., 2007, Effects of total 

dissolved solids on aquatic organisms: A review of literature 

and recommendation for salmonid species: American Journal of Environmental Science v. 3, no. 1, p. 1–6.

Wilson, J.M. and Laurent., P., 2002, Fish gill morphology; 

inside out: Journal of Experimental Zoology, v. 293, 

p. 192–213.

Wurts, W.A., 1998, Why can some fish live in fresh water, 

some in salt water, and some in both?: World Aquaculture, 

v. 29, no. 1, p. 65.
The Chronic Toxicity of Sodium 

Bicarbonate Defined Under Laboratory 

Conditions

By Aïda M. Farag and David D. Harper

Chapter 3 of

The Potential Effects of Sodium Bicarbonate, a Major Constituent of 

Produced Waters from Coalbed Natural Gas Production, on Aquatic 

Life

Edited by Aïda M. Farag and David D. Harper

Prepared in cooperation with Montana Fish, Wildlife, and Parks, U.S. Bureau of 

Land Management, and the U.S. Environmental Protection Agency

Scientific Investigations Report 2012–5008

U.S. Department of the Interior

U.S. Geological Survey
Contents

Introduction...................................................................................................................................................43

Methods.........................................................................................................................................................44

Fathead Minnow .................................................................................................................................44

White Sucker Experiment..................................................................................................................45

Ceriodaphnia Experiment ..................................................................................................................45

Freshwater Mussel Experiment .......................................................................................................49

Results ...........................................................................................................................................................50

Fathead Minnow .................................................................................................................................50

Summary Results Fathead Minnow.................................................................................................54

White Sucker.......................................................................................................................................54

Summary Results White Suckers.....................................................................................................55

Ceriodaphnia Experiment ..................................................................................................................55

Freshwater Mussel Experiment .......................................................................................................57

Discussion.....................................................................................................................................................57

References Cited..........................................................................................................................................59

Tables

3–1. Nominal exposure concentration of sodium bicarbonate, estimated number of eggs 

at the start of the experiment, number hatched and estimated percent hatch during 

chronic fathead minnow (Pimephales promelas), experiment 1 completed in reconstituted Tongue River water..........................................................................................................44

3–2. Mean temperature, dissolved oxygen, conductivity, pH, alkalinity, bicarbonate, calcium, magnesium, sodium , potassium, chloride, and sulfate during chronic toxicity 

experiment 1 with fathead minnow (Pimephales promelas) completed in reconstituted 

Tongue River water.....................................................................................................................46

3–3. Mean temperature, dissolved oxygen, conductivity, pH, alkalinity, bicarbonate, and 

hardness, during chronic toxicity experiment 2 with fathead minnow (Pimephales 

promelas) completed in reconstituted Tongue River water................................................47

3–4. Mean alkalinity expressed as calcium carbonate equivalent, bicarbonate, hardness, 

pH, conductivity, temperature, and dissolved oxygen during 7-day chronic toxicity 

experiment with fathead minnow (Pimephales promelas) less than 48-hours old..........47

3–5. Mean temperature, dissolved oxygen, conductivity, pH, alkalinity, bicarbonate, 

calcium, magnesium, sodium , potassium, chloride, and sulfate during chronic 

toxicity experiment with white suckers (Catostomus commersoni) completed in 

reconstituted Tongue River water............................................................................................48

3–6. Water chemistry measured during a 7-day experiment with Ceriodaphnia (Ceriodaphnia dubia) completed in moderately hard reconstituted water. Exposures presented are 

nominal concentrations of sodium bicarbonate....................................................................49

3–7. Water chemistry measured during a chronic static renewal experiment with 

fatmucket mussel (Lampsilis siliquoidea) completed in reconstituted Tongue River 

water.............................................................................................................................................49

3–8. Percent survival of fathead minnows (Pimephales promelas) completed in reconstituted Tongue River water during chronic experiments 1 and 2. Exposures presented 

are nominal concentrations of sodium bicarbonate.............................................................50
3–9. Mean lengths and weights of fathead minnows (Pimephales promelas) at day 30 or 

37 and day 60 of chronic experiments 1 and 2 completed in reconstituted Tongue River 

water.............................................................................................................................................51

3–10. Mean sodium-potassium adenosine triphosphatase, (N+/K+ ATPase), also known as 

the Na+/K+ pump, activity in micromoles adenosine diphosphate per milligram protein 

per hour in whole body fathead minnows (Pimephales promelas) at day 30 or 37 and 

day 60 during experiments 1 and 2 ..........................................................................................52

3–11. Mean whole body ions in fathead minnows (Pimephales promelas) during experiment 1 

completed in reconstituted Tongue River water with nominal concentrations of sodium 

bicarbonate..................................................................................................................................52

3–12. Mean whole body ions in milligrams per gram dry weight in fathead minnows (Pimephales promelas) on day 30 of chronic experiment 2 completed in reconstituted 

Tongue River water.....................................................................................................................53

3–13. Mean percent survival during a 7-day static renewal chronic exposure of fathead minnow (Pimephales promelas) completed in reconstituted Tongue River water................53

3–14. Mean lengths of fathead minnows (Pimephales promelas) at day 7 of a 7-day static 

renewal exposure completed in reconstituted Tongue River water..................................53

3–15. Mean percent survival of white suckers (Catostomus commersoni) during a 60-day 

(53-day post-hatch) chronic exposure completed in reconstituted Tongue River 

water.............................................................................................................................................54

3–16. Mean percent survival of white suckers (Catostomus commersoni) during a 60-day 

(53-day post-hatch) chronic exposure completed in reconstituted Tongue River 

water.............................................................................................................................................54

3–17. Mean whole-body ions in white suckers (Catostomus commersoni) on day 30 of a 

chronic experiment completed in reconstituted Tongue River water...............................55

3–18. Mean whole-body ions in white suckers (Catostomus commersoni) on day 53 of a 

chronic experiment completed in reconstituted Tongue River water...............................55

3–19. Survival and reproduction of Ceriodaphnia dubia exposed to nominal concentrations of 

milligrams sodium bicarbonate per liter (mg NaHCO3/L) in moderately hard reconstituted water...................................................................................................................................56

3–20. Survival of 10-day old newly transformed fatmucket mussels (Lampsilis siliquoidea) to 

chronic exposures completed in reconstituted Tongue River water.................................57

Introduction

As a result of surface discharge, ephemeral streams in the 

arid Powder River Basin may consist of 100 percent Coalbed 

Natural Gas (CBNG ) produced water effluent during certain 

seasons. Thus, aquatic life inhabiting newly created perennial stream habitats may be exposed continuously to undiluted effluent water throughout the year. Sodium bicarbonate 

(NaHCO3) is the principle salt in CBNG produced water 

discharged at many locations in the Powder River Basin, and 

individual measured concentrations of up to 3,000 milligrams 

sodium bicarbonate per liter (mg NaHCO3/L) have been 

documented at some locations (ALL Consulting, 2003; Patz 

and others, 2004). Whereas acutely toxic effects of NaHCO3

have been investigated (Mount and others 1997, Chapter 2 in 

this report), no adequate studies have been performed to assess 

the potential chronic effects of NaHCO3 exposure. The U.S. 

Environmental Protection Agency (USEPA) guidelines for the 

establishment of water-quality criteria for the protection of 

aquatic life suggest that a minimum of three chronic experiments be completed with one fish species, one invertebrate 

species, and one acutely sensitive freshwater species (Stephan 

and others, 1985). Specific species selected for chronic 

toxicity studies should be easily maintained in a laboratory 

facility for extended periods of time; should be identical to 

species used in acute toxicity experiments for comparisons 

of acute and chronic toxicity; and should be representative of 

species that are found in the region of concern (for example, 

Tongue and Powder River Basins of Montana and Wyoming) 

for regional applicability. However, the use of species with a 

range outside of the local basin could provide useful information for managers throughout the western United States and 

elsewhere that encounter elevated concentrations of NaHCO3. 

In addition to population level effects (for example, 

decreased survival), sublethal effects at the individual organism level may be evaluated to provide information about the 

mechanisms of toxicity related to elevated concentrations of 

NaHCO3. Sodium bicarbonate may disassociate in the water 

column into separate ions of sodium (Na+) and bicarbonate 

(HCO3

–

); two ions that play a key role in acid/base balance 

and concomitant ionoregulation in freshwater organisms. 

The ionoregulatory status of young fish can be assessed by 

measuring whole-body ions (Reid and MacDonald, 1988). 

Additionally, the enzyme sodium-potassium adenosine triphosphatase, (N+

/K+ ATPase), also known as the Na+/K+ pump, 

is responsible for Na+ movement across the basal membrane 

of gills and other organs into the blood (Marshall, 2002). The 

measurement of N+/K+ ATPase concentrations during NaHCO3

exposure could explain a mechanism by which a change in ion 

content within the animal takes place. Moreover, histological 

examinations may provide additional information related to 

the ionoregulatory status of aquatic organisms. For example, 

increases in the number of mitochondrial rich cells has been 

observed when fish are exposed to brackish water, and it is 

these types of cells that are considered responsible for ion and 

acid/base regulation in freshwater organisms (Wilson and Laurent, 2002). Studies designed to include measurements of the 

above endpoints may provide explanations of the mechanisms 

of action that affect survival or growth observed and would 

support the definition of an effect concentration. 

The present study was initiated to assess the chronic 

toxicity of NaHCO3 to aquatic organisms. The objectives of 

the study were to (1) define the chronic effects of NaHCO3, 

a principle component of CBNG production, on aquatic life; 

(2) to define sublethal effects at the individual organism level 

to explain the mechanisms of toxicity during chronic exposure; and (3) to define the effect concentrations in relation to 

concentrations measured in the field so that managers could 

be more aware of the extent that NaHCO3 may or may not 

currently affect aquatic life in the Tongue and Powder River 

Basins of Montana and Wyoming. Multiple aquatic species were used in the assessment of the chronic toxicity of 

NaHCO3, which allows for sensitivity comparisons among 

species and improves the level of confidence in observed 

responses. The exposure of representative species from different phyla, such as fish and aquatic invertebrates, further 

enhances the confidence in experimental results by expanding 

the possible range of potential effects. Further, the inclusion of 

an experimental species that is known to be acutely sensitive 

to NaHCO3 defines concentrations of NaHCO3 that would protect some of the most sensitive species, a goal of established 

water-quality criteria.

The Chronic Toxicity of Sodium Bicarbonate Defined 

Under Laboratory Conditions

By Aïda M. Farag and David D. Harper
44 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Methods

Chronic toxicity experiments were completed with four 

aquatic species to meet the strategy for the selection of test 

species described above. Three chronic experiments were 

completed with fathead minnows (Pimephales promelas, 

FHM), one with white suckers (Catostomus commersoni), one 

with Ceriodaphnia dubia, and one with fatmuckets (a freshwater mussel, Lampsilis siliquoidea). All of these experiments 

could be paired with the acute toxicity experiments completed 

in the present study for comparison of effect concentrations 

and to derive acute/chronic ratios as suggested by the USEPA 

(Stephan and others, 1985; see chapter 6). All four species 

were maintained in the laboratory for extended periods of 

time, though white sucker had to be spawned in the field. The 

four experimental species are resident in the Tongue and Powder River Basins, and are found throughout the United States. 

Sublethal parameters were monitored during two of the FHM 

experiments, and to a lesser extent, during the white sucker 

experiment. 

The FHM (during two of the three experiments) and 

white suckers were exposed to NaHCO3 in a flow-through 

diluter system (Mount and Brungs, 1967). The diluter system 

contained 12, 4-L glass exposure chambers that were replicated 3–4 times and supplied with 250 milliliters (mL) of 

reconstituted Tongue River (as prepared in chapter 2) water 

every 15 minutes to obtain the desired exposure concentrations. Complete water replacement in each chamber occurred 

approximately every 4 hours (h).

Fathead Minnow 

Two flow-through chronic toxicity experiments were 

completed with FHMs exposed to NaHCO3. The FHMs were 

exposed to concentrations that ranged from 500 to 1,400 milligrams sodium bicarbonate per liter (mg NaHCO3

/L) in experiment 1 and from 300 to 625 mg NaHCO3

/L in experiment 2. 

Also, fish were held in a control treatment of reconstituted 

Tongue River water without added NaHCO3. Both experiments were terminated at 60-days-post-hatch (referred to from 

here as dph), a 65-day (d) total exposure. 

Before initiating the experiment, the FHM eggs were 

carefully picked from spawning tiles and combined into a 2-L, 

acid-washed, plastic container of reconstituted Tongue River 

water. Seventy eggs from this container were randomly pipetted into each replicate experimental chamber. This method 

was used to ensure that each replicated exposure chamber contained eggs from several different females. Steps were taken 

to minimize inaccuracies of egg counts (for example, manual 

counters used to physically document eggs as they entered the 

chamber, multiple counts performed by separate researchers 

on an identical chamber); however, the small size of individual 

FHM eggs suggests that an exact number of eggs might be 

difficult to assess. Therefore, the calculated percent hatch 

for control and experimental concentrations was an estimate. 

Though not tested statistically because these were estimates, 

percentage hatch of the eggs at 1,400 mg NaHCO3/L were 

smaller compared to the controls during experiment 1 

(table 3–1). Percent hatch ranged from 44 to 78 percent. Fish 

were fed a ration of 10 cultured brine shrimp/fish twice per 

day. This ration was doubled as fish grew.

Survival, conductivity, dissolved oxygen (DO), and 

temperature were monitored daily in each replicate during the 

experiments. The pH, hardness, and alkalinity were measured 

weekly from alternate replicates so that all were sampled 

at least three times throughout the experiment. Also, water 

samples were collected weekly (from systematically selected 

replicates with a random starting point) in 150-mL certified 

clean I–Chem® vials and shipped to Red Buttes Environmental 

Laboratory in Laramie, Wyoming for analyses of major ions, 

including calcium (Ca), potassium (K), sodium (Na), and 

magnesium (Mg), along with Quality Assurance and Quality 

Control (QA/QC) samples that were measured with an atomic 

absorption spectrophotometer (table 3–2, 3–3). The measured 

concentrations of mg NaHCO3/L were within ±10 percent of 

the nominal concentration and the nominal concentrations 

will be used throughout this report. There was a drop in the 

Ca concentration measured in the water as the amount of mg 

NaHCO3 /L increased. Calcium in the water decreased 21 

percent in the 500 mg NaHCO3/L treatment to 

53 percent in the 1,400 mg NaHCO3/L compared to the control. In fact, white particulate matter was observed on aquaria 

and metering devices used for the larger concentrations during 

the experiment.

At 37-days post median hatch (30-days for experiment 

2), about 50 percent of the fish remaining in each replicate 

exposure chamber were randomly sampled. The lengths and 

weights of these fish were measured and then the whole 

fish were placed in vials with Davidson’s solution added to 

10 times the volume of tissue, for histological examinations. 

At 60 days of exposure, the remaining fish were collected, 

Table 3–1. Nominal exposure concentration of sodium 

bicarbonate, estimated number of eggs at the start of the 

experiment, number hatched and estimated percent hatch 

during chronic fathead minnow (Pimephales promelas), 

experiment 1 completed in reconstituted Tongue River water 

(control). Exposures presented are nominal concentrations of 

sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter]

Exposure 

(mg NaHCO3/L)

Estimated 

number of eggs 

added

Number of 

eggs hatched

Esimated 

percent 

hatched

Control 280 175 62.5

500 280 219 78.2

800 280 194 69.3

1,100 280 197 70.4

1,400 280 123 43.9
Methods 45

weighed, measured for length, and placed in appropriate vials 

for histological analyses. 

Samples for histological analyses were shipped to the 

U.S. Fish and Wildlife Service in Bozeman, Montana, not 

shown, and examined by a procedure similar to Farag and others (2003). 

All anion/cation analyses were accompanied by standard 

QA/QC analyses and were all within acceptable limits. The 

anion/cation charge balance ranged from 0.35 percent to 

4.67 percent difference and the percent recovery of Na in each 

treatment ranged from 94 percent to 104 percent. 

A 7-d FHM static-renewal chronic experiment also was 

conducted with reconstituted Tongue River water (water 

reconstituted with same procedure described in chapter 2). 

Fathead minnows less than (<) 48-h old were received from 

Aquatic BioSystems (Ft. Collins, Colorado) and acclimated 

to increasing concentrations of reconstituted Tongue River 

(control) water for a 1-h time period. Five hundred mL of control and concentrations of 300 to 2,000 mg NaHCO3

/L water 

were added to 1-L chambers and held in a water bath that was 

maintained at 25 degrees Celsius (°C) ±1. The experiment was 

initiated by adding 10 fish to each chamber in a nonsystematic 

order. Fish were fed 200 microliters (μL) of a brine shrimp 

suspension twice daily.

Survival was monitored daily. The experimental waters 

were replaced daily and conductivity, DO, and temperature 

were monitored in a composite of the discarded replicate 

waters. The pH, hardness, and alkalinity were measured from 

alternate replicates so that all were sampled at least three 

times throughout the 7-d experiment. A variable pH alkalinity 

measurement was performed where the sample was titrated 

to pH 8.3 and pH 4.5 to provide HCO3

-

 mg/L measurements 

in addition to total alkalinity as milligrams calcium carbonate 

per liter (mg CaCO3/L) (table 3–4; American Public Health 

Association, 1975). 

White Sucker Experiment 

The white suckers for the experiment were obtained from 

a private pond near Dillon, Montana, not shown. The artificial 

pond receives water from the Beaverhead River, not shown, 

by way of an irrigation ditch. The pond water quality consisted 

of a pH of 8.69, total alkalinity of 136 mg/L as CaCO3 and a 

specific conductance of 416 microsiemens per centimeter (μS/

cm). Sexually mature white suckers were captured in trap nets 

that were set overnight on multiple occasions. Eggs from three 

females were fertilized with sperm from seven males in reconstituted Tongue River water. The fertilized eggs were treated in 

a solution of 4.7 mL of iodine dissolved in 2 L of reconstituted 

Tongue River water for 30 minutes. The eggs were then rinsed 

and transported to the U.S. Geological Survey, Columbia 

Environmental Research Center, Jackson Field Research Station (from here defined as Jackson Field Research Station) in a 

plastic cooler filled with reconstituted Tongue River water. 

Upon arrival at the Jackson Field Research Station the 

eggs were acclimated from 16°C to 18°C and approximately 

50–60 eggs were placed in each of the 4-L chambers in the 

flow-through diluter system described in the second paragraph 

of the methods section above. The white suckers were exposed 

for 63-d (51-dph) to 450, 800, and 1,400 mg NaHCO3/L and a 

control with no added NaHCO3. Each exposure concentration 

was replicated three times and randomly assigned across the 

12 glass aquaria in the diluter. After 30 days of exposure, fish 

were collected from each replicate experimental chamber and 

the number of surviving fish was reduced to <15 fish per 4-L 

replicate chamber. The sampled fish were anesthetized with 

ice, measured for length and weight, and prepared for histological and whole-body ion analyses. Fish were fed a commercial diet at 8 percent body weight per day that was split among 

four rations.

Conductivity, DO, and temperature were monitored daily 

in each replicate. Alkalinity, hardness, and pH were measured 

in a sample collected from each batch of prepared simulated 

Tongue River water and were measured two times in each 

concentration. Water samples were collected six times from 

each concentration for analyses of total NaHCO3. This procedure and chemistry sampling was similar to that used during 

the fathead minnow experiments (table 3–5). The measured 

concentrations of mg NaHCO3/L were within ±13 percent of 

the nominal concentration and the nominal concentrations will 

be used throughout the remaining text.

Ceriodaphina Experiment 

Ceriodaphnia dubia were exposed in a static-renewal 

system to various concentrations of NaHCO3 until 60 percent of the surviving individuals produced three broods of 

offspring. The experiments were completed according to 

the USEPA protocol EPA/600/4–91/002 (U.S. Environmental Protection Agency, 1994). Brood stock C. dubia were 

obtained from Aquatic BioSystems, Fort Collins, Colorado. 

Neonates from individual brood stock cultures were used in 

the experiments. Individuals were cultured in 30-mL plastic 

cups containing 15 mL of reconstituted, moderately hard water 

maintained at 25°C. Individuals were fed a combination of 

yeast, CEROPHYLL®, and Trout chow (YCT), and the unicellular green algae, Selenastrum capricornutum daily. Neonates 

from individual cultures that produced at least 20 offspring 

were used for the experiments.

Experimental concentrations of NaHCO3 were obtained 

by adding the appropriate quantities of NaHCO3 to reconstituted moderately hard culture water. Acute toxicity rangefinding experiments were completed to determine the 

concentrations used in the chronic toxicity experiment. The 

nominal concentrations used were 500, 700, 900, 1,200, and 

1,500 mg NaHCO3/L (table 3–6) plus a control of moderately 

hard reconstituted water with no added NaHCO3. Ten C. dubia 

were placed individually into replicate 30-mL plastic exposure 

containers with 15 mL of experimental water. The exposure 
46 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life Table 3–2. Mean temperature, dissolved oxygen, conductivity, pH, alkalinity, bicarbonate, calcium, magnesium, sodium , potassium, chloride, and sulfate during chronic toxicity experiment 1 with fathead minnow (Pimephales promelas) completed in reconstituted Tongue River water (control). Exposures concentrations presented are nominal concentrations of sodium bicarbonate. [NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; μS/cm, microsiemens per centimeter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, chloride; NO3-, nitrate; SO42-, sulfate; ±, standard error of the mean; ( ), sample size; --, sample size less than 3] Exposure (mg NaHCO3/L) Temperature (°Celsius) Dissolved oxygen (mg/L) Conductivity (μS/cm) pH Alkalinity (CaCO3, in mg/L) Bicar- bonate (mg/L) Hardness (mg/L) Ion concentration (mg/L) Ca2+ Mg2+ Na+ K Cl- NO3

- SO42-

Control 24.2 6.1 651 8.47 203 248 261 46.4 34.3 48.5 0.76 1.77 1.65 150

±0.1 ±0.05 ±1 ±0.02 ±3 ±4 ±61 ±1.95 ±0.34 ±1.3 ±0.19 ±0 ±0.36 ±1

(4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (3) (3) (3) (3)

500 24.5 6.1 952 8.6 391 477 238 36.77 34 136 .7 1.95 2.17 149

±0.1 ±0.06 ±18 ±0.03 ±5 ±6 ±4 ±1.15 ±0.63 ±5.9 ±0.11 -- -- --

(4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (3) (2) (2) (2)

800 24.4 6.1 1,180 8.63 539 657 228 33.8 34.1 207 .76 1.42 1.24 150

±0.32 ±0.5 ±7 ±0.04 ±7 ±9 ±3 ±2.25 ±0.12 ±1.1 -- -- -- --

(36) (4) (4) (4) (4) (4) (4) (4) (4) (4) (2) (1) (1) (1)

1,100 24.4 6.1 1,430 8.71 696 849 208 24.2 34 292 .76 .05 .03 1.54

±0 ±0.02 ±18 ±0.04 ±8 ±10 ±5 ±1.71 ±0 ±1.9 -- ±0.01 -- ±0.04

(4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (2) (3) (2) (3)

1,400 24.1 6.05 1,720 8.72 892 1,090 205 21.6 34.3 382 .76 .05 .03 1.55

±0.3 ±0.02 ±16 ±0.12 ±8 ±10 ±3 ±2.5 ±0.2 ±4.8 -- -- -- --

(4) (4) (4) (4) (4) (4) (3) (4) (4) (4) (1) (2) (2) (2)
Methods 47

Table 3–3. Mean temperature, dissolved oxygen, conductivity, pH, alkalinity, bicarbonate, and 

hardness, during chronic toxicity experiment 2 with fathead minnow (Pimephales promelas) completed in 

reconstituted Tongue River water (control). Exposures presented are nominal concentrations of sodium 

bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; μS/cm, microsiemens per centimeter; CaCO3, calcium carbonate; 

±, standard error of the mean; ( ), number of samples]

Exposure 

(mg 

NaHCO3

/L)

Temperature 

(°Celsius)

Dissolved 

oxygen

(mg/L)

Conductivity 

(μS/cm) pH

Alkalinity

(CaCO3

, in 

mg/L)

Bicarbonate 

(mg/L)

Hardness 

(mg/L)

Control 25.0 6.35 652 8.51 200 316 259

±0.2 ±0.34 ±5 ±0.12 ±6.2 ±7.1 ±5.8

(61) (61) (61) (4) (4) (4) (4)

300 25.0 6.49 773 8.51 277 311 255

±0.2 ±0.30 ±8 ±0.09 ±14 ±8.5 ±7

(61) (61) (61) (4) (4) (4) (4)

400 25.0 6.36 847 8.53 320 306 251

±0.2 ±0.30 ±45 ±0.06 ±13 ±5 ±4

(61) (61) (61) (4) (4) (4) (4)

625 25.0 6.52 1,040 8.55 437 295 242

±0.3 ±0.30 ±11 ±0.05 ±19 ±7.3 ±6

(61) (61) (61) (4) (4) (4) (4)

Table 3–4. Mean alkalinity expressed as calcium carbonate equivalent, bicarbonate, hardness, pH, 

conductivity, temperature, and dissolved oxygen during 7-day chronic toxicity experiment with fathead 

minnows (Pimephales promelas) less than 48-hours old. Completed in reconstituted Tongue River water 

(control). Exposures presented are nominal concentrations of sodium bicarbonate. Sample size is three for 

each.

[NaHCO3, sodium bicarbonate; CaCO3, calcium carbonate; μS/cm, microsiemens per centimeter; ±, standard error of the mean]

Exposure 

(mg 

NaHCO3

/L)

Alkalinity

(CaCO3

, in 

mg/L)

Bicarbonate 

(mg/L)

Hardness 

(mg/L) pH Conductivity(μS/cm)

Temperature 

(°Celsius)

Dissolved 

oxygen

(mg/L)

Control 193 241 253 8.4 631 24.5 5.7

±7 ±18 ±8.1 ±0.01 ±5.2 ±0.1 ±0.2

300 273 335 260 8.5 754 24.5 5.7

±3 ±3 ±4 ±.02 ±4 ±.07 ±.1

500 378 446 233 8.6 914 24.5 5.8

±2 ±8 ±5.8 ±.003 ±14.9 ±.09 ±.1

625 445 519 226 8.6 1,030 24.5 5.5

±0.8 ±8 ±12.5 ±.02 ±3.6 ±.2 ±.2

800 544 636 205 8.6 1,180 24.6 5.63

±4 ±8 ±14.5 ±.07 ±2.8 ±.02 ±.3

1,100 685 792 176 8.6 1,430 24.6 5.6

±7 ±3 ±6.9 ±.05 ±44.5 ±.01 ±.1

1,400 867 985 165 8.6 1,700 24.6 5.6

±67 ±5 ±7 ±.02 ±2.3 ±.07 ±.09

2,000 1,160 1,349 148 8.8 2,200 24.6 5.6

±3 ±8 ±2 ±.01 ±.9 ±.03 ±.1
48 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life Table 3–5. Mean temperature, dissolved oxygen, conductivity, pH, alkalinity, bicarbonate, calcium, magnesium, sodium , potassium, chloride, and sulfate during chronic toxicity experiment with white suckers (Catostomus commersoni) completed in reconstituted Tongue River water (control). Exposures presented are nominal concentrations of sodium bicarbonate. [NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; μS/cm, microsiemens per centimeter; CaCO3, calcium carbonate; Ca2+, calcium; Mg2+, magnesium; Na+, sodium; K, potassium; Cl-, chloride; SO42-, sulfate; N, sample size; ±, standard error of the mean; --, sample size less than 1, standard error not calculated; >, greater than] Exposure (mg NaHCO3/L) Temperature (°Celsius) Dissolved oxygen (mg/L) Conductivity (μS/cm) pH Alkalinity (CaCO3, in mg/L) bicar- bonate (mg/L) Hardness (mg/L) Ion concentration (mg/L) Ca2+ Mg2+ Na+ K Cl- NO3

- SO42--

Control 16.5 7.74 681 8.3 192 234 255 45.3 35.8 48.7 2.18 1.84 1.4 142.14

±1.02 ±0.28 ±17 ±.06 ±3 ±4 ±7.07 ±1.1 ±1.24 ±1.49 ±0.42 -- -- --

N>50 N>50 N>50 N=2 N=2 N=2 N=2 N=13 N=13 N=13 N=4 N=1 N=1 N=1

450 16 7.77 962 8.48 354 430 252 44.6 35.9 133 2.07 1.79 1.44 140

±1.2 ±.26 ±54 ±.01 ±29 ±35 ±0 ±1.12 ±1.8 ±15.63 ±0.18 -- -- --

N>50 N>50 N>50 N=2 N=2 -2 N=2 N=13 N=13 N=13 N=2 N=1 N=1 N=1

800 16.5 7.72 1,240 8.51 527 643 247 42.4 35.9 203 2.22 1.75 1.37 140

±1.1 ±.46 ±181 ±.01 33 40 1 1.8 1.18 15.2 0.11 -- -- --

N>50 N>50 N>50 N=2 N=2 N=2 N=2 N=13 N=13 N=13 N=2 N=1 N=1 N=1

1,400 16 7.66 1,820 8.55 920 1,120 238 34.6 35.3 388 2.53 1.68 1.37 139

±1 ±.44 ±159 ±.12 85 104 3 3.34 1.51 14.45 0.21 -- -- --

N>50 N>50 N>50 N=2 N=2 N=2 N=2 N=13 N=13 N=13 N=2 N=1 N=1 N=1
Methods 49

containers were held in a water bath at 25±1°C. The experiment lasted for 7 to 8 days and the C. dubia were fed YCT and 

algal suspension daily. The water in each replicate experimental container was replaced daily, and the number of surviving 

adult C. dubia and the number of neonates produced were 

recorded daily.

The 3 criteria for a successful experiment based on 

responses observed in the control treatment were; 80 percent 

or more survival of females, an average of 15 or more young 

produced per surviving female, and the production of 3 broods 

by 60 percent of surviving control organisms. The exposure 

period could be extended to 8 days to allow for individuals to 

produce three broods.

Freshwater Mussel Experiment

Advances during the last 10 years in culture and testing methods development with freshwater mussels led to the 

establishment of American Society of Testing and Materials 

International guidelines for conducting acute and chronic 

experiments with freshwater fatmucket mussel, Lampsilis siliquoidea (American Society of Testing and Materials 

International 2006; Wang and others, 2007). Newly transformed juvenile fatmucket mussels were obtained from 

Missouri State University, Springfield, Missouri, USA. The 

juvenile mussels were held in a water bath that contained 

well water maintained at 20°C (range was 20.0–22.8°C) at the 

Jackson Field Research Station. Before starting the experiment, mussels were fed a mixture of commercially prepared 

algae and shellfish diets during a 48-h acclimatization period. 

Five mussels were randomly placed into each of four replicate 

30-mL beakers per exposure concentration. The mussels were 

exposed to 500, 750, 1,250, 1,500, and 2,000 mg NaHCO3/L 

and a control of reconstituted Tongue River water in a 10-d 

static renewal experiment according to the procedures 

described by Wang and others (2007) and American Society 

of Testing and Materials International (2006). The mussels 

were fed twice daily during the experiment. Water from each 

concentration was sampled at the beginning and end of the 

experiment and analyzed as described above (table 3–7). 

The Spearman-Karber method was used to calculate 

LC50s at 96-h and 37-d with TOXSTAT 3.4. Significant differences for various endpoints for the 60-d FHM and white 

sucker experiments were defined by a one-way ANOVA 

Table 3–6. Water chemistry measured during a 7-day experiment with Ceriodaphnia

(Ceriodaphnia dubia) completed in moderately hard reconstituted water (control). Exposures 

presented are nominal concentrations of sodium bicarbonate. 

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; K, potassium; CaCO3, calcium carbonate; Ca2+, 

calcium; Mg2+, magnesium; Na+, sodium]

Exposure 

(mg 

NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L)

Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Sulfate

Control 76 93 15 9 22 8 96

500 319 345 15 13 121 3 97

700 410 446 15 13 182 3 97

900 546 579 14 13 216 3 99

1,200 721 750 15 12 339 2 97

1,500 911 938 14 12 390 3 100

Table 3–7. Water chemistry measured during a chronic static renewal experiment with fatmucket mussel (Lampsilis siliquoidea) 

completed in reconstituted Tongue River water (control). Exposures presented are nominal concentrations of sodium bicarbonate. 

Carbonate, bicarbonate, pH, calcium, magnesium, sodium, potassium, chloride, nitrate, and sulfate provided. Sample size equals 2.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; Ca2+, calcium, Mg2+, magnesium,; Na+, sodium; K, potassium; Cl-, chloride; NO3-, nitrate; SO4

2-, 

sulfate]

Exposure 

(mg 

NaHCO3/L)

Alkalinity

(CaCO3, in 

mg/L)

Carbonate

(CaCO3, in 

mg/L)

Bicarbonate

(mg/L) pH Ion concentration (mg/L)

Ca2+ Mg2+ Na+ K Cl- NO3- SO4

2--

Control 204 11 235 8.72 48 34 46 2.2 2 0.3 150

500 367 26 416 8.62 37 34 129 2.2 2 .2 148

750 511 1 623 8.59 22 33 189 2.2 1.4 .5 152

1,000 672 8 810 8.76 20 33 255 2.2 1 .5 151

1,250 810 31 951 8.69 22 33 318 2 1.2 .5 152

1,500 934 33 1,099 8.84 16 33 367 2.1 1.2 .4 150
50 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

followed by either a Dunnett’s post hoc means comparison for 

survival or Tukey means comparison for N+/K+ ATPase and 

whole-body ion concentrations. Data analyses were performed 

according to the methodology described in U.S. Environmental Protection Agency protocol EPA/600/4–91/002 (1994). 

The Toxicity Relationship Analysis Program (TRAP) (U.S. 

Environmental Protection Agency, 2010) was used to calculate 

the Inhibition Concentration (IC) that affects 20 percent of 

the sample population (IC20) based on mortality. The Linear 

Interpolation Method (Norberg-King 1993) was used to define 

IC20 based on growth or reproduction. Survival data for the 

7-d experiment were analyzed with the TRAP (U.S. Environmental Protection Agency, 2010) to determine the LC50 and 

the 95-percent Confidence Interval (CI). The IC20 calculated 

as mg NaHCO3/L were based on measured rather than nominal 

concentrations, except for the 7-d FHM experiment. The Na 

measurements for the 7-d FHM experiment were not available 

at the time of printing. Many of the IC20 also are reported as 

mg HCO3

-

/L and total alkalinity (expressed as mg CaCO3/L). 

The Trimmed Spearman-Karber Program was used to calculate a 10-d effect concentration on 50 percent of the sample 

population (EC50). 

Results

Fathead Minnow

A 60-d LC50 could not be calculated because of the 

elevated mortality that occurred in the 800, 1,100, and 1,400 

mg NaHCO3/L exposure concentrations by day 37. The FHM 

96-h [± standard deviation (SD)] and 37-d LC50s were 1002 + 

94 mg NaHCO3/L and 877 + 71 mg NaHCO3/L, respectively. 

During experiment 1, FHM survival decreased significantly at 

concentrations >500 mg NaHCO3/L at day 37 (table 3–8). In 

experiment 2, FHM survival decreased significantly in the 625 

mg NaHCO3/L concentration at day 30 (table 3–8). It should 

be noted that survival in the control from 0–30 days was 68 

percent. This is 2 percent below the guideline of 70 percent for 

a successful early lifestage experiment. Freshly fertilized eggs 

were used to start this experiment and the mortalities observed 

in the control happened on days 4–6. Significant mortalities 

were observed in 500 mg NaHCO3/L during experiment 1 

and in 625 mg NaHCO3/L during experiment 2. Because the 

observed effects bracket those observed during experiment 1 

when survival in the control was 89 percent, experiment 2 data 

continued to be used for interpretation. However, an IC20 for 

FHM cumulative 37-d survival was 462 mg NaHCO3/L with a 

95-percent CI of 278–645 mg NaHCO3/L and was calculated 

with data from experiment 1 only. This also can be expressed 

as 368 mg HCO3

-

/L (95-percent CI, 239–497 HCO3

-

/L), and 

total alkalinity expressed as 302 mg CaCO3/L (95-percent CI, 

196–408 CaCO3/L) An IC20 based on survival was not calculated for experiment 2.

No significant differences were observed among treatment groups for weights and lengths (table 3–9). Because a 

T-test revealed no significant difference between lengths and 

weights of control fish at day 37 from experiment 1 and day 

30 from experiment 2, the data were combined for a pooled 

reference. Although no significant difference for growth was 

observed as a result of exposure, a significant increase in 

length and weight was observed at day 60 for the pooled reference when compared to those at days 30 and 37, and these reference data indicate the fish grew appropriately for the course 

of the experiments. An IC20 could not be calculated based on 

growth for FHM from experiment 1 and 2. 

Significant decreases in Na/K ATPase were observed 

during the FHM experiment 1. At day 37, exposures to 

≥625 mg NaHCO3/L resulted in significant Na/K ATPase 

decreases compared to the controls (table 3–10). The decreases 

were not completely concentration dependent because the 

800 mg NaHCO3/L resulted in less of a decrease than the 

625 mg NaHCO3/L exposure. On day 60, all the NaHCO3

treatments were associated with a significant decrease in Na/K 

ATPase and the amount of decrease appeared to be concentration dependent. It should be noted that the sample number 

N=4 for all treatments except the 625 mg NaHCO3/L, where 

N=2. Therefore, data were analyzed additionally with the 

Bonferroni test and the results were similar. However, the 

reader is cautioned about the small sample size for the 625 mg 

NaHCO3/L treatment.

Table 3–8. Percent survival of fathead minnows (Pimephales promelas) completed in reconstituted Tongue River water (control) 

during chronic experiments 1 and 2. Exposures presented are nominal concentrations of sodium bicarbonate. Note: egg mortality 

from “estimated percent hatch” reported in table 3–1 is not included in the percent survival calculations.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; +, sacrificed concentrations at day 37; --, no concentration exposed or data gathered]

Fathead minnow experiment 1: percent survival Fathead minnow experiment 2: percent survival

Exposure 

(mg NaHCO3/L) 96-hours 0 to 37-days 37 to 60 days Exposure (mg NaHCO3/L) 96-hours 0 to 30-days 30 to 60 days

Control 94 96 96 Control 90 68 99

500 83 164 93 300 93 35 100

800 55 133 + 400 90 57 100

1,100 40 121 + 625 60 130 100

1,400 8 12 + -- -- -- --

1

Significant difference at α = 0.05.
Results 51

There were significant increases in whole-body Ca 

and Mg during both FHM experiments (tables 3–11, 3–12). 

Whole-body Ca was elevated in FHM exposed to 500 and 

625 mg NaHCO3/L treatments. Although not significant, a 

trend of elevated Ca was observed in the 800 and 1,100 mg 

NaHCO3/L treatments. Also, whole-body Mg was elevated 

in fish from the 625 mg NaHCO3/L on days 30 and 60, and 

whole-body Na was elevated in fish exposed to 1,100 mg 

NaHCO3/L. The increase in whole-body Ca and Mg may be 

associated with the noted drop in measured Ca in the water 

column (table 3–2). 

It should be noted that, as was the case with Na/K ATPase 

measurements, N=2 for the 625 mg NaHCO3/L treatment. The 

Bonferroni test was used again; however, the repeatability of 

these trends lends confidence to the statistical interpretation 

regardless of the small N for this one treatment.

Rodlet cells were found closely associated with cranial 

meninges in all groups of fish and appeared to increase with 

concentrations or the number of days of exposure. Similarly, 

focal degeneration in ovarian tissue appeared to be more common in fish from the greater NaHCO3

 exposure concentrations, 

but no degenerative changes of this type were observed in fish 

from the control and 500 mg NaHCO3/L treatments.

Gill lesions (epithelial hypertrophy, edema, mucus and 

necrosis) increased in severity with NaHCO3 concentration or 

the number of days of exposure. For example, at 30 d of exposure, fish from the control exhibited no gill necrosis, whereas 

fish from the 1,100 mg NaHCO3/L exposure experienced moderate changes and contained focal areas of necrosis in the gills. 

At 60 d, large necrotic lesions were observed in the gills of 

fish exposed to the control and 500 mg NaHCO3/L treatment, 

but were more numerous in the 500 mg/L NaHCO3 treatment. 

Changes were not observed in the kidney at day 30, but at day 

60 the numbers of regenerating tubules were slightly greater in 

fish exposed to the 500 mg NaHCO3/L treatment compared to 

those in the controls. There were no notations about mitochondrial rich cells during the histological examinations and 

the presence of necrotic cells in the gills may have precluded 

observations of these types of cells. Because swelling was 

present, the small size of these tissues also may preclude the 

observation of mitochondrial rich cells. 

For additional comparisons, a third experiment, a 7-d 

FHM experiment was included. This experiment was not initiated with eggs, but instead was initiated with <2-dph FHM. 

Survival of FHM during the 7-d static renewal experiment was 

reduced significantly at concentrations >1,100 mg NaHCO3/L 

compared to the control (table 3–13). The 7-d LC50 is 1,857 

mg NaHCO3/L (95-percent CI, 1,507–2,108 mg NaHCO3/L). 

An IC20 based on survival at 7-d was calculated as 1,190 mg 

NaHCO3/L (95-percent CI, 536–643 mg NaHCO3/L). The 

IC20 also may be expressed as 903 mg HCO3

-

/L (95-percent CI, 803–1,003 mg NaHCO3

/L) and total alkalinity 

expressed as 780 mg CaCO3/L (95-percent CI, 693–866 mg 

NaHCO3/L). Weights of FHM were reduced significantly at 

>625 mg NaHCO3/L compared to the control (table 3–14). An 

IC20 based on growth was calculated as 591 mg NaHCO3/L 

(95-percent CI, 536–643 mg NaHCO3/L). This can also be 

expressed as 499 mg/L HCO3

-

/L (95-percent CI, 467–531) or 

424 (95-percent CI, 397–454) total alkalinity expressed as mg 

CaCO3/L.

Table 3–9. Mean lengths and weights of fathead minnows (Pimephales promelas) at day 30 or 37 and day 60 of 

chronic experiments 1 and 2 completed in reconstituted Tongue River water (control 1 and control 2). Exposures 

presented are nominal concentrations sodium bicarbonate. Sample size (N) equals four unless indicated otherwise.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; mm, millimeter; mg, milligram; ( ), standard deviation; NS, no sample; --, no 

calculation of standard deviation]

Exposure 

(mg NaHCO3/L)

Length (mm)

day 30 or 37

Weight (mg)

day 30 or 37

Length (mm)

day 60

Weight (mg)

day 60

Control 1 14.93 0.03 25.75 0.18

(.7) (.01) (1.13) (.06)

Control 2 15.2 .03 25.81 .16

(.37) 0 (.7) (.01)

300 15.88, N=3 .04, N=3 28.34 .22

(1) (.01) (3.02) (.07)

400 15.44, N=3 .03 26.2 .18

(0.13) 0 (1.45) (.03)

500 15.44 .04 25.45 .19

(2.28) (.02) (2.9) (.06)

625 15.05, N=2 .03, N=2 24.68, N=2 .16, N=2

(.19) 0 (.11) (.01)

800 15.26 .04 NS NS

(1.68) (.02) -- --

1,100 15.32 .03 NS NS

(1.49) (.01) -- --
52 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Table 3–10. Mean sodium-potassium adenosine triphosphatase, (N+/K+ ATPase), also known as the 

Na+/K+ pump, activity in micromoles adenosine diphosphate per milligram protein per hour in whole 

body fathead minnows (Pimephales promelas) at day 30 or 37 and day 60 during experiments 1 and 2. 

Completed in reconstituted Tongue River water (control) with nominal concentrations of bicarbonate. 

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; μmoles ADP/mg protein/hr, micromoles adenosine diphosphate per milligram protein per hour; ( ), standard deviation; NS, no sample; --, no calculation of standard deviation]

Exposure 

(mg NaHCO3/L) Sample size

Activity day 30 or 37

(μmoles ADP/mg 

protein/hr)

Sample size

Activity day 60

(μmoles ADP/mg 

protein/hr)

Control 8 13.63a 8 22.08a

(.67) (.45)

300 3 12.49a 4 19.32b

(1.13) (.18)

400 3 10.40a 4 17.90b,c

(0.49) (.29)

500 4 11.59a 4 16.22c,d

(.96) (.42)

625 2 4.80b 2 12.26d

(.66) (.59)

800 4 7.37c -- NS

(.80) -- --

1,100 4 3.00b -- NS

(.42) -- --

a,b,cDifferent superscript letters denote significant differences (α = 0.05).

Table 3–11. Mean whole body ions in fathead minnows (Pimephales promelas) during 

experiment 1 completed in reconstituted Tongue River water (control) with nominal concentrations 

of sodium bicarbonate. Sample size equals 4 for all exposures except 800 mg NaHCO3/L where 

sample size equals three. Exposures presented are nominal concentrations of sodium bicarbonate

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), ± standard error of the mean]

Exposure 

(mg NaHCO3/L)

Calcium

(mg/L)

Magnesium

(mg/L)

Potassium

(mg/L)

Sodium

(mg/L)

Day 37

Control 36.86a 2.98a 17.23 6.32a

(2.01) (0.16) (0.82) (0.39)

500 65.95b 3.90b 15.72 6.50a

(6.29) (0.35) (0.42) (0.14)

800 53.91a,b 3.07a,b 17.26 7.66a,b

(4.9) (0.07) (0.51 (0.37)

1,100 43.28a,b 2.92a,b 17.76 8.83b

(0.9) (0.05) (0.33) (0.39)

Day 60

Control 25.33a 2.20a 13.27 9.67

(2.36) (0.15) (0.51) (0.36)

500 54.02b 3.30b 12.08 8.9

(7.21) (0.36) (0.29) (0.33)

a,bDifferent superscript letters denote significant differences (α = 0.05).
Results 53

Table 3–12. Mean whole body ions in milligrams per gram dry weight (mg/g) in fathead minnows 

(Pimephales promelas) on day 30 of chronic experiment 2 completed in reconstituted Tongue River 

water (control). Exposures presented are nominal concentrations of sodium bicarbonate. Standard 

error of the mean is in parenthesis. Sample size equals four for control, three for 300 and 400 mg 

NaHCO3/L and two for 625 mg NaHCO3/L.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), standard error of the mean]

Exposure 

(mg NaHCO3/L)

Calcium

(mg/L)

Magnesium

(mg/L)

Potassium

(mg/L)

Sodium

(mg/L)

Day 37

Control 21.27a 2.20a 11.92 9.18

(3.22) (.13) (.4) (.27)

300 22.13a 2.14a 11.92 9.1

(1.92) (.05) (.23) (.27)

400 28.91a,b 2.23a 12.6 9.63

(2.47) (.05) (.33) (.31)

625 42.26b 2.86b 12.29 8.87

(11.99) (.22) (.57) (.35)

a,bSuperscript letters denote significant differences (α = 0.05).

Table 3–13. Mean percent survival during a 7-day 

static renewal chronic exposure of fathead minnow 

(Pimephales promelas) completed in reconstituted Tongue 

River water (control). Exposure concentrations presented 

are nominal values of sodium bicarbonate. Sample size 

equals three.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per; ( ), standard 

deviation; --, not calculated]

Exposure 

(mg NaHCO3/L)

7-day mean number of individuals Percent survival

Control 10 100

(0) --

300 10 100

(0) --

500 10 100

(0) --

625 9.3 93

(.6) --

800 10 100

(0) --

1,100 17.7 77

(2.3) --

1,400 17.7 77

(1.5) --

2,000 14.3 43

(1.2) --

1

Significant difference at α = 0.05.

Table 3–14. Mean lengths of fathead minnows 

(Pimephales promelas) at day 7 of a 7-day static 

renewal exposure completed in reconstituted 

Tongue River water (control). Exposure 

concentrations presented are nominal values of 

sodium bicarbonate. Sample size equals three.

[NaHCO3,sodium bicarbonate; mg/L, milligrams per liter,; 

mg, milligrams; ( ), standard deviation]

Exposure 

(mg NaHCO3/L) Day 30 weight (mg)

Control 0.005

(.0005)

300 .006

(.001)

500 .005

(.0006)

625 1.004

(.0004

800 1.004

(.00007)

1,100 1.004

(.0008)

1,400 1.003

(.0006)

2,000 .003

(.0009)

1

Significant difference compared to the control 

(α = 0.05). 
54 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Summary Results Fathead Minnow

During the flow-through chronic experiments, the 

survival of FHM was significantly less than the control in all 

concentrations ≥500 mg/L NaHCO3. In the 300 and 400 mg 

NaHCO3/L treatments that did not reduce survival, growth 

was not affected. Na/K ATPase decreased in all treatments 

>625 mg NaHCO3/L at day 37 and was decreased in all treatments in a concentration dependent manner by day 60. The 

severity of the microscopic lesions was related directly to 

NaHCO3 concentration and the duration of exposure. After 

60 d, fish from the 500 mg NaHCO3/L treatment exhibited a 

slight increase in the incidence of kidney damage, including 

a greater number of regenerating tubules and focal degeneration, compared to fish in the control treatment. Similarly, 

the severity of gill lesions was related directly to NaHCO3 

exposure concentration and duration of exposure. Whereas 

significant mortality was not observed in the 7-d static renewal 

experiment until >1,100 mg NaHCO3/L, weights were reduced 

in FHM from this experiment in concentrations > 625 mg 

NaHCO3/L.

White Sucker

Estimated percent hatch of white sucker eggs was 

23 percent in the control, 21 percent in the 450 mg/L NaHCO3 

treatment, 29 percent in the 800 mg/L NaHCO3 treatment, and 

30 percent in the 1,400 mg/L NaHCO3 treatment. After 

30 days, the survival of white sucker fry that hatched was 

54 percent in the control, 80 percent in the 450 mg/L NaHCO3 

exposure, 60 percent in 800mg/L NaHCO3 exposure, and 

51 percent in the 1,400 mg/L NaHCO3 exposure. From 

31–60-d survival was 70–82 percent in the control and 

treatments, except that survival was 45 percent (±29) in 1,400 

mg/L NaHCO3 exposures. 

A second experiment was performed to validate the 

repeatability of the hatching success of white suckers. After 

22 days of the initial white sucker spawning, 10 female and 

14 male white suckers from the Beaverhead River pond 

were spawned and the eggs transported to the Jackson Field 

Research Station. At least 250 eggs were placed in a 4-L glass 

aquaria and exposed to NaHCO3 using the methods described 

in Methods, White Sucker Experiment section above. Hatching success and survival of the second group of eggs spawned 

was similar to the first group (data not presented).

Although survival was unaffected (table 3–15) by the 

exposures to NaHCO3, mean lengths and weights of white 

suckers from all treatments were significantly less than those 

of fish in the control treatment (table 3–16). The responses followed a concentration-response pattern with the smallest mean 

lengths and weights measured in fish exposed to 1,400 mg 

NaHCO3/L. Based on weight, the IC20 for white suckers was 

348 mg NaHCO3/L (95-percent CI, 327–445 mg NaHCO3/L). 

The histological examination revealed bacterial kidney 

disease in control fish, but not in fish exposed to varying concentrations of NaHCO3

. In general the severity of gill, kidney, 

gastrointestinal tract, and liver lesions was related directly to 

NaHCO3 exposure concentration and the duration of exposure. 

Laboratory rearing of wild fish and specific culture conditions 

(for example, feed) may account for some of the variability 

among groups. However, liver lesions clearly progressed 

from cytoplasmic degeneration and cystic change to nuclear 

degeneration and preneoplastic change as exposure concentration and duration of exposure increased. Statistical evaluations 

were not performed on whole-body ion concentrations 

(tables 3–17, 3–18).

Table 3–15. Mean percent survival of white suckers 

(Catostomus commersoni) during a 60-day (53-day posthatch) chronic exposure completed in reconstituted 

Tongue River water (control). Exposure concentrations 

presented are nominal values of sodium bicarbonate. 

Standard deviation is plus or minus (±) mean in 

parentheses and sample size equals three.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per]

Exposure 

(mg NaHCO3/L) Day 0–30 Day 31–60

Control 54 70

(±5.0) (±15)

450 80 72

 (±3) (±20)

800 60 82

 (±17) (±7.7)

1,400 51 45

 (±13) (±29)

Table 3–16. Mean lengths and weights of white suckers 

(Catostomus commersoni) during a 60-day (53-day posthatch) chronic exposure completed in reconstituted 

Tongue River water (control). Exposure concentrations 

presented are nominal values of sodium bicarbonate. 

Sample size equals three.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; mm, 

millimeters; mg, milligrams; ( ), standard deviation]

Exposure 

(mg NaHCO3/L)

Length

(mm)

Weight

(mg)

Control 19.9 0.04

(.4) (.004)

450 117.6 10.024

(1.1) (.004)

800 117.4 10.022

(1) (.05)

1,400 116.4 10.019

(.4) (.002)

1

Significant difference at α = 0.05.
Results 55

Summary Results White Suckers

The estimated percent hatch for white suckers was less 

than 30 percent in all treatments. This percent hatch was less 

than that observed for FHM, but it is not unexpected because 

the white sucker eggs were obtained from wild fish and were 

incubated in the laboratory. Effects on the survival of white 

suckers exposed to ≤1,400 mg NaHCO3/L were not observed. 

However, lengths and weights were reduced in exposures 

>450 mg NaHCO3/L. Though whole-body ion concentrations 

do not explain the mechanism of toxicity, the presence of 

bacterial kidney disease in the controls may have masked the 

usefulness of the whole-body ion measurements. Also, there is 

a question of whether or not the presence of some concentrations of NaHCO3

 minimized the detection of bacterial kidney 

disease in fish or alleviated the disease expression. Histological examinations point to progressive liver lesions as the 

concentrations and duration of exposures continued and these 

lesions may have been linked to the observations of decreased 

lengths and weights. 

Ceriodaphnia Experiment

The survival and reproduction of C. dubia in control 

water was 100 percent and 26 neonates/adult, respectively, 

which meet the requirements for a successful experiment (table 3–19). The estimated 7-d LC50 was 1,192 mg 

NaHCO3/L with a 95 percent CI of 963–1,476 mg NaHCO3/L. 

The number of neonates produced was the largest in the 

moderately hard reconstituted water control and smallest in 

the 1,500 mg NaHCO3/L exposure (table 3–19). The 7-d IC20 

based on reproduction was 359 mg NaHCO3/L (95-percent 

CI=258–506). This can also be reported as 274 mg HCO3

-

/L 

(95-percent CI=190–377) and total alkalinity of 249 expressed 

as mg CaCO3/L (95-percent CI=173–354).

Table 3–17. Mean whole-body ions in white suckers (Catostomus commersoni) on day 30 

of a chronic experiment completed in reconstituted Tongue River water (control). Exposure 

concentrations presented are nominal values of sodium bicarbonate. Sample size equals three.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; mg/g, milligrams per gram; ( ), standard deviation]

Exposure 

(mg NaHCO3/L)

Calcium

(mg/g)

Magnesium

(mg/g)

Potassium

(mg/g)

Sodium

(mg/g)

Control 15.62 1.67 20.49 10.85

(9.75) (.43) (3.45) (2.4)

450 12.91 1.59 18.93 10.47

(3.24) (.44) (4.78) (2.11)

800 16.27 1.63 19.3 10.52

(2.99) (.9) (1.61) (1.3)

1,400 20.2 1.8 20.3 11.12

(9.12) (.41) (3.32) (1.63)

Table 3–18. Mean whole-body ions in white suckers (Catostomus commersoni) on day 53 

of a chronic experiment completed in reconstituted Tongue River water (control). Exposure 

concentrations presented are nominal values of sodium bicarbonate. Sample size equals two 

for all exposures except 450 mg NaHO3/L where sample size equals three. Statistics were not 

performed on these data.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; mg/g, milligrams per gram; ( ), standard deviation]

Exposure 

(mg NaHCO3/L)

Calcium

(mg/g)

Magnesium

(mg/g)

Potassium

(mg/g)

Sodium

(mg/g)

Control 7.62 1.33 17.53 8.44

(0.39) (.01) (1.17) (.29)

450 9.36 1.75 22.46 9.68

(2.12) (0.51) (6.71) (3.51)

800 12.02 1.63 19.52 9.31

(1.76) (.16) (2.09) (1.77)
56 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life Table 3–19. Survival and reproduction of Ceriodaphnia dubia exposed to nominal concentrations of milligrams sodium bicarbonate per liter (mg NaHCO3/L) in moderately hard reconstituted water (control) during chronic static renewal experiments. [mg/L, milligrams per liter; Ad, adults; Neo, neonates; N/A, neonates per adult (number of neonates produced during the 7-day exposure, divided by the number of adults at the initiation of the experiment); --, no data] Day Control 500 mg/L 700 mg/L 900 mg/L 1,200 mg/L 1,500 mg/L Ad Neo N/A AD Neo N/A Ad Neo N/A Ad Neo N/A Ad Neo N/A Ad Neo N/A

0 10 -- -- 10 -- -- 10 -- -- 10 -- -- 10 -- -- 10 -- --1 10 0 -- 10 0 -- 10 -- -- 10 0 -- 8 0 -- 6 0 --2 10 0 -- 10 5 -- 10 -- -- 10 0 -- 8 0 -- 5 0 --3 10 0 -- 10 0 -- 6 -- -- 9 0 -- 8 0 -- 4 0 --4 10 55 -- 10 40 4 6 20 -- 8 17 2.1 7 12 1.7 4 4 --5 10 72 -- 10 61 6.1 6 24 -- 8 22 2.8 7 24 3.4 4 0 --6 10 18 -- 10 31 3.1 6 22 -- 8 27 3.4 7 13 1.9 3 0 --7 10 113 -- 10 60 6 6 63 -- 8 54 6.8 7 55 7.9 2 0 --

Total -- 258 25.8 -- 192 19.2 -- 129 12.9 -- 120 12 -- 104 10.4 -- 4 .2
Discussion 57

 Freshwater Mussel Experiment

The lack of foot movement during a 5-minute observation 

period was the effect endpoint monitored during the mussel 

experiment. Also, mussels that did not show foot movement 

were considered to be dead. Mussels exposed to 1,000 mg 

NaHCO3/L for 4 days had 16 percent survival and 2 percent of 

mussels exposed to 1,500 mg NaHCO3/L survived. Additional 

mortalities were observed in mussels exposed to 1,000 and 

1,250 mg NaHCO3/L, but survival in all other experimental 

concentrations remained constant until day 10 (table 3–20).

The 10-d EC50 for newly transformed juvenile fatmuckets was 1,061 mg NaHCO3

/L (95-percent CI, 990–1,137 mg 

NaHCO3/L). The 10-d IC20 based on foot movement 

was calculated to be 952 mg NaHCO3/L (95-percent CI, 

828–1,076 mg NaHCO3/L). This also can be expressed as 

715 mg HCO3

-

/L (95-percent CI, 626–805 mg HCO3

-

/L) and 

total alkalinity expressed as 605 mg CaCO3/L (95-percent CI, 

508–680 expressed as mg CaCO3/L)

Discussion

Regardless of the species (fish, crustacean, or bivalve) or 

type of effect measured (growth, survival, or reproduction), 

data from the present study demonstrated approximately 500–

1,000 mg NaHCO3/L affects aquatic animals in chronic exposure conditions. Also, this range of NaHCO3 concentrations 

elicited effects regardless of whether animals were cultured in 

controlled conditions in the laboratory (for example, C. dubia; 

FHM) or spawned in the wild (white sucker). Compared to the 

other species tested, freshwater mussels were the least sensitive to NaHCO3 exposure, especially in relation to the IC20 for 

C. dubia. The IC20 for C. dubia and freshwater mussels is 359 

and 952 mg NaHCO3/L, respectively. 

When survival is the only consideration, the FHM appear 

more sensitive than white sucker and incurred significant 

reductions in survival in exposure concentrations >500 or 

>1,100 mg NaHCO3/L depending on the type of experiment, 

whereas no effects on survival of white sucker were observed 

in any of the exposure concentrations. However, growth 

effects were observed in white sucker exposed to concentrations as low as 450 mg NaHCO3

/L compared to 650 mg 

NaHCO3/L for FHM. And the growth effects documented 

in white sucker were substantiated by histological findings 

that revealed progressive liver disease with increased exposure concentrations. If both survival and growth effects are 

considered, the results of the chronic fish experiments define 

effects at the lower end of the range of NaHCO3 concentrations investigated. 

To investigate the mechanisms of toxicity of NaHCO3, 

Na/K ATPase induction and whole-body ion concentrations 

were studied. These measurements along with the pattern of 

mortality and growth effects between the fish species revealed 

changes that may occur with exposure to NaHCO3. Because 

Na/K ATPase is involved, at least in part, in the regulation of 

Na+ in freshwater fish (Marshall, 2002) and the concomitant 

movement of HCO3

-

 once an ion gradient is established, the 

expectation was that concentrations of Na/K ATPase would 

be elevated during NaHCO3 exposures. This expectation was 

based on the hypothesis that an induction of the enzyme system would assist the animal in regulating ion status. Quite to 

the contrary, the concentrations of Na/K ATPase were reduced 

in fathead minnow exposed to elevated NaHCO3. Other 

researchers have documented decreases in ATPase as a result 

of NaHCO3 exposure. For example, a decrease in N-ethylmaleimide (NEM)-sensitive ATPase of the ascending limb and 

outer medullar collecting tubule of the nephron was observed 

in rats as a result of metabolic alkalosis induced by a NaHCO3

exposure (Khadouri and others, 1992). 

In the present study, FHM exposed to 300 or 400 mg 

NaHCO3/L did not exhibit any effects on growth or survival; 

however, Na/K ATPase was reduced significantly at day 60, 

but not day 37 in fish exposed to these concentrations compared to the controls. The reduced Na/K ATPase concentration 

in FHM exposed to the 300 or 400 mg NaHCO3/L was reduced 

to a lesser extent than in fish exposed to concentrations > 400 

mg NaHCO3/L where effects on survival were observed. In 

Table 3–20. Survival of 10-day old newly transformed fatmucket mussels (Lampsilis siliquoidea) 

to chronic exposures completed in reconstituted Tongue River water (control). Exposure 

concentrations presented are nominal values of sodium bicarbonate.

[NaHCO3, sodium bicarbonate; mg/L, milligrams per liter; ( ), number of alive individuals per exposure, in percent]

Exposure 

(mg NaHCO3/L) 0 days 4 days 6 days 10 days

Control 20 (100) 20 (100) 20 (100) 20 (100)

500 20 (100) 20 (100) 20 (100) 20 (100)

750 20 (100) 19 (95) 19 (95) 19 (95)

1,000 20 (100) 16 (80) 13 (65) 13 (65)

1,250 20 (100) 9 (45) 6 (30) 4 (20)

1,500 20 (100) 2 (10) 0 (0) 0 (0)

2,000 20 (100) 0 (0) 0 (0) 0 (0)
58 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

addition, fish had an early onset of significantly decreased 

Na/K ATPase at day 37 in all but the smallest (500 mg/L 

NaHCO3/L) concentration that resulted in significant mortality. 

The significant decrease in Na/K ATPase together with 

the lack of growth effects suggests that the Na/K ATPase 

activity was actually shut down before the onset of death. 

The decrease rather than increase in Na/K ATPase may explain 

why effects on the growth of FHM were not observed. Energy 

for growth was not diverted to enzyme induction. Although 

FHM were tested under NaHCO3 chronic exposure conditions, 

the resulting ionic imbalance may, in fact be an acute response 

mediated through an extreme ionic gradient that overwhelmed 

the animal’s ability to maintain ion regulation. As a result, 

Na/K ATPase no longer functioned in the animal and death 

occurred. 

When the magnitude of Na/K ATPase decreases in 

FHM exposed to NaHCO3 are compared, it appears 

percent decrease in the activity of Na/K ATPase and the age 

of the fish at the onset of the decrease may affect the ability of 

FHM to survive. For example, fish were able to survive 

an 8.12 percent decrease in Na/K ATPase activity in the 

400 mg NaHCO3/L treatment compared to the control when 

this decrease was first documented on day 60. However, fish 

with an 8.50 percent decrease in Na/K ATPase activity first 

documented on day 37 in the 500 mg NaHCO3 /L treatment 

incurred significant reductions in survival and subsequently 

exhibited a 26.5 percent reduction in Na/K ATPase at day 60. 

The effects of NaHCO3 on Na/K ATPase activity and the ability of fish to survive likely are related to the age of the fish at 

exposure. Na/K ATPase was not measured in fish from the 7-d 

FHM experiment where growth effects were observed. Such 

measurements could shed more light on the potential effect of 

Na/K ATPase on NaHCO3 toxicity.

The changes in whole-body ion concentrations appear to 

be related indirectly to NaHCO3 exposure. Significant elevations of whole-body Ca and Mg occurred in FHM exposed to 

the higher range of the NaHCO3/L treatments that appeared 

to be associated with the reduced amount of Ca in solution. 

These changes were noteworthy in FHM and white suckers, 

but the data could not be statistically tested because of small 

sample sizes. A white precipitate was observed on the experimental equipment in the greater NaHCO3

 concentrations. It is 

possible that some of the whole-body Ca measured was actually precipitates that settled onto the fish. The one instance of 

increased whole-body Na in FHM was observed in the greatest 

concentration of 1,100 mg NaHCO3/L on day 37. Thus, wholebody ion concentrations do not appear to be an adequately 

sensitive indicator of NaHCO3 toxicity. Whole-body ion 

measurements do not detect changes among extracellular and 

intracellular ion concentrations, where the ionoregulation 

effects in this situation are likely to occur. 

Several histological anomalies suggested that fish 

were adversely affected as a result of exposure to >450 mg 

NaHCO3/L. For example, necrotic (dying) cells were found in 

gills of FHM and the incidence of necrotic cells increased with 

exposure concentration. The presence of necrotic cells may 

have precluded the observation of a proliferation of mitochondrial rich cells. Changes in kidney, intestine, and livers were 

observed in white sucker as well as progressive liver disease 

in relation to exposure concentration, which may explain the 

observed adverse effects on the white sucker.

The histological investigation also detected the presence 

of bacterial kidney disease in white suckers held in the control 

water during the experiment. This disease may, at least in part, 

explain the low hatch success of white sucker embryos. The 

lack of bacterial kidney disease in the exposed white sucker 

also may be related to the greater percent hatch of white 

sucker embryos exposed to some of the NaHCO3 treatments. 

A white film that appeared to be fungus accumulated around 

the white sucker eggs early in the experiment. The amounts of 

the film that accumulated on eggs, but could not be quantified, 

were related indirectly to the NaHCO3 exposure concentrations; however, the white suckers continued to hatch in the 

presence of this film. Likely the presence of the NaHCO3 salt 

protected the eggs during incubation. Salt has been used by 

hatchery personnel to treat fish during transport to minimize 

stress and disease expression (Piper and others, 1982). However, when the white sucker embryos hatched, the NaHCO3

affected their ability to grow to the same extent as the fish in 

the control treatment, and was associated with progressive 

liver disease. All diluter equipment was completely disinfected 

according to USGS protocol and no white suckers from the 

laboratory were released into the wild.

The natural background concentration of salts in the 

Tongue and Powder Rivers made it necessary to use control 

water for chronic toxicity experiments that mimicked the natural conditions. Simulated Tongue River water was used for all 

the chronic toxicity experiments, with the exception of the C. 

dubia. The C. dubia experiment was completed with moderately hard reconstituted water in accordance with U.S. Environmental Protection Agency (1994) suggested guidelines. 

In theory, the use of the simulated Tongue River water could 

have defined lesser effects compared to this control water 

because animals were exposed to some salt concentrations in 

the control. For this reason, one might consider the use of the 

simulated Tongue River as a conservative approach. However, 

the C. dubia experiment used a moderately hard reconstituted water and the IC20 effects on reproduction was defined 

at 359 mg NaHCO3/L, similar to effects at 450 and 500 mg 

NaHCO3/L noted for FHM and white suckers, respectively. 

Additional work may be initiated with C. dubia in simulated 

Tongue River water to further define any specific differences 

between control waters with this species.

The use of the simulated Tongue River water as a control 

does not, however, preclude the use of these data in other 

watersheds where NaHCO3 may be introduced to a system. 

The repeatability of effects defined on multiple species of 

both invertebrates and fish was noted at concentrations above 

500–1,000 mg NaHCO3/L. The effects were repeatable and 

included sublethal (for example, growth and reproduction) and 

lethal (survival) endpoints; and the pattern of Na/K ATPase 

reduction suggests that the system shuts down before the 
References Cited 59

onset of death. These data can be used for comparisons by 

other regions regardless of the reference water present in that 

particular region.

The overall results of these experiments suggest that there 

is minimal room for NaHCO3 increases in the Tongue River/

Powder River watershed (fig. 1–2) if a goal is to maintain the 

health of aquatic life. In the end, it was difficult to bracket 

effects with no effect concentrations because decreased survival (fathead minnow) at 450 mg NaHCO3

/L and decreased 

growth (white sucker) at 500 mg NaHCO3/L was observed. 

Concentrations during the low-flow period (late summer and 

early fall) are generally 250 and 350 mg NaHCO3/L in the 

main stem of the Tongue and Powder Rivers, respectively. 

To date, concentrations of NaHCO3 determined to adversely 

affect biota in this study have been observed in some tributaries, but have not been observed in the main stem of the Tongue 

and Powder Rivers (Peterson and others, 2010; chapter 4, 

Discussion section). Continued monitoring of water quality throughout the Tongue River/Powder River watershed is 

needed to identify exceedances of the NaHCO3 effect concentrations determined in this study. 

References Cited

American Public Health Association Standard Methods, 1975, 

Alkalinity titration method for water and wastewater (14th 

ed.): Washington, D.C., American Public Health Association, p. 278.

American Society of Testing and Materials International, 

2006, Standard guide for conducting laboratory toxicity 

tests with freshwater mussels: West Conshohocken, Pa., 

American Society of Testing and Materials International, 

E 2455–06, 52 p.

ALL Consulting, 2003, Handbook on coalbed methane produced water—Management and beneficial use alternatives: 

coalbed natural gas resources and produced water management, Tulsa, ALL Consulting, Report to the U.S. Department of Energy, accessed April 2010 at http://www.all-llc.

com/CBNG/BU/index.htm, 44 p.

Farag, A.M., Skaar, D., Nimick, D.A., MacConnell, E., and 

Hogstrand, C., 2003, Characterizing aquatic health using 

salmonid mortality, physiology, and biomass estimates in 

streams with elevated concentrations of arsenic, cadmium, 

copper, lead, and zinc in the Boulder River Watershed, 

Montana: Transactions of the American Fisheries Society, 

v. 132, p. 450–467.

Khadouri, C., Marsy, S., Barlet-bas, C., Cheval, L., and 

Doucet, A., 1992, Effect of metabolic acidosis and alkalosis 

on NEM–sensitive ATPase in rat nephron segments: American Journal of Physiology, v. 262, p., F583–F590.

Marshall, W.S., 2002, Na+, Cl-, Ca2+, and Zn2+ Transport by 

fish gills—Retrospective review and prospective synthesis: 

Journal of Experimental Zoology, v. 293, p. 264–283.

Mount, D.R., Gulley, D.D., Hockett, J.R., Garrison, T.D., and 

Evans, J.M., 1997, Statistical models to predict the toxicity 

of major ions to Ceriodaphnia dubia, Daphnia magna, and 

Pimephales promelas (fathead minnows): Environmental 

Toxicology and Chemistry, v. 16, p. 2,009–2,019. 

Mount, D.I., and Brungs, W.A., 1967, A simplified dosing 

apparatus for fish toxicology studies: Water Research, v. 1, 

p. 21–29.

Norberg-King, T.J., 1993. A linear interpolation method for 

sublethal toxicity—The inhibition concentration (ICp) 

approach (Version 2.0.): Duluth, U.S. Environmental 

Protection Agency, National Effluent Toxicity Assessment 

Center Technical Report 03–93, 39 p.

Patz, M.J., Reddy, K.J., and Skinner, Q.D., 2004, Chemistry of 

coalbed methane discharge water interacting with semi-arid 

ephemeral stream channels: Journal of the American Water 

Resources Association, v. 40, no. 5, p. 1,247–1,255. 

Peterson, D.A., Clark, M.A., Foster, K., Wright, D.L., and 

Boughton, J.K., 2010, Assessment of ecological conditions 

and potential effects of water produced from coalbed natural 

gas development on biological communities in streams of 

the Powder River structural basin, Wyoming and Montana, 

2005–08: U.S. Geological Survey Scientific Investigations 

Report 2010–5124, 84 p.

Piper, R.G., McElwain, I.B., Orme, L.E., McCraren, J.P., 

Fowler, L.G., and Leonard, J.R., 1982, Fish hatchery management: Washington, D.C., U.S. Fish and Wildlife Service, 

317 p.

Reid, S.D., and McDonald, D.G., 1988, Effects of cadmium, 

copper, and low pH on ion fluxes in rainbow trout, Salmo 

gairdneri: Canadian Journal of Fisheries and Aquatic Sciences, v. 45, p. 244–253.

Stephan, C.E., Mount, D.I., Hansen, D.J., Gentile, J.R., Chapman, G.A., and Brungs, W.A., 1985, Guidelines for deriving 

numerical national water quality criteria for the protection 

of aquatic organisms and their uses: Duluth, U.S. Environmental Protection Agency, Office of Research and Development, EPA–PB85–227049, 106 p.

Wang, N., Augspurger, T., Barnhart, M.C., Bidwell, J.R., 

Cope, W.G., Dwyer, F.J., Geis, S., Greer, I.E., Ingersoll, 

C.G., Kane, C.M., May, T.W., Neves, R.J., Newton, T. J., 

Roberts, A.D., and Whites, D.W., 2007, Intra- and Interlaboratory variability in acute toxicity tests with glochidia and 

juveniles of freshwater mussels (unionidae): Environmental 

Toxicology and Chemistry, v. 26, p. 2,029–2,035.
60 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Wilson, J.M., and P. Laurent, 2002, Fish gill morphology— 

Inside Out: Journal of Experimental Zoology, v. 293, 

p. 192–213.

U. S. Environmental Protection Agency, 1994, Short term 

methods for estimating the chronic toxicity of effluents 

and receiving waters to aquatic organisms, in P.A. Lewis, 

Klemm, D.J., Lazorchak, J.M., Norberg-King, T.J., Peltier, 

W.H., and Heber, M.A. eds., Duluth, U.S. Environmental 

Protection Agency, EPA/600/4 -91/002, 315 p.

U.S. Environmental Protection Agency, 2010, The Toxicity 

Relationship Analysis Program: Duluth, U.S. Environmental Protection Agency, Mid-Continent Ecology Division, 

accessed April 25, 2011, at http://www.epa.gov/med/Prods_

Pubs/trap.htm.
The Potential Toxicity Defined In Situ and 

Streamside in the Powder and Tongue River 

Watersheds

By Aïda M. Farag, David D. Harper, and Don Skaar

Chapter 4 of

The Potential Effects of Sodium Bicarbonate, a Major Constituent of 

Produced Waters from Coalbed Natural Gas Production, on Aquatic 

Life

Edited by Aïda M. Farag and David D. Harper

Prepared in cooperation with Montana Fish, Wildlife, and Parks, U.S. Bureau of 

Land Management, and the U.S. Environmental Protection Agency

Scientific Investigations Report 2012–5008

U.S. Department of the Interior

U.S. Geological Survey
Contents

Introduction...................................................................................................................................................65

Methods.........................................................................................................................................................65

In Situ and Site Water Experiment...................................................................................................65

Mixing Zone and Site Water .............................................................................................................68

Results ...........................................................................................................................................................71

Site Water/Mixing Zone Experiments..............................................................................................71

Discussion.....................................................................................................................................................75

Summary........................................................................................................................................................77

References Cited..........................................................................................................................................77

Figures

4–1. Map showing field sites for 96-hour in situ toxicity experiments in the Powder River 

Drainage, Wyoming....................................................................................................................67

4–2. Graph showing percent survival at 96 hours for in situ experiments started with 2-daypost-hatch (2-dph) or 6-day-post-hatch (6-dph) fathead minnows. Powder River 

drainage, July–August 2006......................................................................................................74

4–3. Graph showing percent survival at 96 hours for the mixing zone experiment started 

with 2-day-post-hatch fathead minnows. Receiving water was added to experimental 

waters to provide the percent mixture indicated. Sample waters were collected 

from the Powder and Tongue Rivers watershed, September–October 2007 ...................77

Tables

4–1. Mean alkalinity expressed as milligrams per liter calcium carbonate equivalent, 

bicarbonate, calcium, magnesium, sodium, potassium, chloride, sulfate, and ammonia 

(mg N/L) during in situ experiments with 2- and 6-day-post-hatch fathead minnows 

(Pimephales promelas) performed in the Powder River Basin in Wyoming and 

Montana........................................................................................................................66

4–2. Mean water chemistry recorded with hand-held meters during in situ toxicity experiments with fathead minnows (Pimephales promelas) in the Powder River Basin in 

Wyoming and Montana .............................................................................................................68

4–3. Minimum and maximum temperature, conductivity, dissolved oxygen, and pH measurements recorded every 30 minutes at four sites during in situ experiments with fathead 

minnows (Pimephales promelas) in the Powder River Basin, Wyoming and Montana. 

Data was recorded with YSI 5000TM Data Sonde units ..........................................................68

4–4. Additional trace metal scans analyzed with Inductively Coupled Plasma (ICP) in water 

samples collected from in situ experimental sites for fathead minnows (Pimephales 

promelas). Experiments completed in the Powder River Basin, Wyoming and Montana. 

Sample size is 1 for all constituents ........................................................................................69

4–5. Mean alkalinity expressed as milligrams per liter calcium carbonate equivalent, bicarbonate, sulfate, calcium, magnesium, sodium, and potassium, during in situ experiments with 4- and 6-day-post-hatch pallid sturgeon (Scaphirhynchus albus) performed 

in the Powder River Basin in Wyoming...................................................................................69

4–6. Mean temperature, conductivity, dissolved oxygen, and pH measurements recorded 

with hand-held meters during in situ toxicity experiments with pallid sturgeon (Scaphirhynchus albus) in the Powder River Basin in Wyoming.....................................................70
4–7. Additional trace metal scans analyzed with Inductively Coupled Plasma (ICP) in water 

samples collected from in situ experimental sites with pallid sturgeon (Scaphirhynchus 

albus) completed in the Powder River Basin, Wyoming......................................................70

4–8. Mean concentrations of ammonia, in water from experimental sites during in situ 

experiments performed in the Powder River Basin, Wyoming...........................................71

4–9. Mean alkalinity expressed as milligrams per liter calcium carbonate equivalent, 

bicarbonate, sulfate, calcium, magnesium, sodium, potassium, and total ammonia (mg 

N/L) during 96-hour static renewal exposures of 2-day-post-hatch fathead minnows 

(Pimephales promelas) to mixtures of waters collected in the Tongue and Powder 

River Basins, Wyoming and Montana, September 2007 ......................................................72

4–10. Survival of fathead minnows (Pimephales promelas) during 96-hour in situ experiments 

in the Powder River Basin, Wyoming and Montana. Two experiments were performed 

with fathead minnows of different ages. Fish were 2-day-post-hatch (dph) at the initiation of experiment 1 and were 6-day-post-hatch at the initiation of experiment 2 .........73

4–11. Survival of pallid sturgeon (Scaphirhynchus albus) during 96-hour in situ experiments in 

the Powder River Basin, Wyoming. Fish were 4 or 6-day-post-hatch (dph) at the initiation of the experiments ..............................................................................................................73

4–12. Dye concentrations as measured in transects across the Powder River downstream 

from the confluence of Beaver Creek. Beaver Creek entered on the right bank and 

distances at each transect were measured in meters from the right stream bank to the 

left stream bank .........................................................................................................................74

4–13. Dye concentrations as measured in transects across the Tongue River downstream 

from diffusers injecting untreated coal bed natural gas product water. Diffusers were 

located on the right bank and distances at each transect were measured in meters 

from the right stream bank to the left stream bank...............................................................75

4–14. Dye concentrations as measured in transects across the Tongue River downstream 

from diffusers injecting treated coal bed natural gas product water. Diffusers were 

located on the right bank and distances at each transect were measured in meters 

from the right stream bank to the left stream bank...............................................................75

Introduction 

In situ toxicity experiments provide needed information 

about the ability of fish to survive in the field under real-world 

conditions (Farag and others, 2003; Harper and others, 2008). 

Reduced survival of fish may indicate the inability of a watershed to support an optimal population size (Farag and others, 

2003). Therefore, these measurements can indicate impairment 

at the population level. The placement of sites for these studies 

also can provide indications about the source(s) of impairment. However, the overall goal when managing aquatic life is 

to avert or minimize conditions where the survivability of fish 

is affected. To achieve this management objective, laboratory 

experiments performed in controlled conditions are used to 

define threshold concentrations where effects of an element or 

compound may be observed on aquatic life. These threshold 

concentrations can then be used to alert managers and site 

operators about the potential effects of effluents that may be 

introduced into a water body. 

As part of the effort to define threshold concentrations, 

in situ experiments can provide supporting data for laboratory 

derived thresholds. In situ experiments are especially critical 

when performed at sites where concentrations exceed laboratory derived thresholds, because the ability of fish to survive 

in real-world conditions can be determined. In this sense, field 

data are used to ground-truth laboratory derived concentrations before extensive catastrophic events where populations 

are affected. In situ experiments provide a link between the 

laboratory and the field to further assess potential effects in the 

field. The Powder/Tongue River watersheds provide a unique 

opportunity for field assessments because some tributaries in 

these watersheds were historically ephemeral, but now contain 

water year round as a result of discharge inputs of coalbed 

natural gas produced waters (Patz and others, 2004). As such, 

field sites with 100 percent effluent water could be studied in 

these watersheds.

Streamside experiments performed with site water are 

another alternative to gather field information. During these 

experiments, site water is used, but because the experiment is 

initiated outside of the stream, some physical characteristics 

such as temperature and dissolved oxygen may be controlled 

to reduce potentially confounding factors. Stewart (1996) 

used these types of assays when attempting to assess ambient water conditions. Again, these types of experiments add 

useful information to data gathered from laboratory and in situ 

experiments because they add further evidence to the support 

or refute laboratory or in situ results.

Another opportunity for field studies in the Powder/

Tongue River watersheds is provided by mixing zones where 

tributaries, treated effluent, or untreated effluent flow into 

the main stem Tongue or Powder River. Research related 

to toxicity in mixing zones is limited. However, it has been 

documented that toxicity and related gill lesions were elevated 

in mixing zones that received acid-mine drainage (Henry and 

others, 2001). In this case, toxicity was related to metals that 

precipitated from solution in the mixing zone. In November 

2006, the Aquatic Task Group (ATG) defined an investigation 

of the survivability of fish in mixing zones of the Powder and 

Tongue Rivers as the top priority for research needs still to be 

accomplished.

The main goal of this study was to investigate the survivability of fish in the field, at sites where concentrations of 

NaHCO3 exceeded threshold concentrations defined in the 

laboratory. A second goal of this study was to investigate the 

physical extent of the mixing zones at confluence points in the 

Tongue/Powder River watersheds. 

Methods

In Situ and Site Water Experiments

Experiments to investigate the acute toxicity of produced 

waters in the Powder River Basin, Wyoming and Montana, 

were completed during July–August 2006 and July 2007. 

Early lifestage fathead minnow (FHM) Pimephales promelas

and pallid sturgeon Scaphirhynchus albus (endemic species 

used in the laboratory experiments) were studied during 2006 

and 2007, respectively. An additional static renewal experiment with pallid sturgeon was completed in controlled conditions with site-collected water in 2007. 

The Potential Toxicity Defined In Situ and Streamside in 

the Powder and Tongue River Watersheds

By Aïda M. Farag1, David D. Harper1, and Don Skaar2

1

USGS, Columbia Environmental Research Center, Jackson Field Research 

Station, Jackson, Wyoming.

2

Montana Fish, Wildlife, and Parks, Helena, Montana.
66 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Experiments lasted in situ for 96-hours (h). Fish were 

held in 450 milliliters (ml) cellulose acetate butyrate containers with 149 micrometers (μm) mesh screen covering approximately 40 percent of the surface area. Each container held 10 

fish and 4 replicate containers were deployed per site. In situ 

containers were held in place at each site by strapping the 

individual containers to a vinyl-coated metal dish drying rack 

that was placed in a perforated [118 liters (L)] Rubbermaid 

TM HDPE storage tub. Storage tubs were held in place with 

stakes, rocks, and rope to prevent loss from potential flash 

flooding. At one site, the depth was not adequate to allow the 

use of the storage tub, and the dish drying rack was placed 

directly in the stream channel with a storage tub cover over the 

top to provide shade.

Experiments were initiated with 2-day post-hatch (dph) 

FHM on 26–29 July (experiment 1) and 6-dph FHM on 

30 July–02 August, 2006 (experiment 2). Note: the terms 

“2-dph” and “6-dph” will be used from this point. Newly 

hatched fish were shipped directly to Buffalo, Wyoming from 

Aquatic Biosystems, Fort Collins, Colorado. The brood stock 

were deemed free of disease and kept in isolation at the Colorado facility. All relevant permits were obtained for importation of the offspring. 

For experiment 1, two reference sites and four experimental sites were used; Powder River at Moorhead and Clear 

Creek (reference sites), SA Creek, Upper Beaver Creek, 

Lower Beaver Creek, and Burger Draw (experimental sites; 

fig. 4–1). 

Following the completion of the 96-h experiments with 

2-dph fish, experiment 2 was initiated with 6-dph FHM in situ 

at Clear Creek (reference site) Upper Beaver Creek, Lower 

Beaver Creek, and Burger Draw (experimental sites) with fish 

from the same source.

Survival was monitored every 24 h, and water samples 

were collected daily from all sites. Water samples were placed 

on ice and filtered each evening with a 0.45 μm MilliporeTM

filter. Subsamples for cation analyses were acidified to less 

than (<) pH 2.0 with ultrapure nitric acid and were held at 4 

degrees Celsius (°C) during storage and shipment for analyses. 

Anion samples were collected into bicarbonate rinsed containers and stored at 4°C during storage and shipment. Alkalinity (American Public Health Association Standard Methods, 

1975a), hardness (American Public Health Association Standard Methods, 1975b), and pH were measured each evening 

(table 4–1). Water temperature, conductivity, and dissolved 

oxygen were measured manually at each site at least once 

daily (table 4–2), and monitored remotely with YSITM Data 

Sonde loggers at Powder River at Moorhead, Clear Creek, SA 

Creek and Lower Beaver Creek every 30 minutes (table 4–3). 

Ammonia was measured on-site daily with a HachTM test kit 

(table 4–1). Additional trace metal scans were performed on 

one subsample of water from each site (table 4–4). 

In 2007, 2-dph pallid sturgeon were transported from the 

Montana Fish Wildlife and Parks Miles City, Montana Fish 

Hatchery, to Buffalo, Wyoming. Pallid sturgeon at 2-, 4-, and 

6-dph were exposed in experiments at; Clear Creek (reference 

Table 4–1. Mean alkalinity expressed as milligrams per liter calcium carbonate equivalent, bicarbonate, calcium, magnesium, 

sodium, potassium, chloride, sulfate, and ammonia (mg N/L) during in situ experiments with 2- and 6-day-post-hatch fathead minnows 

(Pimephales promelas) performed in the Powder River Basin in Wyoming and Montana.

[CaCO3, calcium carbonate; mg/L, milligrams per liter; mg N/L, milligrams of nitrogen per liter; ( ), standard deviation error; <, less than; N, sample size; --, 

no calculation of standard error was performed]

Site

Alkalinity 

(CaCO3, in 

mg/L)

Bicarbonate 

(mg/L)

Sulfate 

(mg/L)

Calcium 

(mg/L)

Magnesium 

(mg/L)

Sodium 

(mg/L)

Potassium 

(mg/L)

Total 

ammonia

(mg N/L)

Powder R. at Moorhead 202 196 1,767 197 185 660 32 <0.01

(11) (10) (36) (49) (43) (162) (11) --

N=5 N=5 N=3 N=4 N=4 N=4 N=3 N=5

Clear Creek 198 192 285 74 52 68 5 .05

(12) (11) (12) (13) (9) (5) (.2) --

N=8 N=8 N=5 N=6 N=6 N=6 N=5 N=1

Lower Beaver 1,926 1,740 75 10 27 765 20 .05

(68) (128) (1) (.4) (.4) (5) (0.2) (.03)

N=9 N=9 N=5 N=6 N=6 N=6 N=5 N=5

Upper Beaver 1,759 1,646 257 16 24 664 20 .5

(66) (64) (211) (.4) (.2) (6) (0.4) (.1)

N=9 N=9 N=5 N=6 N=6 N=6 N=6 N=5

Burger Draw 2,535 2,315 27 15 28 1,003 41 1.5

(135) (136) (9) (2) (1) (7) (2) (.3)

N=9 N=9 N=5 N=5 N=5 N=5 N=5 N=5

SA Creek 1,364 1,245 395 17 45 605 16 .13

(53) (44) (18) (.6) (.6) (18) (2) (.04)

N=5 N=5 N=3 N=4 N=4 N=4 N=3 N=5
Methods 67

Powder River

River

Powder

Little Powder River

Beaver Creek

SA Creek

Clear Creek

Hanging

Woman Creek

Burger Draw

Crazy

Woman Creek

Upper Beaver Creek

Lower 

Beaver Creek Burger Draw

Clear Creek

S. A. Creek

Powder River

at Moorhead

Buffalo

106°

45°

44°

MONTANA

WYOMING

Index map

Montana

Wyoming

0 5 10 MILES

0 5 10 KILOMETERS

Base map data from ESRI and Montana State Library

Lambert Conformal Conic projection

North American Datum, 1983 (NAD 83)

EXPLANATION

In-situ toxicity sites

Map produced by

Montana Fish, Wildlife & Parks

Data Services Section

January 24, 2012

Figure 4-1. Field sites for 96-hour in situ toxicity experiments in the Powder River drainage, Wyoming.
68 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

site) Upper Beaver Creek, Lower Beaver Creek, and Burger 

Draw (experimental sites) with the same methods employed 

with FHM during 2006. The water quality monitoring design 

and methods were the same as described for 2006 in situ 

experiments (tables 4–5, 4–6, and 4–7); however, in addition to performing on site ammonia assays with a HachTM test 

kit, water samples were collected and acidified for ammonia 

analyses in the laboratory with the U.S. Environmental Protection Agency 350.1 methodology (U.S. Environmental Protection Agency, 1993) (table 4–8). 

In addition to the in situ experiments completed in the 

field in 2007, a simultaneous experiment was completed with 

site water in more controlled conditions. Water was collected 

at Clear Creek (reference site), Upper Beaver Creek, Lower 

Beaver Creek, and Burger Draw (experimental sites) and held 

in insulated 22-L containers. Four replicates of five fish were 

exposed to water from each site in 530-mL containers. The 

containers were kept in a water bath that fluctuated slightly 

with the room temperature (16–22°C), and water in the containers was replaced every 12 h.

Water samples intended for laboratory analyses were kept 

refrigerated in Buffalo, Wyoming, and transported in ice to the 

U.S. Geological Survey, Columbia Environmental Research 

Center, Jackson Field Research Station, Jackson, Wyoming 

(from here defined as Jackson Field Research Station). 

Samples were then sorted, and shipped in ice to the State of 

Montana Department of Health and Human Services Environmental Laboratory for analyses.

For both experiments in 2006, the survival data were 

analyzed with the Toxstat software package (Toxstat Version 

3.4, 1994). A T-test was performed between the reference sites 

(experiment 1). Because no difference was observed, the data 

were pooled and a one-way ANOVA was performed followed 

by a Bonferroni means comparison. Because one reference 

site was used during experiment 2, no pooling was necessary. 

T-tests were used to compare the survival of 2-dph against 

6-dph FHM at sites. All data met homogeneity and normality assumptions without transformations. In 2007, a one-way 

ANOVA was performed followed by a Bonferroni means comparison for 4-dph and 6-dph pallid sturgeon in situ, and 4-dph 

pallid sturgeon static-renewal experiments. All means were 

tested with a statistical criterion of alpha less than (α <) 0.05.

Potential differences in mean ammonia concentrations 

derived with field assays of chilled samples and the EPA 

method 350.1 laboratory assays were defined with T-tests in 

the Toxstat software package with a statistical criterion of α < 

0.05. No transformations were necessary as all data met homogeneity and normality assumptions without transformation. 

Mixing Zone and Site Water

Mixing zones were characterized at three locations of the 

Powder River/Tongue watersheds: downstream from the confluence of Beaver Creek and the Powder River; downstream 

from the untreated product water diffuser in the Tongue River 

Table 4–2. Mean water chemistry recorded with hand-held 

meters during in situ toxicity experiments with fathead minnows 

(Pimephales promelas) in the Powder River Basin in Wyoming 

and Montana.

[°C, degrees Celsius; μS/cm , microsiemens per centimeter; mg/L, milligrams per liter; ( ), standard deviation error; N, sample size]

Site Temperature

(°C)

Conductivity (μS/cm)

Dissolved 

oxygen 

(mg/L)

pH

Powder R. at 

Moorhead

27.4 3,616 7.6 8.3

(2.3) (131) (.5) (.2)

N=5 N=5 N=5 N=5

Clear Creek 21.6 857 8.9 8.5

(3.2) (49) (2.0) (.3)

N=9 N=9 N=9 N=8

Lower Beaver 21.9 2,843 8.8 9.3

(3.4) (82) (1.0) (.1)

N=9 N=9 N=9 N=4

Upper Beaver 20.3 2,612 8.0 9.0

(2.4) (38) (.08) (.1)

N=9 N=9 N=9 N=4

Burger Draw 21.9 3,649 8.3 9.0

(4.7) (30) (.6) (.1)

N=9 N=9 N=9 N=6

SA Creek 27.6 2,620 9.5 8.9

(2.5) (91) (2.0) (.2)

N=6 N=6 N=6 N=6

Table 4–3. Minimum and maximum temperature, conductivity, 

dissolved oxygen, and pH measurements recorded every 

30 minutes at four sites during in situ experiments with fathead 

minnows (Pimephales promelas) in the Powder River Basin, 

Wyoming and Montana. Data was recorded with YSI 5000TM

Data Sonde units.

[°C, degrees Celsius; μS/cm , microsiemens per centimeter; mg/L, milligrams per liter; NA, not applicable]

Site

Temperature

(°C)

Conductivity (μS/

cm)

Dissolved 

oxygen 

(mg/L)

pH

Powder River at 

Moorhead

21.34–

31.20

3,280–

4,142

NA 7.74–

8.38

Clear Creek 15.12–

29.51

669–891 5.59–

12.33

7.81–

8.73

Lower Beaver 14.13–

31.95

2,390–

2,644

5.14–

10.68

9.23–

9.35

SA Creek 16.21–

29.97

2,095–

2,892

NA 8.50–

8.97
Methods 69

Table 4-4. Additional trace metal scans analyzed with Inductively Coupled Plasma (ICP) in water samples collected 

from in situ experimental sites for fathead minnows (Pimephales promelas). Experiments completed in the Powder 

River Basin, Wyoming and Montana. Sample size is one for all constituents.

[<, less than detection limit; mg/L, milligrams per liter; ( ), date sample was taken (month/day/year); CaCO3, calcium carbonate]

Constituent (mg/L) Powder River at 

Moorhead (7/26/2006)

Clear Creek 

(7/27/2006)

Lower Beaver Creek 

(7/27/2006)

Upper Beaver Creek 

(7/27/2006)

Arsenic <0.001 <0.001 0.004 0.005

Barium .063 .045 .215 .079

Beryllium <.002 <.002 <.002 <.002

Calcium 150.000 88.800 10.600 14.600

Cadmium <.002 <.002 <.002 <.002

Chromium .004 .002 .003 .011

Copper .004 .007 .009 .005

Iron .100 .100 .380 .340

Magnesium 144.000 41.300 27.500 44.700

Manganese .048 .052 .011 .031

Sodium 525.000 69.600 773.000 599.000

Nickel <.010 <.010 <.010 <.010

Lead <.005 <.005 <.005 <.005

Selenium <.005 <.005 <.005 <.005

Zinc .011 .052 .058 .008

Total hardness as CaCO3 968.000 392.00 140.000 220.000

Table 4–5. Mean alkalinity expressed as milligrams per liter calcium carbonate equivalent, bicarbonate, sulfate, 

calcium, magnesium, sodium, and potassium, during in situ experiments with 4- and 6-day-post-hatch pallid sturgeon 

(Scaphirhynchus albus) performed in the Powder River Basin in Wyoming.

[mg/L, milligrams per liter; ( ), standard error; N, sample size]

Site

Alkalinity (mg/L 

as calcium 

carbonate)

Bicarbonate 

(mg/L)

Sulfate 

(mg/L))

Calcium 

(mg/L)

Magnesium 

(mg/L)

Sodium 

(mg/L)

Potassium 

(mg/L)

Clear Creek 145 165 322 84 36 58 5

(17) (16) (41) (8) (3) (6) (0.4)

N=5 N=5 N=5 N=5 N=5 N=5 N=5

Lower Beaver 1,419 1,513 289 16 35 681 19

(55) (59) (33) (1) (1) (17) (0.5)

N=4 N=4 N=4 N=4 N=4 N=4 N=4

Upper Beaver 1,468 1,626 163 16 29 650 20

(26) (11) (10) (1) (0.5) (14) (0.5)

N=4 N=4 N=4 N=4 N=4 N=4 N=4

Burger Draw 2,348 2,561 56 14 23 943 29

(47) (67) (11) (0.2) (0.5) (17) (0.2)

N=5 N=5 N=5 N=5 N=5 N=5 N=5
70 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

near Decker, Montana, not shown; and downstream from the 

treated product water diffuser (Higgins LoopTM Treatment) near 

Decker, Montana. Rhodamine WT dye was added at a constant 

drip into Beaver Creek and into the treated and untreated product water. At the Powder River, transects were spaced at 4, 8, 

16, 32, and 804 meters (m) below the confluence of Beaver 

Creek. On the Tongue River, transects were spaced 15, 30, 60, 

90 and 1,200 m, downstream from the diffusers for treated 

and untreated discharge. The dye concentration was measured 

in the water at evenly spaced locations (0.15–3 m) across the 

river, and spacing was dependent on the width of the river. The 

number of locations across the width ranged from 5–11 locations and the width of the river at site locations ranged from 3 

to 33.4 m. At each point along the transect, dye concentrations 

were measured with a YSI–600 oms sondeTM, equipped with a 

6130 Rhodamine WT optical probe. The concentration of the 

injected dye solution was measured in reference to a dilution 

series of the stock solution (Kilpatrick and Cobb, 1985). 

The depth of the mixing areas (shallow in the Powder 

River and deep in the Tongue River) and the complexity of the 

mixing action, made the logistics of completing an in situ toxicity experiment problematic. For this reason, it was decided 

to collect waters on site and complete site water experiments 

in the laboratory. 

Table 4–6. Mean temperature, conductivity, dissolved 

oxygen, and pH measurements recorded with hand-held 

meters during in situ toxicity experiments with pallid sturgeon 

(Scaphirhynchus albus) in the Powder River Basin in Wyoming.

[°C, degrees Celsius; μS/cm , microsiemens per centimeter; mg/L, milligrams per liter; ( ), standard deviation error; N, sample size]

Site Temperature

(°C)

Conductivity 

(μS/cm)

Dissolved 

oxygen 

(mg/L)

pH

Clear Creek 24.6 747 8.9 8.9

(.4) (53) (.4) (.1)

N=8 N=7 N=7 N=8

Lower Beaver 23.5 2,803 7.1 9.0

(1.8) (80) (.4) (.1)

N=6 N=5 N=5 N=6

Upper Beaver 24.9 2,716 8.8 9.0

(.6) (21) (.4) (.1)

N=6 N=5 N=5 N=6

Burger Draw 22.2 3,553 7.8 8.9

(.5) (158) (.2) (.1)

N=8 N=7 N=7 N=8

Table 4–7. Additional trace metal scans analyzed with Inductively Coupled Plasma (ICP) in water samples collected 

from in situ experimental sites with pallid sturgeon (Scaphirhynchus albus) completed in the Powder River Basin, 

Wyoming. Sample size is one for all constituents.

[mg/L, milligrams per liter; <, less than detection limit; ( ), date sample was taken (month/day/year); CaCO3, calcium carbonate]

Constituent (mg/L) Clear Creek 

(7/11/2006)

Burger Draw 

(7/11/2006)

Lower Beaver Creek 

(7/11/2006)

Upper Beaver Creek 

(7/11/2006)

Arsenic <0.001 0.001 <0.004 0.004

Barium .04 .220 .080 .170

Beryllium <.001 .001 <.001 .001

Calcium 92.800 13.600 15.900 14.600

Cadmium <.002 .002 <.002 .002

Chromium <.002 .002 <.002 .002

Copper .001 .001 .006 .002

Iron .140 .130 .830 .180

Magnesium 40.400 22.300 34.800 28.400

Manganese .029 .017 .017 .006

Sodium 72.400 986.000 71.000 664.00

Nickel <.01 <.010 <.010 .010

Lead <.001 <.001 <.001 <.0010

Selenium <.005 <.005 <.005 <.005

Zinc .010 .005 .012 <.005

Total hardness as CaCO3 398.000 126.00 183.00 154.000
Results 71

Experiments were completed with water collected from 

the Powder River and from Beaver Creek near the confluence with the Powder River, in Johnson County, Wyoming, 

not shown. Water also was collected from the Tongue River 

and two discharge points into the Tongue River in Big Horn 

County, Montana, not shown; one site contained treated (Higgins LoopTM ion exchange) coalbed natural gas (CBNG) product water, the other site contained untreated CBNG product 

water; water was collected immediately above their discharge 

point into the Tongue River. Water collected from all sites was 

placed in acid washed, 20-L HDPE containers, which were 

chilled and transported to the Jackson Field Research Station. 

The waters were used to complete 96-h static renewal experiments with 2-dph FHM September–October, 2007. 

Newly hatched FHM were shipped directly to the Jackson 

Field Research Station from Aquatic Biosystems, Fort Collins, 

Colorado. The brood stock were deemed free of disease and 

kept in isolation at the Colorado facility. The proper permits 

were obtained for importation of the offspring. At the Jackson 

Field Research Station, fish were acclimatized to Tongue River 

water and held at 20°C. 

Static-renewal experiments with the site waters were 

completed in 1-L glass beakers filled with 750 mL of site 

water and maintained in a water bath at 20°C. The fish were 

exposed for 96-h and the experimental water was replaced 

every 24-h; the replacement water was allowed to equilibrate 

to the exposure temperature in the water bath overnight. Water 

from Beaver Creek, untreated CBNG product water, and 

treated CBNG product water were experimented with 100-percent, 75-percent, and 50-percent concentrations. Beaver Creek 

water was diluted with Powder River water to prepare the 

proper concentrations, and the product waters were diluted 

with Tongue River water to prepare the proper concentrations. 

Waters collected from the Powder and Tongue Rivers served 

as reference waters for the experiments. Water temperature 

and dissolved oxygen were monitored twice daily, and water 

samples were collected daily for water chemistry from the 

100 percent (undiluted) and 50 percent diluted treatments 

(table 4–9).

Results

Ammonia concentrations were largest at Burger Draw 

and averaged 1.5–2.0 mg/L in 2006, and 1.17 mg/L in 2007. 

Ammonia concentrations did not exceed 0.5 mg/L at any other 

sites (table 4–8). Ammonia concentrations in samples measured in the field were greater than acidified samples measured 

in the laboratory. Differences were not significant between 

the methodologies at Burger Draw, Lower Beaver and Clear 

Creeks, however, differences were significant at Lower Beaver 

Creek (T=2.57).

Survival of 2-dph FHM in 2006 was 77 percent and 

78 percent in the Powder River at Moorhead and the Clear 

Creek sites, respectively. Because there was no significant 

difference in survival between the two sites, the data were 

combined to create a pooled reference (78 percent as pooled 

reference) for further comparisons. Survival was significantly 

less at all experimental sites (F=2.77) when compared to the 

pooled reference (table 4–10) during experiment 1. Survival of 

6-dph FHM was 90 percent at Clear Creek (reference site) and 

was not significantly different at any experimental site when 

compared to the reference site. 

Survival of 2-dph pallid sturgeon was low at all sites 

including the reference in 2007, and the experiment was terminated at 24 h. The experiment was re-started with 4-dph fish, 

and survival at Clear Creek (reference site) was 95 percent, 

whereas survival in all experimental sites was 0 percent (table 

4–11). Survival of 4-dph pallid sturgeon in a static-renewal 

experiment was similar to the in situ experiment, with reduced 

survival of fish exposed to product water (0 to 15 percent), and 

90 percent survival of fish held in Clear Creek reference water. 

An experiment completed with 6-dph fish provided similar 

results when compared to the 4-dph FHM, though time to 

death was longer with the older fish. 

Site Water/Mixing Zone Experiments

In the Powder River at the Beaver Creek confluence, dye 

concentrations, or otherwise stated as the part of the stream 

comprised of Beaver Creek discharge was greatest on the bank 

closest to the input source (table 4–12). At the first transect 

point, 0.3 m from the bank and 4 m below the confluence, the 

dye concentration was 122 μg/L or approximately 16 percent of the concentration of dye in undiluted Beaver Creek 

water. Dye concentrations across this transect 4 m below the 

confluence were; at 0.6 m, 106 μg/L or approximately 14 

percent; at 0.9 m, 65 μg/L or approximately 8 percent; at 1.2 

Table 4–8. Mean concentrations of ammonia, in water from 

experimental sites during in situ experiments performed in 

the Powder River Basin, Wyoming. Measurements were 

performed with a HachTM test kit in the field and with the EPA 

350.1 methodology (U.S. Environmental Protection Agency, 

1993) in the laboratory.

[mg/L, milligrams per liter; ( ), standard deviation error; N, sample size]

Site Ammonia 

(mg/L, laboratory)

Ammonia

(mg/L, field)

Clear Creek 0.01 (0) 0.01 (0)

N=3 N=5

Lower Beaver .01 (<.01) .4 (.23)

N=3 N=5

Upper Beaver .12 (.06) .29 (.12)

N=4 N=4

Burger Draw 1.05 (.12) 1.17 (.07)

N=5 N=5
72 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

m, 60 μg/L or approximately 8 percent; at 1.5 m, 25 μg/L or 

approximately 3 percent; at 1.8 m, 13 μg/L or approximately 

2 percent; at 2.1 m, 5μg/L or <1 percent. At the first transect 

approximately 22 percent of the river water was considered 

mixed with Beaver Creek water although the concentrations 

vary across the width of the river at this point. 

Dye concentrations at the transect 8 m below the confluence ranged from 58–2.5 μg/L or approximately 8–0.3 percent 

of the concentration of dye in undiluted Beaver Creek water 

(table 4–12). At 8 m below the confluence approximately 

40 percent of the stream was considered mixed with Beaver 

Creek water. At 16 m below the confluence of Beaver Creek, 

dye concentrations across the width of the river ranged from 

48–4.4 μg/L, or approximately 6–0.6 percent of the concentration of dye in undiluted Beaver Creek water. At the 16 m transect, approximately 55 percent of the stream was mixed with 

Beaver Creek water. At 32 m below the confluence of Beaver 

Creek, dye concentrations across the width of the river ranged 

from 37 to less than 1 μg/L, or approximately 5 to less than 

0.1 percent of the concentration of dye in undiluted Beaver 

Creek water. At the 32 m below the confluence, approximately 

75 percent of the stream was considered mixed with Beaver 

Creek water. At 804 m below the confluence of Beaver Creek, 

dye concentrations across the width of the river ranged from 

23–7 μg/L, or approximately 3–1 percent of the concentration of dye in undiluted Beaver Creek water. At 804 m below 

the confluence, approximately 100 percent of the stream was 

considered mixed with Beaver Creek water. 

At the untreated water outflow into Tongue River near 

Decker, Montana, dye concentrations 15 m downstream from 

the diffuser ranged from 55–0 μg/L, and was most concentrated in measurements taken closest to the diffuser, whereas 

three measurements located farthest from the diffuser outfall 

contained no dyed product water (table 4–13). At the first 

transect the Tongue River water was considered approximately 

30 percent mixed with untreated product water. At 30 m below 

Table 4–9. Mean alkalinity expressed as milligrams per liter calcium carbonate equivalent, bicarbonate, sulfate, calcium, 

magnesium, sodium, potassium, and total ammonia (mg N/L) during 96-hour static renewal exposures of 2-day-post-hatch fathead 

minnows (Pimephales promelas) to mixtures of waters collected in the Tongue and Powder River Basins, Wyoming and Montana, 

September 2007. Higgins LoopTM technology was used to treat product water before release at one experimental location. 

[CaCO3, calcium carbonate; mg/L, milligrams per liter; ( ), standard error; N, sample size; --, no standard deviation error when sample size is less than 3; NM, 

not measured]

Site Alkalinity 

(CaCO3, in mg/L)

Bicarbonate 

(mg/L)

Sulfate 

(mg/L)

Calcium 

(mg/L)

Magnesium 

(mg/L)

Sodium 

(mg/L)

Potassium 

(mg/L)

Total 

ammonia

(mg N/L)

Powder River 179 179 858 144 69 374 15 0.08

(4) (4) (37) (5) (3) (18) (1) (.06)

N=3 N=3 N=3 N=3 N=3 N=3 N=3 N=3

Tongue River 236 232 536 64 26 41 4 .03

(15) (17) (493) (2) (16) (17) (.4) --

N=3 N=3 N=3 N=3 N=3 N=3 N=3 N=2

Beaver Creek 1,233 1,097 354 13 29 553 16 .16

(76) (34) (151) -- -- -- -- --

N=3 N=3 N=3 N=2 N=2 N=2 N=2 N=2

Beaver Creek, 50 percent 638 604 586 33 49 499 15 NM

-- -- -- -- -- -- -- --

N=2 N=2 N=2 N=2 N=2 N=2 N=2 --

Treated 89 89 309 106 1.8 106 1.4 .13

(2.1) (2.1) (24) (3.3) (.5) (9.9) (.1) (.02)

N=3 N=3 N=3 N=3 N=3 N=3 N=3 N=3

Treated, 50 percent 199 195 221 81 16 63 2.8 NM

N=2 N=2 N=2 N=2 N=2 N=2 N=2 --

Untreated 1,560 1,420 97 9.7 5.6 678 8.5 2.0

(55) (25) (23) (1.2) (.8) (22) (.4) (.4)

N=3 N=3 N=3 N=3 N=3 N=3 N=3 N=3

Untreated, 50 percent 864 784 110 14 20 381 6.6 NM

N=2 N=2 N=2 N=2 N=2 N=2 N=2 --
Results 73

the confluence, dye concentrations across the width of the 

river ranged from 39–0 μg/L, and one of the transect points 

closest to the opposite stream bank contained no dyed product water. At 30 m, the Tongue River water was considered 

approximately 47 percent mixed with untreated product water. 

At 60 m, dye concentrations across the width of the river 

ranged from 25–0 μg/L, and one transect point closest to the 

opposite stream bank contained no dyed product water. The 

Tongue River water was considered approximately 47 percent 

mixed with untreated product water at this point. At 90 m, dye 

concentrations across the width of the Tongue River ranged 

from 23–3 μg/L, and was considered approximately 67 percent 

mixed with the untreated product water. At 1,200 m, dye 

concentrations across the width of the river ranged from 

19–6 percent and was considered approximately 100 percent 

mixed with untreated product. 

At the treated water outflow, dye concentrations measured 15 m downstream from the diffuser ranged from 

30–0 μg/L, and the dye was most concentrated in the center 

of the stream channel near the diffuser outfall (table 4–14). 

Points along the width of the river that were nearest to both 

stream banks contained no dyed product water. This first 

transect was considered approximately 49 percent mixed with 

treated product water. At 30 m, dye concentrations across the 

width of the Tongue River ranged from 37–0.6 μg/L. At 30 m 

below the confluence, the Tongue River water was considered 

approximately 49 percent mixed with treated product water. At 

60 m, dye concentrations across the width of the river ranged 

from 26–0 μg/L, with one transect point closest to the opposite stream bank containing no dyed product water. At 60 m 

below the confluence, the Tongue River water was considered 

approximately 56 percent mixed with treated product water. At 

90 m, dye concentrations across the width of the river ranged 

from 26–6 μg/L, and 1 transect point closest to the opposite stream bank contained no dyed product water. At 90 m 

below the confluence, the Tongue River water was considered 

approximately 66 percent mixed with untreated product water. 

At 1,200 m, dye concentrations across the width of the river 

ranged from 13–9 percent and at this point the Tongue River 

water was considered approximately 100 percent mixed with 

treated product water.

Survival of 2-dph FHM exposed to 100-percent Beaver 

Creek water and 100-percent untreated product water was 

reduced significantly when compared with Powder River and 

Tongue River reference sites. Survival of all other mixtures 

was not significantly different from the reference sites 

(fig. 4–2). 

Ammonia concentrations were below U.S. Environmental 

Protection Agency water-quality criteria for all exposures and 

dilutions except the untreated CBNG product water (table 4–9; 

U.S. Environmental Protection Agency, 1999). Ammonia concentrations were great enough in the untreated product water 

(mean = 2.0 mg/L) to contribute to mortality.

Table 4–10. Survival of fathead minnows (Pimephales 

promelas) during 96-hour in situ experiments in the Powder 

River Basin, Wyoming and Montana. Two experiments were 

performed with fathead minnows of different ages. Fish 

were 2-day-post-hatch (dph) at the initiation of experiment 

1 and were 6-day-post-hatch at the initiation of experiment 

2. Significant differences were defined between the pooled 

reference and experimental sites for experiment 1 and 

between Clear Creek (reference) and the experimental sites 

for experiment 2.

Site Age

Percent 

survival at

96 hours

Experiment 1

Powder River at Moorhead 2-dph 77

Clear Creek 2-dph 78

Pooled Reference 2-dph 78

SA Creek 2-dph 124

Burger Draw 2-dph 137

Upper Beaver 2-dph 111

Lower Beaver 2-dph 149

Experiment 2

Clear Creek 6-dph 90

Burger Draw 6-dph 73

Upper Beaver 6-dph 75

Lower Beaver 6-dph 75

1

Denotes significant difference [ANOVA (analysis of variance)]; 

(α = 0.05).

Table 4–11. Survival of pallid sturgeon (Scaphirhynchus albus) 

during 96-hour in situ experiments in the Powder River Basin, 

Wyoming. Fish were 4 or 6-day-post-hatch (dph) at the initiation 

of the experiments. Significant differences were defined 

between Clear Creek (reference) and the experimental sites.

Site Age Percent survival at 

96 hours

In situ

Clear Creek 4-dph 95

Lower Beaver 4-dph 10

Upper Beaver 4-dph 10

Burger Draw 4-dph 10

Static renewal

Clear Creek 4-dph 90

Lower Beaver 4-dph 110

Upper Beaver 4-dph 115

Burger Draw 4-dph 10

In situ

Clear Creek 6-dph 80

Lower Beaver 6-dph 10

Upper Beaver 6-dph 10

Burger Draw 6-dph 10

1

Denotes significant difference [ANOVA (analysis of variance)]; 

(α = 0.05).
74 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

0

0

20

40

60

Survival, in percent

80

100

24

Clear Creek 2-day post hatch

Powder River 2-day post hatch

SA Creek 2-day post hatch

Upper Beaver Creek 2-day post hatch

Burger Draw 2-day post hatch

Lower Beaver Creek 2-day post hatch

Clear Creek 6-day post hatch

Upper Beaver Creek 6-day post hatch

Burger Draw 6-day post hatch

Lower Beaver Creek 6-day post hatch

48

Time, in hours

EXPLANATION

72 96

Figure 4-2. Percent survival at 96 hours for in situ experiments started with 2-daypost-hatch (2-dph) or 6-day-post-hatch (6-dph) fathead minnows. Powder River 

drainage, July–August 2006.

Table 4–12. Dye concentrations as measured in transects across the Powder River downstream from the confluence of Beaver 

Creek. Beaver Creek entered on the right bank and distances at each transect were measured in meters from the right stream bank to 

the left stream bank.

[m, meter; μg/L, micrograms per liter; --, no measurment; <, less than]

Transect 1 (4 m) Transect 2 (8 m) Transect 3 (16 m) Transect 4 (32 m) Transect 5 (804 m)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

0.3 122 0.2 55 0.3 47.7 3.0 37 6.1 17

.6 106 .5 48 .6 38.7 4.6 32 7.6 19

.9 65 .8 38 .9 33.8 6.1 23 9.1 22

1.2 60 1.1 44 1.2 29.1 7.6 20 10.6 23

1.5 25 1.4 58 1.5 26.4 9.1 13 12.2 22

1.8 13 1.7 32 1.8 22.4 10.6 8 13.7 18

2.2 5 1.0 12 2.1 10.1 12.2 4 15.2 16

-- -- 2.3 7 2.4 4.4 13.7 3 16.7 15

-- -- 2.6 2.5 -- -- 18.2 <1 18.2 12

-- -- -- -- -- -- -- -- 19.8 11

-- -- -- -- -- -- -- -- 21.3 7
Discussion 75

Discussion

The value of in situ experiments as reviewed by Chappie 

and Burton (2000) cannot be understated for use in the Tongue 

and Powder River watersheds. The limitations of field experiments, including a lack of control for some physical variables, 

such as, temperature and turbidity, are precisely what give the 

experiments strong applicability. The daily fluctuations created by the diurnal cycle are a natural part of life in a prairie 

stream. The in situ experiments performed during this study 

were completed during mid-summer low-flow conditions, 

when days were long and fluctuations in temperature are most 

dramatic. Though care was taken to minimize lethal extremes 

(cover was provided and chambers were suspended in the 

water column), the natural cycle provided the setting that fish 

must endure in natural conditions. 

The results of the field experiments support laboratory findings where fathead minnow and pallid sturgeon had 

reduced survival when exposed to elevated concentrations 

of NaHCO3. The concentrations of NaHCO3 in the various 

sites studied were above 1,800 mg/L, a concentration above 

the determined lethal concentration that will cause 50 percent 

mortality or detrimental effect (LC50) of 1,718 mg/L and 

1,493 mg/L for pallid sturgeon and fathead minnow, respectively (chapter 2, Results). Untreated product water was the 

dominant constituent of water at these sites and likely reduced 

survival.

Table 4–13. Dye concentrations as measured in transects across the Tongue River downstream from diffusers injecting untreated 

coal bed natural gas product water. Diffusers were located on the right bank and distances at each transect were measured in meters 

from the right stream bank to the left stream bank.

[m, meter; μg/L, micrograms per liter; --, no measurment]

Transect 1 (15 m) Transect 2 (30 m) Transect 3 (60 m) Transect 4 (90 m) Transect 5 (1,200 m)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

4.3 35 3.0 39 3.0 23 3.0 23 3.0 19

10.3 30 6.1 22 6.1 25 6.1 21 6.1 16

13.4 55 9.1 29 9.1 23 9.1 16 9.1 15

16.4 0 12.2 10 12.2 13 12.2 9 12.2 12

19.5 0 15.2 0 15.2 5 15.2 10 15.2 14

22.5 0 -- -- 18.2 0 18.2 14 18.2 11

-- -- -- -- -- -- 21.3 11 21.3 9

-- -- -- -- -- -- 24.3 13 24.3 9

-- -- -- -- -- -- 27.4 3 27.4 10

-- -- -- -- -- -- -- -- 30.4 8

-- -- -- -- -- -- -- -- 33.4 6

Table 4–14. Dye concentrations as measured in transects across the Tongue River downstream from diffusers injecting treated coal 

bed natural gas product water. Diffusers were located on the right bank and distances at each transect were measured in meters 

from the right stream bank to the left stream bank.

[m, meter; μg/L, micrograms per liter; --, no measurment]

Transect 1 (15 m) Transect 2 (30 m) Transect 3 (60 m) Transect 4 (90 m) Transect 5 (1,200 m)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

Distance 

(m)

Dye 

(μg/L)

4.6 0 3.0 37 3.0 26 3.0 26 3.0 12

6.1 0 6.1 32 6.1 25 6.1 24 6.1 9.5

9.1 17 9.1 19 9.1 22 9.1 23 9.1 9

12.2 18 12.2 12 12.2 17 12.2 18 12.2 13

15.2 30 15.2 9 15.2 14 15.2 14 15.2 9.5

18.2 0 18.2 3 18.2 10 18.2 12 18.2 13

21.3 0 21.3 .7 21.3 3 21.3 11 21.3 10

-- -- 24.3 .6 24.3 2.8 24.3 10 24.3 9

-- -- -- -- 30.4 0 27.4 6 -- --
76 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

However, when the product water was treated with Higgins LoopTM technology, the acute toxicity of the water was 

removed. The Higgins LoopTM exchanges sodium for hydrogen 

ions (Severn Trent Services, Inc. 2010) and as a result, the 

NaHCO3 was reduced to less than 200 mg/L. This reduction 

accounted for the reduced toxicity. It also should be noted that 

the concentration of ammonia was reduced after treatment 

from 2.0 to 0.13 mg/L (table 4–9). Acute toxicity is minimized by the Higgins LoopTM treatment at the diffuser site near 

Decker, Montana. 

Age and species sensitivity also were documented in 

response to NaHCO3 in the field. Newly hatched 2-dph fish 

were the most sensitive age for FHM, and mortality was 

significantly greater when compared to the reference sites. The 

same species exposed as 6-dph fish were less sensitive, and 

differences in survival between experimental and reference 

sites were not significant. Pallid sturgeon were more sensitive 

than FHM, and 0 percent survival occurred in both 4-dph and 

6-dph pallid sturgeon at experimental sites. Therefore, pallid 

sturgeon had greater overall sensitivity to NaHCO3 (chapter 2 

Results). 

Johnson (2006) also completed in situ experiments with 

FHM during the summer of 2006. The researcher did not 

observe decreased survival at a site approximately 40 kilometers (km) upstream from the Upper Beaver site used during 

this study, although concentrations of NaHCO3 were greater 

than the LC50 defined previously with FHM (Mount and others, 1992; chapter 2). However, Johnson used fish that were 

12-dph to 14-dph. The Johnson results are similar to the lack 

of survival effects observed when 6-dph FHM were used in 

this study. 

Others have documented age sensitivity to various 

contaminants (Rand, 1995), and U.S. Environmental Protection Agency (2002a) guidelines for the assessment of chronic 

toxicity suggest the use of FHM <48 h old. However, current 

guidelines established in 2002 allow the use of FHM as old as 

14-dph to assess acute toxicity (U.S. Environmental Protection Agency, 2002a, U.S. Environmental Protection Agency 

2002b). Based on the results of this study, operators, managers 

and researchers may wish to take care when interpreting and 

comparing results gathered with various aged fish less than 

14-dph. 

Survival of 2-dph FHM exposed to site water collected 

from Beaver Creek in the laboratory was similar to that of 

FHM exposed in the field in 2006, with 40 percent surviving 

in controlled, laboratory conditions (fig. 4–3), and 49 percent 

surviving in the field study (table 4–10). The apparent sensitivity of 2-dph FHM compared with 6-dph FHM highlights the 

importance of experimenting with the most sensitive lifestages 

when evaluating the effects of toxicants.

Ammonia concentrations exceeded U.S. Environmental 

Protection Agency recommended maxima in Burger Draw in 

2006 (1.32 mg/L at pH 9.0) and may have contributed to mortality of FHM (U.S. Environmental Protection Agency, 1999). 

Elevated concentrations of ammonia in Burger Draw also were 

documented by Smith and others (2009) when the researchers calculated lethal concentrations of ammonia in June 2004. 

Smith and others (2009) also cautioned about the existence 

of a diel cycle for ammonia where concentrations would be 

greatest in mid to late afternoon and the smallest in pre-dawn 

hours. Samples collected during this study were collected late 

morning to late afternoon and thus, generally should have 

documented the largest concentrations of the diel cycle.

At all other experimental sites, ammonia concentrations 

were not above chronic criteria concentrations, and survival 

statistically was reduced when compared to the reference 

site. This indicates that although ammonia may have played a 

part in the mortality at Burger Draw, other toxicants were the 

likely causative agent at all other experimental sites for 2-dph 

FHM, and 4-dph and 6-dph pallid sturgeon. To further support 

this conclusion, water samples collected from Beaver Creek 

had 0.16 mg/L of total ammonia during the 2007 mixing zone 

analyses (compared to 0.4 mg/L in the 2006 in situ experiment) and survival was decreased in the experiment completed 

with the 2007 water (table 4–9, fig. 4–3).

Ammonia concentrations were measured with a HachTM 

test kit, and in the laboratory with the U.S. Environmental 

Protection Agency 350.1 methodology (U.S. Environmental 

Protection Agency, 1994) on water samples that had been 

collected and acidified for ammonia analyses. The concentrations were smaller in samples measured in the laboratory, with 

a significant difference between samples collected at Lower 

Beaver Creek (table 4–8). However the mean concentrations 

determined by both methodologies were below U.S. Environmental Protection Agency recommended maxima.

The mixing zone appeared to persist 804 m below the 

main stem of the Powder River and the confluence of Beaver Creek, and 1,200 m downstream from the discharge of 

untreated and treated product water into the Tongue River. 

However, the area containing concentrated untreated product 

water was limited to locations immediately downstream from 

the confluence of Beaver Creek in the Powder River, and 

the diffusers in the Tongue River. In the laboratory experiments with Tongue and Powder Rivers water, the toxicity 

of water from the untreated outflow and from Beaver Creek 

was reduced when the water was diluted to 75 percent of the 

source water with the appropriate receiving water (main stem 

Powder River or Tongue River). In the Powder River below 

Beaver Creek, and the Tongue River below the diffusers, the 

plume of water exceeding more than 75 percent produced 

water was limited during low-flow conditions. If the low 

volume of product water and the relatively large quantity of 

dilution water remain constant, the acute toxicity from individual discharges of product water will be limited. The effects 

of chronic exposures and the cumulative effects from multiple 

sources of product water would likely occur at concentrations 

lower than 75 percent product water, but were not addressed in 

these field experiments. 
References Cited 77

Figure 4-3. Percent survival at 96 hours for the mixing zone experiment started with 

2-day-post-hatch fathead minnows. Receiving water was added to experimental 

waters to provide the percent mixture indicated. Sample waters were collected from 

the Powder and Tongue Rivers watershed, September–October 2007.

0 20

40

60

Survival, in percent

80

100

20

Tougue River water

Powder River water

Beaver Creek

Treated produced water

Untreated produced water

75 percent treated produced water

75 percent untreated produced water

75 percent Beaver Creek water

50 percent treated produced water

50 percent untreated produced water

50 percent Beaver Creek water

40

Time, in hours

EXPLANATION

60 80 100

Summary

The combination of in situ experiments, static-renewal 

experiments performed simultaneously with in-situ experiments, and static renewal experiments performed with site 

water in the laboratory, demonstrated that coalbed natural gas 

(CBNG) product water reduces survival of fathead minnow 

(FHM) and pallid sturgeon. Age affected survival of FHM 

exposed to CBNG product water where fish 2-days-post-hatch 

(dph) were more sensitive than 6-dph fish, but pallid sturgeon 

survival was adversely affected at 4- and 6-dph. Ammonia 

likely contributed to toxicity in Burger Draw.

These results support the laboratory findings and LC50

determinations that will cause 50 percent mortality or detrimental effects defined in chapter 2, Results, and by Mount and 

others (1992). Therefore, the survival of early-lifestage fish, 

especially those <6-dph, likely is reduced significantly in 

the field when concentrations of NaHCO3 rise to more than 

1,500 mg/L. 

The study results also determined that treatment with 

the Higgin’s LoopTM technology and dilution of untreated 

water increased survival in the laboratory. It should be noted 

that both of these situations reduced ammonia in addition to 

concentrations of NaHCO3. Finally, the mixing zones of the 

three outfalls studied ranged from approximately 800–1,200 m 

below the confluence and the areas within these mixing zones 

with acutely lethal concentrations of NaHCO3 (as defined by 

the presence of concentrated dye) are limited and variable 

within each zone. These experiments addressed the acute 

toxicity of effluent waters being added to the main stem rivers, 

but did not address issues related to the volumes of water that 

may be added to the watershed. 

References Cited

American Public Health Association Standard Methods, 

1975a, Alkalinity titration method for water and wastewater, (14th ed.): Washington, D.C., American Public Health 

Association, p. 278.

American Public Health Association Standard Methods, 

1975b, Hardness, Total, (14th ed.): Washington, D.C., 

American Public Health Association, p. 202.

Chappie, D.J., and Burton, G.A, 2000, Applications of aquatic 

and sediment toxicity testing In situ: Soil and Sediment 

Contamination, v. 9, p. 219–245.
78 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Farag, A.M., Skaar, D., Nimick, D.A., MacConnell, E., and 

Hogstrand, C., 2003, Characterizing aquatic health using 

salmonid mortality, physiology, and biomass estimates in 

streams with elevated concentrations of arsenic, cadmium, 

copper, lead, and zinc in the Boulder River Watershed, 

Montana: Transactions of the American Fisheries Society, 

v. 132, p. 450–467.

Harper, D.D., Farag, A. M., and Brumbaugh, W.G., 2008, 

Effects of acclimation on the toxicity of stream water 

contaminated with zinc and cadmium to juvenile cutthroat 

trout: Archives of Environmental Contamination and Toxicology, v. 54, p. 697–704. 

Henry, T.B., Irwin, E. R., Grizzle, J. M., Brumbaugh, W. G., 

Wildhaber, M. L., 2001, Gill lesions and death of bluegill in 

an acid mine drainage mixing zone: Environmental Toxicology and Chemistry, v. 20, p. 1,304–1,311. 

Johnson, Laurie A., 2006, Longitudinal changes in potential 

toxicity of coalbed natural gas produced water along Beaver 

Creek in the Powder River Basin, Wyoming: Laramie, University of Wyoming, Master’s Thesis, 165 p.

Kilpatrick, F.A., and Cobb, E.D., 1985, Measurement of 

discharge using tracers: U.S. Geological Survey Techniques 

of Water-Resources Investigations, book 3, chap. A16, 52 p. 

(Also available at http://pubs.usgs.gov/twri/twri3-a16/.)

Patz, M. J., Reddy, K.J., and Skinner, Q.D., 2004, Chemistry 

of coalbed methane discharge water interacting with semiarid ephemeral stream channels: Journal of the American 

Water Resources Association, v. 40, no. 5, p. 1,247–1,255. 

Rand, G.M., ed., 1995, Fundamentals of Aquatic Toxicology 

(2nd ed.): Philadelphia, Taylor and Francis, 1,125 p.

Severn Trent Services Inc, 2010, DOWEX ion 

exchange resins: Tampa, Severn Trent Services 

Inc, accessed April 25, 2010, at http://msdssearch.

dow.com/PublishedLiteratureDOWCOM/

dh_0032/0901b803800325f0.pdf?filepath=liquidseps/pdfs/

noreg/177-02016.pdf&fromPage=GetDoc, 4 p. 

Smith, R.L., Repert, D.A., and Hart, C.P., 2009, Geochemistry 

of inorganic water released from coalbed natural gas production wells in the Powder River Basin, Wyoming: Environmental Science and Technology, v. 43, p. 2,348–2,354. 

Stewart, A.J., 1996, Ambient bioassays for assessing waterquality conditions in receiving streams: Ecotoxicology, v. 5, 

p. 377–393.

Toxstat Version 3.4, 1994, Cheyenne, Western Ecosystem 

Technology, Inc.

U. S. Environmental Protection Agency, 1993, Determination 

of ammonia nitrogen by semi-automated colorimetry, in

O’Dell, J.W., ed., Method 350.1: Cincinnati, U. S. Environmental Protection Agency, 15 p.

U. S. Environmental Protection Agency, 1994, Short term 

methods for estimating the chronic toxicity of effluents 

and receiving waters to aquatic organisms, in Lewis, P.A., 

Klemm, D.J., Lazorchak, J.M., Norberg-King, T.J., Peltier, 

W.H., and Heber, M.A., eds., Duluth, U.S. Environmental 

Protection Agency, EPA/600/4 -91/002, 315 p.

U.S. Environmental Protection Agency, 1999, Water Quality 

Criteria: Washington, D.C., accessed April 2010 at www.

epa.gov/waterscience/criteria/ammonia.

U.S. Environmental Protection Agency, 2002a, Short-term 

methods for estimating the chronic toxicity of effluents and 

receiving waters to aquatic organisms, (4th ed.): Washington, D.C., U.S. Environmental Protection Agency EPA–

821–R–02–13, 335 p.

U.S. Environmental Protection Agency, 2002b, Methods 

for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms, (5th ed.): 

Washington, D.C., U.S. Environmental Protection Agency, 

EPA–821–R–02–012, 266 p.
Presence/Absence of Native and NonNative Species of Fish in the Powder and 

Tongue River Basins

By David D. Harper, Aïda M.Farag, Trevor Selch, and Don Skaar

Chapter 5 of

The Potential Effects of Sodium Bicarbonate, a Major Constituent of 

Produced Waters from Coalbed Natural Gas Production, on Aquatic 

Life

Edited by Aïda M. Farag and David D. Harper

Prepared in cooperation with Montana Fish, Wildlife, and Parks, U.S. Bureau of 

Land Management, and the U.S. Environmental Protection Agency

Scientific Investigations Report 2012–5008

U.S. Department of the Interior

U.S. Geological Survey
Contents

Introduction...................................................................................................................................................81

Methods.........................................................................................................................................................81

Results ...........................................................................................................................................................83

Discussion.....................................................................................................................................................85

References Cited..........................................................................................................................................92

Figures

5–1. Map showing fish collection and habitat assessment sites in the Tongue and Powder 

Rivers, Wyoming and Montana ................................................................................................82

5–2. Bar graph showing the number of sites where each fish species was collected in 

the Tongue and Powder River drainages, Wyoming and Montana....................................83

5–3. Pie diagram showing proportion of each fish species captured in the Tongue River 

drainage .......................................................................................................................................86

5–4. Pie diagram showing proportion of each fish species captured in the Powder River 

drainage .......................................................................................................................................91

Tables

5–1. Native fish species expected to occur and be present or absent, and non-native fish 

present at sites in the Tongue River drainage in Wyoming and Montana........................84

5–2. Native fish species expected to occur and present or absent, and non-native fish 

present at sites in the Tongue River drainage in Wyoming and Montana........................87

5–3. Habitat characteristics associated with native fish distributions in the Tongue River 

watershed, Montana and Wyoming ........................................................................................88

5–4. Habitat characteristics associated with native fish distributions in the Powder River 

watershed, Montana and Wyoming ........................................................................................89

5–5. Habitat characteristics include the folowing; ability to withstand intermittent flow 

regime stream width, turbidity determined by the ability to see the bottom of a 3-ft 

deep pool during baseflow conditions, substrate composition, presence of cover, 

presence of backwater/side-channels (including those with aquatic vegetation), 

and the absence of predators in a reach ...............................................................................90

5–6. Index of Biotic Integrity (IBI) (Bramblett and others 2005) scores for fish assemblages 

at Powder and Tongue River sampling locations in Wyoming and Montana...................91
Introduction

Before coalbed natural gas (CBNG) development and 

the associated increases of groundwater discharge, fisheries 

assessments in the Tongue and Powder River Basins were 

infrequent. Elser and others (1980), Baxter and Stone (1995), 

and Patton (1997) provided some of the earliest descriptions 

of species composition and habitat associations for species 

found in the Tongue and Powder River Basins. Clearwater 

and others (2002) suggested that an increase in salinity from 

product waters of CBNG production might increase salinity 

of the upper reaches of the Tongue River. They also suggested 

that an increase in discharge might affect the habitat quality 

of these reaches for fishes. Therefore, baseline data of species 

distribution are needed in these watersheds. 

It also follows a study designed to document the presence 

or absence of the fish species that were used in experiments 

would provide valuable information about the current distribution of the test species in the field. It would be optimal to 

repeat the field investigations with time; however, a snapshot 

approach is not without merit. A one-time effort would provide 

information about the status of the species in question during 

that year. These data could then be used to alert researchers 

who may be involved in multiple-year efforts.

The composition and abundance of fish assemblages in 

the Tongue and Powder River Basins have been the focus of 

recent studies (Stagliano, 2006; Peterson and others, 2009; 

Peterson and others, 2010). Those studies were completed 

primarily to document and quantify the existing populations of 

resident and non-native fish species. The purpose of this study 

is to document the presence or absence of fathead minnow 

(Pimephales promelas) (FHM) and white sucker (Catostomus 

commersoni) to determine whether or not the selection of 

these species was appropriate for use in toxicity experiments 

(chapters 2, 3 and 4). Additionally, this research will provide 

a “snapshot” of the fish assemblage in the Tongue and Powder 

River Basins early into the development of CBNG. 

Methods

Fish and habitat surveys were completed in the Tongue 

and Powder River Basins July 19 through July 25, 2004. Fish 

and habitat measurements were taken in general conformity to 

U.S. Environmental Protection Agency Rapid Bioassessment 

protocols (Barbour and others, 1999). The method of fish capture typically was seining, with electrofishing used at a limited 

number of sites. Habitat measurements were modified to 

allow the data to be used in the Wyoming Warmwater Stream 

Assessment (WWSA) (Quist and others, 2006) and Montana 

Prairie Stream Index of Biotic Integrity (IBI) (Bramblett and 

others, 2005). 

The WWSA protocol was used to predict the presence 

or absence of a species at a site based on historic distribution 

and physical habitat. The absence of a species at a site may be 

related to poor physical habitat rather than high salinity. The 

WWSA approach allows qualification of the “absence” side of 

the equation. 

The framework for the WWSA assessment is described 

by Quist and others (2005), and Quist and others (2006). 

These methods provide information on the probable historic native fish distribution in a stream reach, which can be 

compared to the current (or sampled) native fish assemblage. 

An understanding of the expected fish assemblage in a stream 

reach can be used to evaluate factors that may be acting on 

the current fish assemblage. The comparison of predicted 

to observed fish assemblages can indicate changes in community structure because of natural or anthropogenic effects 

(Lipsey, 2001). For example, if a fish species is predicted in 

a stream reach based on its historic distribution, along with 

the elevation, stream size, and habitat requirements, but is not 

represented in a sample of the fish community; other factors 

may contribute to the distribution of that species (for example, 

water quality, predators, land management).

Because fish often have specific habitat requirements, 

their occurrence can be predicted based on the presence 

of habitat characteristics in a stream reach. Lee and others 

(1980), Holton (1990), and Baxter and Stone (1995) were used 

as sources for measurements of general habitat characteristics 

required and preferred by native fishes in the Powder and 

Tongue Rivers. 

Presence/Absence of Native and Non-Native Species of 

Fish in the Powder and Tongue River Basins

By David D. Harper1, Aïda M.Farag1, Trevor Selch2, and Don Skaar2

1

USGS, Columbia Environmental Research Center, Jackson Field Research 

Station, Jackson, Wyoming.

2

Montana Fish, Wildlife, and Parks, Helena, Montana.
82 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Figure 5–1. Fish collection and habitat assessment sites in the Tongue and Powder Rivers, Wyoming and Montana.

Tongue River

at Miles City Powder River at Locate

Tongue River

at Brandenburg

Little Powder River

Powder River

at Moorhead

Tongue River at Birney

Hanging

Woman Creek

Crazy Woman Creek

Clear

Creek

Prairie Dog Creek

Powder River at Sussex

Tongue River at Monarch

Goose Creek

Tongue River at stateline

Tongue River

belowreservoir

Goose Creek

Beaver Creek

Prairie

Dog Creek

Clear Creek

Hanging

Woman Creek

Burger Draw

Bighorn

River

Little Powder River

Crazy

Woman Creek

Big Porcupine

Creek

O'fallon Creek

Mizpah Creek

Rosebud Creek

Tongue River

Powder River

Yellowstone

River

Forsyth

Miles City

Broadus

Busby

Birney Day School

Ashland

Locate

Buffalo

106°

46°

44°

MONTANA

EXPLANATION

Index map

Montana

Wyoming

Sampling site

Map produced by

Montana Fish, Wildlife & Parks

Data Services Section

January 24, 2012

WYOMING

107° 105°

45°

Base map data from ESRI and Montana State Library

Lambert Conformal Conic projection

North American Datum, 1983 (NAD 83)

0 10 20 MILES

0 10 20 KILOMETERS
Results 83

Stream reaches were evaluated based on conformity 

between the habitat requirements of native fishes and the habitat characteristics in the stream reach. This information can be 

used to predict what fishes may be expected in a stream reach. 

As appropriate, the IBI scores were used to help interpret the 

presence/absence analyses. Sites with relatively small IBI 

scores typically are interpreted to have native fish assemblages 

that are affected by anthropogenic influences such as habitat, 

water-quality degradation and the presence of non-native fish 

species. 

Results

Fisheries assessments were performed at 15 sites in the 

Tongue and Powder River Basins. In the Tongue River drainage, six sites were sampled in the main stem and three sites 

in major tributaries. In the Powder River drainage, six sites 

were sampled with three in the main stem, and three in major 

tributaries (fig. 5–1). At all sites combined, 17 native species 

and 8 non-native species were collected and a total of 4,620 

fish were sampled. White sucker, shorthead redhorse, and 

common carp were collected at the most sites (13, 11 and 10, 

respectively; fig. 5–2). When data from both drainages were 

combined, the numbers of fish collected were greatest for sand 

shiners, flathead chub, and white suckers and were (2,221, 

715, and 371, respectively).

In the Tongue River drainage, 16 native and 7 non-native 

species were collected (table 5–1; fig. 5–3). The number of 

species ranged from 12 on the Tongue River at Miles City; 

Tongue River at Brandenburg; and Tongue River at Stateline 

to 4 at Prairie Dog Creek. Number of individuals sampled at 

each site ranged from 262 at the Tongue River at Miles City to 

23 on the Tongue River below the reservoir (because of limited sampling). White sucker, carp and stonecat were collected 

at the most sites (eight of nine sites). In terms of fish numbers 

collected in the Tongue River drainage; white suckers, rock 

bass and flathead chub occurred in the greatest numbers with 

326, 150, and 139, respectively.

In the Powder River drainage, a total of 13 native species 

and 5 non-native species were found (table 5–2; fig. 5–4). 

The number of fish species was greatest in Clear Creek 

(12 species) and smallest in the Powder River at Locate; 

Powder River at Moorhead; and Powder River at Sussex, (5 

species). Sand shiners, flathead chub and white suckers were 

collected at the most number of sites in the Powder River 

drainage (6, 6, and 5 sites, respectively). Sand shiners were the 

most frequently collected species. Total number of individuals 

was greatest at Crazy Woman Creek, (1,216) and smallest at 

Clear Creek, (221). 

White sucker

Shorthead redhorse

Common carp

Flathead chub

Sand shiner

Stonecat

Longnose dace

Green sunfish

Black bullhead

Flathead minnow

Channel catfish

River carpsucker

Smallmouth bass

Rockbass

Yellow bullhead

Plains minnow

Plains killfish

Longnose sucker

Creek chub

Goldeye

White crappie

Pumpkinseed

Spotttail shiner

Western silvery minnow

Emerald shiner

14

12

Number of sites where species were collected 10

8

6

4

2

0

Figure 5–2. The number of sites where each fish species was collected in the Tongue and Powder 

River drainages, Wyoming and Montana. Total number of sample sites was 15.
84 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Table 5–1. Native fish species expected to occur and be present or be absent, and non-native fish present at sites in the 

Tongue River drainage in Wyoming and Montana. 

[( ), number of fish collected]

Site Present Expected/absent Present/non-native

Tongue River Below Reservoir 

(TRBR)

Brown bullhead (1) Carp (4)

White sucker (1) Green sunfish (6)

Yellow bullhead (3) Smallmouth bass (3)

River carpsucker (1)

Tongue River at Monarch (TRM) Shorthead redhorse (2) Mountain sucker Green sunfish (1)

Longnose sucker (1) Fathead minnow Rock bass (81)

Longnose dace (1) White sucker Common carp (1)

Stonecat (2)

Black bullhead (1)

Tongue River at MT/WY stateline (TRSL)Black bullhead (5) Lake chub Rock bass (26)

White sucker (10) Longnose dace Spottail shiner (2)

Shorthead redhorse (1) Fathead minnow Smallmouth bass (5)

Stonecat (2) Channel catfish White crappie (33)

Yellow bullhead (12) Goldeye Common Carp (4)

River carpsucker

Sauger

Emerald shiner

Tongue River at Birney Bridge 

(TRBB)

White sucker (156) Lake Chub

Shorthead redhorse (32) Mountain sucker

Yellow bullhead (5) Longnose dace

Stonecat (1) Fathead minnow

Longnose dace (1) River carpsucker

Sauger

Brassy minnow

Tongue River at Birney Bridge 

(TRBB)

White sucker (156) Lake chub Smallmouth bass (2)

Shorthead redhorse (32) Mountain sucker Rockbass (4)

Yellow bullhead (5) Longnose dace Common carp (5)

Stonecat (1) Fathead minnow

Longnose dace (1) River carpsucker

Sauger

Brassy minnow

Tongue River at Brandenburg 

Bridge (TRBR)

Channel catfish (1) Lake chub Green sunfish (1)

White sucker (109) Goldeye Common carp (23)

Flathead chub (6) Sauger

Longnose dace (2) Shovelnose sturgeon

Sand shiner (4)

Shorthead redhorse (7)

River carpsucker (3)

Yellow bullhead (3)

Fathead minnow (11)

Stonecat (1)
Discussion 85

Habitat assessments completed during fish collections 

are summarized in tables 5–3, 5–4 and 5–5. IBI scores were 

calculated for 14 sites (table 5–6). 

Discussion

White suckers and fathead minnows frequently were captured in the Tongue and Powder River Basins. White suckers 

were the most common species captured in terms of distribution (found at 13 of 15 sites) and numbers of fish collected 

in the Tongue River drainage. Whereas the proportion of 

sample sites with white suckers was not as large in their study, 

Peterson and others (2009, 2010) documented white suckers 

to be relatively abundant and widespread in the Tongue and 

Powder River drainages. This differs from Stagliano (2006), 

who indicated white suckers were not collected in the main 

stem Powder River at six sites in Montana. However, all sites 

sampled by Stagliano (2006) were in the main stem Powder 

River where white sucker are less numerous. Both this study 

and Peterson and others (2009 and 2010) documented that 

that the main stem Powder River had less abundance and 

frequency of occurrence of white sucker when compared to 

tributary sampling sites. 

 Fathead minnows were not as common as white suckers, but were collected at 5 of 15 sites (3 in the Tongue River 

drainage, 2 in the Powder River drainage). They were captured more frequently in tributaries than main stem sites in 

the Tongue and Powder River drainages. This is similar to 

Peterson and others (2009 and 2010) who found FHM to be, 

relatively, common in Tongue and Powder River tributaries. 

Stagliano (2006) did not collect fathead minnow during surveys of the Middle Powder River in Montana.

The presence of white suckers and FHM in tributary 

streams supports their selection for use as experimental organisms in acute and chronic toxicity experiments. Produced 

water entering the main stem Tongue and Powder Rivers is 

rapidly diluted (chapter 4, Results). However, product water 

that enters small tributaries may remain at effect concentrations until diluted upon entering larger bodies of water. Therefore, exposure to elevated concentrations of dissolved salts 

from produced water, such as sodium bicarbonate, is most 

likely to occur in tributaries. 

Fish assemblages in the Tongue and Powder River Basins 

were generally similar to the findings of Stagliano (2006), 

Table 5–1. Native fish species expected to occur and be present or absent, and non-native fish present at sites in the 

Tongue River drainage in Wyoming and Montana.—Continued

[( ), number of fish collected]

Site Present Expected/absent Present/non-native

Goose Creek (GCR) Shorthead redhorse (2) Lake chub Smallmouth bass (3)

White sucker (4) Mountain sucker Common carp (7)

Stonecat (20) Longnose dace Rock bass (39)

Black bullhead (4) Longnose sucker

Fathead minnow (1)

Prairie Dog Creek (PDCR) White sucker (10) Lake chub White crappie (3)

Creek chub (6) Stonecat

Shorthead redhorse (1) Goldeye

Hanging Woman Creek 

(HWCR)

Fathead minnow (123) Lake chub Green sunfish (24)

Black bullhead (2) Yellow bullhead Common carp (79)

White sucker (28) Brassy minnow

Tongue River at Miles City 

(TRMC)

Western silvery minnow (53) Fathead minnow Common carp (4)

Stone cat (24) Black bullhead

Flathead chub (133) Plains minnow

White sucker (8) Sauger

Longnose dace (2) Yellow bullhead

Shorthead redhorse (2)

Sand shiner (27)

Emerald shiner (1)

River carpsucker (1)

Channel catfish (6)

Goldeye (1)
86 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

Peterson and others (2009), and Peterson and others (2010). 

Although site locations were not identical, sand shiners were 

the most frequently collected species in the Powder River in 

all four studies. Generally, rock bass, smallmouth bass, common carp and green sunfish were present at more sites and in 

greater numbers in tributaries than in the main stem sites. The 

species composition in each study was slightly different, but 

these differences may be attributed to the general stochasticity of these systems. The Tongue River and Powder River are 

prairie streams draining watersheds with highly erodible soils, 

and are subject to extreme variations in discharge, which occur 

seasonally (from spring snow melt flow to extreme drought in 

the summer), and as a result of violent prairie thunderstorms 

with intense periods of precipitation (Poff and Ward, 1989). 

In addition, the underlying substrate of both streams is made 

up of, in large part, sand and fine sediments. The extreme 

variability and stochastic nature of the climate, combined with 

the unstable substrate underlying both streams has resulted in 

a fish community that is highly adaptable and variable from 

season to season (Grossman and others, 1982). 

The Wyoming Warmwater Stream Assessment (WWSA) 

uses a series of physical assessments (for example, habitat 

measurements and sampling at representative sites), land 

use and historical data (for example, road density, irrigation 

withdrawal, reservoir construction) to predict the presence and 

absence of native fish species (Quist and others, 2005). The 

Montana Prairie Stream Index of Biotic Integrity (IBI) uses 

fish assemblage data to assess the condition of plains streams 

to assess anthropogenic disturbance (Bramblett and others, 

2005). These attributes include but are not limited to the numbers of native and non-native species found at fish collection 

sites. 

The WWSA and IBI models were in agreement at many 

sites. The Tongue River at Miles City and Crazy Woman 

Creek had large numbers of native fish species, low numbers 

of expected but absent species, and few non-native species. 

These sites also had the largest IBI scores. On the other end 

of the spectrum, Hanging Woman Creek, Tongue River at 

Stateline, and Powder River at Moorhead had few native fish 

species and many expected/absent and or non-native species, 

and these sites had the smallest IBI scores. At other sites, such 

as the Tongue at Brandenburg, Tongue River at Birney, Goose 

Creek, Tongue at Monarch, and the Little Powder River, 

the numbers of native species, expected/absent and present 

White sucker

Shorthead redhorse

Common carp

Flathead chub

Sand shiner

Stonecat

Longnose dace

Green sunfish

Black bullhead

Flathead minnow

Channel catfish

River carpsucker

Smallmouth bass

Rockbass

Yellow bullhead

Longnose sucker

Creek chub

Goldeye

White crappie

Pumpkinseed

Spottail shiner

Western silvery minnow

Emerald shiner

Figure 5–3. Proportion of each fish species captured in the Tongue River drainage. Fish were collected by seining and 

electrofishing .
Discussion 87

Table 5–2. Native fish species expected to occur and present or absent, and non-native fish present at sites in the Tongue 

River drainage in Wyoming and Montana. 

[( ), number of fish collected]

Site Present Expected/absent Present/non-native

Powder River at Sussex (PRSX) Plains minnow (2) Lake chub Plains killifish (24)

Sand shiner (413) Fathead minnow

Flathead chub (31) Burbot

White sucker (5) Black bullhead

Channel catfish

Goldeye

River carpsucker

Sauger

Sturgeon chub

Western silvery minnow

Powder River at Locate (PRLM) White sucker (1) Goldeye Plains killifish (4)

Sand shiner (109)

Flathead chub (350)

Plains minnow (4)

Powder River at Moorhead 

(PRMD)

Sand shiner (37) Goldeye

Flathead chub (176)

Longnose dace (14)

Stonecat (12)

Channel catfish (7)

Clear Creek (CLCR) Sand shiner (138) Lake chub Rock bass (34)

Goldeye (2) Longnose dace Common carp (21)

White sucker (1) Burbot Green sunfish (4)

Stonecat (10) Smallmouth bass (1)

Shorthead redhorse (3)

Creek chub (3)

Flathead chub (2)

River carpsucker (2)

Crazy Woman Creek (CWCR) Sand shiner (1,082) Channel catfish

Fathead minnow (16) goldeye

Black bullhead (3)

White sucker (6)

Longnose dace (35)

Flathead chub (13)

Plains minnow (61)

Little Powder River (LPR) River carpsucker (2) Lake chub Common carp (86)

Channel catfish (4) Black bullhead Green sunfish (6)

Shorthead redhorse (4) Goldeye

White sucker (30) Yellow bullhead

Flathead chub (4)

Longnose dace (1)

Sand shiner (411)

Fathead minnow (235)
88 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life Table 5–3. Habitat characteristics associated with native fish distributions in the Tongue River watershed, Montana and Wyoming. Habitat characteristics include the following: ability to withstand intermittent flow regime stream width, turbidity determined by the ability to see the bottom of a 3-ft deep pool during baseflow conditions, substrate composition, presence of cover, presence of backwater/side-channels (including those with aquatic vegetation), and the absence of predators in a reach. [m, meters; Y, yes; N, no; >, greater than; <, less than; H, high; L, low; SA, sand; GR, gravel; CB, cobble; BL, boulder; AV, aquatic vegetation. Empty boxes indicate not present or not applicable] Species Width (m) Stream characteristics Intermit- tent flow Turbidity Pools (> 0.5 m) Pools (> 0.5 m) Riffles Runs Backwater/ sidechannel Preda- tors absent

Y

N H L SA GR CB BL Cover SA + 

cover

GR + 

cover

CB + 

cover

GR CB BL GR CB BL Present With AV

Black bullhead <20.0 x x x x

Brassy minnow1 <35.0 x x x x x x

Channel catfish >4.5 x x

Creek chub >1.0 x x x x

Emerald shiner1 >29.0 x x x x

Fathead minnow >1.0 x x

Flathead chub >1.5 x x x x x

Goldeye >4.5 x x x

Lake chub <60.0 x x

Longnose dace > 0.5 x x x x x

Longnose sucker >1.0 x x x x x x x x x

Mountain sucker > 0.5 x x x x x x x x x

Northern redhorse >4.5 x x x x x x x x

Plains minnow >4.5 x x x x x

River carpsucker >11.0 x x x x

Sand shiner >1.0 x x x x x

Sauger >8.0 x x

Shovelnose sturgeon >24.0 x x x x x

Stonecat > 1.0 x x

Sturgeon chub >11.0 x x x x x x

Western silvery 

minnow

>11.0 x x x x x x

White sucker >1.0 x x x x x x x x

Yellow bullhead1 <20.0 x

1Indicates fish found only in Montana.
Discussion 89

Table 5–4. Habitat characteristics associated with native fish distributions in the Powder River watershed, Montana and Wyoming. Habitat characteristics include the following: ability to withstand intermittent flow regime stream width, turbidity determined by the ability to see the bottom of a 3-ft deep pool during baseflow conditions, substrate composition, presence of cover, presence of backwater/side-channels (including those with aquatic vegetation), and the absence of predators in a reach.[m, meters; Y, yes; N, no; >, greater than; <, less than; H, high; L, low; SA, sand; GR, gravel; CB, cobble; BL, boulder; AV, aquatic vegetation. Empty boxes indicate not present or not applicable]

Species Width (m)

Stream characteristics

Intermittent flow Turbidity Pools (> 0.5 m) Pools (> 0.5 m) Riffles Runs Backwater/ sidechannel Predators 

absent Y N H L SA GR CB BL Cover SA + 

cover

GR + 

cover

CB + 

cover

GR CB BL GR CB BL Present With AV

Black bullhead <20.0 x x x xBrassy minnow1 <35.0 x x x x x xBurbot >10.5 x xChannel catfish >4.5 x xCreek chub >1.0 x x x xFathead minnow >1.0 x xFlathead chub >1.5 x x x x xGoldeye >4.5 x x xLake chub <60.0 x xLongnose dace >0.5 x x x x xLongnose sucker >1.0 x x x x x x x x xMountain sucker >0.5 x x x x x x x x xNorthern redhorse >4.5 x x x x x x x xPlains minnow >4.5 x x x x xRiver carpsucker >11.0 x x x xSand shiner >1.0 x x x x xSauger >8.0 x xShovelnose sturgeon >24.0 x x x x xStonecat >1.0 x xSturgeon chub >11.0 x x x x x xWestern silvery 

minnow

>11.0 x x x x x x

White sucker >1.0 x x x x x x x xYellow bullhead1 <20.0 x

1Indicates fish found only in Montana.
90 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life Table 5–5. Habitat characteristics include the following: ability to withstand intermittent flow regime stream width, turbidity determined by the ability to see the bottom of a 3-ft deep pool during baseflow conditions, substrate composition, presence of cover, presence of backwater/side-channels (including those with aquatic vegetation), and the absence of predators in a reach. [m, meters; Y, yes; N, no; >, greater than; <, less than; H, high; L, low; SA, sand; GR, gravel; CB, cobble; BL, boulder; AV, aquatic vegetation. Empty boxes indicate not present or not applicable] Species Width (m) Stream characteristics Intermit- tent flow Turbidity Pools (> 0.5 m) Pools (> 0.5 m) Riffles Runs Backwater/ sidechannel Preda- tors absent

Y

N H L SA GR CB BL Cover SA + 

cover

GR + 

cover

CB + 

cover

GR CB BL GR CB BL Present With AV

Tongue River at Monarch, Mont. 21.0 x x x x x x x x x x xTongue River at Mont.-

Wyom. stateline

35.0 x x x x x x x x x x x x

Tongue River Below 

Reservoir, Mont.

50.0 x x x x x x x x x

Tongue River at Birney 

Bridge, Mont.

50.0 x x x x x x x x

Tongue River at 

Brandenburg Bridge, Mont.

26.0 x x x x x x x x x x x

Tongue River at Miles 

City, Mont.

19.0 x x x x x x x x x x x

Goose Creek, Wyom.

om.

15.0 x x x x x x x x x x

Prairie Dog Creek, 

Wyom.

5.1 x x x x x x x x x

Hanging Woman Creek, 

Wyom.

2.9 x x x x x x x

Powder River at Sussex, Wyom. 13.0 x x x x x x x x x xPowder River at Moorhead, Mont. 32.0 x x x x x x x xPowder River at Locate, 

Mont.

8.3 x x x x x x x

Clear Creek, Wyom. 14.0 x x x x x x x x x xCrazy Woman Creek, 

Wyom.

4.8 x x x x x x x x x

Little Powder River, 

Wyom.

7.9 x x x x x x x x x

1Indicates fish found only in Montana
Discussion 91

Shorthead redhorse

Common carp

Flathead chub

Sand shiner

Stonecat

Longnose dace

Green sunfish

Black bullhead

Flathead minnow

Channel catfish

River carpsucker

Smallmouth bass

Rockbass

Plains minnow

Plains killfish

Creek chub

Goldeye

White crappie

Figure 5–4. Proportion of each fish species captured in the Powder River drainage. Fish were collected 

by seining and electrofishing.

Table 5–6. Index of Biotic Integrity (IBI) (Bramblett and others 2005) scores for fish 

assemblages at Powder and Tongue River sampling locations in Wyoming and Montana.

[km2, square kilometer; IBI, index of biotic integrity]

River/section Drainage area (km2) IBI score

Tongue River at Miles City, Mont. 13,998 63

Tongue River at Brandenburg Bridge, Mont. 10,041 52

Tongue River near Birney, Mont. 6,824 53

Hanging Woman Creek, Wyom. 1,210 35

Tongue River at Stateline, Mont. 3,753 34

Prairie Dog Creek, Wyom. 915 56

Goose Creek, Wyom. 1,180 52

Tongue River near Monarch, Mont. 1,134 49

Powder River near Locate, Mont. 33,849 38

Little Powder River at Broadus, Mont. 5,076 56

Powder River at Moorhead, Mont. 20,832 43

Powder River at Sussex, Wyom. 5,470 55

Clear Creek near Arvada, Wyom. 2,991 65

Crazy Woman Creek near Arvada, Wyom. 2,477 66
92 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

non-native were not as clearly associated with the IBI scores. 

Some sites with large numbers of non-native fish species (such 

as Powder River at Sussex or Clear Creek) or few native fish 

species (Prairie Dog Creek) had relatively large IBI Scores. 

In summary, white suckers and FHM were determined to 

be relatively abundant in the Tongue and Powder River drainages. Fish assemblages were similar to sampling completed in 

the Tongue and Powder River drainages by Stagliano (2006), 

Peterson and others (2009) and Peterson and others (2010). 

White suckers were the most common collected species in 

the Powder and Tongue River drainages. White suckers were 

also the most abundant species in the Tongue River drainage, 

but sand shiners were by far the most abundant species in the 

Powder River drainage.

References Cited

Barbour, M.T., Gerritsen, J., Snyder, B.D., and Stribling, J.B., 

1999, Rapid Bioassessment protocols for use in streams and 

wadeable rivers—Periphyton, benthic macroinvertebrates 

and fish, (2d ed.): Washington, D.C., U.S. Environmental 

Protection Agency, Office of Water, EPA 841–B–99–002, 

189 p.

Baxter, G.T., and Stone, M.D., 1995, Fishes of Wyoming, 

Cheyenne: Wyoming Game and Fish Department, 89 p. 

Bramblett, R. G., Johnson, T. R., Zale, A.V., and, Heggem, 

D.G., 2005, Development and evaluation of a fish assemblage index of biotic integrity for northwestern Great Plains 

streams: Transactions of the American Fisheries Society, 

v. 134, p. 624–640.

Clearwater, S.J., Morris, B.A., and Meyer, J.S., 2002, A comparison of coalbed methane product water quality versus

surface water quality in the Powder River Basin of Wyoming, and an assessment of the use of standard aquatic testing organisms for evaluating the potential effects of coalbed 

methane product waters: Laramie, Wy., University of Wyoming, Department of Zoology and Physiology, Report to the 

Wyoming Department of Environmental Quality, 131 p.

Elser, A.A., Gorges, M.W., and Morris, L.M., 1980, Distribution of fishes in southeastern Montana: Helena, Mt., Montana Fish Wildlife and Parks, 111 p. 

Grossman, G. L., Moyle, P.B., and Whitaker, J.O., 1982, 

Stochasticity in structural and functional characteristics of 

an Indiana stream fish assemblage—A test of community 

Theory: American Naturalist, v. 120, p. 423–454.

Holton, G.D, 1990, A field guide to Montana fishes: Helena, 

Montana Department of Fish Wildlife and Parks, 104 p.

Lee, D.S., Gilbert, C.R., Hocutt, C.H., Jenkins, R.E., McAllister, D.E., and Stauffer, J.R., Jr., 1980, Atlas of North 

American freshwater fish: Raleigh, North Carolina Museum 

of Natural History and the U.S. Fish and Wildlife Service, 

Publication #1980–12, 854 p.

Lipsey, T.S.B, 2001, Using elevation, channel slope, and 

stream width to predict the occurrence of native warmwater 

fish species in the North Platte River drainage in Wyoming: 

Laramie, University of Wyoming, Master’s Thesis, 238 p. 

McCormick, S.D., 1993, Methods for nonlethal gill biopsy and 

measurement of Na+, K+ –ATPase activity: Canadian Journal of Fisheries and Aquatic Sciences, v. 50, p. 656–658.

Patton, T.M. 1997. Distribution and status of fishes in the Missouri River drainage in Wyoming—implications for identifying conservation areas: Laramie, Univeristy of Wyoming, 

Ph.D. Dissertation, 53 p.

Peterson, D.A., Wright, P.R.G., Edwards, P., Jr., Hargett, E.G., 

Feldman, D.L., Zumberge, J. R., and Dey, P., 2009, Ecological assessment of streams in the Powder River structural 

basin, Wyoming and Montana, 2005–06: U.S. Geological 

Survey Scientific Investigations Report 2009–5023, 139 p.

Peterson, D.A., Clark, M. A., Foster, K., Wright, D.L., and 

Boughton, J. K., 2010, Assessment of ecological conditions 

and potential effects of water produced from coalbed natural 

gas development on biological communities in streams of 

the Powder River structural basin, Wyoming and Montana: 

U.S. Geological Survey Scientific Investigations Report 

2005–08, 2010, 84 p.

Poff, N.L. and Ward, J.V., 1989, Implications of streamflow 

variability and predictability for lotic community structure: 

A regional analysis of streamflow patterns: Canadian Journal of Fisheries and Aquatic Sciences, v. 46, p. 1,805–1,818.

Quist, M. C., Hubert, W. A., and Rahel, F.J., 2006, Concurrent assessment of fish and habitat in warmwater streams in 

Wyoming: Fisheries Management and Ecology, v. 13,

p. 9–20.

Quist, M.C., Rahel, F.J., and Hubert, W.A., 2005, Hierarchical 

faunal filters—An approach to assessing effects of habitat 

and nonnative species on native fishes: Ecology of Freshwater Fish, v. 14, p. 24–39.

Stagliano, R. S., 2006, Aquatic Surveys and Assessment 

within the Middle Powder River Watershed: Miles City, 

Mt., Bureau of Land Management, ESA010009, Task 

Order #28, 37 p.
Summary and Sample Calculations for 

Water-Quality Criteria

By Aïda M.Farag, Don Skaar, and David D. Harper

Chapter 6 of

The Potential Effects of Sodium Bicarbonate, a Major Constituent of 

Produced Waters from Coalbed Natural Gas Production, on Aquatic 

Life

Edited by Aïda M. Farag and David D. Harper

Prepared in cooperation with Montana Fish, Wildlife, and Parks, U.S. Bureau of 

Land Management, and the U.S. Environmental Protection Agency

Scientific Investigations Report 2012–5008

U.S. Department of the Interior

U.S. Geological Survey
Contents

Summary........................................................................................................................................................95

Water-Quality Criteria ........................................................................................................................96

Numerical Criteria Sample Calculations.........................................................................................96

Suggestions for Future Directions ............................................................................................................99

Conclusions.................................................................................................................................................100

References Cited........................................................................................................................................101

Table

6–1. The species geometric mean acute LC50 values and ranked sensitivity to sodium 

bicarbonate. Dilution waters were reconstituted to simulate Tongue, Powder and 

Yellowstone River water chemistry .................................................................................................97
Summary

As is the case with any element or compound, there is 

a concentration where NaHCO3 will be toxic to aquatic life. 

A key to define the potential toxicity of any substance in the 

field is to complete controlled experiments in the laboratory 

and follow with field experiments and surveys to determine 

the potential exposure of aquatic life to toxic concentrations. 

The comparisons of all of these investigations will lead to a 

realistic interpretation of toxicological results.

In the previous chapters, acute toxicity to aquatic life was 

defined as a lethal concentration that will cause 50 percent 

mortality or detrimental effect at 48-hour (h) or 96-h (LC50 

or EC50). The acute toxicity experiments were completed 

with a suite of organisms, including 7 species of fish, 5 species of invertebrates, and 1 amphibian species. Experiments 

performed on these multiple species resulted in LC50s that 

ranged from 1,120 to greater than (>) 8,000 milligrams sodium 

bicarbonate per liter (mg NaHCO3

/L) (also defined as 769 

to >8,000 milligrams bicarbonate per liter (mg HCO3

-

/L) or 

total alkalinity expressed as 608 to >4,181 milligrams calcium 

carbonate per liter (mg CaCO3/L)) that varied across species 

and lifestage within a species. The results were similar to 

those observed by Mount and others (1997) for Ceriodaphnia 

dubia (C. dubia) where they defined a 48-h LC50 of 1,020 mg 

NaHCO3/L compared to 1,288 mg NaHCO3/L observed during 

this study (both completed in moderately hard reconstituted 

water). Therefore, the current study provides results that are 

comparable to the limited amount data in the literature, and 

provides a broad application of the data because effects are 

defined on a wide range of species. As a result, managers 

located in watersheds with elevated concentrations of NaHCO3

will have benchmarks for comparisons of effects.

The chronic toxicity of NaHCO3 was defined in experiments that lasted from 7–60 days post-hatch. For these 

experiments, sublethal effects such as growth and reproduction, in addition to significant reductions in survival were 

included in the final determination of effects. We observed 

chronic toxicity at concentrations that ranged from 450 to 800 

mg NaHCO3

/L (also defined as 430 to 657 mg HCO3

-

/L or 

total alkalinity expressed as 354 to 539 mg CaCO3/L) and the 

specific concentration depended on the sensitivity of the four 

species of invertebrates and fish exposed. Managers now also 

have data for chronic effects on multiple species.

Sublethal measurements such as sodium-potassium 

adenosine triphosphatase (Na/ATPase) explained some effects 

at the individual level more completely, and defining these 

measurements in light of population level effects (survival) 

allows one to suggest how varying concentrations of NaHCO3

may affect fish. It appears that percent decrease in the activity of Na/K ATPase and the age of the fish at the onset of the 

decrease may affect the ability of fathead minnow (Pimephales promelas) (FHM) to survive. For example, fish were 

able to survive an 8.12 percent decrease in Na/K ATPase 

activity in the 400 mg NaHCO3/L treatment compared to the 

control when this decrease was first documented on day 60. 

However, fish with an 8.50 percent decrease in Na/K ATPase 

activity first documented on day 37 in the 500 mg NaHCO3/L 

treatment incurred significant reductions in survival and subsequently exhibited a 26.5 percent reduction in Na/K ATPase at 

day 60. The effects of NaHCO3 on Na/K ATPase activity and 

the ability of fish to survive likely are related to the age of the 

fish at exposure.

The acute laboratory results were substantiated with 

field experiments where early lifestage fish were held in live 

containers in situ for 96-h. The combination of in situ experiments, static-renewal experiments performed simultaneously 

with in situ experiments, and static renewal experiments 

performed with site water in the laboratory, demonstrated that 

coalbed natural gas (CBNG) product water reduces survival 

of FHM and pallid sturgeon (Scaphirhynchus albus). Multiple tributary sites were associated with reduced survival and 

the concentrations of NaHCO3 at these sites were above the 

laboratory LC50 determined for the species experimented 

with in the field. Trace element analyses of water collected 

from the field did not reveal any other potential cause for the 

toxicity. However, it must be noted that trace organics were 

not measured in the water or sediments from these sites. At 

all but one site (Burger Draw), total ammonia concentrations 

were less than or equal to (≤) 0.5 mg/L and did not appear to 

be elevated to the extent to cause acute mortality. These results 

Summary and Sample Calculations for Water-Quality 

Criteria

By Aïda M.Farag1, Don Skaar2, and David D. Harper1

1

USGS, Columbia Environmental Research Center, Jackson Field Research 

Station, Jackson, Wyoming.

2

Montana Fish, Wildlife, and Parks, Helena, Montana.
96 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

suggest that the concentrations of NaHCO3 in the tributary 

sites, except Burger Draw, caused the significant reductions in 

survival that were observed. Experiments were not performed 

on the main stem of the Powder or Tongue Rivers (except 

that the Powder River at Moorhead was used as a reference 

site). The mixing zone study provided evidence of substantial 

mixing near the outfalls to the main stem [800–1,200 meters 

(m) below the confluence] and also demonstrated a reduction 

in toxicity (based on survival) of water that was treated with 

ion exchange before release into the main stem of the Tongue 

River. 

Data from the field experiments also defined a difference 

in sensitivity with age of the early lifestage fish. Differences 

were noted in sensitivity between 2-day-post-hatch (dph) and 

6-dph FHM. As a result, care should be used when extrapolating results with fish that are greater than 2-dph to younger fish 

that seem to be more sensitive to NaHCO3. Measurements 

were not made of Na/K ATPase in fish from experiments 

completed in situ, but the data from the laboratory experiments suggests that a significant decrease in Na/K ATPase 

may have affected the ability of the 2-dph fish to survive in the 

field. Pallid sturgeon survival was adversely affected at 4- and 

6-dph and suggests that pallid sturgeon are more sensitive to 

NaHCO3, regardless of age.

Water-Quality Criteria

Data provided in this report document a full array of 

effects generated by exposures to NaHCO3. Because NaHCO3 

is a principle component in CBNG effluents, there is interest in defining the potential for NaHCO3 to affect aquatic life 

where surface discharge might occur. One approach to limit 

potential effects of NaHCO3 contained in effluents would be 

to establish numerical NaHCO3 water-quality criteria for the 

protection of aquatic life. Clear sets of policies, guidelines, 

and procedures to establish these criteria have been adopted 

by the U.S. Environmental Protection Agency (Stephan and 

others, 1985; Erickson and Stephan, 1988). Furthermore, once 

a sufficient database is generated to calculate criteria, data and 

decisions will be reviewed by the U.S. Environmental Protection Agency before a criterion would be adopted as part of a 

water-quality standard.

A numerical criterion is the most common, but is one of 

multiple types of criteria that may be implemented by States 

or Tribes. A numerical standard is appropriate when a specific 

element or chemical present in an effluent is known to cause 

toxicity. Other criteria that might be implemented include, 

narrative (may be based on whole effluent toxicity testing), 

biological (based on numbers and kinds of organisms expected 

to be present), nutrient (specifically limits nitrogen and 

phosphorous inputs to a system), and sediment criteria (U.S. 

Environmental Protection Agency 2011a, b, c). 

 Criteria are one part of a water-quality standard that 

might be implemented by States or Tribes. The derivation of 

overall water-quality standards includes three areas of concern 

(1) designations of use for water bodies, (2) establishment of 

criteria, and (3) guidelines related to anti-degradation (Gardner 

and Goss, 2009).

The purpose of the discussion that follows is to present 

sample calculations of numerical water-quality criteria using 

the database of toxicity evaluations that was generated in the 

previous chapters. This discussion will attempt to explain the 

specific calculations for numerical acute and chronic waterquality criteria and demonstrate how they might apply to 

NaHCO3. The final derivation and implementation of such 

criteria is, of course, left to the discretion of the concerned 

management agencies.

Numerical Criteria Sample Calculation

The focus of this study has been NaHCO3 and the sample 

calculations are first performed for this compound. However, 

standards often are established for simple elements and therefore additional calculations are provided for standards based 

on HCO3

-

 because it is the suspected toxic fraction of the 

compound. Finally, calculations also are provided for alkalinity because it is a low cost and easily performed water-quality 

measurement. For this reason, its use can have wide spread 

applicability around this watershed and elsewhere. It should be 

noted that total alkalinity is expressed as “CaCO3 mg/L.” The 

reader is cautioned that although the measurement is expressed 

in this manner, HCO3

-

 is generally the main fraction of total 

alkalinity. 

The criteria can be calculated to protect against acute or 

chronic effects of an element or compound. The calculation of 

an acute standard or criterion maximum concentration (CMC) 

involves several steps including: sensitivity ranking, calculations of geometric means, calculation of final acute value 

(FAV), and the final calculation of the CMC. Once an acceptable database of toxicity evaluations has been constructed, the 

responses (LC50 or EC50) of various species are compared to 

one another. Geometric means are calculated for each species 

then for each genus and the results are ranked to define the 

four most sensitive genera in the dataset. A total of 20 acute 

experiments that resulted in 13 genus mean or species mean 

acute values were performed during this study (table 6–1 and 

chapter 2, Results) (note to reader: Selenastrum capricornutum; plant species experiment data are not included and should 

be rerun, but does not appear more sensitive, so likely will 

not affect results and interpretation). The geometric means 

(table 6–1) for the four most sensitive genera are as follows. 

1. Freshwater mussel (Lampsilis siliquoidea), LC50 = 

1,120 mg NaHCO3/L.

2. Ceriodaphnia (Ceriodaphnia dubia) LC50 = 

1,157 mg NaHCO3/L. 

3. Sturgeon (Scaphirhynchus), LC50 = 1,377 mg 

NaHCO3/L.

4. Fathead minnow (Pimephales promelas), LC50 = 

1,906 mg NaHCO3/L.
Summary 97

These four geometric means are used to determine an 

final acute value (FAV), which predicts sensitivity at the 

probability of 0.05. Thus, data are extrapolated to protect 95 

percent of all species. A computer program was used for this 

calculation (provided courtesy of U.S. Environmental Protection Agency and Bill Stubblefield, Parametrix Inc). 

For this dataset, the FAV=918 mg NaHCO3/L. The FAV 

is divided by two to account for the derivation based on 

50 percent mortality (or other moribund observations, if an 

EC50 rather than an LC50 was used) (Stephan and others, 

1985; Erickson, 1988). Therefore, using this dataset would 

provide a CMC=459 mg NaHCO3/L. 

This citerion mazimum concentration (CMC, also known 

as the acute criteria) defines the magnitude (for example, 

concentration) of NaHCO3 that would be allowed in an effort 

to protect aquatic life. However, this number would then need 

to be defined in terms of duration and frequency. Duration 

suggested by the U.S. Environmental Protection Agency is that 

the average in a 1-hr period should not exceed the suggested 

magnitude more than once every 3 years. However, the applicability of this recommendation would need to be investigated 

by concerned agencies.

To derive a chronic standard or Criterion Continuous 

Concentration (CCC), at least three acute/chronic ratios (ACR) 

must be calculated where the ratio between effects observed 

in a chronic experiment are compared with the acute effects 

observed in the same type of experimental water. For this dataset, the Inhibition Concentration (IC) that affects 20 percent 

Table 6–1. The species geometric mean acute LC50 values and ranked sensitivity to sodium bicarbonate. Dilution waters 

were reconstituted to simulate Tongue, Powder and Yellowstone River water chemistry. 

[LC50, median 50-percent lethal; mg/L, millgrams per liter; >, greater than; --, indicates data were not acceptable to calculate a LC50]

Species Dilution water

(reconstituted) LC50 mg/L Geometric mean LC50 Sensitivity rank

Fathead minnow

Pimephales promelas

Tongue River 2,078 1,906 4

Powder River 2,078

White sucker

Catostomus commersoni

Tongue River 4,494 5,038 8

Powder River 5,648

Rainbow trout

Oncorhynchus mykiss

Tongue River 8,070 8,070 9

Pallid sturgeon

Scaphirhynchus albus

Tongue River 1,295 1,325 3

Powder River 1,356

Shovelnose sturgeon

Scaphirhynchus platorynchus

Yellowstone River 1,430 1,430 3

Sturgeon genus mean is 1,377

Walleye

Sander vitreus

Yellowstone River 3,249 3,249 7

Northern pike

Esox lucios

Yellowstone River >8,000 >8,000 10

Freshwater mussel

Lampsilis siliquoidea

Tongue River 1,120 1,120 1

Powder River

African clawed frog

Xenopus Laevis

Tongue River 1,940 1,940 5

Powder River

Tubifex

Tubifex tubifex

Tongue River 3,297 3,332 9

Powder River 3,367

Amphipod

Hyalella azteca

Tongue River 1,419 >2,338 6

Powder River >3,851

Chironomid

Chironomus dilutus

Tongue River 4,974 6,314 11

Powder River 8,014

Ceriodaphnia

Ceriodaphnia dubia

Tongue River 989 1,157 2

Powder River 1,355

Algae

Selenastrum capricornutum

Tongue River -- -- --
98 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

of the sample population (IC20) chronic effect concentrations 

were as follows: 

1. Fathead minnow (Pimephales promelas) = 527 mg 

NaHCO3/L; mean of 462 from experiment 1 (mortality) 

and 591 from 7-day (d) (growth)

2. C. dubia = 359 mg NaHCO3/L (reproductive effects)

3. Freshwater mussel (Lampsilis siliquoidea) = 952 mg 

NaHCO3/L (moribund lack of foot movement). 

The ACRs that result are as follows:

1. Fathead Minnow (Pimephales promelas)1,749/527 = 3.32.

2. C. dubia 1,288/359 = 3.59.

3. Freshwater mussel (Lampsilis siliquoidea)1,120/952 = 

1.18.

The geometric mean, or the Final Acute/Chronic Ratio 

(FACR) = 2.41.

Finally, using this database, CCC = FAV/FACR = 918/2.41= 

381 mg NaHCO3/L.

The CCC (also known as the chronic criteria) defines the 

magnitude of a continuous NaHCO3 concentration that would 

be allowed in an effort to protect aquatic life. However, this 

number would then need to be defined in terms of duration 

and frequency. Duration suggested by the U.S. Environmental Protection Agency is that the average concentration in a 

24-hr period should not exceed the suggested magnitude more 

than once every 3 years. However, the applicability of this 

recommendation would need to be investigated by concerned 

agencies.

Below, the CMC and CCC are derived for HCO3

-

 and 

total alkalinity expressed as mg CaCO3/L. The geometric 

means for the same four genera expressed as mg HCO3

-

/L are 

as follows: 

1. Freshwater mussel (Lampsilis siliquoidea), LC50 = 

844 mg HCO3

-

/L.

2. C. dubia, LC50 = 768 mg HCO3

-

/L. 

3. Sturgeon (Scaphirhynchus), LC50 = 957 mg HCO3

-

/L.

4. Fathead minnow (Pimephales promelas), LC50 = 

1,334 mg HCO3

-

/L.

FAV = 634 and CMC = 634/2 = 317 mg HCO3

-

/L.

For this dataset, IC20 chronic effect concentrations were as 

follows: 

1. Fathead minnow (Pimephales promelas) = 434 mg HCO3

-

/L; mean of 368 from Experiment 1 (mortality) and 499 

from 7-d (growth).

2. C. dubia = 274 mg HCO3

-

/L (reproductive effects).

3. Freshwater mussel (Lampsilis siliquoidea) = 

715 mg HCO3

-

/L (moribund lack of foot movement). 

The ACRs that result are as follows:

1. Fathead minnow (Pimephales promelas) 1,254/434 = 2.90

2. C. dubia 842/274 = 3.07

3. Freshwater mussel (Lampsilis siliquoidea) 844/715 = 1.18

The geometric mean of the ACR = 2.19 and the CCC = 

634/2.19 = 290 mg HCO3

-

/L.

The geometric means as alkalinity expressed as mg CaCO3/L 

are as follows: 

1. Freshwater mussel (Lamsilis siliquoidea), LC50 = 

670 mg CaCO3/L.

2. C. dubia, LC50 = 819 mg CaCO3/L. 

3. Sturgeon (Scaphirhynchus), LC50 = 794 mg CaCO3/L.

4. Fathead minnow (Pimephales promelas), LC50 = 

1,114 mg CaCO3/L.

Thus, FAV = 583 and CMC = 583/2 = 292 mg CaCO3/L total 

alkalinity.

For this dataset, IC20 chronic effect concentrations were as 

follows:

1. Fathead minnow (Pimephales promelas) = 

463 mg CaCO3/L; mean of 302 from experiment 1 (mortality) and 424 from 7-d (growth).

2. C. dubia = 249 mg CaCO3/L (reproductive effects).

3. Freshwater mussel (Lamsilis siliquoidea) = 

605 mg CaCO3/L (moribund lack of foot movement). 

The ACRs that result are as follows:

1. Fathead minnow (Pimephales promelas) 1,139/463 = 3.07.

2. C. dubia 846/249 = 3.29.

3. Freshwater mussel (Lamsilis siliquoidea) 670/605 = 1.12.

The geometric mean of the ACR = 2.24 and the CCC = 

583/2.24 = 260 mg CaCO3/L total alkalinity.

The identical four species/genus (fathead minnow, C. 

dubia, freshwater mussel, and sturgeon) were used as the four 

most sensitive for all calculations based on NaHCO3, HCO3

-

, 

and total alkalinity. This was done for simplicity. However, if 

sensitivity comparisons are based on HCO3

-

 and total alkalinity; the most sensitive genus changes slightly because amphipods and amphibians appear more sensitive with respect to 

these parameters. 

Field validations and additional laboratory studies can 

be used to validate the acute and chronic findings. First, 

tributary sites were associated with reduced survival and the 

concentrations of NaHCO3

- at these sites were more than the 

laboratory LC50 determined for the species experimented with 

in the field. Second, data from a chronic experiment performed with white suckers (Catostomus commersoni) revealed 

growth effects at 450 mg NaHCO3/L. Histological effects also 

observed during the white sucker experiment supported these 
Suggestions for Future Directions 99

findings. These data were not included into CCC calculation 

because there was a large difference between the acute and 

chronic results for white suckers, which seemed to skew the 

ACR results (ACR for white suckers was >10). Regardless, 

the chronic experiment with white suckers point to an effect in 

the 500 mg NaHCO3/L range much like fathead minnow and 

C. dubia and validate the chronic findings.

In summary, a database of toxicity evaluations of 

NaHCO3 on aquatic life has been constructed. The experiments 

were performed with multiple species and were performed to 

meet U.S. Environmental Protection Agency acute and chronic 

guidelines established for the calculation of water-quality 

criteria. Using these data, acute and chronic criteria of 459 

and 381 mg NaHCO3/L, respectively, can be calculated for the 

protection of aquatic life. These numbers address the magnitude of concentrations, but additional considerations related to 

frequency and duration of allowable exceedance also would 

need to be considered. Finally, field experiments performed in 

tributaries in the Powder River Basin and an additional laboratory chronic experiment validate the findings and add credibility to the calculations performed.

Treatment practices employed in the Tongue/Powder 

River watershed appear to reduce the concentrations of 

NaHCO3 to less than the sample criteria calculations and the 

deionization treatment (Higgins LoopTM, EMIT) appears to 

reduce the toxicity of untreated effluent water. Also, as of 

2011, a treatment system has been located in the Beaver Creek 

tributary where toxicity was documented during this study. 

Although in situ bioassays have not been repeated at this site 

since the implementation of the treatment system, it is likely 

that toxicity of the tributary water is decreased if all inputs to 

the tributary are being treated with deionization. 

Suggestions for Future Directions

The current body of work provided is substantial and 

provides data necessary to define the potential toxicity of 

NaHCO3 (HCO3

-

, total alkalinity, conductivity) on aquatic life. 

The next steps would involve some refinement of the laboratory data, addition of species to the database, and monitoring 

of field sites as managers move to a monitoring and restoration 

phase of activities. 

Although this volume contains an extensive body of work 

related to the effects of NaHCO3 on aquatic life, additional 

laboratory experiments could provide data to further refine the 

conclusions. There are several acute and chronic laboratory 

experiments that could further the research:

1. Selenastrum acute experiment—To further develop the 

acute database, a selenastrum experiment should be 

completed. The exposure completed during this study 

was completed successfully, but the samples collected 

for population counts were not readable with acceptable 

accuracy within the scope available. The researchers 

considered the selenastrum response that was observed to 

be less sensitive than the four species included in the final 

calculation of the sample acute criterion. However, these 

observations must be considered anecdotal and the sensitivity of selenastrum to NaHCO3

 can only be definitively 

characterized with a successful experiment that includes 

accurate population counts.

2. Use of sensitive invertebrate species for acute experiment—During this study, Chironomus (midges), represented Class Insecta (table 1–1). Chironomus is a genus 

within the order Diptera. However, insects from the order 

Plecoptera (stoneflies) and Ephemeroptera (mayflies) 

often are more sensitive to toxicant exposures than Chironomus. Chironomus often is cultured in the laboratory 

and was an important first step to define the sensitivity of 

invertebrates to NaHCO3. As a next step, the sensitivity 

of Insecta could be further refined with the addition of an 

acute assay that utilizes a representative of Plecoptera or 

Ephemeroptera or both.

3. Additional mussel chronic experiment—Freshwater mussels were among the four most sensitive species in the 

acute experiments completed during this study. A 10-d 

chronic experiment also was performed with freshwater 

mussels. The 10-d chronic experiment was performed 

with newly transformed freshwater mussels. When culturing freshwater mussels, newly transformed mussels are 

fairly robust and easy to work with in the laboratory. 

However, from 2 weeks after newly transformed until 

2 months of age, the freshwater mussel cultures may 

encounter excessive mortalities and generally are not used 

as experimental animals. For this reason, a 10-d chronic 

experiment with newly transformed mussels (American 

Society of Testing and Materials International, 2006) 

was completed. The mussel culture was allowed to reach 

2-months old, the age considered acceptable for use in 

a 28-d experiment (American Society of Testing and 

Materials International, 2006). However, the cultured 

animals did not survive until 2 months of age and the 28-d 

experiment was not completed. Although it is generally 

accepted that data from a 10-d experiment will mimic that 

of a 28-d experiment, the U.S. Environmental Protection 

Agency may have some reservations with the use of the 

10-d chronic data in the current sample calculations. For 

this reason, a 28-d chronic experiment with freshwater 

mussels is recommended to be performed with 2-month 

old mussels. 

4. Ceriodaphnia dubia (C. dubia) chronic experiment in 

simulated Tongue River water—Sample calculations for 

chronic numerical criteria were performed with acute and 

chronic experiments completed with C. dubia performed 

in moderately hard reconstituted water. Although this is 

acceptable, an additional chronic experiment performed 

in simulated Tongue River water would strengthen the 

database and be similar to fathead minnow and freshwater 

mussel experimental water quality. 
100 The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life

5. White sucker acute/chronic experiment—The estimated 

percent hatch was 21 percent and control survival 54 

percent at 30-d during the white sucker chronic experiment. These are small numbers but not unexpected 

because of experiments with the progeny of native fish 

that were spawned in the field. The progeny were brought 

into the laboratory for this experiment. The results during the white sucker chronic experiment were not unlike 

those observed during other chronic experiments. What 

was unusual was the large difference between acute and 

chronic effects noted for white sucker. For this reason, the 

acute chronic ratio was not included in the sample calculations provided. To make better use of the white sucker 

data two approaches are suggested. First, a white sucker 

acute experiment should be performed again. Depending 

on the results of this experiment, a second chronic experiment could be performed. Some initial research could be 

conducted to define the usefulness of a shorter chronic 

experiment to limit costs. However, the growth effects 

defined with white sucker were not observed until the end 

of the 60-d experiment and care must be taken to allow a 

chronic experiment sufficient time for observable effects 

to manifest.

6. Chronic experiment with additional species—If a shorter 

chronic experiment is found useful for white sucker, such 

experiments could also be used for additional species. 

For example, sauger is a much valued game species and 

acute and chronic experiments with this species would 

enhance the database. Sauger is a cannibalistic species 

which makes chronic experiments of a shorter duration 

desired so the experiment could end before this behavior 

manifests. 

7. Use of additional amphibian species—The African clawed 

frog was used for this study because the timing necessary 

to collect frog eggs from the field for additional amphibian species was unattainable. Although feral populations 

of African clawed frogs exist in the United States, they 

are not native. The use of a strictly native frog species 

(for example, Leopard Frog) would add to this database. Additional field investigations could be performed 

to gather amphibian eggs for the laboratory studies. If 

field collected individuals are unavailable, there may be 

sources from hatchery programs (for example, redundant 

individuals from captive breeding programs) that might 

be available for breeding use during acute and chronic 

experiments. 

8. Measurements of trace organics—Measurements of trace 

organics were not performed at the field sites or in the 

mixing zones. Trace organics could be measured in sediments or pore waters at these locations to define whether 

or not a source of organics is currently present as a potential pathway to the water column.

9. Monitoring—A monitoring program to provide an early 

alert to resource managers could include measurements of 

NaHCO3, alkalinity, and conductivity in water at key sites 

throughout the Tongue/Powder River watershed. These 

sites should be strategically placed to define the effects of 

effluent on critical tributaries (for example, Beaver Creek, 

Burger Draw) and the main stem of the rivers. When 

possible, sites should also include some locations within 

mixing zones. The monitoring strategy could include 

periodic, possibly a 3–5 year interval, assessments of the 

survival of aquatic life during in situ bioassays. These 

assays should include a plan where multiple species (fish, 

invertebrate, and amphibians) are studied to define the 

extent of effects on sensitive species, should they occur in 

the future.

Conclusions

A combination of in situ experiments, static-renewal 

experiments performed simultaneously with in situ experiments, and static renewal experiments performed with site 

water in the laboratory, demonstrated that coalbed natural gas 

(CBNG) product water reduces survival of fathead minnow 

and pallid sturgeon. More precisely, the survival of earlylifestage fish, especially those less than (<) 6-days post hatch 

(dph), likely is reduced significantly in the field when concentrations of NaHCO3

 rise above 1,500 mg/L. This conclusion is 

supported additionally by laboratory experiments. 

A database of toxicity evaluations of NaHCO3 on aquatic 

life has been constructed. Using these data, acute and chronic 

criteria of 459 and 381 mg NaHCO3/L, respectively, can be 

calculated for the protection of aquatic life. These concentrations are understandably less than 1,500 mg NaHCO3

/L cited 

above because criteria are calculated to protect 95 percent of 

the most sensitive species and are calculated to protect beyond 

significant mortality. 

Sublethal investigations revealed percent decrease in the 

activity of sodium-potassium adenosine triphosphatase (Na/K 

ATPase, an enzyme involved in ionoregulation) and the age 

of the fish at the onset of the decrease may affect the ability of 

fathead minnow to survive exposures to NaHCO3. Increased 

sensitivity to NaHCO3 for younger fathead minnow (2-dph 

compared to 6-dph) also was documented during acute experiments. However, age was not a factor for pallid sturgeon and 

they were sensitive to product water regardless of age.

Treatment with the Higgins LoopTM technology and dilution of untreated water increased survival in the laboratory. 

Both of these situations reduced ammonia in addition to the 

concentrations of NaHCO3. These experiments addressed the 

acute toxicity of effluent waters being added to the main stem 

rivers, but did not address issues related to the volumes of 

water that may be added to the watershed. 

Mixing zones of the three outfalls studied ranged from 

approximately 800–1,200 m below the confluence and the 
References Cited 101

areas within these mixing zones with acutely lethal concentra-

tions of NaHCO

3

 (as defined by the presence of concentrated 

dye) are limited and variable within each zone. 

Areas with concentrations of NaHCO

3

 more than the con-

centrations likely to cause significant mortality, and more than 

the calculated sample water-quality criteria in the Tongue and 

Powder River Basins appear to be limited to tributaries and 

parts of mixing zones with considerable additions of untreated 

effluent.

Suggestions for future directions include performing 

additional acute and chronic experiments with additional spe-

cies to further refine the database and beginning an effort to 

monitor for potential toxicity effects of CBNG product water 

in the field.

References Cited

American Society of Testing and Materials International, 

2006, Standard guide for conducting laboratory toxicity 

tests with freshwater mussels: West Conshohocken, Pa., 

American Society of Testing and Materials International, 

E 2455–06, 52 p.

Erickson R.J., and Stephan, C.E., 1988, Calculation of the 

final acute value for water quality criteria for aquatic organ-

isms—Duluth, U.S. Environmental Protection Agency, 

Environmental Research Laboratory, EPA/600/3–88/018, 

63 p.

Gardner, T, H. and Goss, H., 2009, American Fisheries Society 

Short Course—Introduction to Water Quality Standards: 

accessed March 2009 at www.fisheries.org/units/wqs.

Stephan , C.E., Mount, D.I., Hansen, D.J., Gentile, J.H., Chap-

man G.A., and Brungs, W.A., 1985, Guidelines for deriving 

numerical national water quality criteria for the protection 

of aquatic organisms and their uses: Springfield, Va., Envi-

ronmental Protection Agency, National Technical Informa-

tion Service, PB85–227049, 54 p. Note: 2011 updated .pdf 

available at http://water.epa.gov/scitech/swguidance/stan-

dards/criteria/aqlife/upload/85guidelines.pdf.

U.S. Environmental Protection Agency, 2011a, Basic informa-

tion, water quality criteria: accessed April 2010 at www.epa.

gov/waterscience/criteria/basics.html.

U.S. Environmental Protection Agency, 2011b, What are water 

quality standards? Water quality criteria: accessed April 

2010 at http://water.epa.gov/scitech/swguidance/standards/

crit.cfm.

U.S. Environmental Protection Agency, 2011c, Water quality 

criteria for nitrogen and phosphorous: accessed April 2010 

at www.epa.gov/waterscience/criteria/nutrient/.
Publishing support provided by: 

Rolla Publishing Service Center 

For more information concerning this publication, contact:

Director, USGS Columbia Environmental Research Center

4200 New Haven Road

Columbia, MO 65201

(573) 875–5399

Or visit the Columbia Environmental Research Center Web site at:

http://www.cerc.usgs.gov/

Edited by Farag and Harper—The Potential Effects of Sodium Bicarbonate, a Major Constituent of Produced Waters, on Aquatic Life—Scientific Investigations Report 2012–5008