IN THE MATTER OF :

PROPOSED AMENDMENTS TO:

35 III . Adm. Code 302 .102(b)(6), 302

.102(b)(8)

302

.102(b)(10), 302 .208(g), 309 .103(c)(3),

405 .109(b)(2)(A), 405 .109(b)(2)(B), 406 .100(d);

REPEALED 35 Ill . Adm

. Code 406.203, PART 407 ; and

PROPOSED NEW 35 Ill . Adm. Code 302.208(h)

BEFORE THE ILLINOIS POLLUTION CONTROL BOARD

Dorothy Gunn, Clerk

Illinois Pollution Control Board

100 West Randolph Street

Suite 11-500

Chicago, Illinois 60601

Mathew Dunn

Illinois Attorney General's Office

Environmental Control Division

James R. Thompson Center

100 West Randolph Street

Chicago, Illinois 60601

ALSO SEE ATTACHED SERVICE LIST

PLEASE TAKE NOTICE that I have today filed with the Office of the Clerk of the Pollution Control

Board the

Illinois Environmental Protection A2encv's Additional Information and Documents,

a copy of which is herewith served upon you

.

ILLINOIS ENVIRONMENTAL PROTECTION AGENCY

By:

i~

Sanjay K

. Sofat, Assistant Counsel

Division of Legal Counsel

NOTICE OF FILING

RECEIVED

CLERK'S OFFICE

APR 0 9 2001

R07-09

(Rulemaking - Water)

Marie E. Tipsord

Hearing Officer

Illinois Pollution Control Board

100 West Randolph, Suite 11-500

Chicago, Illinois 60601

Jonathan Fun

Illinois Department of Natural Resources

One Natural Resources Way

Springfield, Illinois 62702-1271

STATE OF ILLINOIS

Pollution Control Board

Dated

: April 6, 2007

Illinois Environmental Protection Agency

1021 North Grand Avenue East

Springfield, Illinois 62794-9276

(217) 782-5544

THIS FILING PRINTED ON RECYCLED PER

* * * * * PC #2 * * * * *

---

BEFORE THE ILLINOIS POLLUTION CONTROL BOARD

IN THE MATTER OF :

)

PROPOSED AMENDMENTS TO:

35 Ill . Adm. Code 302 .102(b)(6), 302 .102(b)(8)

302 .102(b)(10), 302 .208(g), 309 .103(c)(3),

405 .109(b)(2)(A), 405 .109(b)(2)(B), 406 .100(d)

;

REPEALED 35 111

. Adm . Code 406 .203, PART 407 ; and

PROPOSED NEW 35 Ill . Adm. Code 302 .208(h)

ADDITIONAL INFORMATION AND DOCUMENTS

THE ILLINOIS ENVIRONMENTAL PROTECTION AGENCY (the "Agency" or "Illinois

EPA") respectfully submits this additional information and documents for the Illinois Pollution

Control Board's (the "Board") R07-9 rulemaking proceeding

. The Agency files this additional

information and documents in support of the Agency's proposal and to address the questions

raised by Dr. Anand Rao, a Senior Environmental Scientist for the Board, at the March 7, 2007

hearing

. The Agency commends the efforts of the Environmental Law & Policy Center, the

Sierra Club, and Prairie Rivers Network in providing helpful questions at this hearing

. The

Agency also thanks the Board for holding this hearing on this important rulemaking proposal

. In

response to Dr

. Rao's questions, the Agency provides the following responses :

Dr. Rao

: "IEPA has granted wet weather dischargers allowed mixing zones for sulfate and

sometimes chloride with consideration to upstream flows in the past few years

. Can you be a

little more specific and tell us what was the receiving stream and what particular source

received this permit?" Transcript ("hereinafter Tr. ), p.55.

An example of a fac lity receiving this type of mixing consideration is Mine X

. The receiving

stream for the mine effluent is a small channelized ditch

. The hardness value measured in the

RECEIVED

CLERK'S OFFICE

APR 0 9 2001

STATE OF ILLINOIS

Pollution Control Board

R07-09

(Rulemaking - Water)

* * * * * PC #2 * * * * *

---

mine effluent was lower than the values measured in the ditch upstream of the mine discharge

.

The Agency used average effluent hardness of 373 mg/L to calculate the sulfate standard

. To

calculate the chloride concentration downstream of the mine effluent, the Agency used upstream

and effluent chloride values and average effluent and upstream flows

. The calculated chloride

concentration downstream of the mine effluent was 277 mg/L

. The proposed sulfate standard

equation from Section 302

.208(h)(2)(A) was then used to calculate the allowable sulfate

concentration in the ditch, 1903 mg/L

. This concentration is much higher than the maximum

effluent concentration from the mine

; therefore, no mixing is needed to meet the standard .

The average chloride concentration, however, was 567 mg/L in the effluent

. In order to

determine the allowed mixing during wet weather to assess attainment of the water quality

standard of 500 mg/L, actual flow values from the effluent and the ditch were compared . The

lowest dilution ratio during discharge was 1

.29 to I with much higher ratios at other times .

Given the upstream average chloride of 29

.2 mg/L, the allowable effluent chloride concentration

is 1107 mg/L using 100% of the stream flow at this conservative wet weather discharge

condition

. The permit contains a special condition that prohibits the discharge during dry

weather if the effluent has potential to violate any applicable water quality standards .

Dr. Rao

: "Can you tell me how many mine discharge permits currently exist in the State that are

affected by these rules? " Tr., p.73.

There are 19 active coal mines in Illinois at the present time

. The Agency believes that all of

these mines have discharges that have the potential to exceed either the Board's existing sulfate

or the chloride water quality standards in their final effluent

. Other mine related discharges exist

at mine reclamation sites, coal ash disposal sites, and related facilities not associated with one of

the active mines . These sources total approximately 90 NPDES permits, and most of these

2

* * * * * PC #2 * * * * *

---

discharges would also not meet one or both of these standards in the final effluent .

Dr. Rao

: "Has the Illinois State Water Survey identified a map of these 7Q1 .1 streams ; how

many 7Q1 .1 streams are there?" Tr., p.80

.

No, the Illinois State Water Survey has not developed a map depicting the 7Q1 .1 hydrologic

statistics . The Agency, however, does believe that sufficient data and information exist to

identify these 7Ql .1 zero flow streams . As part of the permit application, the applicant will

provide the basic information necessary for identifying these small streams . Where necessary,

the Agency would rely on the expertise of a hydrologist at the Illinois State Water Survey

("ISWS") in identifying the 7Q1

. 1 zero flow streams . The Agency believes that many thousands

of small headwater streams throughout the State would be classified as 7Q1 .1 zero flow streams

.

The exact boundaries of these streams would be determined based on watershed area, rainfall

patterns, and soil type.

Dr. Rao: "Has Dr. Soucek's report undergone peer review? " Tr., p.83.

Dr. Soucek has recently published three peer reviewed papers concerning the research he

conducted under contract from the Illinois EPA, the Illinois Coal Association, and USEPA . The

first paper, Effects of Hardness, Chloride, and Acclimation on the Acute Toxicity of Sulfate to

Freshwater Invertebrates, presents research that was funded by the Illinois EPA and the Illinois

Coal Association. This paper published in Environmental Toxicology and Chemistry in 2005

and is now included as Exhibit 1 . This paper is a summary of Dr . Soucek's final report to the

Illinois EPA and the Illinois Coal Association which was added as Exhibit Q in the "Facts in

Support" document (See Attachment I of the Agency's proposal)

.

Dr. Soucek has also recently published two papers that the Illinois EPA was previously

unaware of, as both papers were published following the first hearing. The paper entitled,

3

* * * * * PC #2 * * * * *

---

Comparison of Hardness- and Chloride-Regulated Acute Effects of Sodium Sulfate on Two

Freshwater Crustaceans,

presents research funded by USEPA and is a summary of Dr

. Soucek's

final report submitted to USEPA, which was included as Exhibit U of the Agency's proposal

.

This publication has now been included as

Exhibit 2 . The second publication entitled,

Bioenergetic Effects of Sodium Sulfate on the Freshwater Crustacean, Ceriodaphnia dubia,

was

published in Ecotoxicology, and is now included as Exhibit 3 .

This paper summarizes research

funded by USEPA and mostly includes data that was reported in the final report to USEPA (See

Exhibit U of the Agency's proposal).

Respectfully Submitted

ILLINOIS ENVIRONMENTALPROTECTION AGENCY

By :

DATED : April 6, 2007

Illinois Environmental Protection Agency

1021 North Grand Avenue East

P.O . Box 19276

Springfield, Illinois 62794-9276

(217) 782-5544

Sanjay K . Sofat

Assistant Counsel

Division of Legal Counsel

4

* * * * * PC #2 * * * * *

---

EXHIBIT

1

* * * * * PC #2 * * * * *

---

/SE

%pBESS/

EFFECTS OF HARDNESS, CHLORIDE, AND ACCLIMATION ON THE ACUTE

TOXICITY OF SULFATE TO FRESHWATER INVERTEBRATES

DAVID JOHN

SOUCEK,s't and ALAN JAMES KENNEDY$

¶Center for Ecological Entomology, Illinois Natural History Survey, Champaign, Illinois 61820, USA

$Analytical Services Incorporated, U .S

. Army Engineer Research and Development Center, Vicksburg, Mississippi 39180

. USA

(Received 17 March 2004 ; Accepted 5 November

2004)

Abstract-The acute toxicity of sulfate to

Ceriodaphnio dubia, Chironomus tentons, Hyalella azleca, and Sphaeriura

simile was

assessed to support potential updates of Illinois (USA) sulfate criteria for the

protection of aquatic life . The mean lethal concentrations

512

(-2,800

to 50%

mg/Lof

mg/L)

a

for

sample

Hand

. azfeca,population

hardness

2,050

(106

(L(SOs),

mg/L

mg/L)for

expressed

.

C.

survival

dubia,as 2,078

ofnag H

.

SOmg/L

azleca

; -/L,

for

was

in

S.

moderately

positively

simile,and

hard

correlated

14.134

reconstituted

mg/L

with

for

chloride

Cwater

. lenrans(MHRW)

concentration

. At

were

constant

as

. Hardnessfollowssulfate

:

also

mgSO,''-was -found

/L at

to

hardness-

ameliorate

90

sodium

mg/L to

sulfate

3,516

toxicity

mg SO,'to

-/L

C .

at

dubiahardness

and H=

. azfeca,484

mg/Lwith

. Using

LC50s

a

forreformulated

C. dubiaincreasing

MHRW with

from

a similar2,05(1

hardness but higher chloride concentration and different calcium to magnesium ratio than that in standard MITRW, the mean LC50

for H. azleca increased to 2,855 mg/L, and the LC50 for C

. dubia increased to 2,526 mg/L . Acclimation of C. dubia to 500 and

1,000 mg SO,'- - /I

. for several generations nominally increased mean LC50 values compared with those cultured in standardMHRW

.

Keywords -Sulfate

Total dissolved solids

Osmoregulation

Hyalella

Toxicity

INTRODUCTION

Aquatic ecotoxicological research has primarily focused on

the impairment of fauna by contaminants that are toxic at

minute concentrations; however, ordinarily benign major ions

(e .g ., sodium, sulfate) can reach concentrations in wastewater

discharges that severely impair sensitive in-stream macroin-

vertebrates and laboratory test organisms [1-5]

. Concentra-

tions of these major ions and therefore, of total dissolved solids

(TDS), which is essentially the sum of the concentrations of

all common ions

(e.g ., sodium, potassium, calcium, magne-

sium, chloride, sulfate, and bicarbonate) in freshwaters, can

be elevated by numerous practices, such as reverse osmosis

systems, pH modifications, and mining operations [6] ; and

investigations of major-ion toxicity have involved irrigation

drainage water [1 ,7-9], inundation of freshwater systems by

brackish water

[3,10], laboratory-formulated salt solutions

[11,12], and mining activities [4,5,13] .

Coal preparation facilities wash coal to reduce sulfur emis-

sions prior to burning in coal-fired power plants and treat waste-

waters for acid-soluble metals . This practice often produces a

waste containing sulfuric acid that is usually neutralized by the

addition of sodium hydroxide or sometimes quicklime (CaO)

prior to release to a receiving system [14] .

The result is an

effluent containing high concentrations of sulfate, sodium, and/

or calcium ions and therefore, TDS

. Other ions potentially pres-

ent at high concentrations because of coal preparation activities

include magnesium and chlorides

; therefore, the interacting ef-

fects of these various ions should be considered . Researchers

have found hardness and multiple "nontoxic" cations in solu-

tion to ameliorate major-ion toxicity ([8,11,15] (ht(p

://s cholar.

lib

.vt.edu/theses/available/ctd-051499-130633/) , and several

" To whom correspondence may be addressed

(d-soucek@ i nhs .uiuc . edu)

1204

Environmental Toxicology

and Chemistry,

Vol 24,

No

. 5, pp .

1204-1210, 2005

C

2005

5ETAC

Printed in the USA

0730.7268/05 $12 00 + 00

studies indicate that calcium is more important than magnesium

in this regard [16-18] .

There are no federal water quality criteria for the protection

of freshwater life for TDS, sulfate, or sodium 119], but several

states, including Illinois, are developing standards for sulfate

to protect aquatic life . Although major-ion

(i .e ., TDS) toxicity

is caused by osmoregulatory stress from the combination of

all cations and anions, chloride standards currently exist, and

Illinois plans to additionally regulate for sulfate in order to

address the major non-chloride component of TDS in these

waters

. Therefore, the objectives of the current study were to

generate lethal concentrations to 50% of a

sample population

(LC50s) and lethal concentrations to 10% of a sample popu-

lation (LCIOs) for sulfate with selected freshwater inverte-

brates (Ceriodaphnia dubia, Chironomus tentans, Hyalella

azleca, and Sphaerium simile) in the U .S . Environmental Pro-

tection Agency

(U .S . EPA)'s [20] moderately hard reconsti-

tuted water (MHRW) and to determine the effects of laboratory

water composition, water hardness, and test organism accli-

mation on the acute toxicity of sulfate . The endpoints generated

are described in terms of sulfate concentrations to address

regulatory issues ; however, it is important to note that in our

exposures, sodium was the major cation, and effects observed

are probably caused by the combination of all dissolved ions .

MATERIALS AND METHODS

Toxicity of sulfate to freshwater invertebrates in "HR W

Four invertebrates were selected for initial testing . Three

of these, C. dubia, H, azleca, and C. tentans,

are standard

U .S

. EPA organisms used to test for either water column or

sediment toxicity [20,21] . The fourth,

S simile, is a fingernail

clam (Bivalvia, Sphaeriidac) that was easily obtained from the

field and represented the phylum Mollusca . Reliable toxicity

data for sodium sulfate have been generated for C

. dubia [1 1],

so this organism was used in the present study for comparative

* * * * * PC #2 * * * * *

---

Sulfate toxicity to freshwater invertebrates

purposes . Additionally, previous studies have found C

. dubia

to be more sensitive to major-ion or TDS toxicity than other

U .S

. EPA-recommended test species (e .g ., Daphnia

magna,

Pimephales promelas) [5,9,11] .

The cladoceran, C . dubia,

was cultured in-house (Soucek

Laboratory, Illinois Natural History Survey) according to U .S .

EPA methods [20] . The mean L.C50

in NaCl reference tests

for these C. dubia

cultures was 2,030 tog NaCl/L, which was

comparable to the value of 1,960 mg/L reported in previous

studies [11] .

The midge, C. tentans,

also was cultured in-house

according to U .S . EPA methods [21]

. Prior to testing, larvae

were fed a diet of ground Tetra Mm

® (TetraWerke, Melle,

Germany) flake food and rabbit pellets (free of antibiotics) .

Aniphipods, H azteca,

were obtained from a commercial

source (Aquatic Research Organisms, Hampton, NH, USA)

and were acclimated to MHRW at 22°C

and a 16:8-h (light :

dark) photoperiod for at least 7 d prior to testing

. Sphaeriid

clams were collected from Spring Creek, near Loda, Illinois,

USA, and acclimated to MHRW at 22°C and a 16:8-h

(light :

dark) photoperiod for 5 to 7 d prior to testing

. Clams were

identified to species by Gerald Mackie (University of Guelph,

Department of Zoology, Guelph, ON, Canada) .

For toxicity testing, a pure (99%) grade of anhydrous so-

dium sulfate (Na 2 SO 4 ) (

CAS 7757-82-6) was obtained from

Fisher Scientific (Pittsburgh, PA, USA) to serve as the source

of sulfate. A concentrated solution of this salt (19,040 mg

SO,' - /L), as well as a sample of laboratory-deionized water,

was acidified to pH <2

.0 and analyzed for priority metal con-

centrations at the Illinois State Water Survey (Champaign, IL,

USA) using inductively coupled plasma-atomic emission spec-

trometry according to U

.S . EPA methods [22] . All metals an-

alyzed were below acute standard levels ([19],

and R . Mosher,

Illinois Environmental Protection Agency, Springfield, IL.,

USA, personal communication) in the concentrated sulfate

sample, and all were below detection limits in the deionized

water sample except for iron (37 ttg/L) and zinc (9 sg/L)

. The

actual metal concentrations have already been reported [23] .

For definitive static, nonrenewal toxicity tests, conducted

according to American Society for Testing and Materials E729-

96 methods

[24], treatments comprised a 75% dilution series

(i .e.,

the 100% concentration was serially diluted by 25%),

rather than the standard 50%, because major-ion toxicity tests

often cause 100% mortality in one concentration and 0% mor-

tality in the next highest concentration if the spread is too

great

. Five to six concentrations were tested using MHRW as

both the diluent and control, with four replicates tested per

concentration . Tests with C. dubia and C.

lentans were con-

ducted for 48 h with a 16 :8-h (light

:dark) photoperiod, with

the C . dubia tests being conducted at 25°C and the C

. tentans

tests at 22

°C . H. azteca and S. simile were exposed for 96 h

at 22°C and a 16 :8-h (light :dark) photoperiod

. C. dubia, C.

tentans, and H. azteca

were exposed in 50-m1 glass beakers

with five organisms per beaker, and for C

.

tentans

and H.

azteca, I

g of quartz sand was added to each beaker to serve

as substrate

. Clam tests were conducted in 150-m1 glass bea-

kers (no substrate) with three to five organisms per replicate,

depending on the animal size . All clams used were juveniles

.

In the first experiment, clams averaged 4

.6 mm in length (an-

terior to posterior margin), whereas in the second and third

tests, they averaged 5 .4 and 8

.3 mm in length, respectively.

This slight difference in size for the last test did not substan-

tially affect toxicity . C dubia

used were <24 h old, C tentans

were 10 d old, and H.

azteca were approximately third insist

Environ. Toeivol . Chem

. 24, 2005

1 20 5

(7-14 d old)

. Percent survival in each replicate was recorded

every 24 h and at the end of the exposure period

. A dissecting

microscope was used to assess survival

of llyalella and

Sphaerium .

Standard water chemistry parameters, including tempera-

ture, fill, conductivity, dissolved oxygen, alkalinity, and hard-

ness, were measured at both the beginning and the end of each

exposure period. The pH measurements were made using an

Accumet® (Fisher Scientific, Pittsburgh, PA, USA) model

AB15 p1I meter equipped with an Accouter gel-filled combi-

nation electrode (accuracy <±0

.05 pH at 25 ° C) . Dissolved

oxygen was measured using an air-calibrated Yellow Springs

Instruments (Yellow Springs, OH, USA) model 58 meter with

a self-stirring biochemical oxygen-demand probe . Conductiv-

ity measurements were made using a Mettler Toledo ®

(Fisher

Scientific) model MC226 conductivity/TDS meter

. Alkalinity

and hardness were measured (beginning of tests only) by ti-

tration as described in work by the American Public Health

Association [25] . Samples from each treatment were analyzed

to confirm sulfate concentrations by ion chromatography at

the Illinois Natural History Survey Aquatic Chemistry Lab-

oratory (Champaign, IL, USA) .

All LC50 values were calculated using either the Spearman-

Karber method or probit analysis . To increase confidence in

LC50 values, three assays were

conducted with each organism,

except that only two were conducted for C

. tenlans because

of their relative tolerance and low variation in LC50s for the

first two tests . This provided a stronger estimate of the mean

LC50 value for each species . Geometric means are reported

because they are less affected by extreme values

. In addition,

LC 10 values were calculated for all species

. With the exception

of those for H. azteca,

all LC50 values presented are geometric

means of the Spearman-Karber LC50s for a given species,

generated from measured sulfate concentrations . The H. azteca

data did not permit use of the Spearman-Karber method, so

probit analysis was used

. The LCIO values presented were

generated using probit analysis (the Spearman-Karber program

does not calculate LCIOs) with the combined data from all

tests for a given species .

Influence of dilution water composition on sulfate toxicity

Based on observations of others that fl azteca

had much

better control survival in water-only whole-effluent toxicity

tests using modified laboratory water [26], experiments were

conducted to determine sulfate LC50 values for C. dubia

and

H azteca using the alternate water type referred to as refor-

mulated moderately hard reconstituted water (RMIIRW)

. Re-

formulated moderately hard reconstituted water is similar to

MHRW with two basic differences : The nominal chloride con-

centration in RMHRW is nearly 18-fold higher than that in

MHRW, and the calcium and magnesium salt concentrations

are adjusted so that RMHRW has a Ca :Mg molar ratio of 3.25 :

1, whereas MHRW has a Ca :Mg molar ratio of 0 .88

:1 (table

1) . A minor modification in the present study was that an-

hydrous CaSO 4 (CAS 7778-18-9) was used for both RMHRW

and MHRW. The nominal concentrations shown in Table I

take this modification into account

. Mean LC50s and LC1Os

were generated for both species in this water using the same

laboratory and calculation methods as described above, with

the only exception being the changed diluent/control water .

An additional experiment was conducted with H

azteca to

attempt to isolate the two basic differences between MHRW

and RMHRW

. In this experiment, only one nominal sulfate

* * * * * PC #2 * * * * *

---

1206

Envtron . Toxicol

. Chem. 24, 2005

Tabletin

.

testing

Nominal

withchemical

Hyalella

composition

azteca andof

two

Ceriodaplmia

laboralorywaters

dubia

used

°

°

mg/L(standard

RMHRW

The

MHRW

average

.

=

=

moderately

reformulated

deviation),

pH for all

hard

and

moderately

treatments

reconstituted

dissolved

during

hard

oxygen

reconstituted

water

all

never

tests

[201dropped

.

was

water8below

.0 t

[26)6

0.2

.5

.

"Conductivity

concentration

Conductivity (S/cm)

and

of

followed

samples

= 1.711

a

in

linear

t

MHRW

[SOL

trend

(mg/L)]

varied

described

+

depending

717,15,

by

r=

the

upon

=

formula0.9963SO;'

.

:

concentration (2,500 mg/L) was tested with various base wa-

ters . The first of these was MHRW

; the second was RMHRW

;

the third, called chloride, had the same chloride concentration

ratio

(33.9

(0mg/L)

.88:1)

as

as

RMHRW

MHRW(Table

; and the

1)

final

but the

medium,

same

called

Ca:Mg

Ca/Mg,molar

had the same Ca

:Mg molar ratio (3 .25

:1) as RMHRW, but the

same chloride concentration (1

.9 mg/L) as MHRW. Hyalella

was exposed to these four treatments for 96 h at 22

° C . Mean

percent survivorship values for each treatment were compared

using analysis of variance with JMP-1N® software

[27] .

Influence of hardness

on the toxicity of

sodium sulfate

In these experiments, we tested the toxicity of sulfate (with

sodium as the major cation) to C . dubia

in six freshwater

solutions having nominal hardness values of <100 (standard

U .S

. EPA MHRW), 200, 300, 400, 500, and 600 mg/L (as

CaCO 3) .

Hardness was increased by adding enough CaSO,

(CAS 7778-18-9) and MgSO, (CAS 7487-88-9) in the same

achieve

molar ratio

the

as

nominal

that in

hardness

U.S

. EPA

valuesMHRW

. Then

(Ca/Mg

Na,S04=

was

0added,.88)

to

as was done with the standard MHRW

. Whole carboys were

made at each elevated hardness level, and this water was used

as both diluent and control

; therefore, each concentration with-

in a given test had the same hardness

(i .e ., [Ca"] and [Mg"]

did not change with dilution)

. The only parameters that varied

within a particular test were sodium, sulfate, and conductivity

.

At least three tests were conducted for each hardness level to

provide a mean LC50 value and standard deviation

. Exposures

were conducted using the same laboratory and calculation

methods described above, with the only exception being the

hardness of the diluent

. An additional assay was conducted

with H. azteca

at only one sulfate concentration (1,460 mg/

L) and three different hardness levels (90, 200, and 300 mg/

L as CaCO,) .

Hyalella was exposed to sulfate at each of these

hardness levels for 96 h, and mean percent survival was com-

pared between treatments using analysis of variance with JMP-

IN (27]

.

Influence of

chloride on the toxicity o('sulfate

In this experiment, we tested the toxicity of sulfate to 11

.

azteca

in six freshwater solutions having nominal chloride

D .1 . Soucek and A

.J . Kennedy

Table 2 .

Toxicity of sulfate to freshwater organisms in MHRW°

•

geometric

Lethal

MHRW =

concentrations

moderately

means of all

hard

Speannan-Karbcr

to

reconstituted

50%

. of a sample

values

waterpopulation

generated

[20] .

(LC5Os)

for a giv-are

en

was

organism

>90% in

using

all exposuresmeasured

sulfate

.

concentrations

. Control survival

`Lethal

were

all tests

generated

concentration

for a given

using

speciesto

probit

10%

.

of

analysis

a sample

with

population

the combined

ft-C10)

data

valuesfrom

'Tests

third test

produced

was not

similar

conductedLC50s

.

and because values were so high, a

concentrations of 1

.9, 10, 15, 20, 32, and 60 mg/L

. Chloride,

as NaCl (CAS 7647-14-5, Fisher Scientific AC42429-0010),

was added at appropriate concentrations to a solution with a

hardness of approximately 106 mg/L (Ca/Mg = 3

.25, molar

ratio) and a nominal sulfate concentration of 2,800 mg/I,

. The

only parameters that varied between treatments were sodium

and chloride

. In general, tests were conducted using the same

laboratory methods as described above for

Hyalella . Sulfate,

chloride, and bromide were measured in test solutions by ion

chromatography

. Hyalella was exposed to sulfate at each of

the six chloride levels for 96 h, and mean percent survival was

compared between treatments using analysis of variance with

JMP-]N[27]

. One additional aspect of this experiment that was

different from others in this study using

Hyalella was that the

organisms were cultured in RMHRW and not acclimated to

MIIRW, as in previous experiments, to potentially improve the

health of the test organisms

[26] . Finally, two test endpoints

were recorded

. Tests were checked for survival under a dis-

secting microscope, and total survival included all living in-

dividuals, even if they were lying on the bottom and only legs

were twitching . Functional survivors included only those in-

dividuals that were active and upright or burrowing .

Influence of acclimation on

the toxicity

of sulfate to

C . dubia

This experiment was designed to determine the effects of

acclimation to relatively high sulfate levels on the response of

MHRW

C. dubia

with

to sulfateNa2SO, .

added

C. dubia

to achieve

were cultured

sulfate concentrationsin

U .S

. EPA

of 500 and 1,000 mg/L

. After two to three generations had

been cultured in these two sulfate concentrations, acclimated

organisms were tested in high sulfate solutions using standard

MHRW as a diluent and control as described above

. Three

replicate tests were conducted for each acclimation level to

provide a mean LC50 value and standard deviation

.

RESULTS

Toxicity of sulfate to freshwater invertebrates in MHRW

Of the four species tested in MHRW, the most sensitive

was 1i. azteca,

with a mean LC50 of 512 mg S042-a /L (Table

2) . C. dubia and the fingernail clam, S

.

Simile,

were similar

in sensitivity, with mean LC50s of 2,050 and 2,078 mg

SO; - /L, respectively

. C

. lentans was tolerant to sulfate ex-

posure, with a mean LC50 of 14,134 mg SO

; -/L. The LCIO

values were calculated by analyzing all tests for each species

Species

n

Mean LC50"

(mg SO,' - /I,)

Range

(i ng SO;/L) (mg

LCI0`SO

; -/L)

Sphaerium

Ilyalella

CeriodaplmiaChironomusaztecasimile

tentan,sdubia

33

32a

14,1342,0782,050

512

141,901-2,3191,869-2,270.123-14,146431-607

11,6821,5021,759262

Component (units)

MHRW

RMHRW'

Mgt*

HCOj

K*

Conductivity

Ca/Mg

Hardness

Na`

Ca"pH`SO'

CI-(mg/L)(mg/L)(mg/L)(mg/L)(mg/If(mg/L)(mg/L)(molar

(mg/L

(S/cm)°ratio)as

CaCO,)

2959069179426120

72L9.9.1.7.3.1.2.688

34110759326926337632.7.25.9.3.1.7.2.1.9

* * * * * PC #2 * * * * *

---

Sulfate toxicity to freshwater invertebrates

Table 3 . Influence of culture/testing water composition on toxicity of sulfate to

//yolella azteca and Ceriodaphnia duhia

simultaneously, and these ranged from 262 mg SO; - /L for

Hyalella

to 11,682 mg SO - /L for C . tentons (Table 2) .

Influence of dilution water composition on sulfate toxicity

Testing H. azteca in RMHRW produced a strikingly dif-

ferent response compared to results of tests in MHRW (Table

3) . The mean LC50 in RMHRW (2,855 mg S024- / L) was more

than 5 .5-fold higher (p < 0 .0001) than that generated using

MHRW (512 mg SO;-/L), with a >8-fold increase in the LCIO

value .

C. dubia also had a significantly different (p = 0 .0205),

though not as striking, response, with the mean LC50 increas-

ing from 2,050 mg S042-/L in MHRW to 2,526 mg SO ; - /L in

RMHRW. The LCIO for C. duhia increased from 1,759 mg /

L in MHRW to 2,216 mg SO; -/L in RMHRW (Table 3)

.

In the experiment with H. azteca

designed to dissect the

effects observed in RMHRW, only 45% and 55% of the test

organisms exposed to 2,500 mg

S042 -/L

were alive in the

MHRW and Ca/Mg treatments, respectively, after 48 h, where-

as 85% and 80% survived in the RMHRW and chloride media,

respectively (Fig- 1)

. After 96 h, all of the organisms had died

in MHRW and Ca/Mg, whereas 80% still survived in RMHRW

and 25% survived in chloride . These data suggest that chloride

played the larger role in protecting H azteca against sulfate

toxicity and that the different Ca :Mg ratio played a smaller

role .

Influence of hardness on the toxicity of sodium sulfate

Increasing water hardness decreased the toxicity of sodium

sulfate to H. azteca (Fig . 2)

. In controls, 90% of test organisms

20

n

MHRW

RMHRW

Chloride

Calm,

Fig, I Effect of various components of reformulated, moderately hard,

reconstituted water (RMHRW) on percent survival ofHyalella azteca

in elevated (2,500 mg SO ;- / L) sulfate solutions . The chloride and Ca/

Mg treatments consisted of standard moderately hard reconstituted

water (MHRW) with chloride or Ca :Mg molar ratio adjusted to match

RMHRW

Envirnn . To.x,col. Chem . 24, 2005

1207

'°

°

Different

LCIO

LC50 == lethal

lethal

capital

concentration

concentration

letters indicate

toto

50%means

10%

of

of

are

a

a

significantly

sample

sample

populationpopulationdifferent

.

.

(p < 0 .05) . Only intraspecific comparisons were tested

.

' MIIRW = moderately hard reconstituted water 1201 .

RMHRW = reformulated moderately hard reconstituted water [26] .

survived in MHRW (no sulfate added), whereas after 96 h, all

organisms were dead in the hardness = 100, SO ; - = 1,460

mg/L treatment . However, in the hardness = 200 and hardness

= 300 mg/L treatments, 15% and 60% of test organisms sur-

vived, respectively.

Whereas the mean LC50 for C. duhia in standard MIIRW

(hardness = 90) was 2,050 mg SO

; L, the mean LC50s sub-

stantially increased at hardness values of 200 and 300 mg/L

(Table 4) . Mean LC50s were even higher at the higher hardness

values of 390, 484, and 578 mg/L, with a maximum of 3,516

mg SO; - /L at a hardness of 484 (Table 4) . The LC10s increased

as well, from 1,759 mg SO ; -/L at a hardness of 90 mg/L to

2,173 mg SO4 /L, and 2,389 mg SO; -/L at hardness values of

200 and 300 mg/L, respectively . Whereas in the 90 through

500 nominal hardness tests, measured sulfate concentrations

were very close to nominal sulfate concentrations, measured

sulfate in the 600 nominal hardness tests was somewhat lower

than nominal sulfate concentrations, suggesting that some pre-

cipitation of CaSO, occurred . Therefore, results may be ques-

tionable at this hardness level . If the mean LC1O at that hard-

ness is excluded, a linear relationship exists between water

hardness and LCIO, described by the formula LCIO (mg

SO ; /L) = 2

.685(hardness) + 1595.5, r2 = 0.959 . When the

LC10 at a hardness of 578 (nominal hardness of 600) is in-

cluded, the relationship is better described by a logarithmic

function with the formula LCI0 (mg SO ;

-/L) _

526 .24(ln[hardness])-574 .81 (rr= 0

.8713)

.

048 hours

•

•

722hous

RC

96 hours

-

control (EPA hardness-IW haNms

00 hardness x300

water)

Fig . 2 . Effect of hardness on toxicity of elevated sulfate to Hyale/la

in moderately hard reconstituted water. Average measured sulfate con-

centration was 1,460 mg/L (standard deviation = 25) fur the three

treatments excluding the control (106 mg/L sulfate) . EPA = Envi-

ronmental Protection Agency . Different upper- or lower-case letters

indicate means are significantly different (p < 0 .05) .

Species

Water type

Mean' LC50s

(mg SOI - /L)

Range

LC10^

(mg SO; -/L)

CC

IL

1/C.

.

duhia"teeaztecaduhia

.

MHRWRMHRW^RMHRWMHRW'

2,050

2,855

2.526512 AHBA

2,436

1,869-2,2702,835-2,876431-607-2,607

2,1852,2161,759262

* * * * * PC #2 * * * * *

---

1208

Environ . Toxicol

. Chem. 24, 2005

Table 4 . Influence of water hardness on toxicity of sulfate to

Ceriodaphnia dvhia in MHRW'

MHRW = moderately hard reconstituted water [20] .

Lethal concentrations to 50% of a sample population (LC50s) are

geometric means craft Spearman-Karber values generated for a giv-

en organism using measured sulfate concentrations,

°Lethal concentration to 10% of a sample population (Lot)

values

were generated using probit analysis with the combined data from

all tests for a given treatment .

Influence of chloride on

the toxicity of sulfate

Sulfate toxicity to H. azteca decreased with increased levels

of chloride when hardness was held constant (Fig . 3) . At the

lowest measured chloride concentration tested (5 mg/L), only

20% of the test organisms exposed to 2,846 ntg

S02 -1L

were

alive after 96 h, and none of these organisms were functionally

alive. At 13 mg CI -

/L, both total and functional survival in-

creased nominally, but not significantly (p > 0 .05) ; however,

significant increases in total and functional survival were ob-

served at and above 18 mg Cl /L (p < 0.05)

. Survival was

85% and 100% in the 36 and 67 mg Cl-/L treatments, re-

spectively . Bromide concentrations in all treatments were be-

low detection limits (<0 .01 mg/L) .

Influence of acclimation on the toxicity

ofsufate to C . dubia

In this experiment, with

C. dubia acclimated for several

generations to either 500 or 1,000 mg S0

42-/L,

nominal in-

creases in mean LC50 values were observed ; however, these

means were not significantly greater (p < 0 .05) than that for

organisms cultured in standard MHRW (Fig . 4) .

too

90

so

70

6

0

20

o total

∎functional

5

36

18

23

chloride (mg/L)

Fig . 3 . Effect of increasing chloride concentrations on sulfate toxicity

to Hya/ells azleca. Mean t standard deviation sulfate concentration

for all treatments was 2,846

'-

80 mg SOj-/L, mean hardness was

106 t 2 mg/L as CaCO3 , and Ca:Mg was 5.4 : 1, Different upper- or

lower-case letters indicate means are significantly different (p < 0 .05) .

Total = all survivors including those lying on bottom barely moving ;

functional = survivors that arc moving about .

67

D .J . Soucck and A .J . Kennedy

500

E

2300 -

2100

1900

E

0

E

1700

1500

I

A

I

A

MHRW

MHRW+500mgIL MHRW11000mg/L

sulfate

sulfate

culture water

Fig . 4 . Effect ofacclimation on sulfate toxicity to

Ceriodaphnia dubia_

Organisms were cultured for at least two generations m moderately

hard reconstituted water (MIIRW) . MIIRW with 500 mg

S04'_,

or

MHRW with 1,000 mg SO ; - . Three tests were conducted with each

population of organisms, Treatments with the same upper-case letters

indicate that means are not significantly different (p < 0 .05) . LC50

= median lethal concentration .

DISCUSSION

Toxicity of ,sulfate to

freshwater invertebrates in MHRW

The geometric mean for the three tests with C. dubia in

this study was 2,050 mg SO; /L (Table 2), which compares

favorably with the value of 3,080 mg Na,SO,/L (equivalent

to 2,082 mg SO ; - /L) generated in previous studies [11] . Values

generated in this study for H . azteca

and S.

simile

were lower

than values generated by others for the fathead minnow, P.

promelas (5,380 mg/L), and D . magna (3,096 mg/L) [7 lj . The

midge, C. tentans, was relatively insensitive compared with

other invertebrates . This finding agrees with the observation

of no significant reductions in relative chironomid abundance

in waters exceeding 3,000 mg SO ; -/L below a coal processing

discharge facility (A .J . Kennedy, unpublished data) . The Brit-

ish Columbia Ministry of Environment, Land and Parks

(BCMELP) has an online database (w lapwww .gov .bc .ca/wa d

wq/BCguidelines/sulphate/index .html) that includes a variety

of sulfate toxicity data for a number species . The values gen-

erated by BCMELP for Hyalella were quite variable and not

similar to that obtained in this study using MHRW ; however,

with the exception of hardness estimates, water quality data

were not presented in the database, so it is difficult to make

comparisons with our study . As will be discussed below, water

quality data, including cations and anions present, are critical

for predicting the responses of freshwater organisms (espe-

cially Hyalella) to elevated sulfate concentrations .

Influence of chloride on the toxicity of sulfate

The composition of dilution water used during testing in

this study had a dramatic effect on the toxicity of sulfate to

Hyalella

. In fact, the 96-h LC50 in RMHRW was 5 .5-fold

higher than that generated using MHRW . Both dilution waters

were similar in terms of hardness (-90-106 mg/L as CaCO,),

alkalinity, and fill, but one potential reason for the difference

in response is the difference in chloride concentrations between

the two media (see Table 1) . Freshwater organisms tend to

osmoregulate hypertonically with respect to the surrounding

medium, achieved by active transport of ions into the hemo-

lymph [28,29] . The principal inorganic anion of crustacean

hemolymph is chloride, and it has been suggested that low

chloride concentrations may limit the distribution of at least

Ilardncss,

nominal

(actual)

n

Mean LC50s

(mg SO;-/L)

Range

(mg SO; - /L)

LCIO ,

(mg S04'- /L)

9o (89)

2,050

1,969-2,270

1,759

200 (194)

3,000

2,706-3,265

2,173

300 (288)

2,946

2,383-3,361

2,389

400 (390)

3,174

3,073-3,369

2,744

500 (484)

3,516

3,338 3,716

2,793

600 (578)

3,288

2,761-4,220

2,547

* * * * * PC #2 * * * * *

---

Sulfate toxicity to freshwater invertebrates

one cut yhaline amphipod (Co, ophiurn crurvispinum) in fresh-

waters [30] . H. azteca is a euryhaline amphipod [7], and per-

haps when encountering high ion (Na` and SO') concentra-

tions in MHRW, it is not able to osmoregulate because of the

relatively low concentration of chloride . This same effect was

observed, to a lesser extent, with C . dubia .

The experiment with Hvalella, in which hardness and sul-

fate were held constant and chloride was variable (see Fig . 3),

supports the hypothesis that chloride has a protective effect

against sulfate toxicity, because incremental increases in chlo-

ride were associated with incremental increases in survival .

Borgmann [31] included bromide as one of the ions required

by Hyalella for long-term survival, stating that chloride is not

required ; however, chloride is chemically similar to bromide,

and results of this study indicate that if chloride is not indeed

required, it does appear to at least provide protection from salt

toxicity . Bromide was not present at measurable concentrations

(<0

.01 mg/L) In our experiments . The results of this study

further support the findings of others that MIIRW may not be

an acceptable medium for water-only testing with Hyalella

[26] .

The fingernail clam, S. simile, had a marginally lower LC10

value for sulfate than that of C, dubia in MHRW, but the former

was not tested in RMHRW because of temporal restrictions

in its availability . It remains unclear whether or not mollusks

will have the same physiological response as two crustaceans

to increased chloride in toxicity experiments with sulfate . In

a field study, 76% of transplanted Asian clams

(Corbicula

/lumlnea) in and below a treated mining discharge survived

sulfate levels of approximately 3,600 mg SO ;- /L with -700

mg CI - /L, although, as will be discussed below

. hardness

(700-800 mg/L as CaCO,) likely played a role in this system

[5] . Chloride is a principal anion in the hemolymph of most

bivalves [32], but others have found that in the unionoidean

Toxolasma texasensis, chloride and bicarbonate are equivalent

anionic components [33] . Because bicarbonate is readily avail-

able via respiration and metabolism, this mussel may not de-

pend on external chloride concentrations for osmorcgulation

to the extent that some crustaceans do . If this is the case, the

effect of chloride observed for Hvalella and Ceriodaphnia

might not be manifested in some unionoidean bivalves, and

further work should be done to clarify this .

Influence of hardness on the toxicity of

sodium

sulfate

Another factor that appears to have a strong effect on the

toxicity of sulfate is the presence of other major cations, in

this case, calcium and magnesium, measured as hardness . In

our sodium-dominated system, increased hardness reduced the

toxicity of sulfate to Hyalella ( see Fig . 2) and had a dramatic

effect on the 48-h LC50 for C

.

dubia (see Table 4)

. Mount et

al . [II] obtained a similar result in that when using only

Na,SO4 , the LC50 for C . dubia was 2,082 mg SO; -/L,

but

when using a L 1 ratio of Na2SO4 and MgSO4 , the LC50 in-

creased to 2,335 mg SO /L . They were careful to point out

that the effect was not necessarily caused by hardness, but

rather by multiple major cations, citing that the LC50 (ex-

pressed as mg CI - /L) for C dubia in NaCI was nearly identical

to that in CaCI,, despite the fact that the two solutions had

very different hardness values . However, increased calcium is

known to decrease the passive permeability of gill epithelia

to water and ions in seawater-adapted fish and crabs [34,35J .

A similar phenomenon may explain the results of the hardness

experiments conducted in this study ; i .e ., we hypothesize that

Environ Toxicol. Chem . 24, 2005

1209

the increased calcium concentrations at higher hardness levels

reduced epithelial permeability, thus reducing passive diffu-

sion and the energy required to osmoregulate and accounting

for the decrease in toxicity . In support of this hypothesis, the

decreased toxicity of sulfate to 1yalella in RMIIRW was not

entirely explained by the increased chloride concentration (see

Fig . I), The different Ca :Mg ratio also appeared to have an

effect, and hardness in RMHRW was similar to that in MHRW

(- 106 and -90 mg/L as CaCO 5 , respectively) . An alternative

hypothesis is that increased calcium is competing for binding

sites in a manner similar to that proposed for metals like copper

[36] ; however, this may be unlikely, because sulfate is an anion

and sodium is a monovalent cation . Further experiments are

required to test these hypotheses .

Others have observed reduced toxicity of saline solutions

because of increased hardness . Dwyer et al . [8] demonstrated

that increasing the hardness of an NaCl-dominated irrigation

return water reduced its toxicity to striped bass and

D . magna .

A similar phenomenon was observed with a coal processing

discharge in Ohio, USA [5,15] . Although this discharge did

include elevated sulfate (3,672 mg/L) and chloride (792 mg/

L) concentrations, the nature of the toxicity was complicated

by other ions in solution . The hardness of the field-collected

effluent (-792 ± 43 mg/L as CaCO,) and several synthetic

solutions of varying hardness appeared to reduce sodium and

sulfate-dominated TDS toxicity in a fashion similar to that

observed in the current study, on both an acute and chronic

scale [5 ; Alan l . Kennedy, unpublished data] . In addition, the

BCMELP database suggests a hardness effect on sulfate tox-

icity for both D . magna and Hyalella . The present study has

shown quantitatively that, in a sodium-dominated system, sul-

fate toxicity is reduced as hardness progressively increases,

although results may require further investigation at the highest

hardness tested (578 mg/L) . Higher hardness levels should be

tested to determine whether the relationship remains linear or

is logarithmic and reaches an asymptote .

Influence of acclimation on the toxicity

of sulfate to C . dubia

We hypothesized that C. dubia acclimated to varying levels

of sulfate would be less sensitive to sulfate than naive organ-

isms, as implied in other studies addressing TDS acclimation

[1,37] and shock [38] . Although the LC50s for the sulfate-

acclimated organisms were nominally higher, the means were

not significantly different from those of unacelimated organ-

isms . Perhaps more generations of exposure are required before

a significant benefit is seen, and further work should be done

in this area .

CONCLUSIONS

In conclusion, sulfate toxicity is a complex issue, and a

number of factors may interact to determine the responses of

various organisms to sodium and sulfate-dominated, saline wa-

ters . We have found that in MHRW, 11. azteca is the most

sensitive to sulfate of the four invertebrates tested, followed

by C. dubia

and

S

. simile, then C. tentans . Furthermore, we

demonstrated that increasing chloride concentration reduces

the toxicity of sulfate to Hyalella, and increasing water hard-

ness ameliorates sodium sulfate toxicity to

Hyalella and C.

dubia . More research is required into the hardness issue to

determine whether it was truly calcium that ameliorated sulfate

toxicity, because only one Ca

:Mg ratio was used in this study

when increasing hardness, and other major cations like potas-

* * * * * PC #2 * * * * *

---

121 0

Environ Toricol . Chem . 24, 2005

sittm were not investigated

. In addition, the actual mechanism

behind the mode of protection from multiple cations should

be studied . Finally, the aforementioned issues should be ex-

amined at a chronic scale using sublethal and/or multigener-

ational endpoints as more accurate indicators of population-

level effects

.

Acknowledgement--This

study was funded by the Illinois Environ-

mental Protection Agency and the Illinois Coal Association

. The au-

thors thank loan Esarey, Center for Ecological Entomology, Illinois

Natural History Survey (INHS), Jens Sandberger, Center for Aquatic

Ecology . INKS, and L

.oretta Skowron, Illinois State Water Survey .

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for conducting acute toxicity tests on test materials with fishes,

macroinvertebrates, and amphibians . P729-96 . In Annual Book

25

.

of

American

ASTMStandards,Public

Health

Vol

Association,

11 .05 . Philadelphia,

American Water

PA, pp

Works

178-199As-

.

sociation, Water Environment Federation . 1998 . Standard Meth-

ods for the Examination of Water and

Wastewater, 20th ed-

American Public Health Association, Washington, DC .

26 . Smith ME, Lazorchak JM, Bern . LE, Brewer-Swa tz 5, Thoeny

WT. 1997

. A reformulated, reconstituted water for testing the

freshwater amphipod, Hyalella azteca . Environ Toxicol Chem

16 :1229-1233 .

27 . Salt J, Lehman A

. 1996 . JMP® Start Statistics . SAS Institute,

Duxbury Press, Belmont, CA, USA .

28 . Greenaway 11. 1979 . Fresh water invertebrates

. In Mutiny G, ed,

Comparative Physiology of Osmoregulation in Animals . Aca-

demic, London, UK, pp 117-162 .

29 .

Environment,Schmidt-Nielsen

5th

Ked.

.

1997Cambridge

. Animal

University

Physiology

Press,

: Adaptation

Cambridge,and

UK, pp 305-314 .

30 . Bayliss D, Harris RR . 1988 . Chloride regulation in the freshwater

amphipod

Corophiun curvispinum and acclamatory effects of

external C1 - . J Comp Physiol 158 :81--90 .

31. Borgmann U . 1996

. Systematic analysis of aqueous ion require-

ments of Hyalella azteca : AA standard artificial medium including

the

363 .

essential bromide ion

Arch

Environ

Contam Toxicol30 :356-

32 . McMahon RF, Bogan AE. 2001 . Mollusea : Bivalvia . In Thorp

JH, Covich AP, eds, Ecology and Classification of North Amer-

icon

Freshwater Invertebrates, 2nd ed Academic, San Diego,

CA, USA, pp 352-353 .

33 .

in

Byme

freshwater

RA, Dietz

bivalvesTI4

.

.

1997

J Exp

. Ion

Blottransport

200:457-465and

acid-base

.

balance

34 . Lucu C, Flik G . 1999 . Na*-K--ATPase and Na`/CH" exchange

activities in gills of hyperregulating Carcinus maenas .AmJPhy-

sial 276 :8490-R499 .

35 . Pie P, Maetz .J . 1981 . Role of external calcium in sodium and

chloride transport in the gills of seawater-adapted Mugil capita .

J Comp Physiol B 141

:511-521 .

36 . Paquin PR, Gorusch JW, Apte 5, Batley GE, Bowles KC, Camp-

bell PGC, Delos CG, Di Toro DM, Dwyer RL, Galvez F, Gen-

semer RW, Goss GO, Hogstrand C, Janssen CR,

McGeer JC,

Naddy RE, Playle RC, Santore RC, Schneider U, Stubblefield

WA, Wood CM, Kuen BW. 2002 . The biotic ligand model : A

historical overview .

Comp Biochem Physiol C 133'.3-35 .

37 . Keel TM, Peterka JJ . 1995 . Survival to hatching of fishes in

sulfate-saline waters, Devils Lake, North Dakota . Can J Fish

Aqua( Sci 52 :464-469 .

38 . Wichard W, Tsui PTP, Komnick H . 1973- Effect of different sa-

linities on the conifonn chloride cells of mayfly larvae . J Insect

Physiol 19 :1825--1835 .

* * * * * PC #2 * * * * *

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EXHIBIT

2

* * * * * PC #2 * * * * *

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/SETAVPNESS/

Environmentat Toxicology and Chemistry, Vol . 26, No . 4, pp . 773-779 . 2007

© 2007 SETAC

Printed in the USA

0730-7268/07 $12

.00 ~

.00

COMPARISON OF HARDNESS- AND CHLORIDE-REGULATED ACUTE EFFECTS OF

SODIUM SULFATE ON TWO FRESHWATER CRUSTACEANS

DAVID JOHN SOUCEK*

Illinois Natural History Survey, Champaign, Illinois 61920, USA

(Received 9 May 2006 ; Accepted 31 October 2006)

Abstract-Based on previous observations that hardness (and potentially chloride) influences sodium sulfate toxicity, the objective

of the current study was to quantify the influence of both chloride and water hardness on acute toxicity to Hyde/lo azteca and

Cerfodaphaia dubia . In addition, observed toxicity data from the present study were compared to toxicity predictions by the salinity/

toxicity relationship (Sfld) model . Hardness had a strong influence on sulfate toxicity that was similar for both crustaceans, and

nearly identical median lethal concentration (LC50)/hardness slopes were observed for the two species over the tested range .

Chloride had a strong but variable influence on sulfate acute toxicity, depending on the species tested and the concentration range .

At lower chloride concentrations, LC50.s for H

.

azteca strongly were correlated positively with chloride concentration, although

chloride did not affect the toxicity of sodium sulfate to C . dubia . The opposite trend was observed over the higher range of chloride

concentrations where there was a negative correlation between chloride concentration and sulfate LC50 for both species . The widely

ranging values for both species and a high correlation between LC50s in terms of sulfate and conductivity suggested that, whether

based on sulfate, conductivity, or total dissolved solids (TDS), attempts at water quality standard development should incorporate

the fact that water quality parameters such as hardness and chloride strongly influence the toxicity of high TDS solutions . The STR

model predicted toxicity to C. dubia relatively well when chloride was variable and hardness fixed at approximately 100 mg/L ;

however, the model did not account for the protective effect of hardness on major ion/TDS toxicity .

Keywords-Sulfate

Total dissolved solids

Hyoleta

Ceriodaphnia

Salinity/toxicity relationship model

INTRODUCTION

Currently no federal water quality criteria exist for the pro-

tection of freshwater life for total dissolved solids (TDS), sul-

fate, or sodium ; however, toxicity due to major ions or TDS

has received increasing attention in recent years [1,2] . Ordi-

narily benign major ions (e .g ., sodium, sulfate) and, therefore,

TDS, which is essentially the sum of the concentrations of all

common ions (e .g ., sodium, potassium, calcium, magnesium,

chloride, sulfate, and bicarbonate) in freshwaters, can reach

concentrations in wastewater discharges that severely impair

sensitive aquatic species [3-7] . Common sources of effluents

with elevated TDS include reverse osmosis systems, pH mod-

ifications of wastewater, agricultural runoff, gas and oil pro-

duction, and coal or metal mining operations [11 ; investigations

of major ion toxicity also have included inundation of fresh-

water systems by brackish water and laboratory-formulated

salt solutions [5-13] .

The fact that TDS toxicity is dependent on the ionic com-

position of a water or effluent has been well established

17,9,12,[4,15] . Mount et al . [l2] developed statistical models

to predict toxicity of high TDS waters to standard test organ-

isms based on ionic composition ; seven major ions (Na', K',

Ca"-', Mg', Cl- , HCO,, and SO2-) were included in the anal-

ysis . In that study, solutions were more toxic when dominated

by particular major ions (i.e., K', Mg', HCO ;), and toxicity

due to several individual ions, including SO ; - , to Cerio-

daphnia dubia and Daplmia magna was reduced when so-

lutions contained more than one cation [12] . Several research-

ers have observed that hardness and/or multiple nontoxic cat-

ions in solution ameliorate major ion toxicity [9,12,15], and

*To whom correspondence may be addressed

( d -soucek@inhs .uiuc .ed u ) .

773

laboratory experiments with synthesized freshwaters have

demonstrated that increasing hardness at a constant calcium-

to-magnesium ratio (Ca :Mg) results in decreased sodium sul-

fate toxicity to C. dubia [14) .

In experiments with sodium sulfate in laboratory-synthe-

sized freshwaters, Soucek and Kennedy [14] observed that,

while composition of dilution water strongly affected sulfate

toxicity to C . dubia, the magnitude of the effect on H. azteca

was even greater. Specifically, whereas the median lethal con-

centrations (LC50s) for

C

. dubia in two diluents with different

chloride concentrations and Ca :Mg ratios ranged from 2,050

to 2,526 mg SO

; -/L,

the LC5os for H. azteca in the same two

diluents were 512 and 2,855 mg SO; /L, respectively [141 .

Freshwater organisms use several different osmorcgulatory

strategies, but most freshwater amphipods and daphnid cla-

docerans regulate hypertonically with respect to the surround-

ing medium ; this is achieved by active transport of ions, prin-

cipally chloride, into the hemolymph (see [ 16-191) . However,

even among amphipods, there is a wide range of sodium and

chloride influx rates and integument permeabilities, which de-

termine osmoregulatory effectiveness [20,21], so the ionic

composition of water may regulate to varying degrees the

response of different species to high levels of sodium sulfate .

The observed contrast in responses of C .

dubia and H .

azteca to sulfate under different water quality conditions [14]

led to the interest in quantifying the relationships between

sulfate toxicity and hardness and chloride concentrations for

the two distantly related freshwater crustaceans

. Therefore, the

objectives of the current study first were to determine the

influence of hardness on sodium sulfate toxicity to H . azteca

and to compare its responses to those of C . dubia

and, second,

to quantify the effects of chloride on acute toxicity of sodium

* * * * * PC #2 * * * * *

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774

Environ . ToricoL CIa-in . 26, 2007

sulfate to H . azieca and C. duhia over a wide range of chloride

concentrations . The data generated in the first two objectives

were useful for investigating the relationship between LC50s

calculated in terms of sulfate and those calculated in terms of

conductivity to determine the potential utility of a conductiv-

ity- or TDS-based water quality standard . The data also pre-

sented an opportunity to test the effectiveness of the salinity/

toxicity relationship (STR) model developed by Mount and

Gulley [221 in predicting acute toxicity of a wide range of

sodium, sulfate, chloride, and hardness combinations to C .

dubia .

MATERIALS AND METHODS

General culturing and testing methods

The cladoceran, Ceriodaphnia dubia, was cultured in the

laboratory according to U .S . Environmental Protection Agency

(U .S . EPA) methods [231 . Amphipods, Hyalella azteca, also

were cultured in the laboratory according to U .S . EPA methods

1241 in a reformulated, moderately hard, reconstituted water

described in Smith et al . [25]

For toxicity testing, a pure (99%) grade of anhydrous so-

dium sulfate (Na,SO,) (Chemical Abstract Service [CAS]

7757-82-6) was obtained from Fisher Scientific (Pittsburgh,

PA, USA) to serve as the source of sulfate . Previous experi-

ments indicated that the salts and deionized water sources used

for our experiments had low to undetectable levels of trace

metal contaminants [14,26] .

For definitive static, nonrenewal toxicity tests, conducted

according to American Society for Testing and Materials E729-

96 methods [271, treatments comprised a 75% dilution series

(i .e ., the 100% concentration was diluted serially by 25%),

rather than the standard 50%, because major ion toxicity tests

often cause 100% mortality in one concentration and 0% mor-

tality in the next highest concentration if the spread is too

great . Five to six concentrations were tested in addition to

controls with four replicates tested per concentration . Tests

with C. dubia were conducted for 48 h with a 16 :8-h light :

dark photoperiod at 25'C, and H. azteca were exposed for 96

h at 22'C and a 16 :8-h light :dark photoperiod . Both organisms

were exposed in 50-ml glass beakers with five organisms per

beaker and, for H . azteca . I

g of quartz sand was added to

each beaker to serve as substrate . Only one of the 63 tests was

fed, and that fed test had an LC50 in the range of two other

tests conducted with the same organism in the same water

without food . Ceriodaphnia duhia used were less than 24-h

old, and H. azteca were approximately third instar (7-14-d

old) . Percent survival in each replicate was recorded every 24

h and at the end of the exposure period

. A dissecting micro-

scope was used to assess survival of H . azteca .

Standard water chemistry characteristics were measured at

both the beginning and the end of each exposure period . Tem-

perature, pH, conductivity, and dissolved oxygen were mea-

sured using appropriate meters, and alkalinity and hardness

were measured (beginning of tests only) by titration as de-

scribed by American Public Health Association et al. [28] .

Samples from each treatment were analyzed to confirm sulfate

concentrations by ion chromatography at the Illinois Natural

History Survey Aquatic Chemistry Laboratory (Champaign,

IL, USA) .

All LC50s were calculated based on both measured sulfate

concentrations and measured specific conductivity values for

each test concentration using the Spcarman-Karber method

[29] . To increase confidence in LC5ns, three to five assays

1) 1 . Soucek

were conducted with each organism for each water quality

combination . This provided a stronger estimate of the mean

LC50 for a given set of water quality characteristics for each

species

. In all, 63 LC5ns were generated .

Influence of chloride on the toxicity of sodium sulfate

In these experiments, the toxicity of sulfate (with sodium

as the major cation) to H. azteca and C. duhia was measured

in freshwater solutions having nominal chloride concentrations

of 1 .9, 10, 15, 20, 25, 33 (H. azteca only), 100, 300, and 500

ntg CI/L . Chloride, as NaCI (CAS 7647-14-5, Fisher Scientific

Catalog AC42429-0010), was added at appropriate concentra-

tions to a solution with a hardness of approximately 100

mg/L (molar ratio of Ca:Mg

= 1.41 ; 2.33 in terms of mass) .

The Ca :Mg ratio was chosen because it is the median value

for water bodies sampled in Illinois (R . Mosher, Illinois En-

vironmental Protection Agency, Springfield, IL, USA . personal

communication)

. Whole carboys were made for each elevated

chloride level, and this water was used as both diluent and

control ; therefore, each concentration within a given test had

the same chloride concentration (i .e . . [Cl - ] did not change with

dilution) . The only parameters that varied within a particular

test were sodium, sulfate, and conductivity . At least three tests

were conducted for each hardness to provide a mean LC50

and standard deviation . Exposures were conducted using the

same laboratory and calculation methods described above.

After LC50s were calculated as described above, regression

analysis was conducted using 1MP® software [301 to determine

the relationship between chloride concentration and sulfate

LC50 for each species ; mean LC50s for each chloride con-

centration were used in these analyses . Because observation

of data scatter indicated two different trends were involved

depending on the chloride concentration range, two separate

analyses were conducted for each species : One for the range

of 5 (1 .9 mg/L nominal concentration) to 25 mg Cl/L and one

for the range of 25 to 500 mg CI/L . Then, multiple regression

analysis with covariance was conducted for the same data

ranges using all individual data points to generate an equation

for both species, and to determine if the curves were signifi-

cantly different for the two species .

Influence of hardness on the toxicity of sodium sulfate

In these experiments, the toxicity of sulfate (with sodium

as the major cation) to H. azteca was tested in six freshwater

solutions having nominal hardness values of <100, 200, 300,

400 . 500, and 600 mg/L (as CaCO,) . Hardness was increased

by adding enough CaSO, (CAS 7778-18-9) and MgSO, (CAS

7487-88-9), at a set molar ratio (Ca :Mg = 1 .41 ; 2 .33 in terms

of mass), to achieve the nominal hardness . Measured hardness

values for all tests were similar to target nominal hardness

values (±2 .2%)

. A chloride concentration of 25 mg/L was used

for all tests investigating the effects of hardness on sodium

sulfate toxicity to H. azteca based on results from the above-

described tests investigating the effects of chloride on sodium

sulfate toxicity to H.

azteca and C dubia . Whole carboys

were made for each elevated hardness level, and this water

was used as both diluent and control ; therefore, each concen-

tration within a given test had the same hardness (i .e ., [Ca2']

and [Mg") did not change with dilution) . The only parameters

that varied within a particular test were sodium, sulfate, and

conductivity . At least three tests were conducted for each hard-

ness to provide a mean LC50 and standard deviation

. Expo-

sures were conducted using the same laboratory and calcu-

* * * * * PC #2 * * * * *

---

Water quality effects on sulfate toxicity

lation methods described above . The LC50s for IL azteca were

compared to previously generated LC50s for C . dubia [ 14],

which were conducted in solutions having a Ca :Mg molar ratio

of 0.88 and [CI-] of 5 mg/L .

After LC50s were calculated as described above, regression

analysis was conducted using 7MPx software [30] to determine

the relationship between hardness and sulfate LC50 for each

species . Mean LC50s for each hardness were used in these

analyses . Then, multiple regression analysis with covariance

was conducted for the same data ranges using all individual

data points to generate an equation for both species and to

determine if the curves were significantly different for the two

species .

Relationship between sulfate LCSOs and conductivity

LC50s

To investigate variability in conductivity at sulfate LC50

concentrations, linear regression analysis was used, and C .

dubia data from Soucek and Kennedy [14] were included in

the analyses .

Comparison of test results with SIR model predictions

To compare results from the present study to toxicity pre-

dictions generated by the STR model [22] . nominal concen-

trations of all constituent ions (except HI and OH - , which are

not required by the model) were calculated at observed mean

sulfate LC50s for CC dubia in each test solution type (CI- _

x, hardness = y, and Ca :Mg =

z) .

The model does not predict

toxicity to H

. azteca

. Ions required by the model are Ca",

Mg", Na', K - , CI - , HCO, and SO' - . These calculations were

possible because adding known salt concentrations to deion-

ized water generated all test solutions . The average of the

absolute value of % difference between nominal and measured

sulfate concentrations was 2 .082% .

The model output includes equivalents of cations and an-

ions and requires that, to have confidence in model output, the

difference between the two be less than 15% [22] . The average

(±standard deviation) % difference between cations and an-

ions for inputs was 0 .09 (±0 .01)%, indicating excellent agree-

ment between cation and anion equivalents . Other model out-

puts included calculated TDS, a NUMCAT value that estimates

effective number of cations, LC50 in terms of % of solution,

and % survival in 100% of solution . To examine the predictive

ability of the STR model over the range of solutions tested,

we created scatter plots of predicted % survival versus either

chloride concentration or water hardness as appropriate for

each species tested . Because all of our input values were ion

concentrations calculated at experimentally observed sulfate

LC50s, the observed % survival was always 50% . Observed

survival data were plotted as a horizontal line for compar-

ison to STR-predicted data.

RESULTS

Influence of chloride on the toxicity of sodium sulfate

Chloride had variable effects on sodium sulfate toxicity to

C. dubia and H. azteca over the range of 5 to 500 mg

Cl- /L . For H. azteca, two different linear trends were observed

depending on the chloride range (Fig . IA and B) . Increasing

chloride concentration from 5 to 25 mg/L resulted in increasing

SO; - LC50s (r2 = 0.8503, p = 0.0258) for H . azteca

(Fig .

IA), although for C . dubia, the slope was not significantly

different from zero over this chloride concentration range (r2

= 0

.4906, p = 0

.1877) . In addition, the LC50s for C

.

dubia

loo) A

1- 13 86Ar a 2085.1

,'=0.49%,,p-571877 f

f

c

IAq

V I(YNI

Chlnrile Oncht .1

'2 X0.1, . 2530 .7

09493,p =0,025 7,

y= .0 .8/5+19(4

.4

r t -n .A75.p

0.0105

n 7t. name

too

Environ. Toxicoi. Chere . 26, 2007

775

1011

3110

cool

Chloride onen .)

25

Figmg/L

. I .

Influence

and (B) 25

of

to

chloride

500 mg/L,

concentration

on toxicity

over

of

two

sodium

ranges,

sulfate

(A) 5

toto

Ceriodaphnia dubia and Hyalella azteca . Hardness was approxi-

mately

for the

100tests

.

mg/lat

5

for

mg

all

Cl-tests

/L (0and

.88)C.

.Mg

LC50

molar

= Median

ratios

lethal

were 1,41

concentra-except

tion .

were higher than those for H. azteca for each chloride con-

centration over this range . When using a combined data set

of individual test LC50s for C. dubia and H. azteca over this

chloride range and at hardness = 100 (n = 33) . in a simple

linear regression analysis with covariance (with species as a

treatment effect and chloride concentration as continuous ef-

fect) . a strong positive relationship was observed (r 1 = 0 .7900,

p < 0.0001) with both the chloride and treatment (species)

effects being significant (p < 0 .0001 ; Table 1) .

Although a positive relationship between chloride concen-

tration and SO LC50 was observed for H. azteca over the

range of 5 to 25 tug Cl - /L, a significantly negative trend (r'

= 0.875, p = 0.0195) was observed over the range of 25 to

500 mg CI - /L . An even stronger negative relationship (r' _

0

.9493,

p = 0.0257) was observed for C . dubia over the same

chloride range . When using the combined data set of individual

test LC50s for C . dubia and H. azteca over this chloride range

and at hardness = 100 (n = 30), in a multiple linear regression

analysis with covariance as described above, a negative re-

lationship was observed (r' = 0 .6539, p < 0 .000 1), with both

the chloride and treatment (species) effects being significant

(p < 0.0001 and p = 0.0003, respectively ; Table 1) .

Effects of hardness on toxicity of sulfate to Hyalella at

chloride = 25 mg/L

A strong linear trend of decreased sulfate toxicity with

increased hardness (r2

= 0 .7092, p = 0 .0354) was observed

for H. azteca (Fig . 2) . The LC50 values increased from less

than 1,900 mg/L at hardness = 100 mg/L, to greater than 4,000

50(1

WO

* * * * * PC #2 * * * * *

---

776

Environ . Toricol .

Chem .

26, 2007

Table I . Results of multiple regression analysis with covariance for

three different subsets of data . Individual median lethal concentrations

were used as data points . Data for both species were included in

analysis

Tenn

Estimate

ICI - ] range = S to 25 nag/I, ; hardness approximately 100 mg/I

r= = 07900, u = 33

au

201q

15(5

1000

v=3.6716,41527.1

r~=0.107.p=0.0354

I(vdAi, ,vI>

P

mg/L at a hardness of 500 mg/L . The mean LC50 value at

600-mg/h hardness was lower than that at 500-mg/L hardness .

It remains unclear how the trend will continue with increasing

hardness above 600 mg/I

.

. When using the combined data set

of individual test LC50s for C. dubia and H. azreca over this

hardness range (n = 38) in a multiple linear regression analysis

with covariance as described above, a positive relationship was

observed (r' = 0.5177 . p < 0.0001 ; Table 1) . The hardness

effect was observed to be significant (p < 0 .0001), but the

treatment (species) effect was not (p = 0 .9046 ; Table 1) .

Relationship between sulfate LC50s and conductivity

LC50s

Conductivity LC50s ranged from 1,071 to 8,449 µmhos/

cm, and LC50s based on sulfate ranged from 512 to 4,345 mg

SO;-

/L (Fig

. 3)

. When including all data from the present study

and C. dubia data from Soucek et al . [14], there was a strong

positive relationship between sulfate LC50s and conductivity

•

C ,Llhta

o rt. a.mn0

0

100

200

7(

.500

50)

015)

700

Fig. 2. Influence of hardness on toxicity of sulfate to Hyatella azre

(a

and Ceriodaphnia dubia The C. duhta data arc from Soucek et al .

[141 . Concentration for all H

. azreca tests was approximately 25

mg/L, and Ca:Mg molar ratio was 1 .41 . LC50 = Median lethal con-

centration .

9000

soot)

7000

6000

I

G E 40(X1

v

9X0-

2(11x)

INK)

•

U. Jahia

o H . avleua

D .1 . Soucek

1 (101

20110

)

4(551

5000

Sulfate at LC50mg/L)

Fig . 3 . Relationship between median lethal concentration (LC50) in

terms of sulfate (mg/L) and in terms of conductivity (µmhos/cm) for

tests with flyalel/a aorta and Ceriodaphnia dubia. In addition to

the 63 new tests generated for the present study, 19 tests from Soucek

et al . [141 with C, dubia were included . Hardness values ranged from

100 to 600 mg/L and chloride ranged from 5 to 500 mg/L . Points

enclosed by the solid oval were conducted at 30(1 mg CI

- /IL and those

in the dashed oval at 5017 mg CI - H . (nominal concentrations) .

LC50s (r2 = 0.9077,

p < 0.0001 ; Fig . 3) . Twelve data points

enclosed in solid and dashed ovals in Figure 3 diverged from

the line farmed by the remaining points, suggesting sulfate

LC50s were lower than would be predicted by conductivity

LC50s

. These points were for tests with C . dubia and H. azteca

when nominal chloride concentrations were 300 mg/L (solid

oval) and 500 nag/L (dashed oval) .

Comparison of test results with STR model predictions

All ion concentrations used as input for the STR model

were concentrations at the observed mean sulfate LC50levels

for each test solution type (CI- = x, hardness = y, and Ca :

Mg = z), so observed percent survival in each case was 50% .

For tests with C . dubia in which hardness was fixed at ap-

proximately 100 mg/L and chloride varied from 5 to 500

mg/L, the STR model predicted % survival values ranging

from 69 .0 to 48

.4% (Fig . 4A) . Most predictions were greater

than 50%, and thus the model slightly underpredicted toxicity

in most cases .

For tests with C. dubia in which chloride was fixed at

approximately 25 mg/I, and hardness varied from 100 to 600

mg/L, the STR model predictions were highly variable, ranging

from 4 .1 to 82 .9% survival (Fig . 4B)

. Only the hardness =

100 mg/L prediction was greater than 50% (82 .9%) ; for hard-

ness values of 200 to 600 mg/L, toxicity was strongly over-

predicted, with % survival predictions of 4 .1 to 21 .8 .

DISCUSSION

Chloride had a strong but variable influence on acute sulfate

toxicity, depending on the species tested and the concentration

range . In multiple linear regression analyses with covariance,

including species as a variable, the species term was significant

over both the lower (5 to 25 mg

Cl-/L) and the higher (25 to

500 mg Cl -/L) chloride ranges

. The difference between the

two species was most striking over the 5- to 25-mg/L chloride

range where LC50s for H. azteca were strongly positively

correlated with chloride concentration and chloride did not

affect the response of C . dubia ( see Fig . 1) . Hyalella appears

Intercept

1,270 .23

<0 .0001

Chloride

35

.14

<0.0001

Species

Term

449 .68

Estimate

<0 .0001

P

ICI 1 range = 25 to 500 mg/L, hardness approximately 100 mg/L

r'=06539,5=30

Intercept

2,189 .48

<00001

Chloride

-1 .46

<0 .0001

Species

178 .92

0 .0003

Term

Estimate

p

Hardness range = 100 to 600, Cl = 25 mg/L

r' = 0.5177, n = 38

Intercept

1,969 .38

<0.0001

Hardness

3 .15

<0.0001

Species

-1038

0.9046

* * * * * PC #2 * * * * *

---

Water quality effects on sulfate toxicity

Pigchloride

. 4 . Percent

(A) and

survival

hardness

of(B) Ceriodaphnia

as predicted

dubia

by the

at

salinity/toxicityvarying

levels of

trations

relationship

served

the horizontal

%

at

survival

nominal

line

(STR)

is

in

sulfate

model

observed

each

Points

case

median

result50%are

lethal

. Model

output

concentrations,

inputs

predicted

were ion

by

making

STR

concen-andob-

to require a minimal amount of chloride for effective osmo-

regulation at high sodium sulfate concentrations . Although

there are several different osmoregulatory strategies used by

freshwater organisms, most freshwater amphipods and daphnid

cladocerans regulate hypersonically with respect to the sur-

rounding medium, and this is achieved by active transport of

ions into the hemolymph [16-19] . The principal inorganic an-

ion of crustacean hemolymph is chloride, and it has been sug-

gested that low chloride concentrations may limit the distri-

bution

of at least one euryhaline amphipod (Carophium rurv-

is'pinum) in freshwaters [20] . Even among amphipods, there

is a wide range of sodium and chloride influx rates and in-

tegument permeabilities that determine osmoregulatory effec-

tiveness [20,21] ; therefore, it might not be surprising that the

responses of H

. azteca and

C. dubia to sodium sulfate were

quite different over the lower range of chloride concentrations .

Although Borgmann [31] suggested that, under low salinity

conditions, bromide was required but chloride was not needed

by H. azteca

for survival, growth, and reproduction, data from

the present study suggest that the chloride is quite important

in determining the effect of elevated levels of sodium sulfate

on that species . Laboratory deionized water and concentrated

sodium sulfate solutions were analyzed previously for bro-

mide, and levels were below detection limits

[26] .

Over the higher range of chloride concentrations (25-500

mg/L), a different trend was observed . Although the slopes of

Envirun . Toxirol. Chem

.

26, 2007 777

the lines for the two crustacean species were different, there

was a negative correlation between chloride concentration and

sulfate LC50 for both species . The trend was stronger for C.

dubia, with a more negative slope (-2 .2) compared to H.

azteca (-0 .875), although r2 values were high and relation-

ships statistically significant for both . These data suggest that,

over this range of chloride concentrations, chloride and sodium

sulfate toxicity are additive . Chloride LC50s (as NaCI) for C .

dubia generally range from 900 to 1,200 mg Cl - /L (e.g ., [12]),

and so the highest two chloride concentrations in the present

study were likely to cause some toxicity without sulfate pres-

ent .

Hardness had a strong influence on sulfate acute toxicity

that was similar for both crustacean species . A number of

studies have provided evidence that increasing hardness ame-

liorates toxicity of waters with high dissolved solids concen-

trations [7,9,12,15] and Soucek and Kennedy [14] showed

quantitatively that, in a sodium-dominated system, sulfate tox-

icity to C. duhia is reduced as hardness progressively increas-

es, albeit with diminishing returns in the hardness = 600

mg/L range . In the present study, the results of multiple linear

regression analyses indicated no difference between the sen-

sitivities of the two species over the hardness range of 100 to

600 mg/L as CaCO, . This was in contrast to the results of the

tests in which chloride was varied, where the two species had

different slopes over both ranges of chloride concentrations

examined . In addition, these results are notable because nearly

identical slopes were observed for the two species despite the

fact that the waters for tests conducted with C. dubia had a

different chloride concentration (5 mg/L) and Ca :Mg molar

ratio (0 .88) than those used for tests with H. azteca (25 mg

Cl -

/L and 1 .41 Ca :Mg molar ratio) . As an explanation for this

phenomenon of hardness ameliorating sulfate toxicity, Soucek

and Kennedy [14] proposed that increased calcium concentra-

tions decrease the passive permeability of epithelial cells to

water and ions in various aquatic organisms [32,33], reducing

passive diffusion and the energy required to osmoregulate and

accounting for the decrease in toxicity . Calcium can mitigate

hydrogen ion toxicity to aquatic organisms by decreasing

membrane permeability to H* and stimulating active Na* up-

take ([see [34]) ; however, Ports and Fryer [35] found that

calcium had little effect on sodium loss in Daphnia magna .

Although data from the present study support this hypothesis,

other explanations are possible and empirical work is needed

to determine the mechanism behind the phenomenon .

In the present study, LC50s in terms of conductivity were

highly correlated with LC50s in terms of sulfate for both spe-

cies, except when extremely high chloride concentrations were

used (300 to 500 mg/L) . The plots of conductivity LC50s and

sulfate LC50s clearly illustrate the contention that knowledge

of the contribution of various major ions is critical to effec-

tively managing produced waters or effluents with high con-

centrations of dissolved solids

[2] . Not only did sulfate L.C50s

range from 512 to 4,345 mg/L, but conductivity LC50s ranged

from 1,071 to 8,449 µmhos/em . These wide ranges were ob-

served for just two species with relatively similar sensitivities .

Clearly, any attempt at water quality standard development,

whether based on TDS, conductivity, sodium, or sulfate,

should incorporate the fact that the water quality parameters

like hardness and chloride strongly regulate the toxicity of

high TDS solutions . Finally, the conductivity/sulfate plots pro-

vide further evidence that chloride and sulfate toxicity are

additive . When chloride was less than or equal to 100 mg/L,

m)

80

0 70

r

2 ts)

50

40

otrxerved F_aurv,v,,l

20

Il0

200

300

400

500

ran

I0)

Chloride

tang/L,

B

90

•

70

90

a0

observed % survisat

t 70

'0

0

Ion

ho

300...

400 . .

500

900

700

Oatdncs,

one/1.t

* * * * * PC #2 * * * * *

---

778

Environ

. Toxicol . Chew . 26 . 2007

sulfate toxicity was strictly related to conductivity

; however,

when 300 and 500 mg Cl-/L

solutions were tested, sulfate

LC50s were lower than predicted by LC50s based on con-

ductivity.

When chloride was variable and hardness fixed at approx-

imately 100 mg/L, the STR model was relatively accurate in

predicting toxicity to C. dubia ; predicted survival ranged from

48 to 69%, and observed survival was 50% in each case be-

cause calculated ion concentrations at observed sulfate LC50

were used as inputs . With one exception (48%), the model

underpredicted toxicity for this data range . This might he be-

cause the STR model largely is based on the results of fed

tests, which the authors acknowledged had a small influence

on test results [12] . However, Soucek ([36] ; h ttp ://www .pea .

state .ten

.us/news/eaw/buffalolake-item23 .pdf) compared 48-h

sulfate 1-C50s in unfed tests using moderately hard, reconsti-

tuted water [23] and reformulated moderately hard reconsti-

tuted water [25] as diluents with 48-h sulfate LC50s obtained

from fed, 7-d chronic tests in the same two diluents . In both

cases

. average LC50s for unfed tests were significantly lower

than those in fed tests

. This factor alone might explain the

discrepancy between predicted and observed results for C .

dubia for these tests .

When chloride was held constant (5 mg/L) and hardness

was varied from 100 to 600 mg/L, the STR model was rela-

tively inaccurate in predicting toxicity to C. dubia, with a trend

of underprediction at hardness = 100 ing/L, followed by in-

creasing degrees of overprediction at hardness = 200 to 600

mg/L . This finding is in agreement with Kennedy et a]

. [15],

who found that the STR model ovetpredicted toxicity to C.

dubia in sodium sulfate-dominated coal-processing effluents

with hardness values in the 700- to 800-mg/L range . The pres-

ent study suggests that the STR

model does not account for

the protective effect of hardness on major ion/TDS toxicity

;

however, because of the presence of a pattern in the inaccuracy,

data from the present study might he useful in improving the

model .

CONCLUSION

In conclusion, chloride had a strong but variable influence

on the acute toxicity of sulfate, depending on the species tested

and the concentration range . Over the 5- to 25-mg/L chloride

range, mortality of H . azteca decreased with increased chloride

concentration and chloride did not affect the response of C .

dubia .

The opposite trend was observed over the higher range

of chloride concentrations (25-500 mg/L) where increasing

chloride concentrations resulted in increased mortality at given

sulfate concentrations for both species . Hardness had a strong

influence on sulfate acute toxicity that was similar for both

crustacean species, and nearly identical LC50/hardness slopes

were observed for the two species despite the fact that test

waters for the two species had different chloride concentrations

and Ca: Mg ratios . The LC50s in terms of conductivity were

highly correlated with LC50s in terms of sulfate for both spe-

cies . The wide range of values for both conductivity and sulfate

LC50s suggests that single-value water quality standards for

TDS, conductivity, sodium, or sulfate are not practical, and

the fact that water quality parameters like hardness and chlo-

ride strongly regulate the toxicity of high TDS solutions should

he incorporated into standard development . In addition, both

the sulfate LC50/chloride plots and the conductivity/sulfate

plots provided evidence that chloride and sulfate toxicity are

additive. The STR model predicted toxicity to C . dubia rel-

D .J- Soucek

atively accurately when chloride was variable and hardness

fixed at approximately 100 mg/L; however, the model does

not account for the protective effect of hardness on major ion/

TDS toxicity . Data from the present study would be a useful

incorporation to the STR model .

Acknowledgement-The present study was funded by the U

.S . En-

vironmental Protection Agency grant CP96543701-0 . Thanks to C .

Stephan of the U.S . EPA and A . Haldeman and J . Sandberger of the

Illinois Natural History Survey

.

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on the acute toxicity of sulfate to freshwater invertebrates . Final

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. American Public Health Association, American Water Works As-

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. Environ Set Technol 11 :714-719 .

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ments of Hyalella azteca : A standard artificial medium including

the essential bromide ion . Arch Eanviron Coma . Toxicol30356-

363 .

32 . Lucn C, Flik G . 1999

. Na'-R`-ATPase and Na'/Ca"' exchange

activities in gills of hyperregulating Carcimu maenas. Am J Phys-

iol 276 :R490-R499 .

33 . Pic P, Maetz J . 1981 . Role of external calcium in sodium and

chloride transport in the gills of seawater-adapted Mugil capim .

J Camp Physiol B 141 :511-521 .

34 . Havas M, Advokaat E . 1995 . Can sodium regulation he used to

predict the relative acid-sensitivity of various life-stages and dii-

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870 .

35 . Potts WTW, Fryer G . 1979 . The effects of pH and salt content

on sodium balance in Daphnia magna and Acanlholeberis car-

vi? ostris (Crustacea : Cladoeera) . J Contra, Phvsiol B 129 :289-294 .

36 . Soucek DJ . 2006 . Effects of water quality on acute and chronic

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and Hyalella azteca, Third Quarterly Report . U .S . Environmental

Protection Agency, Chicago, IL .

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EXHIBIT

3

* * * * * PC #2 * * * * *

---

Ecotoxicology (2007) 16:317-325

DOI 10 .1007/s10646-007-0133- 5

Bioenergetic effects of sodium sulfate on the freshwater

crustacean, Ceriodaphnia dubia

David J . Soucek

Accepted : 10 January 2007 /Published online: 13 March 2007

© Springer Science+Business Media, LLC 2007

Abstract

I tested the hypothesis that if sodium sulfate

alters the bioenergetics of Ceriodaphnia

dubia, con-

centrations that cause reduced fecundity in the short

(7-day) and long (5 generations) term should also cause

changes in feeding rate and/or metabolism, measured

as oxygen consumption . In addition, to test the

hypothesis that an altered bioenergetic level caused

by sodium sulfate exposure will affect the response of

that organism to another toxicant, I measured the

acute toxicity of phenol to C. dubia in the presence and

absence of both food and sodium sulfate . Sodium

sulfate reduced the filter-feeding rate of C

.

dubia,

which was associated with significantly reduced oxygen

consumption . This decreased energy level appeared to

result in a consistent but decreased level of fecundity

over a number of generations and the reproductive

impairment was dose-dependent . These effects

occurred at concentrations much lower than those at

which acute (mortality) effects have been observed, a

finding that may have regulatory implications . In

addition, whereas phenol toxicity to C. dubia

was

exacerbated by the addition of food, increased phenol

toxicity, likely induced by an increase in filtering or

metabolic rate due to food addition, was negated when

sodium sulfate was added to the test medium .

Keywords Sodium sulfate - Fecundity Respiration

Feeding rate Ceriodaphnia dubia

Division

D . J. Soucek

of Biodiversity

(G_a)

and Ecological Entomology, Illinois

Natural History Survey, 1816 South Oak St., Champaign, IL

61820, USA

c-mail : d-soucek@inhs

.uiuc .edu

Introduction

While a wealth of literature exists on the physiological

effects observed in organisms exposed to changes in

salinity when sodium and chloride are the dominant

ions, i.e.,

seawater

(e.g.,

Aarset and Aunaas 1990 ;

Amer and Koivisto 1993 ; Cowgill and Milazzo 1990 ;

Guerin and Stickle 1992; Richmond and Woodin 1999),

only recently has toxicity of effluents dominated by

other "major ions" (particularly sodium and sulfate) in

freshwater systems received increasing attention

(Goodfellow et al

. 2000) . Common sources of effluents

with elevated total dissolved solids (TDS), which in

freshwater is essentially the sum of the concentrations

of all common ions, include reverse osmosis systems,

pH modifications of waste water, agricultural runoff,

gas and oil production, and coal or metal mining

operations (Goodfellow et al . 2000). Sodium and

sulfate are two of the most common dominant ions in

effluents associated with the coal industry, and the fact

that toxicity of TDS in general and sodium sulfate in

particular is dependent on characteristics like hardness,

number of major cations, and chloride concentration of

a water or effluent has been well established (e.g.,

Kennedy et al . 2005 ; Mount et al . 1997 ; Soucek and

Kennedy 2005) .

Despite the increasing interest in acute sodium

sulfate toxicity, few studies have documented chronic

or sub-lethal effects of exposure to sulfate dominated

effluents (Chapman et al . 2000; Kennedy et al . 2004,

2005) . The purpose of this study was to test the

hypothesis that if sodium sulfate alters the bioenerget-

ics of Ceriodaphnia dubia, concentrations that cause

reduced fecundity should also result in changes in

feeding rate and/or metabolism . Bioenergetic models

Springer

* * * * * PC #2 * * * * *

---

318

vary, but most often include a growth or reproduction

endpoint (P) that is influenced by energy intake or

consumption (C),

one or more respiration or metabolic

rates (R),

and excretion/egestion rates (E) according to

the following basic formula : P = C-R-E (e.g., Hayes

et a]

. 2000; McNaught 1989) . While metabolic end-

points like oxygen consumption, ammonia excretion,

and energy substrate utilization (O

:N ratio) have been

investigated in numerous studies with invertebrates,

particularly with respect to starvation or nutritional

status (e .g., Mayzaud and Conover 1988

; Pillai and

Diwan 2002

; Snow and Williams 1971), bioenergetic

based studies of cladoceran responses to contaminants

are rare (Arner and Koivisto 1993 ; Bridgham 1988 ;

McNaught 1989) . To test the above stated hypothesis, I

determined the short (7-day) and long-term (5 gener-

ations) effects of sodium sulfate on C. dubia fecundity,

and measured the effects of sodium sulfate on filter-

feeding and oxygen consumption rates . In addition, the

effects of the presence of food during testing on acute

toxicity of sodium sulfate were evaluated . Based on

results of the first two objectives, I also tested the

hypothesis that if sodium sulfate alters the bioenergetic

state of C. dubia,

it will influence the response of that

organism to another toxicant with a different mode of

action, in this case, phenol . Effects of sodium sulfate on

C. dubia

excretion/egestion rates were not measured in

this study.

Materials and methods

General culturing and water chemistry

The cladoceran, Ceriodaphnia dubia,

was selected as a

test organism because of its ubiquitous use in the

United States for regulatory permit testing, its greater

sensitivity to contaminants compared to other cladoc-

erans, and the large base of data available on its

response to other contaminants (see http://cfpub .epa.

gov/ecotox/) . Previous studies have demonstrated the

repeatability of its response to sodium sulfate (Soucek

and Kennedy 2005 ; Kennedy et al . 2005) . Organisms

were initially obtained from a commercial source

(Aquatic

Research Organisms, Hampton, NH,

www.holidayjunction

.com/aro/) , and then a continuous

culture of the organisms initiated from a single female

was maintained in the laboratory for at least 1 year

prior to commencement of testing . The organism was

cultured in moderately hard, reconstituted water

(MHRW) according to U .S

. EPA methods (2002) .

Details on culturing conditions and response to refer-

ence toxicants may be found in Soucek and Kennedy

i Springer

(2005)

. Over the course of the several months during

which these experiments were conducted, average pH,

conductivity, alkalinity, and hardness for culture water

was 8.0 ± 0.1,

300 ± 7 pS/cm, 62 ± 2 mg/I as CaCO3,

and 92 ± 2 mg/I as CaCO 3, respectively.

For toxicity testing, a pure (99%) grade of anhy-

drous sodium sulfate (Na

2SO4, CAS No . 7757-82-6)

was obtained from Fisher Scientific (Pittsburgh, PA,

USA)

. Previous experiments indicated that the salts

and deionized water sources used for experiments had

low to undetectable levels of trace metal contaminants

(see Soucek and Kennedy 2005)

. Standard water

chemistry characteristics, including temperature, pH,

conductivity, dissolved oxygen, alkalinity and hard-

ness, were measured at both the beginning and the

end of each exposure period according to standard

methods (American Public Health Association et al .

1998)

. With the exception of conductivity, the addition

of sodium sulfate to test solutions had a negligible

effect on the above listed water quality parameters .

Samples from each experimental treatment were

analyzed to confirm sulfate concentrations by ion

chromatography at the Illinois Natural History Survey

Aquatic Chemistry Laboratory, Champaign, IL, USA

.

For longer-term experiments like culturing of C. dubia

in water with 1,000 mg

S042_/I

for five generations,

conductivity was measured daily in culture water and

sulfate concentration was calculated based on the

following formula for sulfate in MHRW obtained in

previous studies: [SO;-] = (0.503

x eonductivity(pS/

cm)) -

135.04, R2 =0.9949, n = 59 (Soucek unpub-

lished data).

Three-brood survival/reproduction bioassays

To generate dose-response curves illustrating the

effects of sodium sulfate on C

. dubia fecundity or

reproduction, 7-day, three-brood chronic bioassays

were conducted according to guidelines described in

American Society for Testing and Materials (ASTM) E

1295-01 (2002a)

. Briefly, one <24-h-old neonate was

placed in each of ten replicate 50-m1 beakers for each

of six sulfate concentrations, including a control (no

additional sulfate added) . Treatments were comprised

of a 75% dilution series (i .e.,

the highest concentration

was serially diluted by 25%)

. Each test organism was

fed at a rate identical to that used for culturing

(U.S.

EPA 2002), and test solutions were renewed daily .

Neonates produced by the first generation test organ-

isms (first brood usually occurred on the third or fourth

day of the test) were counted daily and the tests lasted

until at least 60% of test organisms had produced three

broods (7 days in each case) . Endpoints included the

D .J . Soucek

* * * * * PC #2 * * * * *

---

Bioenergetic Effects of Sodium Sulfate on C . dubia

number of young produced by each first generation

C. dubia,

and survival of first generation C. dubia .

Tests were conducted in two different dilution waters :

MHRW and a "reformulated MHRW" or RMHRW

(Smith et al

. 1997) which had a similar hardness and

pH but different Ca

:Mg ratio and chloride concentra-

tion

. RMHRW was tested in addition to MHRW

because of previous studies indicating that sulfate

toxicity was reduced in this water (Soucek and

Kennedy 2005) . Three tests were conducted in each

water type to obtain average values for percent

survival and reproduction. For survival, Fisher's Exact

test was used to compare treatments to controls and for

reproduction Dunnet's test was used (WEST, Inc . and

Gulley 1996) .

An additional benefit of conducting 7-day chronic

bioassays is that calculation of 48-h LC50s (lethal

concentration to 50% of a sample population) from

these data allowed a comparison of acute toxicity when

tests are fed to previously generated data for the same

two diluents in unfed tests (Soucek and Kennedy

2005)

. Survival was evaluated for each chronic test at

48 h and LC50s were calculated using the Spearman-

Karber method (Hamilton et al . 1977)

. Mean 48-h

LC50s for fed tests in MHRW and RMHRW were

compared to mean LC50s for unfed tests in the same

waters with ANOVA and Student's T-test for post-hoc

comparisons using JMP ® software (Sall and Lehman

1996) .

Long-term measurement of reproductive rate

To investigate the long-term, generational effects of

exposure to sodium sulfate on C. dubia fecundity,

organisms were cultured according to U . S. EPA

(2002) methods with the only modification being that

the culture water contained elevated sodium sulfate .

The concentration of 1,000 mg SO ;-/I was selected

because of its proximity to reproductive impairment

thresholds generated in the three-brood chronic bio-

assays in MHRW

. Control (MHRW) and treatment

(MHRW + 1,000 mg SO4-/I) cultures were initiated

simultaneously using the same cohort of neonates

produced by females cultured in MHRW . One adult

organism was held in each of twenty 50-m1 culture

beakers, and neonates were counted and removed

daily

. Neonates to start a new generation were selected

from a female that had produced at least three broods

and had not reproduced on the previous day . After the

first generation, only neonates produced in MHRW

were used to start a new MHRW culture and likewise

for the MHRW + 1,000 mg

S02_/1

treatment. Five

generations were cultured in each treatment (control,

319

elevated sulfate)

. The endpoint evaluated was the total

number of neonates produced per female after 8 days

(usually three or four broods per female) . Because

C . dubia

neonates are clones, there is dependence

between generations, thus the unit of measure was a

clonal line

. Therefore, a repeated-measures ANOVA

was used to test whether there was a difference

between the treatment and control, and if that differ-

ence varied from generation to generation (the time

component) .

Filter-feeding experiment

To determine the effects of sodium sulfate on C

. dubia

filter-feeding rates, a simple clearing experiment was

conducted

. MHRW was used as a control and

MHRW + 1,000 mg SO;-/l

was the exposure treat-

ment

. 50-m1 beakers were filled with 30 ml of each

test solution ; four replicates were used for each

treatment

. To each beaker, 1 .0 ml of the Pseudokirch-

neriella subcapitata solution used for daily feeding was

added, and the solution was thoroughly mixed by

stirring with a disposable transfer pipet . Next, eight

48-h-old C. dubia were added to each beaker

. Two

additional treatments in the experiment consisted of

the same two solutions prepared as described above

(four replicates each) with no organisms added

. After

24-h, all solutions were again thoroughly mixed and 3-

ml aliquots were removed from the center of each

beaker and placed in cuvettes for measurement of

light absorbance by chlorophyll (665 nm wavelength)

using a Uvikon XL dual-beam spectrophotometer

(BioTek Instruments, Wonooksi, VT)

. Absorbance

readings were fit into a previously calculated regres-

sion equation to determine concentration (dry mass

per unit volume) of algae present

. The difference in

algal concentration between the treatments with and

without organisms for a given test solution was used

as the amount of algae consumed by C. dubia .

Consumption data were expressed on a pg algae h-t

individual-1 basis. Treatment means were compared

with Student's T-test using JMP ® software (Sall and

Lehman 1996).

Oxygen consumption experiment

Oxygen consumption was measured according to

previously published methods (e .g.,

Correa et al .

1985

; Rockwood et al . 1990) by placing the test

organisms into biochemical oxygen demand (BOD)

bottles, and measuring change in dissolved oxygen

concentrations after 24 h

. MHRW was used as a

control and MHRW + 1,000 mg SO

;-/l was the

1 Springer

* * * * * PC #2 * * * * *

---

320

exposure treatment . Solutions were poured into eight

replicate bottles for each treatment as well as three

additional bottles per treatment to which no organisms

would be added . Next, 4

.5 ml of the Pseudokirchneri-

ella subcapitata

solution used for daily feeding was

added to each bottle, and dissolved oxygen measure-

ments were made using an air-calibrated Yellow

Springs Instruments (RDP, Dayton, OH, USA) model

58 meter with a self-stirring BUD probe

. Thirty 48-h-

old C. dubia

then were added to each 300 ml BOD

bottle

. After 24 h, dissolved oxygen was measured

again in each replicate BOD bottle and the number of

surviving organisms recorded

. This exposure period

was chosen because it was the minimum period of

exposure in some previous studies with invertebrates

(Rockwood et al

. 1990), although other experimental

designs have used shorter exposure periods (e.g.,

Correa et al . 1985) . For each treatment, the average

change in oxygen concentration in bottles without

organisms was subtracted from the value for each

bottle containing organisms to correct for potential

differences in oxygen consumption rates not due to test

organisms . Oxygen consumption data were expressed

on a ltg

02

h-t individual - ' basis for C. dubia .

Treat-

ment means were compared with Student's T-test using

JMP® software (Sall and Lehman 1996)

.

Multiple chemical bioassay

To test the hypothesis that if sodium sulfate alters the

bioenergetic state of C . dubia, it will influence the

response of that organism to another toxicant with a

different mode of action, static, non-renewal acute

toxicity tests were conducted according to ASTM E

729-96 methods (2002b) with phenol as the second

toxicant

. Three test media were used for dilution water

and controls: MHRW with no food, MHRW with food

(0.45 ml of a Pseudokirchneriella subcapitata solution

per replicate beaker), and MHRW with food plus

1,000 mg SOQ/1

. Phenol LC50s were generated for

each media type

. Treatments were comprised of a 50%

dilution series of phenol with the highest concentration

being 25 mg/1

. Five phenol concentrations were tested

in addition to controls with four replicates tested per

concentration

. Percent survival in each replicate was

recorded at 24 h and at the end of the exposure period .

Four tests were conducted in each diluent type, and

then data for each diluent were combined

(n = 80

organisms per test concentration) for LC50 calculation .

LC50s with robust 95% confidence intervals were

calculated

using the Spearman-Karber method

(Hamilton et al . 1977) .

Springer

Results

Three-brood survival/reproduction bioassays

At the end of 7 days, average percent survival of

control C. dubia

(no sodium sulfate added) was 96% in

MHRW and 100% in RMHRW, and survival was high

(>80%) in all treatments in both diluents up to

-1,700 mg SO;-/I (Fig

. 1) . For RMHRW, high survival

(90%) was observed at -2,200 mng SO4 -/I as well .

Toxicity was greater in MHRW, which had an average

Least Observable Adverse Effects Concentrations

(LOAEC, defined as the lowest test concentration that

produced a statistically significant effect compared to

the control) of 2,216 mg SO

;-/l, whereas RMHRW had

a LOAEC of 3,000 mg So ,-/1 for survival . The mean 7-

day LC50 in MHRW was 2,049 mg SO4 -/I and in

RMHRW, it was 2,442 mg SO;- /I . In both cases, there

appeared to be a threshold response rather than an

incremental dose-related response .

Comparison of mean 48-h LC50s generated during

7-day chronic tests (fed tests) to those from previously

conducted unfed tests indicated that feeding had a

significant effect on acute toxicity of sodium sulfate to

C. dubia in both diluent types (Fig

. 2). The mean "fed"

LC50 in MHRW (2,446 mg SO4

-/1) was significantly

higher (p < 0 .05) than the respective mean "unfed"

LC50 (2,056 mg SO ;-/I), and in the RMHRW diluent

the mean "fed" LC50 of 3,065 mg SOQ-

/I was signif-

icantly higher than the mean "unfed" LC50 (2,527 mg

SO;- /I).

There was a dose-related response for reproduction

in both diluents, with increasing sodium sulfate causing

C. dubia to produce fewer neonates (Fig

. 3). In fact,

simple regression analysis indicated a significant neg-

ative linear relationship between sulfate concentration

and mean number of neonates per female for both

D .J . Soucek

than those for survival : 899 mg SO

;-/I for MHRW and

1,236 mg SO'4-/ l for RMHRW

. Using these regression

equations to calculate EC50s, values of 1,148 mg SO

;-/

1 and 1,458 mg SO4 -

/I were obtained for MHRW and

RMHRW, respectively .

Long-term measurement of reproductive rate

Organisms that either died due to technician error or

did not reproduce at all (two out of 100 individuals in

the MHRW and three out of 100 individuals in the

MHRW + 1,000 mg SO4-

/I treatment) were excluded

MHRW (y = -0

.0157x + 33 .71, R2 = 0.979) and

RMHRW (y = -0.0134x + 34.76,

R2 = 0.906) . The

LOAECs for reproduction were substantially

lower

* * * * * PC #2 * * * * *

---

Bioenergetic Effects of Sodium Sulfate on C. dubia

120

100

40

20

0

0

0

500

1000

Fig . 1 Mean percent survival of

Ceriodaphnia dubia after 7 days sulfate concentrations

. MHRW = moderately hard reconstituted

at various sulfate concentrations in two different diluents

. Values

water, RMHRW = reformulated moderately hard reconstituted

shown are means (,standard deviation) for three bioassays . water

. LOAEC = least observable adverse effect concentration

Horizontal error bars are standard deviations for measured

Fig. 2 Mean (±standard

3500

deviation) 48-h median lethal

sulfate concentration (LC50)

in fed and unfed tests in two

3000

different diluents

. Different

capital letters indicate means

,4 ,

2500

are significantly different

(p < 0.05) .

g

2000-

MHRW = moderately hard

reconstituted water,

c

U

1500

RMHRW = reformulated

'~

moderately hard

u

1000

reconstituted water

r

500

Fig

. 3 Mean number of

40

neonates produced per

female Ceriodaphnia duhia

after 7 days at various sulfate

35 -

concentrations in two

different diluents . Values

30

shown are means (,standard

deviation) for three bioassays .

°' 25

Horizontal

standard deviations

error bars

forare

0

c

a

20

measured sulfate

concentrations .

x

C

MHRW = moderately hard

m 15

E

reconstituted water,

RMHRW = reformulated

10

moderately hard

reconstituted water .

5

LOAEC = least observable

adverse effect concentration

0

0

1500

2000

2500

sulfate (mg/L)

Dunfed

O fed

•

MHRW, mean LOAEC = 899 mg/L

O RMHRW, mean LOAEC = 1236 mg/L

1

T

1

500

MHRW

3000

Dilucnt

1000

1500

sulfate (mg/L)

3500

RMHRW

2000

321

2500

Springer

* * * * * PC #2 * * * * *

---

322

from analyses .

Ceriodaphnia dubia cultured in

1,000 mg SOq-

/I produced significantly fewer neonates

than those cultured in MHRW according to the

repeated measures ANOVA (F=359

.464, DF = 36,

p < 0 .0001 for treatment effect, Fig

. 4) . In addition,

sulfate affected C. duhia

reproduction differently in

different generations as both the generation term

(F = 43.760, DF = 33, p < 0.0001)

and the genera-

tion x treatment interaction term (F= 4

.018,

DF = 33, p = 0

.0092) were significant . Separate pair-

wise analysis of treatment effects for each generation

also indicated that C. duhia

cultured in 1,000 mg

SO4'-/l

produced significantly fewer neonates after 8 days than

controls (p < 0

.0001 for each generation) . Combining

the five generations, the overall mean (±standard

deviation) number of neonates produced per female

after 8 days in MHRW was 62.2 ± 6.6 (n = 98), and for

the MHRW + 1,000 mg SO

;

A-

it was 50.6 ± 5.3

(n = 97) .

Fig. 4

Mean number

(±standard deviation)

of

neonates produced per

female Ceriodaphnia duhia

70-

(n = 19-20

per treatment per

65 -

generation) after 8 days over

>,

five generations in either

~- 60 -

controls

°O

(MHRW = moderately hard

reconstituted water) or

treatments exposed at to

1,000 mg SOa -/I

. Neonates

produced by generation one

were used for generation two

and so on . Asterisks indicate

means for a given generation

are significantly different

(p

< 0.05,

repeated measures

ANOVA and post-hoc

pairwise tests)

Fig

. 5 Mean (±standard

deviation) algal and oxygen

consumption rates (in pg per

hour per individual) for C.

duhia in controls (MHRW)

and exposed to

1,000 mg

SOa-11

consumption . P-values

shown are for Student's t-test

comparing control and

treatment means for each

endpoint separately

41 Springer

I

Filter-feeding and oxygen consumption

experiments

Solution type (with or without sodium sulfate) did not

have an effect on light absorbance (p = 0.2892)

or

change in oxygen concentration (p = 0

.0739) when no

organisms were added

. However, to further control for

potential differences in oxygen consumption rates not

due to test organisms, the average change in oxygen

concentration in bottles without organisms for a given

treatment was subtracted from the value for each

bottle containing organisms for the same treatment

.

Significant differences were observed between treat-

ments for both algal and oxygen consumption (Fig

. 5) .

The ratio of algae to oxygen consumption (in terms of

fig/h-' individual-) was 2.10

for the controls, and

slightly higher (2

.65) for the sulfate exposed organisms .

This difference is attributable to the fact that while

exposure to 1,000 mg S0,2-/I

reduced feeding rate by

25%, it reduced the respiration rate by 41%

.

D .J . Soucck

2

MHRW

O algae, p = 0.0381

D oxygen, p = 0.0002

MHRW + 1000 S04

* * * * * PC #2 * * * * *

---

Biocnergetic Effects of Sodium Sulfate on C. dubia

Multiple chemical bioassay

The presence or absence of food, and the presence or

absence of sulfate had strong effects on phenol toxicity

to C. dubia .

When combining data from four separate

tests (n = 80 individuals per test concentration), the

LC50 (lower and upper 95% confidence limits) in

MHRW with no food added was 4 .34 (3 .88-4 .85) mg

phenol/I, whereas when food was added to each

beaker, the phenol LC50 was 3 .29 mg/I (3

.04-3 .57) .

When both food and 1,000 mg S0

42-/I

were added to

MHRW, the phenol LC50 was 4.26 mg/I (3 .91-4

.65) .

Discussion

The mean 7-day sulfate LOAECs for C . dubia

survival

in MHRW and RMHRW (2,216 and 3,000 mg SO ;

respectively), compare well with other published val-

ues with sodium sulfate dominated effluents

. Kennedy

et al. (2005) collected effluent from a coal preparation

facility in Ohio, USA, and conducted 7-day chronic

tests with C.

dubia in both the actual effluent and a

laboratory prepared effluent meant to simulate the

field-collected effluent . Treatments were based on

conductivity (pS/cm) and survival LOAECs were in

the range of 4,500 to >6,000 pS/cm. These values

correspond to sulfate concentrations in the range of

2,100 to >2,800 mg SO4-/I according to the regression

equation used in this study generated with sodium

sulfate in MHRW

. However, effluents tested in the

Kennedy et al

. (2005) study contained low levels of

trace metals and aluminum, and had elevated hardness

(650-770 mg/I as CaCO

3), which is known to amelio-

rate sulfate toxicity (Soucek and Kennedy 2005) and

may change the SO4-/conductivity

relationship . While

the results are not directly comparable to those from

the present study because of the differences in ion

composition, survival data from the two studies fall

within the same approximate range

. Additionally,

results of previous work indicating that Ca:Mg

and

chloride concentration influence sulfate toxicity (Sou-

cek and Kennedy 2005) were supported by these

results, as toxicity was consistently lower in RMHRW

than in MHRW .

Comparison of C

. dubia survival results in 7-day

chronic tests to those in previously conducted unfed

48-h acute tests (Soucek and Kennedy 2005) suggests

that feeding organisms during sodium sulfate bioassays

improves survivorship

. The 48-h LC50s generated in

these fed tests were significantly higher than those

from unfed tests

. In fact, the 7-day LC50 for C. dubia in

MHRW during the present study was similar to the

323

mean unfed 48-h LC50 from the Soucek and Kennedy

(2005) study (2,049 and 2,050 mg SO ;-/l,

respectively) .

Similar trends were observed in RMHRW

. Pieters

et al- (2005) documented similar results with

Daphnia

magna exposed to fenvalerate

; comparing high and low

food treatments, effects of fenvalerate on survival and

reproduction were exacerbated by reduced food con-

centration. It is well known that food, dissolved organic

carbon, and other ligands can reduce availability and

toxicity of some metals (e.g., Paquin et al

. 2003), so

feeding is recommended against in acute toxicity

bioassays with most freshwater organisms (ASTM

20026) . However, sodium and sulfate are stable as

ions and unlikely to bind with the algal food used in

these tests

. Therefore, a more likely explanation for

the increased survival in fed tests in the present study is

that fed organisms have more energy to direct toward

ionoregulation than starved organisms .

Reproduction of C. dubia

was strongly influenced by

sodium sulfate in three-brood chronic tests

. Dose-

related, negative relationships between sulfate and

reproduction were observed in both diluents . The slope

for MHRW was nominally steeper than that for

RMHRW, which is consistent with previous findings

that increased Ca :Mg ratio and chloride concentration

reduces sodium sulfate toxicity to C . dubia (Soucek

and Kennedy 2005)

. While LOAECs for reproduction

were less than half the values for survival in this study,

organisms at the LOAEC concentrations still produced

an average of -20 neonates, or 63-70% of the means

for controls .

Kennedy et al

. (2004) observed reduced reproduc-

tion of C

. dubia at conductivities ranging from 3,254

to 4,530 tS/cm, while conductivities at LOAECs in

the present study ranged from 2,050 to 2,700 µS/cm

.

The disparity between the two studies may again be a

result of the fact that the field collected and mock

effluents from the Kennedy et al. (2004) study had

much higher hardnesses (650-770 mg/I as CaCO 3)

compared to those in the present study (90-100 mg/I

as CaCO3).

In this study, the EC50s in terms of mg

Na2SO4/1

were 1,566 and 2,070 for MHRW and

RMHRW, respectively

. Cowgill and Milazzo (1990)

conducted three-brood chronic tests with

C. dubia

using NaCl as a toxicant and obtained an EC50 of

1,794 mg NaCI/l (hardness of their diluent was 90-

110 mg/1)

. This value falls within the range observed

in the present study for Na

2SO4, suggesting that

sulfate and chloride salts of sodium have similar

effects on reproduction

. This is in contrast to the

observation of Mount et al

. (1997) that NaCl is more

toxic than Na

2SO4 in terms of 48-h LC50s (1,960 and

3,070 mg/I for the respective salts)

.

~] Springer

* * * * * PC #2 * * * * *

---

324

Based on reproductive impairment results of the

three-brood chronic bioassays (LOAEC

in

MHRW = 899 mg SO ;-/l), the five-generation expo-

sure was conducted at a concentration of 1,000 mg

SO /1, and this concentration of sulfate had a signif-

icant and consistent effect on CC dubia

reproduction

relative to controls over the course of the five gener-

ations . While the generation and interaction terms

indicated that differences in reproduction between

controls and sulfate exposed organisms were different

from generation to generation, the actual percent

differences between controls and sulfate exposed only

ranged from 12.9 to 23.3 (mean ± SD = 18.4

± 3.7) .

The finding of significant generational differences was

likely due to the relatively high statistical power

(n = 19-20 per treatment per generation)

. The fact

that sulfate-exposed C. dubia

reproduced at a rela-

tively constant but significantly lower rate compared to

controls suggests a decrease in energy allocated to

reproduction due to increased osmoregulation require-

ments, an explanation also suggested by Amer and

Koivisto (1993) working with Daphnia magna in

various NaCl exposure levels .

If the observed decrease in reproductive rate in

C. dubia

exposed to sodium sulfate is due to changes in

energy allocation, one might expect to observe changes

in other

bioenergetic endpoints when similarly

exposed . That was the case in the present study, as

consumption of algae was significantly decreased when

exposed to sulfate, as was metabolic rate, measured as

oxygen consumption

. At the most basic level, following

the bioenergetic model of P = C-R-E, and holding

excretion constant because it was not measured in this

study, a decrease in both algal consumption (C) and

respiration (R) would result in reduced growth or

reproduction (P)

. Respiration and reproduction rates

in freshwater daphnids have been observed to decrease

in response to elevated NaCl (Arner and Koivisto

1993), and 2,2'-dichlorobiphenyl (Bridgham 1988),

although in the latter study, variable results were

obtained in two different respiration experiments

.

Temperature has also been documented to influence

respiration rates in Ceriodaphnia reticulata

(Gophen

1976)

. Both temperature and food concentration influ-

ence daphnid filter-feeding rates as well (Gophen 1976

;

Mourelatos and Lacroix 1990), and several chemicals,

including magnesium, nitrite, I,indane, and zinc have

been shown to reduce food consumption in daphnids

(see McNaught 1989) .

The results of this study imply that C. dubia

populations exposed to sodium sulfate would grow at

slower rates than unexposed populations

. Because this

organism is a low level consumer, this effect may

4) Springer

cascade to higher trophic levels

. However, the impli-

cations of reduced feeding rates observed due to

sodium sulfate exposure are not entirely negative

.

The results of phenol bioassays in the present study

indicated that phenol toxicity increased when food was

present, perhaps due to a higher metabolic rate and/or

a higher filtering rate because filter-feeding increases

with increased food concentration (Mourelatos and

Lacroix 1990) . However, addition of 1,000 mg SO;

-/1 to

the test medium, thereby reducing feeding rate,

negated the effect of food on phenol toxicity

.

Brix et al

. (2001) reported similar results when they

observed that increased sulfate concentrations caused

reduced acute toxicity of selenate to several freshwater

species including C. dubia,

and Hansen et al . (1993)

observed reduced bioconcentration of selenate in

Chironomus decorus and D . magna

at elevated sulfate

concentrations (up to 206 mg SO ;

-/1

for

D . magna and

up to 3,235 mg SO ; /l for C.

decorus. The suggested

mechanism was related to direct competition at the cell

uptake site since the two chemicals were structurally

similar, and much is known about the absorption

antagonism between selenate and sulfate (reviewed in

Hansen et al . 1993), but results from the present study

suggest that a sulfate induced lower filtering rate also

may be a contributing factor to reduced selenate

uptake and toxicity.

In conclusion, this study has shown that sodium

sulfate reduces filter-feeding rate in C

. dubia, which is

associated with, but not necessarily the cause of a

reduced metabolic rate, measured as oxygen con-

sumption

. This decreased energy level appears to

result in a consistent but decreased level of reproduc-

tive output over a number of generations and the

reproductive impairment is dose-dependent

. In addi-

tion, sodium sulfate toxicity appears to be reduced

when test organisms are fed, whereas phenol toxicity

to C. dubia is

exacerbated by the addition of food .

Increased phenol toxicity induced by a likely increase

in filtering or metabolic rate due to food addition, is

negated when sodium sulfate is added to the test

medium .

Acknowledgement This study was funded in part by the U .S .

Environmental Protection Agency Grant

#CP96543701-0 .

Thanks to Alex Haldeman and Jens Sandberger of the Illinois

Natural History Survey for technical assistance.

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* * * * * PC #2 * * * * *

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Winfield RP, Wit KB, DiToro DM (2003) Metals in aquatic

environments : a review of exposure, bioaccumulation, and

toxicity models

. Society of Environmental Toxicology and

Chemistry (SETAC), Pensacola, FL, USA

Pieters BJ, Paschke A, Reynaldi S, Kraak MHS, Admiraal W,

Liess M (2005) Influence of food limitation on the effects of

fenvalerate pulse exposure on the life history and popula-

tion growth rate of Daphnia magna . Environ Toxicol Chem

24 :2254-2259

Pillai BR, Diwan AD (2002) Effects of acute salinity stress on

oxygen consumption and ammonia excretion rates

of the

marine shrimp Metapenaeus

monoceros . J Crust Biol

22(l) :45-52

Richmond CE, Woodin SA (1999) Effect of salinity reduction on

oxygen consumption by larval estuarine invertebrates . Mar

Biol 134 :259-267

Rockwood JP, Jones DS, Coler RA (1990) The effect of

aluminum in soft water at low pH on oxygen consumption

by the dragonfly Libellula Julia

Uhler . Hydrobiologia

190 :55-59

Sall J, Lehman A (1996) JMP start statistics

. SAS Institute,

Duxbury Press, Belmont, CA, USA

Smith ME, Lazorchak IM, Herrin LE, Brewer-Swartz S, Thoeny

WT (1997) A reformulated, reconstituted water for testing

the freshwater amphipod, Hyalella azteca . Environ Toxicol

Chem 16 :1229-1233

Snow NB, Williams PiLeB (1971) A simple method to determine

the O

:N ratio of small marine animals . J Mar Biol Ass UK

51 :105-109

Sauces DJ, Kennedy Al (2005) Effects of hardness,

chloride,

and acclimation on the acute toxicity of sulfate to freshwater

invertebrates. Environ Toxicol Chem 24:1204-1210

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. Environmental Protection Agency (2002) Methods for

measuring the acute toxicity of effluents

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waters to freshwater and marine organisms, 5th edn

. EPA

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® V3 .5 . Western

Ecosystems Technology Inc, WY, USA

0 Springer

* * * * * PC #2 * * * * *

---

STATE OF ILLINOIS

)

COUNTY OF SANGAMON

Dorothy Gunn, Clerk

Pollution Control Board

100 West Randolph Street

Suite 11-500

Chicago, Illinois 60601

(OVERNIGHT MAIL)

Mathew Dunn

Illinois Attorney General's Office

Environmental Control Division

James R. Thompson Center

100 West Randolph Street

Chicago, Illinois 60601

(OVERNIGHT MAIL)

PROOF OF SERVICE

1, the undersigned, on oath state that I have served the attached Illinois Environmental

Protection Agency's Additional Information and Documents upon the persons to whom it is

directed, by placing a copy in an envelope addressed to :

ALSO SEE ATTACHED SERVICE LIST

(FIRST CLASS)

and mailing it from Springfield, Illinois on April 6, 2007, by U .S . Mail with sufficient postage

affixed.

SUBSCRIBED AND SWORN BEFORE ME

THIS 6m DAY OF APRIL 2007.

SS

PRINTED ON RECYCLED PAPER

Marie E. Tipsord

Hearing Officer

Illinois Pollution Control Board

100 West Randolph, Suite 11-500

Chicago, Illinois 60601

(OVERNIGHT MAIL)

Jonathan Fun

Illinois Department of Natural Resources

One Natural Resources Way

Springfield, Illinois 62702-1271

(OVERNIGHT MAIL)

BRENDA

OFFICIAL

BOEHNER

SEAL

NOTARY PUBLIC, STATE OF ILLINOIS

'-:,

MY

•+

COMMISSION

44+ba+a+444444wwe

CWIRES 11

•

.32009

.•• a n .

. .

.

2

* * * * * PC #2 * * * * *

---

http://www .ipcb

.state .il .us/Cool/External/casenotifyNew .asp?caseid=13086&notifytype=Notic e

4/6/2007

Printing Notice List . . . .

Party Name

Sonnenschein Nath & Rosenthal

Role

7800 Sears Tower

City & State

Chicago

Page 1 of 4

Phone/Fax

312/876-8000

Interested Party

233 South Wacker Drive

IL 60606-6404

312/876-7934

Elizabeth Leifel

Katten, Muchin &Zavis

525 West Monroe

Chicago

312/902-5200

Interested Party

Suite 1600

IL 60601-3693 312/902-1061

Nancy] . Rich, Esq .

Interested

U .S . Fish &

PartyWildlife

Service

4469-48th Avenue Court

IL

Rock

61201Island

309/793-5800

Mr . Mike Coffey

Stateside Associates

Interested Party

2300 Clarendon Blvd .

Suite 407

Arlington

VA 22201

t

Illinois Environmental Regulatory Group

Springfield

217/523-4942

Interested Party

3150 Roland Avenue

IL 62703

217/523-4948

Brenda Carter, Project Manager

Deirdre K . Hirner, Executive Director

Caterpillar Inc .

Peoria

309/675-4105

Interested Party

100 N .E

. Adams Street

IL 61629

309/675-5861

Bill Compton, Environmental Affairs

Illinois. Municipal-League

500 E . Capitol

Springfield

217/525-1220

Interested Party

P .O . Box 5180

IL 62705

217/525-7438

Kenneth Alderson

Abbott Laboratories

Dept . 590, Bldg . P-14

North Chicago

Interested Party

1401 Sheridan Road

IL 60064-4000

847/937-8935

Jeffrey Smith

Goodwin & Broms, Inc .

Springfield

217/698-0222

Interested Party

400 Bruns Lane

IL 62707

217/698-0422

Daniel 3

. Goodwin

Dept . of Commerce & Economic

Small Business Office

ODD~portunityInterested

Party

620 East Adams Street, Fifth

SpringfieldIL

62701

217/785-6162

Dan Wheeler

Barnes & Thornburg

Floor

1 North Wacker Drive

Chicago

312/357-1313

Interested Party

Suite 4400

IL 60606

312/759-5646

Fredric P . Andes

Thorn Creek Basin SanitarxD1 trot

Chicago

708/754-0525

Interested Party

700 West End Avenue

IL

Heights60411

708/754-3940

James L . Daugherty, District Manger

Interested

Exxon MobilePartyOil Corporation

1-55 & Arsenal Road East

IL

Channahon60410

815/521-7755

Bob Elvert

Metropolitan Water-Reclamation-District

of-Greater

Interested PartyChicago

100 East Erie

ChicagoIL

60611

312/751-6583

Ron Hill, Principal Assistant Attorney

Richard Lanyon, General Superintendent

Huff & Huff, Inc .

Interested Party

512 West Burlington Avenue

Suite 100

LaGrange

IL 60525

James E . Huff, P.E .

* * * * * PC #2 * * * * *

---

http

://www .ipcb .state .il .us/Cool/External/casenotifyNew

.asp?caseid=13086&notifytype=Notic e

4/6/200 7

t'rinnng Notice List . . . .

Admiral Environmental Services, Inc .

Interested Party

2025 South Arlington Heights

Road Suite 103

Hee Hight

ightss

IL 60005-4141

rage G (n 4

Philip A . Twomey

American Bottoms RWTF

Interested Party

One American Bottoms Road

Sauget

IL 62201

Kay Anderson

Openlands Project

Interested Party

25 East Washington Street

Suite 1650

Chicago

IL 60602

Stacy Meyers

Fox Metro Water Reclamation District

Interested Party

682 State Route 31

Oswego

IL 60543

Mr . Gregg Buchner

Illinois Department of Natural Resources

Interested Party

One Natural Resources Way

Springfield

IL 62702

William Richardson, Chief Legal Counsel

Mr

. Mike Branham

Scott Fo wler, Mines and Minerals

Joel Cross

Interested

Wheaton SanitaryParty

District

P .O . Box 626

W

IL

h eato

60189n

630/668-1515

Robert Clavel, Engineer-Manager

AmerenInterested ServicesParty

One

PO Box

Ameren

66149Plaza

MO

St . Louis63166

314.554.4908

Mr. Michael Bolinger

August Mack Environmental, Inc ..

Indianapolis (317) 579-

Interested Party

8007 Castleton Road

IN 46250

7400

Charles Schnurpel, Project Manager

Illinois

Interested

EPAParty

1021

PO Box

N .

19276Grand

Ave . East

SpringfieldIL

62794-9276

524-5951

Scott Twait

Prairie Rivers_ Network

Interested Party

809 South 5th Street

Champaign

IL 61820

Jean Flemma

Glynnis Collins

Beth Wentzel

Interested

Fox River

PartyWRD

P .O . Box 328

IL

Elgin60121

847-742-2068

Rick Manner, Engineer

Interested

E

nvi

ronmental

PartyLaw-&-Polic

Center

Suite

35 E .

1300Wacker

ChicagoIL

60601

312 795 3707

Albert Ettinger, Senior Staff Attorney

Midwest Generation

440 S . LaSalle Street

Chicago

(312) 583-

Interested Party

Suite 3500

IL 60605

6080

Mr . Bill Constantelos

Exxon

Interested

MobilParty

PO Box 874

JolietIL

60410

815-521-7755

Stacey Ford, NSR Coordinator

ECTInterested

Party

3701 NW 98th street

GainesvilleFL

32606

3523320444

Gary Dalbec

* * * * * PC #2 * * * * *

---

h ttp://www .ipcb .state .il .us/Cool/External/casenotifyNew .asp?caseid=13086&notifytype=Notic e

4/6/2007

I'rintmg Notice List . . . .

Illinois Coal Association

Interested Party

1480 E . 1200th Street

Industry

IL 61440

Page i 014

Greg Arnett

Illinois-A meric an Water Co .

Interested Party

123 S .W . Washington Street

Peoria

IL 61602-1317

Dean Falkner, Chair, Illinois Section

Formosa Plastics

Interested Party

P .O . Box 27

Illiopolis

IL 62539

Ms. Kim Bennett

M RRI

Interested Party

P .O . Box 1642

Murphysboro

IL 62966

Mr . Ron Balch

John Tippy

Illinois Association of Wastewater

Agencies

241 N . Fifth Street

Springfield

Interested Party

IL 62701

Mr . William Cellini

Environmental Consulting and

Technology

3701 NW 98th Street

Gainsville

Interested Party

FL 32606

Gary Dalbec

Illinois Coal Association

Interested Party

P .O . Box 727

Harrisburg

IL 62946

Gerald DeNeal

Illinois Coal Association

Interested Party

8100 E . Main Street

Williamsville

IL 62693

Roger Dennison

Interested

Illinois Coal

PartyAssociation

212 S . Second St .

IL

Springfield62701

217-528-2092

Phil Gonet

Illinois Rural Water Association

Interested Party

P .O . Box 6049

Taylorville

IL 62568

Loy McCart

U.S . EPA

Interested Party

Region 5 (WT-15J)

77 West Jackson Blvd .

Chicago

IL 60604

Dave Pfeifer

Citclo Petroleum

Interested Party

135th Street & New Ave .

Lemont

IL 60439-3569

Brigette Postel

Bolten & Menk, Inc .

2730 Ford Street

Ames

Interested Party

P .O . Box 668

0668IA

50010-

Gregory Sindt

Illinois Natural History Survey

607 E . Peabody Drive

Champaign

Interested Party

IL 61820-6970

David Soucek

Rhodia Inc .

Interested Party

1101 Arnold Street

Chicago

Heights

IL 60411

Kirk Thompson

Akzo N obel

Interested Party

8201 West 47th Street

P .O . Box 1569

McCook

IL 60525

* * * * * PC #2 * * * * *

---

Total number of participants : 59

bttp ://www.ipcb

.state .il .us/Cool/External/casenotifyNew

.asp?caseid=13086&notifytype=Notic e

4/6/2007

Printing Notice List . . . .

rage 4 01 4

George Yanku

Farmland Foods

Interested

Party

1220 N . 6th Street Road

Monmouth

IL 61462

Isaac Yoder

Farmland Foods

7501 N .W . Tiffany Springs

Interested Party

Parkway

Kansas City

MO 64153

Bud Ackerman

Viper Mine

Interested Party

8100 East Main Street

Williamsville

IL 62693

Kayla Primm

IDOT

Interested Party

2300 South Dirksen Parkway

IL

Springfield62764

217/785-4246

Steven Gobelman, Geologic/Waste Assessment Specialist

James Huff

Interested Party

915 Harger Road

Oak Brook

IL 60525

* * * * * PC #2 * * * * *