Environmental Management (2011) 47:10331046

DOI 10.1007/s00267-011-9679-3

Role of a Comprehensive Toxicity Assessment and Monitoring

Program in the Management and Ecological Recovery
of a Wastewater Receiving Stream
Mark S. Greeley Jr. Lynn A. Kszos
Gail W. Morris John G. Smith Arthur J. Stewart

Received: 7 October 2009 / Accepted: 7 April 2011 / Published online: 15 May 2011
Springer Science+Business Media, LLC (outside the USA) 2011

Abstract National Pollution Discharge Elimination Permit (NPDES)-driven effluent toxicity tests using Ceriodaphnia dubia and fathead minnows were conducted for
more than 20 years to assess and monitor the effects of
wastewaters at the United States (U.S.) Department of
Energy Y-12 National Security Complex (Y-12 Complex)
in Oak Ridge, Tennessee. Toxicity testing was also conducted on water samples from East Fork Poplar Creek
(EFPC), the wastewater receiving stream, as part of a
comprehensive biological monitoring and assessment program. In this paper, we evaluate the roles of this long-term
toxicity assessment and monitoring program in the management and ecological recovery of EFPC. Effluent toxicity
testing, associated toxicant evaluation studies, and ambient
toxicity monitoring were instrumental in identifying toxicant sources at the Y-12 Complex, guiding modifications to
wastewater treatment procedures, and assessing the success
of various pollution-abatement actions. The elimination of
untreated wastewater discharges, the dechlorination of

remaining wastewater streams, and the implementation of

flow management at the stream headwaters were the primary actions associated with significant reductions in the
toxicity of stream water in the upper reaches of EFPC from
the late 1980s through mid 1990s. Through time, as regulatory requirements changed and water quality improved,
emphasis shifted from comprehensive toxicity assessments
to more focused toxicity monitoring efforts. Ambient toxicity testing with C. dubia and fathead minnows was supplemented with less-standardized but more sensitive
alternative laboratory toxicity tests and in situ bioassays.
The Y-12 Complex biological monitoring experience
demonstrates the value of toxicity studies to the management of a wastewater receiving stream.
Keywords In situ bioassay
Effluent toxicity
Long-term
monitoring
Ambient toxicity
Biomonitoring

Introduction
The submitted manuscript has been authored by a contractor of the
U.S. Government under contract DE-AC05-00OR22725. Accordingly,
the U.S. Government retains a nonexclusive, royalty-free license to
publish or reproduce the published form of this contribution, or allow
others to do so, for U.S. Government purposes.
M. S. Greeley Jr. (&)
L. A. Kszos
G. W. Morris

J. G. Smith
A. J. Stewart
Environmental Sciences Division, Oak Ridge National
Laboratory, Oak Ridge, TN 37831, USA
e-mail: Greeleyms@ORNL.gov
L. A. Kszos
Neutron Sciences Directorate, Oak Ridge National Laboratory,
Oak Ridge, TN 37831, USA
A. J. Stewart
Oak Ridge Associated Universities, Oak Ridge, TN 37830, USA

When the United States (U.S.) Environmental Protection

Agency (USEPA) applied water quality-based limitations
for toxic pollutants to National Pollutant Discharge Elimination System (NPDES) permits in the 1980s, many industrial facilities and municipalities around the U.S. found they
were unable to immediately meet strict chemical-based
water quality criteria. One such industrial facility was the
U.S. Department of Energy (DOE) Y-12 National Security
Complex (formerly known as the Y-12 Plant and hereafter
referred to as the Y-12 Complex) in Oak Ridge, Tennessee.
The Y-12 Complex, a nuclear weapons component production facility constructed at the headwaters of East Fork
Poplar Creek (EFPC) during the early 1940s as part of the
U.S. Manhattan Project, was by the mid 1980s discharging

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both treated and untreated wastewaters to the stream from

over 200 outfalls (Stewart and others 2011). The NPDES
permit issued to the facility in May 1985 required the Y-12
Complex to establish a Toxicity Control and Monitoring
Program (TCMP) for the purpose of identifying and controlling the release of toxicants to EFPC from permitted
discharges. This NPDES permit was also among the first in
the nation to require the biological monitoring of the
receiving stream, resulting in the establishment of a Biological Monitoring and Abatement Program (BMAP) to
ensure and evaluate the effectiveness of the Y-12 Complex
TCMP and associated water pollution control programs in
protecting the classified uses of EFPC (Loar and others 2011;
Peterson 2011; Peterson and others 2011; Stewart and others
2011).
Whole effluent toxicity testing was the primary investigative tool of the Y-12 Complex TCMP. Effluent toxicity
testing conducted for the TCMP was used extensively
beginning in 1985 to: (1) locate and prioritize sources of
toxicants to EFPC; (2) identify and evaluate toxicants of
concern; (3) guide changes in wastewater treatment operations to facilitate reductions in toxicant loading to the
receiving stream; (4) ensure the efficacy of treatment
modifications; and (5) monitor the toxicity of remaining
wastewaters following toxicant reduction efforts.
Ambient toxicity testing was one of several complimentary bioassessment approachesincluding bioaccumulation monitoring, analyses of bioindicators in sentinel
fish species, and surveys of stream communities
employed in the multi-task BMAP to assess changes over
time in water quality and the ecological health of EFPC as
water pollution controls and associated remedial actions
were implemented at the Y-12 Complex. Testing of EFPC
waters for ambient toxicity was initiated in 1986 and continued to the 2005 renewal of the facilitys NPDES permit.
The diversity and long-term nature of the BMAP, combined with the intensive toxicity assessments conducted for
the TCMP, provide a unique opportunity to examine the
relationship between specific water pollution controls and
remedial actions at a major industrial facility (Loar and others
2011) and resultant changes in ambient toxicity (this paper),
water quality (Stewart and others 2011), pollutant bioaccumulation (Southworth and others 2011), and the health of
aquatic organisms and communities (Hill and others 2010;
Adams and Ham 2011; Ryon 2011; Smith and others 2011) in
a freshwater receiving stream. Elements of the aquatic toxicity testing program for the Y-12 Complex have been discussed previously (Stewart and others 1990; Stewart 1996;
Loar and others 1992; Kszos and others 1992; Hinzman 1993;
Stewart 1996; Kszos and others 1997; Hinzman 1998; Adams
and others 2002; Kszos and Stewart 2003).
Radiological and non-radiological contaminants of historical concern in EFPC include elevated nutrients, chlorine

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Environmental Management (2011) 47:10331046

in process waters, polychlorinated biphenyls (PCBs), and

mercury, uranium, and various other metals from a variety
of industrial process at the Y-12 Complex (Loar and others
2011). Environmental contaminants originate from multiple
point sources within the Y-12 Complex, including wastewater treatment facility outfalls and storm drains, and from
non-point sources such as groundwater inputs and runoff
from contaminated floodplain soils. Beginning in the 1980s
and continuing to the present, the Y-12 Complex committed
to a series of remedial actions and pollution abatement
activities to reduce toxic wastewater discharges to EFPC.
Table 1 summarizes some of the major actions taken by the
Y-12 Complex which could have contributed in the ensuing
decades to significant improvements in the water quality of
EFPC. A more detailed description of these remedial
actions and pollution abatement activities can be found in
Loar and others (2011).
Figure 1 demonstrates the functional relationships
between wastewater toxicity testing performed under the
TCMP and ambient toxicity testing performed for the
BMAP in establishing effluent limitations for new wastewater treatment facilities at the Y-12 Complex (adapted
from Kingrea 1986; Loar and others 1992). From the initiation of these programs, the complimentary TCMP and
BMAP toxicity testing programs acted in tandem to both
guide and evaluate the success of the Y-12 Complexs
toxicity control and pollution reduction efforts.
The goals of the this paper are to: (1) reevaluate, from
the perspective of more than 20 years of toxicity monitoring experience in the EFPC watershed, the relationships
between specific water pollution controls and remedial
actions implemented by the Y-12 Complex and changes in
the measured toxicity of effluent streams and ambient
EFPC waters; (2) explore the relationships between effluent
and ambient toxicity and changes over time in the conditions of stream communities; (3) examine the respective
contributions of effluent toxicity assessments, routine
ambient toxicity monitoring, and related special studies
and toxicant evaluations to facilitating and monitoring of
the ecological recovery of the receiving stream; and (4)
discuss implications of the Y-12 Complex TCMP and
BMAP toxicity assessment experience for the environmental management of receiving waters.

Materials and Methods

Study Sites
For the purposes of orientation, the locations of the Y-12
Complex and the primary BMAP monitoring sites along
EFPC are shown in Fig. 2 (for greater detail on BMAP
sampling locations see Loar and others 2011). EFPC

Environmental Management (2011) 47:10331046

1035

Table 1 Summary of selected pollution abatement and remedial actions at the Y-12 Complex from 1985 through 2000
Dates

Activity

19861992

Source collection and elimination of untreated discharges

1986

Completion of a central pollution control facility

19861987

Relining of sanitary and storm sewers

1988

Replacement of original retention basin (New Hope Pond) with a new lined basin, Lake Reality

Late 1992

Dechlorination of major wastewater discharges (with minor discharges dechlorinated

during 1993 1994)

19962000

Additional relining of sanitary and storm sewers

1996
1996

Completion of central and east end mercury treatment systems

Implementation of flow management (temporary)

Early 1997

Implementation of flow management (permanent)

1996

Lake Reality bypass (temporary)

1998

Lake Reality bypass (permanent)

2000

Bank stabilization project

Adapted from Loar and others (2011)

Fig. 1 Decision tree for
establishing effluent limitations
for wastewater treatment
facilities at the Y-12 Complex.
BAT = Best Available
Technology (modified from
Kingrea 1986, and presented
originally in Loar and others
1992)

BAT Effluent
Limitations

Modify Effluent
Limitations

Yes

Is Wastewater
Toxic?

(TCMP)
No

Yes

Develop Toxicity
Control Plan and/or
Conduct TRE

Yes

Is Wastewater
Toxic?

(TCMP)

No

Are Classified Uses

Being Maintained?

(BMAP)

Yes
Final Effluent
Limitations

originates within the Y-12 Complex in Oak Ridge, Tennessee, where it receives wastewater discharges via underground
outfalls before emerging aboveground just upstream of a
BMAP sampling location at EFPC kilometer 25 (EFK25).
For several years, EFPC flowed through a retention basin
(originally New Hope Pond, which was replaced in 1988 by
Lake Reality) located near the border of the Y-12 Complex
downstream of a BMAP sampling location at EFK24. The
Lake Reality retention basin was permanently bypassed in
1998 as part of the Y-12 Complex pollution control and

management strategy, allowing subsequent free flow of

stream water from upper EFPC within the Y-12 Complex to
lower EFPC downstream of the old retention basins. Upon
exiting the Y-12 Complex just downstream of EFK23, EFPC
flows through the City of Oak Ridge prior to merging with
Poplar Creek approximately 25 km from the stream origin.
The only additional significant wastewater discharge to
EFPC is the municipal Oak Ridge Wastewater Treatment
Facility located near EFK14 (Fig. 2), a facility which also
receives some wastewaters from the Y-12 Complex.

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Environmental Management (2011) 47:10331046

Fig. 2 Locations of biological

monitoring sites on EFPC in
relation to the Y-12 Complex.
EFK = East Fork Poplar Creek
kilometer. Note that only core
BMAP monitoring sites are
shown on the map as point
references for more numerous
toxicity testing sites along
EFPC

Initial ambient toxicity assessments conducted in 1986

with standard aquatic test organismsCeriodaphnia dubia
(C. dubia) and fathead minnows (Pimephales promelas)
evaluated water from 11 sites in lower EFPC downstream
of the retention basin near the boundary of the Y-12
Complex, and one site in upper EFPC immediately
upstream of the retention basin. Because of a need to isolate very specific point sources of potential toxicants, more
sites along EFPC were eventually examined for ambient
toxicity than the five routinely monitored by other BMAP
tasks, and toxicity sampling locations were more specifically delineated (for instance, EFK25.1 for an ambient
toxicity site rather than the less-specific EFK25 designation
used by other BMAP tasks: see Table 2 for EFPC locations
monitored at least once for ambient toxicity). After the
initial toxicity assessments had demonstrated that ambient
toxicity was mainly restricted to upper EFPC, subsequent
ambient tests with C. dubia and fathead minnows focused
on a smaller subset of the originally tested locations within
lower EFPC and added additional testing sites in upper
EFPC (Table 2).
In situ bioassays, which place test organisms such as the
fingernail clam into the stream for exposure purposes (test
described elsewhere in this paper), lack the obvious controls of laboratory toxicity tests. Thus, three reference
streams located off the Oak Ridge Reservation in nearby
valleys were used in the in situ clam tests to provide
comparisons with the conditions expected for EFPC if the
Y-12 Complex had not been constructed. Located in

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watersheds with rural impacts but no industrial contamination, these reference streamsHinds Creek (HCK20),
Cox Creek (CXK0.2) and Brushy Fork (BFK7)have
similar physical and chemical characteristics to EFPC but
are unaffected by Y-12 Complex discharges (Fig. 2).
Toxicity Tests
Effluent Toxicity Testing
Requirements for effluent toxicity testing were included in
both the 1985 and subsequent 1995 NPDES permits issued
to the Y-12 Complex. During the period covered by the
1985 permit, cooling tower blowdown, storm drain discharges, and treatment facility effluents were tested periodically with chronic 3-brood C. dubia tests (initially by
the methods of Horning and Weber 1985; current test
method 1002.0 in USEPA 2002a) and 7-day fathead minnow larvae growth and survival tests (initially by the
methods of Horning and Weber 1985; current test method
1000.0 in USEPA 2002a). Following the 1995 renewal of
the NPDES permit (which remained in effect through
2005), acute 48-h effluent tests based solely on C. dubia
survival (current test method 2002.0 in USEPA 2002b)
replaced the previous chronic effluent testing as a permit
requirement.
By testing various dilutions of wastewater effluents, the
standardized chronic toxicity tests determined a no-effectconcentration (NOEC) of effluent, with either survival and

Environmental Management (2011) 47:10331046

1037

Table 2 C. dubia and fathead minnow chronic toxicity tests conducted on ambient water samples from EFPC through 2005
Sitea

1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Upper East Fork Poplar Creek

EFK25.1

10

10

12

12

11

OF 201

EFK24.6

12

10

10

12

12

11

12

EFK24.1 6

11

11

11

12

12

11

12

Lower East Fork Poplar Creek

EFK23.8 17

13

12

12

10

12

12

11

12

EFK22.8 3

EFK21.9 1

EFK20.5 3

EFK18.2 3
EFK16.1 2

EFK13.8 3

EFK10.0 3

EFK7.6

EFK5.1

EFK2.1

With the exception of EFK 25.1, fathead minnow testing ceased in 1996 after four tests at each of the four upstream sites and one test each at
downstream sites
a
EFK = East Fork Poplar Creek kilometer; OF 201(Outfall 201) is an instream NPDES monitoring site approximately 10 m downstream of
EFK25.1

reproduction (C. dubia) or survival and growth (fathead

minnows) as test endpoints. Acute toxicity tests identified a
concentration (the Lethal Concentration50 or LC50) of
effluent which could be lethal to 50% of the test organisms
over a given test period (usually 48 h). In the case of both
the NOEC and the LC50, the lower the value the greater the
toxicity of the tested effluent. Additional details of sampling protocols, test methods, and data analysis procedures
for the effluent toxicity tests can be found in Stewart and
others (1990) and Stewart (1996).
Toxicity Loading Analyses
To evaluate the relative contributions to EFPC of toxins
from the various Y-12 Complex waste streams, each
wastewaters NOEC was compared to its expected final
concentration in the receiving stream [the instream waste
concentration (IWC)]. If the NOEC was less than the IWC,
the wastewater could be expected to be harmful upon
discharge to the receiving stream. To facilitate comparisons across wastewater streams, the expected ambient
toxicity from effluent discharges was characterized by
instream Toxic Units (iTUc if derived from chronic test
results; iTUa if derived from acute test results), with the
iTUc or a defined as the wastewaters calculated instream
waste concentration (IWC) divided by the wastewaters
NOEC. Thus an instream TUc [ 1 (where the NOEC is

less than the IWC) indicates that discharges have the

potential to be harmful to stream organisms possessing
similar sensitivity to toxicants in the stream water as the
C. dubia and fathead minnow larvae test organisms.
Ambient Toxicity Testing with Standard Test Organisms
Because the toxicity of ambient receiving waters is generally much lower than the toxicity of effluents measured
directly at a wastewater discharge, ambient toxicity testing
was routinely performed with chronic 3-brood C. dubia
tests and 7-day fathead minnow (Pimephales promelas)
(initially by the methods of Horning and Weber 1985;
USEPA 2002a) with an emphasis on test organism
responses to full-strength stream water (Stewart 1996).
Ambient toxicity testing on EFPC waters began in 1986,
when significant remedial actions at the Y-12 Complex
were just underway, and continued at selected monitoring
locations through the 2005 renewal of the NPDES permit
(Table 2).
Supplemental Ambient Tests
To supplement the standardized C. dubia and fathead
minnow toxicity tests, the ambient toxicity testing program
also employed less-standardized laboratory toxicity tests
and in situ bioassays with additional test organisms. Tests

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conducted with species other than C. dubia and fathead

minnows generally addressed specific toxicity-related
issues that could not be studied readily with the standard
toxicity tests, such as examining the potential for continuing low-level ambient toxicity in upper EFPC after
C. dubia and fathead minnows had stopped responding in
routine ambient tests.
Alternative model organisms used in laboratory tests
included the medaka (Oryzias latipes), a small aquarium
fish widely employed in basic research into early vertebrate
development and in studies of the effects of pollutants on
developmental processes. In these tests, newly-fertilized
medaka embryos were individually exposed in 7-ml glass
vials or plastic 24-well plates to water from various locations along EFPC by methods adapted from USEPA
guidelines for a fish developmental toxicity test (Benoit
and others 1991). In this test, survival and the incidences of
developmental abnormalities were assessed daily and test
solutions were renewed every other day.
In situ bioassays conducted in EFPC and nearby streams
employed native species such as the fingernail clam,
Sphaerium fabale (Smith and Beauchamp 2000). In the in
situ fingernail clam test, clams were placed in individual
clear Plexiglas tubes anchored to the bottom of the stream,
and left in situ for up to 85 days. Further details of the
experimental procedures can be found in Smith and
Beauchamp (2000).
Statistical Analyses
Statistical procedures used to estimate a wastewaters
NOEC or LC50 in C. dubia and fathead minnow tests were
performed according to USEPA test guidelines (currently
USEPA 2002a, 2002b). Various alternative statistical
methods for evaluating the results of ambient tests using
these test organisms, including ANOVA, were also used as
described in Stewart (1996). Individual-based medaka test

results were statistically analyzed with Chi-square tests.

Clam bioassays were statistically evaluated by ANOVA
and associated tests as detailed in Smith and Beauchamp
(2000).

Results
Toxicity of Wastewater Discharges from the Y-12
Complex
From 1986 through 1992, 72 chronic toxicity tests with
C. dubia and fathead minnows were conducted to characterize the toxicity of cooling tower blowdown discharges,
various untreated waste streams, and effluents from several
Y-12 Complex wastewater treatment systems. The relative
toxicities of wastewater streams before and after untreated
discharges to EFPC were eliminated in the early 1990s are
presented in Table 3. The average total toxicant loading to
upper EFPC from Y-12 Complex wastewaters before these
untreated discharges were eliminated exceeded 4 iTUc, a
value far greater than the 1 iTUc threshold for expected
instream chronic toxicity. Following the elimination of
these untreated discharges, the average toxicity loading to
the stream was reduced substantially to a relatively low
0.24 iTUc.
Routine toxicity monitoring with C. dubia, and less
frequently with fathead minnows, continued on selected
effluents from Y-12 Complex wastewater treatment facilities, storm drains and cooling towers through 2005 as
specified in the facilitys NPDES permit. Consideration of
the annual worst-case results of quarterly C. dubia toxicity tests performed from 1986 through 2005 on effluents
from the three main wastewater treatment facilities at the
Y-12 Complex (Fig. 3) demonstrated that even these
treated wastewaters occasionally exceeded the threshold
for expected instream toxicity. For example, effluent from

Table 3 Comparison of toxicity loading analyses to East Fork Poplar Creek using results of chronic C. dubia and fathead minnow toxicity tests
conducted from 1986 through 1992 on cooling tower blowdown and various treated and untreated waste streams at the Y-12 Complex before and
after the elimination of untreated waste discharges
Toxicity loading of wastewater discharges
Before elimination of untreated discharges

After elimination of untreated discharges

Wastewater source

Annual flow
(l/Year)

Instream chronic
toxic units (iTUc)a

Annual flow
(l/Year)

Instream chronic
toxic units (iTUc)a

Cooling towers

556 9 106

0.12

354 9 106

0.05

Untreated waste streams

113 9 10

3.82

Treated waste streams

138 9 106

0.23

447 9 106

0.19

Total

807 9 106

4.07

801 9 106

0.24

Instream chronic toxic units (iTUc) = Instream waste concentration (IWC)/No-Observed-Effect-Concentration (NOEC)

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Table 4 Results of chronic Ceriodaphnia dubia and fathead minnow
toxicity tests of ambient water samples collected daily from 10 sites in
lower EFPC in early 1986 prior to the initiation of major remedial
actions by the Y-12 Complex
Ceriodaphnia
a

Fig. 3 Annual worst-case C. dubia toxicity test results for

effluents from the three main wastewater treatment facilities at the
Y-12 Complex. Resultsexpressed as instream Toxic Unitswere
derived from chronic 3-brood tests through 1995 and from acute 48-h
tests after 1995; tests were typically conducted quarterly

the Groundwater Treatment Facility alone contributed [4

iTUc of toxicants to the upper reaches of EFPC on two
separate occasions during chronic tests conducted in 1993
and 1995.
Following the replacement of chronic toxicity testing
requirements with acute testing requirements in the 1995
renewal of the Y-12 Complex NPDES Permit, the measured toxicity of individual wastewaters in these routine
effluent tests never again exceeded an instream toxicity
threshold of iTU [1 (Fig. 3). The implications of this
change in test methods to the toxicity testing programs
ability to predict the effects of wastewater discharges on
the biotic communities in EFPC are discussed in the following section of this paper.

Site

Survival
(%)

Reproduction
(offspring per
female)

EFK23.8

100

27.6

90.0

0.564

EFK22.8
EFK21.9

100
100

28.9
30.3

92.5
95.0

0.382a
0.515

EFK20.5

100

30.6

92.5

0.536

EFK18.2

100

29.8

92.5

0.425b

EFK16.1

90

37.0

87.5

0.481

EFK13.8

90

34.5

90.0

0.583

EFK10.0

100

23.3

90.0

0.468

EFK7.6

100

30.2

80.0

0.495

EFK5.1

90

28.6

85.0

0.479

EFK2.1

100

28.6

87.5

0.639

Control

100

28.6

100.0

0.571

Growth
(mg/
larva)

Test endpoints that differed significantly from controls (ANOVA,

P \ 0.05)

Table 5 Comparison of chronic toxicity to C. dubia and fathead

minnows in tests of different ambient water samples collected in 1986
from upper EFPC (EFK 24.1) and lower EFPC (EFK 23.8) conducted
just prior to the initiation of major remedial actions by the Y-12
Complex
August 1986

September 1986

Survival
(%)

Reproduction/
growth
(mean, SD)

Survival
(%)

Reproduction/
growth
(mean, SD)

Control

100.0

13.2 (3.3)

100.0

20.4 (5.5)

EFK24.1

90.0

80.0

11.7 (4.6)b

EFK23.8

100.0

14.8 (2.6)

100.0

18.0 (3.5)

Site

Ambient Toxicity of the Receiving Stream

Routine toxicity testing of ambient water samples from

EFPC was begun in 1986 as part of comprehensive BMAP
efforts to characterize stream conditions prior to the initiation of extensive remedial actions and pollution abatement
activities planned by the Y-12 Complex in the EFPC
watershed (Table 1). Early ambient toxicity tests (Table 2)
initially focused on the waters of lower EFPC downstream
of the retention basin near the boundary of the Y-12
Complex (Fig. 2). In these early ambient tests, no conclusive evidence was found of toxicity to either C. dubia or
fathead minnows (Table 4), although occasional reductions
in fathead minnow survival or growth (as shown in Table 4
for ambient samples from EFK22.8 and EFK18.2) sporadically occurred in some ambient water samples.
Other ambient tests conducted in the mid to late 1980s
specifically compared the toxicity of water from EFK24.1

Survival
(%)

Adapted from technical report by Loar and others (1992)

a
EFK = East Fork kilometer

Fathead Minnow and C. dubia Tests

Fathead minnow

C. dubia
4.4 (4.5)b

Fathead minnows
Control

92.5

0.53 (0.05)

92.5

0.36 (0.07)

EFK24.1

90.0

0.41 (0.07)

92.5

0.26 (0.08)

EFK23.8

97.5

0.50 (0.06)

75.0

0.26 (0.07)

Adapted from technical report by Loar and others (1992)

a
EFK = East Fork kilometer
b

Test endpoints that differed significantly from controls (ANOVA,

P \ 0.001)

in upper EFPC with the toxicity of water from EFK23.8 in

lower EFPC. Water from EFK23.8 sampled downstream of
the New Hope Pond retention basin had no adverse effect
on either C. dubia or fathead minnows in the two 1986

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ambient toxicity tests shown in Table 5; in contrast, water

sampled from EFK24.1 upstream of the retention basin
significantly reduced C. dubia reproduction in both of these
early tests (ANOVA, P \ 0.001).
Table 6 summarizes the results of a subsequent series of
ambient toxicity tests initiated in 1988 and 1990, respectively, for two additional sites in upper EFPC at EFK24.6
and EFK25.1 within the Y-12 Complex. In tests conducted
prior to 1992, the frequencies of C. dubia test failures due
to reductions in either survival or reproduction were 60%
or greater for both sampling locations. By 1992, the frequencies of test failures for both sites had decreased significantlyto 0% for EFK 24.6 and to 30% for EFK

Table 6 Changes over time in the frequency of chronic C. dubia tests

demonstrating significant decreases in either survival or reproduction
in ambient water from two sites in upper EFPC
EFK24.6

EFK25.1

Time
period

Pre-1992

41 60

Frequency of test
failures (%)

Frequency of test
failures (%)

10 30

Late 1992: dechlorination of major discharges begun

1993

12

12 17

1994

12

12 33

1995

11 27

11 9

Early 1996: initial implementation of flow management

1996

12

4a 0

1997

EFK = East Fork kilometer

a

Toxicity monitoring at site discontinued

Fig. 4 Results of chronic

C. dubia and fathead minnow
toxicity tests of ambient water
sampled from East Fork Poplar
Creek at Outfall 201, an
instream NPDES monitoring
site located just downstream of
EFK 25.1 within the Y-12
Complex (NOEC =
No-Observed-EffectsConcentration)

123

Supplemental Ambient Tests with Alternative Test

Organisms

17 76

Elimination of untreated discharges completed

1992

25.1following the elimination of untreated wastewater

streams in Y-12 Complex discharges. The annual frequencies of test failures for both sites then fluctuated from
0 to 33% before testing was eventually discontinued at
EFK25.1 in 1996 and at EFK24.6 in 1997 (Table 2).
Routine ambient toxicity testing was also conducted
from 1994 through 2005 at a fourth site in upper EFPCa
newer instream NPDES monitoring station (Outfall 201)
located just downstream of EFK25.1 (Table 2 and Fig. 4).
Similar to the situation for the other testing sites in upper
EFPC, toxicity test failures (as indicated in Fig. 4 by
NOEC values \100%) involving water from Outfall 201
were relatively common prior to 1996, particularly in the
case of C. dubia tests. Following the implementation of
flow management in EFPC in 1996, there was only a single
subsequent test failure with either C. dubia or fathead
minnows at this ambient sampling site through 2005
(Fig. 4).

To supplement the standardized C. dubia and fathead

minnow tests, studies of ambient toxicity in EFPC were
also conducted with other native and non-native test
organisms. Results of laboratory tests of water samples
from EFPC using newly-fertilized embryos of the medaka,
a small non-native fish commonly employed as a model for
vertebrate development, are shown in Fig. 5. In tests begun
in 1997, survival of medaka embryos and larvae through
2 days post-hatch was significantly reduced compared to
laboratory controls in water samples from various sites in
both lower and upper EFPC (Chi square tests of individualbased results, P \ 0.01). Embryo survival in these tests

1041

1.75

se (m
Length increa

m)

Environmental Management (2011) 47:10331046

75
50
25
0
trol
Con
13.8
E FK
18.2
EFK

Si

te

23.4
EFK
24.6
EFK
25.1
E FK

2005
2004
2003
2002
2001
2000
1998
1997

1.25
1.00
0.75
0.50
0.25
0.00
20
HCK XK0.2
7
C
BFK 13.8
EFK FK23.4
E
24.4
EFK

ar

Ye

2005
2004
2003
2002
2001
2000
1999
1998

Ye
ar

100

rv
Medaka Su

ival (%)

1.50

Site

Fig. 5 Survival of medaka embryos during toxicity tests of ambient

water from EFPC. EFK = EFPC kilometer

Discussion
The toxicity assessment and monitoring program described
in this paper is but one facet of the extensive water pollution
controls and biological monitoring efforts conducted at the
Y-12 Complex since the mid 1980s. The relationships
between toxicity testing and receiving-water impacts have

100

75

50

2005
2004
2003
2002
2001
2000
1999
1998

ar

25

0
20
HCK XK0.2
7
C
BFK 13.8
EFK FK23.4
E
24.4
EFK

Ye

Survival (%)

gradually increased over time for all tested locations in

EFPC, and by 2003 had approached the levels of laboratory
controls in water samples from monitoring sites in lower
EFPC (EFK13.8 and EFK18.2). However, significant
decreases in embryo survival (P \ 0.05) continued through
2005 in ambient water samples from tested sites in upper
EFPC (EFK24.6 and EFK25.1).
In addition to laboratory tests using alternative test
organisms such as the medaka, in situ bioassays with
species such as the native fingernail clam (Smith and
Beauchamp 2000) were conducted as early as 1988 at
various locations in EFPC and other streams in the Oak
Ridge area. Results of clam bioassays conducted annually
from 1998 through 2005 at three locations in EFPC and
three nearby reference streams unaffected by industrial
discharges are shown in Fig. 6. Clam survival in these
bioassays was consistently high at all three reference
streams; similar to survival in the reference streams at
EFK13.8 in lower EFPC; and significantly reduced at
EFK23.4 and EFK24.4 (ANOVA, P \ 0.05). Clam growth
was also relatively high in the reference streamsalthough
more variable from year-to-year than the survival endpointbut was again significantly reduced (P \ 0.05) at
all three of the tested EFPC locations.

Site

Fig. 6 Growth and survival of fingernail clams during in situ

bioassays in EFPC and reference sites. Reference sites were Hinds
Creek (HCK20), Cox Creek (CXK0.2), and Brushy Fork (BFK7) (see
Smith and Beauchamp 2000). Data are not presented for Cox Creek in
2002 due to loss of bioassay units from vandalism. Test durations
varied from 80 to 85 days

been previously examined in relatively short-term studies

(examples include Eagleson and others 1990; Dickson and
others 1992; Kosmala and others 1999). However, relatively
few long-term studies have examined how toxicity assessments, as implemented through NPDES permitting
requirements in the mid-1980s and continuing to the present,
have been used in conjunction with other approaches such as
bioaccumulation studies and biological surveys to successfully assess the effects of aquatic pollution and monitor the
ecological recovery of receiving waters following an
extensive and prolonged implementation of water pollution
controls.

123

1042

Whole effluent toxicity testing isalong with chemical

analyses and biological surveysa significant component
of the USEPA integrative approach for controlling toxics in
surface waters (USEPA 1991). Considered to be predictive
of the health of receiving waters (Eagleson and others
1990; Grothe and others 1996; DeVlaming and NorbergKing 1999; Maltby and others 2000), effluent toxicity
testing requirements have been routinely included for many
years in NPDES permits issued for industrial facilities and
municipalities under the U.S. Clean Water Act, and have
been specifically included in the Y-12 Complexs NPDES
permits since the mid 1980s. Since effluent toxicity limits
began to be applied to NPDES permits in the 1980s,
compliance of wastewater discharges with toxicity limits
has improved significantly at the Y-12 Complex and
nationwide as well (Ausley 2000).
Chronic effluent toxicity testing conducted for the Y-12
Complex TCMP using the standard aquatic test organisms
C. dubia and fathead minnows conclusively demonstrated
that wastewaters discharged from the facility were sufficiently toxic in the mid 1980swith a calculated total
iTUc [ 4 at expected instream waste concentrationsto
provide a likely explanation for both the observed ambient
toxicity of stream water from upper EFPC and measured
impairments of stream communities (Ryon 2011; Smith
and others 2011). Intensive toxicity testing of numerous
effluent streams throughout the Y-12 Complex over the
period 19861992 was instrumental in identifying two
untreated process wastewaters as the most significant
sources of toxicity to the receiving stream. Before redirection to treatment facilities, these particular discharges
were estimated to be responsible for approximately 94% of
the total toxicity loading to the stream from Y-12 Complex
wastewaters, in only 14% of total discharges. By 1992
once these particular discharges had been rerouted to
wastewater treatment facilities, all other untreated discharges eliminated, and the toxicity of remaining wastewater discharges such as cooling tower and wastewater
treatment facility effluents significantly decreased through
chemical substitutions and process modificationsthe
average releases of toxicants to EFPC from the Y-12
Complex had been reduced nearly twenty-fold to a value
(total iTUc = 0.24) well below the chronic threshold for
expected instream toxicity. However, monitoring of the
chronic toxicity of various wastewater treatment facilities
continued to demonstrate the occasional presence of
detectable toxicants in these effluent streams through at
least 1996 that undoubtedly contributed to the continuing
impairment of stream communities in upper EFPC during
this period of time. Whether this episodic chronic toxicity
of wastewater effluents persisted after 1996 will never be
known, as the mandated change from chronic toxicity
testing of effluents to less sensitive acute toxicity testing in

123

Environmental Management (2011) 47:10331046

the 1995 renewal of the NPDES permit for all practical

purposes negated the ability of the effluent testing program
to effectively monitor such intermittent releases of toxicants to the stream that were not actually acutely lethal to
the test organisms.
In addition to routine monitoring of effluent toxicity,
special toxicant identification studies were also conducted
for the Y-12 TCMP whenever new sources of significant
toxicity were discovered during routine effluent or ambient
toxicity tests. Examples of such studies based on the ultimate chemicals of concern included investigations focused
on nickel (Kszos and others 1992), chlorine (Stewart and
others 1996), uranium (Taylor and others 1987), and lithium (Kszos and others 2003). Whenever sources of toxicity
were identified, environmental management strategies
(typically, best management practices) were invoked to
mitigate the problem (Taylor and others 1987). Specific
examples of remedial actions taken to reduce pollutants
identified through the ambient toxicity testing program
included: (1) installation of a post-treatment carbon filter at
the West End Treatment Facility to lower the effluents
toxic concentrations of nickel; (2) whole-stream dechlorination in upper EFPC by use of sodium metabisulfite; (3)
the removal and containment of urea, used as a deicer, from
an open storage site that was found to be draining to EFPC;
and (4) rerouting from upper EFPC to the Oak Ridge
Wastewater Treatment Facility of sulfate-rich effluent from
a coal-fired boiler unit.
Ambient toxicity testing, although not as commonly
used as effluent toxicity testing, has been suggested to be
possibly a more accurate and relevant predictor of receiving-waters effects (Birge and others 1989; Stewart and
others 1990; Dickson and others 1992). Just as effluent
toxicity indicated the presence of toxicants in discharges
sampled at the pipes, ambient toxicity testing provided
direct evidence of the presence of toxicants in upper EFPC
downstream of the Y-12 Complex outfalls. Ambient toxicity testing initially focused on numerous sampling sites in
lower EFPC downstream of the retention basins and only a
single site in upper EFPC at EFK 24.1, located just
upstream of the basins. In these early ambient toxicity tests
conducted before the first retention basinNew Hope
Pondwas closed in late 1988 and replaced by Lake
Reality, the average reproduction of C. dubia over a total of
nine such tests was 44% lower in water samples from the
upstream EFK24.1 site than in water samples from the
downstream EFK23.8 site (complete results not shown).
These early tests led to several preliminary conclusions
regarding the ambient toxicity of EFPC waters: (1) ambient
toxicity, if present in the lower reaches of EFPC, was
unable to be conclusively detected through the use of
C. dubia and fathead minnow testing [the infrequent and
sporadic reductions in fathead minnow growth or survival

Environmental Management (2011) 47:10331046

occasionally observed in ambient tests of samples from

lower EFPC were attributed to pathogens in the water
samples (Kszos and others 1997)]; (2) the ambient waters
of upper EFPC were persistently toxic to C. dubia but not
to fathead minnows; and (3) passage through the retention
basin was apparently beneficial in reducing the ambient
toxicity of EFPC waters.
Once it became obvious from the results of these early
ambient tests that neither C. dubia nor fathead minnow
tests could reliably detect toxicity in the waters of lower
EFPC, the focus of further ambient toxicity investigations
quickly shifted to upper EFPC. Routine ambient toxicity
monitoring was conducted on at least a quarterly basis
beginning initially at EFK24.1 during 1986, then further
upstream at EFK24.6 in 1988, and at EFK25.1 in 1990. The
latter two sampling sites were eventually replaced for
routine ambient toxicity testing purposes by a new instream
NPDES monitoring location located just downstream of
EFK25.1 at Outfall 201.
By 1992, with the elimination of untreated discharges
and other measures taken to improve EFPC water quality,
water samples collected from upper EFPC at EFK24.1 had
become consistently non-toxic to both C. dubia and fathead
minnows (Adams and others 2002). However, ambient
toxicity to C.dubia continued to be detected well into the
1990s in water samples from sites further upstream within
the Y-12 Complex. Much of this remaining ambient toxicity in upper EFPC was attributed to the presence of total
residual chlorine (TRC) from discharges of cooling tower
blowdown and chlorinated process water (Stewart and
others 1996). The dechlorination of major outfalls to EFPC
began in late 1992 and continued for the next several years
as additional discharges were subsequently treated. However, occasional failures of the dechlorination systems
contributed to fish kills in upper EFPC even after this time
(Etnier and others 1996) and probably also to the continuing ambient toxicity observed at some EFPC locations
through the mid 1990s. With the implementation of flow
management in 1996, which significantly increased the
flow of EFPC at the stream headwaters to volumes similar
to those present before extensive water pollution controls
began in the mid 1980s, ambient toxicity to C. dubia and
fathead minnows largely disappeared from EFPC ambient
waters.
Similar to the toxicant identification studies conducted
for the TCMP, routine monitoring of ambient toxicity for
the BMAP was supplemented by special studies designed
to address specific toxicological concerns. For example,
several of the toxicant evaluations performed for the
TCMP also had associated BMAP special studies that
examined the potential effects of toxicants of concern on
ambient toxicity or specific EFPC biota. Special toxicityrelated studies conducted in support of both the ambient

1043

and effluent toxicity testing programs included: (1) investigations of the production, export, and ecological effects
of bioparticles in the Lake Reality retention basin (Cicerone and others 1999); (2) a demonstration that in-stream
toxicological problems due to chlorine were modified
substantially by environmental conditions (e.g., sunlight
and algal biomass; Stewart and others 1996); and (3) both
in situ and laboratory investigations into the acclimation of
minnows to TRC in EFPC (Lotts and Stewart 1995).
Other tests employing longer exposure durations or
more sensitive test organisms or life stages were also used
in the BMAP program to help monitor the later stages of
stream recovery after C. dubia and fathead minnows had
stopped responding in ambient tests. Among the alternative
tests used for this purpose were a 21-days medaka embryo
development test and an in situ fingernail clam bioassay of
8085-days duration (Smith and Beauchamp 2000). Both
test organisms were affected at locations whereor at
times whenneither C. dubia nor fathead minnow tests
detected ambient toxicity, for example at sites in lower
EFPC throughout the study period and at sites in upper
EFPC following the implementation of flow management.
Another special study involved the use of full life-cycle C.
dubia tests to determine specifically if longer exposure
durations with this standard test organism could reveal
toxicant effects not seen in the shorter 3-brood tests
(Stewart and Konetsky 1998).
Results of the Y-12 Complex toxicity studies, considered together, suggest that effluent and ambient toxicity
testing with C. dubia and fathead minnows, alternative
laboratory toxicity tests with other organisms, and in situ
bioassays were all predictive to some degree of observed
biological impacts in EFPC. For instance, fish community
(Ryon 2011) and benthic invertebrate (Smith and others
2011) surveys conducted in the mid to late 1980s demonstrated impairment of instream aquatic communities at a
time when C. dubia and fathead minnow effluent and
ambient toxicity tests indicated there should be toxic
effects on stream communities. In addition, observed
reductions in the measured toxicity to C. dubia and fathead
minnows of both effluents and the ambient waters of upper
EFPC through the mid 1990s were accompanied by gradual
improvements in EFPC fish and benthic invertebrate
communities. Taken together, this evidence suggests a
certain degree of predictive and causal association between
effluent and ambient toxicity and the ecological condition
of EFPCbut with some caveats. For instance, neither
C. dubia nor fathead minnow ambient tests were sufficiently sensitive even in the early stages of the BMAP in
the mid 1980s to detect toxicity in the waters of lower
EFPC, when the absence of pollution-sensitive fish and
benthic invertebrate species from these communities was
strongly suggestive of toxic impacts in the stream.

123

1044

Furthermore, C. dubia and fathead minnow ambient tests

were also incapable of detecting the apparent continuing
low-level toxicity of stream water from both lower and
upper EFPC following flow management as implied by the
results of alternative ambient tests with medaka and fingernail clams and the continuingalthough also diminishingimpairments of stream communities during this
period. Based on the Y-12 Complex BMAP experience, the
relative sensitivities of the various testing approaches to
assessing ambient toxicity in EFPC were as follows: in situ
tests (clam test/most sensitive) [ alternative laboratory
tests (in this case, the medaka test focused on fish development) [ chronic C. dubia tests [ acute C. dubia tests
and chronic and acute fathead minnow tests (least
sensitive).
With the significant reductions in the toxicity of both
Y-12 Complex wastewaters and EFPC ambient waters that
have occurred over time, additional factors such as habitat
degradation and the continuing presence of excessive
nutrients in Y-12 Complex wastewater may be assuming
increasingly greater significance to the further recovery of
stream communities in EFPC. For example, excessive
nutrients in wastewater effluents, which stimulate periphyton production and thus foster an overabundance of
grazers in both fish and benthic invertebrate communities
(Hill and others 2010; Ryon 2011; Smith and others 2011),
may now be exerting greater influence on the composition
of these communities in the upper reaches of EFPC than
any remaining low-level ambient toxicity, although this
hypothesis remains to be conclusively proven. This
example illustrates the potential difficulties of interpreting
monitoring results from a single line of evidence (for
instance, from only ambient toxicity test results), especially
in the later stages in the recovery of a receiving body of
water, and emphasizes the importance of taking a weight
of evidence or multi-criteria approach (La Point and
Waller 2000) to the implementation of a successful biomonitoring program.

Summary and Environmental Management

Implications
The Y-12 Complex case-study demonstrates the value of a
long-term and diverse toxicity assessment and monitoring
program to a large industrial facility seeking to reduce or
eliminate toxic impacts to receiving waters. Effluent toxicity testing conducted for the Y-12 Complex TCMP
helped identify sources of toxicants and facilitate significant reductions in toxicity loading to the upper reaches of
EFPC in the late 1980s and early 1990s. Decreases in
effluent toxicity documented by the testing program in turn
led to measurable decreases in the ambient toxicity of

123

Environmental Management (2011) 47:10331046

stream water and marked improvements in the biological

health of the stream (Ryon 2011; Smith and others 2011).
Ambient toxicity testing conducted for the Y-12 Complex
BMAP was particularly useful in evaluating the effectiveness of pollution abatement actions and, in conjunction
with other BMAP activities such as instream biological
surveys, in directly assessing and monitoring the ecological
recovery of the receiving stream.
With some exceptions, such as the problematic switch
from chronic to acute testing of wastewater effluents in the
1995 renewal of the NPDES permit, the Y-12 Complex
toxicity assessment and monitoring program is a prime
example of the successful implementation of adaptive
environmental management principles. The program was
diverse, with multiple species used and a variety of sites
tested as appropriate, and generally very adaptable (except
when specifically mandated otherwise by regulatory decisions), with test methods and scope evolving as the situation and information needs changed over time. As the
toxicity of effluents and the receiving stream decreased due
to the success of remedial actions or pollution abatement
measures, testing became more focused, sites were dropped, and more sensitive tests and bioassays were developed
and applied as needed to further evaluate and monitor
wastewater toxicity and subsequent improvements in
stream water quality.
In summary, the Y-12 Complex TCMP and BMAP casestudies show the utility of effluent and ambient toxicity
assessments as environmental management tools. Effluent
and ambient toxicity testing, special toxicological studies,
and toxicity identification studies have been particularly
useful to environmental managers and regulators in
evaluating causal and mechanistic relationships between
environmental contamination, pollution control and environmental remediation activities, and subsequent effects on
the ecological condition of receiving waters. The Y-12
Complex example further illustrates the importance of
combining toxicity studies with other assessment techniques such as chemical analyses, bioaccumulation
assessments, and instream biological surveys in an integrated weight of evidence approach for successfully
assessing and monitoring the ecological health of a
receiving body of water.
Acknowledgments The authors acknowledge the many individuals
who made significant contributions to this project, including Kitty
McCracken, Belinda Konetsky, Linda Wicker, W. Kelley Roy, Peggy
Braden, G. Jayne Haynes, Richard D. Bailey, and numerous intern
students. Logan Elmore provided assistance with figures. The work
was funded by the Environmental Compliance Department of the
Y-12 National Security Complex, which is managed by BWXT Y-12,
LLC for the U.S. Department of Energy under contract number
DE-AC05-00OR22800. Oak Ridge National Laboratory is managed
by the University of Tennessee-Battelle LLC for the U.S. Department
of Energy under contract DE-AC05-00OR22725.

Environmental Management (2011) 47:10331046

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