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Role of a Comprehensive Toxicity Assessment and MonitoringProgram 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 Per-
mit (NPDES)-driven effluent toxicity tests using Cerio-daphnia 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 con-
ducted on water samples from East Fork Poplar Creek
(EFPC), the wastewater receiving stream, as part of a
comprehensive biological monitoring and assessment pro-
gram. In this paper, we evaluate the roles of this long-term
toxicity assessment and monitoring program in the man-
agement and ecological recovery of EFPC. Effluent toxicity
testing, associated toxicant evaluation studies, and ambient
toxicity monitoring were instrumental in identifying toxi-
cant 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 pri-mary 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 regu-
latory requirements changed and water quality improved,
emphasis shifted from comprehensive toxicity assessments
to more focused toxicity monitoring efforts. Ambient tox-
icity testing with C. dubia and fathead minnows was sup-
plemented 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 manage-
ment of a wastewater receiving stream.
Keywords In situ bioassay Effluent toxicityLong-term
monitoring Ambient toxicity Biomonitoring
Introduction
When the United States (U.S.) Environmental Protection
Agency (USEPA) applied water quality-based limitations
for toxic pollutants to National Pollutant Discharge Elimi-
nation System (NPDES) permits in the 1980s, many indus-
trial 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 produc-
tion 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
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
1 3
Environmental Management (2011) 47:10331046
DOI 10.1007/s00267-011-9679-3
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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 con-
trolling 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 thereceiving stream, resulting in the establishment of a Bio-
logical Monitoring and Abatement Program (BMAP) to
ensure and evaluate the effectiveness of the Y-12 Complex
TCMP and associated water pollution control programs in
protectingthe classified uses of EFPC (Loar and others 2011;
Peterson2011; Peterson and others 2011; Stewart and others
2011).
Whole effluent toxicity testing was the primary inves-
tigative 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 ofconcern; (3) guide changes in wastewater treatment oper-
ations 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 compli-
mentary bioassessment approachesincluding bioaccu-
mulation 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 con-
tinued to the 2005 renewal of the facilitys NPDES permit.
The diversity and long-term nature of the BMAP, com-
bined 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 andothers
2011) and resultant changes in ambient toxicity (this paper),
water quality (Stewart and others 2011), pollutant bioaccu-
mulation (Southworth and others 2011), and the health of
aquatic organisms and communities (Hill and others2010;
Adams andHam 2011; Ryon 2011; Smith andothers 2011) in
a freshwater receiving stream. Elements of the aquatic tox-
icity testing program for the Y-12 Complex have been dis-
cussed previously (Stewart and others 1990; Stewart1996;
Loar andothers 1992; Kszos andothers 1992; Hinzman 1993;
Stewart 1996; Kszos andothers 1997; Hinzman 1998; Adams
and others2002; Kszos and Stewart2003).
Radiological and non-radiological contaminants of his-
torical concern in EFPC include elevated nutrients, chlorine
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 waste-
water treatment facility outfalls and storm drains, and from
non-point sources such as groundwater inputs and runoff
from contaminated floodplain soils. Beginning in the 1980sand 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.
Table1summarizes 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).
Figure1 demonstrates the functional relationships
between wastewater toxicity testing performed under the
TCMP and ambient toxicity testing performed for theBMAP in establishing effluent limitations for new waste-
water treatment facilities at the Y-12 Complex (adapted
from Kingrea1986; Loar and others 1992). From the ini-
tiation 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 moni-
toring 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 condi-
tions 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 environ-
mental 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
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originates within the Y-12 Complex in Oak Ridge, Tennes-see, where it receives wastewaterdischargesvia 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 ofstream 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.
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 dechlorinatedduring 1993 1994)
19962000 Additional relining of sanitary and storm sewers
1996 Completion of central and east end mercury treatment systems
1996 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)
BAT EffluentLimitations
Modify EffluentLimitations
Develop ToxicityControl Plan and/or
Conduct TRE
Final EffluentLimitations
Is WastewaterToxic?(TCMP)
Are Classified UsesBeing Maintained?
(BMAP)
Is WastewaterToxic?(TCMP)
Yes
No
Yes
Yes
No
Yes
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)
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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 iso-
late very specific point sources of potential toxicants, moresites along EFPC were eventually examined for ambient
toxicity than the five routinely monitored by other BMAP
tasks, and toxicity sampling locations were more specifi-
cally 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 upperEFPC (Table2).
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 con-
trols 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
watersheds with rural impacts but no industrial contami-
nation, 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 dis-
charges, and treatment facility effluents were tested peri-
odically with chronic 3-brood C. dubia tests (initially by
the methods of Horning and Weber 1985; current test
method 1002.0 in USEPA2002a) and 7-day fathead min-
now larvae growth and survival tests (initially by the
methods of Horning and Weber1985; current test method1000.0 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-effect-
concentration (NOEC) of effluent, with either survival and
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
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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 thetoxicity of the tested effluent. Additional details of sam-
pling 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 compari-
sons 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 o r 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 gen-
erally 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 monitoringlocations through the 2005 renewal of the NPDES permit
(Table2).
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
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 7 10 10 12 12 11 4
OF 201 2 5 4 4 4 4 4 4 4 4 4 4
EFK24.6 9 12 10 10 9 12 12 11 12 2
EFK24.1 6 8 8 11 11 11 9 12 12 11 12 5 4 4 4 4 4 4 4 4
Lower East Fork Poplar Creek
EFK23.8 17 13 12 12 10 9 9 12 12 11 12 5 4
EFK22.8 3 4 3 3 3 4 4 4 4 4 4 1
EFK21.9 1 4 3 3 3 4 4 4 4 4 4 1
EFK20.5 3 4 3 3 3 4 4 4 4 4 4 1
EFK18.2 3 4 3 3 3 4 4 4 4 4 4 1
EFK16.1 2
EFK13.8 3 4 3 3 3 4 4 4 4 4 4 1
EFK10.0 3 4 3 3 3 4 4 4 4 4 4 1
EFK7.6 2
EFK5.1 2
EFK2.1 2
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 sitesa
EFK= East Fork Poplar Creek kilometer; OF 201(Outfall 201) is an instream NPDES monitoring site approximately 10 m downstream of
EFK25.1
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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 contin-
uing 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 testsincluded 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 loca-
tions along EFPC by methods adapted from USEPA
guidelines for a fish developmental toxicity test (Benoit
and others1991). 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 streamsemployed native species such as the fingernail clam,
Sphaerium fabale (Smith and Beauchamp2000). 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 LC50in C. dubiaand 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 charac-
terize 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 theseuntreated 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 facili-
ties, 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 tox-
icity 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 chronicC. dubiaand 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 9 106 3.82 0 0
Treated waste streams 138 9 106
0.23 447 9 106
0.19
Total 807 9 106
4.07 801 9 106
0.24
aInstream chronic toxic units (iTUc) = Instream waste concentration (IWC)/No-Observed-Effect-Concentration (NOEC)
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the Groundwater Treatment Facility alone contributed[4
iTUc of toxicants to the upper reaches of EFPC on twoseparate 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 mea-
sured 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 fol-
lowing section of this paper.
Ambient Toxicity of the Receiving Stream
Fathead Minnow and C. dubia Tests
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 initi-
ation of extensive remedial actions and pollution abatement
activities planned by the Y-12 Complex in the EFPC
watershed (Table1). Early ambient toxicity tests (Table2)
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 con-
clusive evidence was found of toxicity to eitherC. dubiaor
fathead minnows (Table4), although occasional reductions
in fathead minnow survival or growth (as shown in Table 4
for ambient samples from EFK22.8 and EFK18.2) spo-
radically 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
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
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
Table 4 Results of chronic Ceriodaphnia dubiaand 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 Fathead minnow
Sitea
Survival
(%)
Reproduction
(offspring per
female)
Survival
(%)
Growth
(mg/
larva)
EFK23.8 100 27.6 90.0 0.564
EFK22.8 100 28.9 92.5 0.382a
EFK21.9 100 30.3 95.0 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 0.639Control 100 28.6 100.0 0.571
Adapted from technical report by Loar and others (1992)a
EFK= East Fork kilometerb 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
Sitea Survival
(%)
Reproduction/
growth
(mean, SD)
Survival
(%)
Reproduction/
growth
(mean, SD)
C. dubia
Control 100.0 13.2 (3.3) 100.0 20.4 (5.5)
EFK24.1 90.0 4.4 (4.5)b
80.0 11.7 (4.6)b
EFK23.8 100.0 14.8 (2.6) 100.0 18.0 (3.5)
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 kilometerb Test endpoints that differed significantly from controls (ANOVA,
P\0.001)
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ambient toxicity tests shown in Table 5; in contrast, water
sampled from EFK24.1 upstream of the retention basin
significantly reducedC. dubiareproduction in both of these
early tests (ANOVA, P\ 0.001).
Table6 summarizes the results of a subsequent series of
ambient toxicity tests initiated in 1988 and 1990, respec-
tively, for two additional sites in upper EFPC at EFK24.6
and EFK25.1 within the Y-12 Complex. In tests conductedprior to 1992, the frequencies ofC. dubia test failures due
to reductions in either survival or reproduction were 60%
or greater for both sampling locations. By 1992, the fre-
quencies of test failures for both sites had decreased sig-
nificantlyto 0% for EFK 24.6 and to 30% for EFK
25.1following the elimination of untreated wastewater
streams in Y-12 Complex discharges. The annual fre-
quencies 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 2and 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).
Supplemental Ambient Tests with Alternative Test
Organisms
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 individual-
based results, P\ 0.01). Embryo survival in these tests
Table 6 Changes over time in the frequency of chronicC. dubiatests
demonstrating significant decreases in either survival or reproduction
in ambient water from two sites in upper EFPC
EFK24.6 EFK25.1
Timeperiod
n Frequency of testfailures (%)
n Frequency of testfailures (%)
Pre-1992 41 60 17 76
Elimination of untreated discharges completed
1992 9 0 10 30
Late 1992: dechlorination of major discharges begun
1993 12 8 12 17
1994 12 0 12 33
1995 11 27 11 9
Early 1996: initial implementation of flow management
1996 12 0 4a
0
1997 2
a
0
EFK= East Fork kilometera
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-Effects-
Concentration)
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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 OakRidge 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 end-
pointbut was again significantly reduced (P\ 0.05) at
all three of the tested EFPC locations.
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
been previously examined in relatively short-term studies
(examples include Eagleson and others1990; Dickson and
others1992; Kosmala and others 1999). However, relativelyfew long-term studies have examined how toxicity assess-
ments, 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 success-
fully assess the effects of aquatic pollution and monitor the
ecological recovery of receiving waters following an
extensive and prolonged implementation of water pollution
controls.
0
25
50
75
100
1997
1998
2000
2001
2002
2003
2004
2005
EFK25.1
EFK24.6
EFK23.4
EFK18.2
EFK13.8
Control
MedakaSurviva
l(%)
YearSite
Fig. 5 Survival of medaka embryos during toxicity tests of ambient
water from EFPC. EFK= EFPC kilometer
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
1998
1999
2000
2001
2002
2003
2004
2005
EFK24.4EF
K23.4EFK
13.8BFK
7CXK0.2HC
K20
Lengthincre
ase(mm)
Year
Site
0
25
50
75
100
1998
1999
2000
2001
2002
2003
2004
2005
EFK24.4EF
K23.4EFK13.8B
FK7CXK0.2H
CK20
Survival(%)
Year
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 Beauchamp2000). 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
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Whole effluent toxicity testing isalong with chemical
analyses and biological surveysa significant component
of the USEPA integrative approach for controlling toxics in
surface waters (USEPA1991). Considered to be predictive
of the health of receiving waters (Eagleson and others
1990; Grothe and others 1996; DeVlaming and Norberg-
King 1999; Maltby and others 2000), effluent toxicity
testing requirements have been routinely included for manyyears 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 suffi-ciently 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 redi-
rection 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 dis-
charges eliminated, and the toxicity of remaining waste-
water 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
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 toxi-
cants 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 significanttoxicity were discovered during routine effluent or ambient
toxicity tests. Examples of such studies based on the ulti-
mate chemicals of concern included investigations focused
on nickel (Kszos and others 1992), chlorine (Stewart and
others1996), uranium (Taylor and others 1987), and lith-
ium (Kszos and others2003). 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 atthe West End Treatment Facility to lower the effluents
toxic concentrations of nickel; (2) whole-stream dechlori-
nation 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 receiv-
ing-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 tox-
icity 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 ofC. dubiaover 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
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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 earlyambient 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 dischargesand other measures taken to improve EFPC water quality,
water samples collected from upper EFPC at EFK24.1 had
become consistently non-toxic to bothC. dubiaand 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 tox-
icity 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
others1996). The dechlorination of major outfalls to EFPC
began in late 1992 and continued for the next several years
as additional discharges were subsequently treated. How-
ever, 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 contin-
uing 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 toxicity-
related studies conducted in support of both the ambient
and effluent toxicity testing programs included: (1) inves-
tigations of the production, export, and ecological effects
of bioparticles in the Lake Reality retention basin (Cice-
rone 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 others1996); and (3) both
in situ and laboratory investigations into the acclimation ofminnows 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 lowerEFPC 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 Konetsky1998).
Results of the Y-12 Complex toxicity studies, consid-
ered 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 demon-
strated 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 toC. dubiaand 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 suffi-
ciently 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.
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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 fin-
gernail clams and the continuingalthough also dimin-
ishingimpairments of stream communities during this
period. Based on the Y-12 Complex BMAP experience, therelative 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 devel-
opment)[ 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 assumingincreasingly greater significance to the further recovery of
stream communities in EFPC. For example, excessive
nutrients in wastewater effluents, which stimulate periph-
yton production and thus foster an overabundance of
grazers in both fish and benthic invertebrate communities
(Hill and others2010; Ryon2011; Smith and others2011),
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 bio-
monitoring 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 tox-
icity testing conducted for the Y-12 Complex TCMP
helped identify sources of toxicants and facilitate signifi-
cant 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
stream water and marked improvements in the biological
health of the stream (Ryon 2011; Smith and others2011).
Ambient toxicity testing conducted for the Y-12 Complex
BMAP was particularly useful in evaluating the effective-
ness 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 deci-
sions), with test methods and scope evolving as the situa-
tion and information needs changed over time. As the
toxicity of effluents and the receiving stream decreased dueto the success of remedial actions or pollution abatement
measures, testing became more focused, sites were drop-
ped, 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 case-
studies 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 envi-
ronmental 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 tech-
niques such as chemical analyses, bioaccumulation
assessments, and instream biological surveys in an inte-
grated 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.
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