EPA903-R-01-001
CBP/TRS 249/01
January 2001
Application of the 10-d Acute
and 28-d Chronic Leptocheirus
plumulosus Sediment Toxicity
Tests to the Ambient Toxicity
Assessment Program
^
Chesapeake Bay Program
A Watershed Partnership
U.S. EPA Headquarters Library
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Application of the 10-d Acute and 28-d Chronic Leptocheirus plumulosus
Sediment Toxicity Tests to the Ambient Toxicity Assessment Program
2001
Chesapeake Bay Program
A Watershed Partnership
Chesapeake Bay Program
410 Severn Avenue, Suite 109
Annapolis, Maryland 21403
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FINAL REPORT
Application of the 10-d Acute and 28-d Chronic Leptocheir us plumulesus Sediment Toxicity
Tests to the Ambient Toxicity Assessment Program
Prepared by:
Daniel J. Fisher, Ph.D.
Gregory P. Ziegler
Lance T. Yonkos
Bonnie S. Turley
University of Maryland System
Agricultural Experiment Station
Wye Research and Education Center
Box 169
Queenstown, Maryland 21658
Prepared for:
Ms. Kelly Eisenman, Toxics Coordinator
U.S. Environmental Protection Agency
Chesapeake Bay Program Office
410 Severn Avenue
Annapolis, MD 21403
October 3,2000
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FOREWORD
This study was designed to compare the current suite of sediment toxicity test methods used
by the Ambient Toxicity Assessment Program with the 10-d acute and 28-d chronic Leptocheirus
plumulosus test method proposed by the U.S. Environmental Protection Agency's (U.S. EPA) Office
of Science and Technology. A team of scientists from two Chesapeake Bay research laboratories
worked jointly to complete this goal. Mr. Lenwood Hall from the University of Maryland and Dr.
Joe Winfield from Old Dominion University played a critical role in technical assistance and project
coordination. The sediment toxicity tests using the U.S. EPA's methods were conducted by Dr.
Daniel Fisher's research group of the University of Maryland's Wye Research and Education Center.
Sediment collection and the routine Ambient Toxicity Assessment Program's sediment toxicity tests
were managed by Alan Messing of Old Dominion University' s Applied Marine Research Laboratory.
The U. S. Environmental Protection Agency's Chesapeake Bay Program Office supported this study.
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ABSTRACT
The goal of this report is to compare the U.S. EPA approved Leptocheirus plumulosus 10-d
acute and 28-d chronic sediment test methods with sediment toxicity test methods used in the
Ambient Toxicity Assessment Program. The toxicity of estuarine and freshwater sediments was
evaluated during the late summer/early fall of 1998 at four stations in the Choptank River and six
stations in the Anacostia River. Sediment samples from these stations were collected and split
between two laboratories. The Ambient Toxicity Assessment Program methods included the 10-d
sheepshead minnow Cyprinodon variegatus embryo/larval test and 20-d survival and growth tests
(with 10-d survival data) with the estuarine amphipods L. plumulosus and Lepidactylus dytiscus, the
freshwater amphipod Hyalella azteca, and the polychaete worm Streblospio benedicti. These tests
were conducted by Old Dominion University's Applied Marine Research Laboratory (AMRL). The
U.S. EPA L. plumulosus tests were conducted by the University of Maryland's Wye Research and
Education Center (WREC). The 10-d static acute test used mortality as the endpoint while the 28-d
static-renewal exposure used mortality, growth, and reproduction as endpoints.
Results from the WREC testing indicate that the toxicity of the sediments to L. plumulosus
from the Anacostia River was much greater than that from the Choptank River. L. plumulosus
survival was not effected in the Choptank River sediments in the acute test but was significantly
reduced in four of the six stations in the Anacostia River. The 28-d chronic toxicity test was more
sensitive than the 10-d test with survival significantly reduced at all of the Anacostia River stations
and at one Choptank River station. In addition, growth rate and/or reproduction was reduced in the
three middle Anacostia River stations. The growth rate and reproduction endpoints did not detect
any more toxic stations in the 28-d test than the survival endpoint although they did indicate the
severity of the toxicity at the three middle river stations. The acute and chronic toxicity followed
a gradient down river in the Anacostia River with the most toxic stations occurring in the middle
river sections.
The 10-d and 28-d U.S. EPA recommended L. plumulosus test methods produced results
consistent with the 10-d and 20-d L. plumulosus methods used in the Ambient Toxicity Assessment
Program. The AMRL 10-d test showed one additional toxic station in the Anacostia River than die
WREC test but both tests indicated that AR4 was the most toxic station tested. Neither 10-d test
indicated toxicity in the Choptank River. Both chronic methods showed that all stations in the
Anacostia were toxic and that a gradient of toxicity existed with the middle river stations most toxic.
Both the WREC 28-d test and the AMRL 20-d test showed toxicity at one station in the Choptank
River. The AMRL test found slight toxicity at two other Choptank River stations although survival
at these stations was reduced by only about 15% from control survival.
Test results were similar for the L. plumulosus whole sediment tests conducted by WREC
and AMRL and the H. azteca tests conducted by AMRL as all chronic tests indicated all stations in
the Anacostia to be toxic. In contrast to the ranking from the L plumulosus tests, the H. azteca
results showed the two upstream stations to be most toxic. This difference in ranking may be a
function of the salinity adjustments necessary to conduct the estuarine organism tests using
freshwater sediments. Neither the 10-d or 20-d S. benedicti or the 10-d C variegatus sediment test?
were as sensitive as the L. plumulosus or the H. azteca tests. In contrast, the L dytiscus test detected
toxicity at every station tested based on survival and found the greatest toxicity in the Choptank
u
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River. This species appears overly sensitive to muddy sediments and has been dropped from the
Ambient Toxicity Assessment Program.
In conclusion, it appears that the L. plumulosus sediment tests conducted by the U.S. EPA
method and the Ambient Toxicity Assessment Program method give a level of sensitivity necessary
for use as a biomonitoring tool. In addition, both test methods give comparable results. Although
there is some question on the effects of salinity adjustment on toxicity in this species, the test was
still able to detect all of the stations found to be toxic to the freshwater species H. azteca. There
appears to be some difference in the ranking of stations when these two species are compared. When
used in a multi organism/multi variant data analysis procedure (Hall et al., 2000) where toxicity,
sediment chemistry, and benthic community structure are taken into account, bothL plumulosus test
methods would seem to work equally well in detecting toxic stations.
The U.S. EPA feels that its 28-d L. plumulosus method will be the standard whole sediment
estuarine toxicity test in the future since it not only measures survival and growth but also
reproduction. It has undergone a rigorous validation program including round-robin testing with
laboratories throughout the country while the Ambient Toxicity Assessment Program method was
developed and is used at only one laboratory. The U.S. EPA method is also somewhat less labor
intensive because it does not require a test breakdown in the middle of the exposure period. The
similarity between the U.S. EPA 28-d method and the Ambient Toxicity Assessment Program 20-d
method found in this study is important because it gives confidence in comparing the earlier Ambient
Toxicity Assessment Program work with other datasets generated using the U.S. EPA method in
Chesapeake Bay and throughout the East Coast of North America.
111
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TABLE OF CONTENTS
Foreword i
Abstract ii
Table of Contents iv
List of Tables v
Introduction 1
Materials and Methods 2
Sample Stations 2
Sample Collection, Handling, and Storage 2
Sediment Toxicity Tests 2
Data Analysis 3
Results and Discussion 3
Water Quality 3
Reference Toxicant Test 4
Leptocheirus plumulosus Sediment Toxicity Test Results 4
Leptocheirus plumulosus Acute vs. Chronic Toxicity 5
Station Ranks according to Leptocheirus plumulosus Sediment Toxicity 5
Comparison of U.S. EPA Leptocheirus plumulosus Test Results with Ambient
Toxicity Test Program Leptocheirus plumulosus Test Results 5
Comparison of U.S. EPA Leptocheirus plumulosus Test Results with the Other
Ambient Toxicity Test Program Sediment Test Results 6
References 8
Tables 10
iv
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LIST OF TABLES
Table 1 Test conditions for 10-d acute sediment toxicity tests with
Leptocheirus plumulosus 10
Table 2 Test conditions for 28-d chronic sediment toxicity tests with
Leptocheirus plumulosus 11
Table 3 Test conditions for reference toxicity tests with Leptocheirus plumulosus 12
Table 4 Water quality summary for the 10-d acute Leptocheirus plumulosus
sediment toxicity test 13
Table 5 Water quality summary for the 28-d chronic Leptocheirus plumulosus
sediment toxicity test 14
Table 6 Summary of the toxicity test results for 10-d and 28-d Leptocheirus
plumulosus sediment tests conducted at the University of Maryland's
Wye Research and Education Center 15
Table 7 Sediment test toxicity data for the Anacostia River sample stations for tests
conducted at the University of Maryland's Wye Research and
Education Center 16
Table 8 Sediment test toxicity data for the Choptank River sample stations for tests
conducted at the University of Maryland's Wye Research and
Education Center 17
Table 9
Table 10
Comparison of endpoints between Leptocheirus plumulosus toxicity tests
conducted at the University of Maryland's Wye Research and Education
Center and Old Dominion University's Applied Marine Research
Laboratory on samples from the Anacostia River and the Choptank River...
..18
Comparison of sediment toxicity at each station in the Anacostia and
Choptank Rivers versus test method conducted at the University of Maryland's
Wye Research and Education Center (WREC) and Old Dominion
University's Applied Marine Research Laboratory (AMRL) 19
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INTRODUCTION
In 1989, research was initiated to develop a chronic sediment toxicity test with benthic
amphipods indigenous to Chesapeake Bay. This research, sponsored in part by the U.S. EPA
Chesapeake Bay Program and the Office of Science and Technology, culminated in the development
of a draft method for assessing the chronic toxicity of marine and estuarine sediments using the
amphipod Leptocheirus plumulosus (DeWitt et al., 1992). This chronic test measures survival,
growth and reproduction after a 28-d exposure to whole sediments. Currently, the U.S. EPA is in
the process of finalizing protocols for this 28-d test (DeWitt et al., 1996; U.S. EPA, 1998). Few
standardized chronic test methods are available for estuarine and marine sediments, hence, it is
anticipated that the 28-d chronic test with L. plumulosus will have broad applicability in the
environmental testing realm. Already, it has been, and will be, applied in several studies of sediment
contamination both within and outside of the Chesapeake Bay (Emery et al., 1996; McGee and
Fisher, 1999; McGee et al., 1993, Scott et al., 1996).
The 1994 Chesapeake Bay Basinwide Toxics Reduction and Prevention Strategy directs the
Chesapeake Bay Program Signatories to: "Support and conduct the necessary biological and
chemical assessments, including ambient toxicity and community structure, of Bay habitats to ensure
future characterization of all tidal Bay habitats through the Regions of Concern identification
protocol." The current Ambient Toxicity Assessment Program (1990-1999) has generated data
critical for defining geographic areas with existing toxics problems and providing new information
for important living resources habitats where no, or limited, data existed. The sediment toxicity
methods currently used in this program include: 10-d estuarine sheepshead minnow Cyprinodon
variegatus embryo/larval test; 20-d estuarine amphipod Leptocheirus plumulosus survival and
growth test (with 10-d survival data); 20-d freshwater amphipod Hyalella azteca survival and growth
test (with 10-d survival data); 20-d estuarine amphipod Lepidactylus dytiscus survival and growth
test; and 20-d estuarine polychaete worm, Streblospio benedicti survival and growth test (Hall et al..
1994). One weakness of the existing program is that the sediment test methods used in this effort
have not been rigorously evaluated or validated. For example, the 20-d L. plumulosus survival and
growth test method differs significantly from the 28-d chronic test method developed by the U.S.
EPA for the same species. While the U.S. EPA method has undergone a rigorous validation program
including round-robin testing with laboratories throughout the country, the Ambient Toxicity
Assessment Program method was developed and is used at only one laboratory.
The objective of this report is to compare the U.S. EPA approved 10-d acute and 28-d
chronic sediment test with L. plumulosus with sediment toxicity methods used in the Ambient
Toxicity Assessment Program. The sediment tests were conducted in conjunction with the current
Ambient Toxicity Assessment Program using split samples. The incorporation of the two tests into
the current repertoire of tests will broaden the utility of the existing Chesapeake Bay Ambient
Toxicity database in several ways. First, scientist will have the ability to directly compare toxicity
data collected in the Ambient Toxicity Program with other studies conducted in Chesapeake Bay and
throughout the nation using the same species and test method. Second, it will be advantageous to
compare existing methodologies used in the Ambient Program to a U. S. EPA-standardized approach
that has undergone rigorous scientific evaluation and field validation. Finally, some evidence
suggests that the reproductive endpoint in the 28-d test may be more sensitive than the survival and
growth endpoints currently in use.
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MATERIALS AND METHODS
Sample Stations
Ten sampling stations were selected for the 1998 portion of the ongoing Ambient Toxicity
Assessment Program. Six stations were located in the Anacostia River (AR1-AR6) in Washington,
B.C. and four were in the Choptank River (CR59, CR61, CR62, and CR63) on the Eastern Shore
of Maryland. The Choptank River was selected for ambient toxicity testing because it is an
ecologically important eastern shore river that has not been tested previously in the ambient toxicity
program. Stations selected for testing were located in areas that were also tested by NOAA in their
Ambient monitoring program in Chesapeake Bay in 1998. The Anacostia River was selected for
ambient toxicity testing because this is the only "Region of Concern" identified by the Chesapeake
Bay's Toxic Subcommittee where ambient toxicity data collected from the Ambient Toxicity
Assessment Program are lacking. Station depth varied from eight to fifteen feet in the Anacostia and
sixteen to twenty-five feet in the Choptank. The Anacostia overlying water was fresh while the
Choptank had a salinity of approximately 10%o. The coordinates of the stations are as follows: AR1
(N38 55.159 x W76 56.460), AR2 (N38 54.856 x W76 57.213), AR3 (N38 52.478 x W76 58.999),
AR4 (N38 52.263 x W77 00.353), AR5 (N38 51.775 x W77 00.458), AR6 (N38 51.402 x W77
01.276), CR59(N38 43.879 xW76 15.050), CR61 (N3841.262xW76 16.713), CR62(N38 39.785
x W76 13.845), and CR63 (N38 35.889 x W76 07.515).
Sample Collection, Handling, and Storage
General sediment collection, handling, and storage procedures described in Hall et al. (1991)
were used in this study. Samples were collected at each station by Old Dominion University's
Applied Marine Research Laboratory (AMRL) personnel and returned to the laboratory for testing.
Sediments were collected September 23-24, 1998, by petite ponar grab. The top two centimeters
from each grab was used for toxicity testing. Enough sediment was collected at each field replicate
site at each station location to supply both AMRL and the WREC for the various sediment toxicity
tests. True field replicates were maintained separately and transported to both laboratories.
Sediment was collected at each station by first randomly identifying 5 grab sample sites within a 100
meter square grid. At each sample site a discrete field replicate was collected for bioassays and
stored on ice. All samples were transported on ice in coolers, out of direct sunlight. Bioassay
samples were held in refrigerators at 4°C until initiation of the toxicity tests.
Sediment Toxicity Tests
The 10-d static acute test withL plwnulosus used the current U.S. EPA method (U.S. EPA,
1994) with mortality as the endpoint. The chronic sediment toxicity test with this species consisted
of a 28-d static-renewal exposure with mortality, growth and reproduction as the endpoints. The
method followed the most recent U.S. EPA chronic draft method (U.S. EPA, 1998). Summaries
for each test method are contained in Tables 1 and 2. The acute and chronic tests were conducted
September 30 - October 9,1998 and September 29 - October 27,1998, respectively. Test organisms
were obtained from existing cultures at the WREC laboratory by size sorting amphipods through a
600 vm onto a 250 /^m sieve for the chronic test and 710 yum onto a 500 /^m sieve for the acute test.
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Test sediments were press-sieved through a stainless steel 250 /u.m mesh prior to testing to remove
live organisms, particularly indigenous L. plumulosus (U.S. EPA/U.S. ACE, 1994) and facilitate
recovery of neonates (the reproductive endpoint) at the end of the chronic test. Existing ambient
sediment test protocols also require sieving of sediments prior to testing, but only to 500 //m (Hall
et al., 1991). Control sediments, collected from a site in the Magothy River, were also sieved to 250
(Um. These silty-clay sediments are used to culture the amphipods. Overlying water was Wye River
water, filtered to 1 //m and adjusted as necessary with Hawaiian Marine Mix® to 17 to 20%o.
A 96-h water-only reference toxicity test using aqueous cadmium was performed on the same
batch of organisms used in the sediment tests. This is the reference test method used in thelatest
U.S. EPA draft methods and WREC laboratory Standard Operating Procedure (DeWitt et al., 1996;
McGee and Fisher, 1998). A summary of the method is presented in Table 3. Test conditions were
those used routinely by the WREC for reference toxicity tests with this species. The trimmed
Spearman-Karber method was be used to calculate the median lethal concentration (LC50). The
LC50 of an acceptable test must fall within the 2 standard deviation range for the control chart
generated at the WREC laboratory.
The sediment toxicity methods currently used in the Ambient Toxicity Assessment Program
include: 10-d estuarine sheepshead minnow Cyprinodon variegatus embryo/larval test; 20-d
estuarine amphipod Leptocheirusplumulosus survival and growth test (with 10-d survival data); 20-d
freshwater amphipod Hyalella azteca survival and growth test (with 10-d survival data); 20-d
estuarine amphipod Lepidactylus dytiscus survival and growth test (with 10-d survival data); and
20-d estuarine polychaete worm Streblospio benedicti survival and growth test (with 10-d survival
data). Culture, maintenance, and test procedures used for S. benedicti and L. dytiscus are described
in Hall et al. (1991); H. azteca are described in Hall et al. (1992); Cyprinodon variegatus eggs and
L. plumulosus are described hi Hall et al. (1994). These tests were conducted by Old Dominion
University's Applied Marine Research Laboratory (AMRL) on samples split with the WREC.
Data Analysis
Statistical procedures for the analysis of sediment toxicity test data are presented in U. S. EPA
(1994) and U.S. EPA/ACE (1994). The data were analyzed using the statistical package SigmaStat®
2.03 by SPSS, Inc. Data were assessed for normality and homogeneity of variance using the
Kolmogorov-Smirnov test and Levene's Median test, respectively. Survival data were Arc Sine
Square Root transformed prior to analysis. All data met the normality and homogeneity of variance
assumptions. Data were then analyzed via ANOVA followed by comparisons between test
sediments and the control using Fisher's LSD test. The 10-d and 28-d L. plumulosus test endpoints
were compared with other sediment bioassay endpoints obtained by AMRL by assessing the relative
agreement in the toxicity ranking of the sediments.
RESULTS AND DISCUSSION
Water Quality
Measurements for water quality during the acute test are given in Table 4 while those for the
chronic tests are given in Table 5. Overlying ammonia was low in all test beakers, with the highest
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recorded values of 6.0 mg/L from Stations AR2 and AR4 in the acute test and 8.0 mg/L from Station
AR4 in the chronic test. These values are well below the level of 60 mg/L that would be considered
to be a problem by the U.S. EPA (U.S. EPA, 1998). Values for pH, salinity and dissolved oxygen
were within acceptable U.S. EPA ranges (U.S. EPA, 1998).
Reference Toxicant Test
The cadmium chloride reference toxicity test run in conjunction with these sediment tests
resulted in a 96-h LC50 of 0.37 mg/L as cadmium. This value falls within the acceptable range (±
2 standard deviations) for cadmium reference toxicity tests conducted at the WREC laboratory (0.28
to 0.39 mg/L as cadmium).
Leptocheirus plumulosus Sediment Toxicity Test Results
The toxicity of the sediments from the Anacostia River was much greater than that from the
Choptank River (Table 6). L. plumulosus survival in all of the Choptank River sediments in the
acute test was greater than 96.0%. Control amphipod survival was 98.0%. In contrast, amphipod
survival in sediments from four of the six stations in the Anacostia during the acute test was
significantly reduced (AR1, AR3, AR4, and AR5) with the greatest reduction in survival occurring
at Station AR4 (6.5% survival). Stations AR1 and AR3 showed only minimal reductions in survival.
Actual individual replicate data for both the acute and chronic tests at each station can be found in
Table 7 (Anacostia) and Table 8 (Choptank).
L. plumulosus survival was significantly reduced in the chronic test at all of the Anacostia
River stations and in Station CR63 in the Choptank River (Table 6). The worst stations appeared
to be AR3, AR4, and AR5, with AR4 being the worst overall with only 11.0 % survival. The
reduction in survival at the other Anacostia stations and at Station CR63 in the Choptank was not
as dramatic as that in AR3 and AR4.
Control amphipod survival met the performance quality assurance criteria for a valid test in
the acute test (>90%) and the chronic test (>80%) with the exception of one replicate of the chronic
test. There was a serious problem in this replicate. At the midpoint in the test this treatment
developed a thick black mat covering the entire surface and all indications of amphipod
activity/burrows disappeared. No amphipods were alive at the end of the 28-d test period. Since the
other control replicates had high survival and were relatively consistent, it was felt that this treatment
was an anomaly and it was not included in the statistical analyses. Since this study, our laboratory
has conducted experiments which show that light intensity must be kept at between 300 to 500 lux
directly over the entire experimental unit in order to avoid this problem. This appears to have solved
the problem with the occasional formation of these thick mats which are believed to be formed by
a fungus. No problems have been experienced in ten chronic tests since the light intensity was
increased. Previously, this light intensity had been recommended by the U.S. EPA but in the final
draft of the method the light intensity will be emphasized and required.
The control treatment for the chronic tests met the performance quality assurance criteria of
measurable growth and reproduction for the sublethal endpoints. Reproduction was low although
it was measurable. A very small amphipod was used to start this chronic test (250 to 350 //m and
0.036 mg dry weight). It is possible that there was not enough time for an organism of this size to
achieve an optimum reproductive state. In recent tests, larger amphipods (400 to 500 //m) have been
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used to start the test and neonate production in surviving control organisms has been in the 3.0 to
5.0 neonates/organism range.
Analysis of the sublethal endpoints in the chronic test showed a significant reduction in
growth rate at Stations AR4 and AR5 of the Anacostia River but no reduction in growth rate at any
of the Choptank River stations (Table 6). The number of neonates per survivor was reduced at
Stations AR3, AR4, and AR5 in the Anacostia River but there was no reduction in neonates per
survivor at any of the Choptank River stations (Table 6).
Leptocheirus plumulosus Acute vs. Chronic Toxicity
The 10-d acute toxicity test with this amphipod showed four of the six Anacostia River
stations to be toxic but none of the Choptank River stations. The greatest toxicity was found at
stations AR4 and AR5 with only minor toxicity at Stations AR1 and AR3. The 28-d chronic toxicity
test was more sensitive, indicating toxicity at all of the Anacostia River stations and at Station CR63
on the Choptank River. The additional endpoints of growth and reproduction did not detect any-
more toxic stations in the 28-d test than the survival endpoint although these endpoints did indicate
the severity of the toxicity at stations AR3, AR4, and AR5.
Station Ranks according to Leptocheirus plumulosus Sediment Toxicity
The toxicity data allows the stations to be ranked according to severity of toxicity. The
toxicity follows a gradient down river in the Anacostia River. The most toxic stations occurred in
the mid river sections with most of the endpoints showing reductions from control values at Stations
AR3, AR4, and AR5. Survival, growth and reproduction were severely curtailed at AR4 and AR5
and survival and reproduction were reduced at AR3. Only survival was reduced at Stations AR1.
AR2 and AR6 and toxicity seemed to be decreasing at Station AR6 where the Anacostia River meets
the Potomac River. Since only Station CR63 caused toxicity in the Choptank River a ranking
gradient could not be established for this river system.
Comparison of U.S. EPA Leptocheirus plumulosus Test Results -with Ambient Toxicity Test Program
Leptocheirus plumulosus Test Results
The 10-d and 28-d U.S. EPA recommended L. plumulosus test methods conducted by the
WREC produced results consistent with the 10-d and 20-d L. plumulosus methods used by ODU-
AMRL in the Ambient Toxicity Assessment Program (Table 9). These data were provided by
AMRL for use in these comparisons and will be published in their entirety in Hall et al. (2000). The
AMRL 10-d*test showed one additional toxic station in the Anacostia River than the WREC test
(AR2). Both tests indicated that AR4 was the most toxic station tested. Neither 10-d test indicated
toxicity at any of the Choptank River stations.
Both chronic test methods showed that all stations in the Anacostia were toxic to I.
plumulosus. The AMRL 20-d test indicated toxic effects on survival but not growth at these stations-
Stations AR3 and AR4 were found to be most toxic. The WREC 28-d tests showed the most toxic
stations to be AR3, AR4, and AR5 but this test showed severity of toxicity by effects on both growth
and reproduction at Stations AR4 and AR5 and reproduction at Station AR3. Thus, both test
methods delineated a gradient with the most severe toxicity occurring in the middle portion of the
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river. The U.S. EPA method was able to detect differences in the sublethal endpoints of growth and
reproduction at these severely toxic stations although these endpoints were not able to detect any
additional toxic stations since all were detected using the long term chronic survival endpoint.
Both the WREC U.S. EPA 28-d test method and the AMRL 20-d test method showed
toxicity at Station CR63 in the Choptank River. The WREC method indicated toxic effects on
survival but not on the sublethal endpoints while the AMRL data showed toxic effects on growth
(length) but not survival. The AMRL method indicated toxicity at CR59 and CR62 that the WREC
method did not detect. These effects were on survival, not growth. Their statistical procedure was
able to detect a 16.2% reduction from control survival at Station CR 59 and a 13.2% reduction at
Station CR62 as significantly different. It is uncertain what the ecological relevance of such small
reductions in L. plumulosus survival in a long term test would be, although this effect would have
to be considered only slightly toxic.
There were a couple of differences in the test designs that may have played a factor in the
slight differences seen between the two methods. The major difference in the 10-d test methods is
that the amphipods were fed in the AMRL method but not in the U.S. EPA method. Another
difference is that the AMRL method requires sieving the test sediments to 500 //m while the U.S.
EPA method requires sieving to 250 ^m to facilitate neonate collection at the end of the test. This
may have had an effect on the chemical composition of the final test sediments but the similar test
results indicate otherwise.
In conclusion, the two methods produced comparable toxicity results for these split
environmental samples. Rankings of toxicity severity from the two methods were also similar.
There are a couple of advantages apparent in the U.S. EPA chronic method. First, the method gives
a direct measure of effects on reproduction which, in some cases, may prove a more sensitive
indicator of environmental effects and would lend itself to use in population modeling. In addition,
the U.S. EPA 28-d method is less labor intensive although it does involve counting neonates and an
additional eight days of testing. In the 20-d AMRL method there are two separate test breakdowns
after day 10 and day 20. Test breakdown is a labor intensive part of any toxicity test. At the 10-d
breakdown the amphipods have to be sieved, counted, and transferred back into the test sediment
for an additional 10-day s. Extra amphipod handling and manipulation of the sediment during this
10-d breakdown could also cause problems with this test.
Comparison of U.S. EPA Leptocheirus plumulosus Test Results with Other Ambient Toxicity Test
Program Sediment Test Results
Tests results were similar for the L. plumulosus whole sediment tests conducted by WREC
and AMRL and the Hyalella azteca tests conducted by AMRL (Table 10). These data were provided
by AMRL for use in these comparisons and will be published in their entirety in Hall et al. (2000).
The H. azteca test indicated all stations hi the Anacostia River to be toxic both in the 10-d and 20-d
test using survival as the endpoint and in the 20-d test using growth as the endpoint. Thus, the
chronic tests for both species yielded similar results. The ranking of toxicity is somewhat different
though. The H. azteca results showed Stations AR1 and AR2 were most toxic followed by AR5,
AR3, AR4, and AR6. The down river gradient seen in the L plumulosus tests was not seen here,
although toxicity at the mid stations was still significant based on survival and growth. This
difference in ranking may be a function of the salinity adjustment necessary for the freshwater
sediments of the Anacostia in order to test L. plumulosus and the other estuarine species. Salinity
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differences have been shown to have an effect on toxicity, especially with regards to metals (Hall
and Anderson, 1995). The major similarity between the test methods is that all stations were
detected as toxic by both the L plumulosus and the H. azteca test methods.
The toxicity results from the other AMRL test species are not as similar as the L. plumulosus
and H. azteca results (Table 10). These data were provided by AMRL for use in these comparisons
and will be published in their entirety in Hall et al. (2000). Neither the 10-d or 20-d Streblospio
benedicti or the 10-d Cyprinodon variegatus sediment tests were as sensitive as the L. plumulosus
or the H. azteca tests. The S. benedicti 20-d test indicated that Stations AR4 and AR5 were toxic
as did these other two tests but this test only showed one other station as toxic (AR2). There were
no growth effects with this species. The C. variegatus test did not show any of the Anacostia
stations as toxic but did pick up slight toxic hits at CR62 and CR63. This fish test was much less
sensitive to the sediments from the Anacostia River than the invertebrate species. Past performance
of this species in the Ambient Toxicity Assessment Program has shown it to be moderately sensitive
in other river systems so its insensitivity to the Anacostia sediments was surprising. The
Lepidactylus dytiscus test had the opposite problem. It detected toxicity at every station tested based
on survival. No effects were found on growth. It did show severe toxicity at Stations AR4 and AR5
in the Anacostia River similar to the L. plumulosus and the H. azteca tests but it also showed severe
toxic effects at CR59 (9% survival) and CR63 (5% survival)(Hall et al., 2000). In fact, this test
indicated that these two stations were more toxic than any of the stations in the Anacostia River.
This species appears overly sensitive to muddy sediments. In fact, in the ARML mud reference
sediment this species had only 12% survival in the 20-d test. Lepidactylus dytiscus has recently-
been dropped from the suite of organisms used in the Ambient Toxicity Assessment Program.
In conclusion, it appears that the L. plumulosus sediment test conducted by either the U.S.
EPA method or the Ambient Toxicity Assessment Program method gives a level of sensitivity
necessary for use as a biomonitoring tool. Although there is some question on the effects of salinity
adjustment on toxicity in this species, the test was still able to detect all of the stations found to be
toxic to the freshwater species H. azteca. There appears to be some difference hi the ranking of
stations when toxicity to these two species is compared. When used in a multi organism/multi
variant data analysis procedure (Hall et al., 2000) where toxicity, sediment chemistry, and benthic
community structure are taken into account, both L. plumulosus test methods would seem to work
equally well in detecting toxic stations.
-------
REFERENCES
DeWitt, T.H., L.A. Niewolny, S.L. Nieukirk, B. Gruendell, W. Gardiner, and A. Borde. 1996.
Support for Development of a Standard Chronic Sediment Toxicity Protocol with the
Estuarine Amphipod, Leptocheirus plumulosus. Draft Final Report prepared for U.S.
Environmental Protection Agency, Office of Science and Technology, Washington- DC,
under contract 68-C2-0134 by Battelle Marine Sciences Laboratory, Battelle Memorial
Institute, Pacific Northwest Division, Richland, Washington.
DeWitt, T.H., M.S. Redmond, I.E. Sewall, and R.C. Swartz. 1992. Development of a Chronic
Sediment Toxicity Test for Marine Benthic Amphipods. CBP/TRS 89/93. U.S. EPA
Chesapeake Bay Program, Annapolis, MD.
Emery, V.L., Jr., R.B. Wright, J.D. Farrar, and D.W. Moore. 1996. The Use of Chronic Sublethal
Sediment Bioassays to Determine the Spatial Distribution of Toxicity in the Gunpowder
River, MD. 17th Annual Meeting of the Society of Environmental Toxicology and
Chemistry, Washington, D.C.
Hall, L.W., Jr. and R.D. Anderson. 1995. The Influence of Salinity on the Toxicity of Various
Classes of Chemicals to Aquatic Biota. Critical Reviews in Toxicol. 25:281-346.
Hall, L.W., Jr., R.D. Anderson, A. Messing, J. Winfield, A.K. Jenkins, I.J. Weber, R.W. Alden, D.
Goshorn and M. McGinty. 2000. Ambient Toxicity Testing in Chesapeake Bay. Year 8
Report. U.S. Environmental Protection Agency, Chesapeake Bay Program Office-
Annapolis, MD.
Hall, L.W., Jr., M.C. Ziegenfuss, R.D. Anderson, W.D. Killen, R.W. Alden, and P. Adolphson.
1994. A Pilot Study for Ambient Toxicity Testing in Chesapeake Bay. Year 3 Report
CBP/TRS 116/94. U.S. EPA Chesapeake Bay Program, Annapolis, MD.
Hall, L.W., Jr., M.C. Ziegenfuss, S.A. Fischer, R.W. Alden, III, E. Deaver, J. GoochandN. Debert-
Hastings. 1991. A Pilot Study for Ambient Toxicity Testing in Chesapeake Bay. Volume
1 - Year 1 Report CBP/TRS 64/91. U.S. EPA Chesapeake Bay Program, Annapolis, MD.
Hall, L.W. Jr., M.C. Ziegenfuss, S.A. Fischer, R.D. Anderson, W.D. Killen, R.W. Alden, HI E.
Deaver, J. Gooch and N. Shaw. 1992. A Pilot Study for Ambient Toxicity Testing in
Chesapeake Bay - Year 2 report. CBP/TRS 82/92. U.S. Environmental Protection Agency,
Chesapeake Bay Program Office, Annapolis, MD.
McGee, B.L. and D.J. Fisher. 1998. Culturing and Testing Protocols for Conducting Sediment
Toxicity Tests with Freshwater and Estuarine Amphipods. Standard Operating Procedure,
University of Maryland at College Park, Wye Research and Education Center, Queensto\m.
MD.
McGee, B.L. and D.J. Fisher. 1999. Field Validation of the Chronic Sediment Bioassay with die
Estuarine Amphipod Leptocheirus plumulosus in Chesapeake Bay. Final Report. U.S.
Environmental Protection Agency, Office of Science and Technology, Washington. D.C.
McGee, B.L., C.E. Schlekat and E. Reinharz. 1993. Assessing Sublethal Levels of Sediment
Contamination Using the Estuarine Amphipod Leptocheirus plumulosus. Environ, ToxicoL
Chem. 12:577-587.
Scott, K.J., C. Mueller and W. Starkel. 1996. Chronic Response of Leptocheirus plumulosus in
28-d Exposures to Contaminated Estuarine Sediments. 17th Annual Meeting of the Society
of Environmental Toxicology and Chemistry, Washington, D.C.
-------
U.S. EPA. 1994. Methods for Assessing the Toxicity of Sediment-associated Contaminants with
Estuarine and Marine Amphipods. EPA/600/R-94/025. Office of Research and
Development, Washington, D.C.
U.S. EPA. 1998. Draft Method for Assessing the Chronic Toxicity of Sediment-associated
Contaminants with Leptocheirus plumulosus. First Edition. Office of Research and
Development, Washington, D.C.
U.S. EPA/U.S. ACE. 1994. Evaluation of Dredged Material Proposed for Discharge in Waters of
the U.S. - Testing Manual (Draft). Washington, D.C.
9
-------
Table 1. Test conditions for 10-d acute sediment toxicity tests with Leptocheirus plumulosus.
1. Test type Whole sediment, static
2. Temperature 25 °C
3. Overlying water Filtered Wye River water adjusted as necessary with well
water or Hawaiian Marine Mix® to 17 to 20%o
4. Light Ambient laboratory
5. Photoperiod 16:8 (L/D)
6. Test chamber 1 L glass beaker covered with watchglass
7. Sediment volume 175 ml (2 cm)
8. Overlying water volume 800ml
9. Water renewal None
10. Size and life stage of amphipods 2-4 mm, sub-adults
11. Number of organisms/replicate 20
12. Number of replicates 5
13. Feeding None
14. Aeration 1-2 bubbles/sec with 1 ml pipette
15. Water quality Salinity, pH and total ammonia at beginning and end of
test; Temperature and D.O. daily
16. Test duration 10 d
17. Endpoint Survival
4.
18. Performance criteria Control survival > 90%
10
-------
Table 2. Test conditions for 28-d chronic sediment toxicity tests with Leptocheirusplumulosus.
1. Test type Whole sediment, static renewal
2. Temperature 25 °C
3. Overlying water
4. Light
5. Photoperiod
6. Test chamber
7. Sediment volume
8. Overlying water volume
9. Water renewal
Filtered Wye River water adjusted as necessary with well
water or Hawaiian Marine Mix® to 17 to 20%o
Ambient laboratory
16:8 (L/D)
1 L glass beaker covered with watch glass
175 ml (2 cm)
800ml
3 x /week, replace 400 ml
10. Size and life stage of amphipods neonates; size sorted on nested 250 and 500 //m mesh
sieves
11. Number of organisms/replicate
12. Number of replicates
13. Feeding
14. Aeration
15. Water quality
16. Test duration
17. Endpoints
18. Performance criteria
20
TetraMin 3x/week
1-2 bubbles/sec with 1 ml pipette
Salinity, pH and total ammonia at beginning and end of
test; Temperature and D.O. daily
28 d
Survival, growth, reproduction
Control survival > 80%
Measurable growth and reproduction
11
-------
Table 3. Test conditions for reference toxicity tests with Leptocheirus plumulosus.
1. Test type Aqueous, static
2. Temperature 25 °C
3. Toxicant Cadmium
4. Diluent water
5. Light
6. Photoperiod
7. Test chamber
8. Water volume
Filtered Wye River water adjusted as necessary with well
water or Hawaiian Marine Mix® to 8%o.
Ambient laboratory
16:8 (L/D)
300 ml glass beaker
200ml
9. Size and life stage of amphipods 2-4 mm, sub-adults
10. Number of organisms/replicate 10
11. Minimum number of replicates 2
12. Feeding None
13. Substrate None
14. Aeration
15. Water quality
16. Duration
17. Endpoint^
18. Test acceptability
None
D.O., salinity, pH, hardness and alkalinity at the beginning
and end of test; Temperature daily
96 h
Survival
90 % control survival
12
-------
Table 4. Water quality summary for the 10-d acute Leptocheirus plumulosus sediment toxicity test.
Test Type / Date
Treatment
Acute: 9/30-10/9
Control
AR1
AR2
AR3
AR4
AR5
AR6
CR59
CR61
CR62
CR63
Temperature
(°Q
Mean
24.5
24.4
24.5
24.5
24.4
24.4
24.4
24.5
25.1
25.1
25.1
SD
0.
1.
1.
1.
1.
1.
0.
1.
89
11
05
03
04
04
96
03
0.26
0.
0.
23
30
DO (mg/L)
Mean
6.8
6.8
6.7
6.6
6.7
6.6
6.8
6.7
6.7
6.7
6.7
SD
0.28
0.32
0.35
0.47
0.39
0.56
0.34
0.47
0.42
0.49
0.37
pH
Range
7.88
6.91
6.65
6.97
7.51
6.79
7.55
7.84
7.97
7.85
7.66
-8.37
-8.44
-8.27
-8.27
-8.04
-8.08
-7.86
-8.44
-8.55
-8.53
-8.51
Salinity (%o)
Mean
17.3
17.5
17.0
17.1
17.0
17.2
17.0
17.8
18.3
18.3
18.8
SD
0.55
0.55
0.84
0.45
0.45
0.55
1.64
1.48
1.34
1.41
0.89
Ammonia
Overlying
Range
O.I
<0.1
O.I
0.1
1.7
2.7
3.2
O.I
0.1
O.I
0.1
(mg/L)
-0.6
-6.0
-6.2
-4.6
-8.0
-4.8
-5.8
-5.0
-2.1
-6.2
-4.0
-------
Table 5. Water quality summary for the 28-d chronic Leptocheirusplumulosus sediment toxicity test.
Test Type / Date
Treatment
Chronic: 9/29-10/27
Control
AR1
AR2
AR3
AR4
AR5
AR6
CR59
CR61
CR62
CR63
Temperature
Mean
24
.7
24.7
24
24
24
24
24
24
24
24
24
.5
.7
.7
.7
.6
.5
.6
.6
.6
SD
0.63
0.54
0.97
0.62
0.60
0.65
0.66
0.82
0.63
0.64
0.61
DO (mg/L)
Mean
6.8
6.8
6.6
6.7
6.7
6.6
6.6
6.8
6.6
6.8
6.7
SD
0.38
0.37
0.49
0.33
0.42
0.37
0.38
0.23
0.46
0.37
0.32
pH
Range
7.28
6.89
7.34
7.43
6.63
7.63
7.66
7.21
7.96
7.83
7.61
-8.40
-8.14
-8.24
-8.18
-8.29
-8.11
-8.16
-8.10
-8.46
-8.55
-8.22
Salinity (%o)
Mean
18.0
17.8
18.0
18.0
17.8
17.8
18.1
18.7
18.8
18.6
18.5
SD
1.78
1.74
1.56
1.72
1.61
1.62
1.73
2.13
1.51
1.43
1.39
Ammonia
Overlying
Range (mg/L)
<0. 1 - 0
<0.1 -6
<0.1 -6
<0. 1 - 3
<0.1 -6
<0.1 - 3
.7
.0
.0
.2
.0
.0
<0. 1-5.4
0.1-2
0.1-3
O.I -3
O.I -3
.8
.0
.0
.2
-------
Table 6. Summary of the toxicity test results for 10-d and 28-d Leptocheirus plumulosus
sediment tests conducted at the University of Maryland's Wye Research and Education
Center.
Station
Control
AR1
AR2
AR3
AR4
AR5
AR6
CR59
CR61
CR62
CR63
1 0-d survival
(%)
Mean
98.0
75.0*
87.0
79.0*
6.5*
40.0*
96.0
96.0
98.0
98.0
97.0
SD
2.74
20.9
18.9
26.6
7.78
15.40
4.18
6.52
4.47
2.74
4.47
28-d survival
(%)
Mean
93.8
65.0*
55.0*
45.0*
11.0*
55.0*
57.0*
75.0
93.0
95.0
57.0*
SD
6.29
33.00
17.00
7.07
14.30
20.30
23.60
31.20
15.70
8.66
21.10
28-d growth rate
Mean
0.032
0.029
0.029
0.027
0.006*
0.020*
0.030
0.034
0.035
0.028
0.029
SD
0.0036
0.0048
0.0034
0.0030
0.0055
0.0047
0.0034
0.0041
0.0049
0.0024
0.0005
28-d neonates
per survivor
Mean
0.57
0.58
0.56
0.28*
0.03*
0.05*
0.57
0.59
0.49
0.52
0.60
SD
0.184
0.064
0.082
0.192
0.064
0.103
0.156
0.140
0.139
0.147
0.114
^Significantly less than controls (p < 0.05): ANOVA followed by Fisher's LSD
15
-------
Table 7. Sediment test toxicity data for the Anacostia River sample stations for tests conducted
at the University of Maryland's Wye Research and Education Center.
Treatment
Control
AR1
AR2
AR3
AR4
AR5
AR6
Rep
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Position1
8
7
1
13
24
14
39
48
54
21
16
34
22
35
4
12
44
33
43
51
26
38
37
2
23
6
15
46
50
31
25
11
9
3
19
10-d Acute Test
#
Alive
21
19
20
19
20
20
19
12
13
11
20
11
17
19
20
20
20
10
19
10
0
1
1
4
0
10
8
5
5
12
20
19
18
19
20
%
Alive
100
95
100
95
100
100
95
60
65
55
100
55
85
95
100
100
100
50
95
50
0
5
5
20
0
50
40
25
25
60
100
95
90
95
100
28-d Chronic Test
#
Alive
17
19
19
20
O3
19
18
15
10
3
13
7
15
12
8
8
10
10
10
7
7
2
2
0
0
11
4
13
14
13
18
7
7
14
11
%
Alive
85
95
95
100
0
95
90
75
50
15
65
35
75
60
40
40
50
50
50
35
35
10
10
0
0
55
20
65
70
65
90
35
35
70
55
Rep wt
(n\g)
0.904
1.080
0.885
0.839
0
1.079
0.793
0.847
0.719
0.880
0.743
0.807
0.858
0.979
0.895
0.903
0.819
0.820
0.764
0.680
0.344
0.290
0.315
0
0
0.596
0.455
0.652
0.512
0.791
0.772
0.930
0.960
0.906
0.758
growth
rate2
0.031
0.037
0.030
0.029
0.000
0.037
0.027
0.029
0.024
0.030
0.025
0.028
0.029
0.034
0.031
0.031
0.028
0.028
0.026
0.023
0.011
0.009
0.010
0.000
0.000
0.020
0.015
0.022
0.017
0.027
0.026
0.032
0.033
0.031
0.026
#
vounp
14
11
8
9
0
11
9
8
6
2
8
4
7
8
4
4
3
4
2
0
1
0
0
0
0
0
0
0
0
3
7
4
*>
10
8
young/'
-------
Table 8. Sediment test toxicity data for the Choptank River sample stations for tests conducted
at the University of Maryland's Wye Research and Education Center.
Treatment
Control
CR59
CR61
CR62
CR63
Rep
1
2
o
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Position1
8
7
1
13
24
10
30
32
28
45
17
29
40
42
52
18
5
55
36
49
41
27
47
53
20
10-d Acute Test
#
Alive
21
19
20
19
20
20
20
17
19
20
18
20
24
20
20
20
20
20
19
19
20
18
20
20
19
%
Alive
100
95
100
95
100
100
100
85
95
100
90
100
100
100
100
100
100
100
95
95
100
90
100
100
95
28-d Chronic Test
#
Alive
17
19
19
20
O3
11
22
18
6
20
20
21
21
20
13
20
16
19
20
20
13
14
8
6
16
%
Alive
85
95
95
100
0
55
100
90
30
100
100
100
100
100
65
100
80
95
100
100
65
70
40
30
80
Rep wt
(nig)
0.904
1.080
0.885
0.839
0
0.913
1.098
0.983
0.852
1.113
0.973
1.163
1.040
1.134
0.813
0.923
0.790
0.782
0.766
0.789
0.838
0.810
0.809
0.857
0.848
growth
rate2
0.031
0.037
0.030
0.029
0.000
0.031
0.038
0.034
0.029
0.038
0.033
0.040
0.036
0.039
0.028
0.032
0.027
0.027
0.026
0.027
0.029
0.028
0.028
0.029
0.029
#
young
14
11
8
9
0
6
11
9
5
11
10
14
6
9
7
14
10
10
8
7
9
7
6
3
9
young/
survivoi
0.824
0.579
0.421
0.450
0.000
0.545
0.500
0.500
0.833
0.550
0.500
0.667
0.286
0.450
0.538
0.700
0.625
0.526
0.400
0.350
0.692
0.500
0.750
0.500
0.563
'Tests started at same time in separate beakers but used identical randomization for position.
2Growth rate = (Rep weight - initial weight of 0.036 mg)/28.
3Replicate had massive fungal infection that appeared to kill all amphipods. Excluded from
statistical analyses.
17
-------
Table 9. Comparison of endpoints between Leptocheirus plumulosus toxicity tests conducted at the University of Maryland's Wye
Research and Education Center (WREC) and Old Dominion University's Applied Marine Research Laboratory (AMRL) on
samples from the Anacostia River and the Choptank River.
Station
Control
AR1
AR2
AR3
AR4
AR5
AR6
CR59
CR61
CR62
CR63
Acute Tests
WREC 10-d
% survival
98.01
75.0*
87.0
79.0*
6.5*
40.0*
96.0
96.0
98.0
98.0
97.0
AMRL 10-d
% survival
90.7
66.7*
56.0*
57.3*
32.0*
68.0*
82.7
90.7
90.7
92.0
89.3
Chronic Tests
WREC 28-d
% survival
93.8
65.0*
55.0*
45.0*
11.0*
55.0*
57.0*
75.0
93.0
95.0
57.0*
AMRL 20-d
% survival
90.7
45.3*
38.7*
20.0*
8.0*
38.7*
66.7*
76.0*
81.3
78.7*
82.7
WREC 28-d
Growth Rate (mg/d)
0.032
0.029
0.029
0.027
0.006*
0.020*
0.030
0.034
0.035
0.028
0.029
AMRL 20-d
Weight (mg)
0.271
0.262
0.278
0.315
0.232
0.232
0.307
0.242
0.393
0.251
0.193
Length (mm)
6.387
6.447
6.727
6.766
6.313
6.371
6.325
5.968
6.305
5.969
5.835*
WREC 28-d
Young/survivor
0.57
0.58
0.56
0.28*
0.03*
0.05*
0.57
0.59
0.49
0.52
0.60
oo
1 Values are the mean for each station.
* Station significantly different from the control station (p < 0.05)
-------
Table 10. Comparison of sediment toxicity at each station in the Anacostia and Choptank Rivers versus test method conducted at the
University of Maryland's Wye Research and Education Center (WREC) and Old Dominion University's Applied Marine
Research Laboratory (AMRL). The number in parentheses equals the station rank in each River System with 1 being the
most toxic.
Station
AR1
AR2
AR3
AR4
AR5
AR6
CR59
CR61
CR62
CR63
WREC
Leptocheirus
plumulosus
lOd
S2(3)
S(4)
S(l)
S(2)
28d
S(6)
S(4)
S,R(3)
S,G,R(1)
S,G,R (2)
S(5)
S(l)
AMRL
Leptocheirus
plumulosus
lOd
S(4)
S(2)
S(3)
S(l)
S(5)
28d
S(4)
S(3)
S(2)
S(l)
S(3)
S(5)
S(l)
S(2)
G(3)
Hyalella
azteca1
lOd
S(2)
S(l)
S(4)
S(3)
S(4)
S(5)
20d
S,G(2)
S,G(1)
S,G (4)
S,G(5)
S,G(3)
S,G(6)
Lepidactylus
dytiscus
lOd
S(3)
S(5)
S(l)
S(5)
S(2)
S(4)
S(2)
S(3)
S(l)
20d
S(4)
S(4)
S(3)
S(l)
S(2)
S(3)
S(2)
S(4)
S(3)
S(l)
Streblospio
benedicti
lOd
S(2)
S(l)
S(l)
20d
S(l)
S(l)
S(2)
Cyprinodon
variegatus
lOd
S,H(1)
S,H (2)
1 Hyalella azteca only used to test the freshwater sediments of the Anacostia River.
2 Significant effects (p < 0.05) on the various endpoints tested are indicated by letters (S = survival; G = Growth; R = Reproduction;
H = Hatching success).
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