United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
Research and Development
EPA-600/S3-81-025 June 1981
Project Summary
Development of Bioassay
Procedures for Defining
Pollution of Harbor Sediments
Donald A. Bahnick, William A. Swenson, Thomas P. Markee, Daniel J. Call,
Craig A. Anderson, and R. Ted Morris
This research investigates bioassay
methods which may be useful in
assessing the degree of pollution of
harbor sediments. Procedures studied
include 96-hr toxicity tests employing
Hexagenia limbata, Daphnia magna
and Pontoporeia affinis as biological
probes, monitoring cough frequencies
of bluegill sunfish (Lepomis macro-
chirus) in interstitial water derived
from sediments, chemical analyses of
sediment-water systems, and
chemical analysis of chironomids and
Hexagenia limbata exposed to the
sediments. Additional experiments
involved investigation of the degree of
removal of chemical constituents
from sediments due to extraction with
Lake Superior water and the use of
reverse phase liquid chromatography
in detecting the presence of chemical
compounds with high bioaccumula-
tion potential in the sediments.
Sediment-water systems, employ-
ing sediments from the Duluth-
Superior harbor and Lake Superior,
caused little toxicity in 96-hr exposure
tests with Daphnia magna, Hexagenia
limbata and Pontoporeia affinis al-
though Daphnia was the most sensi-
tive of these animals to toxicants.
Similar cough frequencies were
found for bluegill sunfish in dechlorin-
ated city water compared to sediment
interstitial water—Lake Superior
water mixtures but it was observed
that broken opercular patterns
occurred in the interstitial water-
Lake Superior water mixtures.
Most of the sediments used in the
bioassay tests would be considered
polluted according to at least one
chemical parameter. Chemical analy-
ses showed the presence of low
amounts of PCBs in the sediments.
Most of the heavy metals primarily
resided in the residual phase of the
sediments.
Studies on extracting chemical
species from the sediments showed
that only small amounts could be
readily extracted with Lake Superior
water.
Evidence was found that chirono-
mids dwelling in harbor sediments
bioaccumulated PCBs and p,p'-DDE
and possibly some metals.
A general toxicity index was pre-
pared from the chemical data which
indicated that animal survival in the
96-hr acute toxicity tests was
generally lower using sediment
systems from the more industrialized
areas of the harbor.
This Project Summary was develop-
ed by EPA's Environmental Research
Laboratory, Duluth, MN, to announce
key findings of the research project
that is fully documented in a separate
report of the same title (see Project
Report ordering information at back).
Introduction
The need for maintaining vessel
accessibility to our nation's waterways
requires continuous dredging of large
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volumes of sediment by the Corps of
Engineers. Reliable, cost-effective
procedures for identifying the potential
effects of sediment chemical contami-
nants on water quality and aquatic
communities are needed.
Current criteria used for the evalua-
tion of the quality of Great Lakes harbor
sediments are based largely on sedi-
ment chemical parameters. It is recog-
nized that a comprehensive evaluation
procedure should include short-term
bioassay tests in evaluating the toxic
effects of sediments on fish, benthos
and plankton; and also tests which will
assess the bioaccumulation potential of
sediment chemicals in flora and fauna
resulting in magnification in aquatic
food chains.
This research attempts to develop
procedures designed to assess potential
harmful effects of harbor sediments
subjected to dredging. Applicability,
ease of duplication and cost effective-
ness were taken into consideration in
procedure development.
Sediments collected during 1977 and
1978 from six sites in Duluth, Minne-
sota and Superior, Wisconsin harbor
area and one Lake Superior site were
used in preparing systems containing
water overlying a sediment substrate,
containing interstitial water, or
containing elutriate water. Acute 96-hr
toxicity tests were carried out by
exposing Hexagenia limbata, Daphnia
magna and Pontoporeia affinis to
certain of these systems compared to
exposures to identically prepared
controls.
The exposure systems consisted of
sediment, water overlying the sedi-
ment, interstitial water extracted from
the sediment under anaerobic condi-
tions, elutriate water prepared from
Lake Superior water mixed with sedi-
ment which was either exposed to air or
kept under nitrogen prior to elutri-ate
formation and Lake Superior water used
to extract water solubles or colloids
(generated pore water). During 1978,
particulate phase elutriate water was
also used as an animal exposure
system.
All bioassays were conducted in an
environmentally controlled area of the
University of Wisconsin-Superior wet
laboratory under controlled light and
temperature conditions. Oversediment
bioassays were designed to incorporate
the mechanisms for transfer of toxic
substances between sediments and
benthic or planktonic communities
using Daphnia magna and Hexagenia
limbata. Pontoporeia affinis bioassays
were used to measure acute toxicity of
liquid phase elutriate water. Ninety-six
hr toxicity tests employing Daphnia
magna exposed to sediment interstitial
water and elutriate water were also
conducted.
Bluegill sunfish (Lepomis macro-
chirus) were monitored for cough
frequencies and breathing patterns in
mixtures of Lake Superior water with
interstitial water extracted from the
various sediments. Sixteen fish of com-
parable size were used in each test. The
tests were conducted in systems
composed of electrode chambers con-
taining separated compartments. Each
compartment was equipped with stain-
less steel electrodes which detected
action potential resulting from muscu-
lar activity associated with breathing.
The action potentials were recorded bya
Gilson IMP-5H 4-channel physiograph.
The bluegill breathing patterns and
cough responses were recorded for fish
in dechlorinated city water and in Lake
Superior-sediment interstitial water
mixtures.
Sediments and interstitial waters
used in bioassay tests were extensively
analyzed for a variety of chemical
parameters. The chemical testing of
sediments included determinations of
total metals, metals associated with dif-
ferent phases of the sediments, certain
inorganic non-metal substances, parti-
cle size, pH, Eh and some trace organics
(PCBs, pesticides and PAH compounds).
Chemical tests on interstitial water
included a number of the same
chemical parameters as investigated for
sediments.
Chironomids were collected from
various harbor sites by isolating them
from sieved sediment. These chirono-
mids, along with Hexagenia limbata
exposed for 96 hrs to harbor sediments,
were analyzed to -determine body
burdens of specific organic compounds
(PCBs, PAH and pesticides) and some
heavy metals.
Using the results from the animal
toxicity tests and the chemical analyses,
attempts were made to develop a
general index of toxic potential and
chemical quality of harbor sediments in
trying to identify correlations between
sediment chemistry and animal survival
for the acute toxicity tests.
The degree of removal of chemical
constituents from sediments subjected
to mixing with Lake Superior water was
investigated by repetitive additions of
Lake Superior water to sediments,
separating the water from the sedi-
ments by high speed centrifugation and
chemical analysis of the water phases.
The total amount of each chemical
released from the sediments to the
water phase after numerous additions
and removal of water was tabulated.
The concentrations of organic chemi-
cals with high bioaccumulation poten-
tial contained in harbor sediments,
harbor chironomids, and sediment
exposed Hexagenia limbata were inves-
tigated using high pressure reverse
phase liquid chromatography. Evidence
has shown that organic chemicals with
high n-octanol/water partition coeffi-
cients tend to bioaccumulate in the
lipids of aquatic animals. The chemicals
having high partition coefficients tend
to have high retention times on a
reverse phase chromatographic
column. Organic solvent extracts from
harbor sediments, chironomids and
Hexagenia were injected into a high
pressure liquid chromatograph employ-
ing a reverse phase column. Eluted
compounds were detected by a UV
detector.
Conclusions
The acute toxicity tests using I
sediment-water systems resulted in "
generally low toxicity to Hexagenia
limbata, Daphnia magna and Ponto-
poreia affinis. The high survival of test
animals indicated low levels of available
toxicants in the sediments. In 96-hr
bioassays, survival was found to be
significantly lower for test sites com-
pared to controls in only a few tests. The
results demonstrated that the animals
could be successfully maintained in the
complex test systems and that the
sediment-water systems caused low
acute toxicity. This low toxicity in
combination with low precision
between replicates generally resulted in
finding no significant differences in
animal survival between test and
control. Among the test species
employed, Daphnia magna appeared to
be the most sensitive to toxicants.
It was necessary to design the tests
following approved criteria for ecolog-
ical evaluation of dredging and dredge
spoil disposal in marine systems.
Following these criteria, controls for the
bioassay tests were derived from sedi-
ments from Lake Superior and a rela-
tively undisturbed area of the harbor.
Because these sediments contained
varying quantities of many toxic M
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substances, their use negated accurate
determination of the sensitivity of the
bioassay procedures. It is therefore
recommended that future studies aimed
at identifying screening procedures
incorporate more effective controls.
The use of Daphnia magna,
Hexagenia limbata or Pontoporeia
affinis as test organisms for potential
toxic effects of sediments should be
considered further. Of these species,
Daphnia magna appears to be most
suited as a test organism due to ease of
culturing, sensitivity, and the large
amount of information available on the
response of Daphnia to specific chemi-
cals. Further tests would be useful
employing sediment samples having
greater variation in chemical quality.
Recognizing that sediments used in this
study contained large quantities of
potentially toxic heavy metals in
unavailable or non-toxic forms, it is
important to develop better
understanding of the conditions which
would result in transformation to
available forms and the effects that
such transformations woud have.
Comparisons of toxicity results to other
recently developed toxicant screening
techniques such as algal or luminescent
bacteria assays are recommended.
The average cough frequencies of
bluegills in dechlorinated city water
and in interstitial water from the
sampling site sediments mixed with
Lake Superior water were generally
similar. Cough frequencies during the
first 22 to 25 hrs of fish exposure were
found to be elevated above the
frequencies observed for the control for
three of the six sites studied. Bluegill
opercular activity was nearly
continuous in dechlorinated city water
in contrast to broken patterns of activity
observed in interstitial water—Lake
Superior water mixtures formed from
sediments obtained from five of the six
sampling sites.
Based on our observations, bluegill
cough frequencies are difficult to inter-
pret and their usefulness in determining
differences in sediment quality was
limited due to observed similarities in
results for the various sites. The data
suggests that extensive experience in
conducting fish cough response tests is
necessary to interpret the results, and
therefore the technique has limited
application as a general screening
procedure.
Chemical analysis for heavy metals in
he sediments revealedthat the residual
phase of the sediments contained the
highest concentrations of most metals.
However, measurable amounts (>1
mg/kg) of arsenic, cobalt, copper, nickel
and zinc were found in the organic and
sulfide sediment phases for nearly all
the samples, and selenium and
cadmium were found in these phases
for some of the samples.
PCB concentrations in the sediments
ranged from 0.3 to 2.1 mg/kg based on
a dry sediment basis. These values are
not high compared to the 10 mg/kg or
above level for polluted sediments. In
addition to PCBs, low levels of polycyclic
aromatic hydrocarbons were found in
two of the harbor sediments and low
levels (1 to 15 yug/kg) of pentachloro-
phenol were detected.
Chemical analysis of sediment inter-
stitial water showed that many of the
chemical species were probably associ-
ated with very fine (possibly colloidal)
particles in the water. The concentra-
tions of many of the metals were much
lower in filtered water samples com-
pared to non-filtered samples.
The concentrations of chemicals
found in liquid phase elutriate water did
not change greatly when prepared from
an exposed sediment compared to
sediment unexposed to air.
Studies on determining the amounts
of chemical species which would be
released upon flushing the sediments
with Lake Superior water showed that
only about one percent or less of most of
the chemical parameters (COD, Fe, Mn,
Ni, Pb, Cu, Zn, Hg) was removed from
the sediments by water extraction.
These results indicated that the
measured chemical species were not
readily available to water except when
associated with particulates.
Although survival of test organisms
was generally high during the 96-hr
toxicity tests, some correlations of
survival of Daphnia magna in water
overlying sediments (1977 tests) with
chemical parameters were found. Many
of these correlations involved the con-
centrations of metals in the sediment, in
interstitial water removed from the
sediment or in elutriate water formed by
mixing sediment with Lake Superior
water.
Some correlations were found
between Daphnia magna survival in
elutriate water—Lake Superior water
mixtures and individual chemical para-
meters. Correlations were found
between iron concentrations in inter-
stitial water removed from the sedi-
ments and Daphnia survival.
On the basis of one or more chemical
parameters, most of the sediment
samples used in the bioassay tests
would be classified as polluted
according to the currently used sedi-
ment evaluation criteria. Ranking the
sediments according to their concentra-
tions of a large number of metals,
inorganic nonmetals and organic
chemical parameters indicated that
sites located in the Superior harbor
(near the Superior entry to Lake
Superior) and a Lake Superior site were
less polluted than sites located near the
more industrialized zones in the harbor.
A general index of toxic effects,
developed by considering the relative
percentage of low survival for the
various acute toxicity tests, showed that
survival was generally lower in test
systems derived from sediments in the
industrialized areas of the harbor
compared to the less developed areas
and the Lake Superior site. Comparing
the percent low survival values for the
sites to rankings based on sediment
chemical results indicated a positive
correlation (r = 0.80, P > 0.1). A lower
correlation coefficient was found be-
tween rankings based on interstitial
water chemistry and the percent low
survival values. These results indicate
that the combined chemical test for the
sediments were a fair indicator of
general toxicity.
Further analysis of bioassay results
and chemical characteristics of the
sediments is desirable. If correlations
hold for a wide variety of sediments,
additional understanding of the causes
of the observed toxicity results may be
obtained.
Evidence was found that chironomids
dwelling in the harbor sediments had
accumulated PCBs and p,p'-DDE. Com-
pared to PCB levels in the dried sedi-
ment samples, bioaccumulation factors
of 11 to 18 times were found in the
animal tissues. Bioaccumulation
factors for p,p'-DDE in chironomids
compared to dry sediment values
ranged from 10 to 40.
For Hexagenia limbata exposed to
sediments for 96 hrs, tissue/sediment
bioaccumulation factors for PCBs were
4.8 (two harbor sediments) and 8.2
(Lake Superior sediment) while tissue/
sediment bioaccumulation factors for
p,p'-DDE were about 25 to 30.
The amounts of PCBs associated with
ingested sediment in chironomid and
Hexagenia samples were less than 5%
of the total PCBs in these animals.
« US GOVERNMENT PRINTING OFFICE'1M1-757-012/7141
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The results of bioaccumulation
studies of heavy metals in chironomids
indicate that the measured amounts of
most of the metals arose from ingested
inert material contained in their diges-
tive tracts. However, limited evidence
suggested that the observed concentra-
tions of mercury, cadmium and chrom-
ium in chironomids were due to
accumulation in tissues.
Chromatograms of organic extracts
from sediments, chironomids and
Hexagenia limbata, using reverse phase
high pressure liquid chromatography,
showed the presence of organic com-
pounds with high bioaccumulation
potential; although no attempt was
made to identify and quantitate chemi-
cals. This method of screening samples
for the amounts of bioaccumulated
organic compounds is potentially
useful.
Screening of sediment extracts or
extracts of animals exposed to sedi-
ments using reverse phase high
pressure liquid chromatography should
be further investigated. In particular,
the eluent fractions containing organic
compounds with high lipid solubility
should be studied for methods to quan-
titate and perhaps identify highly
bioaccumuable compounds.
Donald A. Bahnick, William A. Swenson, Thomas P. Markee, DanielJ. Call, Craig
A. Anderson, andR. Ted Morris are with the University of Wisconsin, Superior,
Wl 54880.
Richard Anderson is the EPA Project Officer (see below).
The complete report, entitled"Development ofBioassay Procedures for Defining
Pollution of Harbor Sediments," (Order No. PB 81-178 261; Cost: $15.50,
subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
Duluth. MN 55804
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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Fees Paid
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Protection
Agency
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Official Business
Penalty for Private Use $300
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