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SEDIMENT BIOASSAY RESEARCH
AND DEVELOPMENT

R. A. C. PROJECT NO. PDF 03

Environment
Ontario

ISBN 0-7729-7147-1

SEDIMENT BIOASSAY RESEARCH
AND DEVELOPMENT

A. C. PROJECT NO. PDF 03

Prepared for Environment Ontario by:

Gail Krantzberg
Sediment Specialist
Water Resources Branch

OCTOBER 1990
JfK

u

Copyright: Queen's Printer for Ontario, 1990
This publication may be reproduced
for non-commercial purposes with
appropriate attribution.

PIBS 1285

ACKMOWLEDGEMEHT AMD DISCLAIMER

This research was based on a sediment bioassay initiated by A. Hayton. I
gratefully acknowledge W. Scheider, D. Persaud, and T. Lomas for their
constructive advice during the course of this work and thank S. Petro and R. Pope
for assistance in conducting the experimental bioassays. I thank W. Scheider
and D. Bedard for their helpful comments in review of this document.

This report was prepared for the Ontario Ministry of the Environment as part of
a ministry funded project. The views and ideas expressed in this report are
those of the author and do not necessarily reflect the views and the policies
of the Ministry of the Environment, nor does mention of trade names or commercial
products constitute endorsement or recommendation for use.

c Her Majesty the Queen in Right of Ontario as
Represented by the Minister of the Environment

EXECUTIVE SUMMARY

Under the 1987 revision of the 1978 Great Lakes Water Quality Agreement between
Canada and the United States, the parties, m cooperation with state and
provincial governments were assigned the task of cooperatively developing and
implementing strategies for mapping, assessing and managing contaminated
sediment. Adequate assessment of contaminated sediment requires information
beyond bulk sediment chemistry. The biological significance of in-place
pollutants can be measured on the basis of structural or functional modifications
of benthic invertebrate communities, and by demonstrating the bioavailability
of contaminants through a variety of toxicity tests.

Laboratory sediment bioassays are an important component of biological
assessment. Bioassays range from acute lethality tests to chronic, sublethal
tests. Chronic exposures provide information unachievable from acute toxicity
studies. Growth, reproduction, and other physiological parameters have been used
as endpoints in chronic tests. Since benthic organisms can be an important
vector in the transfer of materials from sediment to other compartments of the
ecosystem, the sediment bioassay should also provide information on the extent
to which contaminants may be mobilized into the foodweb.

Biologically based sediment quality guidelines are under development by the
Ontario Ministry of the Environment. These guidelines will, in part, provide
the basis for making decisions on remedial actions. When bulk chemistry exceeds
specified concentrations, the draft guidelines recommend that biological testing,
including sediment bioassays, be conducted to identify whether contaminants are
biologically available.

Sediment bioassays measure the effects of contaminated sediments on the biota.
By far the most frequently described approach is solid phase testing with either
benthic or water column organisms. For the purpose of evaluating the impacts
of in-place pollutants on the biota, as opposed to the consequences arising from
dredging operations, the focus of this study was on the solid phase bioassay.

The principle objective of this study was to contribute to the development of
a methodology for assessing the chronic and acute toxicity of sediments to biota.
This included an examination of the effects of bioassay assembly and sediment
manipulation techniques to the response of the test organisms, and the
sensitivity of growth as a chronic endpoint.

It is reasonable to expect that the exposure of an organism to contaminants will
vary with the state to which the sediment-water system is in equilibrium. I
therefore examined whether an organism's response varied with the length of
settling time of the bioassay assembly proceeding the introduction of the
organism. The duration of exposure required for the response of organisms m
test sediments to differ significantly from the controls was also not known.
As a result, the experiment was designed so that replicates could be harvested
day 10 and day 21. Analysis of the growth response of Hexagenia suggested that
biomass changes were influenced both by sediment type and by the duration of the
period of equilibration. Growth in both test sediments was inversely
proportional to the duration of the equilibration period.

In accordance with the biomass changes noted for mayflies, growth inhibition was
least when the fathead minnows were added 5 days after chamber assembly. There
appeared to be no notable difference between the 6 hr. (5 hr. settling plus 1
hr. aeration) and 1 day equilibration periods with respect to biomass changes.
Metal accumulation was inversely t)roportional to bioassay settling time. The
effects of fish density were variable. Growth inhibition was greater with 15
as compared to 10 fish in some, but not all cases, and density apparently exerted
no influence on biomass changes in the controls. This last finding is of
interest, since it may indicate that the stress of possible overcrowding was
exacerbated by the contaminated sediments.

Exposing fathead minnows to test sediment for 21 days without food introduces
additional stress which could exaggerate the adverse effects of contaminants.
Feeding, however, could alter contaminant accumulation, and gut clearance may
be important for estimating true tissue concentrations pf contaminants. A
feeding experiment was conducted which revealed that feeding had little effect

on accumulation of trace metals and organic contaminants, however, significant
decreases in concentrations of Kn, Fe, Al, Pb, and Ni resulted when fish were
held for 24 h to purge their guts. Only for control organisms was growth improved
by feeding.

Current methods for assembly of sediment bioassays often involve sieving and
homogenizing the sediment. This effectively exposes the organisms to a uniform
dose of contaminants that is in reality a mean dose of the heterogeneously
distributed contaminants. In some cases, the extensive aeration of the sediment
also results in a transformation of chemical species to forms that are of greater
or lesser bioavailability. I examined the question of sediment homogenization
by using diver-collected cores.

Intact sediment from one station resulted in higher mortality and poorer growth
than homogenized sediments for mayfly nymphs, but did not significantly influence
mortality or growth in fathead minnows. Intact sediment from a second station
resulted in the reverse, better growth for mayfly nymphs and substantial
mortality for fathead minnows. In a third station (sandy sediment),
homogenization resulted in higher mortality than m the intact cores for
Hexagenia. This was most likely caused by the elimination of the surface layer
of fine-grained material (present in intact cores) and therefore, the elimination
of suitable substrate for burial and feeding. Homogenization did not effect
growth of fathead minnows, and may have ameliorated toxicity as measured by
mortality.

Sediment manipulation, bioassay assembly, organism density, and bioassay duration
were important determinants of the final endpoints measured. Further efforts
devoted at refining substrate and feeding requirements of test organisms would
assist in calibration of the bioassays.

The results of this research support the use of a 21 day sediment bioassay with
organisms introduced no more than 24 h after bioassay assembly. Several test
organisms should be included in a comprehensive assessment. This work focused
upon the mayfly nymph Hexagenia limbata and the fathead minnow Pimephales

promelas. Growth inhibition was demonstrated to be a sensitive indicator of

sediment toxicity.

Further research into the development of full or partial life cycle tests that
include reproduction as an endpoint is warranted, and establishment of cultures
for all test species, along with the use of reference toxicants, would be of
great value for ensuring repeatability of bioassay results.

Integrated strategies for sediment management must include a consideration of
biological effects observed both in the laboratory and in sztu. An array of
biological test methods can provide an integrated approach to the determination
of the toxicological qualities of sediment contaminated with a variety and
sometimes unknown chemicals. Sediment bioassays are a valuable vehicle for
the assessment of sediment and provide information that reflects the biologically
relevant forms of mixtures of contaminants.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS i

EXECUTIVE SUMMARY ii

l.C INTRODUCTION 1

2.0 OVERVIEW OF SEDIMENT BIOASSAY METHODS 2

2.1 Matrices Used for Biological Assessment

of Sediments 3

2.2 Endpoints 4

2.2.1 Acute toxicity bioassay 4

2.2.2 Sublethal chronic sediment bioassays 4

2.2.3 Bioaccumulation 5

2.3 Sediment Bioassay Design 5

3.0 EXPERIMENTAL DESIGN, RESULTS, AND DISCUSSION 6

3.1 Experiment 1 - March 1 to March 25, 1988 7

3.2 Experiment 2 - March 31 to April 25, 1988 8

3.3 Experiment 3 - April 26 to May 18, 1988 10

3.4 Experiment 4 - June 30 to July 22, 1988 11

4.0 RECOMMENDATIONS FOR RESEARCH PRIORITIES 14

4.1 Chronic Bioassays 14

4.2 Reference Toxicants 15

4.3 Sediment Manipulation 15

4.4 Invertebrate Cultures 16

4.5 Organism Age and Size 17

4.6 Substrate Properties and Colonization Potential 18

4.7 Sediment to Water Ratio 18

4.8 Flow-through, Static-Renewal and Static

Water Regimes 19

4.9 Field Comparison 19

4.10 Bioaccumulation

20

5.0 RECOMMENDED PROTOCOL FOR LABORATORY SEDIMENT
BIOASSESSMENT

21

6.0 OTHER ACTIVITIES RELATING TO SEDIMENT BIOASSAY
DEVELOPMENT

25

7.0 REFERENCES
TABLES
Table 1 - Marine and freshwater organisms used

in acute toxicity tests with sediments

26

Table 2 - Chronic endpoints employed for various
organisms used in sediment bioassays

37

Table 3 - Sample location and sediment composition
of substrates used in sediment bioassays

39

Table 4

Contaminant concentrations in sediment
used for bioassays

Table 5 - Effects of settling time on growth of
Hexagenia limbata

41

Trace organics in oligochaetes in
relation to bioassay settling time and
exposure duration

42

Metals in oligochaetes in relation to
bioassay settling time and exposure
duration

45

Table 8 - Effect of settling time on growth of

Pimphales promelas 46

Table 9 - Metals in fathead minnows in relation to
organism density, bioassay duration and
gut clearance 53

Table 10 - Trace organics m fathead minnows in

relation to organism density, bioassay
duration and gut clearance 62

Table 11 - Effect of feeding on growth of
Pimphales promelas

62

Table 12 - Effect of feeding and gut clearance on

metal concentrations in fathead minnows 63

Table 13 - Effect of feeding and gut clearance on
trace organic concentrations in
fathead minnows 64

Table 14 - Comparison of intact cores and homogenized
sediments with reference to growth and
mortality of Hexagenia limbata and
Pimephales promelas 67

FIGURES
Figure 1

Biomass changes in Hexagenia limbata
nymphs in 10 and 21 day bioassays

Figure 2

Biomass changes in Pimephales promelas
in 10 and 21 day bioassays

69

Figure 3 - Cr, Cu and Pb concentrations in mayflies
and fathead minnows in beaker and intact
core bioassay assemblies 70

Figure 4 - As and Ni concentrations m mayflies

and fathead minnows in beaker and intact
core bioassay assemblies 71

Figure 5 - Cd and Hg concentrations m mayflies

and fathead minnows in beaker and intact
core bioassay assemblies 72

Figure 6 - Zn and Mn concentrations in mayflies

and fathead minnows in beaker and intact
core bioassay assemblies 73

Figure 7 - Al and Fe concentrations in mayflies

and fathead minnows in beaker and intact
core bioassay assemblies 74

l.C INTRODUCTION

The principle objective of the research conducted under the Research Advisory
Committee, Ministry of the Environment Post-Doctoral Grant PDF03 was to develop
a methodology for assessing the chronic and acute toxicity of sediment to biota
and to determine the bioavailability of sediment bound contaminants by measuring
tissue retention of polar and nonpolar substances.

To achieve this goal, the following studies have been conducted:

Examination of the effects of bioassay assembly and sediment
manipulation techniques upon the response of the test organisms.

Assessment of the relative sensitivities of a series of encpoints,
including:

growth inhibition

bioaccumulation

mortality

Comparison of the responses of organisms occupying different ecological
niches including:

benthic infauna e.g. mayfly

water column organisms e.g. fathead minnow

Establishment of an interim recommended protocol for multiple
organism/endpoint bioassays to be conducted in concert with field
collections of benthic macroinvertebrates for the assessment of
community structure and physiochemical analyses of in situ
contaminants.

In addition, the following studies are recommended:

Estimation of the comparative sensitivity of the amphipod Hyallela

azteca and Chironomus as alternate bioassay organisms, and the
establishment of invertebrate cultures for these organisms and for
Hexagenia limbata.

Development of full or partial life cycle tests with reproductive
success as an additional endpoint

Introduction of the routine use of reference toxicants as a means of
ensuring comparable sensitivity of test organisms for each bioassay
conducted

Calibration of growth on the basis of substrate conditions

Further investigations into sediment manipulation with consideration
of the sensitivity of endpomts to static, static-renewal, and flow
through systems

2.C OVERVIEW OF SEDIMENT BIOASSAY KETHODS

Currently, th<= "-••erature contains several approaches to sediment bioassessment
with reference • range of benthic and water column organisms and various
lethal and nonletnai endpoints. It is useful to summarize ongoing research m
the field, to better understand the context within which this report applies.

Sediment bioassays measure the effects of contaminated sediment on biota.
Examining this broad statement more closely reveals the potential for a myriad
of test designs. The substrates which have been explored include sediment
elutriates, pore waters, and whole, sieved or suspended sediments. Chamber
construction ranges from petri dishes and test tubes to 4CL aquaria with static,
static/renewal, flowthrough, and recycling water regimes. Test organisms are
benthic infauna or epifauna, macrophytes, fish or plankton. The bioassay
responses or endpoints vary from acute lethality to nonletnal impacts during
acute or chronic exposures. These range from the molecular and cellular levels
such as biochemical deviations and induction of carcinomas, to the organismal

level such as growth and reproductive inhibition. To avoid confusion, it is
worthwhile noting that the terms acute and chronic refer to exposure duration
while the enpoints may be lethal or nonlethal. The most sensitive assays are
those that utilize chronic exposure intervals and monitor for nonlethal toxicity
reponses of critical life stages of sensitive organisms. The bioassay assembly
can also be applied to monitor bioaccumulation of pollutants by the test organism
in order to estimate contaminant bioavailability and potential for foodweb
mobilization of potential toxicans.

2.1 Matrices used for biological assessment of sediments

Sediment elutriates have been prepared as liquid phase matrices, principally to
assess the impacts of dredging activities on water column organisms (Gannon and
Beaton 1969, Lee et al . 1975, Shuba et al. , Munawar et al. 1983). For exam.ple,
the USEPA/US Army Corps of Engineers (1977) describe a 48h toxicity test that
exposes Daphnia to the elutriate.

Pore waters have been considered as an alternate liquid phase to examine the
effects of contaminated sediments on the burrowing infauna and to identify the
route of exposure of different organisms to different pollutants (Bahnick et al.
1980, Rodgers, J.H., Jr., N. Texas State Univ., pers. comm.). The interstitial
waters may be acquired by squeezing sediment, centrif ugation followed by
filtration, or through the use of dialysis membranes.

By far the most frequently described approach is solid phase testing with either
benthic or water column organisms (e.g., Swartz e^ al. 1985a, Malueg e^ al.
1984, Cairns et^ al. 1984, Ingersoll and Nelson, 1987). For the purpose of
evaluating the impacts of m-place pollutants on the biota, as opposed to the
consequences arising from dredging operations, this research program centred on
the solid phase bioassay. Studies based on pore waters are scant, although of
potential significance particularly with respect to understanding the mechanisms
involved m the bioaccumulation of contaminants. Nevertheless, the remainder
of this review will be dedicated to solid phase testing with a variety of
organisms primarily due to its conceptual simplicity and relatively greater
compatibility with field conditions.

2.2 Endpoints

2.2.1 Acute Toxicity Bioassay

Sediment bioassay reviews by Nebeker (1984), Buikeman (1982), Munawar et al.
(1984), Craig (1984) and Lamberson and Swartz (1985) all demonstrate the
preponderance in the literature of relatively short-term exposures with
mortality as an endpoint. Acute tests typically measure the lethality of
the test sediment relative to a reference substrate. Acute tests are
frequently conducted by placing a specified volume or depth of sediment in
a beaker, jar or aquarium. This is followed by the addition of a volume of
water, often at a ratio of 4:1 {v:v) of water to sediment (Nebeker et al.
1984). The assembly is either static, flowthrough or recycling, and aeration
is normally indicated. Several organisms are added to replicate units and
mortality is tabulated by the end of the exposure. Exposure times vary among
organisms and for the same organisms being employed by different authors.
Table 1 lists a number of acute bioassays that have been described for
freshwater and marine fauna.

2.2.2 Sublethal Chronic Sediment Bioassays

Chronic tests provide critical information which cannot be secured from acute
toxicity studies, especially where the contaminants are materials with delayed
action and of bioaccumulative potential. When an organism's life stage
influences its degree of sensitivity to pollutants, chronic studies are
capable of detecting significant adverse biological impacts of polluted
sediment that would not be observed in an acute test. In some instances,
behavioral modification may result from exposure to contaminated substrates
(Swartz 1985a) and this can have consequences for an organism's ability to
compete for resources, reproduce, and/or avoid predation in nature.

Growth, reproduction, and other physiological parameters have been used as
endpoints in chronic bioassays (Nebeker et al. 1984) as have behavioral
activity such as burrowing (Swartz 1985a) and preference/avoidance (Gagnon

and Beeton, 1969, 1971). Table 2 summarizes some of the endpoints which have
been considered for a number of different taxa.

In addition to the endpoints listed m Table 2, biochemical, enzymatic,
histopathological and morphological changes have also been measured. These
have been reviewed by Beak (1987) and will not be considered m detail here.
While these latter options can provide sensitive early indications of organism
stress, the approaches currently require a degree of technical skill,
expertise, laboratory specialization, and financial resources that are
sufficiently great as to preclude their integration into a routine protocol.

2.2.3 Bioaccumulation

Measurements of tissue concentrations of polar and nonpolar substances can
be used to demonstrate that contaminants in sediment are biologically
available and have the potential to enter the food web. Nebeker et^ al. (1984)
include tissue analysis of chironomids, mayflies and amphipods in their
bioassay approach. Since benthic invertebrates can be an important vector
in the transfer of materials from sediment to other compartments of the
ecosystem (Krantzberg 1987) the sediment bioassay should provide information
on the extent to which contaminants may become mobile.

Currently, the toxicological significance of tissue residues m invertebrates
and many vertebrates is virtually unknown. Unfortunately, assessing this
linkage falls beyond the scope of this research, at present. It should be
stated, however, that for an organism to metabolize or detoxify contaminants,
there is undoubtably a physiological cost in the production of enzymes and
other proteins. Energy diverted from normal metabolic pathways for these
purposes could be quantified and the ramifications at the level of the
organism should be established.

2.3 Sediment Bioassay Design

Having outlined the variety of test organisms and endpoints of potential use,
it is instructive to explicitly consider the many decisions that modify the end

result of the bioassay. From a biological perspective, factors such as organism
size, age, sex, reproductive status, and history of exposure to pollutants can
alter the response of an organism to a given sediment (Luoma 1983, Krantzberg
and Stokes 1989). Clearly, interspecific variability should also be expected
and IS often observed. This has led to recommendations that several taxa be used
for each bioassay (e.g. Nebeker et^ al. 1984). From a population standpoint,
density dependent effects may also be postulated and should be confirmed or
refuted. From a physical and chemical perspective, different methods of sediment
storage, manipulation, chamber construction and assembly, could effect the
outcome of the bioassay. Sediment handling necessarily disrupts sediment
physicochemistry which has direct implications for contaminant activitiy.

These biological, physical and chemical processes were recognized in the
development of a research program directed at on the importance of bioassay
design. Recommendations for the application of the results are presented in
the form of a detailed protocol (Section 5.0) .

3.0 EXPERIMENTAL DESIGN, RESULTS, AND DISCUSSION

The first set of experiments examined the effects of equilibration time of the
bioassay assembly and bioassay duratation on mortality, growth and
bioaccumulation by the mayfly nymph, Hexagenia limbata and the oligochaete
Tubif ex tubifex. The second experiment considered the effects of organism
density, chamber equilibration time and bioassay duration on mortality, growth,
and bioaccumulation of contaminants by juvelines of the fathead minnow,
Pimephales promelas. The third series of test bioassays examined the
consequences of feeding with respect to mortality, growth and bioaccumulation
of contaminants by P^ promelas. The fourth bioassay assessment compared the
toxicity of homogenized sediments with that of sediment collected by diver and
maintained as intact cores. Test organisms for the latter experiment were mayfly
nymphs and fathead minnows. For all investigations, pH and dissolved oxygen were
routinely measured.

3.1 Experiment 1 - March 1 to March 25, 1988

Purpose: To determine the effects of settling time, following the
addition of sediments and water to the bioassay container, on sediment
toxicity to oligochaetes and mayflies. To evaluate the importance of
bioassay duration on identification of toxic sediment. To determine
trace metal and trace organic bioaccumulation by oligochaetes as a
function of bioassay design.

It IS reasonable to expect that the exposure of an organism to contaminants will
vary with the state to which the sediment-water system is m equilibrium. I
therefore examined whether an organism's response varied with the length of time
for which the bioassay assembly was left to settle prior to the introduction of
the test species. The duration of exposure required for the response of
organisms in test sediments to differ significantly from that of the controls
was also not known. The experiment was therefore designed so that half of the
replicates could be harvested at day 10 and half could be harvested at day 21.

The two test sediments were from a silty site m the vicinity of the Toronto Main
STP outfall and a sandy site at Rice Lake. The control or reference sediment
was a silty substrate from Honey Harbour. Two litre widemouth glass jars were
filled to a depth of 3 cm with sediment (surface area = 100 cm^) and
dechlorinated tap water was gently added. Organisms were introduced at 3 time
intervals; 5 hours settling plus 1 hour of aeration, 1 day settling plus 1 hour
aeration and 5 days settling plus 1 hour aeration. At each time interval, either
8 mayflies weighing approximately 25 mg/mdividual (wet weight) or approximately
1.5 gm (wet weight) of oligochaetes (c.a. 150 individuals) were introduced into
the chambers. Each treatment had 4 replicates. Preliminary tests indicated no
density dependent effects for mayflies at this density.

Sediment bulk chemistry and physical properties were measured at the onset of
the experiment (Tables 3 and 4) . Oligochaetes were held in dechlorinated water
overnight to evacuate their gut contents and were frozen and submitted for metal
and trace organic analysis. Mayflies were weighed and counted, but were of

insufficient biomass for analysis.

Analysis of the growth response of Hexagenia indicated that biomass changes were
influenced both by sediment type and by the duration of the period of
equilibration (Table 5). Growth in both test sediments was greatest when the
mayflies were added when 5 days had elapsed following chamber assembly. Growth
was poorest when organisms were added when 6 hours had elapsed following assembly
(1 hour after aeration) .

Mayfly biomass increased most in nymphs exposed to control sediments, followed
by organisms exposed to Rice Lake sediment, and increased least in mayflies
subjected to sediment from the vicinity of the Toronto Main STP. Growth
inhibition relative to controls was more pronounced by day 21 (p < 0.05), as
compared to day 10 (Fig 1).

Due to difficulties m assuring retrieval of all oligochaetes, bioamass changes
were not considered as an endpoint in the bioassay. Bioaccumulation results were
interpreted by analysis of variance. Trace organic concentrations in
oligochaetes (Table 6) did not vary significantly with experimental settling time
and bioassay duration. Manganese and Al concentrations in oligochaetes (Table
7) were significantly different from time zero, with Al increasing with exposure
time and Mn decreasing with exposure time. One hypothesis is that with
sufficient aeration of the bioassay chamber, Mn became less bioavailable with
time due to the formation of insoluble oxyhydroxides. The increase in Al
concentrations in oligochaete tissues with exposure time may reflect the effect
of sediment ingestion which was not mirrored by other trace metals either due
to their relatively low concentrations in sediment, poor bioavailability, or
active metabolic regulation by oligochaetes.

3.2 Experiment 2 - March 31 to April 25, 1988

Purpose: To determine the effects of settling time and bioassay
exposure interval on sediment toxicity to fathead minnows and to
establish whether toxicity is dependent on organism density.

The test design and bioassay assembly parallelled Experiment 1. The two test
sediments were from a sandy site in the vicinity of the Toronto Mam STP outfall
and a silty site in St. Mary's River. Three to four month old juvenile fathead
minnows weighing approximately 0.5 gm per individual were added to each bioassay
chamber at a rate of 10 or 15 individuals per replicate. Four replicates of each
treatment were harvested after 10 or 21 days exposure. Fish were reweighed and
half were immediately frozen for metal and trace organic analyses. The remainder
were held for one day in dechlorinated water to clear their guts and to
illuminate the significance of short term depuration.

In accordance with the biomass changes noted for mayflies, growth inhibition
was least if the fathead minnows were added when 5 days had elapsed following
chamber assembly (Table 8). There was no notable difference between the 6 hr.
(5 hr. settling plus 1 hr. aeration) and 1 day equilibration periods with
respect to biomass changes, and the effects of fish density were variable.
Growth inhibition was greater with 15 as compared to 10 fish in some, but not
all cases, and density apparently exerted no influence on biomass changes in the
controls. This last finding is of interest, since it may indicate that the stress
of the exposure to contaminated sediments could be exacerbated by possible
overcrowding .

By day 21, all fish had decreased in weight (Fig 2). Minnows from the test
sediment lost more weight than did those from the control sediment and analysis
of variance revealed that this outcome was significant by day 21 (p < .05) .

Trace metal concentrations in fathead minnows were responsive to several bioassay
treatments (Table 9) . Less Pb was accumulated in the presence of 15 individuals
than in the presence of 10 individuals, while the reverse was true for Al and
Cd. The explanation for this remains unclear may warrant further examination.
More gamma-chlordane was accumulated by fish at the higher density as compared
with the lower density (Table 10). This phenomenon remained true when tissue
concentrations were corrected for lipid content.

With the exception of Cd, metal accumulation was significantly greater (p <
0.01) when organisms were added to the bioassay chamber after 5 hrs of settling

plus one hour of aeration, as compared with longer settling intervals. In
addition. As continued to increase with exposure duration while Cr decreased
significantly with bioassay duration. No other significant differences in tissue
residues were observed as a consequence of bioassay duration. Concentrations
of trace organics in fathead minnows did not vary with settling time or exposure
duration (Table 10) .

The importance of permitting an interval for gut clearance in the final
measurement of contaminant residues in fathead minnows is discussed under section
3.3.

3.3 Experiment 3 - April 26 to May 18, 1988

Purpose: To determine the effects of feeding on growth,
bioaccumulation and mortality of fathead minnows in sediment bioassays.

Based on the results of Experiment 2, it was apparent that even control organisms
were experiencing stress due to starvarion. In addition, there was evidence that
the expression of toxicity was density dependent. Therefore, a feeding
experiment was initiated employing 10 fish in each chamber, to be introduced to
the chambers when 6 hours had elapsed after assembly. Contaminated sediments
were from a silty site at Canagagigue Creek, a coarse sandy site from the Algoma
slip (St. Mary's River), a silty site from Rice Lake and sandy site from St.
Mary's River. Silty Honey Harbour sediment was used as a reference substrate.
Bioassay design was as above, with a subset of the test organisms retained for
24 hours to depurate gut contents.

For each sediment, 4 replicates were fed ad libitum every second day and 4
replicates were not fed for the duration of the 3 week bioassay. Average biomass
was c.a. 0.3 - 0.4 gms per individual (wet weight) at the onset of the
experiment.

Both the Canagagigue Creek and Algoma slip sediments were lethal with mortality
occurring from day 1 to day 14. No relationship between time until mortality
and feeding was evident.

10

Since fathead minnows ingest sediment and consume detritus, biomass changes may
be linked to substrate properties that are independent of contaminant loads.
For chronic studies, then, standardizing food availability may be necessary.
Introducing food, however, may complicate the interpretation of results,
particularly with respect to bioaccumulation. The only organisms to show an
increase in biomass were the minnows receiving food in the Honey Harbour
treatment, while biomass was lost in the Honey Harbour, unfed minnows (Table 11).
Interestingly, for the other contaminated sediments, feeding did not affect the
extent of biomass lost, perhaps indicating that the presence of the pollutants
significantly influenced fish metabolism. This finding is in accordance with
the hypothesis that the additional stress imposed upon an organism by
contaminants can amplify the importance of naturally encountered stresses such
as food or habitat limitation.

Highly significant {p < 0.001) concentrations of Kn, Fe, Al and Pb, and Ni (p
< 0.02) were lost when fish were allowed to purge their gut contents. The only
effect of feeding was an increase in Ni concentrations in fathead minnows (Table
12). Trace organic concentrations did not differ among treatments (Table 13).
The loss of Mn, Fe and Al may well reflect the importance of these elements in
sediment composition and their correspondingly high concentrations relative to
tissue residues in fish. The loss of Al agrees with the findings for depuration
effects on oligochaete tissue residues. While metal physicochemistry was not
measured, Pb and Ni are known to associate with Fe and Mn oxyhydroxides. Lead
and Ni may have followed pattern of Fe and Kn for this reason.

3.4 Experiment 4 - June 30-July 22, 1988

Purpose: To compare the toxicity of intact sediment cores and
homogenized sediment to mayfly nymphs and fathead minnows.

The current MOE method for assembly of the sediment bioassay involves sieving
and homogenizing the sediment. This effectively exposes organisms to a uniform
dose of contaminants that is in reality a mean dose of the heterogenously

11

distributed contaminants. A positive ramification of homogenization is that it
most likely results in less variability among replicates than would be observed
for intact sediment. In contrast, there are probably many circumstances where
the extensive aeration of the sediment also results m a transformation of
chemical species to forms that are of greater or lesser bioavailability. One
approach to examining the question of how toxicity is influenced by sediment
homogenization involves a comparison of endpoints achieved when organisms are
exposed to diver-collected cores and to homogenized sediment.

The cores used were acrylic tubes of surface area comparable to the 2L glass
jars. Organisms were introduced into the cores and into homogenized sediments
from the same site as those where cores were collected.

Eight Hexagenia nymphs (c.a. 40 mg/individual net weight) or 10 juvenile fathead

minnows (c.a. 400 mg/individual net weight) were the test organisms. Four

replicate diver-collected cores and triplicate jars of homogenized sediment were

used for each organism for each of three test substrates. Mortality, biomass

changes,

and bioaccumulation over three weeks were the endpoints examined. pH and

dissolved oxygen were monitored in all chambers.

One sandy and two fine-grained sites in the vicinity of the Toronto Main* STP
outfall were sampled. Honey Harbour sediments were used for controls. In Site
A (fine) , intact sediment resulted in higher mortality and poorer growth than
homogenized sediment for mayfly nymphs, but did not significantly influence
mortality or growth in fathead minnows (Table 14) . Intact sediment from Site B
(fine) resulted in better growth for mayfly nymphs than homogenized sediment.
Mortality was <10% in both treatments. Homogenization resulted in substantial
mortality for fathead minnows (87% vs 20% in intact cores). In Site C (sandy)
homogenization resulted in higher mortality than in the intact cores for
Hexagenia. This was most likely caused by the elimination of the surface layer
of fine-grained material (present in intact cores) and therefore, the elimination
of suitable substrate for burial and feeding. Homogenization did not effect
growth of fathead minnows, and may have ameliorated toxicity as measured by
mortality. A point of interest is the variability in response between the two

12

test species, which suggests differential modes of action of contaminants upon
organisms with vastly different metabolic pathways and ecological niche
requirements.

The results the chemical analyses of tissue residues for trace metals are
illustrated in figures 3 through 7. While there was a tendency for some metals
to bioaccumulate to a greater extent in beakers as compared to core exposures,
the results were not statistically significant. Considering the high cost of
diver collected cores, this result is encouraging, in the sense that the beaker
assay may be fairly representative, if not more conservative than intact core
bioassays. The finding that intact cores were more toxic to mayflies at station
A but not at station B should be perceived as justification to pursue this line
of investigation and to broaden the range of sediment tested.

13

4.C RECOMMENDATIONS FOR RESEARCH PRIORITIES
4.1 Chronic Bioassays

Recommendation

I strongly urge that the development of chronic, nonlethal bioassays be the
central focus of future research. This has begun with a consideration of a
21 day growth-inhibition test with mayflies and fathead minnows. Clearly,
mortality is also documented and many other potential endpoints could be adopted.

Since sediment organic content will effect growth, independent of the level of
contamination, additional studies on feeding must be performed for fathead
minnows in particular. Food availability has been shown to modify the
bioaccuffiulation of pollutants and studies designed to further assess various
feeding regimes for chronic bioassays are strongly recommended.

The chironomid full or partial life cycle test is worth pursuing. Survival,
changes in biomass, emergence, and reproductive success can all be measured in
10 to 28 days. Several organisms can be introduced into a single chamber (with
replication) or individual organisms can be held in test-tube assembly. These
two approaches could be compared.

Hyallela azteca is also amenable to a full or partial life cycle test, but
ensuring that individual sensitivity remains constant during culturing may be
somewhat more difficult for the amphipod than for the chironomid.

A sensitive and more rapid chronic bioassay measures growth in larval or egg-
sac stage fathead minnows. This test has been considered for effluent testing
and could be applied to sediment bioassessment .

Another interesting class of endpoints to consider is behavioral in nature.
The two most often cited are preference-avoidance and inability (or refusal) to
burrow. I have observed oligochaetes to remain on the surface of noxious

14

sediment. Mayflies also elicit this response, although less frequently. Others
have demonstrated reburial failure in amphipods transferred to clean substrates
following exposure to contaminated sediments. These endpoints, however, my be
more difficult to interpret in terms of their ecological significance than the
partial life cycle tests and should be investigated once the former tests have
been established.

4.2 Reference Toxicants

Recommendation

At least 1 polar (e.g. Cd) and 1 nonpolar (e.g. PCP) compound should be tested
and effect concentrations established. These pretests, or positive controls,
can be conducted as ECj,s or LCj,s in aqueous phase bioassays or developed as a
spiked sediment bioassay.

Reference toxicants have been of value m examining seasonal changes in an
organism's sensitivity, and changes related to age, reproductive status, and
history of exposure to contaminants. In order to ensure that the test organism's
response to contaminants is uniform and does not vary among bioassays (or among
laboratories) , reference toxicants should be incorporated as a pretest in all
bioassays. Loss of vigour in cultured organisms has been demonstrated and could
confound interpretation of sediment bioassay results. When an organism response
does vary, one might justify the application of a correction factor.

4.3 Sediment Manipulation

Recommendation

Further comparisons between intact sediments and homogenized sediments should
be initiated to assess the effect of sediment manipulation on acute and chronic
endpoints. The effects of sediment storage on toxicity should also be
investigated.

15

Many existing protocols, including the current MOE method for assembly of the
sediment bioassay involve sieving and homogenizing the sediment. For many
sediments extensive aeration will result in a transformation of chemical species
to forms that are of greater or lesser bioavailability. One approach to
examining the question of sediment homogenization involves comparisons of
toxicity with diver-collected cores. The cores should be of comparable surface
area as the glass jars being used for routine testing. Organisms can be
introduced into the cores and into sediment which will be sieved and homogenized
and collected from the same site as those where cores were retrieved. Since
the objective of the bioassay is to assess the extent of contamination of the
sediment directly, without the complicating factor of potentially contaminated
site water, dechlorinated laboratory water should replace the water in the cores
and should be used for the jar test as well. I conducted one such experiment
(Section 3.4) and the results should be verified for other sediment types and
additional bioassay organisms.

As an alternative to toxicity testing using diver collected cores, the contents
of the ponar grab can be "cored" on board with minimal mixing of the test
sediment. These pseudo-cores can be returned to the laboratory and subjected
to the same bioassessment as the diver-collected cores and the homogenized
sediment. Again, it would be useful to conduct the above experiments using a
variety of organisms and a broader range of contaminants.

Due to logistical constraints sediment has in the past been stored at 4°C for
several months prior to conducting the bioassay. To establish a maximum
acceptable storage interval bioassays could be repeated using the same sediment
after short and long-term storage (e.g. 2 weeks, 1 month, 2 months, 6 months
storage) .

4.4 Invertebrate Cultures

Recommendation

To facilitate the development of a multi-species approach to sediment
bioassessment, invertebrate cultures should be initiated. The construction of

16

facilities at MOE for culturing Hyalella azteca, Hexagenia, Chironomus, and
oligochaetes should be expanded and actively maintained. Protocols for culturing
these invertebrates have been published by, for example, S.G. Lawrence (ed)
1981, as a Canadian Special Publication of Fisheries and Aquatic Sciences #54.
It would be worthwhile for a full time technician and/or scientist to adopt
responsibility for the maintenance of the cultures.

Since sensitivity to contaminants varies among species and one species is not
necessarily the most sensitive to all classes of pollutants, MOE should adopt
an approach that incorporates the responses of several taxa. The test organisms
should represent different ecological functional groups such as filter-feeders,
burrowing infauna, and benthic foraging fish, for example. Chironomus riparius
can be easily cultured and used for a partial life cycle test. Collection of
sufficient quantities of Hexagenia that are of a prescribed age and size is
problematic and labour intensive. Establishing mayfly cultures, then, would be
of great value. Hyalella has been successfully maintained m stock cultures and
reproduction can be induced easily by controlling the photoperiod. Partial life
cycle tests with the Hyalella would be facilitated by establishing laboratory
cultures although maintaining consistency in c\ilture vigour must be verified.
Oligochaetes can be used to measure reproductive success and contaminant
bioavailability as reflected by bioaccumulation. Tubificids are easily
maintained in the laboratory.

4. 5 Organism Age and Size

Recommendation

I have compared the response of several size classes of mayflies and fathead
minnows exposed to contaminated sediments. These experiments should be conducted
for all test species.

Sensitivity to contaminants and contaminant bioaccumulation has been shown to
vary with age and body size for several taxa. For purposes of tissue analysis,
larger organisms provide greater biomass. However, sensitive life stages are

17

generally early life forms. Increased replication with smaller organisms may
satisfy both needs.

4,6 Substrate Properties and Colonization Potential

Recommendation

The range of substrates that the test species can potentially colonize, in the
absence of contamination, must be determined. Growth rates in sediment of
different organic content should be established.

Organisms such as chironomids and burrowing mayflies exhibit substrate
preferences. The absence of Hexagenia from a coarse-sandy sediment, for example,
may be due to its inability to thrive in such a substrate, regardless of how
pristine it may be. Extremely soft oozy mud may be too unstable a substrate for
chironomids. The organic content of a given sediment can be critical for growth
and survival. In order to formulate decision criteria for the selection of test
organisms based on substrate properties (independent of contamination) , it is
important to establish the range of substrates that the test species can
potentially colonize.

For growth to be used as an endpoint in sediment bioassays, biomass changes
should be calibrated against the same range of sediment types for uncontammated
substrates. One approach is to collect clean, fine sediments and perform a
serial dilution with fine and coarse sand. These experiments should apply to
all test species.

4.7 Sediment to Water Ratio

Recommendation

The effect of increasing or decreasing the currently adopted 1:4 ratio (volume
sediment:volume water) should be measured to ascertain that this ratio is
adequate.

18

The most frequently cited sediment to water ratio in a static test is 1:4 (v:v).
Experimental manipulation of this ratio may reveal a graded response to the test
organism. That is, toxic effects may increase when there is proportionally more
sediment (or less water) m the system but may be independent of the ratio at
relatively high volumes of water.

4.8 Flow-through, Static-Renewal and Static Water Regimes

Recommendation

A comparison of these three approaches, particularly in the case of chronic
tests, would assist in verifying that the observed endpoints best reflect the
biological significance of sediment contamination.

Static beaker tests are generally considered to represent a "worst case" or
"conservative" scenario. If contaminants increase in the water- column during
the exposure interval, it is intuitively logical that some organisms will
demonstrate toxicity responses that would not be observed in a flow-through
system. It is possible, however, that under certain circumstances, flow through
conditions may in fact underestimate the potential impacts of the test sediment
on some organisms as would be observed in nature.

The build-up of metabolites during a chronic bioassay may complicate the
interpretation of the results. A consideration of the static-renewal approach
would therefore be of great utility.

4.9 Field Comparison

Recommendation

Following laboratory investigations on the ramifications of various bioassay
design options, a thorough program of field testing should be instituted.

19

Field experiments should be designed to substantiate that laboratory tests are
a meaningful representation of field conditions. In situ chambers containing
the same species used in the laboratory can be positioned in the sediment. The
endpoints measured would be those evaluated in the laboratory sediment bioassay.
Careful consideration of chamber design, its introduction into the sediment,
water flow and/or exclusion, controls for chamber effects, and other potential
sources of artifact is integral to the success of the field validation program.

4.10 Bioaccumulation

Recommendation

It would be extremely beneficial to devote research efforts towards illuminating
the relationship between the tissue retention of contaminants and their
physiological significance to the exposed organism.

The relationship between the rate and extent of bioaccumulation of contaminants
and their physiological effects are poorly understood. As a consequence, the
ecological significance of tissue retention of pollutants cannot be established
for many contaminants. Such information would be of great value in the
establishment and implementation of sediment and biota guidelines.

20

5.0 RECOMMENDED PROTOCOL FOR LABORATORY SEDIKENT BIOASSESSMENT

The following recommendations for establishing a MCE protocol for sediment
bioassays should be considered as a framework which will be further modified
based upon ongoing research efforts in the development of the assays. It is
anticipated that these recommendations will be complemented by recent findings
of D. Bedard and A. Hayton, and that an MCE sediment bioassay protocol document
will be prepared by these individuals and myself, based on available
information. Further, the sediment bioassays should be recognized as an
important component of a sediment management strategy that includes extensive
biological assessment as well as chemical measurements.

Sample Collection

Sediment is collected by Ponar grab from test and control sites and the surficial
3 cm are placed in polyethylene-lined plastic buckets. These are returned to
the laboratory and may be stored at 4°C for no more than 2 weeks.

Preparation of the Bioassay Chamber

In the laboratory, sediment is air-sieved through a 2 mm mesh to remove large
particles, stones and other debris, and is then thoroughly homogenized. For
the experimental bioassays, 2 L glass widemouth jars, acid washed and hexane
rinsed, are filled to a depth of 3 cm with sediment (surface area = 100 cm^).
Dechlorinated water is gently added at a ratio of 4:1 water to sediment (v:v) .
Care must be taken to avoid having sediments adhere to the glass above the 3 cm
level. Resuspended sediment is allowed to settle for 5 hours (more conservative)
or 24 hours (logistically more practical), after which the overlying water is
aerated for 1 hour by inserting airlines through the lids of the glass jars and
securing the lids in place. Water loss due to evaporation is replaced as
necessary in order to maintain a water to sediment ratio of 4:1. pH and
dissolved oxygen in the overlying water are monitored routinely for the duration
of the experiment.

21

Test Organisms

Presently, organisms for the experimental bioassays are three to four month old
juvenile fathead minnows (Pimephales promelas) (acquired from OMOE Rexdale
laboratory cultures) weighing approximately 0.5 gm wet weight and first year
mayfly nymphs (Hexagenia limbata) weighing about 30 mg wet weight, collected at
a clean reference site (Honey Harbour, Georgian Bay, Ontario) or cultured in the
laboratory. Nymphs collected from the field can be maintained m the laboratory
in glass aquaria containing reference site sediments with gentle aeration of the
overlying water. The aquaria may be kept at <10°C, and gradually brought to 20°C
as mayflies are required. Alternatively, mayflies can be cultured in the
laboratory and harvested upon demand. Depending upon culture availability,
second instar Chironomus tentans may also be used.

Several hours prior to experimental exposure, mayflies are removed from their
aquaria by sieving small volumes of sediment through a 500 urn mesh. Nymphs are
randomly allocated to beakers containing dechlorinated water until each beaker
has 10 individuals of similar size (c.a. 25 mg/ individual, wet weight). The
nymphs are weighed after blotting them on several layers of "Kim Wipes" or acid
rinsed filter papers to remove adhering water. Alternately, when a large number
of assays are run concurrently, 5 to 7 aliquots of 10 individuals each can be
weighed, and the mean bioraass used as an estimate of initial biomass.

Similarly, juvenile fathead minnows are randomly allocated to beakers containing
dechlorinated water until each beaker has 10 individuals of similar size (c.a.
0.5 gms/individual, wet weight). Wet weight of each group of 10 individuals is
measured, or 5 to 7 representative samples of ten individuals each can be
wieghed, as above.

Chironomid length, weight, and head capsule widths can be measured for a
representative subsample of individuals at the onset of the experiment.

22

Conducting the Bioassay

A minimum of 3 replicate jars of each bioassay organism. Organisms are added
when 5 to 24 hours of settling followed by 1 hour of aeration have elapsed.
Aeration persists for the duration of the 21 day exposure interval. All control
and test jars are maintained at 20°C (a water bath may be required) at ambient
light.

During the 21 day bioassay period, mortality is noted and dead organisms are
removed. The presence of mayfly exuvia should also be recorded. As stated
above, pH and dissolved oxygen are monitored and water lost due to evaporation
is replaced. At day 21, organisms are removed from the bioassay jars by passing
the entire contents of each jar through a 500 iim sieve and retrieving the biota.
Recoveries are noted and the remaining organisms are reweighed and measured using
the procedure described above.

Chemical Analyses

Mayflies and fathead minnows are placed in hexane-rmsed alummum-f oil for
analysis of organic residues, or in plastic (e.g. "whirlpak" bags) for metal
analysis. Depuration is currently not recommended for trace organics, however,
the importance of gut clearance prior to sample analyses may be of relevance for
some trace metals. If feasible, a subsample of test organisms are held in
dechlorinated tap water for 24 hours to permit the depuration of gut contents.
Samples are frozen until chemical treatment can be conducted. Biota must be
handled gently, using teflon-coated or nylon forceps.

Concurrent with biota analyses, measurement of sediment physical and chemical
characteristics should be conducted. Aliquots of the homogenized sediment can
be analyzed for trace metals, a range of trace organics (e.g. PCBs, pesticides),
the extent of oil and grease contamination, particle size distribution, and
organic carbon and other measures of nutrient status. The selection of the
parameters of interest will necessarily be site specific and depend upon
available information on the pollutant source (s).

23

Data Interpretation

Mortality in test sediment is compared with controls. Control mortality should
not exceed 10%. Growth inhibition, indicative of chronic exposure to
unfavourable sediment, can be identified by comparing biomass changes of test
organisms with those of control organisms. Analysis of variance should be
applied to appropriately transformed data (if necessary to achieve a normal
distribution), particularly if data are expressed in the form of ratios (e.g.
percent reduction m growth of test organisms relative to controls). Similar
statistical analyses are used to examine differences in tissue retention of
contaminants following the 21 day exposure to sediment that vary in their
chemical composition. Significance at p <Ci.05 is proposed as an indicator of
sediment eliciting unacceptable biological responses.

24

6.0 OTHER ACTIVITIES RELATING TO SEDIMENT BIOASSAY DEVELOPMENT

In conjunction with developing a protocol for the KOE, it has been an advantage
to have the opportunity to discuss many of the growing concerns over sediment
bioassays with others doing research in this area. The conference on
Environmental Risk: Recognition, Assessment and Management, coordinated by the
Society of Environmental Toxicology and Chemistry (SETAC) in Pensacola, Florida
November 9 to 12, 1987 was of great benefit. Members of the American Society
for Testing Materials also held a special session on sediment bioassay protocols.
The meetings were of assistance in setting research priorities.

As a consequence of attending SETAC, I was made aware of the research efforts
being conducted at North Texas State University (NTSU) . After speaking with the
coordinator of the NTSU research and the Liaison Officer for this project, it
was agreed that visiting the NTSU facilities and gaining access to their
voluminous "grey" literature would be a worthwhile investment.

As a direct result of that visit, a strategy for culturing and testing
chironomids was prepared, and plans for future research on lab/field comparisons
were formulated. Many of the documents acquired for MOE are an excellent source
of information that would otherwise have been virtually inaccessible.

Attendance at the first International Conference on Environmental Bioassay
Techniques and Their Application, Lancaster England, was most worthwhile. The
title alone demonstrates the direct relevance of this meeting to my own and MOE
research objectives. In becoming familiar not only with the ongoing research
but with the individuals sharing common research goals, the sediment bioassay
techniques that are currently recommended for development were substantiated.

Subsequent to the completion of the experiments under the RAP PDF03, additional
studies were conducted on bioassay duration and the effects of chemical treatment
on sediment toxicity. This work was done in 1989 in support of the Hamilton
Harbour Remedial Action Plan and in conjunction with the National Water Research
Institute. A paper has been submitted to a peer review journal for
consideration.

25

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Munawar, M., R.L. Thomas, H. Shear, P. McKee, and A. Mudroch, 1984. An
Overview of Sediment - Associated Contaminants and their Bioassessment . Can.
Tech. Report Fish Aquatic Sci. No. 1253.

Nebeker, A.V., M.A. Cairns, J.H. Gakstater, K.W. Malueg, G.S. Schuytema and
D.F. Krawczyk, 1984. Biological methods for determining toxicity of
contaminated freshwater sediments to invertebrates. Environ. Toxicol. Chem.
3:617-630.

30

Nebeker, N.V., S.T. Onjukka, MA. Cairns and D.F. Krawczyk, 1986. Survival
of Daphnia magna and Hyalella azteca in cadmium-spiked water and sediment.
Environ. Toxicol. Chem. 5:933-935.

Nebeker, A.V. and F.A. Puglisi, 1974. Effects of polychlorinated biphenlys
(PCB's) on survival and reproduction of Daphnia, Gammarus, and Tanytarsus.
Trans. Amer. Fish Soc. 103:722-728.

Nebeker, A.V., C. Savonen, R.J. Baker and J.K. KcCrady, 1986. Effects of
copper, nickel and zinc on the life cycle of the caddisfly Clistoronia
magnifica. Environ. Toxicol. Chem. 3:645-649.

Phelps, H.C., J.T. Marey, W.H. Pearson and C.W. Apts, 1983. Clam burrowing
behaviour: Inhibition by copper-enriched sediment. Mar. Poll. Bull.
14:452-455.

Powlesland C. and J. George, 1986. Acute and chronic toxicity of nickel to
larvae of Chironomus riparis. Environ. Pollution 42:47-64.

Prater, B.L. and Anderson, M.A., 1977a. A 96-hour bioassay of Duluth and
Superior harbour basins (Minnesota) using Hexagenia limbata, Asellus communis,
Daphnia magna, and Pimephales promelas as test organisms. Bull. Environ.
Contam. Toxicol. 18:159-168.

Prater, B.L. and Anderson, M.A., 1977b. A 96-hour bioassay of Otter Creek,
Ohio. J. Water Poll. Control Fed. 49:2099-2106.

Prater, B. and Hoke, R.A., 1980. A method for the biological and chemical
evaluation of sediment toxicity. Contaminants and Sediments Volume 1,
pp. 483-499. Ann Arbor Science Publishers.

Scherer, E. , 1979. Avoidance testing for fish and invertebrates. Toxicity
Tests for Freshwater Organisms. Canadian Special Publication of Fisheries
and Aquatic Sciences. Volume 44, pp. 160-170.

31

Shuba,P.J., H.E. Tatem, and J.H. Carroll, 1978. Biological assessment methods
to predict the impact of open-water disposal of dredged material. Dredged
Material Research Program Tech. Rep. D-78-50,

Swartz, R.C., DeBen, W.A., and Cole, F.A., 1979. A bioassay for the toxicity
of sediment to marine marcobenthos. J. Water Pollut. Control Fed. 51,
pp. 944-960.

Swartz, R.C., DeBen, W.A., Jones, J.K. P., Lamberson, J.O., and Cole, F.A.,
1985a. Phoxocephalid amphipod bioassay for marine sediment toxicity. Aquatic
toxicology and hazard assessment. Seventh symposium, hSTy. STP 854.
pp. 284-307.

Swartz, R.C., Ditsworth, G.R., Schults, D.W., and Lamberson, J.O., 1985b.
Sediment toxicity to a marine infaunal amphipod: Cadmium and its interaction
with sewage sludge. Marine Environ. Res. 18:133-153.

Swartz, R.C., D.W. Schults, E.R. Ditsworth and W.A. DeBen, 1984. Toxicity
of sewage sludge to Rhepoxynius abronius, a marine benthic amphipod. Arch.
Environ. Contam. Toxic. 13:207-216.

Tsai, L., J. Welch, K. Chang, J. Schaefer, and L.E. Cronin, 1979. Bioassay
of Baltimore Harbour Sediments. Estuaries 2:141-153.

USEPA, 1988. Preliminary report on the toxicity and chemistry of sediments
from Toronto and Toledo Harbours. Preliminary draft. Sediment Criteria Team,
Freshwater Division, Corvallls Environmental Research Lab.

U.S. Environmental Protection Agency/U.S. Army Corps of Engineers, 1977.
Ecological evaluation of proposed discharge of dredged materials into ocean
waters. Environmental Effects Laboratory, U.S. Army Corps of Engineers,
Waterways Experimental Station, Vicksburg, MS.

Wentsel, R.A., Mcintosh, A., and Atchison, G., 1977. Sublethal effects of

32

heavy metal contaminated sediment on midge larvae (Chironomus tentans) .
Hydrobiol. 56:153-156.

Wentsel, R., A. Mcintosh, and W.P. McCafferty, 1978. Emergence of the midge
Chironomus tentans when exposed to heavy metal contaminated sediment.
Hydrobiol 57:195-196.

White, D.S., 1984. Redistribution of sediment-bound toxic organics by benthic
invertebrates. In: The cycling of toxic organic substances in the Great
Lakes Ecosystem. University of Michigan, University of Minnesota, Michigan
State University, Algonne National Lab and Oak Ridge National Lab. Annual
Report to NOAA pp. 76-78.

Wiederholm, T., A. Wiederholm, and G. Milbrink, 1987. Bulk sediment bioassays
with five species of freshwater oligochaetes. Water Air Soil Poll.
36:131-154.

33

TABLE 1: Marine and Freshwater Organisms Used
in Acute Toxicity Tests with Sediments

SPECIES

DURATION
(d=days)

APPARATUS

REFERENCE

MARINE

Rhepoxynius abronius

lOd

Static,

Swartz et al . 1979

Amphipods

f lowthrough

Rhepoxynius abronius

Static

Swartz et al . 1984

Parahausterius sp.

Static

Swartz et al. 1985b

Parahausterius sp.

lOd

Static

Shuba et al. 1978

Copepods

Arcartia tonsa

4d

Static

Shuba et al . 1978

Isopods

Tigriopus californicus

Shuba et al. 1978

Sphaeroma

quadrldentatum

lOd

Static

Shuba et al . 1978

Shrimp

Palaemonetes puglo
P. vulgaris

4-15d

Static

Shuba et a1 . 1978

Crangon septemsplnosa

4-12d

Static

Hcleese & Metcalfe 1980

Polychaetes

Nereis virens

4-12d

Static

Mcleese et al. 1982

Glycinde picta

lOd

Flowthrough

Swartz et al . 1979

Bivalves

Macoma inquinata

lOd

Flcwthrough

Swartz et al. 1979

Protothaca staminea

lOd

Flowthrough

Swartz et al . 1979

Mya arenaria

2d

Static

Tsai et al. 1979

Fishes

Fundulus heteroclltus

Leiostomus xanthurus

2d

Static

Tsai et al. 1979

34

TABLE 1: Marine and Freshwater Organisms Used
in Acute Toxicity Tests with Sediments (Cont'd)

SPECIES

DURATION
(d=days)

APPARATUS

REFERENCE

FRESHWATER

Daphnia magna

2d

Recycl ing

Malueg et al . 1983

Water Fleas

D^ magna

2d

Static

Cairns et al . 1984

D^ magna

3d

Recycling

Prater & Hake 1980

D. magna

3d

Recycling

Prater & Anderson 1977

D. magna

2d

Recycl ing
Static

USEPA .1988

Amphipods

Hyallela azteca

lOd

Static

Nebeker et al . 1984

Pontoporeia affinis

3d

Static

Nebeker et al . 1986

Gammarus lacustris

lOd

Static

Nebeker et al. 1984

OUgochaetes

unspecified

3d

Static

Keilly et al . 1988

Mayflies

Hexagenia limbata

lOd

Static

Lomas & Krantzberg 1988

Hexagenia limbata

Sd

Recycling

Malueg et al . 1983

Hexagenia limbata

3d

Recycling

Prater & Anderson 1972

Hexagenia rigida

7d

Static

Friesen et al . 1983

Midges

Chironomus

lOd

Static

Nebeker et al . 1984

Chironomus

lOd

Static

Cairns et al . 1984

Chironomus

Static

Marking et al. 1981

Chironomus

Static

Gagnon & Beaton 1971

Crayfish

Orconectes virilis

lOd

Static
Flowthrough

Leonhard 1979

Oligochaetes

Tubifex tubifex
Limnodrilus

hoffmeisteri

3d

Static

McMurtry 1982

?R

TABLE 1: Marine and Freshwater Organisms Used
in Acute Toxicity Tests with Sediments (Cont'd)

SPECIES

DURATION
(d=days)

APPARATUS

REFERENCE

FRESHWATER (Cont'd)

4d

Static

Birge et al. 1987

Fish Micropterus salmoides

Carassius auratus

Salmo gairdneri
Pimephales pronelas

lOd

Static

Lomas & Krantzberg 1988

P. promelas

3d

Recycling

Prater & Anderson 1977b

P. promelas

lOd

Flowthrough

Mac et al. 1984

C. auratus

6-7d

Static

Francis et al. L984

M. salmoides

FABLE 2: Chronic Endpoints Employed

For Various

Orcc'isms Used in Sediment

Jioassays

ENDPOINT

ORGANISM

AUTHOR

iGrowth Inhibition

Midges, Chironomus decorus

Kosalwat & Knight 1987

C. tentans

Wentsel et al. 1977

C. tentans

Nebeker et al. 1984

C. riparis

Powlesland i George 1986

Paratanytarsus parthenogeneticus

Hatakeyama & Yusuno 1981

Tanyarsus dissimilis

Anderson et al. 1980

Oligochaetes, Tubifex tubifex,

Wiederholm et al. 1987

Potamothrix hammoniensis

Limnodrilus hoffmeisteri

L. udekemianus, L. claparedeanus

Mayflies. Hexagenia limbata

Krantzberg (unpubl. data)

Amphipods, Hyallela azteca

de March 1979

Fish, Pimephales promelas

Brungs et al. 1976

Salmo gairdneri

Macdonald 1979

Coregonus clupeaformis

P. prone la

LeBlanc & Suprenant 1985

^ull or Partial

Life Cycle

Waterfleas Daphnia magna

Nebeker et al . 1986

Completion

0. magna

Biesinger & Christensen 1972

Midges C. tentans

Wentsel et al . 1978

P. parthenogeneticus

Hatakeyama & Yasuno 1981

P. parthenogeneticus

LeBlanc & Suprenant 1985

C. riparis

Powlesland & George 1986

C. tentans

Nebeker et al. 1988

C. riparis

Ingersoll et al. 1987

37

TABLE 2: Chronic Endpoints Employed for Various
Organisms Used in Sediment Bioassays (Cont'd)

ENDPOINT

ORGANISM

AUTHOR

Full or Partial

Life Cycle (Cont'd)

Amphipods H. azteca

Ingersoll et al. 1987

Gammarus pseudol imnaeus

Nebeker & Puglisi 1974

Oligochaetes, Niad

Rodgers (pers. comm.)

Caddisflies, Clistoronia magnifica

Nebeker et al. 1984b

Fish Cyprinodon variegatus

Hansen et al . 1987

Burrowing

Oligochaetes, Stylodrilus
heringianus, Potamothrix
vejdovski, Limnodrilus
hoffmeistri

White 1984

Clams, Protothaca staminea

Phelps et al. 1983

Amphipods, Rhepoxynius abronius

Swartz et al 1985

Avoidance

Amphipods, Pontoporeia hoyi
Gammarus

Gagnon & Beeton 1971

Midges, Chironomus

Gagnon & Beeton 1971

C. tentans

Wentsel et al. 1977

Oligochaetes Tublfex tublfex

McMurtry 1982

L. hoffmeistri

Bivalves Macoma balthica

McGreer 1979

Fish Coregonus clupeaformis

Scherer 1979

Salmo gairdneri

Respiration

Oligochaetes, T. tubifex

Brkovic-Popovic and
Popovic 1977

Crayfish Orconectes virils

Anderson et al . 1978

38

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40

TABLE 5 Effect of Settling Time on Growth of Hexagenia 1 imbata
Values in parentheses are standard deviations

SEDIMENT

TORONTO STP

RICE LAKE

CONTROL

KEASUREMENT

SEHLING TIME

5h

24h

120h

5h

24h

120h

5h

24h

Initial

11

19

19

16

20

17

22

23

B i or.a s s

(3)

(4)

(1)

(2)

(4)

(2)

(2)

(3)

(mg)

Percent Biomass

-3

-2

25

17

30

25

103

113

Change

(0.9)

(0.1)

(7)

(2)

(6)

(10)

(8)

(3)

(Day 10)

Growth

103

102

78

93

73

32

n.a.2

n.a.

Inhibition

(Day 10)^

Percent

4

6

12

0

0

0

0

0

Mortality

(5)

(7)

(10)

(0)

(0)

(0)

(0)

(0)

(Day 10)

Percent Biomass

4

18

69

42

77

129

163

159

Change

(9)

(10)

(3)

(9)

(25)

(50)

(10)

(15)

(Day 21)

Growth

97

89

57

74

52

19

n.a.

n.a.

Inhibition

(Day 21)

Percent

7

12

12

8

8

0

0

0

Mortality

(9)

(10)

(10)

(11)

(11)

(0)

(0)

(0)

(Day 21)

* relative to controls
2 not applicable

41

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TABLE 11: Effect of feeding on growth of Pimephales promelas

exposed to clean and contaminated sediments.
Values in parentheses are standard deviations

SEDIMENT

RICE

LAKE

ST. MARYS

CONTROL

MEASUREMENT

TREATMENT

FED

UNFED

FED UNFED

FED

UNFED

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392

366

353 332

344

368

(tng)

(47)

(79)

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5

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(2)

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