FIELD VERIFICATION PROGRAM
(AQUATIC DISPOSAL)
TECHNICAL REPORT D-85-8
APPLICATION OF LABORATORY POPULATION
RESPONSES FOR EVALUATING THE
EFFECTS OF DREDGED MATERIAL
by
John H. Gentile, K. John Scott, Suzanne Lussier,
Michele Redmond
Environmental Research Laboratory
US Environmental Protection Agency
Narragansett, Rhode Island 02882
September 1985
Final Report
Approved For Public Release; Distribution Unlimited
Prepared for DEPARTMENT OF THE ARMY
US Army Corps of Engineers
Washington, DC 20314-1000
and US Environmental Protection Agency
Washington, DC 20460
Monitored by Environmental Laboratory
US Army Engineer Waterways Experiment Station
PO Box 631, Vicksburg, Mississippi 39180-0631
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Destroy this report when no longer needed. Do not return
it to the originator.
The findings in this report are not to be construed as an official
Department of the Army position unless so designated
by other authorized documents.
The contents of this report are not to be used for
advertising, publication, or promotional purposes.
Citation of trade names does not constitute an
official endorsement or approval of the use of
such commercial products.
The D-series of reports includes publications of the
Environmental Effects of Dredging Programs:
Dredging Operations Technical Support
Long-Term Effects of Dredging Operations
Interagency Field Verification of Methodologies for
Evaluating Dredged Material Disposal Alternatives
(Field Verification Program)
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SUBJECT: Transmittal of Field Verification Program Technical Report Entitled
"Application of Laboratory Population Responses for Evaluating the
Effects of Dredged Material"
TO: All Report Recipients
1. This is one in a series of scientific reports documenting the findings of
studies conducted under the Interagency Field Verification of Testing and
Predictive Methodologies for Dredged Material Disposal Alternatives (referred
to as the Field Verification Program or FVP). This program is a comprehensive
evaluation of environmental effects of dredged material disposal under condi-
tions of upland and aquatic disposal and wetland creation.
2. The FVP originated out of the mutual need of both the Corps of Engineers
(Corps) and the Environmental Protection Agency (EPA) to continually improve
the technical basis for carrying out their shared regulatory missions. The
program is an expansion of studies proposed by EPA to the US Army Engineer
Division, New England (NED), in support of its regulatory and dredging mis-
sions related to dredged material disposal into Long Island Sound. Discus-
sions among the Corps' Waterways Experiment Station (WES), NED, and the EPA
Environmental Research Laboratory (ERLN) in Narragansett, RI, made it clear
that a dredging project at Black Rock Harbor in Bridgeport, CT, presented a
unique opportunity for simultaneous evaluation of aquatic disposal, upland
disposal, and wetland creation using the same dredged material. Evaluations
were to be based on technology existing within the two agencies or developed
during the six-year life of the program.
3. The program is generic in nature and will provide techniques and inter-
pretive approaches applicable to evaluation of many dredging and disposal
operations. Consequently, while the studies will provide detailed site-
specific information on disposal of material dredged from Black Rock Harbor,
they will also have great national significance for the Corps and EPA.
4. The FVP is designed to meet both Agencies' needs to document the effects
of disposal under various conditions, provide verification of the predictive
accuracy of evaluative techniques now in use, and provide a basis for deter-
mining the degree to which biological response is correlated with bioaccumula-
tion of key contaminants in the species under study. The latter is an
important aid in interpreting potential .biological consequences of bioaccumu-
lation. The program also meets EPA mission needs by providing an opportunity
to document the application of a generic predictive hazard-assessment research
strategy applicable to all wastes disposed in the aquatic environment. There-
fore, the ERLN initiated exposure-assessment studies at the aquatic disposal
site. The Corps-sponsored studies on environmental consequences of aquatic
disposal will provide the effects assessment necessary to complement the EPA-
sponsored exposure assessment., thereby allowing ERLN to develop and apply a
hazard-assessment strategy. While not part of the Corps-funded FVP, the EPA
exposure assessment studies will complement the Corps' work, and together the
Corps and the EPA studies will satisfy the needs of both agencies.
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SUBJECT: Transmittal of Field Verification Program Technical Report Entitled
"Application of Laboratory Population Responses for Evaluating the
Effects of Dredged Material"
5. In recognition of the potential national significance, the Office, Chief
of Engineers, approved and funded the studies in. January 1982. The work is
managed through the Environmental Laboratory's Environmental Effects of
Dredging Programs at WES. Studies of the effects of upland disposal and
wetland creation are being conducted by WES and studies of aquatic disposal
are being carried out by the ERLN, applying techniques worked out at the
laboratory for evaluating sublethal effects of contaminants on aquatic organ-
isms. These studies are funded by the Corps while salary, support facilities,
etc., are provided by EPA. The EPA funding to support the exposure-assessment
studies followed in 1983; the exposure-assessment studies are managed and
conducted by ERLN.
6. The Corps and EPA are pleased at the opportunity to conduct cooperative
research and believe that the value in practical implementation and improve-
ment of environmental regulations of dredged material disposal will be con-
siderable. The studies conducted under this program are scientific in nature
and will be published in the scientific literature as appropriate and in a
series of Corps technical reports. The EPA will publish findings of the
exposure-assessment studies in the scientific literature and in EPA report
series. The FVP will provide the scientific basis upon which regulatory
recommendations will be made and upon which changes in regulatory implementa-
tion, and perhaps regulations themselves, will be based. However, the docu-
ments produced by the program do not in themselves constitute regulatory
guidance from either agency. Regulatory guidance will be provided under
separate authority after appropriate technical and administrative assessment
of the overall findings of the entire program.
Choromokos, Jr77~?h.D. , P.E.
Director, Research and Development
U. S. Army Corps of Engineers
Bernard D. Goldstein, M.D.
Assistant Administrator for
Research and Development
U. S. Environmental Protection
Agency
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Unclassified
SECURITY CLASSIFICATION OF THIS PAGE (When Data Entered)
REPORT DOCUMENTATION PAGE
READ INSTRUCTIONS
BEFORE COMPLETING FORM
. REPORT NUMBER
Technical Report D-85-8
2. GOVT ACCESSION NO
3. RECIPIENT'S CATALOG NUMBER
4. TITLE (and Subtitle)
APPLICATION OF LABORATORY POPULATION RESPONSES
FOR EVALUATING THE EFFECTS OF DREDGED MATERIAL
5. TYPE OF REPORT & PERIOD COVERED
Final report
6. PERFORMING ORG. REPORT NUMBER
7. AUTHORf*.)
John. H. Gentile, K. John Scott, Suzanne Lussier,
Michele Redmond
8. CONTRACT OR GRANT NUMBERf*)
9. PERFORMING ORGANIZATION NAME AND ADDRESS
US Environmental Protection Agency
Environmental Research Laboratory
Narragansett, Rhode Island 02882
10. PROGRAM ELEMENT, PROJECT, TASK
AREA & WORK UNIT NUMBERS
Field Verification Program
(Aquatic Disposal)
11. CONTROLLING OFFICE NAME AND ADDRESS
DEPARTMENT OF THE ARMY, US Army Corps of Engineers,
Washington, DC 20314-1000 and US Environmental
Protection Agency, Washington, DC 20460
12. REPORT DATE
September 1985
13. NUMBER OF PAGES
86
14. MONITORING AGENCV NAME A ADDRESSf/f different Irom Controlling Olllce)
US Army Engineer Waterways Experiment Station
Environmental Laboratory
PO Box 631, Vicksburg, Mississippi 39180-0631
IS. SECURITY CLASS, (ol thle report)
Unclassified
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Available from National Technical Information Service, 5285 Port Royal Road,
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19. KEY WORDS (Continue on reverie tide If necenary and Identify by block number)
20. ABSTRACT (Canttuu* «n rcrar** etja U n*cw««7 and Identity try block number)
Studies were conducted to determine the effect of Black Rock Harbor (BRH)
dredged material on the survival, growth, reproduction, and population
responses of the benthic amphipod, Ampelisca abdita, and the epibenthic shrimp,
Mysidopsis bahia. Exposure system designs are described that permit continuous
dosing of suspended solids at concentrations of 300 mg/1 while proportionally
mixing contaminated and reference sediments with reliability and precision.
(Continued)
FORM ....
» JAM 73 1*73
EDITION OF » NOV 6S IS OBSOLETE
Unclassified
SECURITY CLASSIFICATION OF THIS P*r»E (Whan Data Entered)
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Unclassified
SECURITY CLASSIFICATION OF THIS PAGEfWi*" Data Entered)
20. ABSTRACT (Continued),
Ninety-six-hour LC50 values were 290 mg BRH/1 for M. bahia and 82 rag BRH/1
for A., abdita, with reproducibility and precision being excellent. Chronic
exposure indicated that survival was significantly decreased at 150 mg BRH/1
for M. bahia and at 12.5 mg BRH/1 for A. abdita. Growth was a sensitive indica
tor of stress for A. abdita whose effects were reflected in delays in repro-
duction in A. abdita but not for M. bahia. Reproduction was the most sensitive
chronic response measured for both species. The number of ovigerous females
of A. abdita were significantly reduced at 4.0-5.0 mg BRH/1, while the number
of young produced in M. bahia was reduced at 32 mg BRH/1. The population
parameters, intrinsic rate of growth, and multiplication rate per generation
measured for _M. bahia and A. abdita were significantly depressed at 42 and
4.7 mg BRH/1 sediments, respectively.
This investigation is the first phase in developing field-verified bio-
assessment evaluations for the Corps of Engineers and the US Environmental
Protection Agency regulatory program for dredged material disposal. This
report is not suitable for regulatory purposes; however, appropriate assessment
methodologies that are field verified will be available at the conclusion of
this program.
Unclassified
SECURITY CLASSIFICATION OF THIS PAGEfHTlon Data Entered)
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PREFACE
This report describes work performed by the U.S. Environmental
Protection Agency (EPA), Environmental Research Laboratory, Narragansett,
R.I. (ERLN), as part of the Interagency Field Verification of Testing
and Predictive Methodologies for Dredged Material Disposal Alternatives
Program (Field Verification Program - FVP). This program is sponsored by
the Office, Chief of Engineers (OCE), and assigned to the U.S. Army
Engineer Waterways Experiment Station (WES), Vicksburg, Mississippi. The
OCE Technical Monitors for FVP were Drs. John R. Hall and William L. Klesch.
The objective of this interagency program is to field, verify existing
predictive techniques for evaluating the environmental consequences of
dredged material disposal under aquatic, wetland, and upland conditions.
The aquatic portion of the FVP study is being conducted by ERLN, with the
wetland and upland portion being conducted by WES.
The principal ERLN investigators for this aquatic study and authors of
this report were Drs. John H. Gentile and K. John Scott and Ms. Suzanne
Lussier and Ms. Michele Redmond. Technical support was provided by Mr. John
Sewall and Ms. Ann Kuhn. A special note of recognition is extended to Dr.
Clifford Katz for his assistance in designing the appropriate life-cycle
graphs, defining the data requirements, and performing the linear matrix
analysis. The authors wish to thank Dr. Hal Caswell for providing the
theoretical and conceptual framework for using life-cycle graphs with the
species used in this study. Appreciation is also extended to Ms. Martha
Marcy for the use of her computer program for life-table analysis; Dr.
James Keltshe for statistical design; Mr. Jeffery Rosen and Ms. Pam Sherman
for data management; and Ms. Catherine Leavene for manuscript preparation.
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The EPA Technical Director for the FVP was Dr. John H. Gentile; the
Technical Coordinator was Mr. Walter Galloway; and the Project Manager was
Mr. Allan Beck.
The study was conducted under the direct WES management of Drs. Thomas M.
Dillon and Richard Peddicord and under the general management of Dr. G. Richard
Lee, Chief, Contaminant Mobility and Criteria Group; Mr. Donald L. Robey,
Chief, Ecosystem Research and Simulation Division; and Dr. John Harrison,
Chief, Environmental Laboratory. The FVP coordinator was Mr. Robert L. Lazor,
and the EEDP Managers were Mr. Charles C. Calhoun, Jr., and Dr. Robert M.
Engler.
COL Tilford C. Creel, CE, and COL Robert C. Lee, CE, were Commanders and
Directors of WES during the conduct of the study. COL Allen F. Grum, USA,
was Director of WES during the preparation and publication of this report.
Mr. Fred R. Brown and Dr. Robert W. Whalin were Technical Directors.
This report should be cited as follows:
Gentile, J.H., et al. 1985. "Application of Laboratory
Population Responses for Evaluating the Effects of Dredged
Material," Technical Report D-85-8, prepared by US Environ-
mental Protection Agency, Narragansett, R.I., for the US Army
Engineer Waterways Experiment Station, Vicksburg, Miss.
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CONTENTS
PREFACE ...................... . ....................................... 1
LIST OF FIGURES [[[ 4
LIST OF TABLES [[[ 5
PART I : INTRODUCTION ........................ . ....................... 6
PART II: METHODS AND MATERIALS ...................................... 10
General Methods ......................... . ....................... 10
Test Methods for Mysidopsis bahia ........ . ...................... 15
Te st Methods for Ampelisca afadita ............................... 26
PART III: RESULTS AND DISCUSSION .................................... 34
Mysidopsis bahia ................................................ 34
Ampelisca abdita ................................................ 46
PART IV: CONCLUSIONS ................................................ 67
REFERENCES ............................................. . ............. 70
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LIST OF FIGURES
No. Page
1 Central Long Island Sound disposal site and South
reference site. 10
2 Black Rock Harbor, Connecticut, source of dredged material.... 11
3 Sediment dosing system with chilled water bath and argon
gas supply 14
4 Suspended sediment feedback control loop and strip chart
recorder 16
5 Suspended sediment proportional diluter for M.
bahia. 17
6A Proportional diluter distribution chamber configuration
for short-term tests with M. bahia 19
6B Proportional diluter distribution chamber configuration
for long-term tests with M. bahia 19
7 Exposure chamber design for M. bahia....» 20
8 Suspended sediment proportional diluter system used for
short- and long-term studies with A. abdita 27
9 Acute exposure chamber for short-term tests with A^. abdita.... 29
10 Chronic exposure chamber for long-term tests with A. abdita... 30
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LIST OF TABLES
No. Page
1 Experimental approach for short-term studies 12
2 Results of definitive short-term 96-hr acute toxicity
tests with juvenile M. bahia 35
3 Analysis of treatment differences for short-term
acute tests with juvenile M._ bahia. 38
4 Growth and reproductive results for chronic tests with
M. bahia. 39
5 Population responses for life cycle tests with M.. bahia 44
6 Ampelisca abdita 96-hr mortalities and suspended particulate
concentrations for REF and BRH sediments 47
7 Dry weight and Ampelisca mortality for an 18-day exposure
to BRH in the suspended phase preliminary chronic test 48
8 Suspended particulate dry weight, standard deviation, and
Ampelisca mortality for 28- and 45-day exposure to BRH
suspended phase in Chronic Test 1 49
9 Suspended particulate dry weight and standard deviation for
Chronic Test 2 at exposure durations of 32 and 58 days 50
10 Suspended particulate concentrations in the dosing system 50
11 Estimated A. abdita mortality in Chronic Test 2 53
12 Number of A. abdita in each sex category for 28- and 45-day
exposures Ifo BRH suspended particulates in Chronic Test 1 57
13 Number of A. abdita in each sex category for 32- and 58-day
exposures tfo BRH suspended particulates in Chronic Test 2 58
14 Mean percent of A. abdita in each sex category for 32- and 58-
day exposures to~BRH suspended particulates in Chronic Test 2 59
15 Mean size (mm) of A. abdita exposed to BRH suspended
paticulates in Experiments 1 and 2 60
16 Mean number of eggsVovigerous females ± standard error for
A. abdita exposed to BRH suspended particulates for 45 days in
Experiment 1 and 58 days in Experiment 2 63
17 Population responses for life-cycle tests with A. abdita 65
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APPLICATION OF LABORATORY POPULATION RESPONSES
FOR EVALUATING THE EFFECTS OF DREDGED MATERIAL
PART I: INTRODUCTION
1. The regulation of potential pollutants in aquatic environments is
generally based upon toxicological information involving the quantification
of a biological response with a pollutant concentration for some finite
period of exposure. Traditionally, decisions have been made utilizing
acute toxicity data where the exposure period is 96 hrs and the measured
biological response is lethality (Sprague 1976). It is well recognized
that this type of information while useful is insufficient to identify
acceptable nontoxic concentrations that are protective of chronic effects
on an organism's growth and reproduction (Mount 1968; Sprague 1971, 1976).
This limitation has been addressed by the development of chronic toxicity
tests designed to assess pollutant effects on growth, survival, and repro-
duction over long periods of exposure, often an entire life cycle. Even
though chronic toxicity tests measure effects on survival, growth, or
reproduction over longer time intervals (Rand 1980; Hansen and Carton
1982), until these endpoints are coupled in a predictive way to popula-
tions they are still tests at the individual species level of biological
organization.
2. The National Research Council's report on "Testing for the
Effects of Chemicals on Ecosystems" (1981) recommends that appropriate
and relevant decisions regarding the release of potentially toxic chemicals
into the environment be based upon a hierarchy of biological tests with the
population being particularly crucial to such an assessment. Relating
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short-term toxicant effects to population dynamics poses two problems:
one due to time scales and one due to the diversity of toxicant effects.
Since population changes take place on a time scale of generations, the
direct approach of simply applying a toxicant to a population and observing
the resulting dynamics is feasible only for the most rapidly growing
organisms. Even when such an approach is possible, it reveals very
little about the mechanisms generating the population response. The only
way around this difficulty is to infer the population consequences from
data on the responses of individual organisms to the toxicant. Since
population growth is obviously in some sense the result of the survival,
growth, maturation, development, and reproductive rates of individuals,
this Inferential approach has some promise.' Using it, however, requires
a determination of which individual characteristics are relevant, and how
their population consequences are to be inferred. By adapting demographic
techniques originally developed for the study of human populations,
population ecologists can calculate a variety of population statistics
from data collected on individual organisms (Hutchinson 1978).
3. Classical demography originated in the seventeenth century with
the introduction of the life table as a means of integrating age-specific
mortality and fecundity. Mathematically, separate measures of survival
and fecundity may be linked together and used to estimate the intrinsic
growth rate of a population.
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This type of analysis has been frequently used in studies of the responses
of both natural and laboratory populations to different environmental
conditions (Birch 1948; Deevey 1947; Frank 1960; Hutchinson 1978), as well
as in studies of human demography (Keyfitz and Flieber 1968). The use of
life-tables for assessing the effects of chronic concentrations of pollut-
ants, however, is limited to only a few studies (Marshall 1978; Hummon 1974;
Winner and Farrell 1976; Winner et al. 1977; Daniels and Allan 1981; Gentile
et al. 1982; Gentile et al. 1983).
4. The unique feature of demographic theory is that it solves the
problems of time scale and diversity of effects. It is no longer necessary
to follow the dynamics of the population for multiple generations. The
estimation of a cohort life table requires only a single generation of
observation, and there are techniques to speed up this process in cases
where individuals can be aged and marked (Caughley 1977). The Euler
equation for r also solves the problem of the diversity of life history
effects by specifying exactly how survival and fecundity information must
be combined to obtain an index of population growth.
5. There are three primary objectives in the aquatic portion of
the EPA/CE Field Verification Program (FVP). The first objective is to
demonstrate the applicability of the intrinsic rate of population growth
as a measure of effects of dredged material and to determine the degree of
variability and reproducibility inherent in the procedure. We are
proposing to apply this technique to Mysidopsis bahia, an epibenthic
crustacean, and Ampelisca abdita, an infaunal crustacean. This phase of
the study, Laboratory Documentation, will involve exposing these organisms
throughout their entire life cycle to suspended particulate and solid
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phases of Black Rock Harbor (BRH) dredged material and Is the subject of
this report. The second objective is to field verify the response observed
in the laboratory and thus to determine the accuracy of the laboratory
prediction. Consequently, this portion of the study is referred to as
the Field Verification Phase. The third objective is to determine the
degree of correlation of tissue residues resulting from the bioaccumulatlon
of contaminants from dredged material and ecologically significant altera-
tions in organism viability as observed in both the laboratory and the
field. The second and third objectives will be combined in a final report
as appropriate for the FVP due in September 1985.
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PART II: METHODS AND MATERIALS
General Methods
Sediment collection, processing, and storage
6. Two sediment types were utilized to conduct the solid and sus-
pended particulate phase tests of these studies. The reference sediment
(REF) was collected from the South reference site in Long Island Sound
(40°7.95'N and 72°52.7'W) by a Smith-MacIntyre grab (0.1 ra2), press
sieved through a 2-mm sieve, and stored at 4°C until used (Figure 1).
BLACK ROCK ft.
HARBOR
SOUTH REFERENCE
• SITE
Figure 1. Central Long Island Sound disposal site
and South reference site
Prior to dredging, contaminated sediment was collected from Black Rock
2
Harbor (BRH) (41°9'N and 73°13'W) with a gravity box corer (0.1 m ) to a
depth of 1.21 m, thoroughly mixed, press sieved through a 2-mm sieve, and
10
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refrigerated (4°C) until used (Figure 2). In all experiments, sediments
were allowed to reach test temperature and mixed prior to use.
BRIDGEPORT
N
4OOrn
Figure 2. Black Rock Harbor, Connecticut,
source of dredged material
Experimental design
7. To meet the objectives of this study, a series of initial
experiments was conducted to characterize those aspects of exposure
analogous to the field exposure conditions at the disposal site. The
intent, however, was not to simulate environmental exposure conditions,
but rather to include the necessary exposure components, which reflect a
level of resuspension containing a mixture of contaminated and uncontam-
inated sediments.
8. Table 1 summarizes the experimental approach for the initial
short-term testing. These studies were conducted to characterize the
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Table 1
Experimental Approach for Short-Term Studies
Task Description
Suspended Solids
Concentration
Reference Sediment
Concentration
BRH Sediment
Concentration
Solid Phase
Determine the effect of
variable uncontaminated
(reference) suspended
solids concentrations
on measured biological
response
Variable over a
range of
10-400 mg/1
Variable over a
range of
10-400 mg/1
None
Reference sediment
Determine the effect of
variable contaminated
(BRH) suspended solids
over a range of concen-
trations previously
determined (above) to
produce no effect on
the measured biological
response
Variable over a
previously deter-
mined no-«ffect
range of concen-
trations
None
Variable over
no-effect range
from previous
experiment
Reference sediment
or no-effect con-
centration of BRH
and/or REF mixture
Determine the effect of
a range of BRH sediment
concentrations coupled
with reference sediment
to maintain a constant
suspended solids con-
centration
Fixed concen-
tration
Variable in pro-
portion with BRH
sediment
Variable in
proportion with
REF sediment
Reference sediment
or same proportion
as the suspended
solids tests
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potential contribution of two interrelated exposure variables, total
suspended particulates and proportion of contaminants, on the measured
biological responses. The first task was to determine the concentration
of uncontaminated total suspended solids that each test species could
tolerate. This was necessary to ensure that if biological effects were
observed, they were caused by the contaminated dredged material and not by
the particle density. Selecting a "no-effect" concentration from this
study, a second test was conducted using only BRH-contaminated sediments
to determine a contaminant dose-response relationship. The final test in
this series was analogous to a field exposure which would have a fixed
suspended particulate concentration, with variable amounts of BRH and
reference sediments. The highest proportion of BRH sediment represents
the center of the disposal mound with decreasing BRH sediment toward the edge
where primarily reference sediment would predominate. These experiments
form the basic design employed in the long-term chronic exposure from
which population responses were determined for the epibenthic mysid
shrimp, Mysidopsis bahia, and the infaunal amphipod, Ampelisca abdita.
Statistical analysis
9. Acute toxicity data were analyzed using probits, moving average,
binomial, and graphical methods, as appropriate (Stephen 1977). Analysis
of variance was used to analyze survival, growth, and reproductive data
from all tests. Significant treatment differences were identified from
Dunnett's and Tukey-Kramer's pairwise comparison tests (Snedecor and
Cochran 1980). In addition, reproductive data for A. abdita were evaluated
by analysis of covariance•to account for differences in female size.
13
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Sediment dosing system
10. Implementation of the experimental design required the con-
struction of two identical sediment dosing systems to simultaneously
provide either BRH or REF material as suspended sediment. The dosing
systems (Figure 3) consisted of conical-shaped slurry reservoirs placed
SEPARATORY
FUNNEL
DELIVERY
MANIFOLD
DOSING
VALVE
TO EXPOSURE
SYSTEM
RETURN
MANIFOLD
SLURRY
RESERVOIR
Figure 3. Sediment dosing system with chilled water bath
and argon gas supply
in a chilled fiberglass chamber, a diaphragm pump, a 4-1 separatory
funnel, and several return loops that directed the particulate slurry
through dosing valves. The slurry reservoirs (40 cm diam x 55 cm high)
contained 40 1 of slurry comprised of 37.7 1 of filtered seawater and 2.3
1 of either BRH or REF sediment. The fiberglass chamber (94 cm x 61 cm x
79 cm high) was maintained between 4° and 10°C using an externally chilled
water source. (The slurry was chilled to minimize microbial degradation
during the test.) Polypropylene pipes (3.8 cm diam) placed at the bottom
14
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of the reservoir cones were connected to the diaphragm pumps (16 to 40
1/min capacity) that had Teflon® diaphragms. These pumps were used to
circulate the slurry but minimize abrasion so that the physical properties
and particle sizes of the material remained as unchanged as possible.
11. The separatory funnel was connected to the pump and returned
to the reservoir by polypropylene pipes. The separatory funnel served
two functions: (a) to ensure that a constant head pressure was provided
by the overflow, and (b) to serve as a connection for the manifold located
4 cm below the constant head level. The manifold served to distribute
the slurry by directing a portion of the flow from the funnel (through
6-mm inside diameter polypropylene tubes) through the Teflon® dosing
valves (Figure 3) and back to the reservoir. At the dosing valves, the
slurry was mixed with seawater to provide the desired concentrations for
the toxicity tests. Argon gas was provided at the rate of 200 ml/min to
the reservoir and separatory funnel to minimize oxidation of the sediment/
seawater slurry. Narragansett Bay seawater filtered (to 15 u) through sand
filters was used. The dosing valves were controlled by a microprocessor
that was connected to a transmissometer (Figure 4) in the preliminary
toxicity studies. The microprocessor can be programmed to deliver a pulse
with a duration of 0.1 sec up to continuous pulse delivery and at intervals
from once every second to once every hour.
Test Methods for M. bahia
Culture
12. Mysidopsis bahia is an epibenthic crustacean important in estu-
arine and marine food webs. The life cycle of this species lends itself
to population studies for two reasons: the life cycle is short, being
15
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STRIP CHART
RECORDER
MICRO-
PROCESSOR
CONTROL
BOX
SLURRY -*•
P^
DOSING VALVE
RETURN TO
RESERVOIR
SOLENOID
EXPOSURE SYSTEM
Figure 4.
TRANSMISSOMETER
Suspended sediment feedback control loop
and strip chart recorder
completed in 25 days at 25°C, and because the young are carried in a
brood pouch, reproductive processes can be easily monitored and quantified*
13. Mysidopsis bahia were cultured in flow-through 76-1 glass
aquaria continuously supplied with filtered (15 u) natural seawater
at a salinity of 28 + 2 ppt and 25 + 2°C. A photoperiod of 14LJ10D was
maintained by microprocessor to simulate dawn and dusk. Flow rates of
200 ml/min provided a 99 percent volume exchange every 24 hr. Sub-gravel
16
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filters were used to provide aeration and a feeding current with a 25.4-mm-
deep dolomite substrate.
14. Cultures were fed continuously, ad libitum, with 24-hr posthatch
Artemia salina (reference strain, Sorgeloos 1981) at a rate of 7 x 10^
nauplii/day for each 76-1 culture.
Exposure system design
15. The suspended sediment proportional diluter (Figure 5) was
(from 3-way valves)
BRH-s lurry REF-s lurry
solenoid
valve
sea water
cpunter
i
relay switch"
distribution
chambers
"Z-float chamber
float switch
Figure 5. Suspended sediment proportional diluter
for M. bahia
17
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designed to mix small quantities of concentrated slurries of suspended
marine sediments (10-20 g/1 from the sediment dosing system with seawater
to produce two dilute sediment suspensions in the mg/1 range. It then
combines slurries of different types (e.g. , REF and BRH sediment suspen-
sions) proportionally to maintain the same concentration of suspended
particulates with different ratios of the two sediments. It can also
function with one sediment diluted with seawater to produce a range of
suspended solids concentrations. The diluter delivers two replicates for
each of five treatments and a seawater control.
16. The distribution chamber (Figure 6) is partitioned into two
cells and works by dividing the two volumes of suspension among a number
of capillary tubes thereby delivering different volumes of each suspension
to the five splitters depending upon the number of capillary tubes draining
into them.
17. Each of the cells of the collection chamber drains its contents
of a proportioned suspension into one of the five splitters (Figure 5).
Each splitter contains two self-priming siphons, each of which takes half
the volume of suspension and delivers these portions of suspension to the
replicate animal exposure chambers.
18. In summary, the diluter system employed for quantitatively
delivering suspended solids to the population tests consists of four tiered
components. The first tier consists of the water cells which measure a
predetermined volume of seawater and three-way valves which deliver micro-
processor quantities of slurry from the sediment dosing system. The
second tier consists of the mixing chambers which combine the slurry and
the seawater to produce the desired concentrations of suspended sediments.
18
-------
BRH
suspension
V V I
a. Short-term studies
T
suspension
collection
chamber
partition
BRH
suspension
capillary tube
y^ilicone seal
uspension
collection
"chamber
b. Long-term studies
Figure 6. Proportional diluter distribution chamber
configuration for M. bahia
19
-------
The third tier includes the distribution and collection chambers where the
REF and BRH slurries are proportionally mixed to produce the five treatment
concentrations. The fourth and final tier includes the splitters where
each treatment concentration is divided into two replicates and delivered
to the exposure chambers.
Exposure chamber design
19. The animal exposure chambers (Figure 7) consist of wide-mouth
hanger-^ I
Nilex
screen
(250/j)
silicone tab-^
PVC-cenlering /^
+i
L
^ uenveiy iiuiu
water
stirring bar "ow
1\
T
C I ) ^-^'
\ outflow [JlZI s,jrrfir
Z-.umhrelln siphon
=rr outflow
m
11 inflow I
~~^^.
manifold
Figure 7. Exposure chamber design
for M. bahia
glass jars, 8.5 cm high x 7.5 cm wide with two 3.2-cm holes drilled in
opposite sides and centered 4.2 cm from the bottom. These holes are
screened with 250-u Nitex® screen netting glued with clear silicone
sealant to the inside to eliminate a ridge where test organisms could be
trapped. Four glass cups are suspended in each of twelve crystallizing
dishes (190 mm x 100 mm) by a small glass tab glued to the top rim. To
maintain the vertical position of each jar, a small drop of silicone
20
-------
sealant is placed at the bottom side of each cup just under the tab.
Each treatment (six total) consists of two replicate crystallizing dishes;
each replicate contains four observation cups for a total of eight per
treatment.
20. Suspended sediment from the diluter flows through glass delivery
tubes which empty into the center of each exposure chamber at the surface.
Drainage from each chamber is accomplished by an enclosed umbrella siphon.
When a chamber fills to about 1 cm from the top, the siphon is primed and
drains about one third of the dish. This excursion of the water level
ensures proper water circulation through the exposure cups. The enclosure
around the umbrella siphon forces water outflow to be.from the bottom of
the water column. Inflow at the top and outflow from the bottom help elim-
inate the potential for vertical size partitioning of suspended particulates,
21. The twelve exposure chambers are held in position on a fiber-
glass grating in a temperature-controlled water bath by plastic rings
3.2 cm high by 20.3 cm diameter. These rings center the dishes over
water-driven magnetic stirrers (embedded in the grating) which are used
to spin 6.4-cm x 0.64-cm Teflon®-coated spin bars in the dishes to keep
the sediment in suspension. The magnetic stirrers are driven by a mani-
fold supplied with deionized water from the temperature control bath by
Teel, epoxy-magnetic submersible pumps, Model #1P681A. This also serves
to circulate the bath to ensure uniform temperature.
22. The position of the dishes in the bath is randomized. Bath
temperature is maintained with Teflon9 heat-exchanging coils under micro-
processor control. Microprocessor controlled lighting is designed to
simulate natural day/night cycles with the florescent lights growing
21
-------
gradually brighter at dawn and dimmer at dusk. The construction materials
that contact the test solutions or the animals are glass, silicone rubber
(to cement and seal), Nitex® screen,and Teflon®.
System monitoring
23. Bioassays were conducted at a temperature of 25° + 2°C, salinity
of 28° + 2 ppt, and illumination of 1000 lux on a 14L:10D cycle. Dissolved
oxygen was measured daily with a YS1 dissolved oxygen probe. Three times
each week suspended particulate concentrations from the control and ex-
posure chambers were analyzed by dry weight determination conducted
according to Standard Methods (American Public Health Association (APHA)
1976) with the following modifications: the filters were washed with a
50-ml aliquot of delonized water before sample filtration, and then with
three 10-ml rinses of deionized water immediately after sample filtration
to remove salt.
Biological design
24. Tests were initiated with 24- to 30-hr postrelease juvenile M.
bahia which were randomly distributed into three exposure cups of five
animals each in two replicate exposure chambers randomized among the
exposure concentrations. In the second chronic test, an individual pair
of males and females was placed in each exposure cup.' Each cup was fed
24-hr posthatch reference Artemia daily. Each cup was removed and moni-
tored daily for mortality in the acute tests and both mortality and
reproduction in the life cycle tests. Test organisms could be seen most
easily over the sediment by shining an intense beam of light horizontally
from the side of the test cup at the sediment/water interface.
25. For the solid phase portion of the test exposure, sediment at
22
-------
room temperature from the reference site and from Black Rock Harbor was
stirred and shaken vigorously before being mixed to obtain the desired
percentage of BRH sediment for each treatment. The sediment was added to
the exposure cups to a depth of 2 cm and allowed to equilibrate for 1 hr
in the bioassay system in flowing test solution before animals were
introduced.
Short-term tests
26. There are two variables in these tests which can be responsible
for producing a biological response: suspended solids concentration and
contaminant concentration. In order to ensure that the reported responses
were the result of BRH contaminants, the sensitivity of M. bahia to a
range of suspended solids concentrations had to be determined using a
relatively uncontaminated reference sediment. This was followed by
assessing effects of BRH contaminants within the range of suspended solids
determined acceptable, and then combining REF- and BRH-contaminated sedi-
ments to provide a constant suspended solids concentration while varying
the BRH contaminant concentration.
27. Preliminary 96-hr flow-through range-finding tests were con-
ducted to determine the appropriate suspended particulate concentration
for the definitive acute and chronic life cycle assays. The first test
used only reference suspended participates to determine the concentration
of suspended particulate which would produce no effect on M. bahia juve-
niles. Previous tests (Rogerson et al. 1984) had determined that 25
mg/1 of reference suspended particulates had no effect on the survival of
juvenile M. bahia. Therefore, the treatments chosen for the first assay
were 102, 76.5, 51, and 25.5 mg/1 reference suspended particulates over a
23
-------
solid phase of reference sediment with a seawater control (no sediment).
28. Subsequent preliminary tests used only BRH suspended material
to determine the concentration which would produce an effect on M^. bahia
juveniles in a 96-hr exposure period. The treatments chosen were: 200,
150, 100, and 50 mg/1 BRH suspended particulate over a solid phase of BRH
sediment with a seawater control (no sediment). The experiment was re-
peated in order to obtain accurate and consistent dosing and mixing of
the suspended sediment.
29. The definitive acute test used a 300 mg/1 suspended particulate
concentration which consisted of individual or combinations of reference
and BRH material with a matching solid phase sediment in the following
percentages: 100%BRH/0%REF, 75%BRH/25%REF, 50%BRH/50%REF, 25%BRH/75%REF,
0%BRH/100%REF, and a seawater control (no sediment).
Long-term tests
30. Replicate life cycle tests were conducted to assess the effects
of suspended particulate exposures of percentage combinations of BRH and
REF sediments on the survival, growth, and reproduction of M. bahia. To
determine which response parameters were the best indicators of stress,
the developmental stages related to reproductive functions were examined
in detail. Specifically, time to sexual maturity, appearance of embryos
in brood sacs, and time to first brood release were determined for all
treatments. Two approaches were used to quantify productivity. First,
the number of juveniles released per female was determined for each treat-
ment. From this the fecundity and reproductive variability between
females could be determined. A second more integrative approach was to
determine the total productivity for each treatment. To estimate total
24
-------
productivity, it was necessary to normalize differences in the number of
females available per exposure concentration. The number of available
female reproductive days (AFRD) was calculated by multiplying the number
of sexually mature females by the number of days each survived during the
reproductive period. Productivity was estimated by calculating the
following ratio: number of young produced per AFRD.
31. Life tables were used to calculate age-specific survivorship
for controls and exposure concentrations (Birch 1948). Starting with an
initial number of newborn females, the percentage of this initial popu-
lation alive at every age was calculated by sequentially subtracting the
percentage of deaths of each age. The fraction surviving at age x gives
the probability that an average newborn will' survive to that age (which is
designated lx ).
32. Age-specific fecundity m^ is the number of female offspring
produced by a female of age x during a designated age period. Specifically,
the types of data collected included the number of juveniles released per
day and the number of sexually mature females.
33. Age-specific survivorship lx and fecundity m^ were used to
calculate, by successive approximation, the intrinsic rate of population
growth r from the Euler equation:
Where
lx «• the probability of a female surviving to age x
mx - the number of female offspring per female of age x
produced during the interval x to x + 1
e « the natural logarithm
r - the intrinsic rate of population growth
x = the age class
25
-------
Test Methods for A. abdita
Collection
34. Ampelisca abdita is a tube-dwelling amphipod which constructs
a soft, upright, membranous tube 3 to A cm long in surface sediments. It
occurs from Maine to Louisiana from the intertidal zone to depths of 60 m.
Ampelisca is a particle feeder, ingesting either surface-deposited particles
or particles in suspension. These amp hi pods brood their young after mating
and fertilization occurs in the water column. The egg-carrying females
return to the sediment and young are released into the surrounding sediment'
They are reproductively active at 5 mm and grow to a maximum length of 7 to
9 mm. Available information indicates that each female will reproduce only
once.
35. Ampelisca were collected from tidal flats in Narrow River, a
small estuary flowing into Narragansett Bay, R.I. The sediment
containing the amphipods was immediately transported to the laboratory.
The sediment was sieved through a 0.5-mm screen and the Ampelisca collected
by flotation from the air/water interface. Since collection temperatures
were close to the experimental temperatures, no acclimation or holding
was necessary for the first long-term chronic test. For the second long-
term test, amphipods were collected from Narrow River and acclimated at
l°C/day up to 20°C. During acclimation, these animals were fed, ad libidum,
laboratory-cultured Phaeodactylum tricornutum. After the animals were
sieved from the sediment, they were sorted by size and randomly placed in
100-ml plastic beakers for subsequent distribution to the appropriate
exposure chamber.
26
-------
Exposure system design
36. The composite dosing system supplied suspensions of REF and BRH
sediments as previously described (Figure 3). The appropriate amount of
material was delivered to the amphipod dosing system via a three-way valve
which was controlled by a microprocessor. For the acute tests, each
slurry was delivered to a mixing chamber (glass 4-1 reagent bottle)
where it was initially diluted with seawater at a preset flow rate (right
side of Figure 8). The diluted suspension then passed from the bottom of
from CDS
ram COS
seawaler
|_ _ return
BRH
siphons
milling chamber
£
1.1 IL
1) '
distribution chamber
magnetic
si trier
to eiposura chambe
Figure 8.
Suspended sediment proportional diluter system used
for short- and long-term studies with A. abdita
the bottle to a distribution chamber (17 cm diam x 9 cm high) fitted with
a standpipe to maintain a constant water level. The suspension then
flowed through a siphon at a flow rate controlled by head pressure to a
collection funnel which then distributed the material to the exposure
27
-------
system. Each collection funnel had an umbrella siphon which acted as a
flow accelerator to rapidly mix materials collected in the funnels. For
the short-term acute tests, the mixing and distribution chambers with
siphons were set up in duplicate and, using either REF or BRH sediment,
one mixing chamber setup was dosed with the test sediment and the other
was fed filtered seawater only.
37. To achieve a test concentration of 100 mg/1, where the mixing
chamber concentration was 200 mg/1, the sediment mixing chamber siphon
and the seawater mixing chamber siphon would be set at equal flow rates,
e.g., 40 ml/min of each. Likewise, to achieve 50 mg/1 of the test sedi-
ment, the seawater-siphon flow rate would be three times the suspension
flow rate, e.g., 60 ml/min seawater and 20 ml/min sediment suspension.
For the chronic tests, a constant suspended particulate density was desired*
where BRH sediments were diluted with REF sediment instead of with seawater-
To achieve this, REF sediment was dosed to one mixing chamber and BRH was
dosed to the other so that there was an equal particle concentration in
each distribution chamber. As for the seawater dilutions, equal siphon
flow rates yield a 50-percent BRH concentration, a 3 to 1 REF to BRH
yields a 25-percent BRH concentration, and so on.
38. The limiting factor of this dosing design is the minimum flow
rate, 10 ml/min, that can readily be attained from the siphons. If each
exposure chamber is to receive 20 ml/min, the total flow to the collection
funnel is AO ml/min; or for a 25-percent BRH concentration, 30 ml/min of
REF sediment and 10 ml/min of BRH sediment. At 25-percent BRH, toxicity
levels were high, so a modified design was implemented (left side of
Figure 8) where the BRH concentration in the distribution chamber was set
28
-------
to 20 percent by feeding that chamber 100 ml/min of REF and 25 ml/min of
BRH at equal particle densities. The BRH concentrations of 10 and 5 per-
cent were subsequently achieved as described above.
39. The collection funnels fed the exposure chambers through a poly-
propylene tube which was fitted with a polypropylene tee with 5-nun outside
diameter glass elbows to split the flow to two exposure systems.
Exposure chamber design
40. The exposure chambers for this study were of two types, an acute
test chamber and a chronic test chamber. The acute chamber consists of a
capped glass jar with screened holes in the sides (Figure 9). Reference
EXPOSURE
CHAMBER
TO DRAIN
SEDIMENT
SUPPORT RACK
MAGNETIC
STIRRER
Figure 9. Acute exposure chamber for short-term tests
with A. abdita
sediment was filled to the bottom of the holes. The test suspension flowed
from the "funnel-tubing-tee" delivery system to a gallon jar containing
two exposure chambers. The suspension passed through the screens and out
of the cap via a glass elbow to a water trap and then to drain. The
sediment was kept in suspension by a magnetic stirrer.
29
-------
41. The chronic test chamber was a gallon jar filled with 0.75 1
of REF sediment (5 cm) (Figure 10). The test suspension was fed to the
gallon jar from the tee and was maintained in suspension by aeration. The
suspension was removed from the jar by a siphon which collected material
from just above the sediment surface. The effluent entered a water trap
with a screened standpipe which permitted monitoring of mating activity.
from dosing system
air
to drain
Figure 10. Chronic exposure chamber for long-term tests
with A. abdita
42. The flow rate to each gallon jar was 40 ml/min for the acute
tests and was reduced to 20 ml/min for the chronic tests in order to
maximize the concentration of food supply. The diatom Phaeodactylum
tricornutum was cultured (Guillard and Ryther 1962) and delivered to each
gallon jar at 1 ml/min using a Harvard peristaltic pump.
System monitoring
43. The total suspended solids concentration in the exposure system
was monitored using total dry weight as milligrams per liter. One-hundred-
milliliter samples were taken biweekly from each gallon jar using a free-
standing siphon to minimize disturbance.
30
-------
Biological design
44. As the Ampelisca were being sorted for distribution to each test
chamber, an extra container with the requisite number of Ampelisca was set
aside for initial size measurements. In both the short-term test and the
preliminary chronic test, juvenile (immature) amphipods were used. In the
two chronic tests, the dosing and exposure system design were identical;
however, the life stages that were used at the start of the test were
different. In the first test 100 subadult amphipods were used in four
replicates of each of three treatments, while in the second test 15 egg-
bearing females were used in four replicates of each of three treatments.
In the second chronic test, the number of eggs of each of 30 females were
enumerated from an initial sample. The first chronic test lasted 45 days
with an interim harvesting of two replicates from each treatment at 28 days.
The second test ran for 58 days with an interim harvesting of two repli-
cates at 32 days.
45. During the acute tests, each container was checked daily and
the number dead were enumerated and removed. In the chronic tests, the
overflow water trap was checked daily and the amphipods were enumerated
and sexed when possible. At the end of each test, all containers were
sieved and the amphipods were enumerated. Any animals not accounted for
(either removed earlier or recovered on sieving) were considered dead.
46. In addition, animals from the two chronic tests were sexed and
measured by use of a computerized digitizer and camera lucida device. The
data collected at the interim sampling period in both chronic tests in-
cluded: survival, mean size, and proportion of amphipods that were mature.
In addition, the number of eggs from each ovigerous female were enumerated
31
-------
and sized. At the termination of the test, these data were collected
along with the number of young amphipods produced per chamber. In Chronic
Test 2, any sex class having greater than 60 amphipods was subsampled for
size measurements using a Folsom plankton splitter which randomly splits
the sample in half. A minimum sample size of 30 was selected by
examining the size variability that was found In Chronic Test 1 and by
determining the sample number necessary to observe a treatment effect at
the 5-percent probability level.
Short-term tests
47. A 96-hr range-finding test was run for REF and BRH sediments
individually. The nominal concentrations for each run were 200, 100, 50,
25 mg/1 suspended sediment, and a seawater control. The solid phase
treatment for all tests described in this report was REF sediment. For
each treatment, there was a comparison of the acute and chronic test
chambers; therefore, each treatment had one chronic chamber with 50
Ampellsca and one gallon jar with two acute chambers, with 25 Ampelisca/
chamber*
Long-term tests
48. A preliminary chronic test was run for 18 days at a constant
particle load of 50 mg/1, a seawater control, and concentrations of BRH
suspended sediment of 75, 50, 25, and 0 percent. There were two chronic
replicates per chronic treatment, each replicate containing 100 Ampelisca.
The nominal particle load for the two definitive chronic tests was 50 mg/1
and, due to high mortalities in the preliminary chronic test, the BRH concen-
trations were reduced to 10 and 5 percent with REF sediment. There were
four containers/treatment; two replicates were sieved after 28 and 32 days
32
-------
and the other two replicates were sieved at 45 and 58 days, respectively,
which coincided with the production of young in the controls.
33
-------
PART III: RESULTS AND DISCUSSION
Mysldopsis bahia
Preliminary tests
49. Prelimninary tests were conducted to determine the effect of
contaminated (BRH) and uncontaminated (REF) suspended solids concentrations
on the survival of M^ bahia. A 4-day exposure to REF suspended solids
concentrations of 300 mg/1 did not cause mortality. A similar exposure
to BRH-contaminated suspended solids resulted in 73-percent mortality, and
a 96-hr LC50 for BRH suspended solids of 245 mg/1. Since a concentration
of 300 mg/1 of REF suspended solids produced no observable acute effects,
this concentration of total suspended solids was used in the design of
the definitive short-term tests. In the latter tests, the total suspended
solids concentration was held constant while the contaminant concentration
was varied by altering the proportions of BRH and REF sediments in each
treatment.
Short-term tests
50. The results of three definitive short-term tests are summarized
in Table 2. The suspended solids diluter developed for these studies
provided a consistent and reproducible exposure environment for all treat-
ment combinations at a nominal suspended solids concentration of 300 mg/1.
Analysis of variance detected no statistically significant differences
(P = 0.05) in total suspended solids concentrations between treatments
within an experiment, nor within specific treatments between experiments.
51. Dissolved oxygen concentrations were within an acceptable range
(4.9 to 7.0 mg/1) for all experiments and treatments. In Experiments 1 and
2, dissolved oxygen decreased with increasing percentages of BRH sediment.
34
-------
Table 2
Results of Definitive Short-Term 96-hr Acute Toxicity Tests
Treatment
100%REF/0%BRH
75%REF/25%BRH
50%REF/50%BRH
25%REF/75%BRH
0%REF/100%BRH
100%REF/OZBRH
75ZREF/25%BRH
50%REF/50%BRH
25%REF/75ZBRH
0%REF/100%BRH
100%REF/OZBRH
75%REF/25%BRH
50%REF/50%BRH
25%REF/75ZBRH
0%REF/100%BRH
Suspended
Solids
mg/1
266.8 ± 52.1
310.2 ± 49.6
337.4 ± 36.5
358.0 ± 92.6
358.0 ± 26.7
297.3 ± 30.2
260.2 ± 39.6
309.7 ± 51.7
366.8 ± 161.7
311.5 ± 30.2
193.5 ± 64.5
348.6 ± 106.5
202.4 ± 23.5
209.4 ± 29.5
396.4 ± 126.1
with
Dissolved
Oxygen
mg/1
6.3 ± 0.2
5.7 ± 0.4
5.4 ± 0.5
4.9 ± 0.6
4.0 ± 0.5
6.4 ± 0.8
6.4 ± 0.8
5.7 ± 0.6
5.3 ± 0.6
5.2 ± 0.4
6.7 ± 0.1
6.6 ± 0.1
6.7 ± 0.1
6.7 ± 0.1
6.6 ± 0.1
Juvenile M. bahia
Temperature
°C
Experiment 1
25
Experiment 2
25
Experiment 3
25
Salinity Percent
mg/kg Mortality
30.0 ± 0 0.0
10.0
16.7
10.0
33.3
30.0 ± 0 2.0
5.0
2.0
31.0
81.0
30.0 ± 0 0.0
6.0
6.0
18.0
48.0
LC50
mgBRH/1
>358
290
410
-------
52. Test temperatures and salinities of 25°C and 30 mg/kg were
constant throughout all experiments.
53. The acute mortality patterns were similar in each experiment
(Table 2). The ninety-six hour mortalities at approximately 300 mg/1
total solids and 100% BRH are 33, 81, and 48 percent. This variability
in acute mortalities is similar to ranges reported in intercalibration
studies with M. bahia (Schimmel 1981; McKenney 1982). The estimated
96-hr LCSO's for three definitive tests are >358, 290, and 410 mg BRH/1
respectively. These results illustrate good reproducibility for
the acute toxicity test method with M. bahia using BRH dredged material.
54. The acute mortality data can be utilized to examine the possibl*
interactions between BRH contaminant-induced responses and the synergistic
and antagonistic interactions resulting from the presence of reference
sediment. In the preliminary test, where only BRH sediment was used, 16-
percent mortality occurred at a concentration of 150 mg/1. In the three
definitive tests, the 50%REF/50%BRH treatment at 300 mg/1 total solids
provides an analogous 150-mg BRH/1 exposure,but in this case there is an
equivalent amount of reference sediment present. The mortalities in the
definitive tests were 16.7, 2.0, and 6.0 percent, respectively. Although
these values are at the lower end of the'dose-response curve and, conse-
quently, subject to greater inherent variability, they do not indicate a
strong interaction between the sediment types and acute mortality.
55. As previously stated, the principal objectives of the Laboratory
Documentation of this program are to determine the applicability of the
short-term acute tests with M. bahia for measuring the effects with
dredged material, determine the sensitivity of the method, and assess the
36
-------
reproducibility of the test method using BRH sediments. The results
discussed above demonstrate that this method works well with dredged
material. To assess the method's reproducibility and sensitivity, the
following statistical analyses were conducted.
56. The first hypothesis examined was to determine if there were
differences in the acute mortality patterns in the three experiments as a
function of the total suspended solids exposure concentrations. Analysis
of variance results indicate that there were no significant differences
(P » 0.05) between mortality and suspended solids concentration within and
between the experiments. The second hypothesis was to determine if mor-
tality patterns were related to the BRH sediment treatment combinations.
Statistically significant differences (P<0.05) in mortality patterns were
found with treatments. In order to address the question of experimental
reproducibility, a two-way analysis of variance of experiments and treatment
combinations was conducted. The results of this analysis indicated that
there were no significant differences in the mortality patterns between
the experiments. Since significant differences were detected between
treatments, we were able to address the issue of sensitivity. Treatment
differences were analyzed by Tukey-Kramer's pairwise comparison test using
the 100%REF/0%BRH as the control. The results of this comparison are sum-
marized in Table 3. These results indicate that there were statistically
significant differences (P<0.05) in mortality when the BRH sediment concen-
tration reached 25%REF/75%BRH which is equivalent to 225 mg/1 BRH sediment.
57. In summary, the short-term test with juvenile M. bahia performed
Satisfactorily when applied to dredged material. The reproducibility of
this method was acceptable as Judged from the range of estimated 96-hr LC50
37
-------
values and from statistical analyses of mortality data (Table 2).
Table 3
Analysis of Treatment Differences for
Short-Term
Acute
Tests with Juvenile M.
bahia
Treatment
100%REF/0%BRH
75%REF/25%BRH
50%REF/50%BRH
25%REF/75%BRh
05£REF/100%BRH
N
6
6
6
6
6
Mortality*
0.0375
0.2379
0.2327
0.4607
0.8333
Grouping**
A
A
A
B
C
*Arcsine transformation of mortality data.
**Same letters are not significantly different.
Long-term tests
58. Replicate long-term tests were conducted to determine the effect
of BRH sediment on the survival, growth, and reproduction of M. bahia ex-
posed throughout an entire life cycle. Data on survival and reproduction
were then used to calculate the intrinsic rate of population growth and
other population parameters.
59. The chronic dosing and exposure system proved to be reliable
when operated at a nominal total suspended solids concentration of up to
300 mg/1 for 30 days. The precision of the dosing system was within
15 percent at 300 mg/1 total solids in the first chronic experiment
and 22 percent at 200 mg/1 total solids in the second chronic experiment.
The mean dissolved oxygen concentration ranged from 6.3 to 7.0 mg/1 and
6.3 to 6.8 mg/1 for Experiments 1 and 2, respectively. Dissolved oxygen
decreased slightly with increasing concentrations of BRH sediments. Both
long-term tests were conducted at 25°C and 30 + 0.5 mg/kg salinity.
38
-------
60. The growth and reproductive results of the long-term chronic
tests are summarized in Table 4. The growth of M. bahia, as measured by
dry weight, was similar in all the long-term chronic experiments. Analysis
of variance indicated that growth did not differ significantly (P » 0.05)
between experiments nor were there significant differences resulting from
exposure to BRH sediments in any of the experiments.
Table 4
Growth and Reproductive Results for
Chronic Tests with M. bahia
BRH-sediment Growth
__ mg/1 mg dry wt.
Zero*
65
155
275
311
Zero*
87
156
396
Zero*
43
95
0.61
0.63
0.56
0.69
0.54
0.52
0.57
0.56
0.49
+ 0.08
+ 0.16
± 0.19
± 0.09
+ 0.08
± 0.12
+ 0.02
+ 0.02
± 0.02
Sexual Initial Young
maturity reproduction per
days days , AFRD
Experiment 1
13
13
19
19
Experiment 2
12
15
20
Experiment 3
11
12
16
19
20
23
19
22
24
16
18
22
0.12
0.17
0.03
0.20
0.01
0.31
0.10**
0.01**
AFRD
EC50
mg/1
125
47
32
* REF Sediment Control
** Significantly different from REF sediment control (P
0.05)
61. The three measures of reproductive function quantified in the
long-term chronic tests were the times to sexual maturity and initial
reproduction, and the number of young produced per available reproductive
-------
day (AFRD) (Table 4). The times to reach sexual maturity in the REF
controls were 13, 12, and 11 days, respectively, for Experiments 1, 2, and 3.
In all experiments, the time to reach sexual maturity increased with in-
creasing concentrations of BRH sediments. If we assume that the range in
times to sexual maturity reported for the controls is indicative natural
variability, then the 6^day delay at 155 mg/1 in Experiment 1, the 8-day
delay at 156 mg/1 in Experiment 2, and the 5-day delay at 95 mg/1 in Exper-
iment 3 represent significant deviations from the control variability. No
estimates of sexual maturation times were reported for the 311- and 396-mg/J
treatments because of complete female mortality.
62. The times to reach initial reproduction in the REF were
19, 19, and 16 days,respectively, for Experiments 1, 2, and 3. The time to
initial reproduction increased with increasing concentrations of BRH sedi-
ment for those concentrations where reproduction occurred. Major deviation6
from the controls occurred at 155, 156, and 95 mg/1 BRH sediment in Experi-
ments 1, 2, and 3, respectively. The brood duration for the REF sediment
ranged from 5-7 days. At the 155-mg/l treatment in Experiment 1,
the brood duration was 4 days resulting in only 3 young produced, while in
the 156-mg/l treatment in Experiment 2, the brood duration was also 4 days
and resulted in only 1 young being produced.
63. Production, estimated from the number of young per AFRD, in the
REF sediment was 0.12, 0.20, and 0.31 for Experiments 1, 2, and 3,
respectively. There was a distinct decrease in production with increasing
BRH sediment concentration in all experiments and particularly at the 155-
and 156-mg/l treatments in Experiments 1 and 2 where only 3 and 1 young
were reproduced,respectively. The concentrations of BRH sediment that
40
-------
produced a 50% decrease in the AFRD (EC50) were determined by graphical
interpolation. The treatment AFRD values were converted to percent of
the REF (100%) and graphed on the BRH sediment concentrations.
The BRH sediment concentration at the intercept of the AFRD slope and the
50% response axis is the estimated EC50 concentration. Using this pro-
cedure, the EC50 values for Experiments 1, 2, and 3 are 125, 47, and
32 mg/1 BRH sediment, respectively.
64. The reproducibility of the chronic responses was generally ac-
ceptable. There were no significant (P « 0.05) effects on growth between
treatments nor between experiments. The times to sexual maturity and
intitial reproduction, while differing slightly in absolute value between
experiments, consistently decreased with increasing concentrations of BRH
sediment. Treatment differences in these parameters were consistent and
reproducible occurring at 155, 100, and 95 mg/1 BRH sediment.
65. Comparisons of production (young/AFRD) from estimated EC50 con-
centrations provides an estimate of the reproducibility of this response
Parameter. The high to low ratio, 3.9, for the range of ECSO's (125-32)
concurs with interlaboratory calibration data for chronic testing with M.
bahia (McKenney 1982). These studies reported high to low ratios of 5.7
and 3.6 for chronic test results conducted by six laboratories with the
heavy metal, silver, and the pesticide endosulfan, respectively.
66. It is difficult to assess the reproducibility of productivity
data, due to the high degree of variability inherent in this parameter. Ini-
tial analyses of variance of the number of female young per available repro-
ductive days for Experiments 1 and 2 indicated there were no significant
(P - 0.05) differences between treatments even though the actual total
41
-------
production varied by an order of magnitude between the REF and the
155- to 156-mg/l exposure concentrations, respectively. The inability to
detect statistical differences was directly attributable to the following
sources of variability: the number of reproducing females, the number of
replicates in the exposure design, and the size of the broods. The decisic"1
to modify the experimental design for Experiment 3 can be criticized for
jeopardizing the true replicability needed to assess test method reproduci-
bility within Laboratory Documentation. However, the authors believed that
it was more important to improve the statistical limits of detectability
for the reproductive portion of this test method than collect data that
would be difficult to interpret because of limitations of the test method.
67. The design modification consisted of increasing the number of
replicates from six to thirty, and reducing the number of exposure concen-
trations. The maximum nominal exposure concentration chosen for Experiment
3 was 100 mg/1 BRH sediment based upon the 40-percent mortality and almost
complete cessation of reproduction at 158 mg/1 BRH sediment in Experiments
1 and 2. Statistical analysis of data from Experiment 3 revealed signifi-
cant (P <0.05) treatment differences between the REF control at both the
95-mg/l and 43-mg/l concentrations of BRH sediment. Thus, the improved
experimental design enabled the statistical discrimination of reproductive
changes in Experiment 3 that were undetected in Experiments 1 and 2.
68. In summary, the reproducibility of the chronic reproductive
data developed within the Laboratory Documentation phase of this study
with M. bahia is consistent with the expected variability of the test
method. This is very encouraging in view of the complex contaminant
profile of the dredged material and affirms the applicability of the
42
-------
long-term chronic test method with M. bahia for assessing the effects of
dredged material contaminants.
Population analyses
69. Population response parameters were calculated from life tables
(Tables Al, A2, A3) that utilized age-specific survival and reproduction
data from the whole life cycle long-term tests. The three response param-
eters examined are intrinsic rate of growth, r , the net reproductive
value or multiplication rate per generation, and the mean generation time.
The patterns of these parameters measured for M. bahia exposed to BRH
sediments in three separate experiments are summarized in Table 5.
70. The differences in the absolute values between the three exper-
iments for the intrinsic rate of growth, r , measured for the REF sediment
are the result of differences in the growth rates of the popula-
tions in the two experiments. Values for r that are positive represent
increasing population size, while values for r that are at or close to zero
represent populations whose births and deaths are balanced, resulting in
maintenance of the population. Strongly negative values for r are indic-
ative of populations whose death rates greatly exceed the birth rates and
ultimately would lead to extinction.
71. Negative values in Experiment 1 and 2 of -0.015 and -0.018,
respectively, are low and are indicative, of populations which are slowly
moving toward extinction. This is in contrast to the positive value of
+0.070 determined for the REF sediment in Experiment 3. In pre-
vious studies, r-values for laboratory populations of M. bahia were greater
than +0.030 (Gentile et al. 1983). Because of the negative r-values in
the first two experiments, the authors changed the experimental design for
43
-------
Table 5
Population Responses for
BRH-sediment
mg/1
Zero
65
155
275
311
Zero
87
156
396
Zero
43
95
Life
Cycle Tests with M. bahia
Intrinsic Multiplication
rate of growth rate per
r generation
-0.015
-0.002
-0.099
-0.018
-0.107
+0.070
+0.038
-0.078
Experiment 1
0.72
0.96
0.13
Experiment 2
0.722
0.062
Experiment 3
4.52
2.16
0.17
Generation
time
days
22.15
23.40
21.00
18.31
26.00
21.59
20.69
22.36
EC50
for r
mg/1
110
42
47
Experiment 3 by increasing the replication and decreasing the number of
treatments.
72. Although the r - values were slightly negative in Experiments 1
and 2, there was still a definable decrease (negative increase) in the r-
value as a function of BRH. sediment concentration. A similar pattern was
clearly discernable in the r - values for Experiment 3. Substantial decreases
in r were observed at 155, 87, and 95 mg/1 in Experiments 1, 2, and 3,
respectively. At these concentrations of BRH sediments, the respective
populations were moving toward extinction much more rapidly than the REF
sediment population. EC50 values for r in Experiments 1, 2, and 3 of 110,
44
-------
42, and 47 mg/1 BRH sediments, respectively, were estimated using the
graphical interpolation method previously described for the reproductive
parameters.
73. The second population response parameter, multiplication rate
per generation, showed a similar pattern of response to that described for
the intrinsic rate of population growth. Specifically, values for the
multiplication rate per generation decreased with Increasing concentrations
of BRH sediments. The absolute values in Experiments 1 and 2 were less
than 1.0,indicative of a population that was not replacing itself, and the
value of 4.52 for Experiment 3 denotes a four-fold increase in population
size for each generation. There were no significant changes in the mean
generation time for those treatments that reproduced successfully in any
of the experiments, indicating that this parameter was not a sensitive index
of stress.
74. Comparison of the range of EC50 values (42-110) for the in-
trinsic rate of population growth, r , indicates a high to low ratio of
2.6 for the three experiments that is well within the expected range of
variability for a chronic life cycle test with M. bahia. In contrast,
the negative values for r in Experiments 1 and 2 indicate that
these populations were not growing satisfactorily, which necessitated a
change in the experimental design for Experiment 3. The latter design
changes resolved both the problems with the intrinsic growth rate and the
problems associated with reproductive variability, thus improving the
Power of statistical analysis. The results of these studies with M.
bahia indicate that: (a) the use of life cycle chronic tests are applicable
for use in evaluating the impact of dredged material contaminants, (b) this
45
-------
species is among the most sensitive to the contaminants in BRH sediments
of the species tested to date, and (c) the reproducibility of the reproductive
and population response parameters is within acceptable levels.
Ampelisca abdita
Exposure system monitoring
75. Table 6 shows monitoring data for the 96-hr range-finding tests.
All the measured concentrations are close to the nominal, although in some
cases standard deviations were as high as 50 percent of the mean, especially
at the high concentrations. In these range-finding tests, particle concen-
tration was affected by the design of the exposure system. The exposure
system using the acute chambers had lower total solids concentrations than
did the chronic test chambers. The presence of two exposure chambers in
each acute exposure system (Figure 9) resulted in obstructed circulation
and increased sedimentation. There was, however, no particle settling in
the acute exposure chambers themselves. At the high concentrations (100
and 200 mg/1), there was 3 to 5 mm of sedimentation in the chronic exposure
chambers; consequently, the acute exposure chambers appear to represent a
truer water column exposure.
76. The monitoring data for the preliminary chronic test (18 days)
is shown in Table 7. Replication was good, although there was higher vari-
ability at the REF and 25% BRH level than in the other treatments.
This variation was caused by intially high levels of REF sediment at the
beginning of the test.
77. Table 8 shows the particle concentrations for Chronic Test 1.
Mean values ranged from 31.8 to 43.8 mg/1 and those concentrations are the
replicate values for the 28-day exposure of 10% BRH. The amount of BRH
46
-------
Table 6
Concentrations (x ±
SD) for REF and BRH Sediments, comparing
two exposure chambers, acute
Nominal
Concentration
mg/1
0
Seawater control
25
50
100
200
Exposure
Chamber
A
C
A
C
A
C
A
C
A
C
REF
Meas. Cone.
mg/1
x ± SD
29.0 ± 18.4
22.5 ± 1.8
41.8 ± 18.3
60.2 ± 31.0
93.1 ± 46.5
120.2 ± 25.2
199.2 ± 54.4
233.6 ± 46.7
(A) and chronic (C)*
Mortality
2
6
2
2
6
10
8
0
4
4
BRH
Meas. Cone.
mg/1
x ± SD
32.4 ± 4.8
33.9 ± 10.8
47.0 ± 0.9
55.0 ± 9.2
74.9 ± 12.2
92.0 ± 44.9
186.8 ± 9.6
239.2 ± 30.1
Mortality
2
4
24
20
18
20
42
50
92
88
*N for dry weight determinations » 2, N for % mortality » 50. Sizes (mm)
of A. abdita (x ± SD) N = 25 : REF - 3.54 ± 0.65, BRH - 3.39 ± 0.47.
sediment in each treatment is also shown and ranges from 1.8 to 1.9 mg/1
for the 5-percent BRH exposures and from 3.2 to 4.4 mg/1 for the 10 percent
exposures. In Chronic Test 2, the 3-way valves were adjusted to provide
a slightly higher suspended participate concentration at about 5 mg/1 above
the concentration in Test 1. Total suspended load ranged from 39.7 (control)
to 50.1 mg/1 (10 percent BRH) over all treatments and the resultant BRH
sediment concentrations were 2.0 to 2.3 mg/1 at 5 percent BRH and 4.3 to
5.0 mg/1 at 10 percent BRH (Table 9). The exposure concentrations were
less variable in Test 2; only replicate 4 at 5-percent BRH had a co-
efficient of variation greater than 30 percent. The variability of all
the 58-day exposure concentrations was higher than the 32-day concentration
47
-------
Table 7
Dry Weight and Ampelisca Mortality for an 18-day Exposure to BRH in
the Suspended Phase Preliminary Chronic Test*
Treatment
Seawater
control
100%
0%
25%
50%
75%
REF
BRH
BRH
BRH
BRH
Replicate
1
2
1
2
1
2
1
2
1
2
Dry Weight
mg/1
x ±. SD
4
4
56
52
50
51
50
55
55
53
.6
.0
.3
.5
.1
.2
.5
.8
.9
.8
+
±
±
+
±
±
±
±
±
±
3.
3.
27
35
22
23
12
8.
6.
7.
2
8
.5
.9
.2
.3
.0
9
4
0
N
3
3
6
6
6
6
6
6
6
6
BRH
Sediment
mg/1
0
0
0
0
12.
12.
25.
27.
41.
40.
5
8
3
9
9
4
Mortality
%
13
5
14
9
90
98
100
100
100
100
*The number of amphipods per replicate is 100. Initial mean size and
SD = 3.11 ± 0.48 mm (N=«91).
variability because of 3-way valve malfunctions which occurred during the
last week of the test. Because the exposure design for the long-term
chronic tests was different than that for the short-term tests (Figure 1),
the suspended solids concentrations in the two distribution chambers and
the BRH mixing chamber were checked weekly in Test 1 and twice per week
in Test 2 (Table 10).
78. Temperature was measured daily and salinity was checked every
other day. Temperature ranged from 19.5eto 20°C in Chronic Test 1 and
from 19.5°to 21°C in Chronic Test 2.' Salinity ranged from 28 to 32 ppt
in both tests.
48
-------
Table 8
Suspended Particulate Dry Weight, Standard Deviation,and Ampelisea Mortality
for 28- and 45-day Exposure to BRH Suspended Phase in Chronic Test 1*
Treatment
REF Control
5% BRH
10% BRH
Exposure
Duration
days
28
45
28
45
28
45
Repli-
cate
1
2
3
4
1
2
3
4
1
2
3
4
Dry Weight
mg/1
43.0 ± 15.0
40.4 + 11.4
36.3 ± 10.3
41.0 ± 13.1
37.3 + 8.4
35.9 ± 12.4
36.5 ± 9.8
38.8 ± 12.7
31.8 + 7.8
43.8 ± 13.2
42.6 + 14.8
34.5 ± 9.5
N
8
8
11
11
8
8
11
11
8
8
10
11
BRH
Sediment
mg/1
0
0
0
0
1.9
1.8
1.8
1.9
1.9 ±
3.2
4.4
4.3
3.5
3.9 +
Mortality %
Overflow
0
1
2
12
0
0
3
34
0.1
0
0
13
2
0.6
Total
1
5
18
25
3
12
17
61
4
9
35
21
*Number of amphipods per replicate is 100. Overflow mortalities are those
found in the overflow water trap.
49
-------
Table 9
Suspended Particulate Dry Weight and Standard Deviation for
Chronic Test 2
at Exposure
Durations of 32
and 58
Days
Exposure
Duration
Treatment days
REF control 32
58
5% BRH 32
58
10% BRH 32
58
Replicate
1
2
3
4
1
2
3
4
1
2
3
4
Dry Weight
mg/1
42.5 + 6.2
40.7 ± 8.5
39.7 H- 9.6
42.9 ± 11.1
40.8 + 5.0
40.4 + 7.0
45.5 + 12.3
45.1 ± 19.9
46.5 + 6.8
42.5 ± 7.4
50.1 + 13.7
49.1 ± 13.1
N
9
9
17
17
9
9
17
17
9
9
17
17
BRH
Sediment
rag/1
0.0
0.0
0.0
0.0
2.0
2.0
2.3
2.3
2.2 ± 0.2
4.7
4.3
5.0
4.9
4.7 + 0.3
Table 10
Suspended Particulate Concentrations in the Dosing System
Chronic Test Number
Experiment 1
mg/1 + SD(N)
Experiment 2
mg/1 + SD(N)
REF Distribution Chamber
BRH Mixing Chamber
84.6 ± 19.8(8)
91.3 ± 24.7(9)
93.3 + 35.0(16)
78.3 ± 26.4(17)
50
-------
Short-term tests; mortality
79. The REF and BRH 96-hr range-finding tests were designed to
determine threshold mortalities for each sediment when diluted with sea-
water. The chronic and acute test chambers were each used in these short-
term tests for comparative purposes to ensure that chamber design would
not cause mortalities in the chronic tests.
80. The REF suspended phase, at nominal total solids concentrations
of 200 mg/1 and below, caused no significant mortalities (Tables Bl and
B2). The BRH suspended phase did cause significant mortalities (Tables
6, B3, and B4). Mortality patterns in the two chamber types were compa-
rable. The 96-hr LC50 value for the acute exposure chambers was 84.2 mg/1
with 95-percent confidence limits of 72.8 to 97.4 mg/1. For the chronic
exposure chambers, the LC50 value was 90.9 mg/1 with 95-percent confidence
limits of 76.2 to 110.7 mg/1. These LC50 values are not different from
each other as shown by complete overlap of the 95-percent confidence inter-
vals. The replication is good, especially considering that two different
chamber designs were used, and that the suspended particulate concentra-
tions were not exactly the same. Ainpelisca's acute response to BRH
sediments in the solid phase, which has been reported elsewhere (Rogerson
et al. , 1984), was also consistent among several series
of tests.
81. Based on these mortality patterns, a preliminary chronic test
was designed to have a constant total particle concentration of approxi-
mately one half the LC50 values and a BRH contaminant gradient was achieved
by proportionally diluting the BRH sediment with REF sediment. The BRH
suspended particle proportions were 75, 50, 25,and 0 percent. As Table 7
51
-------
shows, significant mortalities were obtained at all BRH exposures.
82. The mortalities in these short-term tests are not surprising
since Ampelisca is a particle feeder (Mills 1967) and the target organs
affected by BRH sediment are the digestive tract and hepatopancreas. The
absolute mortalities found in the range-finding tests and the 18-day pre-
liminary chronic test are not strictly comparable, however. The diatom £.
tricornutum was supplied to the amphipods in the 18-day test and not in the
96-hr tests. It is not known if the presence of food stimulates feeding,
thereby increasing exposure to contaminated particles, or if the food supply
ameliorates toxicity by providing a nutritional food source in the presence
of a potentially toxic material.
Long-term tests
83. As there were significant mortalities at 25% BRH (12.5 mg/1 BRH
sediment) in the 18-day test, the exposure concentrations for the long-term
test were decreased. The experimental design for the long-term tests
included three treatments: 10- and 5-percent proportions of BRH sediment
and a reference control at approximately 50 mg/1 total suspended solids.
Two additional replicates were added to each treatment in order to evaluate
growth and survival at two time intervals, 25 and 45 days in Test 1 and
32 and 58 days in Test 2.
Mortality
84. Chronic mortalities for the 28-day exposure in Experiment 1 were
not significant (Table 8) at 10% BRH and approximately 40 mg/1 total sus-
pended solids (3.8 mg/1 BRH). In Chronic Test 2, survival was estimated
by comparing the number of Ampelisca harvested on day 32 with the estimated
initial number of young, assuming 100-percent survival of all eggs of the
52
-------
ovigerous females that were introduced into the containers at day 0. These
females had a mean egg number of 17.7 eggs (SD *> 6.73) to yield 266 initial
young. As shown in Table 11, survival in the control and 5-percent expo-
sures was better than at 10-percent BRH. However, the 95-percent confidence
interval around the mean egg number/female is 15.2 to 20.2 eggs which would
yield between 227 and 303 initial young; only replicate 2 at 10-percent BRH
falls significantly outside this range (Table 11). The mortality threshold
appears to occur at 4.5 mg/1 BRH sediment based upon the 32-day data.
Table 11
Estimated A. abdita Mortality in Chronic Test 2*
Treatment Replicate
REF 1
2
5% BRH (2.0 mg/1) 1
2
10% BRH (4.5 mg/1) 1
2
REF 3
4
5% BRH (2.3 mg/1) 3
4
10% BRH (5.0 mg/1) 3
4
No.
Harvested
32 Days
240
245
278
307
227
190
56 Days
159
127
73
118
178
206
Mortality
%
9.8
7.9
0
0
14.7
28.6
40.2
52.3
72.6
55.6
33.1
22.6
*Assumming 100% hatching success of eggs from 15 egg-bearing females with
with a mean egg number/female (+ SD) of 17.7 + 6.73 eggs to yield 266
initial young.
53
-------
Based upon the results of the preliminary chronic (18-day) and the two
definitive (28- and 32-day) chronic tests, the chronic mortality threshold
for BRH suspended participates is between 4 and 12.5 mg/1 for A. abdita
exposed for 18 days or longer. By converting the proportion of BRH sedi-
ment to milligrams per liter of BRH sediment, the mortalities from the 18-
and 28-day exposure in Chronic Test 1 were analyzed to estimate an LC50
value. The 32-day data in Chronic Test 2 was not used because the mortality
data are only estimates. Although the 28-day exposure is longer, the mor-
talities for an 18-day exposure are at least no greater than those for 28
days. This chronic LC50, calculated using the binominal test, is 7.03 mg/1
with 9 Si-percent confidence limits from 4 to 12.5 mg/1. The 96-hr LC50 for
the chronic exposure chambers was 90.9 mg/1, yielding an acute mortality:
chronic mortality ratio of 12.9.
85. The mortalities for the 45-day exposure in Chronic Test 1 in-
creased, ranging from 18 to 51 percent over all treatments (Table 8). Of
the total mortality in this experiment, 66 of 167, or 40 percent, were
found in the overflow cups (Figure 3) that were checked daily. Of these
66 Ampelisca, 50 were mature males, and over half of these were found in
the 5-percent BRH replicate 4. The cause of these mortalities does not
appear to be BRH sediment since the mortalities were mostly adult males
which frequently experience natural senility and die after mating (Mills
1967). In Test 2, the 58-day estimated mortality pattern was reversed
(Table 11) with the greatest survival occurring in the 10-percent BRH
exposure.
86. As will be shown below, natural mortality occurs across all
treatments and is indicative of the stage of development of the population.
54
-------
As such, only the 28-day, Test 1, mortality data were used to calculate
the acute:chronic ratio. The goal of the exposure system design, to ex-
pose Ampelisca to nonlethal concentrations of BRH suspended particulates,
was achieved in both chronic tests. The natural life span of Ampelisca
at 20°G is 4 to 6 weeks, a period that is encompassed by the second two
replicates of each test.
Population structure
87. Ampelisca abdita individuals can be easily sexed and classified
into stages of sexual maturity according to morphological characteristics.
Morphologically, adult males (M) are the most distinctive with a carinate
urosome and elongated second antennae (Bousfield 1973). Males are
specially adapted for swimming, which facilitates mating in the water
column. They do not molt again and presumably die after mating. Whether
they mate with more than one female is unknown.
88. Female A. abdita are more difficult to sex, but they can be
divided into five progressively distinct groups: FDV, FE, FOV, FS, and OTH,
The earliest stage, after sexual differentiation, is here termed developing
female (FDV). This stage animal is distinguished by the presence of a
brood plate on the interior of each of the first five coxal appendages.
As the female grows, the brood plate increases in size and eggs begin to
develop in the oviduct which is dorsal to the digestive tract, and which
can be seen through the exoskeleton. This stage is termed developing egg
female (FE). The females go through a series of molts until one of the
final molts, at which time the brood plates develop long setae and the
eggs are deposited through a gonopore into a fully developed brood pouch.
This stage is termed ovigerous female (FOV). Presumably mating occurs at
55
-------
this time. Since all eggs in a brood pouch are in the same stage of
development, all eggs in the oviduct are assumed to be deposited at once.
After releasing their young, the females do not immediately die but remain
in the population for some undetermined time. They do not have eggs in
the oviduct or in the brood pouch but do retain the setose oostegites.
They are termed spent females (FS).
89. There is one other group, termed undifferentiated (OTH). These
include juveniles, undifferentiated females, and undifferentiated males.
In most cases where this group was encountered, the animals were larger
than 4.0 mm. These animals are probably males since females can be dis-
tinguished at a smaller size, when the brood plates first develop.
90. In Chronic Test 1, after the 28- and 45-day exposures, two
replicates from each treatment were sieved to 0.5 mm and the amphipods
were sex classified and measured. Development of the female population
was progressively retarded as the BRH concentration increased (Table 12)
at 28 days, as evidenced by the greater numbers of ovigerous females (FOV)
in the REF and their total absence in the 10% BRH treatment.
Conversely, the earliest stage of development (FDV) was the predominant
developmental stage at the highest BRH concentration. The same results
were evident for the adult males (M) and the undifferentiated (OTH)
groups, where there were more males in the control treatment than in the
BRH exposed groups. These data suggest that exposure to BRH sediment
caused a delay in maturation and development over the 28-day exposure
period. After a 45-day exposure, there does not appear to be any differ-
ence among the treatments, although in the 10-percent treatment there was
a greater proportion of the undifferentiated (OTH) group, again indicating
56
-------
Table 12
Number of A. abdita in Each Sex Category for 28- and 45-Day Exposures
Treatment
REF
5% BRB (1
10% BRH (3
REF
5% BRH. (1
10% BRH (3
*Initial N
to BRH Suspended
Replicate
1
2
mean
.9 mg/1) 1
2
mean
.8 mg/1) 1
2
mean
3
4
mean
.9 mg/1) 3
4
mean
.9 mg/1) 3
4
mean
for each replicate
Particulates in Chronic Test
FOV
28 Days
18
9
13.5
4
1
2.5
0
0
0
45 Days
22
17
TsiTs
27
14
20.5
13
21
17.0
FE
18
26
22.0
35
7
21.0
12
3
7.5
18
23
20.5
19
10
T975
7
11
9.0
was 100 Ampelisca.
FDV
6
7
6.5
2
22
T270
33
40
36.5
2
2
2.0
2
0
1.0
13
2
7.5
See text
1*
M
25
16
20.5
18
1
9.5
0
3
1.5
29
29
29.0
30
13
"2175
6
35
20.5
for desl
OTH
32
37
34.5
38
57
1775
48
48
48.0
4
2
3.0
2
0
~~T7o
23
10
16.5
gnatior
of classes.
that development of the males is also impacted. Chi-square tests of the
mean proportions of each group showed significant treatment effects at
P < 0.05.
91. Similar results were obtained in the second chronic test, but
since the test was initiated with ovigerous females, the major effects
occurred at the 58-day sampling rather than at 32 days (Tables 13, 14).
57
-------
Table 13
Number of A. abdita in Each Sex Category for 32- and 58-Day Exposures
to BRH Suspended Particulates in Chronic Test 2*
Treatment
REF
5% BRH (2.0
10% BRH (4.5
REF
5% BRH (2.3
10% BRH (5.0
Replicate
1
2
mg/1) 1
2
mg/1) 1
2
3
4
mg/1) 3
4
mg/1) 3
4
FOV
32
22
3
10
18
2
1
58
46
39
30
53
23
2
FE
Days
37
32
66
31
36
6
Days
15
25
9
17
25
5
FDV
22
86
53
82
60
45
20
22
7
5
29
97
FS M
35
3
32
28
1
0
19 27
8 15
15 5
17 13
3 31
0 1
OTH
124
121
117
148
128
138
32
18
7
13
67
101
*See text for designation of classes.
At 32 days, the populations were similar, mainly because of the replicate
variability. For example, the REF replicate 2 is similar to both
10% BRH replicates, in having low numbers of ovigerous females and adult
males (Table 13). There is a trend, however, for more ovigerous females
and adult males to be present in the control and 5% exposures. This trend
becomes more dramatic at 58 days. The population exposed to 10% BRH had
fewer ovigerous females and more developing females (FDV) and subadult
males (OTH) than in the lower treatment conditions. Chi-square again
showed significant treatment effects at P < 0.05. These results also
suggest a delay in the maturation of the population in Chronic Test 2.
58
-------
Table 14
Mean_Percent of A. abdita in Each Sex Category for 32- and 58-Day Exposures
to BRH Suspended Particulates in Chronic Test 2*
Treatment
REF
5% BRH
10% BRH
REF
5% BRH
10% BRH
(2.0
(4.5
(2.3
(5.0
mg/1)
mg/D
mg/1)
mg/1)
FOV
5.2
4.8
0.7
29.7
43.5
6.5
FE
32 Days
14.2
16.6
10.1
58 Days
14.0
13.6
7.8
FDV
22.3
23.1
25.2
14.7
6.3
32.8
FS M
7.8
10.3
0.2
9.4 14.7
16.8 9.4
0.8 8.3
OTH
50.5
45.3
63.8
17.5
10.5
43.8
*See text for designation of classes.
Growth
92. The mean sizes and variability estimates for each development
stage and replicate for Chronic Tests 1 and 2 are shown in Tables A8 and A9,
respectively. Analysis of variance tests were done to determine treatment
effects for the 28- and 45-day samples in Test 1 and for the 32- and 58-day
samples in Test 2. Although some of the stage-classified samples were split
to reduce the sample size, it was felt that these smaller subsamples could
be included in the ANOVA because they would reflect a conservative estimate
of the statistical differences.
93. The initial mean size and standard deviation of Ampelisca for
Test 1, measured on a sample of 100 animals from the original pool of test
organisms, was 3.30 + 0.51 mm. The survival (Table 8) and general growth
of these animals (Table 15) was good for all treatments. In most of the
treatment comparisons, the sizes of the 5% BRH-exposed Ampelisca were
slightly larger than the control animals, but none of these differences
59
-------
Table 15
Mean size (mm) of A. abdita Exposed to BRH Suspended Particulates
in Experiments 1 and
Treatment
FOV
FE
2*
FDV M
OTH
Experiment 1
28 Days
REF
5% BRH (1.9
10% BRH (3.8
mg/1)
mg/1)
6.71
7.09
—
7.15**
7.39
6.94
6.35 6.5.
5.18 6.8J
5.79 6.51
> 6.64
J 5.95
J 6.11
45 Days
REF
5% BRH (1.9
10% BRH (3.9
mg/1)
mg/1)
7.46
7.54
7.01
7.68**
7.97
7.49
6.43 6.7(
6.63 6.8
6.15 6.5
3 7.37**
I 7.94
L 6.55
Experiment 2
32 Days
REF
5% BRH (2.0
10% BRH (4.5
mg/1)
mg/1)
6.14
6.34
6.75
6.37
6.39
6.04
5.29 5.5(
5.22 5.5!
4.67 5.2.
3 5.74
J 5.47
5 4.42
58 Days
REF
5% BRH (2.3
10% BRH (5.0
mg/1)
mg.l)
6.89
6.25
6.12
7.58
6.99
6.69
7.18 6.4(
6.88 6.2'
3 7.33
'4 7.23
5.80 5.83 6.15
*Sizes connected by the same line are not significantly different from
each other at P < 0.05.
**Control and 10% BRH are not significantly different at P < 0,05.
were statistically significant. There were no significant treatment or
time differences among male mean sizes, indicating that adult males were
the same size regardless of the length of time that the growth took.
Therefore, even though it took longer for the males to mature as a result
of exposure to BRH sediment (Table 12), they were the same size at matura-
tion across all treatments. When all females were pooled, there was a
60
-------
treatment effect at 28 days between REF and 10% BRH with the reference
females being significantly larger (t = 8.167, df = 157). As noted
above, the females in the 5% BRH treatment were not significantly different
from those in REF at 28 days because of the large size variation
between replicates (Table Cl). This variation is a reflection of the
developmental stage difference between the two replicates. Analysis of
variance, with replicates pooled, does show that the developing females
(FDV) are significantly larger in the reference (P < 0.05). The same is
true for the undifferentiated group (OTH) indicating that the growth of
males and females is slowed.
94. At the 45-day exposure, the differences in growth are not as
great, although there is a consistent trend in all classes for the 10%
BRH Ampelisca to be the smallest. There was a significant (P < 0.05)
difference among the ovigerous females with the reference and 5% BRH
treatments being consistently larger than the 10% BRH exposed amphipods.
95. In Experiment 2, the statistical differences in sizes among the
classes are more prevalent than those found in the first test. As expected
from the results in Test 1, at 32 days there were no treatment effects on
ovigerous females or adult males. The ANOVA showed that the 10% BRH
exposure produced significantly smaller nonovigerous females and subadult
males, indicating an adverse impact on growth. By day 58, all development
stages showed significant treatment effects on size. In all cases, the
REF were larger than the 10% BRH exposure condition. Additionally,
the REF ovigerous females (FOV) and females with eggs in the oviducts
(FE) were significantly larger than those in the 5% BRH exposure (Table 15).
96. The size data, in conjunction with the data on population
61
-------
structure, presented in the previous section, has shown that a major effect
of BRH suspended particulates is the retardation of growth and subsequent
maturation. The more dramatic effects on growth (Table C2) that were found
in Experiment 2 are the probable result of a more complete exposure of
Ampelisca throughout its life cycle to BRH sediments. The results of the
longer exposure condition also suggest that the earlier part of the life
cycle may be more sensitive. Notwithstanding these differences, the
replication of the growth dose response to BRH suspended particulates is
very good.
Fecundity and productivity
97. The number of eggs produced per female in A. abdita is a
function of female size (Mills 1967). Treatment effects were assessed
using analysis of covariance, with female size as the dependent variable.
Once the quantitative relationship between egg number and female size is
determined, the differences in egg number/female among treatments can be
analyzed by statistically adjusting the fecundity estimates based on the
regression relationship.
98. Analysis of covariance showed positive correlation of egg
number with size (F » 30.26, P < 0.001) for the 45-day data in Experiment
1. There was a trend for more eggs to be produced/female in the REF
and 5% BRH treatments than in the 10% BRH exposure. However, when
egg number/female was adjusted for female size in the covariance analysis,
there were no differences among treatments (Table 16). Identical results
were found for the day 58 ovigerous females in Experiment 2 (Table 16).
Again the egg number/female tended to be greater in the REF and
5% BRH animals than at 10% BRH, but the differences were not significant
62
-------
Table 16
Mean Number of Eggs/Ovigerous Female ± Standard Error for A. abdita
Exposed to BRH Suspended Participates for 45 days in Experiment 1
and 58 days in Experiment 2
Treatment
N
_Raw
x ± SE
Size_ Adjusted
x ± SE
Total
Young
Produced
REF 39
5% BRH (1.9 mg/1) 41
10% BRH (3.9 mg/1) 34
Initial 30
REF 68
5% BRH (2.3 mg/1) 59
10% BRH (5.0 mg/1) 16
Experiment 1
19.8 ± 2.08
21.8 ± 2.06
14.4 ± 1.89
Experiment 2
17.7 ± 1.23
13.6 ± 1.17
12.3 ± 1.21
9.2 ± 1.23
18.5 ± 1.85 (A) Present
19.4 ± 1.86 (A) Present
18.3 ± 2.12 (A) Absent
17.0 ± 1.52 (A)
12.44 ± 1.04 (B) 1212
13.54 ± 1.12 (B,A*) 318
10.93 ± 2.11 (B) 0
*Raw data and egg number adjusted for female size. Means with the same
letter are not significantly different at P < 0.05.
when fecundity was adjusted for female size. In Chronic Test 2, all of
the experimental fecundities were significantly lower than the egg produc-
tion found in the INITIAL sample which was the original pool of ovigerous
females at the start of the experiment. Although it was not statistically
tested, the Test 2 fecundities also appear lower than the fecundities at
all treatments in Experiment 1.
99. A possible explanation for the lowered fecundity in Experiment
2 may be that the Experiment 1 animals were the Fl laboratory generation,
whereas, the Experiment 2 Ampelisca were an F2 laboratory generation. To
test this hypothesis, all control ovigerous females were submitted to the
63
-------
covariance analysis, including the 28-day Experiment 1, and the INITIAL
and the 32-day Experiment 2 amphipods. The analysis revealed that, in
fact, the 58-day Experiment 2 fecundities (Table 16) were significantly
lower (P < 0.05) than all other control groups, except for the 28-day
Experiment 1 animals. Lowered fecundities of an F2 generation probably
result from the lowered nutritional quality of the laboratory food source
(Phaeodactylum), which does not simulate the diverse detrital component
found in nature.
100. The number of young produced in Experiment 2 is also shown in
Table 16. The REF produced more young (F3) than the 5% BRH
treatment and no young were found in the 10% BRH exposure. The causes
are twofold: fewer ovigerous females were present at the highest BRH
concentration (Tables 13 and 14), and they tended to carry fewer eggs
(Table 16). In Experiment 1, a larger sieve size (0.5 mm mesh) was used
and the young (F2) were not quantified; however, they were present in the
REF and 5% BRH and were absent from the 10% BRH exposure.
Population analyses
101. As is the case with M. bahia, population response parameters
were calculated from life tables using age-specific survival and repro-
duction data (Tables A4, A5). To construct these Ampelisca life tables,
the test period was broken into 14-day intervals and survival was estimated
for each age interval by knowing the initial number of amphipods, those
harvested at the interim sampling, either day 28 or 32, and those found at
the final sampling, either day 45 or day 58 for Experiments 1 and 2,respec-
tively. The reproduction data for Experiment 1 were estimated based on
the mean number of eggs per ovigerous female since the total number of
64
-------
young produced was not quantified. In Experiment 2, the number of eggs
per ovigerous female were included and combined with the actual number of
young produced for each treatment. The population responses in the two
experiments are summarized in Table 17. The responses shown are intrinsic
rate of growth, r , the multiplication rate per generation, and generation
time.
Table 17
Population Responses for Life-Cycle Tests with A. abdita
Treatment
Intrinsic Multiplication
rate of growth rate per
r generation
Generation
time
(days)
REF
5% BRH (1.9 mg/1)
10% BRH (3.9 mg/1)
REF
5% BRH (2.2 mg/1)
10% BRH (4.7 mg/1)
Experiment 1
.045 5.65
.038 4.93
.021 2.46
Experiment 2
.023
.009
.023
3.50
1.60
0.28
38.5
41.7
43.0
54.0
51.6
54.9
102. Both the experiments show the same dose response in intrinsic
rate of growth and multiplication rate per generation; each response
decreases with increasing BRH concentration. These results indicate
that, although the absolute values of these responses are different,
exposure to BRH sediments elicits a similar dose response pattern. This
is an important consideration from a toxicological viewpoint since these
two experiments are not true replicates in that the population stage was
65
-------
different at the beginning of the two tests. In Test 1, field-collected
subadults were used to initiate the tests, and therefore the early part
of the life cycle was not exposed to BRH sediments. This would account
for the proportionately greater number of ovigerous females across all
treatments. Conversely, in Test 2, all Ampelisca were exposed from hatching
and brood release. The steeper response pattern for r that was found in
Test 2 results from fewer ovigerous females developing and the subsequent
decreased contribution of young to the population. The differences in
actual BRH sediment concentrations do not appear great enough to cause
the observed drop in r. The longer generation time in Experiment 2 prob-
ably reflects the longer duration of the experiment. In addition to the
fewer number of ovigerous females, as noted in the previous section, the
number of eggs per female is lower in Experiment 2. The total reduction
in fecundity is a probable result of the fact that the Ampelisca used in
this test were an F2 generation laboratory stock that were reared on
Phaeodactylum.
103. The differences in the experimental conditions of the two
tests, i.e., the initial propulation stage, appear sufficient to explain
the absolute differences in the intrinsic rate of growth and multiplication
rate per generation between Test 1 and Test 2. More importantly, the
magnitude of the differences is not great; e.g., for the controls, the
tests differ by a factor of less than 2. The observed dose response of
the population parameters is a good indication that population responses
can be used to evaluate the long-term effect of dredged material in the
suspended phase.
66
-------
PART IV: CONCLUSIONS
104. The objectives of the Laboratory Documentation phase of the
Field Verification Program are to demonstrate the applicability of using
the chronic responses growth, reproduction, and intrinsic rate of population
to determine the precision and reproducibility of the test methods and the
response parameters measured.
105. The results of this study demonstrate the feasibility of con-
ducting flow-through suspended solids whole life cycle toxicity tests with
eplbenthic and infaunal crustaceans for periods of 60 days. The suspended
solids dosing systems developed for these studies were capable of propor-
tionally mixing contaminated sediment (BRH) with reference sediment (REF)
to produce a graded contaminant profile with consistency and precision.
106. Short-term tests with M. bahia resulted in 96-hr LCSO's of
358, 290, and 410 mg/1 BRH sediment while similar tests with A. abdita pro-
duced, values of 84 and 91 mg/1. These results provide an estimate of the
reproducibility of the short-term test methods developed for these species.
107. Long-term test methods were successfully developed to evaluate
the effect of suspended solids on the growth, reproduction, and intrinsic
rates of population growth, r , with M. bahia. Growth of M. bahia was not
significantly affected by exposure to BRH sediments. Two measures of re-
productive function, the times to sexual maturity and initial reproduction,
while differing slightly in absolute values between experiments, consistently
decreased with increasing concentrations of BRH sediments. Treatment differ-
ences in these parameters were consistent and reproducible occurring at 155,
156, and 95 mg/1 BRH sediment. The third measure of reproductive function,
the number of young per available reproductive day (AFRD), decreased with
67
-------
increasing concentrations of 8RH sediments in all experiments. The EC50
values for this parameter were 125, 47, and 42 mg/1 for the three experi-
ments, which is within the expected range of variability for the chronic
test method with M. bahia. The measures of population response, intrinsic
rate of population growth,r , and multiplication rate per generation, de-
creased with exposure to increasing concentrations of BRH sediments. The
EC50 values for these parameters were 100, 42, and 47 mg/1 for the three
long-term experiments. These studies demonstrate the successful application
of long-term chronic test methods using dredged material suspended solids
exposure with M. bahia. Growth, reproductive, and population parameters
measured using this method responded in a consistent and reproducible manner*
The variability of these response parameters measured was within a factor of
3.0 for the three experiments.
108. The results of the long-term chronic tests with A. abdita in-
dicate that this species is very sensitive to BRH sediment in the suspended
phase. The BRH concentrations used for the chronic tests did not cause
significant mortalities; however, effects on growth and reproduction were
dramatic. In both replicates of the chronic tests, growth was inhibited at
4 to 5 mg/1 BRH sediments (10% BRH) causing a delay in the maturation of
adult females. Although there were test replicate differences in fecundity
that were related to the generation of Ampelisca used, there were no treat-
ment effects on the number of eggs produced per female. The effects of
slower growth and delayed maturation were evident in the calculation of r ,
the intrinsic rate of population growth. In both tests, a consistent dose
response was found for this parameter. The results of these studies demon-
strate that the long-term chronic test method when applied to evaluating
68
-------
the effects of dredged material suspended solids using the infaunal amphi-
pod, A. abdita, gave a consistent and reproducible dose response for growth,
population structure, and intrinsic rate of population growth.
69
-------
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72
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APPENDIX A
Life Tables for M. bahia and A. abdita
-------
Table Al
Life Tables for M. bahia Exposed to BRH Sediments in Experiment 1
No.
Age/ Day s Females
REF
1- 5 18.
6-10 18.
11-15 18.
16-20 18.
21-25 18.
26-30 18.
31-35 0.
65 mg/1 BRH Sediment
1- 5 13.
6-10 13.
11-15 13.
16-20 13.
21-25 12.
26-30 11.
31-35 0.
155 mg/1 BRH Sediment
1- 5 12.
6-10 12.
11-15 12.
16-20 12.
21-25 12.
26-30 11.
31-35 0.
* lx » the probability of a
** nig » female offspring per
lv*
1.000
1.000
1.000
1.000
1.000
1.000
0.000
1.000
1.000
1.000
1.000
0.923
0.846
0.000
1.000
1.000
1.000
1.000
1.000
0.917
0.000
female surviving
Female
Young
0.0
0.0
0.0
3.0
4.0
6.0
0.0
0.0
0.0
0.0
3.0
0.5
9.0
0.0
0.0
0.0
0.0
0.0
1.5
0.0
0.0
to age x
mv**
0.000
0.000
0.000
0.167
0.222
0.333
0.000
0.000
0.000
0.000
0.231
0.042
0.818
0.000
0.000
0.000
0.000
0.000
0.125
0.000
0.000
female per age interval
-------
Table A2
Life Tables for M. bahia Exposed to BRH Sediments in Experiment 2
Age/Days
REF
1- 5
6-10
11-15
16-20
21-25
26-30
31-35
36-40
87 mg/1 BRH
1- 5
6-10
11-15
16-20
21-25
26-30
31-35
36-40
No.
Females
18.
18.
18.
17.
13.
12.
8.
0.
Sediment
16.
16.
16.
16.
14.
12.
11.
0.
* lx - the probablility of a
** m» • female ofJ
rsorine oer i
lx*
1.000
1.000
1.000
0.944
0.722
0.667
0.444
0.000
1.000
1.000
1.000
1.000
0.875
0.750
0.687
0.000
female surviving
female oer aze inl
Female
Young
0.0
0.0
0.0
8.0
4.5
0.0
0.5
0.0
0.0
0.0
0.0
0.0
0.5
0.0
0.5
0.0
to age x
terval
rax**
0.000
0.000
0.000
0.471
0.346
0.000
0.062
0.000
0.000
0.000
0.000
0.000
0.036
0.000
0.045
0.000
-------
Table A3
Life Tables for M. bahia Exposed to BRH Sediments in Experiment 3
No.
Age/Days Females
REF
1- 5 28.
6-10 28.
11-15 28.
16-20 28.
21-25 24.
26-30 19.
31-35 10.
36-40 0.
43 mg/1 BRH Sediment
1- 5 34.
6-10 34.
11-15 34.
16-20 34.
21-25 34.
26-30 31.
31-35 24.
36-40 0.
95 mg/1 BRH Sediment
1- 5 32.
6-10 32.
11-15 32.
16-20 32.
21-25 32.
26-30 27.
31-35 24.
36-40 0
* lx » the probability of
** nu » female offspring p<
lx*
1.000
1.000
1.000
1.000
0.857
0.679
0.357
0.000
1.000
1.000
1.000
1.000
1.000
0.912
0.706
0.000
1.000
1.000
1.000
1.000
1.000
0.844
0.750
0.000
a female surviving
Female
Young
0.0
0.0
0.0
51.5
25.0
33.5
16.5
0.0
0.0
0.0
0.0
33.5
22.0
7.0
11.0
0.0
0.0
0.0
0.0
0.0
4.0
1.5
0.0
0.0
to age x
****
0.000
0.000
0.000
1.839
1.042
1.763
1.650
0.000
0.000
0.000
0.000
0.985
0.647
0.226
0.458
0.000
0.000
0.000
0.000
0.000
0.125
0.056
0.000
0.000
ar female per age Interval
-------
Table A4
Life Tables for A. abdita Exposed to BRH Sediments in Experiment 1
No.
Age/Days Females lx*
REF
1-14
15-28
29-42
43-56
57-70
5% BRH (1.85 mg/1)
1-14
15-28
29-42
43-56
57-70
10% BRH (3.85 mg/1)
1-14
15-28
29-42
43-56
57-70
50
46
42
42
0
50
45
40
40
0
50
47
43
33
0
* lx » the probability of
** m.. = female offsorine o<
1.000
0.920
0.840
0.840
0.000
1.000
0.900
0.800
0.800
0.000
1.000
0.940
0.860
0.660
0.000
a female surviving
ar female oer acre ii
Female
Young
0.0
0.0
90.0
192.5
0.0
0.0
0.0
23.0
223.5
0.0
0.0
0.0
0.0
123.0
0.0
to age x
iterval
mx**
0.000
0.000
2.143
4.583
0.000
0.000
0.000
0.575
5.588
0.000
0.000
0.000
0.000
3.727
0.000
-------
Table A5
Life Tables for A. abdita Exposed to BRH Sediments in Experiment 2
Age/ Day s
REF
1-14
15-18
29-42
A 3-56
57-70
71-84
5% BRH (2.15 mg/1)
1-14
15-28
29-42
43-56
57-70
71-84
10% BRH (4.73 mg/1)
1-14
15-28
29-42
43-56
57-70
71-84
No.
Females
133
133
101
101
100
0
133
133
133
103
76
0
133
133
92
92
92
0
* lx » the probability of a
** ro~ * female offsi
lv*
1.000
1.000
0.759
0.759
0.756
0.000
1.000
1.000
1.000
0.774
0.571
0.000
1.000
1.000
0.692
0.692
0.692
0.000
female surviving
Female
Young
0.0
0.0
0.0
100.0
365.0
0.0
0.0
0.0
0.0
82.5
130.0
0.0
0.0
0.0
0.0
5.5
31.5
0.0
to age x
mv**
0.000
0.000
0.000
0.990
3.650
0.000
0.000
0.000
0.000
0.801
1.711
0.000
0.000
0.000
0.000
0.060
0.342
0.000
prlng per female per age interval
-------
APPENDIX B
Acute Toxicity Data for A. abdita
-------
TABLE Bl
ACUTE TOXICITY DATA SHEET
COE/ERLN FVP
STUDY PLAN: B
INVESTIGATOR: SCOTT/REDMOND
EXPERIMENT DESCRIPTION: SUSPENDED DATE OF TEST:
TEST NUMBER: 1 ACUTE CHAMBER SPECIES: AMPELISCA SP
HH» EXPERIMENTAL CONDITIONS »•»
830701
RANGE: 19. 30 - 22. 00
RANGE:
TEMPERATURE: 20. 00 DEGREES CENTIGRADE
SALINITY: 31. 00 PARTS PER THOUSAND
EXPOSURE DURATION: 4 DAYS
PHOTOPERIOD: 14 HOURS
FLOW RATE: SO MLS/MIN VOLUME ADDITIONS/DAY 3O
NUMBER OF ANIMALS/REPLICATE: 23
NUMBER OF REPLICATES/TREATMENT: 2
ANIMAL'S LIFE STAGE: JUVENILE. AGE: DAYS SIZE: MILLIMETERS
CONTROLS: SEAUATER/SOUTH REFERENCE SOLID
FOOD USED NONE
NUMBER DEAD
AT DAY
234
EXPOSURE CONCENTRATIONS ( 1 )
NOMINAL
•mmmimmmmmmmmmmm
100MQ REF/RCF
200MG REF/REF
2SMG REF/REF
30MO REF/REF
SEAWATER/REF
MEA«URED<2)
mmmmmmmmmmmmm
93.09
199. 19
29. OO
41. 79
OXYGEN
MG/L
ANIMALS
USED PER
TREATMENT
4 04
SO
SO
SO
SO
SO
*Y 10
*
4
2
1
3 t
10
96 HOUR LC30 NOT CALCULATED NO EFFECT CONCENTRATION 199.2MG/L REF/REF
ORGANISMS COLLECTED NARROW R. 830630, 3.34 +/- 0. 6SMM PERCENT FOR SOLID PHASE TESTS
-------
TABLE B2
ACUfE TOXICITY DATA SHEET
COE/ERLN FVP
STUDY PLAN: 8 INVESTIGATOR: SCOTT/REDMOND
EXPERIMENT DESCRIPTION: SUSPENDED DATE OF TEST:
TEST NUMBER: 2 CHRONIC CHAMBER SPECIES: AMPELISCA SP
830701
** EXPERIMENTAL CONDITIONS
TEMPERATURE: 20. 00 DECREES CENTIGRADE
SALINITY: 31. OO PARTS PER THOUSAND
EXPOSURE DURATION: 4 DAYS
PHQTOPERIOD: 14 HOURS
FLOW RATE: 9O MLS/MIN VOLUME ADDITIONS/DAY
NUMBER OF ANIMALS/REPLICATE: SO
I-4UMBER OF REPLICATES/TREATMENT: 1
ANIMAL'S LIFE STAGE: JUVENILE ACE: DAYS SIZE:
CONTROLS: SEAWATER/SOUTH REFERENCE SOLID
FOOD USED NONE
RANGE: 19. 9O - 22. OO
RANGE:
30
NOMINAL
•••••••••
100MG REF/REF
200MO REF/REF
23MO REF/REF
30MC REF/REF
SEAWATER/REF
•RATIONS (1)
MEASURED (2)
^R^BUM^BHMHM^BIBflMI
120. 19
233.60
22. 30
60. 20
OXYGEN
M0/L
ANIMALS
USED PER
TREATMENT
4 DAY IO
«•»•;•••••
30 i
1
1
30 i
1
1
so :
so :
!
30 '',
MILLIMETERS
»••••• NUMBER DEAD
*—— AT DAY
I 2 i 3 4 1C
o
2
1
3
3
96 HOUR LCSO NOT CALCULATED NO EFFECT CONCENTRATION 233. 6MG/L REr/REr
ORGANISMS COLLECTED NARROW R. 830630, 3.34 +/- 0. 63MMCN-23). SOLID PHASE RSr
SEDIMENT -ATCH 3, BOTTLES 3, 4. MO/L DETERMINED BY DRY WEIGHT MEASUREMENTS.
U> PERCENT FOR SOLID PHASE TESTS
(25 MILLIGRAMS/LITER FOR SUSPENDED PARTICULATE TESTS; DRY WEIGHTS OR CCULTSS
COUNTS OR BOTH.
-------
TABLE B3
ACUTE TQXICITY DATA SHEET
COE/ERLN FVP
STUDY PLAN: 8 INVESTIGATOR: SCOTT/REDMOND
EXPERIMENT DESCRIPTION: SUSPENDED DATE OF TEST:
TEST NUMBER: 1 ACUTE CHAMBER SPECIES: AMPELISCA SP
** EXPERIMENTAL CONDITIONS »*
830711
RANGE: 20. OO - 20. 30
RANGE:
TEMPERATURE: SO. 00 DEGREES CENTIGRADE
SALINITY: 32. OO PARTS PER THOUSAND
EXPOSURE DURATION: 4 DAYS
PHOTOPERIOD: 14 HOURS
FLOW RATE: 40 MLS/MIN VOLUME ADDITIONS/DAY
NUMBER OF ANIMALS/REPLICATE: 23
NUMBER OF REPLICATES/TREATMENT: 2
ANIMAL'S LIFE STAGE: JUVENILE ACE: DAYS SIZE:
CONTROLS: SEAWATER/SOUTH REFERENCE SOLID
FOOD USED NONE
MILLIMETERS
EXPOSURE CONCENTRATIONS (1)
NOMINAL i MEASURED<2)
100MG BRH/REF
200MO BRH/REF
23MG BRH/REF
30MG BRH/REF
SSAUATER/REF
74. 9O
186.90
32. 40
47. 00
OXYGEN
MO/L
ANIMALS
USED PER
TREATMENT
4 DAY 10
30
30
3O
SO
i 3O
NUMBER DEAD
•mmmmm AT DAY
1234
21
46
12
9
1
96 HOUR LC3O 84. 2(72. 8-97. 4JMG/L NO EFFECT CONCENTRATION LESS THAN 32.4 MG/L
ORGANISMS COLLECTED NARROW R. 830711, 3.39 */- 0. 47MM(N»a3). SOLID PHASE PEF
SEDIMENT ?ATCH 3. 30TTLES 3, 4, 12. MG/U DETERMINED BY DRY WEIGHT MEASUREMENTS.
<1> PERCENT FOR SOLID PHASE TESTS
<2> MILLIGRAMS/LITER FOR SUSPENDED PARTICIPATE TESTS; CRY WEIGHTS OR CQULTES
COUNTS CR BOTH.
-------
TABLE B4
ACUTE TOXICITY DATA SHEET
COE/ERLN FVP
STUDY PLAN: 8 INVESTIGATOR: SCOTT/REDMOND
EXPERIMENT DESCRIPTION: SUSPENDED DATE OF TEST:
TEST NUMBER: 2 CHRONIC CHAMBER SPECIES: AMPELISCA SP
*» EXPERIMENTAL CONDITIONS *»
S3O711
TEMPERATURE: 20. 00 DECREES CENTIGRADE
SALINITY: 32. 00 PARTS PER THOUSAND
EXPOSURE DURATION: 4 DAYS
PHQTOPERIOD: 14 HOURS
FLOW RATE: 4O MLS/MIN VOLUME ADDITIONS/DAY
NUMBER OF ANIMALS/REPLICATE: 3O
NUMBER OF REPLICATES/TREATMENT: 1
ANIMAL'S LIFE STAGE: JUVENILE ACE: DAYS SIZE:
CONTROLS: SEAMATER/SOUTH REFERENCE SOLID
FOOD USED NONE
RANGE: 20. 00 - 20. 90
RANGE.
13
MILLIMETERS
NOMINAL
mmmmmmmmm
IOOMG BRH/REF
2OOMG BRH/REF
29MG BRH/REF
3OMG BRH/REF
SEAWATER/REF
MTRATIONS <1>
MEASURED<2)
92. OO
239. 19
33. 79
39. OO
i
1 OXYGEN
MG/L
mmmmmmmm
ANIMALS
USED PER
TREATMENT
4 DAY 10
»—•!-—-
30 !
1
9O i
4
1
90 :
9O i
1
4
so :
•»•••!
•»mmm
I
......
NUMBER DEAD
• AT DAY
21314
29
44
10
10
2
10
96 HOUR LC30 9O. 9(76. 2-110. 7JMG/L NO EFFECT CONCENTRATION LESS THAN 33.8 MG/L
ORGANISMS COLLECTED NARROW R. 830711, 3.39 */- 0. 47MM(N»23>. SOLID PHASE P.EF
SEDIMENT r-'-TCH 3. 3QTTLES 3,4-12. MG/L DETERMINED BY DRY WEIGHT MEASUREMENTS.
CD PERCENT FOR SOLID PHASE TESTS
(2) MILLIGRAMS/LITER FOR SUSPENDED PARTICULATE TESTS; DRY WEIGHTS OR COULTSS
COUNTS OR BOTH.
-------
APPENDIX C
Growth of A. abdita in Chronic Tests
-------
Table Cl
Mean Length (mm) and Standard Deviation of A. abdita
Exposed
to BRH Suspended Farticulates
for 28 and 45
Days in Chronic Test 1*
Treatment Replicate FOV
FE
FDV
M
OTH
28 Days
REF
5% BRH (1.9 mg/1)
10% BRH (3.8 mg/1)
REF
5% BRH (1.9 mg/1)
10% BRH (3.9 mg/1)
1
2
1
2
1
2
3
4
3
4
3
4
6.81 ±
6.53 ±
6.71 ±
7.30 ±
6.26
7.09 ±
7.55±
7.34 ±
7.46 ±
7.65 +
7.30 ±
7.54 ±
6.92 ±
7.07 ±
7.01 ±
0.58
0.58
0.58
0.34
0.55
0.51
0.39
0.47
0.44
0.40
0.46
0.42
0,56
0.51
7.32 ±
7.03 ±
7.15 ±
7.71 ±
5.81 ±
7.39 ±
7.13 ±
6.18 ±
6.94 ±
45
7.64 ±
7.72 ±
7.68 ±
8.14 ±
7.66 ±
7.97 ±
7.06 ±
7.77 ±
7.49 ±
0.52
0.48
0.51
0.67
0.53
0.96
0.52
0.93
0.70
Days
0.44
0.76
0.63
0.60
0.64
0.65
0.59
0.67
0.72
6.79 ±
5.97 ±
6.35 ±
6.35 ±
5.07 ±
5.18 ±
6.10 ±
5.54 ±
5.79 ±
7.32 ±
5.53 ±
6.43 ±
6.63 ±
6.63 ±
6.16 ±
6.08 ±
6.15 ±
0.49
0.69
0.72
1.18
0.59
0.71
0.66
0.56
0.67
0.26
2.79
1.92
0.89
0.89
0.64
0.89
0.64
6.54 ±
6.56 ±
6.55 ±
6.92 ±
6.15
6.88 ±
6.58 ±
6.58 ±
6.69 ±
6.72 ±
6.70±
6.92 ±
6.56 ±
6.81 ±
6.42 ±
6.52 ±
6.51 ±
0.47
0.49
0.47
0.43
0.45
0.08
0.08
0.50
0.49
0.49
0.49
0.45
0.50
0.37
0.41
0.40
6.82 ±
6.48 ±
6.64 ±
7.06 ±
5.20 ±
5.95 ±
6.45 ±
5.76 ±
5.76 ±
7.74 ±
6.66 ±
7.38 ±
7.94 ±
7.94 ±
6.36 ±
6.98 ±
6.55 +
0.61
0.45
0.55
0.46
0.56
1.06
0.83
0.58
0.79
0.41
0.85
0.75
0.57
0.57
0.51
0.63
0.61
*N for each measurement is shown in Table 12.
Initial size of 100 A. abdita is 3.30 mm ± 0.51.
-------
Table C2
Mean Length (mm) and Standard Deviation of A. abdita
Exposed to BRH Suspended Particulates for 32 and 58 days in Chronic Test 2*
Treatment Replicate FOV
FE
FDV
M
OTH
32 Days
REF
5% BRH (2.0 mg/1)
10% BRH (4.5 mg/1)
REF
5% BRH (2.3 mg/1)
10% BRH (5.0 mg/1)
1
2
1
2
1
2
3
4
3
4
3
4
6.09 ±
6.53 ±
6.14 ±
6.67 ±
6.15 ±
6.34 ±
6.50 ±
7 OA
6.75 ±
7.16 ±
7.26 ±
7.21 ±
6.65 ±
6.89 ±
6.73 ±
6.65 ±
6.05 -
6.57 ±
0.24
0.38
0.29
0.64
0.78
0.76
0.63
0.62
0.62
0.65
0.63
0.79
1.05
0.87
0.38
0.42
6.39 ±
6.33 ±
6.37 ±
6.40 ±
6.35 ±
6.39 ±
5.96 ±
6.49 ±
6.04 ±
58
7.44 ±
7.66 ±
7.58 ±
6.96 ±
7.01 ±
6.99 ±
6.85 ±
5.90 ±
6.69 ±
0.70
0.77
0.73
0.65
0.85
0.72
0.50
0.85
0.58
Days
0.63
0.51
0.56
0.63
0.41
0.48
0.58
0.69
0.69
5.33 ±
5.28 ±
5.29 ±
5.21 ±
5.24 ±
5.22 ±
4.60 ±
4.76 ±
4.67 ±
7.02 ±
7.32 ±
7.18 ±
6.82 ±
6.96 ±
6.88 ±
6.36 ±
5.45 ±
5.80 ±
0.27
0.38
0.36
0.50
0.41**
0.46
0.53
0.51
0.53
0.83
0.48
0.67
0.89
0.64
0.77
0.54
0.53**
0.69
5.51 ±
5.33 ±
5.50 ±
5.60 ±
5.57 ±
5.59 ±
5.25
5.25
6.31 ±
6.58 ±
6.40 ±
6.54 ±
6.13 ±
6.24 ±
5.84 ±
5.43 -
5.83 +
0.41
0.31
0.41
0.43
0.31
0.36
0.60
0.63
0.62
0.10
0.58
0.53
0.45
0.45
5.80 ±
5.67 ±
5.74 ±
5.49 ±
5.46 ±
5.47 ±
4.67 ±
4.27 ±
4.42 ±
7.37 ±
7.26 ±
7.33 ±
7.38 ±
7.15 ±
7.23 ±
6.56 ±
5.46 ±
6.15 ±
0.52**
0.54**
0.53
0.58**
0.53**
0.55
0.63**
0.74**
0.73
0.45
0.51
0.47
0.99
0.48
0.69
0.56
0.56**
0.77
* N for each test is shown in Table 13.
**Indicates those samples which were subsampled with the Folsom plankton splitter.
------- |