EPA -
                     CBP/TRS 89/93
                     December 1992
Development of a Chronic
    Sediment Toxicity Test
       for Marine Benthic
              Amphipods

           December 1992
Chesapeake Bay Program
                      i Printed on recycled paper

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 Development of a Chronic
   Sediment Toxicity Test
      For Marine Benthic
            Amphipods
              Theodore H. DeWitt
              Michele S. Redmond
                John E. Sewall
               Richard C. Swartz
               U.S. EPA - ERLIN
            Pacific Ecosystems Branch
           2111 S.E. Marine Science Dr.
            Newport, OR 97365-5260
        Cooperative Agreement #CR-816299010
                   and
              Contract #68-CO-0051

                Project Officer
               Robert C. Randall
         Office of Research and Development
            Pacific Ecosystems Branch
            2111 S.E. Marine Science Dr.
            Newport, OR 97365-5260

Printed by the U.S. Environmental Protection Agency  for the Chesapeake Bay Program

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DISCLAIMER









      This material has been funded in part by the U.S. Environmental Protection Agency under




Contract #68-CO-0051 and Cooperative Agreement #CR-816299010. It has been subjected to




the Agency's review,  and it has been approved for publication as an EPA document.  Mention




of trade names or commercial products does not constitute endorsement  or recommendation for




use.
      This report is Contribution No. N-240 from EPA's Environmental Research Laboratory-




Narragansett.

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                       TABLE OF CONTENTS
EXECUTIVE SUMMARY

INTRODUCTION

ACKNOWLEDGEMENTS
  111

   x

 xiv
CHAPTER I:  Collection, Handling, and Culture of the Amphipods
       Leptocheirus  plumulosus.  Ampelisca abdita,   Lepidactylus
       dytiscus, and Monoculodes edwardsi

       Introduction
       Leptocheirus plumulosus
       Ampelisca abdita
       Lepidactylus dytiscus
       Monoculodes edwardsi
       Figures
 1-1
 1-3
1-12
1-23
1-27
1-33
CHAPTER II: The Acute and Chronic Sensitivity of the Estuarine
      Benthic Amphipod, Leptocheirus plumulosus, to Chemically-
      Contaminated Sediments

      Introduction
      Materials and Methods
      Results
      Discussion
      Conclusions
      Figures and Tables
2-01
2-03
2-17
2-25
2-40
2-43
CHAPTER III: Development  of a Chronic Sediment Bioassay with
      Ampelisca abdita

      Introduction
      Materials and Methods
      Results and Discussion
      Figures and Tables
3-01
3-02
3-09
3-23
APPENDIX A:  Literature  Review  of  Selected  Chesapeake  Bay
      Amphipods
 A-l

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APPENDIX B: Procedures to Minimize the Risk of Releasing Non-
      Indigenous Amphipods, Pathogens, Waters, or Sediments into
      Local Waters or Watersheds
                                                                         n
B-l
APPENDIX C: Leptocheirus plumulosus Annex to the ASTM E1367-90
      Document
C-l
APPENDIX D: Research Methodology to Assess Chronic Toxicity of
      Marine and Estuarine Sediments with the Benthic Amphipod,
      Leptocheirus plumulosus
D-l
APPENDIX E: Ampelisca abdita: Generic Life Cycle Test Design
E-l
REFERENCES
R-l

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                                                                                Ill



          DEVELOPMENT OF A CHRONIC SEDIMENT TOXICITY TEST




                      FOR MARINE BENTHIC AMPHIPODS
EXECUTIVE SUMMARY








      Most marine sediment toxicity bioassays presently test only the acute mortality of




benthic organisms exposed for short periods of time to contaminated sediment. However, the




contaminant concentration needed to induce mortality may be considerably greater than the




concentration needed to slow somatic growth, or reproductive output. Benthic organisms in




the field are generally chronically (not acutely)  exposed to contaminated sediments, and




benthic populations may be exposed to contaminants for more than one generation. Response




criteria are  needed  that reflect both the lethal and sublethal consequences of long-term



exposure to contaminated sediment.








      Research to  develop a chronic  sediment  test for marine benthic amphipods was



initiated in fall  1989 as a cooperative effort between researchers at the U.S. Environmental




Protection Agency and Oregon State University. A workplan for this research was developed




in conjunction with the EPA Chesapeake Bay Liaison Office, the EPA Office of Puget Sound,




the EPA Office of Science and Technology, and researchers from several laboratories in the




Chesapeake Bay and Pacific Northwest regions. The sequence of work proposed was to (1)




select several amphipod species that were abundant in Chesapeake Bay and showed promise




of being good candidates for use in toxicity tests based on previous research, geographic




distributions relative to  urban or industrial centers,  or  taxonomic  affinity with  other




toxicologically sensitive amphipods; (2) collect these amphipods from Chesapeake Bay, ship

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                                                                                  IV



them, to the EPA Environmental Research Laboratory in Newport, OR, and attempt to culture




each species; (3) conduct short-term, comparative toxicity experiments to select the most




sensitive species; (4) select one or more species for further development based on ease of




culture and handling and toxicological sensitivity; (5) develop a chronic toxicity test method,




including appropriate controls; (6) conduct chronic, concentration-response, sediment toxicity




experiments with chemical-spiked sediment; and (7) conduct a chronic sediment toxicity test




with field-collected, chemically contaminated sediment from Chesapeake Bay.








       We report here the results of this research effort which culminated in the development




of a research method for assessing the chronic toxicity of contaminated marine and estuarine




sediments using the benthic amphipod, Leptocheirus plumulosus.  The report is presented




in three  chapters followed by five appendices.  The first chapter describes our efforts at




collecting, handling, and culturingfour estuarine amphipods from Chesapeake Bay, including




L. plumulosus.  This chapter includes maps of the distribution and abundance of these




amphipods within Chesapeake Bay and methodologies for establishing cultures of amphipods




which  could be readily adopted by other laboratories.   The second chapter reports  the




development of acute and chronic sediment toxicity test methods for  L. plumulosus. its




sensitivity to non-contaminant environmental variables, cadmium, two polynuclear aromatic




hydrocarbons, and contaminated sediment from Baltimore Harbor, MD. The third chapter




reports our attempts to develop a chronic sediment toxicity test with Ampelisca abdita. This




effoi't was not  as successful as that with JL. plumulosus, primarily because we could  not




determine satisfactory conditions for its reproduction.  The L. plumulosus and A.  abdita




chronic sediment toxicity tests were developed independently, and, thus, different conditions




were necessary under which the experiments and final test protocols were conducted. The




different experimental conditions reflected the different ecologies of the two amphipods.

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Chapter  1:  Collection,  Shipping,  Culture, and  Handling  of the  Amphipods
Leptocheirus   plumulosus,  Ampelisca   abdita,  Lepidactylus  dytiscus,   and
Monoculodes edwardsi.
Leptocheirus plumulosus: This burrow-building  aorid amphipod was found throughout

Chesapeake Bay in medium- to fine-grained sediments in waters of ca. 5-25%o.   It was

tolerant of handling and shipping, and quite receptive to culturing.  Cultures started in

March, 1990, were still thriving and expanding in August, 1992.  Static-renewal cultures

were maintained in plastic dishpans with a <1 cm layer of sediment and 10-15 cm layer of

seawater at 20%o and 20°C. The water was replaced three times per week, at which time the

amphipods were fed with a mixture of cultured phytoplankton and a small amount of a dry

food mixture.  Cultures were thinned when the  density of adults exceeded ca. 1.5 cm"2, and

new cultures  started with ca. 100  adults and  200  juveniles.  Generation time for Li.

plumulosus was approximately 4 wk and females produced multiple broods. High numbers

of sub-adult and newborn age-classes were available for sediment toxicity tests at all times

of the year.
Ampelisca abdita: This tube-building ampeliscid amphipod was found in relatively saline

waters (i.e., >20%o) of Chesapeake Bay adjacent to seagrass beds in sandy-mud sediments.

High densities of A. abdita were difficult to obtain in Chesapeake Bay, so animals from

Narragansett, RI, were used also.  This species was more difficult to ship, handle, or culture

than L_. plumulosus. Culture conditions were similar to those for L_. plumulosus, with the

exceptions that A. abdita were fed only algae (i.e., no dry food), the salinity was maintained

at 30%o, and the depth of the substrate was 4 cm.  Our success in culturing A. abdita was

highly variable: some cultures thrived, but most had little reproduction. No environmental

factors could be identified that consistently regulated culture success.

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                                                                                            VI
           Lepidactvlus dytiscus: Only one population of this  haustoriid amphipod was  found in

           Chesapeake Bay during our efforts to collect the animal. It was tolerant of being shipped

           across the country, and was maintained in laboratory culture from March, 1990, through

           August, 1992 under conditions described for L. plumulosus. with the exception that the

           substrate was fine sand and the salinity was 32%o.  Reproduction occurred in spring and

           summer, and was reduced or absent in fall and winter. Additionally, the generation time for

           this species was approximately 1 yr.  For these two reasons, cultures of L. dytiscus were not

           sufficiently productive to supply the numbers of animals, on a year-round basis, that were

           needed for sediment toxicity tests.



           Monoculodes edwardsi:  Populations  of this oedicerotid  amphipod  were found in sandy

           sediments at mesohaline salinities  (i.e. 10-20%c) in Chesapeake Bay.  Mortality during

           shipping was high.  M. edwardsi was cultured under conditions similar to L. plumulosus.

           with the exception that fine sand was used for the substrate, and small cultures have been

           maintained for over 2 yr in the laboratory.  However, generation time appears to be >1 mo.,

           and only low and highly variable population densities (ca. 10-40 animals/pan) were sustained

           under these conditions. Relative to L. plumulosus. it was not practical to attempt to produce

           numbers of M. edwardsi as were needed for the experiments.
           Chapter 2: The Acute and Chronic Sensitivity of the Estuarine Benthic Amphipod,
           Leptocheirus plumulosus, to Chemically-Contaminated Sediments
                 Two sediment toxicity test methodologies, one for acute exposures and the other for

           chronic exposures, were designed using the benthic, estuarine amphipod,  Leptocheirus
_

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                                                                                  vii



plunmlosus.  Both methods used animals from laboratory cultures as were described in




Chapter 1. The 10-d acute sediment toxicity test utilized sub-adult sized animals (i.e., 2-4




mm) under static conditions, whereas the 28-d method used 0-d old newborn L/. plumulosus




under static-renewal conditions.  The procedures for both tests were very similar to sediment




toxicity test  procedures established for other marine and  estuarine, amphipods (ASTM,




1990b). Procedures for reference toxicant controls and negative controls were also established




for both toxicity tests.








       The mortality,  growth, and  fertility of  P0 L. plumulosus were affected by  28-d




exposures to high concentrations of sediment-associated phenanthrene and field-collected




sediment from a highly contaminated site in Chesapeake Bay. Shorter-term exposures (i.e.,




10-d) of sub-adult L. plumulosus to sediment-associated acenaphthene, phenanthrene and the




polluted Chesapeake Bay sediment also affected mortality and growth; reproduction was not




recorded in the 10-d exposures since the test was designed to minimize the likelihood that




broods would be released during the exposure. The sensitivity of the 10-d and 28-d tests were




similar, particularly with respect to mortality and growth. Fertility, the number of juveniles



produced per female in an exposure chamber, was considerably more sensitive than mortality




or growth in one experiment, but not in a second experiment.








       The acute and chronic ~L. plumulosus sediment toxicity tests are sufficiently developed




to be used to assist in the evaluation of sediment quality, but the methodologies should be



viewed as interim in development until their limitations are  better defined.  This is




particularly true for the 28-d test method for which several uncertainties remain.  Chief




among these is the interaction between  nutrition and toxicological sensitivity, but also




requiring attention are the effects of salinity,  temperature, and grain size on response

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                                                                                 Vlll
sensitivity, its sensitivity to other chemicals, and its sensitivity relative to other toxicity tests.



Reference toxicity tests also need to be developed for the sub-lethal responses of growth and




fertility.
Chapter 3: Development of a Chronic Sediment Bioassay with Ampelisca abdita








       Research with Ampelisca abdita sought to develop culture methods and a chronic




bioassay for this species. Bioassay development built on the research of Scott and Redmond




(1989), who showed that A. abdita could be used to test chronic and population endpoints.




Culturing methods and results are described in Chapter I.  The approach to chronic test




development was to 1) establish cultures, 2) estimate optimum  temperature and salinity



regimes, 3) outline  a proposed chronic test design, 4) evaluate the chronic test design with




uncontaminated sediment,  and 5) evaluate the chronic  test design with contaminated




sediment. The experiments conducted addressed points 2-4. Both cultures and the controlled




experiments described in this section utilized amphipods from Narragansett, RI.
       A workable draft test protocol for a generic, 35-day chronic sediment toxicity test with




this species was developed.  However, successful reproduction in laboratory-held A. abdita




was inconsistent.   Juvenile  amphipods of a known age were  successfully isolated from




brooding females held in seawater only.  Although it was  feasible to initiate a test with




newly-released juveniles, 8-10 day old amphipods were easier to work with. Sex ratio of




juvenile amphipods used to start a test was determined from daily and final observations.




A survival curve for an acceptable test control could be distinguished from that showing




unacceptable control mortality.  Significant differences in growth were also detected under

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                                                                                   IX



control conditions in 10-d to 14-d exposures for this species, and a short-term growth test may




be a viable sublethal toxicity test for A. abdita.









      There are still unresolved problems with the culture and chronic testing  of this




species.  In controlled experiments with uncontaminated sediment, the amphipods grew,




looked healthy,  and produced eggs and sperm, but rarely reproduced.  Replicate culture




containers with the same density, light cycle, salinity, temperature, sediment, and renewal




and feeding regimes performed drastically differently, regardless of container type. Shipping




and handling stress may be particularly important in determining the success of subsequent




toxicity test responses.  Offspring of field-collected and shipped females with broods showed




poorer survival after 10-d than did offspring from cultured females. Juveniles developing in




the maternal brood pouch may be a very sensitive life stage for A. abdita. This species may




require a flow-through system with frequent volume replacements, a different photoperiod




and temperature regime, or may not be culturable in some waters.









       To complete the development of a sublethal sediment tests with this species, the low




reproduction  problem must  be  resolved,  successful  life  cycle  tests  conducted  in




uncontaminated sediment to firmly establish performance under control conditions,  and




finally  chronic  and  short-term growth  tests  conducted with contaminated material.




Intel-laboratory comparisons of the test methodology will be vital to ensure that this chronic




test can be conducted successfully in other regions of the country.

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 INTRODUCTION








       Nearly all chemical contaminants entering coastal waters eventually accumulate in



 the sediments.  Most toxic contaminants (including heavy metals, chlorinated pesticides,




 PAH's, PCB's, TBT, oil and grease) bind to particulate matter in the water column and sink




 to the sediment surface. Thus, concentrations of toxic chemicals in sediments can be several




 orders of magnitude higher than in the  water column.  As many of these  chemical




 contaminants are persistent and can exert toxic effects to both benthic and demersal biota



 for years after their initial discharge, sediments have become both a sink and a source of



 contamination in many marine ecosystems.








       The impact of sediment pollution on marine ecosystems is reflected in changes in




 macrobenthic community structure and function (Pearson and Rosenberg, 1978; Swartz et al,



 1985b, 1986a; and others). Benthic infauna are good indicators  of sediment contamination



 because of their proximity and  long-term  exposure (as residents of sediments)  to toxic




 materials in polluted sediments.  Benthic community responses to organic enrichment are




 predictable (Pearson and Rosenberg, 1978; Mearns and  Word, 1982),  but comparable




 predictive models of the response of the benthos to chemical contaminants have not been




 developed.  Major obstacles to the development of such models are (1) our inability to



 discriminate between organic enrichment and contaminant effects and (2) uncertainty in the



long-term responses of benthic fauna to chemical contaminants.
      Little  is known  of the  toxicological  responses of most marine benthic taxa to




contaminated sediment.  Only a few marine species have been examined, and most data




concern only  acute mortality (Swartz,  1987).  Nonetheless, the acute mortality of certain

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                                                                                  XI
benthic species is being used as an assessment of the environmental impact of contaminated




sediment (Chapman and Long,  1983).  Surveys of the relative toxicological sensitivities of




taxonomically diverse benthic  species are necessary to validate the current choices of




sediment toxicity test species.  Furthermore, virtually nothing is known of the long-term




effects of contaminated sediments on benthic populations, such as effects on individual




growth rates, reproductive output, or rate of population growth. New sediment toxicity tests




are needed to predict the long-term and sublethal impacts of chronic exposure of benthic




invertebrates to low levels of sediment contamination. These issues must be addressed if the




effects of contaminated sediments on  marine ecosystems are to be assessed and protective




sediment quality criteria developed.








       Most marine sediment toxicity bioassays presently test only the acute mortality of




benthic organisms exposed for short periods of time to contaminated sediment. However, the




contaminant concentration needed to induce mortality may be considerably greater than the




concentration needed to slow somatic growth, or reproductive output. Benthic organisms in




the  field are generally chronically (not acutely) exposed to contaminated sediments,  and




benthic populations may be exposed to contaminants for more than one generation. Response




criteria are needed that reflect both the  lethal and sublethal consequences  of long-term




exposure to contaminated sediment.








       Research to develop a  chronic sediment test for  marine benthic  amphipods  was




initiated in fall 1989 as a cooperative effort between researchers at the U.S. Environmental




Protection Agency and Oregon State University. This project was funded in part by the EPA




 Chesapeake Bay Liaison Office, EPA Office of Puget Sound, and EPA Office of Science and




 Technology with the understanding that  the new sediment toxicity test would be directly

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 applicable to sediments from Atlantic coast and Pacific Northwest estuaries. A workplan for




 this research was developed in conjunction with these EPA offices and researchers from




 several laboratories in the Chesapeake Bay and Pacific Northwest regions. The sequence of




 work proposed was to (1) select several amphipod species that were abundant in mid-Atlantic




 estuaries, particularly Chesapeake Bay, and showed promise of being good candidates for use




 in toxicity tests based on previous research, geographic distributions relative to urban  or




 industrial centers, or taxonomic affinity with other toxicologically sensitive amphipods; (2)




 collect these  amphipods from Chesapeake Bay, ship them to the EPA  Environmental




 Research Laboratory in Newport, OR, and attempt to culture each species; (3) conduct short-




 term, comparative toxicity experiments to select the most sensitive species; (4) select one  or




 more species for further development based on ease of culture and handling and toxicological




 sensitivity; (5) develop  a chronic toxicity test method, including appropriate controls; (6)




 conduct chronic, concentration-response, sediment toxicity experiments with chemical-spiked




 sediment; and (7) conduct a chronic sediment toxicity test with field-collected, chemically




 contaminated sediment from Chesapeake Bay.
       We report here the results of this research effort which culminated in the development




of a research method for assessing the chronic toxicity of contaminated marine and estuarine




sediments using the benthic amphipod, Leptocheirus plumulosus.  The report is presented




in three chapters followed by five appendices.  The first chapter describes our efforts at




collecting, handling, and culturing four estuarine amphipods from Chesapeake Bay, including




JL plumulosus.  This chapter includes maps of the distribution and abundance of these




amphipods within Chesapeake Bay and methodologies for establishing cultures of amphipods




which could be readily adopted by  other laboratories.  The second chapter reports the




development of acute and chronic sediment toxicity test methods for _L. plumulosus, its

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sensitivity to non-contaminant environmental variables, cadmium, two polynuclear aromatic




hydrocarbons, and contaminated sediment from Baltimore Harbor, MD. We believe these




methods will find wide utility in Chesapeake Bay and throughout much of the country.  The




third chapter reports our attempts to develop a chronic sediment toxicity test with Ampelisca




abdita.  This effort was not as successful as that with L. plumulosus, primarily because we




could not determine satisfactory conditions for its reproduction. The appendices include (A)




a literature review of the biology and ecology of the amphipods initially considered for the




sediment toxicity test development; (B) a protocol for handling and disposing materials that




come into contact with non-indigenous amphipods, sediments or waters; (C) a methodology




for testing the acute toxicity of contaminated sediment with L. plumulosus; (D) a methodology




for conducting chronic sediment toxicity tests with K plumulosus; and (E) the design of a life




cycle sediment toxicity test with A. abdita.

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                                                                               XIV
ACKNOWLEDGEMENTS
       Many aspects of this project could not have been completed without the help of several




people. We thank Ray Alden, Rich Batiuk, Faith Cole, Janet Lamberson, Beth McGee, Chris




Schlekat and John Scott for helping to improve this manuscript through their thoughtful




reviews.  We also thank Richard Batiuk, Jack Gakstatter, Chris Zarba, Barry Burgan, Ray




Alden, Bob Diaz, Tuck Hines, Fred Holland, Rom Lipcius, Harriet Phelps, Eli Reinharz, and



John Scott for guidance in developing the research plan and for providing logistical support




during the collection of amphipods from Chesapeake Bay and Narragansett Bay. We thank




John Brezina, Emily Deaver, Paul Gerdes, Tammy Tonare, and Tom White for their




assistance in the field. We thank Beth McGee and Chris Schlekat for collecting sediment and




preparing the sediment dilution series and Claudia Walters for providing QA/QC assistance




in the experiment with field sediment. We thank Dave Hansen for providing AVS and SEM



analyses.  We also thank Mary Culver, Linda Lip trap, and Sharon Nieukirk for assistance




in preparation of species distribution maps. And finally, we wish to thank our US EPA and



AScI colleagues in Newport, OR, for their tireless assistance in the culturing, toxicology, and




chemistry laboratories: Michael Becerra, Wally DeBen, George Ditsworth, Steve Ferraro,




John Frazier, Laura Hoselton, Jill Jones, Janet Lamberson, Bob Ozretich, Don Schults, and




Rob  Singleton.  This  project was supported in part by US EPA cooperative  agreement




CR816299010 to Oregon State University.  Funds  for this research were provided, in part,




by the EPA Chesapeake Bay Liaison Office, the EPA Region 10 Office of Puget Sound, and



the EPA Office of Science and Technology.

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                                  Chapter I








    COLLECTION, HANDLING, CULTURE OF THE AMPHIPODS




       LEPTOCHEIRUS PLUMULOSUS, AMPELISCA ABDITA,




  LEPIDACTYLUS DYTISCUS, AND MONOCULODES EDWARDSI
1.1 INTRODUCTION








      The development of acute and chronic sediment toxicity tests with Chesapeake Bay




amphipods began with the identification and collection of candidate species and quickly




followed with the culture  of these species. Six species were selected for consideration for




sediment toxicity test development from a list of 60 amphipod species (Appendix A) based on




their distribution relative to sediment contamination, taxonomic and ecological similarity to




other amphipods currently used in sediment toxicity tests, and their relative abundance and




ecological importance in  Chesapeake  Bay.   Of these,  five  species were collected from




Chesapeake  Bay sediments  in  March, 1989, and shipped west to the EPA ORD ERL-N




Newport laboratory for development as sediment toxicity test species.  Culture techniques




needed to be developed for  each species so that sufficient numbers of animals could be




available for experiments.  Methods for field collection, handling and shipping, and culturing




four species are presented below.
      These five amphipod species  (i.e.,  Leptocheirus plumulosus,  Ampelisca abdita,




Lepidactylus dytiscus, Monoculodes edwardsi, and Corophium lacustre) were collected from






                                       1-1

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                                                                                  1-2
 various localities between Baltimore, MD,  and Virginia Beach, VA, by T.H. DeWitt and
 assistants during a trip that extended from 19 to 28 March, 1990.  Shortly after collection,
 each species was sorted, held in native habitat sediment in running (or frequently changed)
 seawater, placed in shipping containers and shipped via Federal Express (overnight delivery)
 to the EPA laboratory in Newport, OR. A sediment sample from each collecting site was also
 sent to Newport for grain size analysis. One of the original candidate species (Neohaustorius
 schmitzi) was not collected for logistical reasons (i.e., uncertainty of collecting locations), and
 an alternate species, G. lacustre. was substituted.  However, G. lacustre failed in culture,
 subsequent attempts to have it collected and  shipped to Oregon failed, and it was deleted
 from the remainder of the research effort.  Ampelisca abdita was also collected from
 Narragansett, RI, by personnel from the EPA  Environmental Research Laboratory.

       Leptocheirus plumulosus showed the  best promise  for mass culturing,  although
 populations of Ampelisca abdita. Monoculodes edwardsi and Lepidactylus dytiscus have been
 sustained for nearly  two years in the laboratory.  Static-renewal culture conditions were
 employed for all species because of previous success at culturing amphipods in this manner
 (DeWitt, 1987)  and in order  to minimize the amount of waste-water which had  to be
 sterilized.  Because these species were not indigenous to Oregon estuaries, special efforts
 were made to chlorine-bleach-sterilize  or autoclave all materials (e.g., sediment, water,
 glassware, adsorbent materials, culturing and handling equipment, etc.) that came into
 contact with the amphipods before  otherwise used  or discarded.  These materials  were
 sterilized to minimize the risk  of accidentally introducing non-indigenous amphipods or
pathogens into local waters. Procedures for handling and sterilization of these material are
presented in Appendix B. We strongly believe that these quarantine handling and culturing
practices must be adopted by all laboratories using any non-indigenous toxicity test organism

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                                                                                1-3
or sediment.  Additionally, some States require permits and special containment procedures
for handling and culturing non-indigenous species; the procedures described below may or
may not be sufficient for another laboratory's permit to work with such materials.
1.2 Leptocheirus plumulosus

1.2.1  SUMMARY

Leptocheirus plumulosus was collected in large numbers from the field and was easily mass-
cultured in the laboratory. This species was very tolerant of being handled and survived
shipping well.   Cultures were reared under static renewal  cultures  using  equipment
commonly  available  in  aquatic  laboratories.   Further research  into the  nutritional
requirements of this species and simplification of handling methods should further reduce the
cost and effort involved in culturing these amphipods, and may make culturing preferable to
field collections even in areas with large natural populations.

1.2.2  OVERVIEW OF THE SPECIES

       Leptocheirus  plumulosus  is a euryhaline amphipod of the family Aoridae found
throughout the mesohaline portions of Chesapeake Bay (Fig. 1-1).   Under  laboratory
conditions, it is fast growing and can mature in less than 25 days at 25°C.  The size of the
first brood is typically 10-20 young for a healthy female; larger mature females can produce
over 40 young in a single brood (see Chapter 2). At 25°C, the minimum interval  of time
between broods is less than 10 days.  Females may live for  over 100 days and produce at

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                                                                                           1-4



          least 6 broods in that period, and potentially may live longer and produce more broods.




          Reproduction of animals in the cultures continues uninterrupted year-round under constant




          culture  conditions.  The sex ratio  of the young  is assumed to be  approximately 1.0




          (females/males). The newly released young are about 1.5 mm long, and, at 25°C, they can



          double in size in about 10 days and triple their size (or more) in 14 days.  Large adults are




          over 1 cm long. Eggs first  appear in the ovaries of females by age 12-d (i.e.,  days since




          leaving the maternal brood pouch), and eggs may be seen in the brood pouch by age 14-d.








             L. plumulosus constructs a U-shaped burrow in the soft, organically rich sediment that




          it seems to prefer.  The burrow walls have little cohesive structure to them, and the burrows




          disintegrate during sieving.  The animal pumps water through the burrow and may filter out




          suspended particles  for food.  It also pulls in sedimentary particles surrounding the tube




          opening, apparently scraping the surface of mineral particles for food or tearing pieces  of




          organic material into small  enough pieces to ingest.  Animals will occasionally leave their




          tubes to roam the sediment  surface,  apparently  picking up  pieces of  food material or




          searching for mates. This  feeding mode may allow individuals to live in water without




          sediment for extended periods of time if particulate food is available. The regular spacing




          of burrow openings suggests that this species  may be territorial.  Males may compete for



          mates as evidenced by their  high mortality, due to fighting, when held in culture containers




          in the absence of females. Survival of both sexes may be >90% in mixed sex culture between




          0-4 wk of age.
                 Please refer to Appendix A (Literature  Review  of Selected  Chesapeake  Bay




          Amphipods) for further details and references concerning the natural history of Leptocheirus




          plumulosus.
_

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                                                                                1-5
1.2.3  FIELD COLLECTION
      Leptocheirus plumulosus were collected in shallow water (1-2 m deep) by hand using




a small grab sampler and a suction dredge; the latter was far more efficient at collecting this




amphipod.  Approximately 50 amphipods were  collected in a 2 h period by T.H. DeWitt on




3/21/90 from muddy sediment at the end of the fishing pier at Fort Armitage Park in




Baltimore Harbor, MD, with a small Ponar sampler in 1-2 m depth (salinity ca 5%o). This




was not a good site for collecting L. plumulosus.  Approximately 1000 animals were collected




rapidly (i.e., < 0.5 h) by T.H. DeWitt and Tom White (Virginia Institute of Marine Science)




on 3/24/90 from muddy substrate in Queens Creek, York River, near Williamsburg, VA, in




shallow water (ca. 1 m deep) by suction dredge (salinity ca 14%o); this was a very good site




to collect L_. plumulosus in March, 1990.  Scientists at the Maryland  Department of




Environment have routinely collected this species from Corsica River  and  Magothy River




subestuaries in northeastern Chesapeake Bay (B.  McGee  and C.  Schlekat,  personal




communication).  However, the  abundance of  this amphipod can be variable at any site




(including Queens Cr.  and Corsica R.), ranging from highly abundant  to absent  (Emily




Deaver and Ray Alden, Old Dominion University, pers. comm.).
       Leptocheirus plumulosus is widely distributed in Chesapeake Bay (Fig. 1-1), and is




therefore potentially widely available year-round. However, this amphipod is highly motile




especially nocturnally, at which time many individuals may be found swimming in the nekton




(Dauer et al, 1982). Furthermore, L.. plumulosus populations boom in the  early spring and




bust in the summer following the return of predatory fish to Chesapeake Bay (Hines et al,




1986).  Thus, population densities at specific collection sites will probably vary considerably

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                                                                                           1-6
          throughout the year, and some sites will be ephemeral in  quality while others (such as
          Corsica R., apparently) may be quite dependable as sources of this amphipod.
           1.2.4 SHIPPING

                 Leptocheirus plumulosus were shipped successfully from Chesapeake Bay to Newport,
           OR, in March, 1990, and from Newport, OR,  to Narragansett, RI, in June, 1991.  Field-
           collected animals were held overnight or longer in running or frequently changed bay water
           at a salinity and temperature close to that where the animals were collected. Dead or injured
           animals were removed prior to packing. The choice of salinity depended on the ambient
           salinity from which the animals were taken: ~15%o from the field, and  20%c from the
           laboratory cultures. Amphipods were shipped at densities of 50-100 per plastic container (i.e.,
           a 250-1000 ml sandwich box or ice cream tub) containing water and substrate (i.e., 0.2-1.0
           cm layer of marsh grass detritus) or just water (i.e., 15-25%o).  Field-collected animals were
           shipped with substrate and cultured amphipods without substrate.
                 Several plastic containers were placed in an insulated cooler along with 3 or 4 freezer
          packs (such as blue ice) to keep the temperature cool, but above freezing, and then the cooler
          was sealed and immediately shipped by overnight delivery. The field-collected L. plumulosus
          suffered approximately 25% mortality, whereas very few of the laboratory-cultured animals
          died during shipment. Mortality may have been due to lack of oxygen caused by the BOD
          of the higher organic-content substrate included with the field-collected amphipods.  Future
          shipments of L. plumulosus should (1) minimize the amount of substrate included in the
          shipping containers, (2) use low organic-content or sterile substrates, or (3) omit substrate
_

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                                                                                1-7
altogether.  Shipping success might be further enhanced if the amphipods were shipped in
oxygen-saturated water.

       Upon arrival at the laboratory, the unopened containers of amphipods were placed in
a water bath and slowly acclimaited to 20°C.  The containers were then opened, the overlying
water was decanted for chlorination and disposal, and the containers were refilled with 20°C
water at the same salinity as the packing water.  Later, the amphipods were sieved from the
packing substrate and transferred to tubs for culturing.
1.2.5  CULTURING

       Leptocheirus plumulosus was cultured in inexpensive polyethylene tubs measuring
29.2 cm x 34.3 cm x 13.3 cm (depth) (i.e., 11.5" x 13.5" x 5.5"), holding about 13 L (3.5 gal)
of seawater (11-12 cm deep) with a <1 cm thick sediment layer. This configuration held
several hundred mature animals and facilitated the handling of individual culture containers
for sieving, water replenishment and moving. The tubs were held in shallow seawater-table
trays which served the dual purposes of catch basins for any water spilled from the tubs and
water baths to  maintain the appropriate culture temperature.  The amphipods were also
cultured in the  tubs placed on shelves with the room air temperature maintained at 20°C.

   The cultures were  maintained at 20°C and a salinity of 20%c. The culture sediment was
a muddy sand from South Beach, Yaquina Bay, OR, that had been sieved through a 0.5 mm
or 0.25 mm mesh sieve. Photoperiod was maintained at 16:8 hr light:dark.  The tubs were
gently aerated constantly. About 60% of the water in each tub was changed every other day,

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                                                                                1-8
except on weekends. This was accomplished by pouring off the old water and refilling the
tub from a plastic pitcher with fresh seawater laden with the algal food. The stream of
incoming water was directed onto a flat piece of glass held at the water surface to disburse
the flow and minimize disturbance of the sediment bed.  The renewal water consisted of
seawater (ca. 32%o), cultured phytoplankton and deionized water which were combined to a
salinity of 20%o and ca. 106 algal cells per ml. The algae used are Pseudoisochrysis paradoxa
and Phaeodactylum tricornutum in equal portions by volume.  The  cultures were also fed
about 0.5 gr. of a dry food (i.e., "gorp") just after the water change. Gorp consisted of 48.5%
Tetra min®, 24% dried alfalfa, 24% dried wheat leaves and 4.5% Neo-Novum® (a maturation
feed for shrimp mariculture; Argent Chemical Laboratories, Redmond, WA), combined and
ground to a fine powder.  The gorp was sprinkled on the water surface.

      An important aspect of maintaining culture health was  to prevent overcrowding.
Densities should be maintained below 1500 per  tub  (e.g., <1.5  cm'2).  The occurrence of
overcrowding was marked by cultures with a large number of animals of small size and few
gravid females. Those females that were gravid bore only a very small number of eggs (e.g.
<5). Under these  conditions, newly released young were very difficult to obtain,  and  the
animals in the tubs appeared to be stressed from food or space limitation. The number of
adults (i.e., animals >4 mm long) should not exceed ca. 400 per tub.  To avoid overcrowding,
cultures were thinned.approximately every two months by sieving through a 1 mm mesh
sieve. This allowed the young to pass through and remain in the sediment. Only about 100
healthy adults were returned to the culture tub.  The rest were used to start new cultures
or disinfected and discarded.

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                                                                                  1-9



    L. plumulosus has been in culture in the Newport, OR, EPA laboratory since March, 1990.




The performance of the cultures and the potential of this species for mass production of




animals are excellent.  Twenty culture tubs are currently maintained at 20°C which has




consistently provided  sufficient numbers of animals  to conduct 50-  to  80-beaker acute




sediment toxicity experiments.  These cultures provided up to 1000 newly released young per




month for chronic sediment toxicity tests. Densities of >1500 animals per tub (i.e., >1.5 cm'2)




were readily achieved in the culture tubs, but this was an overcrowded density leading to




reduced growth and fecundity as described previously. L. plumulosus does not seem sensitive




to seasonal changes when maintained under constant culture conditions,  and can provide




animals for toxicity tests year-round.
1.2.6  HANDLING









       The contents of culture tubs were gently sieved through a 0.5 mm mesh to obtain




subadult L. plumulosus (i.e., 2-4 mm long) for acute toxicity tests. This allowed some of the




very smallest animals to pass through, but retained all of the animals over a few days old.




Larger  animals were excluded by gently  sieving the animals  through a  1 mm screen.




Animals were  rinsed free of sediment  and washed into  a shallow glass  picking dish.




Subadults 2-5 mm long were transferred by pipette into a smaller glass dish for acclimation




to the test  temperature and  salinity.   Water for  sieving and rinsing were the  same




temperature  and salinity as the cultures (e.g., 20%o) to minimize stress.  Unused cultured




animals were returned to the culture tub after the sediment bed had settled for a few hours.

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                                                                                           1-10



                  Cultured amphipods used in the 28-d chronic sediment toxicity test were of uniform




           age, 1- to 2-d post brood-release.  To obtain newly-released juveniles, contents of the culture




           bins were sieved through a 1 mm screen to isolate adults, and gravid females were selected




           from the mass of adults and transferred to fresh culture tubs with sediment so that they




           could acclimate to the temperature and salinity of the toxicity test. The females were fed in




           the same  manner as the general cultures during this acclimation period.  Gravid females




           were isolated for 8 days before the start of the toxicity test if the test was run at 20°C, or for




           5 days in advance if the test was conducted at 25°C. Gravid females were easily recognized




           by the dark egg mass in the brood pouch; as the eggs approach hatch, the mass turned a tan




           color and became somewhat translucent which was more difficult to see without a dissecting




           microscope. Three days before the start of a toxicity test, the females were sieved from the




           isolation tub using a 1 mm mesh, rinsed well and placed in a glass dish with water only (e.g.,




           no sediment) at the test temperature and salinity and fed with the algal suspension used to




           feed the general cultures.  The females were inspected to insure that no previously released



           young were transferred to the glass dish. All debris was removed from the bottom of the dish




           since this could conceal young. The following day, the adult females were separated from the



           young they had released using a 1 mm screen, and the young left behind were ready for



           experimental use. This process was repeated on the succeeding two days in order to provide




           additional animals in the event that insufficient numbers of young were produced on the day




           of the toxicity test. The isolated juveniles were maintained in a glass dish with sediment pre-




           sieved to <0.25mm  and were fed with the algal suspension. Exposures were initiated only



           with the <24hr-old juveniles.  If insufficient numbers of these young were available, some




           replicates of the experiment were set aside to be started the following day with newly isolated




           juveniles.  This procedure was judged to be superior to mixing together juveniles produced
_

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                                                                                 1-11
on different days on the basis that variability in sensitivity or growth within replicates would
be reduced if all test animals were of the same age.

       The performance control (e.g., culture sediment) survival of these young was >90%.
Although each female can produce up to 40 young, we isolated one gravid female for each
newborn that is needed for a toxicity test.  This seeming excess was required because of the
uncertainty of the timing of brood release for each female.  After producing young, the
females were returned to the cultures; the processes of culture thinning and isolating newly
released young were often combined into a single effort.
1.2.7 CONCLUSION


   Leptocheirus plumulosus was well adapted for laboratory culture. Cultures consistently
produced large numbers of animals within narrow age brackets required for research or
routine acute or chronic toxicity tests.  Field-collected animals may have also been used for
acute and chronic sediment toxicity tests, although the utility of these approaches was not
evaluated in this study.  Further work in improving culture techniques should be directed
at defining the minimum diet required to maintain highly productive cultures, obtaining
greater synchrony in brood release among gravid females, and simplifying the  process of
obtaining newly released young needed for chronic toxicity tests.

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                                                                               1-12



1.3 Ampelisca abdita








1.3.1  SUMMARY








      This species has been maintained in culture for several generations, but with great




variability in culturing success.  Periodic reference toxicant tests with cadmium chloride




showed that the sensitivity of cultured animals was comparable to that of field-collected




animals from the source population in Narragansett, RI.  Several hundred animals were




maintained at 20°C in each of several plastic dish bins, with about 4 cm of Yaquina Bay,




Oregon, sediment, and about 13 cm of overlying seawater. Half of the overlying seawater was




renewed 3-5 times per week with a mixture of seawater (30%o) and algae. Amphipods were




sieved from the culture bins when needed for testing, or when density exceeded 2000 animals




per bin. With more research, this species may hold the potential to be routinely cultured.




However,  the productivity of these cultures was too variable to consistently provide the large




numbers of animals needed for frequent toxicity tests.
1.3.2 OVERVIEW OF THE SPECIES
       Ampelisca abdita is a tube-dwelling amphipod belonging to the family Ampeliscidae,




found mainly in protected areas from the low intertidal zone to depths of 60 m.  It ranges




from central Maine to south-central Florida and the eastern Gulf of Mexico (Mills  1964,




Bousfield 1973), and has also been introduced into San Francisco Bay, CA (Nichols and




Thompson 1985). In Chesapeake Bay, A. abdita has been reported from moderate- to high-




salinity waters (e.g., >20%o) (Fig. 1-2).  Where A. abdita are present, they are often dominant

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                                                                                1-13
 members of the benthic community, with densities up to 110,000 m'2 (Nichols and Thompson
 1985, Stickney and Stringer 1957, Santos and Simon 1980).  This species generally inhabits
 sediments from fine sand to mud and silt without shell, although it may also be found in
 relatively coarser sediments with a high organic content (Stickney and Stringer 1957).

       Ampelisca abdita is a particle feeder, feeding both on particles in suspension and on
 those from the surface of the sediment surrounding its tube.  Gut contents of field-collected
 specimens have been found to include algal material, sediment grains, and organic detritus
 (Mills  1967,-Stickney and Stringer 1957).

       In the colder waters of its range, A. abdita produces two generations per year, an
 overwintering generation which breeds in the spring and a second which reproduces in mid
 to late summer (Mills 1967, Nichols and  Thompson 1985).  Each female produces one brood,
 and males die shortly after mating.  Sex ratio  of the population at  breeding times is
 approximately 1:1.  In New England, breeding of the overwintering generation begins when
 the water temperature is about 8°C, but in warmer waters south  of Cape  Hatteras, NC,
 breeding might be continuous throughout the year.  Adults mate in the water column, and
 intense breeding activity is correlated with the full moon and spring tides.  Juveniles are
 released after approximately two weeks  in the brood pouch, at about 1.5 mm in length.  It
 then takes 40-80 days for newly released juveniles to become breeding adults (Mills 1967).
 Females in a population from Barnstable Harbor, MA, were found to carry a mean of 26 eggs
(Mills 1967),  and a population from North Carolina a mean of 13.7 (Nelson  1980).  In the
laboratory, A. abdita will breed all year,  although large numbers of individuals are needed
to ensure the ability to harvest sufficient numbers for testing. At 20°C,  its full life cycle is
approximately 6 to 8 weeks (Scott and Redmond 1989).

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                                                                               1-14



      A.  abdita has been collected in waters of -2°C to 27°C  (Redmond and Scott,




unpublished data). It is euryhaline, and has been reported in waters which range from fully




marine to 10%o salinity (Bousfield 1973). This species is photonegative, and has been found




to have  a strong mortality response when exposed to sunlight  (Redmond and Scott,




unpublished data).








      Please  refer  to  Appendix A (Literature  Review of Selected Chesapeake  Bay




Amphipods) for further details and references concerning the natural history of A. abdita.
1.3.3  FIELD COLLECTION
       Ampelisca abdita were collected by hand (e.g., shovel and sieve) and with a suction




dredge from intertidal and shallow subtidal sediments, respectively.  Personnel from the EPA




Environmental Research Laboratory and SAIC in Narragansett, RI, collected several hundred




amphipods by shovel and sieve from the intertidal in Pettaquamscutt Cove (Pettaquamscutt




River, Narragansett, RI) on 1/30/90, 8/29/90, 3/26/91, and 7/31/91.  They regularly collect A.




abdita in this manner from this site, although winter collections sometimes require chopping




through ice to access the sediment.  T.H. DeWitt (with help from Paul Gerdes, Virginia




Institute of Marine Science) collected approximately 150 A. abdita on 3/24/90 with a suction




dredge from muddy sand substrate in Zostera beds off the lee side of Allen's Island, VA, near




the mouth of York River (salinity ca 25%o). This method allowed rapid collection of many




amphipods over a large area of the benthos. However, many other amphipod species were




simultaneously collected requiring considerable post-collection sorting.  Subtidal populations

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                                                                                1-15
 (3-10 m) were collected from several sites in San Francisco Bay on 7/30-31/91 with a small
 grab sampler and bucket dredge from a boat (John Brezina, personal communication).

       A. abdita is widely distributed along the U.S. eastern seaboard (including the lower
 reaches of Chesapeake Bay, Fig. 1-2) and San Francisco Bay, CA, and is therefore potentially
 widely available for field collection. Intertidal populations may be seasonally ephemeral, with
 mass emigrations occurring in response to disturbance from other macrofauna or storms
 (Mills, 1967; Grant,  1965).  However,  the intertidal and shallow subtidal population at
 Pettaquamscutt R. (Narragansett, RI) has proven to be a dependable, year-round source of
 A. abdita for several years (M.S. Redmond, unpubl.  data).
1.3.4 SHIPPING
       Ampelisca abdita have been shipped around the country in several different ways,
each with mixed success.  Field-collected animals were held overnight or longer in running
or frequently changed bay water at a salinity and temperature  close to that where the
animals were collected. Dead or injured animals were removed prior to packing. Am phi pods
were shipped from Chesapeake Bay in March, 1990, packed 25-50 individuals per water-filled
(20%c) plastic container (i.e., 250-1000 ml sandwich boxes or ice cream tubs) with a 3-5 mm
thick, silty-sand substrate. Amphipods were shipped from Narragansett, RI, in (1) sandwich
boxes or cubitainers with A. abdita in seawater only, (2) sandwich boxes with a 2 cm layer
of mud, (3) sandwich boxes filled with mud and >1 cm layer of seawater, or (4) 4-1 jars with
a 2 cm layer of mud.  The various  containers were topped-off with water or had a 1 cm
headspace of air. Several plastic containers were typically placed in an insulated cooler along

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                                                                               1-16



with 3 or 4 freezer packs (such as blue ice) to keep the temperature cool, but well above




freezing, and the cooler was sealed and immediately shipped by overnight delivery. A. abdita




in their tubes only were shipped from San Francisco Bay in large plastic bags filled to 1/2




capacity with water, with a 5-6" air headspace.  Each bag was packed in  an insulated




cardboard box with a single ice pack for cooling.








       Survival of shipped A. abdita was variable. Inclusion of ice packs was very important:




most of the amphipods died in one shipment that omitted cooling.  Shipping success might




be further enhanced if the amphipods were shipped in oxygen-saturated water.








       Upon arrival at the laboratory, the containers of amphipods were opened, placed in




a water bath with aeration,  and slowly acclimated to 20°C.  The overlying  water in the




containers was decanted for chlorination and disposal, and the containers were refilled with



20°C water at the same salinity as the packing water. Later, the amphipods were sieved




from the packing substrate and transferred to tubs for culturing.
1.3.5 CULTURING








       Cultures of Ampelisca abdita were initially maintained in 1-gal glass jars which held




ca. 4 cm sediment and ca. 20 cm overlying water. The water was constantly gently aerated




from a glass pipette attached to a filtered air supply.  More recently, we have maintained




cultures in plastic dish tubs (ca. 27cm x 30cm x 17cm deep) with a 4 cm layer of sediment




and 13 cm column (ca. 10.5 L) of overlying water.  The water is also constantly aerated. The




tubs are held in shallow seawater-table trays which serve the dual purposes of catch basins

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                                                                               1-17



for any water spilled from the tubs and water baths to maintain the appropriate culture




temperature.









       The culture sediment was collected from tide flats in Yaquina Bay, OR, and wet-sieved




through a 250 um sieve before use. The fine sediment facilitated recovery of small juvenile




animals when cultures were sieved through a 0.25 mm  screen.   The  photoperiod was




maintained at 16 h light: 8 h dark. This mid-summer photoperiod has been found to sustain




reproductive activity in other amphipod species (Arthur, 1980).









       The culture water was renewed 3-5 times per week, at which time the cultures were




also fed. Prior to renewal, aeration was stopped, the sides of the tubs rinsed down, and any




amphipods trapped on the water surface  pushed underwater with a glass rod. The floaters




were given some minutes to burrow into  the sediment.  Then, ca. 1/3 to 1/2 of the overlying




seawater in  each bin was carefully poured off and replaced with an algae-seawater mixture.




The renewal water was added using a turbulence reducer (glass dish attached to a glass rod)




held just at  the water surface, so that the sediment was not disturbed.  The renewal water




was  a mixture  of filtered seawater (28-35%o),  a culture  of the diatom, Phaeodactylum




tricornutum, and a culture of the golden-brown flagellate, Pseudoisochrysis paradoxa, in the




ratio of approximately 1.5:1:1.  The salinity of the  mixture was adjusted to ca. 30%o with




deionized water if necessary.  About 3.5 liters  of the renewal water were added to each




amphipod culture bin.








       Cultures  were maintained at 20°C and  28-35%o  which was the routine sediment




toxicity test temperature and salinity for A. abdita (Scott and Redmond 1989). A. abdita will




tolerate lower temperatures, but it grows more slowly and will probably not reproduce until

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                                                                               1-18



the temperature reaches about 10°C. We tried to culture this species at 25°C, hoping this




would shorten the life-cycle and increase productivity. This is a temperature that shallow-




water populations would encounter in summer.  However, after a few generations, culture




production declined. Similarly, while Bousfield (1973) reported A. abdita present at 10%o, we




had some data that suggested that culture productivity was worse at 20%e than at 30%o. We




do not know if the higher temperature or lower salinity was the cause of poor culture




performance since there was considerable variation among replicates. Our decision to use




20°C  and 28-35%c as routine culture conditions was based on the results of a preliminary




experiment  and our judgement that the higher  temperature and  lower salinity might




represent marginal environmental conditions for populations in the field.








      From January - August, 1990, most A. abdita were maintained at 25°C in glass gallon




jars with screened overflows and fed only Pseudoisochrysis paradoxa, mixed with seawater,




3 times per  week. These cultures were productive initially, but reproduction decreased by




the fourth generation.  Temperature was then decreased to 20°C,  and the amphipods  were




fed a mixture of three algal  species (e.g., IP. paradoxa, Phaeodactylum tricornutum, and




Chaetoceros calcitrans) 5 times per week. Experimental data (see Chapter 3) indicated that




A. abdita grew better when fed a mixture of algal species than if they were fed P. paradoxa




alone. Samples taken from culture jars in October 1990 (after ca.  6 generations), indicated




that reasonable numbers of individuals were again being produced.  However, the cultures



crashed in late November, possibly  due to a late-fall water quality problem (e.g., natural




release  of toxic compounds during the  decomposition of dying macrophytes or a bloom of




dinofiagellates in Yaquina Bay), and  the cultures had to be replenished with animals shipped




from Narragansett, RL

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                                                                                1-19
       Since the January, 1991, shipment of amphipods, A. abdita have been cultured in
plastic tubs instead of gallon jars, because tubs are more convenient to work with and do not
need to be thinned as often. A culture tub is usually started with 400-1000 animals (e.g., 0.5-
1.2 cm"2).  In one instance, approximately 4000 healthy animals (e.g., 5 cm"2) were recovered
from one tub. This is the maximum density we have obtained in our cultures; field densities
of 110,000 m"2 (e.g., 11 cm"2) have been reported (Santos and Simon, 1980).

       Regular estimates of female fecundity were not obtained from the cultures due to the
sensitivity of the animals to sieving. The brood sizes of eight first generation females ranged
from 1-27 eggs, with a mean clutch of 13.5 (7.2 SD). Field-collected A. abdita had average
brood sizes of 26 (Mills, 1967) and 13.7 (Nelson, 1980). Scott and Redmond (1989) obtained
means of 13.6 and  15.8 eggs/female in A. abdita produced and reared to maturity in the
laboratory. Thus, it is possible to achieve reasonably natural brood sizes in cultured animals,
albeit inconsistently.

       Production data from our A. abdita cultures are ambiguous or inconsistent. Maximum
production was about 5 times the original number of animals added to  a tub,  as was
described previously.  Populations crashed beyond recovery in other culture tubs, despite all
attempts to maintain consistent and constant conditions  among  all tubs.   Variation in
production of cultured A. abdita did not appear to be correlated to the density or life stages
of the animals with which cultures were initiated, season (except in case of possible late-fall
water  quality problems), type of container, or sediment  source.  In  experiments with
uncontaminated sediment, animals survive, grow, look healthy, and produce eggs and sperm
but rarely produced offspring (see Chapter 3).  There appears to be  some unidentified
factor(s) which causes reproduction to be inconsistent in this species.  One possibility is a

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                                                                                1-20
natural microbial or viral pathogen present in low density in Yaquina Bay water or sediment
to which A. abdita is sensitive, although we  have no direct evidence to support this
hypothesis.

       Ampelisca abdita may be sensitive to some aspect of our culturing regime, such as the
lack of flowing water in the culture tubs, the omission of a critical nutritional ingredient in
the diet, or the lack of seasonal changes in photoperiod or temperature.  No factors were
discover-ed that distinguished healthy cultures from mediocre or failed cultures. Some culture
containers were quite productive, but the majority were not.  A flowing seawater system for
delivery of seawater and algal food daily would be less labor-intensive, and this species might
perform better in  a flow-through  system.  A. abdita has sometimes  shown  increased
sensitivity to toxicants in a static system compared to flow-through (Word et al.,  1989).  In
previous efforts to develop a chronic sediment toxicity test with A. abdita. Scott and Redmond
(1989) got this species to reproduce with a 14 h light: 10 h dark photoperiod, which might be
a better approximation of their summer breeding photoperiod in Rhode Island. It also may
be possible to stimulate  higher production  and synchronize  reproduction  by mimicking
overwintering: maintain low temperature (e.g., <10°C) and a shorter diurnal period  (e.g., 8
h light: 16 h dark) for a few weeks until reproduction is desired, and then gradually increase
the temperature and diurnal period to simulate the onset of spring and, hopefully, stimulate
reproduction.

       Further efforts to culture A. abdita might be best conducted  at a laboratory  near a
natural supply of the species, since one could use flow-through culture conditions and
constantly administer suspended-particulate food. Neither were practical at our laboratory

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                                                                               1-21



due  to the large volume of wa.ter that would have required treatment  to prevent the




accidental release of pathogens or non-indigenous amphipods into the local  environment.
1.3.6  HANDLING









      Animals for acute sediment toxicity tests were obtained by sieving the contents of each




culture tub through a 0.5 mm screen.  Larger adults were excluded by sieving with a 1 mm




sieve. Animals swimming or crawling in the culture tub immediately prior to sieving were




removed by dip net; these were probably either reproductively active adults or stressed




animals. Sieving extracted only about half of the amphipods in a tub; the rest remained in




their tubes.  More animals could be coaxed from the tubes by allowing the tube mat to "rest"




for 20-30 min between bouts of sieving. Animals also could be forced to leave from individual




tubes by gently working a probe along the tube toward the opening.  This was done under




a dissecting microscope and was very time consuming. The animals were then gently washed




from the sieve into a shallow, flat bottomed glass dish for picking using 28-30%o seawater at




20°C for sieving and washing to minimize stressing the animals.









      Healthy A. abdita were  light pink and often remained tightly curled. Unhealthy




animals  tended to be translucent  white  and uncurled.  As with most other amphipods,




individual amphipods were transferred from the  picking dish using a wide bore pipette




(ASTM, 1991).  Handling and time-held-without-sediment were minimized. Before use in a




test, amphipods had food available on a daily basis.  General observations suggested that A.




abdita were stressed by repeated sieving, and cultures were not used if the culture tub had

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                                                                                 1-22



been sieved recently. Detailed handling information on setting up an acute bioassay with




this species is provided in ASTM (1991) and Redmond et al. (1991).
1.3.7 CONCLUSION








       Due to some unidentified factor(s) that seem to inhibit reproduction, we have been




unable to produce large numbers of A. abdita on a consistent basis. We have had spectacular



successes followed by complete  failure of cultures, with no  discernable pattern to explain




these inconsistencies.   Further research on culture methods for  this organism may be




worthwhile, since it is widely used in acute sediment toxicity tests, and it may potentially be



used to test the  chronic toxicity of sediments (see Section  III of this  report).   Successful




culture methods would allow the development of multiple sources of the animals for toxicity




tests, and eliminate the difficulty of obtaining animals in winter, when they are available but



frequently difficult to collect. Culture research might be more successfully conducted in a lab




near a naturally occurring population of this species, since until the problems in culturing




are identified, large numbers of field-collected animals are required to support investigations.
1.4 Lepidactylus dytiscus








1.4.1 SUMMARY








       Lepidactylus dytiscus was collected in high densities from one location near Virginia




Beach, VA, and may be available in high densities in other locations within Chesapeake Bay.

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                                                                                1-23



It was possible to maintain L. dytiscus in the laboratory, but cultures expanded only very




slowly due to the long time to first reproduction in this species. Furthermore, reproductive




activity was apparently suspended during the winter months despite maintenance of constant




temperature and photoperiod in the laboratory cultures.  This increased the time required




for  culture expansion and limited the  rate at which  animals  could be harvested  for




experiments. Small cultures have been maintained in our laboratory since March, 1990. The




purpose of culturing JL. dytiscus was to assist the development of chronic sediment toxicity




tests, but the prolonged life cycle of this species made JL. dytiscus less convenient than other




amphipod species for that purpose. However, this species may be suitable for acute sediment




toxicity tests, and animals from our collections were used successfully in a comparative acute




toxicity-test experiment.   Adequate numbers of young _L. dytiscus may be  produced in the




spring and summer, but reproductive activity was substantially lower or non-existent in fall




and winter. While further research may reveal conditions that would enhance the production




rate of this amphipod, the culturing approach used in this project did not successfully produce




sufficient numbers of animals  for experiments on a year-round basis.








1.4.2  OVERVIEW OF THE SPECIES








    Lepidactylus dytiscus is a free-burrowing, estuarine, haustoriid amphipod found in fine




sand sediments. They are widely but sporadically distributed in Chesapeake Bay (Fig. 1-3),




and are  tolerant of salinities  ranging from ca. 5-30%c (Ray  Alden & Emily Deaver (Old




Dominion Univ.),  pers.  comm.).   In  laboratory cultures, L. dytiscus preferred a  sandy




sediment to organic-rich  mud.  They appear to be deposit feeders, and do not form tubes in




the sediment or filter particles from the overlying water.

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                                                                               1-24
       L. dytiscus is a slow growing species and not amenable to the rapid production of large
numbers of offspring. Time to first reproduction seems to be greater than 6 months: a group
of 19 cultured newborns held for six months had 100% survival, but little  growth and no
indication of sexual development. Adult (i.e., sexually mature) animals vary greatly in size
(e.g., 4-12+ mm) and may live  for more than a year.  Although we have not carefully
documented the life-cycle of this  amphipod, females can apparently produce more than one
brood of offspring, but  the period between  broods is probably several weeks or months.
Reproduction in the cultures ceased during winter months even though constant physical
conditions were maintained (e.g., temperature, salinity and photoperiod).
1.4.3 FIELD COLLECTION

       Several hundred L. dytiscus were collected by T.H. DeWitt and Emily Deaver (Old
Dominion Univ.) on 3/26/90 with a shovel and sieve from intertidal and shallow subtidal (ca -
.25 m) sandy sediments in the Lynnhaven River estuary near Virginia Beach, VA (salinity
ca 28%o). Recent searches for other populations of L. dytiscus in the lower Chesapeake Bay
area have not been successful (E. Deaver and R. Alden, personal communication), but high
densities of this amphipod have been reported elsewhere in the estuary, particularly near the
Calvert Cliffs in Maryland (Fig 1-3).

1.4.4 SHIPPING

       Lepidactylus dytiscus were shipped successfully from Chesapeake Bay to Newport, OR,
in March, 1990. Approximately 50-100 animals were packed per plastic container (i.e., a 250-

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                                                                               1-2.5



1000 ml sandwich box or ice cream tub), which held a 1-2 cm layer of fine sand substrate and




filled to the top with 28%o estuarine water.  Field-collected animals were held overnight or




longer in running or frequently changed bay water at a salinity and temperature close to that




where the animals were collected. Dead or injured animals were removed prior to packing.




Several plastic containers were placed in an insulated cooler along with 3 or 4 freezer packs




(such as blue ice) to keep the temperature cool, but above freezing, and then the cooler was




sealed and immediately shipped by overnight delivery. Mortality was low among the shipped




amphipods. Shipping success might be further enhanced if the amphipods were shipped in




O2 saturated water.
1.4.5  CULTURING









       Lepidactylus dytiscus was cultured in inexpensive polyethylene tubs (i.e., dishpans)




measuring 29 cm x 34 cm x 13 cm (depth) and holding about 13 L. The sediment bed was




a 2 cm thick layer of fine sand (<0.5 mm) from Ona Beach State Park (Seal Rock, OR) which




was overlaid with water about 11-12 cm deep.  Temperature was maintained at 20°C and




salinity at 20%o.  This configuration held ca.  500 animals and lent  itself to  handling




individual culture containers for sieving, water replenishment and moving.  The tubs were




held in shallow seawater-table trays which served the dual purposes of catch basins for any




water spilled from the tubs and water baths to maintain the appropriate culture temperature.




The amphipods were also cultured in tubs placed on shelves with the room air temperature




maintained at 20°C.  Each tub was aerated constantly via a thin glass pipette connected to




a filtered air supply.  The tubs were illuminated by banks of fluorescent  room-lights




suspended from the ceiling of the culture lab on a 16 h light: 8 h dark photoperiod. All of

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                                                                               1-26



the overlying water in each tub was changed every other day, except on weekends. This was




accomplished by pouring off the overlying water  and flocculent organic matter on the




sediment surface, and refilling the tub from a plastic pitcher with fresh seawater laden with




the algal food.  The stream of incoming water was moved across  the bottom of the tub to




slightly agitate and aerate the sediment. The fresh, renewal water consisted of seawater (ca.




32%o), cultured phytoplankton and deionized water which were combined to achieve a salinity




of 20%o and ca 10$ algal cells per ml. The algae used were Pseudoisochrysis paradoxa and




Phaeodactylum tricornutum in equal portions by volume. The cultures were also fed about



0.5 g of a dry food (i.e., "gorp") during the water change.  Gorp consisted of 48.5% Tetra min®,




24% dried alfalfa, 24% dried wheat leaves and 4.5% Neo-Novum® (a maturation feed for




shrimp mariculture; Argent Chemical Laboratories, Redmond, WA), combined and ground to




a fine  powder.  The gorp was sprinkled on the sediment surface after the old water was




poured off and before the tubs were refilled.
1.4.6  HANDLING








       Culture bins were gently sieved through a 0.5 mm mesh to obtain subadult L,. dytiscus




(i.e., 2-4 mm long) for acute toxicity tests. This screen size retained all of the animals in a




culture.  Larger animals were excluded by gently sieving the animals through a 1.5 mm




screen. Animals were rinsed free of sediment and washed into a shallow glass counting dish.




Subadults of a uniform size were selected and transferred by pipette into a smaller glass dish




for acclimation to the test temperature and salinity.  Water for sieving and rinsing was




maintained at the same temperature and salinity as the cultures to minimize stress to the

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                                                                                1-27
animals.  Unused animals were returned to the culture tub after the sediment bed had
settled.
1.4.7  CONCLUSION

       Lepidactylus dytiscus was a hardy amphipod, well suited for acute sediment toxicity
tests in most respects. It is distributed throughout Chesapeake Bay with apparent high
densities near the Calvert Cliffs of Maryland (Fig. 1-3).  Ij. dytiscus can be cultured, but their
slow somatic growth and long time to first reproduction resulted in low culture productivity.
The culturing approach described here was not successful in producing sufficient numbers
of L. dytiscus for routine use in sediment toxicity tests. Furthermore, the long lifespan and
slow growth of this species preclude its utility in chronic sediment toxicity tests for which
growth or reproduction are desirable endpoints.  Further research might reveal factors to
enhance culture productivity, such as the discovery of a limiting nutrient, or the simulation
of a shortened annual cycle (e.g., changing temperature, photoperiod and, possibly, salinity
to mimic seasonality) to stimulate more frequent reproduction.
1.5 Monoculodes edwardsi

1.5.1  SUMMARY

      Monoculodes edwardsi was easily collected in shallow water in Chesapeake Bay and
has some potential for being cultured.  Several small culture tubs of M. edwardsi  were

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                                                                               1-28
maintained from March, 1990, through September, 1992, but only at a  relatively low
population density (e.g., 0.01 cm"2). The animals were very active burrowers and swimmers,
and appeared to require a large amount of space. Females produced eggs frequently but did
not always produce juveniles; the eggs possibly were not fertilized or the embryos did not
develop. Further research might reveal means to enhance productivity, but culturing M.
edwardsi under current conditions failed to produce adequate numbers of animals for routine
sediment toxicity tests.
1.5.2 OVERVIEW OF THE SPECIES

       Monoculodes edwardsi is a very active, free-burrowing oedicerotid estuarine amphipod
found in subtidal sandy sediments.  It leaves the sediment at night, as evidenced by trails
made on the surface of the sediment.  M. edwardsi is distributed from the Gulf of St.
Lawrence to Georgia/NT. Florida, and is found also in the Gulf of Mexico. It is an omnivorous
predator, that will opportunistically feed on living or dead animal prey as well as microalgae
and possibly detritus. It has been observed feeding on the remains of conspecifics, but it is
not known if this is evidence of cannibalism (i.e., killing conspecifics for nourishment)  or
indiscriminant scavenging.
      First reproduction occurs approximately at age 32-41 days, and average brood size is
5.7. Females can apparently produce several broods.  Individuals probably do not live longer
than a year, but the life-cycle of this species has not been fully documented.

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                                                                              1-29
1.5.3  FIELD COLLECTION
      Approximately 150 Monoculodes edwardsi were collected with a dipnet and sieve in




shallow water (l-2'm)




off Calvert Cliffs, MD, by Tammy Tonare (VERSAR, Columbia, MD) on 3/12/90.  The dipnet




procedure was quite simple: the net was rapidly scraped across the sediment surface, just




skimming the top 1-3 mm of substrate.  The contents of the net were sieved through the




mesh to concentrate the amphipods. Attempts at collecting the amphipods in shallow water




with a shovel were unsuccessful (relative to the dipnet approach) because the animals were




winnowed from the upper millimeters of sediment as the shovel was drawn to the surface.




 M. edwardsi is widely distributed in Chesapeake  Bay and should be available for  field




collection at many locations (Fig. 1-4).  However, M. edwardsi is highly motile, especially




nocturnally, and local population densities might fluctuate substantially within short periods




of time.
1.5.4  SHIPPING
       Monoculodes edwardsi were shipped successfully from Chesapeake Bay to Newport,




OR, in March, 1990.  Approximately 50-100 animals were packed per plastic container (i.e.,




a 250-1000 ml sandwich box or ice cream tub), which held  a 1-2 cm layer of fine sand




substrate and filled to the top with 10%o estuarine water.  Field-collected animals were held




overnight or longer in running or frequently changed bay water at a salinity and temperature




close to that where the animals were collected. Dead or injured animals were removed prior

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                                                                               1-30
to packing.  Three plastic containers were placed in an insulated cooler along with 3 or 4
freezer packs (such as blue ice) to keep the temperature cool, but above freezing, and then
the cooler was sealed and immediately shipped by overnight delivery. All animals died in one
of the three shipping containers, but survival was high in the remaining containers.  The
reason for the failure of one container could not be determined, but this container held mostly
gravid females. Shipping success might be further enhanced if the amphipods were shipped
in oxygen-saturated water.
1.5.5  GULTURING
       Monoculodes edwardsi cultures were maintained under static/renewal conditions at
20°C and 20%o seawater on a 16hr light : 8hr dark photoperiod.  Each culture was kept in a
29 cm x 34 cm x 13 cm (depth) (i.e., 11.5" x 13.5" x 5.5") plastic tub filled with a 10 cm deep
layer of seawater and a 1.5 cm layer of sand (sieved to <0.25 mm diameter) on the bottom.
The water was constantly  aerated with a gentle flow of filtered air.  Feeding and water
renewal were conducted simultaneously three times per week.  This consisted of a 50-75%
replacement of the old water column with a 1:1 mixture (v/v) of the cultured microalgae
Pseudoisocrysis paradoxa and Phaeodactylum tricornutum at a density of ca. 106cells/ml.
Cultures were initially also provided with 15 ml of frozen Artemia nauplii, but this was
discontinued with no apparent ill effect. In addition, 0.5 g of "gorp" (e.g., 48.5% Tetra min®,
24% dried  alfalfa, 24% dried wheat leaves and 4.5% Neo-Novum®  [a maturation feed for
shrimp mariculture; Argent Chemical Laboratories, Redmond, WA] combined and ground to
a fine powder) was sprinkled on the water's surface once a week.  It is not known if this was

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                                                                                 1-31



an optimal or even sufficient diet for the cultures, but the cultures have been maintained for




longer than 18 mo on this diet.









       Culture densities of M. edwardsi were low relative to the  other amphipod species




considered in this study. The highest density observed was 0.1 cm"2 (e.g., 130 animals per




bin), but typical densities averaged an order of magnitude lower (e.g., 0.01 - 0.03 cm"2; 10-40




animals per bin).  The sex ratio in December, 1991, was 1.4 females/males. Brood size ranged




from 1-12 eggs per gravid female, and  females  could produce several clutches.  Offspring




became sexually  mature within 5-7 weeks.  Sometimes clutches  of eggs appeared to die




within a female's brood pouch: eggs that appeared healthy (e.g., green) one week may turn




black the next. Those eggs may have been unfertilized or did not develop. Juvenile mortality




also may  have been high in  these cultures,  possibly due to  cannibalism by  adults.




Maintaining cultures that could produce large  numbers of animals  for routine sediment




toxicity tests (e.g., several hundred juveniles per week) would seem to require a large amount




of space.  Further work is needed to better define the culture conditions for this species:




cultures  should be able to attain  higher densities with this level of fecundity and the




relatively short time to first reproduction.  In addition to providing large amounts  of space




per individual, better  methods for isolating juveniles from adults might  reduce juvenile




mortality.
1.5.6  HANDLING









       Juvenile M. edwardsi for toxicity tests were extracted from the cultures by sieving the




sediment through a 0.25 mm screen; adults were retained on a 0.5 mm screen. Animals were

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                                                                               1-32



rinsed free of sediment and washed into a shallow counting glass dish from which individuals




of a uniform size were selected and transferred by pipette to a smaller glass acclimation dish.




Water for sieving and rinsing was maintained at the same temperature and salinity as the




cultures  to  minimize the stress to  the  animals.  M. edwardsi were very active, often




swimming and flicking around the counting dish at high speeds.  Negatively phototaxic and




individuals could be coaxed to one side of a dish for collection by placing a light source at the




opposite side.  Unused animals were returned to the culture tub after the sediment bed had




settled.
1.5.7 CONCLUSION








       Monoculodes  edwardsi should be readily available for collection in many parts of




Chesapeake Bay.  However, laboratory cultures were unable to sustain high population



densities, and they apparently suffered from high rates of juvenile mortality. Current culture



conditions were not capable of producing adequate numbers of animals for routine sediment




toxicity tests.  Culture productivity might  be improved  with  knowledge of (1) whether



survival or reproduction were really density dependent as observations suggested, (2) whether




this density dependence was due to space or food limitation, and (3) less stressful means of




separating juveniles from adults.  Secondarily, methods should be developed to constrain or




at least slow-down the animals once they are removed from the cultures in order to assist the




distribution of animals to toxicity-test chambers.

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          WASHINGTON, DC
          RappatiannocK^RZ,
                                                DISTRIBUTION AND ABUNDANCE  OF
                                                   Leptocheirus plumulosus
                                                     No.  Individuals/m2
                                                          Kilometers

                                                         A None

                                                           10-100

                                                           100-1,000

                                                           1,000-10,000

                                                           Present

                                                           Abundant
                                                                                  1-33
Figure 1-1. Distribution and abundance of Leptocheirus plumulosus in Chesapeake Bay
estuary.  Data compiled from several sources, including the US Environmental Protection
Agency's Maryland and Virginia Chesapeake Bay Benthic Monitoring Programs, Dauer et al
(1987), Diaz (1989), Feeley and Wass (1971), Hines and  Comtois (1985), Holland (1985)
Holland et al (1977, 1987, 1988), Jordan and Button (1984), Marsh (1988), Mountford et al
(1983), Reinharz and O'ConneU (1983), and Schaffner et al (1987). Data from the USEPA's
Maryland and Virginia Chesapeake Bay Benthic Monitoring Programs have been condensed
to average  densities per site  over  the 1984-1988 sampling period. The "Present" and
"Abundant" population density designations were subjectively assigned to sites based  on
qualitative descriptions from the literature, personal communications  or experience.

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          WASHINGTON, DC
          Rappa.ha.nnoclT'-Rh
                                                DISTRIBUTION AND ABUNDANCE OF
                                                       Ampelisca abdita
                                                      No. Individuals/m2
                                                           None

                                                         O io-
                                                           100-1,000

                                                           1,000-10,000

                                                           Present

                                                           Abundant
                                                                                 1-34
Figure 1-2.  Distribution and abundance of Ampelisca abdita in Chesapeake Bay estuary.
Data compiled from several sources, including the US Environmental Protection Agency's
Maryland and Virginia Chesapeake Bay Benthic Monitoring Programs, Boesh (1973), Dauer
et al (1984), Feeley and Wass (1971),  Holland et  al (1988), Marsh (1973), Orth (1973),
Reinharz and O'Connell (1983), and Schaffner et al (1987). Data from the USEPA's Maryland
and Virginia Chesapeake Bay Benthic Monitoring Programs have been condensed to average
densities per site over the 1984-1988  sampling period.  The  "Present" and "Abundant"
population density designations were subjectively assigned to sites based on qualitative
descriptions from the literature, personal communications or experience.

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           WASHINGTON, DC

          39 00'
                                                 DISTRIBUTION AND ABUNDANCE OF
                                                      Lepidactylus dytiscus
                                                       No. Individuals/mS
                                                           Kilometers

                                                          A None

                                                          O 10-100
                                                            100-1,000

                                                            1,000-10,000

                                                            Present

                                                            Abundant
                                                                                   1-35
Figure 1-3. Distribution and abundance of Lepidactylus dvtiscus in Chesapeake Bay estuary.
Data compiled from several sources, including the US Environmental Protection Agency's
Maryland and Virginia Chesapeake Bay Benthic Monitoring Programs, Diaz (1989), Jordon
and Button (1984), Lippson et al (1979), and Mountford et al (1977).  Data from the USEPA's
Maryland and Virginia Chesapeake Bay Benthic Monitoring Programs have been condensed
to average densities per site  over the 1984-1988 sampling period.  The "Present" and
"Abundant" population density designations were subjectively assigned to sites based  on
qualitative descriptions from the literature, personal communications or experience.

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           WASHINGTON, DC
                                                 DISTRIBUTION AND ABUNDANCE OF
                                                     Monoculodes edwardsi
                                                      No. Individuals/m2
                                                           Kilometers

                                                          A None

                                                            10-100

                                                            100-1,000

                                                            1,000-10,000

                                                            Present

                                                            Abundant
                                                                                 1-36
Figure  1-4.  Distribution  and abundance of Monoculodes edwardsi in Chesapeake Bay
estuary. Data compiled from several sources, including the US Environmental Protection
Agency's Maryland and Virginia Chesapeake Bay Benthic Monitoring Programs, Ewing and
Dauer (1982), Feeley and Wass (1971), Holland et al (1987),  Loi and Wilson (1979), and
Moutford et al (1977).  Data from the USEPA's Maryland and Virginia Chesapeake Bay
Benthic Monitoring Programs have been condensed to average densities per site over the
1984-1988 sampling period. The "Present" and "Abundant" population density designations
were subjectively assigned to sites based on qualitative descriptions from the literature,
personal communications or experience.

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                                 CHAPTER II








               THE ACUTE AND CHRONIC  SENSITIVITY



              OF THE ESTUARINE BENTHIC AMPHIPOD,



                     LEPTOCHEIRUS PLUMULOSUS,




           TO CHEMICA3LLY-CONTAMINATED SEDIMENTS
2.1 INTRODUCTION









      Sediment toxicity tests are a widely used method for estimating the response of




benthic  organisms to contaminated sediments.  While some researchers have examined




sublethal responses  of various benthic taxa to contaminated sediments,  most sediment




toxicity tests presently evaluate only the acute mortality of benthic organisms exposed for




short periods of time to contaminated sediment (Swartz, 1987). However, the contaminant




concentration needed to induce mortality may be considerably greater than that needed to




slow somatic growth, reproductive output, or population growth. Benthic organisms living




in contaminated sediments are usually exposed to chemical toxicants for much of their life




cycle, if not for generations.  Toxicity tests that reflect both the  lethal and sublethal




consequences of long-term exposure to contaminated sediment would thus provide important




information to assist in environmental risk assessment of this polluted sediments.
                                      2-1

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                                                                                 2-2


      We describe here the development of a chronic sediment toxicity test using a benthic


estuarine amphipod. We focused our attention on benthic amphipod crustaceans because of


their known sensitivity to sediment contaminants and their intimate association with the


substrate.  Amphipods are among the most toxicologically sensitive of taxa used to assay


sediments (Nebeker et al., 1984; Reish, 1987; Swartz et al, 1982), and amphipod population


densities decline along pollution gradients in the field (Bellan-Santini, 1980; Chasse, 1978;


Notrini,  1978; Sanders et al., 1980; Seng et al., 1987; Swartz et al., 1982, 1985b).  Five


amphipod species were selected for consideration for sediment toxicity test development based


on an extensive literature review of the most abundant amphipods species  of mid-Atlantic


estuaries, especially Chesapeake Bay (see Appendix A).  Of these species, the burrowing


aorid, Leptocheirus plumulosus, showed especially great promise based on its abundance,


short life cycle (Marsh, 1988), and its distribution relative to major sources of chemical


contamination (see Appendix A). After preliminary tests with all of the amphipod species,


we selected L. plumulosus, for further development based on its culturability, hardiness in
          t

the laboratory, broad salinity  tolerance, and apparent sensitivity to chemical contaminants


in the field. The progression of the research program to develop a chronic sediment toxicity


test with L. plumulosus was to (1) determine the appropriate conditions under which toxicity


tests could be conducted with this species by measuring its sensitivity to non-contaminant


variables, such as sediment grain size, TOG, and absence of food; (2) measure its short-term


(i.e., acute) and long-term (i.e., chronic) sensitivities to chemical contaminants spiked into


sediment; and (3) compare its acute and chronic sensitivities to real-world contaminated


sediment. Two 10-d acute responses (i.e., mortality and size) and four 28-d chronic responses


(i.e., mortality, size, fertility,  and sex ratio) were examined.  We report here the results of


experiments to determine the (1) acute sensitivity of L. plumulosus to sediment geophysical


variables (i.e., grain size, organic carbon, water content, and Eh), different feeding regimes,

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                                                                                 2-3
 and three common sediment contaminants (i.e., cadmium, phenanthrene, and acenaphthene);
 and (2) its chronic sensitivity to phenanthrene-spiked sediment and a dilution series of
 contaminated sediments  from Baltimore Harbor.   The acute and chronic toxicity-test
 methodologies were based on the widely used 10-d amphipod sediment toxicity tests described
 in Swartz et al. (1985a), DeWitt et al. (1989), and ASTM (1990b), and the new techniques
 should be straight forward for other laboratories to adopt.
2.2 MATERIALS AND METHODS

Toxicity-Test Procedures:


       The amphipods were obtained from cultures derived from field-collected Leptocheirus
plumulosus (see Chapter 1).  For the 10-d exposures, pre-reproductive individuals 2-4mm in
length were isolated from the cultures by first sieving the amphipods through a 0.5mm screen
(e.g., to remove smaller juveniles), then through a 1mm screen (e.g., to remove larger adults),
and finally selecting smaller animals from the remainder for toxicity testing.  For the 28-d
exposures, newly released 1-d old juveniles were obtained from gravid females (see Chapter
1 for handling procedures).


       In both types of exposures (i.e., 10-d and 28-d), 20 randomly selected amphipods were
distributed to holding dishes from which the  animals were transferred to the exposure
chambers.  The amphipods were always double counted prior to the initiation of exposure.
One to three subsets of 20 amphipods were set aside during set-up for measurement (i.e., size

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                                                                               2-4



at T0); these animals were relaxed with carbonated water and preserved in 70% EtOH. The




static exposures were conducted in 1-L beakers containing 175 ml of test sediment and 725




ml of overlying water. Unless otherwise noted, the interstitial and overlying water salinities




were adjusted to 20%«. The compositions of the test substrates varied with each experiment



and are described below.  The exposure chambers were placed in temperature-controlled




water baths within vented cabinets. The exposures were conducted at 25°C with a 16-h:8-h




lightidark photoperiod.  Each exposure chamber was aerated constantly.  Each exposure




beaker was monitored daily for proper temperature, aeration, and amphipod emergence, and




to submerge any amphipods trapped at the water surface.








      Amphipods in the 10-d and 28-d exposures were fed 0, 3, 5, or 7 times per week,




depending on the experiment.  Feeding included either 400 ml of an algal suspension (106




algal cells/ml, 1:1 v/v mixture of Pseudoisochrysis paradoxa and Phaeodactylum tricornutum)




or 10 ml of a finely-ground dry  food (i.e., "gorp": 48.5% TetraMin®, 24% dried alfalfa, 24%




dried wheat leaves, and 4.5% Neo-Novum® [a maturation feed for shrimp mariculture; Argent



Chemical Laboratories, Redmond,  WA]) in suspension in  20%o seawater, or both.   The




amphipods were fed algae at the time each beaker's overlying water was renewed; 400 ml of




the old water was siphoned off and 400 ml of the algal suspension siphoned in.  The gorp was




added as 1 ml of a suspension of dry gorp in seawater at a concentration of 10 mg/ml.








       After  10-d exposure, the sediment from each beaker was sieved through a 0.5mm




screen to collect the remaining amphipods.  These animals were transferred to glass sorting




dishes from  which  survivors  were counted, relaxed with ca. 10% carbonated water, and



preserved in 70% EtOH for later measurement.  After 28-d exposures, the sediments were




sieved through 0.5mm and 0.25mm  screens to  retain adults  and juveniles,  respectively.

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                                                                                 2-5
 Adults were counted and preserved as in the 10-d exposures. Juveniles were too numerous
 to count at the time of bioassay breakdown, so the material remaining on 0.25mm screen and
 in  the  sorting dishes was transferred to a  vial,  stained overnight with  a few ml of
 concentrated rose bengal in 20%o seawater, preserved the next day in 70% EtOH, and held
 until juveniles could be counted at a later time.

       Amphipod size was determined as the length of the curved line running dorsally from
 the base of the first antennae to the base of the urosome (i.e.,  posterior end of the third
 abdominal segment; Fig. 2-1). The measurements were made with a computer-based image
 analyzer connected to a dissecting microscope-mounted video camera.  Even though size of
 each survivor was measured, only the mean size of all survivors from a replicate (i.e., one
 exposure chamber) was used as the size-response endpoint.

      Adult  amphipods in the 28-d exposures were sexed at the time of measurements.
 Revealing sexual characteristics were the presence of eggs in the oviducts or brood pouch
 (females), brood plates  (females), gnathopod morphology (i.e., a notched palm on the dactyl
 and stout 5th and 6th segments) (male), or the presence of penile papillae (males; only visible
 in dead animals) (Bousfield, 1973; Fig. 2-1).

      The reproductive response of the cohort in the 28-d exposures was reported as fertility,
 or the average number of daughters produced per surviving mother. This was calculated
from the number of juveniles and female adults retrieved  at the termination of the 28-d
exposures using the following equation:

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                                                                                2-6
                             Fertility = :
                                        No,Juveniles/2
                                     No. SurvivingFemales




This equation presupposes a juvenile sex ratio of 1.0 (females/males). Since juveniles cannot




be sexed presently, the sex ratio must be based on an estimate for the population and will



be a constant in this equation.  Thus, the choice of the sex ratio parameter will only affect




the absolute estimate of fertility, and will not affect the relative comparison of fertilities




among experimental treatments.
Control Treatments:








       Three types of control treatments were used in the sediment toxicity tests. The first




type was a performance control which tested the response of the amphipods in the absence



of contaminant stress and  under the best possible conditions for the amphipods.  The




performance control used culture sediment as the test substrate and maintained the same




temperature and salinity as the experimental  treatments (i.e., 20%c or 28%o, and 25°C).




Culture sediment was collected from a sandflat adjacent to the lab in Yaquina Bay (South




Beach, OR), sieved to <0.25mm, and stored at 4°C; sediment for the performance controls was




obtained from cold storage, not from the culture bins. The exposure periods were 10- and 28-



d for the acute and chronic toxicity test performance controls, respectively.  Performance




controls were used for QA/QC, to assure that the test organisms were healthy.
       The second control was a reference toxicant control which tested the sensitivity of the




 animals to a single toxicant under repeatable exposure conditions.  The reference toxicant




 control consisted of 96-h,  water-only exposures to cadmium chloride at 20%o and 25°C.




 Animals for these controls were selected from the same population as the test animals.  The

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                                                                                2-7
reference toxicant controls for the acute sediment toxicity tests were conducted within 4 d of
the start of the sediment toxicity test. The reference toxicant controls for the chronic tests
were initiated  1 wk after the start of the  sediment toxicity test because the newborn
amphipods could not survive 96-h without sediment or food, having been released from their
mothers' marsupiuna for less than  1 d.  In this case, the newborns were placed in culture
sediment and fed in the same manner as the amphipods in culture (see Chapter 1) for this
1 wk period, at which time they were sieved from the sediment and randomly allocated to the
different cadmium concentrations. The cadmium concentrations for the these controls ranged
from 0.19 - 6 mg/L, although  30 mg/L was used as the highest Cd concentration for one
experiment.  The reference toxicant  control  was also employed for QA/QC, to determine
whether the sensitivity of the test animals was consistent among experiments.

       The third control was a carrier or site  control in which the substrate was not spiked
with  contaminants,  but was  manipulated  in  all other ways the same  as the other
experimental treatments. This included sieving, salinity adjustment, addition of the toxicant
carrier, rolling, and storage.  These controls were included as the uncontaminated treatment
against which the toxicity of the other experimental treatments were compared statistically.
Geotechnical Analyses:

Sediment particle-size was measured by the sieve/pipette method (Buchanan, 1984), and
sediment water content was measured as the percentage of weight lost upon drying overnight
at 90°C.

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                                                                               2-8
Chemical Analyses:
      Substrates for chemical analyses were spooned into separate 1-L beakers at the same




time as the toxicity-test beakers were filled.  Chemistry beakers were removed from the




exposure tables on day 0 of the exposures immediately prior to adding amphipods to the




toxicity-test beakers, and the water overlying the sediment was aspirated off. Approximately



25 g of wet substrate was collected and stored frozen in glass vials for later measurement of




total-sediment organic contaminants and total organic carbon (TOG) concentrations.  For




metals analyses in experiments using field-collected contaminated sediment, a sample of the




substrate for acid volatile sulfide (AVS) and simultaneously extracted metals (SEM) was




withdrawn from the beaker into the open barrel of a 10 cc plastic syringe.  Parafilm was




secured over the open end of the syringe, and the sample was  frozen.  The  sample was




shipped frozen to the EPA laboratory in Narragansett, RI, for analysis of AVS and SEM.








       Total-sediment organic contaminants were extracted from stored sediment samples




by the method of Ozretich and Schroeder (1986) utilizing acetonitrile, sonication and cleanup




on C-18 solid phase extraction cartridges.  Sediment samples were spiked prior to extraction




with d,0-acenaphthene, d10-phenanthrene, or other deuterated organic compounds (depending




on the experiment and chemicals being measured) allowing quantitation by the method of




surrogate internal standards.   Quantitation of PAH was accomplished using a  Hewlett-



Packard 70B Gas Chromatograph-Mass Selective detector equipped with a 0.25mm ID x 30m




or 60m fused silica, DB-5 coated, capillary column (J & W Scientific).
       Substrate TOG  was  determined using  high temperature  combustion thermal




 conductivity detection with a Perkin Elmer Model 2400 CHN  analyzer.  Samples were

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                                                                                2-9



acidified to pH <2 prior to measurement (to liberate inorganic carbonate as CO2), and the




TOG measurements were calibrated using NBS acetanilide as the standard.









       The AVS was determined by converting the solid-phase sulfide to hydrogen sulfide




(H2S) using cold 6 M HC1. The released H2S was trapped in sulfide anti-oxidant buffer, and




the  sulfide measured with a sulfide-specific electrode.  The SEM were  determined by




inductively coupled plasma spectrometry from a filtered sample of the sediment/acid solution




after the AVS was released (Di Toro et al, 1990).









       Substrate Eh was measured at the beginning and end of the exposure period in each




treatment to determine if the sediment remained aerobic. Eh was measured with a platinum




redox electrode which was inserted ca.  1 cm below the sediment surface and allowed to




equilibrate for 1-2 minutes, until the reading stabilized. Dissolved oxygen levels of the water




overlying the sediment was also measured at the beginning and end of the exposure using




a DO electrode.
EXPERIMENTS:









Sensitivities of Sub-Adults and Newborns to Non-Contaminant Variables









      Two sets of experiments  examined the effects of sediment variables and feeding




regimes on survival and growth of Leptocheirus plumulosus. The first experiment examined




the sensitivity of sub-adult amphipods (i.e., those used in 10-d acute sediment toxicity




exposures) to sediments collected at 12 sites in the Yaquina R. and Alsea R. estuaries in

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                                                                               2-10



Lincoln Co., OR. These sediments ranged from a fine sand to very silty mud, encompassing




TOG concentrations from 0.4% to >4%. Each treatment was replicated three times, and the




exposure period was 10-d. Sensitivity of mortality to several sediment variables (i.e., median




grain size, percent fines [< 63 um], water content, TOG content) was assessed by correlation




analysis.
       The second experiment examined the sensitivity of 1-d old newly released juveniles




(i.e., the age class used in the 28-d chronic sediment toxicity exposures) to sediment from five




sources (e.g., culture sediment [from a sandflat in South Beach,  OR], Eckman Slough,




McKinney Slough, East Log Pond, and Curt's Mud Hole [the latter two were from South




Beach, Yaquina Bay]) and five feeding regimes (e.g., 106 algal cells/ml, 105 algal cells/ml, 104




algal cells/ml, 5 mg gorp, and no food). The field-collected sediments were sieved through a




0.25mm screen; additionally, sediment from Eckman Slough and Curt's Mud Hole was sieved




through only a 1.0mm screen, creating two more sediment treatments.  This difference in



processing allowed us to examine whether forcing sediment through the 0.25mm  screen




altered its suitability to the amphipods.  Culture sediment sieved through a 0.25mm screen




was used in the feeding treatments. Each sediment source, handling, and feeding treatment




was replicated  three times.  The reference toxicant control LC50 could not be determined




since all newborns died. In this experiment, 1-d old newborns were added to the water-only




beakers; later experiments called for newborns to be held for 1 wk under culture conditions




prior to exposure to the reference toxicant  control conditions.

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                                                                                2-11
 The Life History and Demography of Leptocheirus plumulosus
       This experiment followed the full lifespan of a cohort of L. plumulosus in order to




 chronicle the life history of this amphipod.  One hundred newborn amphipods were randomly




 distributed among 5 beakers holding culture sediment.  These beakers were fed microalgae




 and gorp ad libidum daily. After 14 d, and every 7 d thereafter, the amphipods were sieved




 through a 0.5mm sieve and a 0.25mm sieve to separate adults and newborns, respectively.




 Newborns were preserved in 70%  EtOH.  The adults were measured alive with an image




 analyzer connected to a dissecting-scope-mounted video camera,  and then returned to the




 beaker with  the same culture sediment from which they had been  sieved.  In  this  way,




 weekly mortality, production of offspring, and size distribution of adults was monitored.
Acute Toxicity of Cadmium
       The  first experiment compared  the  acute  cadmium  sensitivity  of sub-adult




Leptocheirus plumulosus with two Pacific coast amphipods (Rhepoxynius abronius and




Eohaustorius estuarius) and three Atlantic coast amphipods (Ampelisca abdita. Lepidactylus




dytiscus, and Monoculodes edwardsi) in simultaneous, static, 96-h, water-only exposures. The




purpose of the water-only exposure was to compare these species' relative sensitivities to a




reference toxicant under similar contaminant bioavailability regimes. Since salinity is known




to modify the bioavailability of cadmium through regulation of free ion concentration, three




species (Eohaustorius, Lepidactylus, and Leptocheirus) were exposed to  cadmium at two




salinities (i.e.,  28%o and 20%o); the other species  were exposed only  at  28%o salinity.




Furthermore, since temperature may modify a  species' sensitivity by altering its metabolic

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                                                                              2-12



rates, Leptocheirus was exposed to cadmium at two temperatures (i.e., 15°C and 20°C); all




other species were exposed at 15°C.








      The second experiment examined the acute Cd sensitivity of newborn L. plumulosus




that had been held for 1 wk after release from their mothers' marsupium. In essence, this




experiment established the protocol for the 28-d reference toxicant control for the chronic




sediment toxicity test.  The experimental conditions were the same as described previously




for that control treatment. The nominal Cd concentrations were 0, 18.8, 37.5, 75, 150, 300,




and 600 ug/L. Two replicates were run for each concentration.
Acute Toxicity of Acenaphthene








       These experiments examined the sensitivity of L. plumulosus to acenaphthene-spiked




sediment in 10-d  static exposures.  This polynuclear aromatic hydrocarbon (PAH) is  a




common contaminant of sediments near urban and industrial areas, being derived from




petroleum or the  combustion of organic materials.  In the first sediment exposure, L.



plumulosus was exposed to seven treatments (six nominal acenaphthene concentrations  and




a carrier control)  in each of three sediments.  The  three sediments covered a range of



sediment textures and TOG concentrations, and were selected to examine the effect of TOG




on thebioavailability of acenaphthene. The three sediments were collected from the Yaquina




R. and Alsea R. estuaries in Lincoln Co., OR: South Beach (SB: very fine sand, poorly sorted;




TOG = 0.8-1.6%), McKinney Slough (medium silt, poorly sorted; TOG = 2.4-2.5%  ),  and




Eckman Slough (medium silt, very poorly sorted; TOG = 3.0-3.7%). Each sediment was sieved




through a 500 um screen to remove macrofauna, adjusted to 28%o, and stored at 4°C for 6-d

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                                                                               2-13
 at which time acenaphthene was added. Preliminary flow-through, water-only exposures at
 25°C and 28%o with acenaphthene  indicated that the  10-d LC50 for L. plumulosus in
 seawater was 678 ug/L (Swartz et al., unpublished data). The spiking concentrations for each
.sediment were calculated from equilibrium partitioning equations (assuming log Koc= 3.511)
 so that the sediment  interstitial water (IW) concentration  of  the median treatment
 concentration was approximately the LC50. Each sediment was split into aliquots and spiked
 with acenaphthene to achieve the following nominal sediment concentrations: South Beach:
 0, 7.0, 11.7, 19.4, 32.4, 54.0, and 90.0 mg/dry kg; McKinney SI: 0, 19.4, 32.4, 54, 90, 150, 250
 mg/dry kg; Eckman SL:  0, 32.4, 54, 90, 150, 250, 416 mg/dry kg.  Spiking was accomplished
 using the methods of Ditsworth et al. (1990) by rolling sediment at room temperature
 intermittently over a 24-hr period in ca. 2-L glass jars which had the requisite acenaphthene
 plated onto the inside walls of the jars. The spiked sediments were then stored for 8 d at 4°C
 to allow acenaphthene  to equilibrate between particulate-sorbed and IW phases.  Each
 treatment had two replicates for toxicity and one for chemistry. The  amphipods were fed
gorp daily in this experiment. It would have been a better decision not to feed the amphipods
in  this experiment  for  more  direct comparison with  the  other  acenaphthene and
phenanthrene acute toxicity  experiments.

      A second acenaphthene-spiked sediment exposure was conducted in which  L.
plumulosus was exposed to higher concentrations of acenaphthene-spiked sediment since high
mortalities were not observed in the initial experiment. The sediments and nominal total
sediment acenaphthene  concentrations were: South Beach (0,  150 and 250 mg/dry kg),
McKinney SI. (0 and 416  mg/dry kg), and Eckman SI. (0 and 693 mg/dry kg).  The amphipods
were not fed in this experiment.

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                                                                               2-14
Acute Toxicity of Phenanthrene
      These experiments examined the sensitivity of L. plumulosus to phenanthrene-spiked




sediments in 10-d static exposures. The experimental design was virtually identical to the




acenaphthene-sensitivity experiment described above, including exposure to sediments from




South Beach (TOG = 0.8-2.0%), McKinney Slough (TOG = 2.4-2.5%), and Eckman  Slough




(TOG = 3.0-3.6%).  The phenanthrene concentrations were likewise selected to bracket L.




plumulosus's 10-d, flow-through LC50 for phenanthrene at 25°C and 28%o (i.e., 180 ug/L;




Swartz et al., unpublished data) using the equilibrium partitioning model (Di Toro et al.,




1991), the TOG concentration for each sediment, and a log Koc = 4.065 for phenanthrene. The




three sediments  were  spiked  to  the following nominal, total-sediment  phenanthrene




concentrations: South Beach (0, 7.0, 11.7, 19.4, 32.4, 54, and 90 mg/dry kg), McKinney SI. (0,




19.4, 32.4, 54, 90, 150, and 250 mg/dry kg), and Eckman SI. (0, 32.4, 54, 90,150, 250, and 416




mg/dry kg). Sediments were sieved through a 0.5mm screen and adjusted to 28%o, spiked




with phenanthrene, stored at 4°C  for 13-15 d to equilibrate, at which time the exposure




beakers were loaded with substrate.  Each  experimental treatment had two replicates for




toxicity and one for chemistry. The amphipods were not fed in this experiment.
       A second phenanthrene-spiked  sediment experiment was conducted  in which L.



jglumulosus was exposed to higher concentrations of the PAH since high mortalities were not




observed in the initial experiment. The sediments and nominal total sediment phenanthrene




concentrations were: South Beach (0 and 150 mg/dry kg), McKinney SI. (0 and 416 mg/dry




kg), and Eckman SI. (0, 416,  and 693 mg/dry kg).  The amphipods were not fed in this




experiment.

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                                                                               2-15
Acute and Chronic Toxicity of Phenanthrene-Spiked Sediment
       The objective of this experiment was to compare the 10-d and 28-d responses of




Leptocheirus plumulosus to phenanthrene-spiked sediment and to determine whether




handling amphipods after 10-d of exposure affected their sensitivity to phenanthrene after




28-d of exposure.  One day-old amphipods were exposed to 9 concentrations of phenanthrene




spiked into fine-grained sediment from Eckman Slough (Alsea R. estuary, Lincoln Co., OR;




TOG = 2.44%) for 10 d or 28 d.  The nominal total-sediment phenanthrene concentrations




were 0, 25, 35, 50, 72, 103, 147, 210, and 300 nag/dry kg. Three replicate beakers were set




up for each concentration. The IW and overlying water were maintained at 20%o and 25°C.




The amphipods were fed algae and gorp every other day during this experiment.
       One replicate of each of the 9 concentrations was exposed for 10 d to measure acute




toxicity, after which the survivors were transferred to replacement beakers containing fresh




sediment for an additional 18-d exposure to the same phenanthrene concentrations. The




replacement beakers were set up at the same time as all other beakers, but amphipods were




not added until day 10.  L. plumulosus in these beakers would be used to measure chronic




toxicity plus the effects of handling on toxicity. L_. plumulosus in two other sets of replicate




beakers were exposed undisturbed for 28 days to measure chronic toxicity. At the end of the




10-d and 28-d exposures, the amphipods were sieved from each beaker through  a 0.25mm




screen, and the following data  were collected:  1) survival of the initial cohort, 2) sizes and




sexes of the survivors, and 3) the number of offspring. Sediment chemistry samples were




collected on day 0 for all concentrations (except 210 mg/dry kg at 28 d). Chemical analyses




were conducted to measure total sediment and IW phenanthrene, TOG, Eh, and overlying




water DO.

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                                                                               2-16
Acute and Chronic Toxicity of Field-Collected Sediment
      The purpose of this experiment was to compare the sensitivities of the 10- and 28-d




responses of Li. plumulosus to field-collected, contaminated sediment from Chesapeake Bay.




Since the toxicities of field sediments in Chesapeake Bay were generally unknown, a dilution




series was prepared from mixtures of a chemically contaminated sediment from Curtis Cr.,




Baltimore Hbr., MD, and uncontaminated sediment from Corsica R., MD. The Curtis Cr.




sediment  was known to  be heavily contaminated with  a complex mixture of metals  and




organic chemicals and acutely toxic to field-collected JL. plumulosus, and the IW salinity and




median sediment grain size of Curtis Cr. sediment was expected to be similar to that of the




Corsica R. sediment (Schlekat et al., 1992; McGee et al., in press; E. Reinharz, B. McGee (MD




Dept. of  Environment)  pers. comm.).   Personnel from the Maryland Department of




Environment prepared six concentrations for the dilution series: 100% (Curtis Cr.), 50%, 25%,




12.5%, 6.25%, and 0% (= 100%  Corsica R.).  The sediment  IW salinity was ll%o for all




dilution treatments.  The substrates were color coded by Claudia Walters  (EPA Quality




Assurance officer) so that the test was conducted in a blind fashion; only she knew the cipher




until the termination of all toxicity tests.  Sufficient sediment to test three replicate samples




of each substrate with each toxicity test was  shipped on ice to our laboratory and stored at




4°C. Substrate was added undisturbed (i.e., no homogenization) to each exposure chamber




within 7-d of dilution; the overlying water was ll%o to match the sediment IW salinity.  The




performance and reference toxicant controls were conducted at 20%o, and the site control (i.e.,




the 0% Curtis Cr. treatment) and dilution treatments were conducted at ll%o. The dilution




treatments and controls were  conducted at 25 °C, with the exception of the reference toxicant




controls which were conducted at 20°C. The reference toxicant controls for both the 10-d and

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                                                                                2-17
 28-d sediment toxicity tests consisted of 96-h, water-only exposures at 0, 0.18, 0.50,  1.40,
 3.90, 10.8, 30.0 mg/L Cd.  L. plumulosus in the 10-d exposures were not fed, but those  used
 in the 28-d exposures were fed algae and gorp three times per week.

       One additional beaker for each dilution treatment was loaded with sediment for
 chemistry samples. Samples for total-sediment organic chemicals, AVS, and SEM analyses
 were collected from the chemistry  beakers immediately prior to  starting the exposures.
 Sediment IW chemical analyses were not conducted.
2.3 RESULTS


Sensitivities of Sub-Adults and Newborns to Non-Contaminant Variables


       Although there was some variability in the survival of Leptocheirus plumulosus sub-
adults among sediments from 12 sites in the Yaquina R. and Alsea R. estuaries (Table 2-1),
mortality after 10-d was not significantly correlated with any sediment variable (Table 2-2).
Mortality was >15% in 14 of 36 replicates.  Mean mortality was >15% in 5 sediments (i.e.,
East Long Pond 3 and 5, Eckman SI. 2, McKinney SI., and South Beach "Old Log").  Mean
mortality in all 12 sediments was  17.9%.  All sites were believed to be substantially free of
chemical contamination, based on previous chemical analyses (R. Ozretich, unpubl. data) and
the lack of local industrial activity. The cause of the higher mortality was not apparent, and
was not explained by the sediment variables we measured. The reference toxicant control
LC50 for sub-adults = 3.15 mg/L Cd (95% CI: 2.33-4.25 mg/L).

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                                                                              2-18



      Mortality of newborn L. plumulosus was <17% for all but one of the seven substrates




in the second sediment source and handling experiment, but mortality was not significantly



different among the treatments (Table 2-3).   The size  of the newborn amphipods




approximately doubled during the 10-d exposure, but also was not significantly different




among the sediment source and handling treatments.  Neither mortality nor growth was




correlated with any sediment variable, although size was negatively correlated with mortality




(Table 2-4).  The highest and most variable mortality and slowest and most variable growth



was found in the sandiest substrate, Curt's Mud Hole 
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                                                                               2-19
The Life History and Demography of Leptocheirus plumulosus
      The five replicates of the cohort of L. plumulosus showed very similar life history




behavior over  12 wk (Fig. 2-2), which was  somewhat surprising since  feeding was




unintentionally suspended after week 8. Mortality was very low over the first 3-5 wk, then




senescence, and possibly starvation, led to the slow but steady decline in abundance. Growth




was very rapid over the first 4 wk, then asymptoted at approximately 6.5mm. The first brood




was produced at approximately 3 wk of age, and fertility (i.e., the number of female offspring




born per female) increased with the age of the amphipods.  Despite the discontinuation of




feeding, L.. plumulosus continued to produce large broods through weeks 10 and 11.  It is




probable that longer lifespans and prolonged reproductive periods could have been achieved




had feeding been continued.
Acute Toxicity of Cadmium
       Six marine and estuarine amphipod species, including sub-adult L. plumulosus, had




 comparable acute sensitivity (i.e., within an order of magnitude) to cadmium in seawater




 under certain temperature-salinity conditions when the toxicant was adjusted to its free ion




 concentration (Table 2-6).  JL. plumulosus, Ampelisca abdita, Rhepoxynius abronius, and




 Monoculodes edwar.dsi all had free ion LC50's between 0.01 and 0.09 mg/L Cd2+, although the




 environmental conditions differed under which these highest sensitivities were achieved for




 each species.  One-week old 3L.  plumulosus  were approximately ten times more acutely




 sensitive to Cd in water than were sub-adult L. plumulosus (compare Tables 2-6 and 2-7),




 although the exposure with the 1-wk-old amphipods was conducted at a higher temperature

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                                                                                2-20



(i.e., 25°C vs 20°C).  Note that the sources of the species varied (i.e., R. abronius and E_.




estuarius were  collected from  the field within 4 d of testing, A. abdita and 1-wk-old L.




plumulosus were taken from cultures, and sub-adult L. plumulosus, M. edwardsi, and L.




dytiscus were taken from the stock used to start cultures, several weeks after collection from




the field) and the toxicological comparison was made only once. The relative sensitivities of




these species might vary seasonally or between field-collected and cultured animals.
Acute Toxicity of Acenaphthene








       In 10-d exposures  with sub-adult Ij.  plumulosus, mortality increased and  size




decreased as acenaphthene concentrations increased in three sediments (Tables 2-8 and 2-9).



Mortality and size showed comparable statistical sensitivity to acenaphthene concentration:




in each sediment, one or two acenaphthene concentrations were found to cause significantly




higher mortality  or  lower growth than the  carrier control treatment.  The LC50 of




acenaphthene increased with sediment TOG content for at least two of the sediments (i.e.,



McKinney Slough and Eckman Slough), but the LC50 for acenaphthene in South Beach




sediment could not be calculated (Fig. 2-3). Body size declined as a function of acenaphthene




concentration, but as the TOG content of the sediment increased, higher concentrations of



acenaphthene were required to cause a decrease in growth (Fig. 2-4).








      The reference toxicant control LC50 for the first experiment could not be calculated




due to high mortality in all concentrations. The reference toxicant  control LC50 for the



second experiment was 0.69 mg/L Cd (95% CI:  0.49-0.97 mg/L).

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                                                                               2-21
Acute Toxicity of Phenanthrene
      As with acenaphthene,  mortality  was  enhanced  and  size  depressed  as the




concentration of phenanthrene increased (Tables 2-11 to 2-13).  Mortality and size showed




comparable  statistical sensitivity to phenanthrene  concentration in two sediments (i.e.,




McKinney Slough  and Eckman Slough; Tables 2-12 and 2-13), but size was significantly




depressed in four of seven phenanthrene concentrations in the South Beach sediment while




mortality was  significantly higher  in  only  one concentration  (Table  2-11).    As with




acenaphthene, The LC50 of phenanthrene increased with sediment TOG content (Fig. 2-5)




as was expected from equilibrium partitioning models. Body size declined as a function of




phenanthrene concentration, but relatively higher concentrations of the PAH were needed




to elicit a decrease in growth as the TOG content of the sediment increased (Fig.  2-6).








      The reference toxicant control LC50 for the first experiment was 0.90 mg/L Cd (95%




CI: 0.61-1.33 mg/L), and 0.69 mg/L (95% CI: 0.49-0.97 mg/L) for the second experiment. Note




that the reference toxicant control for the second acenaphthene acute sediment toxicity test




was the same control used for the second phenanthrene acute sediment toxicity test.
Acute and Chronic Toxicity of Phenanthrene-Spiked Sediment
       Mortality of newborn L. plumulosus increased as a function of the concentration of




phenanthrene after 10-d and 28-d exposures to spiked culture sediment (Table 2-14).  The




concentration-mortality responses of the amphipods were very similar despite the nearly 3-




fold difference in exposure period: the 10-d and 28-d LC50's were 161.20 mg/dry kg (95% CI:

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                                                                                 2-22



106.4-244.3) and 177.02 mg/dry kg (95% CI: 165.60-189.22).  Most, if not all, of the mortality




apparently occurred during the first 10 d, since mortality did not change between day 10 and




28 in those replicates that were observed on day 10, transferred to new sediment, and



observed again on day 28 (Fig. 2-7).  Mortality after 28-d was significantly different from the




carrier control mortality only in the highest phenanthrene  concentration (i.e.,  184 mg/ dry




kg) (Table 2-14).








      Size was affected very little by the concentration of phenanthrene after either 10-d or




28-d of exposure (Table 2-14, Fig. 2-7), and was not significantly different  in the spiked-




sediment treatments relative to the carrier control.  Under the performance and  carrier




control conditions, the newborn amphipods doubled in length after 10-d and more than tripled




in length after 28-d. The slopes of concentration-size response were virtually flat for both the




10-d and 28-d exposures and  were not significantly different, although the  y-intercepts




differed because animals in the 28-d exposures had time to grow larger.








       Fertility was significantly lower in all phenanthrene concentrations,  relative to the




carrier control (Table 2-14).  Fertility was reduced by approximately 30-40% in the lower four




phenanthrene concentrations and by 45-60% in the highest four phenanthrene concentrations,



but no distinct concentration-response was observed beyond that (Fig. 2-7). Fertility was




nearly an order of magnitude more sensitive to phenanthrene than was mortality or growth.
       Sex ratio did not vary significantly with phenanthrene concentration (Table 2-14), nor




was sex ratio significantly different from 1.0 within any treatment.  However, females




outnumbered males in 66% of the 32 beakers used for the whole experiment for a grand mean

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                                                                                2-23



sex ratio of 1.45 (0.79 SD) which was significantly different from 1.0 (G-test: pooled G = 6.64,




1 df, p<0.05; Sokal and Rohlf, 1981).









       Handling the amphipods did not affect the toxicological sensitivity of the amphipods.




Sieving, counting, and measuring the amphipods after 10 d exposure did not change the slope




or y-intercept of the concentration-mortality,  -growth or -fertility regressions relative to




animals that were  not  handled during the experiment (Fig. 2-8;  analysis of covariance:




p»0.05).









       An LC50 for the reference toxicant control could not be calculated in this experiment




because mortality was >80% for all concentrations including 0 mg/L Cd. The high mortality




was probably due to starvation, since the newborns used in these water-only exposures were




not provided contact with sediment subsequent to their release from the maternal brood




pouch. Subsequent to this experience, newborn JL. plumulosus were allowed to grow for 1 wk




under culture conditions prior to conducting the reference toxicant exposure.
Acute and Chronic Toxicity of Field-Collected Sediment









       Curtis Cr. sediment was considerably more heterogeneous in texture and chemical




content than was the Corsica R. sediment that was used as a dilutant in this experiment.




The Curtis Cr. sediment was characterized as a very poorly sorted medium silt punctuated




with gravel, a high TOC content (Table 2-15), a strong diesel oil smell, and an oily sheen.




The Corsica R. sediment was characterized as very fine silt lacking gravel, a moderate TOC




content (Table 2-15), and no chemical odor or appearance. Extremely high concentrations of

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                                                                                2-24



PAHs (Table 2-16) and metals (Table 2-17) were found in the 100% Curtis Cr. sediment,




while the dilutant sediment from Corsica R. (i.e., 0% Curtis Cr. treatment) had much lower




concentrations of both classes of contaminants.  The concentrations of the PAHs in the




remaining substrates increased in direct proportion to the amount of Curtis Cr. sediment that




had been added.  The SEM concentrations increased as a function of the  concentration of




Curtis Cr. sediment except in the 0% and 6.25% treatments.  The total-SEM/AVS ratio was




well below  1.0 for all six experimental substrates, and thus would not be expected to exert




substantial toxicological impact (Di Toro et al, 1990).
       For both the acute and chronic sediment toxicity tests, mortality increased and both




size and fertility decreased as a function of the percentage of Curtis Cr. sediment and




chemical contaminants in the substrate (Fig.  2-9).  In the  10-d exposure, L. plumulosus




mortality was significantly higher in the 50% and 100% Curtis Cr. substrates, relative to the




site control (i.e., 0% Curtis Cr. = 100% Corsica R. sediment), but size was not significantly



different among the treatments (Table  2-18).  In the 28-d exposure, the 100% Curtis Cr.



sediment caused  significantly  higher  mortality and decreased  size and fertility  of




Leptocheirus plumulosus relative to the site control (Table 2-18). However, no other chronic




test responses were  significantly different from  the site  control  for any  of the  other




treatments, with the exception of decreased size in 50% Curtis Cr. sediment. Since there was




an  obvious and significant trend between Curtis Cr. sediment concentration  and 28-d



mortality, size, and fertility (Fig. 2-9), the failure of anova to  detect  differences among



treatments was probably partly due to low statistical power due to insufficient replication




(i.e., N=3) and high variability in the responses, particularly for mortality and fertility which




had coefficients of variation  (CV)  ranging from  10.3-87%  and  12.7-710%,  respectively.




Fertility in the carrier control was only half that of the performance control.  The two

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                                                                                2-25



treatments differed with respect to sediment source (i.e., Chesapeake Bay sediment and




South Beach, OR, sandflat sediment, respectively) and interstitial salinity (i.e., ll%o and




20%e, respectively).









      The sex ratio of L. plumulosus exposed for 28-d did not differ significantly among the




sediment dilution treatments (Table 2-18), nor was sex ratio different from 1.0 within any




treatment.  However, as in the 28-d phenanthrene-spiked sediment experiment, females




outnumbered males in 73% of the 22 exposure chambers, and the grand mean sex ratio for




this experiment was 1.64 (0.89 SD) which was significantly different from a 1:1 ratio of




females to males (G-test: pooled G = 10.42, 1  df, p«0.05).









      The reference toxicant control, 96-h LC50 for the 10-d exposure using sub-adults was




0.61 mg/L Cd (95% CI: 0.43-0.66 mg/L).  The reference toxicant control LC50 for the 28-d




exposure was 0.27 mg/L Cd (95% CI: 0.20-0.38).  The 28-d reference toxicant control used




newborns from the same cohort used in the sediment exposures which were then held for 1




wk in culture sediment prior to the water-only reference toxicant control exposure.
2.4 DISCUSSION








       The mortality, growth, and fertility of newborn Leptocheirus plumulosus were affected




by 28-d exposures to high concentrations of sediment-associated phenanthrene and field-




collected sediment from a highly contaminated site in Chesapeake Bay.  Shorter-term




exposures  (i.e., 10-d)  of sub-adult L. plumulosus  to sediment-associated acenaphthene,

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                                                                               2-26



phenanthrene and the polluted Chesapeake Bay sediment also affected mortality and growth;




reproduction was not recorded in the 10-d exposures since the test was designed to minimize




the likelihood that broods would be released during the exposure. The sensitivity of the 10-d




and 28-d tests were similar, particularly with respect to mortality and growth. Fertility, the




number of juveniles produced per female in an exposure chamber, was considerably more




sensitive than mortality or growth in one experiment, but not in a second experiment. These




findings culminate in the establishment of acute and chronic sediment toxicity tests for the




estuarine, benthic amphipod, Leptocheirus plumulosus.
The Sediment Toxicity Test Methodologies:








       Procedures to  conduct 10-d acute- and 28-d chronic sediment toxicity tests with




Leptocheirus plumulosus were developed from the research reported here.  The 10-d acute




sediment  toxicity test method has  since been developed as an  appendix to  the  ASTM




"Standard guide for conducting solid-phase 10-day static sediment toxicity tests with marine




and estuarine amphipods" (ASTM, 1990b). This protocol was coauthored by B.L. McGee and




C.E. Schlekat (Maryland State Department of Environment, Baltimore, MD) with assistance




from T.H. DeWitt, (Oregon State University, Newport, OR), M.S. Redmond and J.E.  Sewall




(AScI, Newport, OR),  and J.O. Lamberson (U.S. EPA, Newport, OR), and  is presented in



Appendix C  of this report.  The 28-d chronic  sediment toxicity test method has been




developed as a "Research Methodology to Assess the Chronic Toxicity  of Marine  and




Estuarine Sediments with the Benthic Amphipod, Leptocheirus plumulosus" (Appendix D).




Both methods used animals from laboratory cultures, and the procedures for culturing L.




plumulosus were described in Chapter 1.  The bioassay procedures used within some  of 10-d

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                                                                                2-27



and 28-d experiments  reported  here  varied  somewhat from these proposed "Research




Methodologies" with respect to the salinity and feeding regime, but these differences reflect




the evolution of the techniques which was the goal of this research.









       The  bioassay procedures  using  the  10-d and  28-d experiments  were  simple




modifications of the  standard amphipod sediment toxicity test procedures (ASTM, 1990b).




The 10-d sediment toxicity test primarily used sub-adult amphipods (i.e., 2-4mm long) from




cultures (although newborn juveniles were used instead in some experiments), in static




exposures, usually without feeding. Schlekat et al. (1992) described a similar 10-d sediment




toxicity test with L. plumulosus. except that they used only 10 animals that were larger (i.e.,




4-8mm) and collected from  the field, 1 qt jars instead of 1 L beakers were used for the




exposure chambers,  the exposure temperature was 20°C, and their Cd 96-hr water-only




reference toxicant controls were conducted at 6%o.  Our two laboratories have designed a




single 10-d sediment toxicity test which has been approved by ASTM and will be included in




the next issue of the ASTM amphipod sediment toxicity test guidelines, i.e., ASTM E-1267-92,




which should be published in 1993. The 28-d sediment toxicity test required 0-d old newborn




juveniles in static-renewal exposures, and the animals were fed three to seven times per week




on a mixture of cultured phytoplankton and/or dried food (i.e., "gorp"). The sediment from




the 28-d bioassay was sieved through a 1.0mm and a 0.25mm screen to capture the surviving




adults (F0-generation) and their offspring, respectively,  whereas the sediments in the 10-d




test were only washed through a 0.5 mm screen to capture survivors.  Juveniles captured on




the 0.25mm screen were relaxed, stained, and preserved prior to counting, whereas the adults




were counted live before preservation. The body lengths of the initial cohort used to seed the




exposure beakers and the adult  survivors  were measured in most of  the 10-d and 28-d




experiments.  In the 28-d exposures, the F0 survivors were  also sexed. These methods

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                                                                               2-28



entailed modest modifications of the standard amphipod sediment toxicity tests, and should




not be difficult to implement  in other laboratories that have experience with amphipod




sediment toxicity tests.








Response to Control Conditions








Performance Controls: Mean mortality in the performance controls ranged from 0% to 11.7%




in the 10-d exposures and from 8% to 12% in the 28-d exposures (Table 2-19).  Mortality was




as high as  15% in one replicate in almost all of the performance control trials, and as high




as 20% to 30% in one replicate each of two  28-d exposures.  Performance control mortality




did not vary significantly as a function of salinity, feeding regime, exposure period, or age at




T0 (anova; p>0.05) Preliminary studies suggest that newborn Leptocheirus plumulosus were




very sensitive to temperature changes (J.E.  Sewall, AScI, pers. comm.). Thus, maintaining



a constant 25°C while selecting and handling  the amphipods during bioassay set up may




reduce performance control mortality for the 28-d test.  The performance control mortality




of the 10-d and 28-d toxicity tests should decrease as more experience is gained with these




tests and this species, as has frequently been the case for other amphipod sediment toxicity




tests (J.O.  Lamberson, EPA, and M.S. Redmond, AScI, pers. comm.). ASTM guidelines for




amphipod acute sediment toxicity tests allow for up to 20% performance control mortality in



individual  replicates as long as the mean mortality among the replicates is <10% (ASTM,




1990b). No general guidelines  have been established for chronic amphipod bioassays, and




while it is not possible to predict whether the criteria used for the 10-d test will apply to the




28-d test, it seems likely  that  low  control  mortality can be achieved with the chronic L.




plumulosus bioassay.

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                                                                                 2-29




       Size of amphipods in the performance controls varied from 2.8 to 7.6mm (Table 2-19),




which was largely a reflection of the initial size of the amphipods' and the duration of the




exposure.  Either salinity or the  addition of food (irrespective of quantity,  quality, or




frequency) significantly affected 10-d performance control growth (i.e., the difference in size




between amphipods at the start and end of the exposure) (Fig. 2-10 and 2-11). Salinity and




food presence were confounded (i.e., 28%o « No Food, 20%o ~ Food Added) in most of the 10-d




experiments  (Table 2-19), so it  was not possible to identify the dominant factor affecting




growth.   In the  28-d performance  controls,  salinity was 28%o  and food was  added.




Performance control fertility was quite consistent among the three, 28-d exposures in which




it was measured  (Table  2-19)  and not obviously affected by any  of  the  environmental




variables measured.








       The performance control is used as a QA/QC control for the health of the amphipods.




Obviously, K plumulosus was  sensitive  to changes in either the feeding regime  or the




salinity, and could also be affected by any of several other factors. In future experiments, the




environmental conditions of the performance control must be held constant, as is specified




in Appendices C and D, in order that the background mortality,  growth, and fertility may be




compared among experiments.  More experience is needed with these two sediment toxicity




tests before absolute criteria can be set for performance control mortality, growth, or fertility




for either the 10-d or 28-d tests.








Reference Toxicant Controls: Juvenile JL plumulosus were  more sensitive than sub-adults to




Cd in the 96-h water-only exposures that comprised the reference toxicant control treatments




for the 28-d and 10-d sediment toxicity tests, respectively.  In preliminary experiments with




subadult L. plumulosus (i.e., sediment grain size sensitivity and cadmium sensitivity),  the

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                                                                                2-30



reference toxicant LCSOs ranged from 2.75-2.79 mg/L Cd. Salinity and temperature were




maintained at 28%o and 20-25°C in these two reference toxicant controls. For the rest of the




study, the 10-d sediment toxicity reference toxicant controls were conducted at 20%o and 25°C,




and the reference toxicant LCSOs for subadults dropped to 0.61-0.90 mg/L Cd. The reference




toxicant controls for the 28-d sediment toxicity test used newborns that were held for 7-d




under culture conditions prior to exposure to the 96-h water-only conditions (also 20%c and




25°C). The LC50 for these tests were 0.27 and 0.28 mg/L Cd. More reference toxicant control




exposures need to be conducted before quality control criteria can be established for either




the 10-d or 28-d L,. plumulosus sediment toxicity tests.  However, these preliminary runs




provide initial guidance for the expected ranges of response of subsequent reference toxicant




control runs.








      No reference toxicant control procedures have been developed for growth or  fertility,




but this would be a useful avenue for further research.
Sensitivity of Leptocheirus plumulosus to Non-Contaminant Variables:








       Sub-adults were tolerant of a broad variety of sediment types from fine sand to silty,




high TOG muds.  They  build burrows more  readily in mud than  in  sand,  and more




individuals may be seen out of their burrows in sandy sediments than in mud. Similar wide




tolerance  for different sediment types was  also  seen for  field-collected L;. plumulosus




(Schlekat et al., 1992).

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                                                                                 2-31



       This amphipod also has  a broad salinity tolerance, ranging  from 1.5%o to 32%o




(Schlekat et al.,  1992).  No experiments were conducted in this study to determine the




salinity tolerance of the cultured amphipods, but informal trials and other observations




confirm that adults and sub-adults tolerated salinities from <5%o to >35%o, and withstood




rapid changes in salinity  without acclimation.   This may not be true for newborns.  A




posteriori analysis of the performance control data from all 10-d exposures indicated that




reduced growth may have been associated with high salinity (i.e., 28%o) (Fig. 2-10), although




high salinity was confounded by the absence of food (Table 2-19). It was also possible that




low salinity (i.e., ll%o) was responsible for reduced fertility in  uncontaminated Corsica R.




sediment relative to fertility in the culture sediment performance control which had  an




interstitial salinity of 20%o (Table 2-18). Clearly, further examination of the effect of salinity




on growth, fecundity, juvenile mortality, and contaminant sensitivity is needed.









       Sub-adult amphipods tolerated 4 to 10-d periods without food or sediment (i.e., the




reference toxicant controls and unpubl. data) with very little mortality (i.e.,  <15%), but




newborns required food and/or sediment to survive even 4-d. If fed, sub-adults grew as much




as 35% in 10-d,  but grew  very little (i.e., <3%) if they were not fed.   If fed, newborn L.




plumulosus doubled in size after  10-d  and tripled in size after 28-d.  Growth of sub-adults




and newborns in 10-d performance controls was possibly affected by the presence of food (Fig.




2-11), but since the presence of food was confounded with salinity, it  was not possible to




distinguish which of these  was more important in stimulating growth.   However, based on




other experiments with food quantity  and quality, we suspect that food was probably the




controlling variable. Size increased with the density of phytoplankton  (i.e., food)  provided,




and the dry food, gorp, also promoted rapid growth and high survival.   However, gorp can




stimulate patches of bacterial mat growth on the sediment surface if it is not eaten, and for

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                                                                               2-32



this reason, our laboratory continues to feed cultures and experimental amphipods with a




mixture of gorp and cultured phytoplankton.  More work should be directed to defining a




simple-to-prepare, nutritious diet for L. plumulosus.








      This species was also amenable to considerable handling during the course  of an




exposure with no apparent effect on its concentration-sensitivity.  Newborns exposed to a




wide concentration range of phenanthrene were sieved from the sediment in their exposure




chambers, counted, transferred to depression slides and measured under an microscope, and




returned  to sediment  without any  change  in  their  mortality-,  growth- or  fertility-




concentration relationship relative to amphipods that were not handled. Furthermore, it was




remarkable that amphipods as young as <24 h old could be used reliably to start sediment




toxicity tests with low  control mortality.  This tolerance of handling will help make L.




plumulosus a durable test organism.
Mortality as an Endpoint:








       Mortality demonstrated consistent concentration-responsiveness in 4-d, 10-d and 28-d




exposures for a variety of chemical contaminants, including acenaphthene, phenanthrene,



cadmium, and a complex mixture of chemicals from a heavily polluted site in Chesapeake




Bay.   The  sensitivity  of L. plumulosus  mortality to acenaphthene and  phenanthrene



decreased as the organic content of the sediment increased, as has been seen for other PAHs




(Swartz et al., 1991) and unpolarized hydrophobic organic chemicals  (DiToro et al., 1990).




Mortality was approximately as sensitive to contaminant concentrations as growth (Table 2-




20).  As compared to fertility, mortality was less sensitive to phenanthrene but equally

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                                                                               2-33



sensitive to Curtis Cr. (Baltimore Hbr.) sediment.  Mortality might have been more sensitive




if variability within-treatments been lower or had more replicates been used. For example,




the average, among-treatment coefficient of variation for 28-d mortality in the Chesapeake




Bay dilution series experiment was 55.1%, which was approximately 12 times higher than




the mean CV of growth in that experiment.









       The duration of the exposure did not greatly affect the sensitivity of mortality in two




experiments that compared 10-d and 28-d exposure periods (i.e., the phenanthrene-spiked




culture sediment and the Chesapeake Bay  sediment dilution series experiments).  If




anything, amphipod mortality in 10-d exposures was slightly more sensitive than mortality




of newborns in 28-d exposures. In the phenanthrene-spiked culture sediment experiment,




newborn amphipods were used for both exposure periods, and the survivors of the 10-d




exposure were returned to their respective experimental treatments for the remaining 18-d




for  comparison with amphipods that were not handled during the 28-d exposure.  The




phenanthrene sensitivity of the handled amphipods  did not differ from  that  of the




unmanipulated amphipods  after 28-d of exposure, and  was not  different from their




phenanthrene sensitivity after 10-d of exposure. Thus, it appears that the lethal toxicity of




phenanthrene was exerted within the first 10-d of exposure, and perhaps within a shorter




period of time.  Mortality of sub-adults in 10-d exposures was more sensitive than newborn




mortality in 28-d exposures when exposed to a dilution-series of Baltimore Hbr. sediment.




However, the sub-adults in the 10-d exposure were not fed, whereas the newborns in the 28-d




exposure  were fed.  It seems likely that the sub-adults  may have  been stressed from




nutritional deficiency which may have rendered L. plumulosus more sensitive to chemical




contaminants, or the amphipods consumed more contaminated sediment when offered less

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                                                                                2-34



food. As will be shown below for growth, the interaction between nutrition and contaminant




stress is clearly an important issue to examine in future research.
Size as an Endpoint:








       Growth of Leptocheirus plumulosus decreased in a concentration-dependant manner




in response to PAHs and contaminated sediment from Baltimore Hbr. As with mortality, the




concentration of PAH necessary to reduce growth increased with the sediment TOC content,




as is predicted by equilibrium partitioning (DiToro et al., 1990). Body length (i.e., size) at the




termination  of  10-d and 28-d  exposures  showed comparable  sensitivity  to  chemical




concentration as mortality (Table 2-20), but was equally or less sensitive than fertility.




Within treatments, size was not highly variable, particularly in comparison with mortality




or fertility.  Thus, increasing the number of replicates probably would not have led to an




appreciable increase in the concentration-sensitivity of growth. However, the "failure" of size




or growth to exceed mortality or fertility in sensitivity could be a reflection of the toxicants




used in these experiments (i.e., predominantly PAHs), and growth should continue to be




measured in exposures with other chemicals and field-collected sediments.  Furthermore,




since males seem to grow faster than females (T.H. DeWitt and R. Singleton, AScI, unpubl.




data),  some of the sensitivity of size may have been masked by differential growth rates.




Future research should examine whether the contaminant-sensitivity of growth is  sex-




dependent, or whether differential growth rates of the two sexes masks the concentration-




response of growth for the two sexes combined.

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                                                                                 2-35
       The chemical-sensitivity of size did not change consistently with the duration of
 exposure to contaminants, and the differences in sensitivity that were observed may have
 been due to nutritional deficiency.  In the phenanthrene-spiked sediment experiment, size
 after 10-d and 28-d exposures declined with PAH concentration (Table 2-14); however, due
 to low statistical power (i.e., low replication), size was not significantly smaller at any
 concentration relative to the carrier control.  In  the Chesapeake Bay sediment dilution
 experiment, size at the 50% and 100% Curtis Cr. sediment treatments was significantly
 reduced in the 28-d exposure, but not in the 10-d exposure (Table 2-18). Furthermore, size
 did not decrease  appreciably with concentration  in  the Chesapeake Bay 10-d exposure.
 However, the sub-adults in this acute exposure were not fed  and very little growth was
 observed in either the carrier control (i.e., Corsica  R.  sediment) or the performance control
 (i.e., culture sediment). Conversely, the newborn L. plumulosus in the 28-d exposure were
 fed and size was reduced as the concentration of Curtis Cr. sediment increased. Since there
 was little growth to begin with in the non-fed, 10-d exposure, there was little opportunity for
 chemical contamination to  reduce growth, unless it were to cause the amphipods to shrink,
 which has been observed (unpubl. data).  Thus, as with mortality, nutrition apparently
 interacts with the concentration-response and/or sensitivity of size in both 10-d and 28-d
 exposures.
Fertility as an Endpoint:

       The fertility of female JL. plumulosus decreased in a concentration-responsive manner
in experimental exposures to phenanthrene-spiked culture sediment and a dilution series of
two Chesapeake Bay sediments. Fertility ranged from 10.3 to 0.08 female offspring per

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                                                                                2-36



female  survivor.   The highest  fertility was  observed in a  performance  control (i.e.,




approximately culture conditions), and the lowest was observed in 100% Curtis Cr. sediment




from Baltimore Hbr.  Fertility was more sensitive to phenanthrene-spiked sediment than




either mortality or growth, but equal in sensitivity to both of these endpoints after 28-d




exposure to the Chesapeake Bay sediment dilution  series.  Fertility was highly variable




within treatments, relative to size: for the Chesapeake Bay sediment dilution experiment, the




average, within treatment CV was 132% (or 36% if the 100% treatment is excluded) (Table




2-18). The statistical power of fertility as a sub-lethal response would clearly increase if more




than 3 replicates were used per treatment.
       Fertility was sensitive to one or more uncontrolled factors.  In the Chesapeake Bay




sediment dilution experiment, fertility in the site control (i.e., the 0% Curtis Cr. treatment




= 100% Corsica R. sediment)  was half that of the performance control.  Both treatments




received food equally. The Corsica R. sediment was collected from a site sustaining a year-




round population of L.  plumulosus  (B. McGee, MD  Dept.  Environment,  pers. comm.).




Although metal  and PAH contaminants were low in  this sediment, it is possible  that




unmeasured chemicals were present that inhibited reproduction. Alternatively, fertility may




have responded to the different interstitial salinities present in the two sediments (i.e., ll%o




in the Corsica R. sediment and 20%o in the South Beach, OR,  sediment). The sensitivity of




fertility to salinity is uncertain: Schlekat et al. (1992) found reduced reproductive production




at 5-15%o relative to 25-32%o after 20-d exposure under non-contaminant conditions, but the




difference was not significant after 28-d of exposure. The effect that food quantity or quality




has on JL. plumulosus fertility could not be determined since the amphipods were fed in all




of the 28-d experiments.  However,  the feeding regime should be expected to  affect the




magnitude and possible contaminant-sensitivity of fertility as it apparently does mortality

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                                                                                2-37



and growth.  Clearly, the effects of non-contaminant environmental variables and food on




reproduction of JL. plumulosus must be high priority for further research in the development




of a chronic sediment toxicity test protocol with this amphipod.









       Finally, the mechanism(s) responsible for reduced fertility need to be determined.  As




measured here, fertility was the number of juvenile females produced per surviving female.




Reduction of fertility could have been caused by reduced egg production, death of embryos




held in the maternal brood pouch, or death and decomposition of offspring shortly after




release from the brood pouch. Examination of the number and condition of eggs and embryos




in the brood chambers of females under contaminant-stress and control conditions should the




necessary information.
Sex Ratio as an Endpoint:









       The sex ratio of surviving L. plumulosus was not affected by the concentration of




chemical contaminants in either experiment in which it was measured.  Thus, it is of no




direct utility as a toxicological endpoint.  However, the sex ratio of the surviving members




of the F0-cohort  must be  measured in every test  so that fertility may be  accurately




determined. Surprisingly, the sex ratio of L. plumulosus was not 1.0 as expected, but the




cohorts were comprised of 60-70% females on average (Fig. 2-12).  While this difference in




relative abundance of the  two sexes appeared in two experiments, possible  alternative




hypotheses  to  explain this  sex ratio are 1) females were unconsciously favored in the




supposed random picking of newborns during the setup of the experiment, 2) females left the




maternal brood pouch slightly before males did and were therefore over represented in the

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                                                                                2-38



pool of ^1-d old newborns, 3) males were more sensitive to handling or contaminant stress



than females, or 4) males died younger than females, possibly due to aggressive interactions




among males.  Most of these alternate hypotheses cannot be tested with these data, but




differential contaminant sensitivity of the sexes, with respect to  mortality, can be rejected




since sex ratio did not change with contaminant  concentration in either experiment.




However, since males grow faster than females, the sex ratio of the  animals within a




replicate could affect the response of growth to chemical contamination by 1) increasing the




variation in size among animals within the replicate, or 2) skewing the estimate of growth




toward the numerically dominant sex. Once the concentration-growth response is established




for each sex, it will be necessary to determine whether the sex ratio obscures or skews this




response for the growth rate of both sexes combined.








       One  significant consequence of the skewed sex ratio concerns  the  calculation  of




fertility. Not only must the proportion of female survivors of the F0-cohort be determined,



but so also should be the proportion of females among the offspring (i.e., Frcohort). Since



L> plumulosus sex cannot be determined morphologically until sexual maturity (i.e., 14-20




days old), the sex ratio of the  offspring in this study was estimated to be 1.0.  However,



future determinations of fertility might be more accurate if the sex  ratio of offspring was




assumed to equal the average adult survivor sex ratio, i.e., 1.53 (SD=0.82, n=54).
 Areas for Further Research








       Many applied and fundamental research issues remain to be pursued in connection




 with the development of acute and chronic sediment toxicity tests with L. plumulosus. The

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                                                                                  2-39



practical, applied issues are 1) development of reference toxicant control methods for growth




and fertility, 2) establishing the ranges of response of mortality, growth, and fertility under




performance and reference toxicant control conditions, 3) determining the sensitivities of




growth and fertility to natural environmental variables such as sediment gram, size, organic




content, and salinity, and 4) determining the sensitivity of all response endpoints to other




classes of chemicals in addition to the PAHs tested in this study.  The more fundamental




issues include  1) determining the influence of nutrition on the toxicological sensitivity of L.




plumulosus and methods to  either measure or control  the nutritional condition of the




bioassay organisms, 2) comparing the relative toxicological sensitivity of L. plumulosus to




other marine and estuarine species, 3) measuring the relative sensitivities of cultured and




field-collected L. plumulosus, and 4) establishing the ecological significance of the acute and




chronic responses.  Some of the practical issues will be resolved as this species is applied in




research and regulatory sediment toxicity tests. The fundamental research questions and the




development of reference toxicant control methods for growth and fertility will require a more




concerted research  effort.









       The  dominant issue to be resolved is the  influence  of nutrition on  the range of




response for mortality, growth, and fertility under controlled conditions, and the interaction




between nutrition and chemical concentration on these response variables. If L,. plumulosus




was not fed during  a 10-d exposure, it hardly grew.  Had the amphipods not been fed in the




28-d exposures, it is likely that growth, fertility, and possibly mortality would also have been




affected.  The nutritional quality of sediments is not of great environmental concern,




especially relative to the impact of chemical contaminants, but sediment toxicity tests with




unfed L. plumulosus might be unable to discriminate between sediments of low nutritional




value and high chemical  contamination.  However, if the amphipods were  to be fed ad

-------
                                                                                2-40



libidum, they might  reduce their exposure to chemical contamination by preferentially




ingesting nutritious food over polluted sediment, or avoidance of the latter. Such selectivity




has been demonstrated for several benthic deposit feeders,  including aorid amphipods




(DeWitt, 1987), and is an important consideration in the determination of bioaccumulation




rates in deposit-feeding bivalves (Lee et al., 1990).  There is no obvious solution to this




dilemma short of independently determining the nutritional quality of the sediment or the




nutritional health of the amphipods. This problem is hardly unique to L. plumulosus as it




plagues all bioassay organisms, especially those for which the qualitative and quantitative




nature of the food are unknown. Considerable effort has been expended trying to identify and




quantify the food of deposit-feeding invertebrates (Lopez and Levinton, 1987; Lopez et al.,




1989), and  it is  not  yet possible to  independently determine the nutritional quality  of




sediments except by  the response of a  bioassay organism.  Furthermore, the nutritional




quality and quantity of the diet has been shown to affect the sensitivity of mysids, another




peracarid crustacean, to zinc (P.M. Vance, OR State Univ., unpubl. data). The influence of




nutrition on bioassay responses has  been a latent problem  that is  now emerging as  an




important research issue for chronic water and sediment toxicity tests.
2.5 CONCLUSION








       The capacity of the estuarine amphipod, Leptocheirus plumulosus, for use in acute




(i.e.,  10-d  exposure) and chronic (i.e.,  28-d  exposure)  sediment  toxicity  tests  was



demonstrated.  New acute and chronic toxicity-test  methodologies developed from the




experiments reported here, and are similar to those described in the ASTM guidelines for

-------
                                                                                 2-41



amphipod sediment toxicity tests (ASTM, 1990b).  Laboratory-cultured L_. plumulosus were




used in this study, but other researchers have successfully conducted sediment toxicity tests




with field collected animals. Mortality and growth had comparable concentration-sensitivity




for both 10-d and 28-d exposure periods, while fertility was more sensitive than mortality or




growth in one 28-d exposure, but only equally sensitive in another.  These responses were




observed in laboratory experiments with chemically-spiked sediments and with a dilution-




series of a highly contaminated sediment from Baltimore Hbr. mixed with an uncontaminated




sediment from the eastern shore of Chesapeake Bay. The 10-d sediment toxicity test was




effective across a wide range  of sediment grain sizes and organic contents, and neither




mortality or size were significantly correlated with any sediment parameter.  L_.  plumulosus




had a very wide salinity tolerance and  probably could  be used to  test sediments with




interstitial water salinities ranging from ca.  2%0 to 30%e,  although the effect of salinity on




chronic sensitivity to contaminants has not been examined. The concentration-sensitivity of




L. plumulosus varied with the bioavailability of the toxicant, as was seen for cadmium in




water  of  different salinities, and both for acenaphthene and phenanthrene  spiked into




sediments with different  organic carbon contents.  This  study provides  the bases for




developing acute and chronic sediment  toxicity test protocols with L. plumulosus. Several




important issues remain to be resolved, including  the  determination  of  the ranges of




responses under control conditions, sensitivities to different contaminants, development of




reference  toxicant controls for  growth and fertility, and the influence of nutrition on the




sensitivity of L_.  plumulosus to  contaminated sediments.  Experience gained  through use of




the new sediment toxicity test methods developed herein will provide much of the necessary




information on  the expected ranges of responses under both performance  and  reference




toxicant control conditions. Future research should focus on the remaining problems, which




are, in order of priority:      1) the influence of non-contaminant variables on toxicological

-------
                                                                                 2-42




sensitivity, particularly nutrition (e.g., quality and quantity), but also temperature, salinity,




sediment grain size, and perhaps ammonia and hydrogen sulfide; 2) development of reference




toxicant method(s) for growth and fertility; 3) determination of the relative sensitivity of




cultured and field-collected animals to chemical contaminants in sediment; 4) simplification




of the culture and feeding methods; 5) comparison of the relative sensitivity of L. plumulosus




to other species (acute and chronic); 6) development of a toxicological database, including both




pure compounds in spiked-sediment exposures and field-collected contaminated sediments;




7) conducting an inter-laboratory comparison study to determine inter-laboratory variability




in toxicity test responses; and 8) conducting field validation studies to determine whether the




methods are predictive of benthic population, community, habitat, or ecosystem responses to




chemical contamination.

-------
                                         2-43
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                                                               2-44
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Figure 2-2.  Survival, growth, and fertility  of 5 replicates of a cohort of new-born
Leptocheirus plmnulosus over 12 wk.

-------
                                                                   2-45






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Figure 2-7.  Mortality, growth,  and fertility of L. plumulosus  in phenanthrene-spiked
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performance controls.

-------
                                                                    2-50
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 Figure 2-8.   Effect of day-10 handling on the mortality, size, and fertility of L. plumulosus
 after 28-d of exposure to phenanthrene-spiked culture sediment.

-------
                                                                         2-51

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-------
                                                                          2-52
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Figure 2-10.  Influence of salinity on mortality and growth (mean ± SD) of L. plumulosus in
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in each experiment is labeled above each bar.

-------
                                                                        2-53
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Figure 2-11.  Influence of feeding on mortality and growth (mean ± SD) of L. plumulosus in
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-------
                                                                         2-54



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Figure 2-12.  Sex ratio (#females:#males) of surviving Fj-generation Li. plumulosus plotted
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                                                                                          2-56

Diameter
% Fines
%TOC
% Water
Eh
Mortality
-0.01
0.02
-0.11
-0.15
0.17
Table 2-2. Product-moment correlation coefficients ("r") for mortality of Leptocheirus plumulosus sub-
adults and sediment variables. Data obtained from 12 estuarine sediments from Oregon. None of the
coefficients were statistically significant (i.e., p>0.05).

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                                                                              2-58
Variables
%Mortality
Amphipod Size
Median Particle
Diameter
%Fines
%Water
%TOC
%Mortality
1.00
-0.61*
0.25
-0.31
-0.29
-0.12
Size
-0.61*
1.00
-0.19
0.17
0.19
0.14
Table 2-4. Pearson product-moment correlations between sediment variables and mortality
and size of L. plumulosus newborns. * = p<0.05.

-------
                                                                                2-59
Feeding
Treatment
No Food
104 Algal cells/ml
105 Algal cells/ml
106 Algal cells/ml
Gorp Only
%Moi
Mean
26.7A
20.0B
6.7B
11.7AB
6.7B
•tality
SD
7.6
5.0
2.9
2.9
11.5
Size
Mean
2.17A
2.30A
2.87B
4.50°
3.49°
(mm)
SD
0.16
0.20
0.14
0.13
0.06
Table 2-5. Effect of feeding regime on mortality and size of newborn Leptocheirus plumulosus.
Differences in mortality and growth among the treatments were tested with anova and
Tukey's multiple-comparisons t-test; treatment means that were not significantly different
were labeled with the same letter. Mean size at T0 = 2.05 mm.

-------
          2-60
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                                                                         2-61
Cd Come
(mj
Nominal
0.0
0.19
0.38
0.75
1.5
3.0
6.0
sntration
?/L)
Measured
0.0
0.21
0.45
0.87
1.69
3.47
6.46
%Mo
Rep. 1
10
40
65
100
100
100
100
rtality
Rep. 2
0
45
70
60
100
100
100
                LC50NOMINAL = 0.25 mg/L Cd (0.16-0.39 = 95% CI)
               LC50MEASURED = 0.28 mg/L Cd (0.18-0.46 = 95% CI)
Table 2-7. Acute mortality of newborn Leptocheirus plumulosus  after 96-h static
exposure to Cd in seawater (20%o salinity, 25°C).

-------
                                                                        2-62
Total Se<
Acenaph
(mg/dr
Nominal
250B
150B
90A
54A
32A
19A
12A
?A
0A
QB
Performance
Control
Performance
Control8
TA
0
m B
*-0
iiment
thene
ykg)
Measured
192.8
121.1
57.5
45.8
22.6
15.8
7.5
3.6
0.0
0.0
0.0
0.0

—
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Mean
20.0*
2.5
7.5
0.0
2.5
0.0
2.5
0.0
0.0
0.0
7.0
5.0

—
tality
SD
5.7
3.5
10.6
0.0
3.5
0.0
3.5
0.0
0.0
0.0
5.7
6.1

—
Size <
Mean
3.63*
3.74*
4.21
4.26
4.35
4.42
4.60
4.42
4.57
4.30
4.57
4.89
3.37
4.73
;mm)
SD
0.11
0.18
0.30
0.04
0.12
0.19
0.04
0.18
0.04
0.09
0.22
0.15
0.59
0.19
Table 2-8.  Effects of acenaphthene-spiked South Beach "Old Log" sediment on
mortality and growth of sub-adult Leptocheirus  plumulosus after 10-d exposure.
Differences in mortality and size among the treatments, relative to the O mg/kg
treatment,  were tested with anova and Dunnett's multiple-comparisons t-test for
exposures conducted in February 1991 (= A) or June 1991 (= B); * = p<0.05. N=2,
except for Performance Controls and T0A. LC50 = >192.8  mg/dry kg.

-------
                                                                         2-63
Total Se<
Acenapt
(mg/dr
Nominal
416B
250A
150A
90A
54A
32A
19A
0A
0B
Performance
Control
Performance
Control8
rp A
-"-fl
rp B
-••o
liment
ithene
7kg)
Measured
422.5
209.7
124.1
67.5
42.2
24.3
14.3
0
0
0
0

—
%Moi
Mean
75.0*
60.0*
2.5
2.5
7.5
2.5
0.0
2.5
0.0
7.0
5.0

—
•tality
SD
7.1
7.1
3.5
3.5
3.5
3.5
0.0
3.5
0.0
5.7
6.1

—
Size (
Mean
2.90*
3.83
4.14
4.40
4.38
4.48
4.47
4.31
6.58
4.57
4.89
3.37
4.73
jnm)
SD
0.09
0.56
0.47
0.08
0.40
0.30
0.11
0.41
0.05
0.22
0.15
0.59
0.19
Table 2-9. Effects of acenaphthene-spiked McKinney Slough sediment on mortality
and growth of sub-adult Leptocheirus plumulosus after 10-d exposure.  Differences
in mortality and size among the treatments, relative to the O mg/kg treatment, were
tested with anova and Dunnett's multiple-comparisons t-test for exposures conducted
in February 1991 (= A) or June 1991 (= B);  * = p<0.05. N=2, except for Performance
Controls and T0A for which  N=5.  LC50 = 209.3 mg/dry kg (95% CI: 166.7-262.8
mg/dry kg).

-------
                                                                         2-64
Total Se<
Acenapl
(mg/dr
Nominal
693B
416A
250A
150A
90A
54A
32A
0A
0B
Performance
Control
Performance
Control13
TA
0
TB
0
iiment
ithene
7kg)
Measured
501.4
356.2
206.4
121.0
63.3
39.7
23.9
0
0
0
0

—
%Moi
Mean
77.5*
45.0*
15.0
15.0
0.0
5.0
15.0
7.5
0.0
7.0
5.0

_
-tality
SD
17.7
21.2
7.1
7.1
0.0
0.0
7.1
3.5
0.0
5.7
6.1

—
Size (
Mean
5.41*
3.89
4.02
4.22
4.27
4.48
4.51
4.42
6.30
4.57
4.89
3.37
4.73
;min)
SD
0.14
0.50
0.57
0.26
0.10
0.04
0.06
0.06
0.14
0.22
0.15
0.59
0.19
Table 2-10. Effects of acenaphthene-spiked Eckman Slough sediment on mortality and
growth of sub-adult Leptocheirus plumulosus after 10-d exposure.  Differences in
mortality and size among the treatments, relative to the O mg/kg treatment, were
tested with anova and Dunnett's multiple-comparisons t-test for exposures conducted
in February 1991 (= A) or June 1991 (= B); * = p<0.05. LC50 = 373.0 mg/dry kg (95%
CI: 321.2-433.1 mg/dry kg).

-------
                                                                         2-65
Total Se<
Phenant
(mg/dr
Nominal
150B
90A
54A
32A
19A
12A
?A
0A
0B
Performance
Control
Performance
Control3
m A
-•-o
TB
0
liment
lirene
y. kg)
Measured
107.7
70.6
48.8
27.0
19.1
11.0
6.4
0
0
0
0

—
%Moi
Mean
60.0
35.0*
5.0
0.0
5.0
7.5
2.5
2.5
0.0
8.0
5.0

—
•tality
SD
56.7
7.1
7.1
0.0
0.0
3.5
3.5
3.5
0.0
4.5
6.1

—
Size (
Mean
3.09
4.14*
4.45*
4.51*
4.67*
5.09
5.30
5.34
4.30
3.82
4.89
3.74
4.73
;mm)
SD
0.00
0.06
0.19
0.12
0.13
0.01
0.23
0.05
0.08
0.38
0.15
0.06
0.19
Table 2-11. Effects of phenanthrene-spiked South Beach "Old Log" sediment on
mortality and growth  of sub-adult Leptocheirus plumulosus after 10-d exposure.
Differences in mortality and size among the treatments, relative to the O rag/kg
treatment, were tested with anova and Dunnett's multiple-comparisons t-test for
exposures conducted in May 1991 (= A) or June 1991 (=B); * = p<0.05. LC50 = 91.9
(76.2-110.8 = 95% CI) mg/dry kg.

-------
                                                                        2-66
Total Sec
Phenant
(mg/dr
Nominal
416B
250A
150A
90A
54A
32A
19A
0A
0B
Performance
Control
Performance
Control8
TA
0
TB
0
iiment
Jtirene
ykg)
Measured
270.0
173.2
120.0
77.1
49.4
30.3
19.0
0
0
0
0

_
%Moi
Mean
80.0*
60.0*
10.0
5.0
7.5
15.0
5.0
2.5
0.0
8.0
5.0

—
•tality
SD
7.1
0.0
7.1
7.1
3.5
0.0
7.1
3.5
0.0
4.5
6.1

_
Size (
Mean
2.99*
2.37
4.07
3.95
4.12
3.79
3.92
3.97
6.58
3.82
4.89
3.74
4.73
;mm)
SD
0.44
0.31
0.20
0.01
0.17
0.00
0.05
0.21
0.05
0.38
0.15
0.06
0.19
Table 2-12. Effects of phenanthrene-spiked McKinney Slough sediment on mortality
and growth of sub-adult Leptocheirus plumulosus after 10-d exposure. Differences
in mortality and size among the treatments, relative to the O mg/kg treatment, were
tested with anova and Dunnett's multiple-comparisons t-test for exposures conducted
in May 1991 (= A) or June  1991 (=B); * = p<0.05.  LC50 = 170.1 (150.9-191.6 =
95%CI) mg/dry kg.

-------
                                                                         2-67
Total Se
Phenanl
(mg/dr
Nominal
693B
416B
416A
250A
150A
90A
54A
32A
0A
0B
Performance
Control
Performance
Control8
rp A
A0
rp B
•"•O
diment
iirene
ykg)
Measured
346.4
273.2
76.6
174.9
105.0
61.0
44.1
27.5
0
0
0
0

—
%Moi
Mean
80.0*
52.5*
12.5
22.5
17.5
17.5
17.5
12.5
7.5
0.0
7.0
5.0

—
"tality
SD
7.1
3.5
10.6
10.6
3.5
10.6
3.5
10.6
3.5
0.0
4.5
6.1

—
Size
Mean
4.65*
5.36*
4.07
3.93
3.74
4.04
4.04
3.89
4.18
6.31
3.82
4.89
3.74
4.73
(mm)
SD
0.07
0.12
0.11
0.19
0.19
0.04
0.16
0.01
0.06
0.13
0.38
0.15
0.06
0.19
Table 2-13. Effects of phenanthrene-spiked Eckman Slough sediment on mortality
and growth of sub-adult Leptocheirus plumulosus after 10-d exposure.  Differences
in mortality and size among the treatments, relative to the O mg/kg treatment, were
tested with anova and Dunnett's multiple-comparisons t-test for exposures conducted
in May 1991 (= A)  or June  1991 (=B); * = p<0.05.  LC50 = 254.8 (229.3-283.1 =
95%CI) mg/dry kg.

-------
                                                                              2-68
Exposure
Duration
(days)
10
10
10
10
10
10
10
10
10
10
28
28
28
28
28
28
28
28
28
28
Total Sec
Phenant
(mg/dr
Nominal
Performance
Control
0
25
35
50
72
103
147
210
300
Performance
Control
0
25
35
50
72
103
147
210
300
jment
hrene
/kg)
Measured
0.00
0.00
20.26
26.34
41.54
51.62
73.32
105.47
141.25
183.97
0.00
0.00
20.26
26.34
41.54
51.62
73.32
105.47
141.25
183.97
%Mortality
0.0
5.0
5.0
0.0
0.0
5.0
5.0
0.0
45.0
55.0
8.3
(6.8)
1.7
(2.9)
11.7
(7.6)
1.7
(2.9)
3.3
(5.8)
8.3
(5.8)
3.3
(2.9)
21.7
(22.5)
18.3
(23.6)
60.0*
(5.0)
Size1
(mm)
3.42
3.58
-
-
3.33
-
3.14
3.27
-
2.80
7.60
(0.27)
7.41
(0.05)
7.57
(0.19)
7.41
(0.31)
7.24
(0.44)
7.40
(0.25)
7.51
(0.56)
7.36
(0.06)
7.30
(0.35)
7.07
(0.55)
Fertility
-
-
-
-
-
-
-
-
-
-
9.13
(0.51)
9.68
(1.52)
5.96*
(0.18)
6.44*
(1.54)
6.25"
(1.20)
6.99*
(1.09)
4.42*
(0.45)
4.20*
(1.78)
4.50*
(0.99)
5.36*
(1.83)
Sex Ratio
(P:M)
-
-
-
.
-
-
-
-
-
-
1.05
1.61
0.94
1.61
1.43
1.63
1.95
1.05
1.71
1.42
Table 2-14.  Mean (SD) responses of newborn Leptocheirus plumulosus to phenanthrene-
spiked culture sediment after 10 and 28 d of exposure. Statistical significance of responses
among the treatments, relative to the O mg/kg treatment, was tested with  anova and
Dunnett's multiple-comparisons t-test; * = p<0.05. N=l for 10-d exposures and N=3 for 28-d
exposures,  except  for 28-d Performance  Control (N=6)  and size in the 147  mg/dry kg
treatment (N=2). Size at T0= 1.75 (0.16 SD) mm. 1Animals were not measured in some of the
acute-exposure treatments because of time constraints.

-------
                                                                                                  2-69
Sediment Variable
Median Diameter (um)
% Gravel
%Sand
% Silt
%Clay
% Water
%TOC
0
4.2
0.0
2.6
49.8
47.6
67.7
1.82
6.25
6.0
1.8
4.9
49.9
43.5
67.7
2.39
% Curtis Cre
12.5
4.6
0.6
7.0
46.3
46.1
66.8
2.66
ek Sediment
25
5.0
2.1
10.0
44.1
43.9
66.6
2.54
50
8.1
1.6
17.0
37.1
44.3
68.0
3.58
100
21.1
6.1
33.2
22.6
38.1
67.6
4.23
Table 2-15.  Grain size analysis, water content, and TOG content for six substrates from a dilution series of
sediment from Curtis Cr. (Baltimore Hbr., MD) and Corsica R., MD.

-------
                                                                                                 2-70
Compound
(ug/dry kg)
Naphthalene
2-Methyl naphthalene
1-Methyl naphthalene
Biphenyl
2,6 Methyl naphthalene
2,3,5 Dimethyl naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Methyl phenanthrene
Fluoranthene
Pyrene
Benz(a)anthracene
Chrysene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(e)pyrene
Benzo(a)pyrene
Perylene
Benzo(ghi)perylene
0
28
89
bdl
bdl
bdl
bdl
bdl
bdl
bdl
77
bdl
bdl
104
105
85
110
93
bdl
bdl
58
11
bdl
(.
6.25
137
271
311
108
170
90
94
1080
892
1950
398
256
3620
2420
1240
1380
570
bdl
444
599
136
257
Jo Curtis Cre
12.5
149
213
348
139
162
119
bdl
1480
1480
2710
589
245
4830
3070
1130
978
569
bdl
377
454
97
228
sk Sediment
25
196
375
394
220
300
255
214
2440
2940
9220
2200
751
14300
9540
4270
4500
2740
2000
1330
1740
377
704
50
935
1120
1230
737
822
799
523
8500
10300
50000
6010
2640
30700
22400
10800
12200
7690
5100
3880
4940
1360
2290
100
1170
1980
2580
1640
1590
1450
1000
21300
14100
71800
14300
5830
71800
46600
23800
28500
20200
12200
9410
12800
4360
5640
Table 2-16. Total-sediment concentrations of selected PAHs measured in a six substrates from a dilution series
of sediment from Curtis Cr. (Baltimore Hbr., MD) and Corsica R., MD.  bdl = below detection limits.

-------
                                                                                                 2-71
Compound
AVS (umole/dry g)
Cd (ug/dry g)
Cu ()ig/dry g)
Ni (fig/dry g)
Pb (ug/dry g)
Zn (ug/dry g)
Total SEM (umole/dry g)
Total SEM/AVS
0
35.8
o.is
25.0
46.4
77
176
4.25
0.12
6.25
20.5
1.15
26.9
29.4
63
109
2.91
0.14
% Curtis Cre
12.5
42.2
0.23
38.6
25.1
114
153
3.92
0.09
ek Sediment
25
72
0.30
87.6
56.8
196
263
7.31
0.11
50
86.3
0.97
148
30.1
243
419
10.43
0.12
100
491.3
2.95
368.9
89.2
817
1264
30.62
0.06
Table 2-17. Acid-volatile sulfides (AVS) and simultaneously extracted metals (SEM) measured in six substrates
from a dilution series of sediment from Curtis Cr. (Baltimore Hbr., MD) and Corsica R., MD.

-------
                                                                                2-72
Exposure
Duration
10
10
10
10
10
'10
10
Treatment
(% Curtis Cr.)
Performance
Control
0%
6.25%
12.5%
25%
50%
100%
%Mortality
0.0
(0.0)
1.7
(2.9)
15.0
(5.0)
0.0
(0.0)
21.7
(10.4)
40.0*
(18.0)
65.0*
(15.0)
Size
(mm)
3.33
(0.11)
3.09
(0.11)
3.28
(0.07)
3.26
(0.13)
3.21
(0.20)
3.23
(0.13)
3.14
(0.29)
Fertility
-
-
-
-
-
-
-
Sex Ratio
-
-
-
-
-
-
-

28
28
28
28
28
28
28
Performance
Control
0%
6.25%
12.5%
25%
50%
100%
12.0
(12.5)
9.2
(3.8)
10.0
(5.0)
11.7
(7.6)
16.7
(5.8)
25.0
(21.8)
86.7*
(2.9)
6.16
(0.11)
6.53
(0.67)
6.90
(0.10)
6.23
(0.15)
6.30
(0.17)
5.60*
(0.26)
5.17*
(0.40)
10.32
(1.71)
5.20
(1.57)
5.53
(3.52)
6.97
(2.08)
4.70
(0.60)
3.13
(1.99)
0.10*
(0.71)
1.87
(0.76)
0.91
(0.42)
1.73
(0.94)
1.72
(1-13)
1.15
(0.46)
2.38
(1.47)
1.50
(0.71)
Table 2-18. Responses of JL plumulosus to a Chesapeake Bay sediment dilution series after
10-d and 28-d exposure periods. Statistical significance of responses among the treatments,
relative to the O% treatment, was tested with anova and Dunnett's multiple-comparisons t-
test; * = p<0.05. N=3 for all treatments except Performance Controls (N=5).  Size at T0 = 2.94
(0.19 SD) mm for 10-d exposures, and 1.83 (0.08 SD) for 28-d exposures.

-------
                      2-73
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-------
                                 CHAPTER III








         DEVELOPMENT OF A CHRONIC SEDIMENT BIOASSAY WITH



                              AMPELISCA ABDITA
3.1 INTRODUCTION









       Research with Ampelisca abdita sought to develop culture methods and a chronic




bioassay for this species. Bioassay development built on the research of Scott and Redmond




(1989), who showed that A. abdita could be used to test chronic and population endpoints.









       Culturing methods  and results are described in Chapter I above. The approach to




chronic test development was to 1) establish cultures, 2) estimate optimum temperature and




salinity regimes, 3) outline a proposed chronic test design, 4) evaluate the chronic test design




with uncontaminated sediment, and 5) evaluate the chronic test design with contaminated




sediment.   The experiments we  conducted addressed points 2-4.  Both cultures and the




controlled experiments described in this section utilized amphipods from a Narragansett, HI,




source population; details of collection and handling were presented in Chapter 1.
                                        3-1

-------
                                                                              3-2



3.2 MATERIALS AND METHODS








General Methods








      An. overview of the six experiments conducted with A. abdita is shown in Table 3.1.




With the exception of #1 (Temperature and Salinity Effects), all experiments were conducted




at approximately 30%o salinity. In all experiments, the photoperiod was maintained at 16 hr




light: 8 hr dark. All experiments used uncontaminated sediment collected from Yaquina Bay




(OR) tide flats sieved through a  0.5mm or 0.25mm screen.   Grain size analyses were




conducted on the processed sediments.  In Experiment #5 (Sediment- and Animal-Source




Effects), an additional uncontaminated sediment was tested.   In  all but Experiment #6




(Container, Aeration, and Nutrition Effects), testing was conducted in temperature-controlled




water baths.








      Unless otherwise noted, the following daily observations were made:  temperature;




salinity; numbers,  sex,  and life stage of amphipods caught on  the water surface tension;



number of amphipods on the sediment surface; number and sex of dead amphipods.  Dead




amphipods were removed daily except where noted.
      At the conclusion of each experiment, the contents of each exposure container were



sieved through a 0.25mm screen and the recovered amphipods counted and sexed. Two types



of survival calculations were made: 1) percent survival and 2) percent survival corrected for



senescence mortality.  A. abdita males die shortly after mating, and females  die at some



indeterminate time after completing their reproductive cycle.  Males and spent females were



easily recognized when dead individuals  were examined under a dissecting microscope.

-------
                                                                                3-3
Treatment effects may be more easily detected by counting these individuals as live in the
survival calculations, thus correcting for mortality due to senescence.

       In Experiments #2, 3, and 5 (Life Cycle at 25°C, Temperature and Nutrition Effects,
and Sediment and Animal Source Effects, respectively), recovered amphipods were preserved
in 70% ethanol with glycerin for later length measurements. Amphipods from Experiments
#2 and 3 were measured with an ocular micrometer and those from Experiment #5 with an
image analysis system. Length was measured along the dorsal surface, from the base of the
first antennae to the base of the telson.
Experiment #1: Temperature and Salinity Effects

       The goal of this experiment was to estimate optimum temperature and salinity for A.
abdita culture and testing. Thirty adult amphipods from the initial field collection (Jan. 1990
- see Chapter I) were placed in sediment in each of 12 one-gallon jars. There were three jars
in each of the following treatments:  20°C, 20%o; 20°C, 30%0; 25°C, 20%c; and 25°C, 30%o. The
amphipods were acclimated gradually to the test temperatures and salinities.  Three times
per week, approximately 75% of the  overlying water in each jar was siphoned out for renewal
and replaced with 500-1000 ml of a salinity-adjusted suspension of the alga Pseudoisochrysis
paradoxa and seawater of the appropriate salinity and temperature. Algal density was not
measured, but undiluted algal cultures ranged between 106-107 cells/ml.

       During the eighth week (days 52-56), the contents of the first jar in each treatment
were sieved and the-material remaining on the sieve examined, with the amphipods still

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                                                                                3-4



alive. Contents of the second jar in each treatment were sieved on day 66 and preserved for




later examination, and contents of the third were examined live during week 10 (70-73 days).




The results were compared non-statistically, since there were no true replicates (jars of the




same temperature/salinity treatment sampled at the same time).  Those amphipods not




sacrificed were returned to the cultures.
Experiment #2: Life Cycle at 25°C








       A draft chronic sediment toxicity test design for A. abdita (Appendix E) was tested in




this experiment. Another goal of this experiment was to define the life cycle of A. abdita at




25°C. Methods essentially followed the chronic test design except:  1) all exposure chambers




were under control conditions; treatments were predefined sampling times, 2) there were only



3 replicates per sampling time, and 3) the last replicates were sieved after 49 days. The 20




juveniles (8-10 days old) used to initiate the experiment were obtained from cultured females.




A flowing seawater delivery system was set up to supply one volume replacement of an algae-




seawater mixture per day per jar. The mixture was prepared so as to supply 100 ml algal




culture plus 500 ml filtered seawater in each jar's volume replacement.  The proportion of




algae to seawater in the overlying water was the same for these exposure chambers as it was




in the chambers used in the previous experiment. The algal density was not measured, but




the undiluted culture  density ranged between  106-107  cells/ml.  Results were examined




graphically.

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                                                                                3-5
Experiment #3: Temperature and Nutrition Effects
       This experiment examined the effects of temperature and food type on reproduction.




Two temperatures  were compared, 20°C  and 25°C.  Feeding treatments were: no food




controls; Pseudoisochrvsis paradoxa:  an  algal mixture of P. paradoxa. Phaeodactvlum




tricornutum. and Chaetoceros calcitrans (1:1:1 by volume; se Table 3-2 for algal density); and




the algal mixture plus approximately 4 mg of finely ground (<125um) Neo-Novum® pellets




sprinkled onto the water surface daily. Static exposure with daily renewals was used because




of the variety  of feeding and temperature regimes necessary. No water was siphoned out;




seawater-food  mixtures (100 ml algal culture mixture  and 500 ml seawater per jar) were




added with a funnel system that delivered water into jars approximately 3/4 of the way down




in the water column and the displaced water exited the  chamber  through an screened




overflow port.  Disturbance of the sediment surface was prevented by the use of a plastic "T"



at the end of the delivery tube.
       The test was initiated with newly-released juveniles, to examine the feasibility of




using that life stage to start a test. The juveniles were released in seawater only from




brooding females that had been collected in Narragansett, RI, shipped to Newport, Oregon,




and held in the gallon jar culture system with feeding until their broods were near release.




A staggered start, adding juveniles to replicate jars, 10/replicate, as they were released, was




used due to the limited number of young produced on any given day.   Cell  counts and




volumes of algal cultures used, and weight of dry food material added were measured daily.




 Twenty replicates were sieved after 14 days to count, preserve, and measure length of




survivors, and the remaining 18 were sieved, preserved, and examined after 41 days.  Five




samples of amphipods taken on day zero (<1 day old) were also preserved and measured.

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                                                                               3-6



      Lengths of 15-day-old amphipods were compared with those of initial animals using




t-tests.  T-tests were also used to compare the lengths of amphipods from identical food




treatments at different temperatures.  Within each temperature treatment, an analysis of




variance followed by Tukey's Studentized Range Test was used to  determine differences




between feeding treatments.
Experiment #4: Density Effects








      This experiment examined the potential effects of amphipod density on reproduction




at 20°C.  There were three replicates each  at 10, 20,  and 40 amphipods per jar. The




experiment was initiated with juveniles 8-10 days old, as in Experiment #2 (Life Cycle at



25°C), and was terminated after 38  days.  Newly released juveniles were collected in




seawater only from females which were obtained from the field in RI, shipped to Oregon, and




held under culture conditions until they carried broods. All treatments were fed the 1:1:1



algal mixture  with the  quart' jar  renewal system  as described for Experiment #3




(Temperature and Nutrition Effects),  with a mixture of 100 ml algal culture and 500 ml




seawater added daily per jar,  plus approximately 5 mg ground Neo-Novum® daily.  Fifty




milligrams of ground Neo-Novum® were stirred with 10 ml of seawater, and 1 ml of the slurry




was added to each replicate. No statistical analyses were performed.

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                                                                               3-7
Experiment #5: Sediment and Animal Source Effects
       This experiment examined the hypotheses that 1) Yaquina Bay sediment might be




sublethally toxic to A. abdita, and 2) offspring of cultured animals might show poorer survival




and reproduction than those of field-collected animals.  Two sediments were tested: the




Yaquina Bay culture sediment, and sediment from central Long Island Sound. The latter,




which has been and continues to be used as a reference sediment in A. abdita tests at the




EPA-Narragansett, RI, laboratory, was collected on Dec. 5, 1989, from the South Reference




site described in  Scott and Redmond (1989), and pressed through a 2 mm  sieve.  Both




sediments were held at 4°C until used.








      There were 4 treatments, each with 6 replicates, all conducted at 20°C: offspring of




cultured animals  tested in Yaquina Bay sediment, offspring of cultured animals tested in




Long Island Sound sediment, offspring of field amphipods tested in Yaquina Bay sediment,




and offspring of field amphipods tested in Long Island sediment. Because of an abundance




of amphipods, an additional treatment with 3 replicates was added with the offspring of field



amphipods in Yaquina Bay sediment at  15°C.
      This experiment was conducted using the quart jar renewal system as described for



Experiment #3 (Temperature and Nutrition Effects), with a mixture of. 100 ml algae and 500




ml seawater added daily per jar. All treatments were fed a 1:1 mixture by volume of P.




paradoxa and P. tricornutum. plus 1 ml/jar of a brine shrimp (Artemia salina) naupk'i




suspension (density not measured) at regular intervals when amphipods became large enough




to capture the nauplii and were approaching sexual maturity. Preliminary observations

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                                                                                3-8



indicated that A. abdita would capture and eat A. salina nauplii if they came within capture




range. Cell counts of algal cultures were taken daily.








       To maximize the hypothesized sediment effect, brooding females and their offspring




were held in the same sediment in which they were to be tested, and fed the algal mixture




plus brine shrimp nauplii. Also, females were allowed to release their young in sediment



rather than in seawater, hi case the juveniles had been stressed by the water- only releases




in previous experiments.








       The experiment was initiated with amphipods which were 1-6 (field offspring) or 1-7




(cultured offspring) days old, with 20 amphipods per test container. Treatments testing




cultured offspring were initiated 9 days after treatments testing field offspring, since females




from the two sources released their young at different times.  Fifteen replicate jars were




sampled after 10 days to examine survival and growth endpoints, and the remainder ended




after 42 days.  Amphipods from the 10 day sampling and initial samples were preserved and




measured to determine growth in a 10-day period.  T-tests were conducted to determine




significant differences between 10-day treatments.
 Experiment #6: Container, Aeration, and Nutrition Effects








       This experiment examined the potential effects of container type, aeration, and the




 amount of food on reproduction.   The experiment was initiated with cultured juvenile



 amphipods of indeterminate age, at 20°C, and was terminated after 56 days. There were four




 treatments with three replicates of 30 amphipods each: 1) aerated quart jar  exposure

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                                                                                 3-9
containers which were fed a 1:1 by volume algal mixture of P. tricornutum: P. paradoxa: 2)
nonaerated quart jars fed the algal mixture; 3) aerated quart jars fed the algal mixture plus,
twice per week, blended A. salina nauplii (<48 hours post-hatch, blended 10-30 sec. in
seawater); and 4). aerated plastic bins (12 cm high x 17 cm diameter) fed the algal mixture.
The quart jars were renewed daily using the renewal system described for Experiment #3
(Temperature and Nutrition Effects) above. Bins were renewed by pouring off about 2/3 of
the overlying water and replacing it with the algal mixture. Each jar received approximately
250 ml algal mixture and 350 ml seawater daily; each bin received 300 ml algal mixture and
400 ml seawater. The amount of algae provided was greatly increased to determine whether
the amounts (i.e., density) used in previous experiments had be sufficient. Algal density was
not measured, but typically ranged from 106-107 cells/ml in the undiluted stock culture.
Minimal daily biological observations were made, and dead amphipods were not removed
daily, due to limited visibility in the containers. No statistical analyses were performed.
3.3 RESULTS AND DISCUSSION

      Table 3.2 summarizes the physical and feeding  data for all  six  experiments.
Temperature and salinity variation was  minimal.   Where cell counts were taken, the
estimated number  of cells per exposure  container was  approximately the same in all
experiments, 3 to 4 x 108 cells/replicate/day, and exceeded values used in previous successful
long-term experiments with A. abdita.  Scott and Redmond (1989) and Gentile et al (1987)
reported delivering 10s - 109 cells P. tricornutum per day to each of their gallon jar exposure
containers. The gallon jars had approximately a 3000 ml water volume, yielding 108/3000 to
109/3000 = 3.3 x  104 to 3.3 x 105 cells/ml. The quart jars in our study have approximately a

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                                                                               3-10
600 ml water volume, and the algal cell density in each exposure chamber was projected to
be approximately 107-108 cells/replicate/d [i.e., 3.3xl04 - 3.3xl05 cells/ml x 600 ml/replicate «
2xl07 -  2xl08 cells/replicate], which was comparable to  cell  densities  used by Scott and
Redmond (1989).
Experiment #1; Temperature and Salinity Effects

       Live recovered amphipods appeared healthy, since they exhibited normal pink
coloration and were active.  The population in each jar increased from 4 to 17 times its
original value of 30. Table 3.3 shows the number and life stage of animals recovered for
each sampling period. Terms for the various life stages were taken from Scott and
Redmond (1989): females with oostegites just developing were called developing females
or FdV, females with eggs in the oviduct FE, females with eggs or developing young in
the brood pouch FOV or ovigerous (brooding) females, females which have released their
young spent  females  or FS, males M,  and undifferentiated, including juveniles and
subadult males and females, UD.

       Higher temperature accelerated the timing of life cycle events.  Jars at  25°C
produced the first Fa juveniles (F0 designates the adults initially added to the jars), the
first observed sexually mature Fx individuals, and at least some F2 juveniles by week 8
(earliest sampling time). There were no F2  individuals  in the 20°C treatments at 8
weeks.

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                                                                            3-11
         The day 66 and day 70-73 data suggested that the combination of 25°C and 20%,
  was not a good long term condition for amphipods from this  population.   At both
  sampling times the lowest recovery was found in that treatment (Table 3.3). Subsequent
  experiments were conducted with ambient salinity seawater (28 - 35%0).


        Previous experiments conducted at EPA's Narragansett laboratory,  however
  (Redmond and Scott, unpublished data), suggested 20%0 might be an acceptable short-
  term salinity for amphipods from this field source.  In one experiment, amphipods
  collected at 18°C and 30%0 were immediately tested for 96 hours without acclimation,
  under static daily renewal conditions in jars with no sediment or aeration. There was
  0% survival at 5 and 10%,,48% at 15%0) and greater than 98% survival at 20,25, 30, and
  35%,. A second experiment utilized amphipods coUected at -1°C and 27%,, acclimated
  to 20°C at a salinity of 30-31%,, then exposed for 96 hours to a range of salinities.
 Exposure jars contained uncontaminated sediment in which the pore water salinity had
 been adjusted with deionized water to that of the salinity treatment.  The overlying
 water in each exposure jar was renewed daily. Results resembled those of the first
 experiment: 0% survival at 10%0, 60% at 15%,, and greater than 97% at 20 and 30%0.
Experiment #2: Life Cycle at 25°f!


       Survival was >90% in replicates sieved at day 14, and <10% of the individuals
were unaccounted for in any replicate (Table 3.4). The 14-day data would thus meet the
standard criteria for acceptable control survival in a 10-day acute test with this species
(ASTM 1990).  Additionally, nonsenescent survival for all sampling times was S90% in

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                                                                           3-12



all but one replicate.  Amphipods were observed feeding during the experiment, and




survivors were active and had normal healthy coloration.  The survival curve (Figure




3.1) resembles the one  hypothesized for control survival in the chronic test design




(Appendix E): high survival in the early part of the life cycle, followed by senescence and




death in the later weeks.








      A preliminary outline of the  A. abdita life cycle  was  obtained  from this




experiment. Amphipods grew (Figure 3.2), became sexually mature, and produced eggs.




A female with eggs in the oviduct was first observed when amphipods were 18-20 days




old, a male at 20-22 days old, and ovigerous females at 23-25 days old.  (The daily




observation data were  qualitative in the sense that not  all life cycle events were




necessarily observed). Young were released in only one jar, when initial animals were




34-36 days old.








      Ovigerous females observed to be brooding eggs in an early stage (dark brown,




no gut or eyes formed) did not necessarily produce young.  For instance, no young or




brooding females were recovered from the first replicate sampled at 21 days (Table 3.4).




However, at least one  brooding female  was observed in this jar on  day 15 of the




experiment, and one spent female was  recovered.   The brooding female's eggs were




apparently not fertilized and probably disintegrated.








      When  young were produced  or eggs  were obviously fertilized  (advanced



development), mean number of young per female was 9.8 - 19.5 ([13+26]/4 - [13+26]/2)




(Table 3.5).  Scott and Redmond  (1989) reported means of 13.6 and 15.8 eggs/female in




laboratory-produced control females from their chronic tests, amphipods from the same

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                                                                            3-13
 source locality as in our study. Their ovigerous females were larger than ours, and since
 number of eggs per female was related to female size (Mills 1967), our fecundity data
 from successfully reproducing amphipods were not unreasonable. However, successful
 reproduction was only observed in 2-4 females in the whole test (Table 3.5).

       Newly released amphipods could easily be obtained by holding brooding females
 in an aerated beaker of seawater and harvesting juveniles within 24 hours of their
 release, as described  in Appendix E.  Juveniles collected in this manner were held in
 sediment for 8-10 days and survival at the end of that time was 90.5%. A test could
 therefore be started with newly released juveniles, although 8-10 day old juveniles were
 easier to work with.  The most  time-consuming step  in obtaining newly-released
juveniles was the isolation of ovigerous females, and if healthy producing cultures could
be established, that effort could be reduced.

       It was possible to accurately determine the number of females in each replicate
despite starting  the  experiment was with  juvenile animals and variability  in the
experience of laboratory personnel with life stages and sexes of A. abdita.  Only 6 out
of 181 amphipods initially added to the experiment  were not accounted for, or 3.3%
(Table 3.4).  Undifferentiated (UD) amphipods recovered on sampling days  14, 21, and
28, introduced some  error into the sex ratios.  However, in an  actual chronic test
sampling  would not take place until  test  day 35,  and the numbers of UD recovered
would be reduced. UD's were likely immature males, since developing females were
identified even at the earliest sampling time. The ratio of malesrfemales in the exposure
chambers varied from 4:6 to 8:2 if UD's were assumed to be immature  males.

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                                                                           3-14
Experiment #3: Temperature and Nutrition Effects
      Two factors potentially responsible for low reproduction in Experiment #2 (Life




Cycle at 25°C) were examined in this experiment. Either the diet provided (food source




and ration) or the high temperature (25°C),  or both together,  could have  produced




detrimental effects which might not have appeared until after several generations. Some




nutritional factor might have been  lacking,  or  a long period of time at a high




temperature could have increased nutritional needs.








      Unfortunately, survival in this experiment was  poor, and survival differences




could not be definitively related to temperature or feeding treatments (Tables 3.6 and




3.7, Figure 3.3).  The algal mix with Neo-Novum® seemed to produce higher survival




after 14 days than the other food source treatments (Table 3.6), but that pattern did not




continue in the 41-day nonsenescent survival data (Table 3.7).
       Survival of newly-released juveniles in Experiment #2 (Life Cycle at 25°C) after




8-10 days was 90.5%; after 10 days in this experiment, survival was 13-77% (Figure 3.3).




Similarly, nonsenescent survival for amphipods 43-59 days old in Experiment #2 was 90-




100%, but only 0-70% for 41-day-old amphipods in this experiment. The 41-day survival



data showed the expected senescent mortality pattern (i.e.,  a dead male was first




observed on day 19), but much poorer survival in the early portion of the life cycle than




in Experiment #2 (Life Cycle at 25°C; Figure 3.1, Table 3.4).  The percentage of missing




amphipods ranged from 10-90%. Missing amphipods were assumed to have died in the




first portion of the experiment, when they were very small and not easily observed.

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                                                                            3-15



       Initial shipping and handling stresses on test amphipods might account for the




survival difference between the two experiments.  Experiment #2 (Life Cycle at 25°C)




used laboratory-produced brooding females, whereas this  experiment utilized field-




collected females.  The field-collected females had been shipped in seawater-only, under




poor shipping conditions (ice packs omitted); most arrived dead.  Those which carried




early stage broods and were still active on arrival were used to isolate juveniles for the




experiment, after the broods matured for 2 weeks. Even though females survived and




broods matured, the initial shipping stress on the developing broods may have been




significant.









       Tested juveniles may have been further stressed in this experiment because at




least some of them had been in the brood pouch under no-sediment conditions for 3 days.




In Experiment #2 (Life Cycle at 25°C), females were sieved from holding jars and placed




into beakers with seawater over a 3-d period.  During this  time, juveniles  were




harvested daily from the beakers and placed in holding jars until the test began.




Frequently, females  bearing late-stage broods released their young soon after being




removed from the sediment. Thus, most of the test animals were probably without




sediment for <24 h.  In this experiment,  females were sieved from the  sediment and




placed in seawater beakers on the first day; juveniles were harvested from the beakers




over the next 3 d and placed directly into  the experimental chambers. Thus, juveniles




harvested on the second or third day had been in the brood pouch under no-sediment




conditions for ca. 48-72 h.  The 14-day treatments receiving the algal mixture plus dry




food were the only 14-day treatments started with animals isolated during the first 24




hours, and had  the highest survival.  Those 41-d  treatments with  the  lowest




nonsenescent survival (25°C, P. paradoxa: 25°C, mix+dry) had been started with animals

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                                                                           3-16



isolated during the second day; other treatments had been started with amphipods




isolated on the first day.








       Possibly the water-only isolation affected survival because shipped broods were




already stressed.  The double stress could partially explain reasonable production in



culture jars started with late stage brooding females that had survived shipment; late




stage broods may also have been less stressed by shipping. However, it does not explain




why "extra" newly-released test juveniles, collected after the experiment had been set




up, grew  and produced a new generation in a culture jar.  In that case, sufficient




numbers of juveniles may have been added (about 100) for reproduction to occur even




if there was poor survival.








       Differences in lengths of amphipods recovered were detected after the 14-day test




period (Table 3.6, Figure  3.4).  Newly  released amphipods were uniform  in size.




Amphipods in all fed treatments were significantly larger than initial animals and unfed




controls. At 20°C, amphipods were significantly larger when fed the algal mixture or the



mixture with Neo-Novum® than when fed P. paradoxa alone. Amphipods in the fed 25°C




treatments were significantly larger than those in the corresponding 20°C treatments.




(In all cases, p<0.05).  Increased growth in the algal mix and  algal mix with Neo-




Novum® treatments was most likely due to increased nutritional diversity.  Because food




was supplied in excess, differential treatment survival presumably did not affect growth.



These data should, however, be considered preliminary in light of the survival problem




in this experiment.

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                                                                           3-17
       The  factor(s) responsible for low reproduction  were not identified with this
 experiment. No young were produced, although males, females with eggs in the oviduct,
 and brooding females were observed.
Experiment #4: Density Effects


       Amphipod survival in this experiment was poor (Table 3.8).  Only 28% of the
juvenile amphipods isolated survived the 8-10 day pretest holding period.  Up to 35% of
the tested amphipods were unaccounted for in some replicates, suggesting high mortality
initially when dead amphipods were small and difficult to see. Nonsenescent survival
was still only 40-70%.  Recovered amphipods had healthy coloration and were active, and
both males carrying sperm and females carrying eggs in the brood pouch were observed.
However, reproduction did not occur in any of the replicates, so the question of how
amphipod density in an exposure container might affect reproduction was  not resolved.
Experiment #5: Sediment and Animal Source Effects
      Broods of the field-collected amphipods were  evidently stressed  during the
processes  of  shipping, acclimation, and holding.   There was  no apparent survival
difference at  10 days between the two sediment treatments (Yaquina Bay and Long
Island Sound), but survival of the cultured amphipods  (>95%) was better than that of
the offspring of field-collected animals (65-85%) (Table 3.9).  After 43 days, survival

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                                                                           3-18



varied from 20-85% (Table 3.10). Nonsenescent survival of field offspring was 35-70%,




whereas that for cultured amphipods was 85-100%.  There were also more animals




missing among the field amphipods, likely small animals which died unobserved early




in the test.








       A treatment-related length difference was detected in this test after only 10 days,




even though the ages of initial amphipods varied by 5 days.  Graphical examination of




the length data (Figure 3.5, Table 3.9) indicated that the only potential growth difference




was between the 15°C  and 20°C treatments with offspring of field animals in Yaquina




Bay sediment.  A T-test showed that those animals tested at 20°C were significantly




larger.








       None of the amphipods in this experiment reproduced, so the potential effects of




sediment and animal  source on reproduction were not defined.  Also, although  all




juveniles tested were released from females held in sediment rather than in seawater




only,  elimination of the hypothesized water-only stress didn't result  in successful




reproduction, or in control-level survival (>90%) across all treatments.
Experiment #6: Container, Aeration, and Nutrition Effects








       Survival  data from this  experiment are shown in Table 3.11.  Since  dead




amphipods were not removed daily, no conclusions could be drawn regarding treatment




vs. natural senescent mortality.  However, since no amphipods in the nonaerated jars



survived, it was clear that aeration was required in tests with this species, even if

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                                                                            3-19
overlying water and food material were renewed daily.   Tested animals did not
reproduce, so effects of the tested parameters on reproduction could not be determined.
General Discussion

       The expected long-term control survival pattern for A. abdita was demonstrated
in Experiment #2 (Life Cycle at 25°C, Figure 3.1), which stands in contrast to those of
later experiments (e.g., Experiment #3, Temperature and Nutrition Effects, Figure 3.3).
Unacceptably high numbers of amphipods died early in the life cycle, before senescence,
in Experiments #3 and #4 (Temperature and Nutrition Effects and Density Effects) and
in the portion of Experiment #5 (Sediment and Animal Source Effects) which tested
offspring of field-collected animals. Early deaths were indicated by poor recovery after
pre-experimental holding periods (#4 and #5), by poor survival in 10-day and  14-day
treatments (#5 and #3, respectively), and in all three experiments by the relatively large
number of missing amphipods (probably small animals which died unobserved), and the
nonsenescent survival values.

       Clearly some factor or factors involved in the process of shipping, acclimation, and
holding could affect survival of this species in laboratory experiments. This process was
a suspect stress in Experiment #3 (Temperature and Nutrition Effects), and results of
Experiment #5 (Sediment and Animal Source Effects) showed that offspring of cultured
animals  survived  better than offspring  of recently  shipped animals.  The  results
particularly suggest that brooding A. abdita should not be shipped, unless the stress(es)
could be identified and eliminated, and that individuals developing in the brood pouch

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                                                                           3-20



may be a very sensitive stage in the life cycle. Other factors besides shipping and




acclimation probably also affected survival, since success of cultures was variable.








       Growth of recovered amphipods in Experiments #2,3, and 5 (Life Cycle at 25°C,



Temperature and Nutrition  Effects,  and Sediment and Animal Source  Effects,




respectively) was within the range of values seen in the literature (Figure 3.6).  Mills




(1967) developed length-age curves for summer and winter generations of A. abdita from




Barnstable Harbor, Massachusetts, over two years; he derived his curves  from length-




frequency distributions of field samples.  Lengths of amphipods in our study, and those




from the control treatments in the chronic tests conducted by Scott and Redmond (1989),




fall within the range of the summer curves. As the growth data from Experiment #3




(Temperature  and Nutrition Effects) showed, these data were affected by  factors,



including nutrition and temperature.  Differences in the methods of age estimation and




measurement also introduce some error into the figure.








       The life cycle appears to be shorter at 25°C than at 20°C (Figure 3.7, Tables 3.3




and 3.4). We have no definitive data to show whether 25°C (or a salinity  of 20%o) was




harmful over the long term.
      The question of why this species reproduced so little in this series of experiments




still  has not been  resolved.  Recovered amphipods  generally appeared  healthy in




coloration and were active.  Amphipods grew, became sexually mature, and produced




eggs and sperm, but young were produced only in the first two experiments. We tested




temperature, type and amount of food, type of sediment, type of container, amount of




aeration, density  of amphipods in the exposure container, collection of juveniles from

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                                                                            3-21



females held in sediment rather than in seawater only, and offspring of cultured vs.




field-collected ovigerous females. Reproduction was not improved by changing any of the




listed variables, although any or all of them might prove to be important once the critical




factor or factors for reproduction were identified.









       Chronic tests have been run previously with this species  (Scott and Redmond




1989; Gentile et al. 1985, 1987) yet our success with culture and chronic testing of this




species has been inconsistent.  Previous tests utilized Fl or F2 laboratory generations of




field-collected organisms, which needed no shipment and sometimes no acclimation, and




thus may have been healthier than our test organisms. As discussed in Chapter I above,




production might be improved by manipulation of photoperiod and temperature to more




closely simulate natural conditions. There could be some natural, noncontaminant factor




in the water from our laboratory area to which this Atlantic population was sensitive or




susceptible,  e.g., a bacterial disease, although we have no evidence that this was the
case.
       Experiment #2 (Life Cycle at 25°C) showed results very close to what would be




hypothesized with a healthy control population of A. abdita: survival and growth were




good, and there was some reproduction at the proper time. Therefore it seems that the




best approach to further research with this species would be to repeat that experiment,




taking care to correct the problems which were identified in this study. A mixture of




algae should be fed  daily, rather than the single species  (P.  paradoxa) used in




Experiment #2,  since results of Experiment #3 (Temperature and Nutrition Effects)




showed that a mixed food  source produced better growth.  Based on the  number of




cells/ml/day provided, as well as the general health, growth, and production of eggs and

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                                                                            3-22



sperm in tested animals, it would appear that the mixed food source we provided was




adequate. If a laboratory conducting a life cycle experiment was not located close to a




source of field animals, offspring  of cultured animals rather than those of shipped




animals could be used. Otherwise the experimental design should be  as specified in




Appendix E.  It might be advisable to try using a flow-through system which delivered




more than 1 volume replacement/day, e.g., 5 volume replacements/day.








      It would also be advisable to conduct a series of experiments examining factors




involved in the shipping, acclimation, and laboratory handling processes in more detail.








      Once  further research  identifies the  factors  involved  with  poor laboratory




reproduction with A. abdita. and successful tests have been conducted under control




conditions, the next step would be to test the chronic test design and the 10-day growth




tests using a contaminated material. Interlaboratory testing should be initiated to verify




that this species could be tested in seawater from various regions of the country.

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                                                                               10
                                                                               CT5
                                                                               -<*
                                                                                    coco
                                                                                                    03

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                                                                                                    o
                                                                                                   I
                                                                                                             3-33
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                                                                                                     03
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                                                                                                    to

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                                                                                                    CUD
                                                                                                    Ofl
                                                                                                    03
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                                                                                               03
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                                                                                                 O -rft
                                                                                             -0  03 -
                                                                                              ^  > T3
                                                                                             C  ?  w
                                                                                             '•gas
                                                                                              03  C«  ®

                                                                                                 C
                                                                                                -
                                                                                              a  S ^

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                                                                                              >  to  tuD
                                                                                              Q3  03  C
                                                                                             T3 rr,  S

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                                                                                                 g2
                                                                                                 8  c

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                                                                                                 03  g.

                                                                                                 
                                                                                                         ns
                                                                                              C  (— i
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-------
                                                                        3-34

Table 3.5. Fecundity of brooding female Ampelisca abdita recovered in Experiment
#2, Life Cycle at 25°C. "Egg" indicates a very early stage of potential young in the
brood pouch, and "gut" indicates that the gut  has formed in  the  developing
amphipods.
     Test   Amphipod  Repli-  #eggs/
     day   age        cate#   female
egg
stage  #young
21 29-31


28 36-38


35 43-45

49 57-59

2
3
3
1
2
3
2
2
1
1
7
2
3
1
1
1
13
1

1
egg
egg
egg
egg
egg
egg
gut
' egg
26*
egg
     * 3 spent females were recovered.

-------
                                                                              3-35

Table 3.6. Ampelisca abdita recovered from various feeding treatments in Experiment #3
(Temperature and Nutrition Effects) after 14 days at the indicated temperatures. P.p. =
Pseudoisochrysis paradoxa at approximately the same concentration as in Experiment #2
(Life Cycle at 25°C).  "Mix"=P. paradoxa, Phaeodactylum tricornutum. and Chaetoceros
calcitrans in a 1:1:1 mixture. "Mix+dry" = the algal mix plus about 4mg/day of ground Neo-
Novum®.  Mix+dry treatments were started two days before other fed treatments.  Ten
amphipods were tested in each replicate.
Treatment
Live
recovered
Missing
 %sur-    Mean%   Length in
 vival     survival   mm, x±SD(n)
20°C,no food



20°C, P.p.



20°C, mix



20°C,mix+dry



25°C,no food


25°C, mix



25°C,mix+dry
    6
    3
    3

    5
    3
    7

    6
    3
    5

   10
   10
    9

    4
    6

    2
    4
    5

   10
    9
   11
  4
  7
  7

  5
  7
  3

  4
  7
  5

  0
  0
  1

  6
  4

  8
  6
  5

  0
  1
  0
 60
 30
 30

 50
 30
 70

 60
 30
 50

100
100
 90

 40
 60

 20
 40
 50

100
 90
100
40.0     1.09+0.07(3)



50.0     2.15±0.20(3)



46.7     2.91±0.21(3)



96.7     3.02±0.13(3)



50.0     1.06±0.01(2)


36.7     3.69±0.15(3)



96.7     3.84±0.08(3)
initial samples
                                            1.08±0.03(5)

-------
                                                                         3-36

Table 3.7. Ampelisca abdita recovered from various feeding treatments in Experiment
#3 (Temperature and Nutrition Effects) after 41 days at the indicated temperatures.
P.p.  = Pseudoisochrysis paradoxa at approximately the  same concentration as in
Experiment #2 (Life Cycle at 25°C). "Mix"=P. paradoxa, Phaeodactylum tricornutum,
and Chaetoceros calcitrans in a 1:1:1 mixture. "Mix+dry" = the algal mix plus about
4mg/day of ground Neo-Novum®. Ten amphipods were tested in each replicate.
Treatment
20°C, P.p.


20°C, mix


20°C,mix-fdry


25°C, P.p.


25°C, mix


25°C,mix+dry


Live
recovered
5
3
1
1
0
4
2
5
3
0
0
0
1
0
0
0
0
0
Missing
4
6
5
2
5
3
5
2
4
8
9
8
3
3
1
7
7
6
%
survival
50
30
10
10
0
40
20
50
30
0
0
0
10
0
0
0
0
0
% survival w/o
senescence3
60
30
40
40
50
70
50
60
50
20
0
10
70
60
70
30
0
30
* Those individuals which were assumed to have died as a result of senescence (i.e.,
males  and spent females) were  not counted as dead  for the purposes  of this
calculation.

-------
                                                                         3-37

Table 3.8.  Percent survival of amphipods, Ampelisca abdita, in Experiment #4,
Density Effects Experiment, after 38 days at 20°C, with varying densities in exposure
containers. Amphipods were 46-58 days old when the experiment was terminated.
Treatment
10 per
replicate

20 per
replicate

40 per
replicate

Live
recovered
4
4
7
8
5
8
14
22
21
Missing
3
3
2
0
4
7
11
6
11
%
survival
40
40
70
40
25
40
35
55
52.5
% survival w/o
senescence3
50
40
70
70
65
50
40
62.5
65









a Those individuals which were assumed to have died as a result of senescence (i.e.,
males  and  spent females)  were not counted  as  dead for the purposes  of this
calculation.

-------
                                                                              3-38

Table 3.9. Experiment #5, Sediment and Animal Source Effects. Percent survival and length
of recovered amphipods, Ampelisca abdita, from two sources after 10 days in the indicated
sediments.  "Field" animals = offspring of ovigerous females from the field; "cultured" animals
= offspring of cultured ovigerous females. At the end of the 10 day exposure period, field
animals were 11-17 days old, cultured animals 11-18 days old. Twenty amphipods were
tested in each replicate.
Animal      Sediment        Live re-      Miss-     %sur-          Length in mm
source	treatment	covered	ing	vival   mean%  (mean±sd:n=3)
field
cultures
Long Island
             Yaquina Bay
             Yaquina Bay
             15°C
Long Island
             Yaquina Bay
13
13
13

14
16
17

14
15
16

20
19
19

20
19
20
6        65    65.0      3.35 ± 0.22
5        65
5        65

6        70    78.3      3.44 ± 0.09
4        80
2        85

6        70    75.0      2.90 ± 0.11
5        75
3        80

0       100    96.7      2.81 ± 0.06
0        95
1        95

0       100    98.3      3.00 ± 0.16
1        95
0       100

-------
                                                                        3-39

Table 3.10. Experiment #5, Sediment and Animal Source Effects. Percent survival
of recovered amphipods, Ampelisca abdita, from two sources after 43 days in the
indicated sediments.  "Field" animals = offspring of ovigerous females-from the field;
"cultured" animals = offspring of cultured ovigerous females. At the end of the 43-day
exposure period, field animals were 44-49 days old, cultured animals 44-50 days old.
Twenty amphipods were tested in each replicate.
Animal
source
Sediment
treatment
Live re-
covered
Miss-
ing
% sur-
vival
% survival
w/o senescence3
field
Long Island
7
9
4
4
5
8
35
45
20
60
70
35
            Yaquina Bay
cultured    Long Island
            Yaquina Bay
                   8
                   8
                   9

                  15
                  13
                  14

                  12
                  17
                   8
        4
        8
        5

        3
        3
        1

        1
        0
        3
        40
        40
        45

        75
        65
        70

        60
        85
        40
          70
          55
          45

          85
          85
          95

          100
          100
          85
a Those individuals which were assumed to have died as a result of senescence (i.e.,
males and  spent  females) were  not counted as dead for the purposes of this
calculation.

-------
                                                                           3-40



Table  3.11. Amphipods,  Ampelisca  abdita. recovered after 56  days at 20°C  in
! (Container, Aeration, and Nutrition Effects Expe
•e exposed in each of three replicates per treatment.
Treatment
aerated
bins
aerated
jars
nonaerated
jars
aerated jars
fed Artemia

Live
recovered
18
4
15
8
3
10
0
0
0
13
7
16
% survival
60.0
13.3
50.0
26.7
10.0
33.3
0
0
0
43.3
23.3
53.3
Mean
%survival
41.1
23.3
0
40.0

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                                   APPENDIX A
    LITERATURE REVIEW OF SELECTED CHESAPEAKE BAY AMPHIPODS
INTRODUCTION








       The ecological and life history characteristics of over 60 amphipod species which have




been reported from the Chesapeake Bay are summarized in Table A-l.  Further detail of




these characteristics for five amphipod species (Lepidactylus dytiscus, Ampelisca abdita,




Leptocheirus plumulosus. Monoculodes edwardsi and Neohaustorius schmitzi) are presented




in the text below and summarized in Table A-2.  These species were selected as possible




candidates for development as sediment toxicity test organisms for Chesapeake Bay region.




A sixth species (Hyalella azteca) is listed in Table A-2, but its ecological and life history




characteristics are not further discussed in the text because this is an established sediment




toxicity species, and information concerning H. azteca may be found in ASTM (1990a).
                                         A-l

-------
                                                                                A-2
                     Characteristics of Selected Species of Interest
                               Lepidactylus dytiscus
                                (Figures A-l,A-6,A-7)

 Habitat. Distribution, and Ecology


    L.dytiscus is found from the upper Chesapeake Bay to the Florida Atlantic coast, typically
 in the intertidal zone, but also subtidally to 3m, often sympatric with other haustoriids such
 as Neohaustorius schmitzi, and others of the genera Acanthohaustorius. Parahaustorius.
 Protohaustorius. Haustorius, and Pseudohaus'torius (Grant and Lazo-Wasem 1982, Bousfield
 1970, Dexter 1967, Croker 1967a).
    This species burrows freely in clean to muddy sand, typically sand with a high silt or
organic content (Mountford et al 1977, Dexter 1967, Grant and Lazo-Wasem 1982), but not
excessive silt-clay (Grant and Lazo-Wasem 1982). Deaver and Adolphson (1990) found that
in short-term experiments, survival was slightly better in 95% sand (90% survival) than in
50%sand/50% mud (79% survival) or > 85% silt/clay (77% survival). The burrowing pattern
of L. dytiscus is almost identical to that of Neohaustorius schmitzi (Howard and Elders 1970).
L. dytiscus may be found ranging from estuarine sands to exposed beaches, to a depth of 9
cm, but is most common in sheltered sand habitats (Grant and Lazo-Wasem 1982, Croker
1967a, Fox and Bynum 1975), in approximately the upper 5 cm (Croker 1967a). The amount
of light available may have an effect on depth of burrowing (Howard and Elders  1970). In
North Carolina, it has been reported in densities of up to 1500/m2, concentrated at the mid-

-------
                                                                                A-3



tide level (Dexter 1967), and may be commonly taken in estuarine plankton (Dexter 1967, Fox




and Bynum 1975).
Feeding and Nutrition









   L. dytiscus is reported to be a suspension-feeder (Croker 1967a, Bousfield 1970).  Gut




contents of field-collected specimens included diatoms, detritus and algae (Croker 1967a).




In the laboratory, L. dytiscus fed on materials in field-collected beach sand, and on a slurry




composed of beach sand detritus, diatoms, and crushed fecal pellets of the ghost shrimp




Callianassa major (Croker 1967a).
Reproduction









    Reproduction in this species may take place year-round, with maximum activity in the




spring and summer (Grant and Lazo-Wasem 1982, Croker 1967a, Dexter 1967). In Georgia,




females were usually dominant in the population, and an annual life cycle was reported




(Croker 1967a). Dexter (1967) reported the mean length of gravid females to be 5.42 mm,




with a mean egg number of 11.0, and young released at 1.36 - 1.52 mm.

-------
                                                                                A-4
Physical Tolerances
    This amphipod tolerates salinity conditions from fresh water to fully marine, and may




occur in brackish or virtually fresh water (Grant and Lazo-Wasem  1982).  It has been




reported from study areas of 5 - 30%o in North Carolina (Dexter 1967), and 7 - I8%o in the




Chesapeake Bay (Mountford et al 1977). Temperature at the latter site ranged from -0.3 to




27.5°C. Deaver and Adolphson (1990) reported >90% survival when L.  dytiscus were tested



for 14 days in salinities ranging from 5  to 40%o, and held and tested them in the laboratory




at 20°C.  JL.  dytiscus is reported to be fairly tolerant of desiccation and high temperature,



and has a negative response to light (Croker 1967a).
Distribution and Abundance in the Chesapeake Bay








   In the Chesapeake Bay, L_. dytiscus was reported to be dominant in 3 m sand communities




at Calvert Cliffs, with a mean summer density of 151/m2 (Mountford et al 1977), and at sand-




bottom stations in the James River (Jordan and Button 1984, Diaz 1989). Other researchers



have also reported it in the Bay (Loi and Wilson 1979, Feeley and Wass 1971).
Other Notes
   Ecotypic plasticity has been noted in size and morphology of JL. dytiscus, and its name




means "scaly-fingered diver." (Grant and Lazo-Wasem 1982). This species may be abundant

-------
                                                                                A-5



in the plankton at times of the new moon (Williams and Bynum 1972).   Ray Alden (Old




Dominion University, Norfolk, VA) has attempted to culture this species and has used it in




several sediment toxicity test exposures (pers. comm.); Deaver and Adolphson (1990) reported




successfully testing this species in 96-hour seawater-only acute toxicity tests with cadmium




and fluoranthene.









      Marcia Nelson (U.S. Fish and Wildlife, Columbia, MO) and Scott Carr (U.S. Fish and




Wildlife, Corpus Cristi, TX) have also worked with a related species (L. triarticulatus) (pers.




comm.); M. Nelson cultured this amphipods by feeding Cerophyll or rabbit chow about once




a week, and reports their life cycle to be 3-4 months at 20°C.
                                Ampfelisca abdita




                               (Figures A-2, A-6, A-7)









Habitat, Distribution, and Ecology









   Ampelisca abdita is a tube-dwelling amphipod belonging to the family Ampeliscidae,




found mainly in protected areas from the low intertidal zone to depths of 60m.  It ranges




from central Maine to south-central Florida and the eastern Gulf of Mexico (Mills 1964,




Bousfield 1973), and has also been introduced into San Francisco Bay (Nichols and Thompson




1985).   Where A. abdita are present, they are often dominant members of the benthic




community with  densities up to 110,000 m"2 (Nichols and Thompson 1985, Stickney and




Stringer 1957, Santos and Simon 1980). This species generally inhabits sediments from fine

-------
                                                                                A-6
 sand to mud and silt without shell, although it may also be found in relatively coarser
 sediments with a sizable fine component (Mills 1967).   A.abdita  is often abundant  in
 sediments with a high organic content (Stickney and Stringer 1957).

    This amphipod is a common food source for fish.  "Ampelisca sp.," probably A.abdita, was
 reported  in gut  contents of silversides,   Menidia menidia,  and juvenile  flounder
 Pseudopleuronectes americanus in the lower Pettaquamscutt River, Rhode Island (Mulkana
 1966), and A. abdita was reported to be food for spot Leiostomus xanthurus and star drum
 Stellifer lanceolatus (Stickney et al 1975).
Feeding and Nutrition

   Ampelisca abdita is a particle feeder, feeding both on particles in suspension and on those
from the surface of the sediment surrounding their tubes.  Gut contents of field-collected
specimens have been found to include algal material, sediment grains, and organic detritus
(Mills 1967, Stickney and Stringer 1957).
Reproduction


   In the colder waters of its range, A.  abdita produces two generations per year,  an
overwintering generation which breeds in the spring and a second which reproduces in mid
to late summer (Mills 1967, Nichols and Thompson 1985). In New England, breeding of the

-------
                                                                                A-7



overwintering generation begins when the water temperature is about 8°C, but in warmer




waters south of Cape Hatteras, breeding may be continuous throughout the year.  Adults




mate in the water column, and intense breeding activity is correlated with the full moon and




spring tides.  Females in a population from Barnstable Harbor, Mass., were found to carry




a mean of 26 eggs. Juveniles are released after approximately two weeks in the brood pouch,




at about 1.5 mm in length.  It then takes 40-80 days for newly released juveniles to become




breeding adults (Mills 1967).
Physical Tolerances









   A. abdita has been collected in waters of -2 to 27°C (Redmond and Scott, unpublished




data).  It is euryhaline, and has been reported in waters which range from fully marine to




10%o salinity (Bousfield 1973). This species is photonegative, and has a strong mortality




response when exposed to sunlight (Redmond and Scott, unpublished data).
Distribution and Abundance in the Chesapeake Bay









    A. abdita has been reported to be present in several areas of the lower Chesapeake Bay




(Reinharz and O'Connell 1983, Boesch 1977, Marsh 1973, Orth 1973, Schaffner et al 1987),




and in some cases it is abundant or dominant (Dauer et al 1984, Boesch 1973, Holland et al




1988).  Lippson et al (1979) reported Ampelisca spp. from the Potomac, and Lynch and




Harrison (1969) reported A. abdita from the York River, Virginia.

-------
                                                                                A-8
Other Notes








   An acute test procedure with this species is well-established, as is its sensitivity to a




variety of contaminated materials (ASTM 1990b, Redmond et al in prep., Scott et al in prep.,



DiToro et al  in press, Breteler et al 1989, Yevich et al 1986,  Rogerson et al 1985, Botton




1979). Chronic tests have also been conducted, and this amphipod can be maintained in the




laboratory with an algal diet (Scott and Redmond  1989, Gentile et al 1985).
                             Leptocheirus plumulosus




                               (Figures A-3, A-6, A-7)
Habitat, Distribution,'and Ecology
   Leptocheirus plumulosus ranges from Massachusetts to Florida, from the intertidal zone




to water approximately 5 m in depth. It builds an unlined, U-shaped burrow of sand grains




and debris in the upper 5-7 cm of sediment, and is typically found in mud to sandy mud and




detritus, especially in areas with a current (Bousfield 1973, Sanders et al 1965, Holland et




al 1977, Jordan and Sutton 1984, Reinharz and O'Connell  1983, Holland et  al  1987).




Shoemaker (1932) reports it at depths between 3 and 12 m in Chesapeake Bay.

-------
                                                                                A-9



   JL. plumulosus was reported in gut contents of the American eel Anguilla rostrata and the




blue crab (Callinectes sapidus) in the James, York, and Rappahannock Rivers (Wenner and




Musick 1975).  Marsh (1988) found that in the Patuxent River,  there was close timing




between the late  spring to early summer increase in production of L.. plumulosus and an




increase in abundance of predators,  which consisted  primarily  of juvenile fish.  He




determined that this was an opportunistic species with "boom and bust" population dynamics.




In winter,  population growth was limited  by low temperatures,  and in  early spring by




nitrogen availability.  In late spring to early summer, most reproduction and population




growth occurred, correlated with warmer temperatures and increased nutrient availability.




By late summer, juvenile survival, growth, and reproduction appeared to be limited by lack




of essential micronutrients.

-------
                                                                              A-10
Feeding and Nutrition
    L. plumulosus is reported to be a surface deposit feeder (Holland et al 1988, Marsh 1988),




and may leave its burrow to forage on the sediment surface (Sanders et al 1965). McGee et




al. (1990) maintained this species in laboratory experiments for 28 days on 6mg Tetramin fed



3 times per week.
Reproduction








   This species has an annual reproductive cycle, with ovigerous females most abundant




from May to September, and two broods per female (Bousfield 1973). Marsh (1988) reported



that in the Patuxent River, Maryland, during 1984 - 1986, this species reproduced primarily



in May and June. Toward the end of this period, growth of juveniles, mean body size of



gravid females, and fecundity decreased.  Fecundity decreased even when normalized for




female body size and it was concluded that the population dynamics were controlled by the




food supply at that time.  C. Schlekat and B. McGee collected gravid JL. plumulosus as late




as December and as early as February from subestuaries in northern Chesapeake Bay (pers.



comm.).
Physical Tolerances

-------
                                                                               A-11



   Although able to tolerate salinities from 3 - 31%o (Sanders et al 1965), this species is




generally found in low to mid salinity areas (Boesch et al 1976, Reinharz and O'Connell




1983), and may be termed an estuarine endemic species (Holland et al 1987). Holland et al




(1988) classify this  amphipod as one of a group of organisms tolerant of a wide range of




salinities and sediment types, but note that it had high production values in 5-10%o habitats




in Baltimore Harbor and the Chester River. Similarly, Marsh (1988) reported L. plumulosus




as a dominant species at a Patuxent  River site where salinity varied  from  10 to 14%o.




Schlekat et al (1992) reported no significant differences in adult survival in salinities ranging




from 2 to 32%o in 10 to  28 day laboratory  experiments.  It has been collected in water




temperatures of 0-30°C (Holland et al 1977, Marsh 1988, Jordan and Sutton 1984).  Marsh




(1988) reported that this species was inactive when temperatures were less than 5°C, and




suggested that its optimum temperature for reproduction was probably between 10 and 20°C.




Feeley and Wass (1971) collected Ij. plumulosus from a variety of substrates, and Schlekat




et al. (1992) reported good adult  survival in the  laboratory in sediments with a large




variation in particle size  distributions and organic  content.  The latter authors  also




successfully tested these amphipods using artificial seawater.
Distribution and Abundance in Chesapeake Bay









   L. plumulosus is widely distributed in the upper Bay and tidal tributaries (Jordan and




Sutton 1984, Holland et al 1987, Mountford et al 1977, Holland 1985, Shoemaker 1932) and




is frequently abundant or dominant (Reinharz and O'Connell 1983, Hines and Comtois 1985,

-------
                                                                              A-12



Dauer et al 1987, Schaffner et al 1987, Diaz 1989, Holland et al 1988, Holland et al 1977,




Marsh 1988).








   Holland et al (1988) observed that the abundance of this species in Baltimore Harbor has




increased over the past decade, to a maximum of approximately 15,000/m2 in  1987 from




essentially zero in 1970, apparently due to a lower contaminant load in that area. They also




reviewed power plant impact studies  from the Chesapeake,  which indicated that L.




plumulosus had higher abundance in thermal impact areas of Chalk Point and Wagner plant




discharges, which they conclude is due to organic enrichment from entrainment mortalities.




This agrees with the findings of Diaz (1989), who reported that L. plumulosus was dominant




in a soft-bottom community in the immediate area of a large sewage outfall.  In the thermal




impact area of the Morgantown plant, reproduction in this species started and ended earlier




than normal (Holland et al 1988).
Other Notes








   L. plumulosus may be more abundant in the plankton at times of the new moon (Williams




and Bynum 1972).  Schlekat et al (1992) successfully tested this species in 4-day seawater-




only tests and 10- to 28-day sediment tests.

-------
                                                                               A-13
                              Monoculodes edwardsi
                                (Figures A-4, A-6, A-7)

Habitat, Distribution, and Ecology


    Monoculodes edwardsi is widely distributed, ranging from the Gulf of St. Lawrence to
Cape Cod and the mid-Atlantic to north Florida and the Gulf of Mexico (Bousfield 1973). It
burrows freely in fine and silty sands from the low intertidal to 75  m (Bousfield 1973,
Mountford et al 1977, Bousfield 1970, Watling and Maurer 1972, Fox and Bynum 1975).
Feeley and Wass (1971) reported taking it at all depths, from the upper layers and surface
of the bottom, and in both sand and mud with considerable detritus. Van Dolah and Bird
(1980) suggest that it probably burrows partially exposed at the sediment-water interface.
   M- edwardsi has been reported in gut contents of young-of-the-year striped bass, Morone
saxatilis in the James, York, and Rappahannock Rivers (Markle and Grant 1970); of the
American eel Anguilla rostrata in the York River (Wenner and Musick 1975); and of the
Atlantic croaker Micropogon undulatus. the spot Leiostomus xanthurus and the star drum
Stellifer lanceolatus in estuaries from South Carolina to Georgia (Stickney et al 1975).

-------
                                                                              A-14
Feeding and Nutrition
   This species is reported to be a suspension feeder (Dexter 1969), although Bousfield (1970)




notes that the mouthparts in this genus are of a non-specialized, omnivorous feeding type




rather than being specialized for filter-feeding.
Reproduction








   The reproductive cycle for M. edwardsi is annual, with ovigerous females most abundant




May to September and several broods produced per female (Bousfield 1973, Holland 1985).




M. edwardsi may sometimes be abundant in plankton tows, and this may be related to its



breeding cycle (Fish 1925), to temperature  changes (Whitely 1948), or to the presence of a



new moon (Williams and Bynum 1972).  Whitely (1948) collected egg-bearing females in the




plankton on Georges Bank in  June, and concluded that primary reproduction took place in




the summer.   Van  Dolah and Bird (1980) examined specimens of this species from the




collections of the National Museum of Natural History (location not stated), and reported that




females of 5-7 mm long carried a large number of small eggs relative to other  amphipod




species examined (Y = 3.69 e°-34L, r = 0.81 where Y = egg number and L = female length).
Physical Tolerances

-------
                                                                               A-15



    This species is found in waters which range from fully marine to oligohaline and brackish




(Bousfield  1973).  Mountford et al (1977) collected it in abundance at a study site in




Chesapeake Bay where the salinity ranged from 7 to 18%c, and temperature from 1 to 30°C




(Holland et al 1977).
Distribution and Abundance in Chesapeake Bay








   M. edwardsi has been reported from several areas in the Chesapeake (Boesch 1977, Ewing




et al 1982, Holland et al 1987, Loi and Wilson 1979), and Mountford et al (1977) reported it




to be dominant in some 3m sand communities. Holland et al (1988) noted that Monoculodes




sp. had consistently high abundances in habitats approximately 5-18%o, and reported it at a




maximum of 3200/m2 at Calvert Cliffs, with year-to-year fluctuations in abundance.
                             Neohaustorius schmitzi




                                 (Figures A-5, A-6)








Habitat, Distribution, and Ecology









   This species is found from Cape Cod to Georgia and southern Florida It coexists with




other haustbriid  species, such  as Lepidactylus dytiscus, in the  intertidal  zone (see




above)(Bousfield 1973, Bousfield 1970, Croker 1967a, 1967b, Dexter 1967, 1971).

-------
                                                                             A-16



   N. schmitzi burrows freely in clean to muddy sands, fine to medium in particle size




(Bousfield 1973, Dexter 1967, Croker 1967a). It prefers cleaner sand to that with more silt,




debris, and shell material (Croker 1967a).  Although this species can burrow up to 10 cm, in



North Carolina 97% of the population was found in the upper 5 cm, and 83% in the top 2.5




cm (Dexter 1971). Croker (1967a) similarly found it to be most abundant in the upper 2.5 cm




in Georgia.  The amount of light may have an effect on depth of burrowing (Howard and




Elders 1970).








   In North Carolina, N. schmitzi was found to be most  abundant on sheltered beaches in



sounds (Fox and Bynum 1975) or on sandy beaches of inlets (Dexter 1967, 1971).  At some




sites this amphipod was present at an average density of 800/m2 (Dexter 1967).  N. schmitzi




was reported most abundant  in the upper intertidal zone in Georgia (Croker 1967a) and




North Carolina (Fox and Bynum 1975, Dexter 1967), middle and lower intertidal in South




Carolina (Knott et al 1983), and mean low tide (0-2 hrs exposure/ tidal cycle) to high tide zone




(8-10 hrs exposure) in North Carolina (Dexter  1971).
   The population may show an  aggregated  distribution.  The amphipods are also




concentrated lower in the tidal zone in winter than in summer, and this may be an avoidance



response to increased wave action and lower temperatures (Dexter 1971). The sexes may be




separated in the tidal zone; females (Bousfield 1970,  1973), particularly gravid females




(Dexter 1971) have been generally reported higher, but Croker (1967a) reported males higher




in the zone.  The juveniles located lower in the intertidal zone may be a dispersal stage




(Bousfield 1970).

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                                                                                A-17
Feeding and Nutrition
    N. schmitzi is reported to be a suspension-feeder (Bousfield 1970; Croker 1967a, 1967b,




Dexter 1969) which feeds for short periods at frequent intervals in the laboratory and during




periods of tidal immersion in nature (Croker 1967b). Croker (1967b) reported gut contents




of this species to consist  of detrital masses which include flagellates, ciliates,  diatoms,




unicellular chlorophytes, and bacteria, as well as small amounts of fine sand. Diatoms of the




genera Navicula, Hantzschia, Nitzschia. Coscinodiscus. Grammatophora, and Cocconeis were




ingested.  The size range  of ingested items was 0.5  - 78.5 urn. Ivester and Coull (1975)




reported similar results of gut content analysis, but a larger size range of material of 0.5 -




300um, and noted that zooplankters  such  as  copepods  were occasionally found.  These




authors also indicate Rhodes (personal communication) has observed N. schmitzi feeding on




polychaetes and other amphipods.









    In the laboratory, N. schmitzi was reported to feed on materials in field-collected beach




sand, and on a slurry composed of beach sand detritus, diatoms, and crushed fecal pellets of




the ghost shrimp Callianassa major (Croker 1967a), or on ghost shrimp fecal pellets alone




(Frankenberg 1967).
Reproduction
   N.  schmitzi  is reproductively active for most  of the year  (Dexter 1969),  with the




reproductive period variously reported to extend from May to September (Bousfield 1973), or

-------
                                                                              A-18



February to October in North Carolina, with peaks in spring (April) and summer (August)




(Dexter 1967, 1971). Knott et al (1983) reported peak densities of animals in February and




May in South Carolina. In North Carolina, there are two generations  per year: a winter




generation which lives about 8 months  and reproduces  in the  spring, and a summer




generation which lives about 4 months and produces the overwintering individuals.  The




mature individuals in the summer generation are smaller than those in the winter generation




(Dexter 1971). In Georgia, maximum reproductive activity occurred in April, with fecundity




decreasing as autumn approached and the summer generation began to age (Croker 1967a,




1968b).
   The maximum range in brood size reported is 2 - 14 eggs per female, with egg number




depending on size of the female and the generation to which she belongs (Dexter 1971). Van




Dolah and Bird (1980) reported the relationship between female length (L) and egg number




(Y) as Y = 1.63L - 2.52, from Georgia specimens in the National Museum of Natural History.




Dexter (1967) reported the mean length of gravid females in North Carolina to be 3.6 mm,




with a mean egg number of 5.69; juveniles were 1.4mm at release. Females appear to produce




only one brood in their lifetime (Dexter 1971), and females have been reported to be dominant




in the population (Dexter 1971, Croker 1967a). Juveniles (Croker 1976a) and some adults




(Williams and Bynum 1972) have been taken in plankton tows, but generally haustoriids in




this subfamily are scarce in the plankton, so although little is known of the mating behavior,




it is not likely that they mate in the water column (Dexter 1971). The young may crawl back




into  the brood pouch one or more times before their final release (Croker 1968a).

-------
                                                                               A-19
Physical Tolerances
   Living high in the intertidal zone, this species appears to be relatively tolerant of high




temperature and desiccation.  Croker (1967a) measured temperatures of > 39°C in the upper




2.5 cm of Georgia sands with N. schmitzi present, and reported that most animals exposed




to 40°C for 2 hours in the laboratory lived for several months afterward at approximately




27°C. Dexter (1971) collected this species in substrate temperatures from 9-33 °C. Animals




exposed to air for 20 min lived for several months once returned to seawater (Croker 1967a).




Gravid females have been found to be more resistant to desiccation than non-gravid females,




males, and juveniles, which may be associated with their concentration in higher tide zones




(Dexter 1971).









   This  species is reported from 6%o to fully marine conditions in the Chesapeake Bay




(Bousfield 1973), and was collected in 16.5 to 244%o in North Carolina (Dexter 1971).








   N. schmitzi is strongly photonegative and very  active, and  sympatric species may be




separated from collections partially by the speed of their reaction to light (Croker 1967a).
Abundance and Distribution in Chesapeake Bay
   This species is reportedly found throughout the Bay (Lippson and Lippson 1984, Bousfield




1973), but thus far no community studies have been located which mention its occurrence.

-------
                                                                               A-20



Holland (unpublished data cited in Lippson et al 1979) reported this species from the high




mesohaline to near polyhaline areas in the Potomac River.

-------
                                                                                                                          A-21
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-------
                                   APPENDIX B








  PROCEDURES TO MINIMIZE THE RISK OF RELEASING NON-INDIGENOUS




             AMPHIPODS, PATHOGENS, WATERS, OR SEDIMENTS




                    INTO LOCAL WATERS OR WATERSHEDS
General Principles:









1)     No non-indigenous animals or sediment will be released into the environment;




2)     All water coming into contact with non-indigenous amphipods or sediment in the




       laboratories will be sterilized prior to disposal;




3)     All equipment  or materials (i.e., glassware,  paper, plastic, etc.) contacting non-




       indigenous  amphipods or sediment will be contained for proper sterilization;




4)     All non-disposable materials will be sterilized or confined to the culture room.
Containment Protocol:
       Culture, holding, handling, and experimentation of non-indigenous species is restricted




to "non-indigenous laboratory rooms" separate from  those used to hold or culture native




species. Any material (living or dead) or equipment used in "non-indigenous laboratories" are




considered as potentially infected and are treated accordingly. Non-indigenous sediments are




kept in clearly marked, clean sealed containers in a refrigerator and opened only in a "non-






                                        B-l

-------
                                                                                  B-2



indigenous laboratory". Access to the "non-indigenous laboratory" is limited to trained and




authorized personnel.  All drains from "non-indigenous laboratories" are either sealed off or



directed to separate designated holding tanks in which the liquid waste can be sterilized prior




to disposal.  The amphipods are cultured in a static-renewal manner to minimize the amount




of water that must be treated. As fresh seawater is added to each culture bin, the displaced




seawater is directed to storage barrels or tanks and treated with chlorine bleach (i.e, 0.5%




chlorine) for sterilization.








       Materials for sterilization of animals, sediment or equipment are kept in the "non-




indigenous  laboratory".  These materials include  disinfectant soap  (for cleaning hands),



chlorine bleach (for sterilizing all non-human materials and surfaces), mops, sponges, and




buckets (for cleaning floors and surfaces), a bleach dip bath (for sterilizing glassware), and




a labelled trash can (for disposal of contaminated paper, gloves, etc.).  All spills are cleaned




immediately.








       Personnel wear lab coats while  in the "non-indigenous laboratory" and these coats



remain in the laboratory.  Hands must be washed with disinfectant soap prior to leaving the




"non-indigenous laboratory".








       No equipment leaves the "non-indigenous laboratory" without first being sterilized by




dipping or wiping with bleach.  Glass-  or plasticware that has been in direct contact with




non-indigenous species, sediment or associated water are held overnight in the chlorine




bleach  dip.   Water,  sediment, and non-indigenous materials  are sterilized  by  either




autoclaving or soaking overnight  in a chlorine bleach solution.  This material is later

-------
                                                                                  B-3



neutralized with sodium thiosulfate and then disposed down  a sanitary drain to the




municipal sewage system, unless it is also chemically contaminated.  Paper and plastic




discarded in the labelled trash can is autoclaved prior to disposal to a municipal landfill.

-------

-------
                                  APPENDIX C








              METHODOLOGY TO ASSESS THE ACUTE TOXICITY




                  OF MARINE AND ESTUARINE SEDIMENTS




       WITH THE BENTHIC AMPHIPOD. LEPTOCHEIRUS PLUMULOSUS
(Leptocheirus plumulosus annex to the ASTM E1367-90 Document, Draft no. 3, May 1992.




Contacts: Beth L. McGee, Christian E. Schlekat, Maryland Department of the Environment,




Ecological Assessment Division, 2500 Broening Highway, Baltimore, Maryland, 21224. phone:




(410) 631-3782, Fax: (410) 631-4105).









      A5.1  Ecological  Requirements - Leptocheirus  plumulosus  (family Aoridae) is  an




infaunal amphipod distributed subtidally along the east coast of the United States from Cape




Cod, Massachusetts to northern Florida (Bousfield, 1973). In Chesapeake Bay, L. plumulosus




is indigenous to oligohaline and mesohaline regions (Feeley and Waas, 1971; Jordan and




Button,  1984; Holland et al., 1988), though it can tolerate an even broader salinity range,




from near 0 to 33%o (Feeley and Waas, 1971; Jordan and Sutton, 1984; Schlekat et al, 1992).




This species constructs U-shaped burrows in sediments ranging from fine sand to silty clay




(Jordan and Sutton, 1984; Holland et al., 1988; Schlekat et al, 1992).  Due to its broad




salinity and sediment  tolerances, it  is a desirable test  species for east coast  estuarine




sediments and has been used successfully in the assessment of contaminated  sediments in




Chesapeake Bay (MDE 1991a,b; Pinkney et al., 1991; see  Chapter  2).
                                        C-l

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                                                                                 C-2
       A5.2 Collecting and Handling Techniques - Leptocheirus plumulosus is most abundant
in the upper 2 cm of sediment, rarely penetrating to depths below 5 cm (Reinharz, 1981).
Amphipods can be collected with benthic grab samplers (e.g., Peterson, Ponar) from various
tributaries of Chesapeake Bay. The contents of each grab are sieved through a 0.5-mm mesh
screen and the retained material  is gently rinsed into polyethylene buckets,  containing
collection site sediment and water. These containers are transported to the laboratory where
they are aerated. It is  desirable to sort amphipods from collection  site debris within 12
hours. A 0.5-mm mesh sieve can be used to separate amphipods from transport sediment.
The material retained on the screen can be rinsed into sorting trays containing collection site
water. Healthy, active amphipods can be removed from detritus by using a bulb pipette of
a suitable size (e.g., one with a 5-mm diameter bulb).

       A5.2.1 For acclimation, L.  plumulosus  can be placed in an aquarium (e.g., 40-L)
containing a 1-2 cm deep layer of  0.5-mm sieved  collection site sediment at a density of
approximately 200 to 300 per aquarium. Aeration should be vigorous.  Two to three days are
sufficient for acclimation to the test environment. A gradual change from collection site
water to test water is desirable. This can be  accomplished by gradually increasing the
proportion of test water in the tanks oyer 2 to 3 days.

      A5.2.2 Culture techniques have been developed (see Chapter 1).  Presently, laboratory
populations can be maintained through several generations in shallow plastic tubs or glass
aquaria containing a 1-2 cm layer of fine grained sediment from the amphipod collection site
or a texturally  similar sediment (Pfitzenmeyer, 1975; see Chapter 1). Water exchange is
static-renewal, with 30-100% of water volume in each container replaced 2 to 4 times per

-------
                                                                                C-3

week.  Culture containers are aerated, maintained at a temperature of approximately 20°C,

a salinity of 20 g/kg and a photoperiod of 16h light:8h dark. Cultures receive a mixture of

.micro-algae (e.g.,  Pseudoisochrysis paradoxa, Phaeodactylum  tricornutum. Tetraselmis

suecica) and approximately 0.1 g of amphipod "gorp" (a mixture of fish food flakes, yeast,

alfalfa powder, ground cereal leaves and shrimp maturation feed) 2-3 times per week (see

Chapter 1). Amphipods can be separated from acclimation or culture sediments using a 0.5

mm sieve immediately prior to initiating the toxicity test.         . .    •.  ;




       A5.3 Toxicity Test Specifications - The effects of different physical conditions on the

sensitivity of L. plumulosus to toxic materials are currently under investigation. This species

is routinely tested at 20°C or 25°C1. Salinity of overlying water will depend on the objectives

of the study.  Toxicity test seawater can be diluted to the same salinity as the  interstitial

water of the test sediment, the ambient bottom salinity at the test site or a selected test

salinity in the range of 2 to 32%o.  Laboratory investigations indicate Leptocheirus is.tolerant

of a range of sediment types (Schlekat et al, 1992); however, a grain size reference should

be included for coarse sediments since these may be somewhat  stressful.  Fine grained

sediments from the amphipod collection site or laboratory cultures  are desirable  as  the

negative control. The exposure chamber routinely used to test Ij. plumulosus is  a 1-L glass

beaker. The exposure chamber should be covered with a watch glass to reduce contamination

of the contents and evaporation of the water and test materials. Aeration can be provided

to each test chamber through a 1-mL glass pipette positioned not closer than 2 cm from -the

sediment surface.  Each test chamber should contain a 2-cm deep layer of sediment and
       1A  test  temperature of  25°C  is  recommended  in this  report  for
 consistency with  test  conditions  in  the  chronic  sediment toxicity
 test.

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                                                                                C-4
enough, overlying water to create approximately a 4:1 (v/v) water to sediment ratio. Sediment
and water should be added to the test chambers the day before the amphipods are added to
allow suspended sediment  particles  to  settle, and to  allow  time for equilibration of
temperature and the sediment-water interface.

      A5.3.1 After overnight equilibration of the test chambers, amphipods can be randomly
distributed to each of the containers. It is desirable to sacrifice a random sample of at least
20 animals from those being sorted on day 0 to provide an initial size range estimate of test
animals. Twenty amphipods should be tested per replicate. Animals caught on the water's
surface can be gently pushed under using a glass rod. Amphipods should be allowed 5 to 10
min to burrow into the test sediments.  Amphipods that have not burrowed within that time
should be replaced with healthy animals, unless the amphipods are repeatedly burrowing into
the sediment and immediately emerging in an apparent avoidance response.  In that case,
the amphipods are not replaced.   Amphipods are not removed from the surface of test
sediments during the course of the toxicity test  even if they appear  dead, since  some
amphipods that seem dead might actually be alive and might later rebury into test substrate.

      A5.3.2  The toxicity test can be terminated after 10 days by sieving amphipods from
test sediments using a 0.5-mm mesh screen. Mortality is the endpoint for this short-term test.
Burrows generally disintegrate during sieving and animals can be transferred to a sorting
tray for enumeration. The ability of surviving amphipods to rebury into clean sediments can
be used as a sublethal test endpoint.

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                                                                                 C-5



       A5.3.3 Other Testing - Partial life cycle tests (28 - 30 days) initiated with juveniles are




being conducted with this species, with amphipod length, reproduction, and survivorship as




viable endpoints. Research is currently underway to determine the optimum conditions for




these tests.









       A5.4 Life Cycle and Age Classes - Leptocheirus plumulosus is an annual species




capable of producing a least two broods, with peak periods of reproduction in early to mid




spring and in the fall (Schlekat et al., 1992; Ray, 1982).  Gravid females have been observed




in Chesapeake Bay as late  as December and as early as February, indicating that timing of




reproduction varies yearly depending on climatic conditions. In cultured populations, females




produce multiple broods and gravid females are available year round (Sewall et al., 1991; see




also Chapter  1).  Size  range  of field-collected test organisms  might depend on the size




structure of the  field population, as the mean size of amphipods collected in early spring is




generally greater than  those collected in the  summer or fall. Size range  of cultured




amphipods is less variable  seasonally.  Immature and adult amphipods, approximately 3  to




5 mm as measured from the base of the first antenna to the end of the third pleon segment




along the dorsal surface, should be  used in toxicity tests because they are easy to handle and




count.  The potential effects  of age,  size, sex, and seasonal variation of field collected




organisms on the sensitivity of JL. plumulosus to contaminants is currently being examined.




Evidence to date indicates  mixed-sex populations within the recommended size range show




consistent responses to field-collected contaminated sediments and 96 h water only exposures




to cadmium (Schlekat et al., 1992; MDE 1991a,b; Pinkney et al., 1991).

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                                                                                  C-6



       A5.5  Control Survival - Mean control survival using Leptocheirus must be at least




90% for the toxicity test to be considered valid.









       A5.6  Sensitivity - Leptocheirus plumulosus is tolerant of handling and a range of




sediment types and salinities.  The sensitivity of this species is comparable to Hyalella azteca




in 96 h water only exposures to cadmium (Schlekat et al., 1992; Pinkney et al., 1991). A




review of benthic surveys and sediment contamination  in Chesapeake Bay indicates a




negative correlation between the presence of L_. plumulosus and the degree of contamination




(Reinharz, 1981; Pfitzenmeyer, 1975).









       A5.7 Interpretation - When interpreting the results of acute toxicity tests, it should




be kept in mind that the early life stage, the reproductive ability, or the long-term survival




of Li. plumulosus might be affected by contaminants at concentrations lower than those that




produce a lethal response.  Partial life cycle sediment toxicity test procedures are under




development for L_. plumulosus and should resolve these questions.

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                                  APPENDIX D








      RESEARCH METHODOLOGY TO ASSESS THE CHRONIC TOXICITY




                   OF MARINE AND ESTUARINE SEDIMENTS




       WITH THE BENTHIC AMPHIPOD. LEPTOCHEIRUS PLUMULOSUS
Abstract








      A generic chronic sediment toxicity test with the amphipod Leptocheirus




plumulosus is described.  This is a draft design which has not been fully tested.  This is a




static test conducted at 25°C, 20%o, and with a 16:8 h light:dark photoperiod. Food is




provided three times per week.  The experiment begins with 20 juveniles (i.e., <1 day old)




per replicate, 5 replicates per treatment, and is terminated after 28 days.  At termination,




the contents of each exposure chamber are sieved through two sieves to collect adults and




offspring. Endpoints are mortality of the initial cohort, body length of the survivors (i.e.,




size), and number of female offspring produced per female survivor (i.e., fertility).
Exposure Conditions




      Exposure chambers are 1-L glass beakers.  Each chamber receives either test




sediment, performance control sediment (i.e., culture sediment), or reference toxicant in




water. Test sediments may be contaminant-spiked sediment, field sediment, or dilutions




of field sediments.  The position of the test and control beakers are randomized within the






                                        D-l

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                                                                                 D-2
water table or room in which the toxicity test is conducted. If there are sufficient
personnel available, the test can run blind, with test and control beakers coded so that
personnel monitoring the test have no knowledge of the identity of treatments in the
exposure chambers.

       The recommended temperature is 25°C, which is an acceptable culture and testing
temperature for this species. The life cycle of L. plumulosus is also shorter at 25°C than
at 20°C. This amphipod has been tested at salinities within the range of 1.5 - 35%o
(Schlekat et al., 1992), and the salinity of the overlying water should be adjusted to match
that of the sediment pore water (and not vice versa). However, if this test is to be used
with spiked or reconstituted sediments, 20%o is recommended for comparability with other
tests. This is  also the recommended culture salinity.

       A photoperiod of 16 h light and 8 h dark was selected to approximate conditions
existing during summer when reproduction in the field is expected to be high. The same
photoperiod is maintained for the JL. plumulosus cultures.  This photoperiod has been
shown to consistently maintain reproductive activity in Hyalella azteca (de March 1977,
cited in Arthur 1980).

       In nature, JL. plumulosus is found in a wide range of sediment types, from  mud or
detritus to sand. The performance control uses culture sediment, which is very fine
grained.

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                                                                               D-3



      The exposure container receives 175 ml of test sediment and 725 ml of overlying




water which is aerated constantly.  The sediment is added to each beaker the day prior to




starting the exposure.  This allows the settling of sediment suspended by the addition of




the overlying water and equilibration of the test sediment and water to  the exposure




temperature.  Each exposure chamber is covered with a glass plate or evaporating dish to




reduce evaporation of the water overlying the sediment.  The exposure chambers are




placed in a constant temperature water bath throughout the exposure.









      Food is provided three times per week (Monday, Wednesday, and Friday) by using




a screened siphon tube to remove approximately 400ml/beaker of overlying seawater, and




replacing this with a salinity-adjusted, algal-seawater mixture and lOmg of a dry food




mixture ("gorp"). A glass disk attached to a glass rod is used to prevent disturbance of




the sediment while the algae is added. The algal is prepared as a 1:1 v/v mixture of




Pseudoisochrysis paradoxa and Phaeodactylum tricornutum to a final density of 106




cells/ml.  The gorp is a finely ground, dry mixture of 48.5% TetraMin®, 24% dried alfalfa,




24% dried wheat leaves and 4.5% Neo-Novum® (a maturation feed for shrimp mariculture;




Argent Chemical Laboratories, Redmond, WA) suspended in 20%o seawater at a




concentration of lOmg/ml; 1ml of the gorp suspension is pipetted into each exposure




chamber at the time of water renewal and feeding.

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                                                                                D-4
Controls
      Three types of control treatments may be used the sediment toxicity test; two are




mandatory (i.e., the QA/QC performance and reference toxicant controls), and the third




(i.e., the experimental) is optional, but highly desirable. The performance control



measures the responses of Ij. plumulosus in the absence of contaminant stress and under




the best possible conditions for the amphipods. The performance control uses culture




sediment as the test substrate and is conducted at 20%o and 25°C.  The exposure periods




is 28-d and is conducted in all ways the same as test sediments. Performance controls are




used for QA/QC, to assure that the test organisms are healthy.  The performance control




is replicated five times.








      The reference toxicant control tests the sensitivity of the animals to a single



toxicant under repeatable exposure conditions. The reference toxicant control consists of




96-h, water-only exposure to cadmium chloride at 20%o and 25°C.  The reference toxicant




control for  the chronic test is initiated 1 wk after the start of the sediment toxicity test




because the newborn amphipods cannot survive 96-h without sediment or food, having




been released from their mothers' marsupium for less than 1 d. A subset of the newborns




used for the test sediment treatments are placed in culture sediment and fed in the same



manner as amphipods in culture a 1 wk period. After this growing-out period, they are




sieved from the sediment and randomly allocated to the different cadmium concentrations.




The cadmium concentrations for the this control typically range from 0.19-6  mg/L.  The




reference toxicant control is also employed for QA/QC, to determine whether the

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                                                                                 D-5



sensitivity of the test animals is consistent among experiments. Only one replicate of




each concentration is used.









       The third control is an experimental control in which one non-contaminant




environmental parameter (such as grain size, TOG, temperature, a carrier solvent, etc.) is




allowed to vary from the standard environmental condition to the same extent that this




parameter varies within one or more of the test sediments. This control thus allows for




an estimation of the effect of this non-contaminant parameter on the response of ]L.




plumulosus.  This control is treated the same as the other test sediments in all other




ways.  These controls are  included as the uncontanainated treatment against which the




toxicity of the other test treatments are compared statistically.
Exposure condition modifications which may affect the test results









1. Temperature. Growth and fertility will decrease as temperature is lowered. Higher




temperatures may increase the magnitude of these responses, but also may stress the




amphipods and so introduce variability. Newborn K plumulosus may be very sensitive to




temperature change, and every effort should be made to maintain them at constant




temperature (i.e., 25°C) during all phases of bioassay setup (i.e., release from maternal




brood pouch, isolation from adults, sorting, and transfer to test beakers).  Generally, the




test temperature should be 25°C. If another temperature is to be used, one or more




temperature control treatments would be recommended.

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                                                                                 D-6



2. Salinity.  Choice of test salinity will generally depend on the necessity to match the




salinity of overlying water to that of the test sediments. Experiments conducted under




control conditions showed no significant difference in survival over 10-28 days in salinities




ranging from 1.5-25%o, and no apparent differences in number of young per female after



28 days when exposed to salinities from 5-32%0 (Schlekat et al.,  1992). However, the




response of the organism to a toxic substance might vary depending on salinity selected,




particularly metals for which bioavailability changes with salinity. In general, the



salinity of the performance and reference toxicant controls should remain constant (i.e.,




20%o), and this salinity should be used unless there are compelling reasons to do




otherwise.  If other salinities are used, a salinity control may be advisable.








3. Physical characteristics of test sediment.  In 20-day exposures, Schlekat et al. (1992)




reported no significant differences in survival or number of young per female in



uncontaminated sediments ranging from 98.1% sand to 96.5% silt/clay. However,




Maryland Dept. of the Environment (1991) indicated coarse sediment texture might have




been a factor contributing to mortality in acute sediment tests with this organism.




DeWitt et al (in prep.) reported mortality or growth of sub-adult and newborn L.




plumulosus were not correlated with sediment grain size, total organic carbon content,



sediment water content, or Eh.  If there is reason to believe sediment characteristics




might affect the amphipod's response, an experimental control for these factors should be




included.








4. Type of exposure.  Some amphipod species have shown increased sensitivity to




toxicants in a static system compared to flow-through or static daily renewal (eg. Word et

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                                                                                 D-7



al 1989). No research has been conducted to compare the sensitivities of Li. plumulosus




under different water-renewal-rate exposure systems.









5. Amount and type of food provided.  10-d growth is reduced if food is withheld, and




mortality, growth, and reproduction probably would be significantly altered if L.




plumulosus was not fed during a 28-d exposure. Comparisons of feeding regimes




employed by Schlekat et al. (1992) and DeWitt et al. (in prep.) suggest that omission of




live algae from the diet  may lead to substantially reduced production of offspring.  It




seems likely  that nutrition may have a significant impact on toxicological sensitivity, as is




suggested in recent work by McGee et al (in prep.). At this time, changing the diet from




that described above may substantially affect the repeatability, variability, and magnitude




of the toxicological responses.









6. Age of the amphipods. Newborn juveniles (i.e., 
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                                                                                D-8
Logistics for Conducting the Test
      The test is initiated with juveniles which are <24 hours old.  Juveniles are




harvested within one day of their release from the brood pouch; their exact age is known,




and their size is essentially uniform. Measurement of an initial sample of juveniles added




to the test will confirm the size range.
       A large number of ovigerous females are isolated 5 days before the test is due to




start (i.e., day -5), and only a small percentage of these females need to release their



young on day zero to produce an acceptable supply of test organisms.  Experience with




some cultured populations has shown that when  ovigerous females are isolated without




regard to developmental stage of the brood, a peak release of young will occur after about



5 days. Cultured populations are sieved through a 1.0mm mesh screen to isolate adults.




Ovigerous females are then selected by examining the adults in a culture dish containing




20%o seawater at 25°C. Approximately 1 gravid female should be isolated for each



juvenile needed for the toxicity test.  Presence of a brood can easily be detected with the




naked eye. The isolated females are transferred  to a holding container with culture




sediment and seawater 25°C  and 20%o salinity. The fe'males are fed in the  same manner




as amphipods in other culture containers.  After  3 days (day -2), the amphipods are sieved




from the culture sediment with a 1.0mm screen using water at the same temperature and




salinity,  the females are transferred to a large glass dish at a density of about 300



amphipods/dish with seawater at the test temperature and salinity, and algal food is




added as in culture containers; however, no sediment is added to the dish.  Females



placed in the dish should be carefully inspected to insure that no young have been

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                                                                                D-9



transferred with them. Juveniles released overnight (i.e., by day -1) are collected and set




aside in a culture tub containing culture sediment pre-sieved to 0.25mm; these juveniles




have not been used in the 28-cl test to, but might be available as a back-up if necessary or




to establish new cultures. Care must be taken to be sure that all juveniles released are




separated from the females. The females are returned to the sediment-free glass dishes




until the next day.  Juveniles released over the next 24 h (i.e., between day -1 and day 0)




are used to start the test; these juveniles will be <24 hours from brood release.  There




should be more young produced than gravid females initially isolated. If insufficient




numbers of newborns are produced for an experiment, it is advisable to delay starting the




exposure of some beakers for 1-d until more 
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                                                                               D-10
offspring (i.e., Ft generation) may be produced in each beaker. After 28 d, the contents of
each exposure chamber are sieved through 1.0mm and 0.25mm sieves to recover the
surviving adults (i.e., F0 generation) and their offspring (i.e., Fj generation), respectively.
Some adults may pass  through the 1.0mm screen (especially if the treatment retards
growth) and must be separated from the young. The surviving adults are counted, and
then measured, alive or preserved.  If preserved, it is recommended that the adults be
relaxed with magnesium chloride, CO2, or other means before preservation to minimize
curling of the dead animal; curling can stretch the animals and lead to larger apparent
size than for animals measured alive.  Body length, from the rostrum to the junction
between the abdomen and urosome, may be measured with an optical micrometer or
computer-assisted digital image analyzer. Each surviving adult should be sexed in order
to determine the number of females which is required for estimating fertility.  Males may
be identified by a notched palm on  the distal segment of the gnathopod or by the presence
of penile papillae ventrally on the abdomen (only visible in preserved animals). Females
are identified by the presence of eggs in the ovaries or brood pouch.  Females lack the
notch on the palm of the last segment of the gnathopod and by the presence of oostigites
(brood plates).

       Juveniles may be counted at the termination of the exposure, but it is more
convenient to stain, preserve, and count them at a later time. The material retained on
the 0.25mm screen is transferred to a sample jar, stained overnight  with a few ml of
concentrated Rose Bengal in seawater, and then preserved for several weeks in 70%
ethanol until resources permit  enumeration.  Care should be taken not to dilute the 70%

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                                                                                D-ll
 ethanol with seawater in the vials by pipetting off all of the stain solution prior to adding
 the alcohol.
Endpoints


       Mortality is measured as the percent of the F0 L/. plumulosus that were not
recovered alive at the end of 28 d. Most of the survivors of the F0 cohort are collected on
the 1.0mm screen on day 28, although some may pass through to be collected on the
0.25mm sieve if growth rate was severely reduced. Mortality should not exceed 10-20%,
as senescence for this species at 25°C is not  observed until after 6 wk of age (DeWitt et al.
in prep.).  Dead amphipods are not removed from  the exposure chambers on a daily basis,
but they are noted as on the daily observation record.

       Fertility is  measured as the number of female offspring produced per surviving
female in the exposure chamber.  The sex ratio of the offspring must be estimated as 1.0
(females to males) since it is not possible, at this time, to sex newborn L. plumulosus.
Thus, the number of female offspring is  0.5 x the number of juveniles collected on the
0.25mm screen.  The number of surviving females can be determined directly by sexing
each animal using the morphological characteristics described above.

       Size of surviving F0-generation L. plumulosus is measured as the length of the
body (in mm) from the tip of the rostrum to the base of the urosome (posterior end of the
third abdominal segment; a major point  of articulation between the abdomen and the

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                                                                               D-12



urosome). Rate of growth may be related to timing of reproduction and thus to population




parameters (Scott and Redmond 1989). All recovered individuals are measured and sexed.




In some treatments, there may not have been sufficient growth and development to allow




for production of young during the test.  In these cases, the state of sexual maturity of the




recovered adults may be important.
Acceptability of the Test








       There should be no more than 20% mean mortality among the performance-control




replicates during the first 28 days of the test. This criterion is only a suggestion as there




are insufficient data to establish definitive criteria for any of the responses.
Statistics



       The final statistical endpoint is the MATC (Maximum Acceptable Toxicant




Concentration) range for the most sensitive endpoint. The upper bound of the MATC




range is the lowest concentration that shows a statistically significant effect (=Lowest




Observed Effect Concentration = LOEC), and the lower boundary is the highest




concentration that shows no statistically significant effect (=Highest No Observed Effect




Concentration = NOEC). If an analysis of variance (or its nonparametric equivalent)




shows no significant results, then the NOEC is the highest concentration included in the




analysis.  (References: Gelber et al. (1985), Weber et al. (1988; appendices), Capizzi et  al.




(1985), Daniel (1978), Sokal and Rohlf (1981))

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                                                                                D-13



       If initial examination of the data indicates that there may be significant lethal or




sub-lethal effects within treatments, the following analyses are conducted:









       1) Tests (Shapiro-Wilks, Bartlett's) are conducted to establish if the assumptions of




       normality and homogeneity of variances are met.









       2) Arc sin square root transformation of the proportional mortality data is




       conducted to stabilize the variance and more closely approximate a normal




       distribution if the data are found to be non-normal or the variances non-




       homogeneous.  Square root or log transformation of the size or fertility data may




       be required for the same reasons.  Re-test for normality and homogeneity of




       variance.
      3) If parametric assumptions are met, an analysis of variance (ANOVA) is




      conducted, followed by Dunnett's procedure if the ANOVA shows a significant




      result.  If parametric assumptions are not met, Steel's Many-One Rank Test is




      used to compare treatments with the control. This test does require equal




      variances, but is fairly insensitive  to such deviations from homogeneity. Another




      non-parametric analysis that may  be conducted is the Kruskal-Wallis test which is




      analogous to ANOVA, and is used  to determine whether significant differences




      exist among treatments, followed by a non-parametric multiple-comparisons




      procedure, such as Dunn's test.  Remember to test treatment means against the




      experimental control (i.e., carrier control, reference sediment, site control, salinity




      or temperature control, etc.), not the performance control (i.e., culture sediment).

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                                                                              D-14
CULTURE METHOD SUMMARY
Physical requirements

      Tubs:
      Salinity:
      Temperature:
      Photoperiod:
      Lights:
      Sediment:
      Water:
      Water Change:
      Aeration:
      Density:
12" x 14" x 6" plastic dishpans
20°C
16hr light : 8hr dark (fluorescent)
Fluorescent, ceiling mounted; Natural, N. facing skylight
<1 cm layer; mud or muddy-sand sieved <0.5mm; 1-3% TOC
Seawater diluted with deionized water; 10-12 cm layer
Static-renewal: 50% vol. water change 3x/wk
Constant bubbling
300-400 adults/tub (« 0.3-0.4 adults/cm2)
Feeding
       3x/wk, at time of water change
             Algal mixture: -7-L per culture tub
                    Pseudoisochrysis paradoxa (chrysophyte)
                    Phaeodactylum tricornutum (diatom)
                    1:1 v/v mixture,  final cone. 106cells/ml, 20%o
             Gorp: dry food mixture; fine powder; 0.5 g per tub
                    48.5% TetraMin®
                    24% dried alfalfa
                    24% dried wheat leaves
                    4.5% Neo-Novum® (shrimp maturation feed; Argent Chem. Lab.)
Culture Renewal
       Start culture with ca. 100 adults and 200 juveniles
       Thin cultures every 6-8 wk
       Inspect culture sediment for worms, copepods; discard if present
       Replace sediment at least every 6 mo.
Special Considerations

       Non-indigenous Species Laws and Practices
             Chlorination or sterilization (autoclave) all materials used

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                                                                               D-15
Leptocheirus plumulosus Chronic Sediment Toxicity Test Design: SUMMARY

Exposure Conditions
       Chamber:
       Sediment Vol.:
       Overlying Water Vol.:
       Water Source:
       Salinity:
       Aeration:
       Temperature:
       Photoperiod:
       Duration:
       Life stage:
       No./replicate:
       Feeding:
       Performance Control:
       Ref. Tox. Control:
1-L glass beakers
175ml
725 ml
Seawater diluted with deionized water
20% or match test sediment IW salinity
Constant
25°C
16hr light : 8hr dark
28 days
<24-hr old newborn (F0-generation)
20
3x/wk, 400ml algae @ 106cells/ml; 10 mg gorp
28-d, culture sediment, 20%o, 25°C; <24-hr old
96-h, Cd in water; 20%o, 25°C; 1-wk old amphipods (F0)
Handling & Recovery

      Obtaining Test Animals
             Isolate newborn (F0) from gravid females in dishes w/out sediment
                   Isolate gravid females from cultures 5d before T0
             Maintain constant 25°C temperature while handling
      Seeding Exposure Chambers
             Transfer by pipette; newborns very fragile
             Double or triple count at T0
             Preserve >1 subsets of 20 newborns
                   Size at T0
      Recovery at Test Termination
             1.0mm sieve - F0-generation (adults)
                   Count, measure, sex survivors
             0.25mm sieve - regeneration (offspring)
                   Stain & preserve, count under magnification

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                                    APPENDIX E
           AMPELISCA ABDITA; GENERIC LIFE CYCLE TEST DESIGN
 Abstract









       A generic life cycle sediment toxicity test with the amphipod Ampelisca abdita is




 described.  This is a draft design which has not been fully tested, and the procedures




 outlined have not always produced high survival or reproduction under control conditions.




 This is a flow-through test conducted at 20 - 25°C, 30%o, and 16 hours light and 8 hours




 dark.  Test sediment is mud to sandy mud, and algal food is delivered daily with




 seawater.  The experiment begins with 10 juveniles, 8 to 10 days old, per replicate, 5




 replicates per treatment, and is terminated after 35 days when the contents of each



 container are sieved and examined. Endpoints are initial mortality,  time to first observed



juvenile tubes (brood release), mimber of surviving young produced per female, number




 and size of animals recovered, arid life stage of animals recovered.
Exposure Conditions
      Test treatments, typically 5 concentrations plus negative (uncontaminated




sediment) and positive (reference toxicant) controls, are based on acute toxicity test data.




Treatments can be "spiked" sediment, field sediment, or dilutions of field sediments. If
                                        E-l

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                                                                                 E-2




there are sufficient personnel available, the test can be set up "blind," i.e., so that




personnel monitoring the test have no knowledge of the treatments in the test chambers.









       The recommended temperature is 20°C, since it has been shown to be an




acceptable culture and testing temperature for this species. The life cycle is slightly




shorter at 25°C vs. 20°C, but it has not been definitively established that the higher




temperature is acceptable for A. abdita. This organism has been maintained and tested




at salinities within the range of 28 - 35%o.








       A light cycle of 16 hours light and 8 hours dark was selected to approximate




conditions existing during summer breeding.  This photoperiod has been shown to




consistently maintain reproductive activity in Hyalella azteca (de March 1977, cited in




Arthur 1980). Feeding (Mills 1967) appears not to be affected by the laboratory light cycle.








       In nature, this species is found in fine sand to silt without shell, is abundant where




the major proportion of sediment is >0.05 mm and <0.25mm, and may also be found in




coarse sediments with a considerable fine fraction (Mills 1967).  Test sediments should




approximate these characteristics, i.e., mud or sandy mud.
       The exposure container used is an aerated 900 ml glass "canning" jar, which was




selected as a smaller version of the gallon "pickle jar" used for maintaining these



amphipods in the laboratory and for previous chronic tests (Scott and Redmond 1989); it




is inexpensive and easily drilled. A 250 um screened overflow hole prevents escape of




juvenile amphipods during a flow-through test. This exposure container has a reasonably

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                                                                                  E-3



high water column, allowing the animals to swim during their mating activities, and




sloping "shoulders" to allow for efficient circulation of suspended food material. Mills




(1967) indicates that turbidity and currents are likely stimulants for feeding in A. abdita.




Sediment depth in the exposure container is 3.5-4 cm, which is the maximum tube length




of A. abdita.









       A flow-through system is used as  a closer approximation to a natural situation




than a static system. The number of volume replacements per day needed has not been




determined.  Algal food is delivered with the seawater so as to ensure an even distribution




to all exposure containers. The  amount added is  measured by cells/ml in the culture used,




and the delivery rate to each exposure container.
Exposure condition modifications which may affect the test results









1. Temperature.  If a temperature other than 20°C is used, the duration of the test will




change (e.g., at 25°C the duration would be <35 d) and the response of A. abdita to




chemicals might change.









2. Salinity. Choice of test salinity will generally depend on the seawater supply available




to a laboratory, and whether or not it is necessary to match the salinity to that of the test




sediments. It may be possible to acclimate A. abdita to lower salinities, since it is reported




in the literature down to 10%o (Bousfield 1973), but successful  acclimation to low salinities

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                                                                                 E-4



has not been demonstrated experimentally.  The response of the organism might vary




depending on salinity selected.








3. Particle size of test sediment. This species can be tested in coarser materials for acute




tests, but some preliminary information indicates that sandy sediment could result in




adverse effects when exposure is for longer periods of time (Redmond and Scott




unpublished). If sediment characteristics might affect the amphipod's response, a grain




size control should be included.








4. Type of exposure. This species has sometimes shown increased sensitivity to toxicants




in a static system compared to flow-through or static daily renewal (e.g., Word et al 1989).








5. Amount and type of food provided: The quality and quantity could affect growth rate,




fecundity, and exposure to bedded contaminants.
Biological Design








       A schedule for conducting a typical A. abdita life cycle sediment toxicity test is




shown in Table E-l. The test is initiated with juveniles which are 8 to 10 days old.




Newly-released juveniles are collected in containers with no sediment from ovigerous




females carrying broods in a late stage of development. Late-stage broods can easily be




identified under a dissecting scope.  If juveniles are harvested immediately after release,



their exact age and size are known, since newly-released juveniles are essentially uniform

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                                                                                  E-5
 in size at about 1.5 mm in length (Mills 1967, also this report). A large number of
 ovigerous females are isolated before the test is due to start. Only a small percentage of
 these females need to release their young overnight to produce an acceptable supply of
 test animals. Juveniles collected with this "no-sediment" procedure are transferred to
 aerated containers with a small amount of sediment and held, with feeding, for 8 to 10
 days.   With this procedure, questions of natural mortality (which has not been
 quantified) and initial mortality due to release of the young in no sediment are
 eliminated, but age of the organisms is still known. Also, older juveniles are easier to
 work with than newly-released individuals, e.g., daily mortalities  are more easily
 observed.  If it is not possible to obtain enough newly-produced young overnight, young
 may be collected  over a period of 1 to 3 days, with only a small variation in test size
 range.  In either  case, measurement of an initial sample of juveniles added to the test will
 confirm the size range.


       During the course of the test, juveniles added to experimental chambers grow,
 molt, reproduce, and  die (especially males, i.e., following reproduction). Exposure to
 chemical contaminants might alter these responses and their timing relative to the
 amphipods' life history patterns in the control beakers. Thus, exposure and control
 containers  must be checked daily.  Sex ratio in Ampelisca abdita populations is
 approximately 1:1 at breeding times (Mills 1967).  The test begins with 10 juvenile
 amphipods. If 50% of the original  amphipods are females, and each female produces  10 -
20 young, the approximate number of young produced in the F! generation in each jar will
be 50 - 100. After 35 days the contents of each container are sieved and the amphipods

-------
                                                                               E-6

preserved in 70% ethanol with 5% glycerin1 for later examination.  Scott and Redmond

(1989) started replicates at 20°C with ovigerous females and observed differences in the

abundance of F2 generation animals after 56 d.  This test design starts with week-old

juveniles, thus eliminating 2 wk of development in the brood pouch and 1 wk of growth,

and results in a test with a 5 wk duration.



Endp pints



       Several endpoints may be measured in this test (Table E-2). Initial mortality is

measured as the number of juveniles initially added minus the number of the initial

generation recovered. Mortality in the initial generation is expected, since males die after

mating and females die at some point after releasing a brood.  Dead amphipods are

removed every day and examined immediately to  determine sex and reproductive

condition. By removing dead individuals daily, the timing of their death can be precisely

determined, and important demographic data can be recorded before the bodies

decompose. These demographic data are necessary in order to estimate the sex ratio

within each beaker.  Also, mortality of prereproductive animals due to toxicants can be

separated from what may be natural or background mortality.  Dead females are

preserved in 70% alcohol and glycerin, and later are measured in order to relate size of
    J. Alcohol is less hazardous than formalin but may not be an adequate
 preservative for storage of specimens longer than several weeks; buffered formalin
 is better.  Glycerin is added to prevent specimens from becoming brittle.  Seawater
 transferred with the amphipods should be removed because it will dilute the
 alcohol and salt crystals may precipitate.

-------
                                                                                E-7



females to the number of young produced. Time to brood release is determined by the




first appearance of juvenile tubes in each exposure chamber.









       After final sieving and preservation, the young in each replicate are counted. The




number of young is divided by the number of females that have produced young




(determined by removal of dead animals as described above, and by examination of sieved




and recovered individuals), to produce a mean number of surviving young per female per




replicate.  The number of juvenile tubes observed in each exposure jar are also counted.




The utility of counting the number of tubes observed is that high mortality in the Fj




juveniles may be observed. For example, if 20 juvenile tubes are observed during the




course of the test, but at sieving only 5 young are recovered, then there has been an acute




effect on the juveniles in that container. The number that died cannot be quantified




accurately since the small tubes  are difficult to count and juveniles may construct >1




tube, but the presence of a large effect can be observed.









       Size of recovered individuals is determined as the length in  mm, from the base of




the first antenna to the base of the telson. All recovered individuals  are measured and




sexed.  In some treatments, there may not be sufficient growth and development to allow




for production of young during the test.  In these cases, the state of sexual maturity of the




recovered adults is particularly important. When ovigerous females are recovered, they




are preserved individually. Females often release  their eggs in preservative, and




individual preservation allows an accurate determination of the number of eggs carried by




a particular female.  The number of eggs can then be directly related to the length of that

-------
                                                                                 E-8
individual.  Growth rate is related to timing of reproduction and thus to population
parameters (Scott and Redmond 1989).
Acceptability of the Test

       There must be no more than 10% mean mortality in prereproductive control
individuals during the first 10 days of the test.  (There is insufficient data to  support
control mortality criteria for longer time periods.) Also, the controls should develop,
mature, and reproduce normally, which at this point in the test development  is a
qualitative judgement made by the researcher.
Statistics

       The final statistical endpoint is the MATC (Maximum Acceptable Toxicant
Concentration) range for the most sensitive endpoint. The upper bound of the MATC
range is the lowest concentration that shows a statistically significant effect (=Lowest
Observed Effect Concentration = LOEC), and the lower boundary is the highest
concentration that shows no statistically significant effect (=Highest No Observed Effect
Concentration = NOEC). If an analysis  of variance (or its nonparametric equivalent)
shows no significant results, then the NOEC is the highest concentration included in the
analysis. (References: Gelber et al. (1985), Weber et al. (1988; appendices), Capizzi et al.
(1985), Daniel (1978), Sokal and Rohlf (1981)).

-------
                                                                                 E-9
      If initial examination of the data indicates that there may be significant lethal or
sub-lethal effects within treatments, the following analyses are conducted:


      1) Tests (Shapiro-Wilks, Bartlett's) are conducted to establish if the assumptions of
      normality and homogeneity of variances are met.


      2) Arc sin square root transformation may be applied to the proportional mortality
      data.  This transformation may be required in some applications as a general
      practice, but in other cases, it might only be used (e.g., to stabilize variances and
      more closely approximate  a normal distribution) when the data are found to be
      non-normal or the variances non-homogeneous.  Square root or log transformation
      of the size or fertility data may be required for the same reasons. Re-test for
      normality and homogeneity of variance.

      3) If parametric assumptions are met, an analysis of variance (ANOVA) is
      conducted, followed by Dunnett's procedure if the ANOVA shows a significant
      result.  If parametric assumptions are not met, Steel's Many-One Rank Test is
      used to compare treatments with the control.  This test does require equal
      variances, but is fairly insensitive to such deviations from homogeneity. Another
      non-parametric analysis that may be conducted  is the Kruskal-Wallis test which is
      analogous to ANOVA, and is used to determine whether significant differences
      exist among treatments, followed by a non-parametric multiple-comparisons
      procedure, such as Dunn's test. Remember to test treatment means against the

-------
                                                                                E-10



      experimental control (i.e., carrier control, reference sediment, site control, salinity




      or temperature control, etc.), not the performance control (i.e., culture sediment).
Survival








       Initial evaluation of data involves determination of effects on survival. Survival in




each treatment can be graphically represented. Figure E-l depicts the survival curve of



A. abdita under control  conditions: low mortality over the first 2-wk (i.e., ca. <10% after




10-d), followed by increasing mortality due to the death of post-reproductive amphipods.




If there is significant mortality of individuals in some treatments which is not due to




natural (post reproductive) mortality, these treatments should be removed from further




analysis of "sublethal" effects.
Other parameters








       Time to first appearance of juvenile tubes in test-days, and length of young




produced are analyzed as described above.  The number of young produced is divided by




the number of females present which have released a brood (determined by the number of




amphipods initially added and the daily observations), and analyzed as above except that




an analysis of covariance is used, with female length the covariate.

-------
                                                                             E-ll
         SUN   MON
          TUES    WED   THURS
FRI
SAT
WEEK1
isolate    collect
ovigerous  juveniles
females
WEEK 2
                           sediments
                           into test
                           containers
amphipods
are 8-10
days old;
time-zero
Table E-l. A. abdita: example of organism collection schedule for life cycle test.

-------
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-------
           100-
            90-
            80-
            70-
            60-
  % alive   50-
            40-
            30-
            20-
            10-
             0-
                 ****
                                                                     E-13

***

                     5    10  15   20   25   30   35

                     time in days (0=test start date)
Figure E-l. Mortality of A. abdita under typical control-exposure conditions.

-------

-------
                                                                                 R-l
 REFERENCES
American Society for Testing and Materials. 1990a. Standard guide for conducting sediment




       toxicity tests with freshwater invertebrates.  E1383-90. In: Annual Book of ASTM




       Standards, Water and Environmental Technology. Vol  11.04. American Society for




       Testing and Materials, Philadelphia, PA.









American Society for Testing and Materials. 1990b. Standard guide for conducting solid-




       phase 10-day static sediment toxicity tests with marine and estuarine amphipods.




       E1367-90. In:  Annual  Book of ASTM  Standards,   Water and  Environmental




       Technology. Vol 11.04. American Society for Testing and Materials, Philadelphia, PA.









Arthur, J.W. 1980. Review of freshwater bioassay procedures for selected amphipods. p. 98-




       108 In:  Buikema, A.L. Jr. and J. Cairns Jr., eds. Aquatic Invertebrate Bioassays.




       ASTM STP 715, American Society for Testing and Materials, Philadelphia, PA.









Bellan-Santini, D. 1980. Relationship between populations of amphipods and pollution. Mar.




       Poll. Bull. 11: 224-227.









Boesch, D.F. 1973. Classification and community structure of macrobenthos in the Hampton




       Road area, Virginia. Mar. Biol. 21: 226-244.
Boesch, D.F. 1977. A new look at the distribution of benthos along the estuarine gradient. In:




       B.C. Coull (ed.), Ecology of Marine Benthos. Univ. of South Carolina Press, p. 245-266.

-------
                                                                                R-2
Boesch, D.F. and R.J. Diaz. 1974. New records of peracarid crustaceans from oligohaline
       waters of Chesapeake Bay. Chesapeake Sci. 15: 56-59.

Boesch, D.F., R.J. Diaz and R.W. Virnstein. 1976. Effects of tropical storm Agnes of soft-
       bottom macrobenthic communities of the James and York estuaries and the lower
       Chesapeake Bay. Chesapeake Sci. 17: 246-259.

Botton, M.L. 1979. Effects of sewage sludge on the benthic invertebrate community  of the
       inshore New York Bight. Estuar. Coastal Mar. Sci. 8: 169-180.

Bousfield,  E.L.  1970.  Adaptive radiation in sand-burrowing  amphipod crustaceans.
       Chesapeake Sci. 11: 143-154.

Bousfield, E.L. 1973. Shallow-water  Gammaridean Amphipoda of New England. Cornell
       University Press, Ithaca, NY.

Breteler, R.J., K.J. Scott, and S.P. Shepherd. 1989. Application of a new sediment toxicity
       test using marine amphipods, Ampelisca abdita, to San Francisco Bay sediments. In:
       U.M. Cowgill and L.R. Williams,  eds. Aquatic Toxicology and Hazard Assessment:
       Twelfth Volume,  ASTM  STP  1027.  American Society  for Testing and Materials,
       Philadelphia, PA.

Buchanan, J.B. 1984. Sediment analysis. In: N.A. Holmes and A.D. Mclntyre, eds. Methods
       for the Study of Marine Benthos. Blackwell Scientific Publications, London, pp. 41-64.

-------
                                                                                R-3



Capizzi,  T., L.  Oppenheimer,  H. Mehta,  H. Naimie,  and J.L.  Fair.  1985.  Statistical




      Considerations in the Evaluation of Chronic Aquatic Toxicity Studies. Environ. Sci.




      Technol. 19: 35-43.









Chasse, C. 1978. The ecological impact on and near shores by the Amoco Cadiz oil spill. Mar.




      Poll. Bull. 9: 298-301.









Croker, R.A. 1967a. Niche diversity in five sympatric species  of intertidal amphipods




      (Crustacea: Haustoriidae). Ecol. Monogr. 37: 173-200.









Croker, R.A.  1967b. Niche  specificity  of Neohaustorius schmitzi  and Haustorius  sp.




      (Crustacea: Amphipoda) in North Carolina. Ecology 48: 971-975.








Croker, R.A. 1968a. Return  of juveniles to marsupium in the amphipod Neohaustorius




      schmitzi Bousfield. Crustaceana 14: 215-216.









Croker, R.A. 1968b. Distribution and abundance of some intertidal sand beach amphipods




      accompanying the passage of two hurricanes. Chesapeake Sci. 9: 157-162.








Daniel,  W.W. 1978. Applied Nonparametric Statistics. Houghton Mifflin Co., Boston.









Dauer,  D.M., R.M. Ewing, and A.J.  Rodi, Jr. 1987. Macrobenthic distribution within the




       sediment  along an  estuarine  salinity  gradient.  Benthic studies  of the lower




       Chesapeake Bay. 8. Int.  Rev. Gesamt. Hydrobiol. 72: 529-538.

-------
                                                                                R-4



Dauer, D., R.M. Ewing, J.W. Sourbeer, W.T. Harlan and T.L. Stokes, Jr. 1982. Nocturnal




      movements of the macrobenthos of the Lafayette River, Virginia. Int. Revue Ges.




      Hydrobiol. 67: 761-775.








Dauer, D.M., T.L. Stokes, H.R. Barker, R.M. Ewing, and J.W. Sourbeer. 1984. Macrobenthic




      communities  of the lower Chesapeake Bay. 4. Bay-wide transects  and the inner




      continental shelf. Int. Rev. Gesamt. Hyrdrobiol. 69: 1-22.









Deaver, E., and P.C.  Adolphson. 1990. Evaluation of the amphipod Lepidactylus dytiscus as




      a sediment toxicity test organism. Poster presented at the 1990 Annual Meeting of the




      Society of Environmental Toxicology and Chemistry, Washington, DC.









DeWitt, T.H. 1987. Microhabitat selection and colonization rates of a benthic amphipod. Mar.




      Ecol. Prog. Ser. 36: 237-250.








DeWitt, T.H.,  R.C. Swartz and J.O. Lamberson. 1989.  Measuring the acute toxicity of




      estuarine sediments. Environ. Toxicol. Chem. 8: 1035-1048.









Dexter, D. 1967. Distribution and niche diversity of haustoriid amphipods in North Carolina.




      Chesapeake Sci. 8: 187-192.









Dexter,  D.M. 1969. Structure of an intertidal sandy-beach  community in North Carolina.




      Chesapeake Sci. 10: 93-98.

-------
                                                                                R-5



Dexter, D.M.  1971. Life history  of the  sand-beach amphipod Neohaustorius  schmitzi




       (Crustacea: Haustoriidae). Mar. Biol. 8: 232-237.









Di Toro, D.M., J.D. Mahoney, D.J. Hansen, K.J. Scott, M.B. Hinks, S.M. Mayr, and M.S.




       Redmond. 1990. Tqxicity of cadmium in sediments: the role of acid volatile sulfide.




       Environ. Toxicol. Chem. 9: 1487-1502.









Di Toro, D.M., C.S. Zarba, D.J. Hansen, W.J. Berry, R.C. Swartz, C.E. Cowan, S.P. Pavlou,




       H.E. Allen, N.A. Thomas, and P.R. Paquin. 1991. Technical basis for establishing




       sediment  quality criteria for  nonionic  organic  chemicals by  using equilibrium




       partitioning. Environ. Toxicol. Chem. 10: 1541-1583.









Diaz, R.J.  1989. Pollution and  tidal benthic communities of the James River estuary,




       Virginia. Hydrobiologia 180: 195-211.









Ditsworth, G.R., D.W. Schults, and J.K.P. Jones. 1990. Preparation of benthic substrates for




       sediment toxicity testing.  Environ. Toxicol. Chem. 9: 1523-1529.









Edmunds,  W.M.  and A.H. Bath.  1976. Centrifuge  extraction  and chemical analysis of




       interstitial waters. Environ. Sci. Technol. 10: 467-472.








Ewing, R.M. and D.M. Dauer. 1982. Macrobenthic communities of the lower Chesapeake Bay.




       I. Old Plantation  Creek, Kings Creek, Cherrystone Inlet and the adjacent offshore




       area. Int. Rev.  Gesamt. Hydrobiol. 67: 777-791.

-------
                                                                                R-6
Feeley, J.B.  and  M.L. Wass.  1971.  The  distribution and ecology of the gammaridea
       (Crustacea: Amphipoda) of the lower Chesapeake estuaries. Virginia Institute of
       Marine Science, Special Papers in Marine Science No. 2.

Pish, C.J. 1925. Seasonal distribution of the plankton of the Woods Hole region. Bull. U.S.
       Bureau Fish. 41: 91-179.

Fox, R.S. and K.H. Bynum. 1975. The amphipod crustaceans of North Carolina estuarine
       waters. Chesapeake Sci. 16: 223-237.

Prankenberg, D., S.L. Coles, and R.E. Johannes. 1967. The potential  trophic significance of
       Gallianassa major fecal pellets. Limnol. Oceanogr. 12: 113-120.

Gelber, R.D., P.T.Lavin, C.R. Mehta and D.A.Schoenfeld. 1985. Statistical analysis, p. 110 -
       123, In: Rand, G.M. and S.R. Petrocelli, eds., Fundamentals of Aquatic Toxicology.
       Hemisphere Publishing Corporation, Washington, D.C.

Gentile, J.H., K.J.  Scott, S.M. Lussier, and M.S. Redmond. 1985. Application of laboratory
       population responses for evaluating the effects of dredged material. Technical Report
       D-85-8, prepared by the U.S.  EPA, Narragansett, RI, for the U.S.  Army  Corps of
       Engineers Waterways Experiment Station, Vicksburg, MS.

-------
                                                                                R-7



Gentile, J.H., K.J. Scott, S.M. Lussier, and M.S. Redmond. 1987. The assessment of Black




       Rock Harbor dredged material impacts on laboratory population responses. Technical




       Report D-87-3, prepared by the U.S. Environmental Protection Agency, Narragansett,




       Rhode Island, for the U.S. Army Engineer Waterways Experiment Station, Vicksburg,




       Mississippi.









Grant, D.C. 1965. Specific diversity in the  infauna of an intertidal sand community. Ph.D.




       Thesis, Yale University, New Haven, CT.









Grant, J. and E.A. Lazo-Wasem. 1982. Systematics and ecology of the estuarine amphipod




       crustacean Lepidactylus dytiscus Say, 1818 (Haustoriidae). Can. J. Zool. 60: 2039-




       2045.









Hamilton, M.A., R.C. Russo and R.V. Thurston. 1977. Trimmed Spearman-Karber method for




       estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol.




       11: 714-719. Correction. Environ. Sci. Technol. 12: 417 (1978).









Hines, A.H. and K.L. Comtois. 1985. Vertical distribution  of infauna in sediments, of a




       subestuary of central Chesapeake Bay. Estuaries 8: 296-304.









Hines, A.H., P.J. Haddon, J.J. Miklas,  L.A. Wiechert and A.M, Haddon. 1986. Estuarine




       invertebrates and fish: sampling design and constraints for long-term measurement




       of population dynamics. In: T.P.  Boyle, ed., New Approaches to Monitoring Aquatic




       Ecosystems. STP  940, American Society for Testing  and Materials, pp.

-------
                                                                              R-8



Holland, A.F. 1985. Long-term,  variation of macrobenthos in a  mesohaline region  of




      Chesapeake Bay. Estuaries 8: 93-113.








Holland, A.F., A.T. Shaughnessy, and M.H. Hiegel. 1987. Long-term variation in mesohaline




      Chesapeake Bay macrobenthos: spatial and temporal patterns. Estuaries 10: 227-245.








Holland, A.F., A.T. Shaughnessy, L.C. Scott, V.A. Dickens, J.A.  Ranasinghe and J.K.




      Summers.  1988. Progress  report: Long-term benthic monitoring and  assessment



      program for the Maryland portion of Chesapeake Bay (July  1986 - Octoberl987).




      PPRP-LTB/EST-88-1. VERSAR, Inc., Columbia, MD.








Holland, A.F., N.K. Mountford, and J.A. Mihurshy. 1977. Temporal variation in upper bay



      mesohaline benthic communities. I. The 9-m mud habitat. Chesapeake Sci. 18: 370-




      378.








Holland, A.F., N.K. Mountford, M.H.  Hiegel, K.R. Kaumeyer  and J.A.  Mihursky.  1980.




      Influence of predation on infaunal abundance in upper Chesapeake Bay, USA. Mar.




      Biol. 57: 221-235.








Howard, J.D. and C.A. Elders. 1970. Burrowing patterns of haustoriid amphipods from




      Sapelo Island, Georgia. In: Trace Fossils. Geological Journal Special Issue 3, J.P.




      Grimes and J.C. Harper, eds. Seel House,  Liverpool, p.243-262.








Ivester, M.S. and B.C. Coull. 1975. Comparative study of ultrastructural morphology of some



      mouthparts of four haustoriid amphipods.  Can. J. Zool. 53: 408-417.

-------
                                                                                R-9



Jordan, R.A. and C.E. Sutton. 1984. Oligohaline benthic invertebrate communities at two




       Chesapeake Bay power plants. Estuaries 7: 192-212.









Knott, D.M., D.R. Calder,  and R.F. VanDolah. 1983. Macrobenthos of sandy beach and




       nearshore environments at Murrells Inlet, South Carolina, USA. Estuar. Coast. Shelf




       Sci. 16: 573-590.









Landrum, P.F., S.R. Nihart, B.J. Eadie and W.S. Gardner. 1984. Reverse-phase separation




       method for determining pollutant binding to Aldrich humic acid and dissolved organic




       carbon in natural waters. Environ. Sci. Technol. 18:  187-192.









Lee, H.E.,II, B.L. Boese, and R.C. Randall. 1990. A method for determining gut uptake




       efficiencies of hydrophobic pollutants in a deposit-feeding clam. Environ.  Toxicol.




       Chem. 9: 215-219.








Lippson, A.J. and R.L.  Lippson. 1984. Life in the Chesapeake Bay. The Johns Hopkins




       University Press, Baltimore, Maryland.  230pp.









Lippson, A.J., M.S. Haire, A.F.  Holland, F. Jacobs, J. Jensen, R.L. Moran-Johnson, T.T.




       Polger,  and  W.A. Richkus.  1979.  Environmental atlas of the Potomac estuary.




       Environmental Center, Martin Marietta Corporation, Baltimore, Maryland. Prepared




       for Maryland Department of Natural Resources, Power Plan Siting Program. 280 pp.









Loi, T.N., and B.J. Wilson.  1979. Macroinfaunal structure and effects of thermal discharges




       in a mesohaline habitat of Chesapeake Bay, near a nuclear plant. Mar. BioL 55: 3-16.

-------
                                                                               R-10



Lopez, G.R., and J.S. Levinton. 1987. Ecology of deposit-feeding animals in marine sediments.




      Quart. Rev. Biol. 62: 235-260.








Lopez, G.R., G. Taghon and, J.S. Levinton (Eds). 1989. Ecology of Marine Deposit Feeders.




      Lecture Notes on Coastal and Estuarine Studies. Springer-Verlag, New York.








Lynch, M.P. and W.Harrison. 1969. Sedimentation caused by a tube-building amphipod. J.




      Sed. Petrol. 40: 434-435.








Markle, D.F. and G.C. Grant. 1970. The summer food habits of young-of-the-year striped bass



      in three Virginia rivers. Chesapeake Sci. 11: 50-54.








Marsh, G.A. 1973. The Zostera epifaunal community in the York River, Virginia. Chesapeake




      Sci. 14: 87-97.








Marsh,  G.A.  1988.  Seasonal  dynamics of a mesohaline, soft-bottom, benthic community:



      secondary  production, sedimentation,  predation,  and  nutrition.  Ph.D.  Thesis,




      University of Maryland.  167 pp.








Maryland Department of the Environment, Ecological Assessment Division. 199 la. Biological




      assessment of marina-related impact in Chesapeake Bay: a pilot study. Final Report



      to U.S. Fish and Wildlife Service, Chesapeake Bay Estuary Program, Annapolis,




      Maryland.

-------
                                                                               R-ll



Maryland  Department of the  Environment,  Ecological Assessment  Division.  199 Ib.




       Assessment  of ambient toxicity of Chesapeake  Bay sediments. Final Report  to




       Maryland Department of Natural Resources, Annapolis, Maryland.









McGee, B., C. Schlekat and E. Reinharz. 1990. A method for testing toxicity of estuarine




       sediments using the amphipod Leptocheirus plumulosus. Poster presented at the 1990




       Annual  Meeting  of the  Society of Environmental  Toxicology and  Chemistry,




       Washington, DC.









Mills, E.L. 1964. Ampelisca abdita, a new amphipod crustacean from eastern North America.




       Can. J. Zool. 42: 559-575.









Mills, E.L.  1967. The biology of an ampeliscid amphipod crustacean sibling species pair. J.




       Fish. Res. Ed. Canada 24: 305-355.









Mountford, N.K., A.F. Holland, and J.A. Mihursky. 1977. Identification and description of




       macrobenthic communities in  the Calvert Cliffs  region of the Chesapeake Bay.




       Chesapeake Sci. 18:  360-369.









Mulkana, M.S.  1966. The growth and feeding habits of juvenile fishes in two Rhode Island




       estuaries. Gulf Res. Rept. 2: 97-163.








Nebeker, A.V., M.A. Cairns, J.H. Gakstatter,  K.W.  Mauleg, G.S.  Schuytema  and D.F.




       Krawczyk.  1984.  Biological  methods for determining toxicity  of  contaminated




       freshwater sediments to invertebrates. Environ. Toxicol. Ghent. 3: 617-630.

-------
                                                                               R-12



Nelson, W.G. 1980. The biology of eelgrass (Zostera marina L.) amphipods. Crustaceana 39:




       59-89.








Nelson, W.G. 1980. Reproductive patterns of gammaridean amphipods. Sarsia 65: 61-71.









Nichols, F.H. and J.K. Thompson. 1985. Persistence of an introduced mudflat community in




       South San Francisco Bay, California. Mar. Ecol. Prog. Ser. 24: 83-97.








Nichols, F.H. and J.K. Thompson. 1985. Persistence of an introduced mudflat community in




       South San Francisco Bay, California. Mar. Ecol. Prog. Ser. 24: 83-97.









Notrini, M. 1978. Long-term effects of an oil spill on Fucus macrofauna in a small Baltic bay.




       J. Fish. Res. Bd. Can.  35: 745-753.








Orth, R. J. 1973. Benthic infauna of eelgrass, Zostera marina, beds. Chesapeake Sci. 14: 258-




       269.








Ozretich, R. J. and W.P. Schroeder. 1986. Determination of selected neutral priority organic




       pollutants in marine sediment, tissue and reference materials utilizing bonded-phase




       sorbents. Anal. Chem.  58: 2041-2048.








Pfitzenmeyer, H. 1975. Benthos. In: T. Koo, ed., A Biological Study of Baltimore Harbor.




       Contribution No. 621.  Center for Environmental and Estuarine Studies, University




       of Maryland.

-------
                                                                               R-13
 Pinkney, A.E., S. Gowda and E. Rzemien. 1991. Sediment toxicity testing of the Baltimore
       Harbor and C &  D Canal  approach channels with the amphipod,  Leptocheirus
       plumulosus.   Final  report  to the Maryland Department of Natural  Resources.
       VERSAR, Inc., Columbia, Maryland.


 Ray, G.L. 1982. The ecology of benthic macro-invertebrates  in two New Jersey salt marsh
       waterways. Rutgers University, Ph.D. Thesis.


 Redmond, M.S., K.J. Scott, KM.  McKenna,  and D.L. Robson.  1991. ERL-N standard
       operating procedure for conducting acute toxicity tests using Ampelisca abdita.  In:
       Petrocelli, E.A., C.Mueller, W.R.Munns, Jr., D.J.Cobb, G.G.Pesch andR.K. Johnston,
       eds. Standard operating procedures for the conduct of marine environmental sampling
       and  analysis.  U.S.  Environmental Protection  Agency  Environmental Research
       Laboratory, Narragansett, RI. ERL-N contribution no. 1263.

 Redmond, M.S., K.M.  McKenna, E.A. Petrocelli, K.J. Scott, P.A. Crocker, and C.R. Demas.
       in preparation. Use of toxicity tests with the amphipod Ampelisca abdita to determine
       sediment toxicity in the lower Calcasieu River estuary, Louisiana.

Reinharz, E. 1981. Animal Sediment Relationships: A Case  Study of the Patapsco River.
       Open file No. 6, Maryland Geological Survey, Baltimore, Maryland.


Reinharz, E. and A. O'Connell. 1983. Animal-sediment relationships of the upper and central
       Chesapeake Bay. EPA-600/3-83-33.

-------
                                                                               R-14



Reish, D.J. 1987. The use of toxicity testing in marine environmental research. In:  (D.F.




       Soule and G.S. Kleppel, eds.) Marine Organisms as Indicators. Springer-Verlag, NY.




       pp. 231-245.








Reish, D.J. and J.A. LeMay. 1988. Bioassay Manual for Dredged Sediments, U.S. Army of




       Engineers, Los Angeles District, 180 p.








Rogerson,  P.F.,  S.C.  Schimmel, and  G. Hoffman.  1985.  Chemical and  biological




       characterization of Black Rock Harbor dredged material. Technical Report D-85-9,



       prepared by the U.S. EPA, Narragansett, RI, for the U.S. Army Corps of Engineers




       Waterways Experiment Station, Vicksburg, MS.








Sanders, H.L., J.F. Grassle, G.R. Hampson, L.S. Morse, S. Garner-Price and C.C. Jones.



       1980. Anatomy of an oil spill: long-term effects from the grounding of the barge



       Florida off West Falmouth, Massachusetts. J. Mar. Res. 38: 265-380.








Sanders, H.L.,  P.C. Mangelsdorf, Jr., and G.R.  Hampson.  1965. Salinity and faunal



       distribution in the Pocasset River, Massachusetts. Limnol. Oceanogr. lO(SuppL): R216-




       R229.








Santos, S.L. and J.L. Simon. 1980. Response of soft-bottom benthos to annual catastrophic




       disturbance in a south Florida estuary. Mar. Ecol. Prog. Ser. 3: 347-355.








SAS Institute Inc.  1985. SAS®  User's  Guide: Statistics. Version five edition. Gary, North




       Carolina.

-------
                                                                               R-15
 Schaffner, L.C., R.J." Diaz, C.R. Olseh, and I.L. Larsen. 1987. Fauna! characteristics and
       sediment accumulation processes in the James River estuary. Virginia. Estuar. Coast.
       Shelf Sci. 25: 211-226.


 Schlekat, C.E., B.L. McGee, andE. Reinharz. 1992. Testing sediment toxicity in Chesapeake
       Bay with the amphipod Leptocheirus plumulosus: an evaluation. Environ. Toxicol.
       Chem.  11: 225-236.

 Scott, K.J. and M.S. Redmond. 1989. The effects  of a contaminated dredged material on
       laboratory populations of the tubicolous amphipod Ampelisca abdita. In: (U.M. Cowgill
       and L.R. Williams, eds.) Aquatic Toxicology and Hazard Assessment, Twelfth Volume,
       ASTM STP 1027, American Society for Testing and Materials, Philadelphia, PA, pp.
       289-303.

 Scott, K.J., M.S. Redmond  and R.J.  Pruell.  in preparation. The acute response of the
       amphipod, Ampelisca abdita. to contaminated sediments from New Bedford Harbor,
       MA.

 Seng, L.T., L.Y.  Kwong, H.S. Chye, K.K. Huat, K.S.  Pheng, S. Hanapi, W.T. Meng, R.S.
       Legore, W. De Ligny,  and G.T. Tan. 1987. Effects of a crude oil terminal on tropical
      benthic communities in Brunei. Mar. Poll. Bull. 18: 31-35.

Sewall, J.E., T.H. DeWitt, and R.C. Swartz. 1991. Laboratory Culture of Chesapeake Bay
      Amphipods  for Chronic and Acute Sediment Quality Testing. Abstract of poster
      presented at SETAC annual meeting, Seattle, WA.

-------
                                                                              R-16



Shoemaker, C.R. 1932. A new amphipod of the genus Leptocheirus from Chesapeake Bay. J.




      Wash. Acad. Sci. 22: 548-551.








Sokal, R.R. and F.J Rohlf. 1981. Biometry. 2nd Ed. W.H. Freeman and Co., San Francisco.









Stickney,  A.P.  and L.D. Stringer. 1957. A study of  the invertebrate  bottom fauna of




      Greenwich Bay, Rhode Island. Ecology 38: 111-122.








Stickney,  R.R., G.L.  Taylor and D.B.  White.  1975. Food habits of five  species of young




      southeastern United States estuarine Sciaenidae. Chesapeake Sci.  16: 104-114.









Swartz, R.C., W.A. DeBen, J.K.P. Jones, J.O. Lamberson and F.A. Cole. 1985a. Phoxocephalid




      amphipod bioassay for marine sediment toxicity. In: (R.D. Caldwell, R. Purdy and R.C.




      Banner, eds.) Aquatic Toxicology and Hazard Assessment: Seventh Symposium, STP




      854, American Society for Testing and Materials, Philadelphia, PA, pp.284-307.









Swartz, R.C., W.A. DeBen, K.A. Sercu and J.O. Lamberson.  1982. Sediment toxicity and




      distribution of amphipods in Commencement Bay, Washington, USA. Mar. Poll. Bull.




      13: 359-364.




Swartz, R.C., D.W. Schultz, G.R. Ditsworth, W.A. DeBen, and F.A. Cole. 1985b. Sediment




      toxicity, contamination, and macrobenthic communities near a large sewage outfall.




      In: (T.P. Boyle, ed.) Validation and Predictability of Laboratory Methods for Assessing




      the Fate and  Effects of Contaminants in Aquatic Ecosystems.  STP 865, American




      Society for Testing and Materials, Philadelphia,  pp 152-175.

-------
                                                                               R-17
 U.S.EPA. 1983. Chesapeake Bay: a profile of environmental change. U.S. EPA Region 3,
       Chesapeake Bay Program.

 Van Dolah, R.F. and E. Bird. 1980. A comparison of reproductive patterns in epifaunal and
       infaunaLgammaridean amphipods. Estuar. Coast. Mar. Sci. 11: 593-604.

 Watling, L. and D. Maurer. 1972. Marine shallow water amphipods of the Delaware Bay
       area, USA. Crustaceana (Suppl.) 3: 251-266.


 Weber, C.I., W.B.Horning II, D.J. Klemm, T.W. Neiheisel, P.A.Lewis, E.L. Robinson, J.
       Menkedick and  F.KessIer. 1988. Short-term methods for  estimating the chronic
       toxicity of effluents and receiving waters to marine and estuarine organisms. EPA-
       600/4-87/028.


Wenner, C.A. and J.A. Musick. 1975. Pood habits and seasonal abundance of the American
       eel, Anguilla rostrata. from the lower Chesapeake Bay. Chesapeake Sci. 16:  62-66.

Whitely, G.C. 1948. The distribution of larger planktonic Crustacea on Georges Bank. Ecol.
       Monogr.  18: 235-264.


Williams, A.B. and K.H. Bynuna. 1972. A ten-year study of meroplankton in North Carolina
       estuaries: Amphipods. Chesapeake Sci. 13: 175-192.

-------
                                                                                 R-18



Word, J.Q., J.A. Ward, B.Brown, B.D.Walls and S. Lemlich. 1989. Relative sensitivity and




       cost of amphipod bioassays. In: Oceans '89, an international conference addressing




       methods for understanding the global ocean, Volume 2. Marine Technology Society,




       Ocean Engineering Society of the Institute of Electrical and Electronics Engineers,




       sponsors.








Yevich, P.P., C.A. Yevich, K.J. Scott, M.S. Redmond, D. Black, P. Schauer, and C.E. Pesch.




       1986. Histopathological effects of Black Rock Harbor  dredged material  on marine



       organisms: a laboratory investigation. Technical  Report D-86-1, prepared by  the




       U.S.EPA, Narragansett, RI, for the  U.S.  Army  Corps  of Engineers Waterways




       Experiment Station, Vicksburg, MS.
                                                         •U.S. Government Printing Office: 1993 — 720-118/80113

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