520-1-88-004
                                                                                    UCRL15988

                                                                                    DE89  006053
                                        The  Effects of Chronic  Radiation  on
                                             Reproductive  Success of the
                                    Polychaete  Worm Neanthes arenaceodentata
                                              Florence  L.  Harrison  and
                                                   Susan L,  Anderson
                                          This  paper was  prepared  for  the
                                            Office  of Radiation  Programs
                                      U.S.  Environmental  Protection Agency
                                                     December,  1988
  This is * preprint of a paper intended for publication in a journal or proceedings. Since
  changes may be made before publication, this preprint is made available with the
  understanding that it will not be cited or reproduced without the permission of the
  author.
                           DISCLAIMER


This report was prepared as n account of work sponsored by an agency of the United States
Government.  Neither the United States Government nor any agency thereof, nor any of their
employees, makes any warranty, express or implied, or assumes any legal liability or responsi-
bility for the accuracy, completeness, or usefulness of any information, apparatus, product, or
process disclosed, or represent* that its use would not infringe privately owned  rights. Refer-
ence herein to any specific commercial product, process, or service by trade name, trademark,
manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recom-
mendation, or favoring  by the United States Government or any agency thereof. The views
and opinions of  authors expressed herein do  not necessarily state or  reflect those of the

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                               DISCLAIMER

This document was prepared as an account of work sponsored by an agency of the
United Stales Government. Neither the United States Government nor the University
of California nor any of their employees, makes any warranty, express or implied, or
assumes any legal liability or responsibility for the accuracy, completeness, or useful-
ness of any information, apparatus, product, or process disclosed, or represents that
its use would not infringe privately  owned  rights. Reference herein to any  specific
commercial products, process, or service by trade name, trademark, manufacturer, or
otherwise, does not necessarily constitute or imply its endorsement, recommendation.
or favoring by the United States Government or the University of California. The
>iews and opinions of authors expressed herein do not necessarily  state or reflect
those of the United States Government or the  University of California..and shall not
be used for advertising or product endorsement purposes.

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THE EFFECTS OF CHRONIC RADIATION ON REPRODUCTIVE SUCCESS

    OF THE POLYCHAETE NORM NEANTHES ARENACEOOENTATA
       Florence L. Harrison and Susan L.  Anderson

            Environmental Sciences Division
         Lawrence Livermore National  Laboratory
                  Livermore, CA  94550
                    Project Officer

                   Marilyn E. Varela
                Report prepared  for the
              Office of Radiation Programs
          U.S. Environmental Protection Agency
                 Washington, DC   20460

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                                   FORENORD

     In response to the mandate of Public Law  92-532,  The Marine Protection,
Research, and  Sanctuaries  Act of  1972,  as  amended,   the  Environmental
Protection Agency (EPA) has developed  a program to promulgate  regulations  and
criteria to control  the  ocean  disposal  of low-level  radioactive wastes.  The
EPA seeks to understand  the mechanisms  for  biological response  of marine
organisms to the  low levels of radioactivity  that may arise from the release
of these wastes  as  a result of ocean disposal  practices.   Such information
will  play  an  important  role  in determining  the  adequacy of environmental
assessments provided to  the   EPA  in   support  rf  any  disposal  permit
applications.   Although  the EPA requires packaging of  low-level  radioactive
wastes to prevent release during radiodecay  of the materials,  some  release  of
radioactive material  into  the  deep-sea  environment  may occur  if a package
deteriorates.   Therefore, methods for  evaluating the  impact on  biota are being
evaluated.

     Mortality and  phenotypic  responses  are  not anticipated at  the expected
low  environmental   levels  that might  occur  if radioactive materials  were
released from the  low-level waste  packages.   Therefore, traditional bioassay
systems  are unsuitable  for assessing  sublethal effects on biota in  the marine
environment.  The EPA Office of Radiation Programs has  had  an  ongoing  program
to examine  sublethal  responses at the  cellular  level,  using  cytogenetic
endpoints.

     The  present  study  examines  the  effects  of  chronic  radiation  on  the
reproductive success  of  the marine polychaete, Neanthes  arenaceodentata,   a
low-fecund  invertebrate species.  Data  were  generated through the second
filial generation on brood  size, abnormal "development,  and  numbers  of  embryos
living,  dying, and dead  following lifetime exposure to radiation.

     The results of  this research may be  useful  in evaluating ocean disposal
of other materials  because many other pollutants are also mutagenic.  Cellular
level  endpoints  and those indicative  of reproductive  success,  and  therefore
predictive  of  population-level impacts,  could  ultimately be used to compare
the risks of several pollutant  classes.

     The Agency  invites  all readers of this  report  to send any comments  or
suggestions to David E.  Janes,  Director, Analysis and Support Division,  Office
of  Radiation  Programs   (ANR-461),  U.S.  Environmental  Protection  Agency,
Washington, DC  20460.
                               Richard J. Guimond, Director
                          Office of Radiation Programs (ANR-458)
                                  n  /Hi

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                               TABLE  OF CONTENTS

Foreword	Hi

List of Figures	v

List of Tables	vi

Abstract	I

).   Introduction	2

2.   Materials and Methods...	3
     2.1  Experimental Approach	3
     2.2  Animal Sources, Culture Conditions, and Irradiation./	3
     2.3  Brood Analysis	7

3.   Results	11
     3.1  Total Doses Received	.11
     3.2  PI  Hatch Size	M
     3.3  F]  Brood Size	12
     3.4  Living Embryos in Fj Broods	14
     3.5  Abnormal Embryos in F] Broods	17
     3.6  Reduced Survival of f] Embryos	17

4.   Discussion	20

Acknowl edgments	32

References	33

Appendix: Data Base from the Experiment to Determine the
            Effects of Chronic Radiation on Reproductive
            Success of Neanthes arenaceodentata	,	A-l
                                       IV

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                                LIST OF  FIGURES

1.    Summary of the life-history stages  and  of the steps  in the
     procedure followed to determine  the effects  of chronic
     radiation on reproductive success of Neanthes arenaceodentata.

2.    Schematic diagram of the radiation-exposure  facility.
     The 60rjQ source and the control  zone were shielded heavily
     wi th 1ead	

3.    Embryo abnormalities identified  in  sacrificed broods.
     Normal  cleavage pattern (a), atypical  cleavage pattern (b),
     and embryos with void regions (c>  are shown	
4.   Broods subjected to a trypan-blue-exclusion test were
     differentiated into embryos that were (a)  alive
     (free of blue color), (b) dying (partially stained blue),
     and (c) dead (totally stained blue)	10

5.   The percent of broods from the FI  mated pairs in each
     of four categories (n > 751, 75% > n _> 501, 501 > n > 25%,
     and n < 25%) of percent living embryos in  the brood	16

6.   The percent of broods from F^ mated pairs  in each
     of four categories (n > 150, 150 > n _> 100, 100 > n _> 50,
     and n < 50) of numbers of abnormal embryos in the brood	19

7.   The percent of broods in each of four categories
     (n > 75%, 75% > n > 50%, 50% > n > 25%, and n < 25%)
     of percent abnormal embryos i n the brood	21

8.   The percent of broods from the F-j  mated pairs in each
     of four categories (n > 150, 150 > n _> 100, 100 > n > 50,
     and n < 50) of actual and estimated numbers of hatchlings	23

9.   The percent of broods in each of four categories
     (n > 75%, 75% > n > 50%, 50% > n > 25%, and n < 25%)
     of percent survival to hatching of the embryos
     i n the brood	25

10.  Mean percent survival of embryos (expressed as percentage of
     the survival fraction of the controls) as a function of  chronic
     dose.  Data from broods that hatched or that were
     harvested before day 3 were excluded	26

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                                LIST OF TABLES

1.    Steps In the procedure used to harvest the broods  from
     the F]  mated pairs.   The harvest was  performed about 4 to 6 d
     after spawning	8

2.    Mean radiation dose  received by worms in each radiation-exposure
     group.   The mean duration of the exposure is in parentheses	12

3.    Number of embryos from parental (P^)  and first filial  (Fj)
     generations in control and radiation-exposed groups.  The brood
     from the F] generation was sacrificed before hatching occurred	13

4.    Number of embryos in broods from the  control and radiation-exposed
     F]  mated pairs.  The broods were sacrificed before hatching
     occurred and were assigned to one of  four categories
     (n  > 150, 150 > n _>  100, 100 > n >_ 50, and n < 50),  according
     to  the number of embryos i n the brood	14

5.    Results from the trypan-blue-exclusion test of the living,
     dying,   and  dead  ?2  embryos  in the  broods  from the  control  and
     radiation-exposed f] mated pairs.  The broods were sacrificed
     before hatching occurred and were assigned to one of four
     percentage categories (n _> 75%, 75% > n >_ 50%, 501 > n > 25%,
     and n < 25%), according to the percent of living embryos
     i n  the brood	15

6.    Results from the analysis of the normal and abnormal
     embryos in the broods from the control and radiation-
     exposed FT mated pairs.  The broods were sacrificed
     before hatching occurred and were assigned to one of four
     categories (n ^ 150, 150 > n ^ 100, 100 > n .> 50, and n < 50),
     according to the number of abnormal embryos in the brood	18

7.    Results from the analysis of normal and abnormal embryos
     in  the broods from the control and radiation-exposed mated
     pairs.  The broods were sacrificed before hatching occurred,
     the number of normal and abnormal embryos determined, the
     percent of abnormal  embryos calculated, and then the broods
     were assigned to one of four categories (n ^ 75%,
     75% > n > 50%, 50% > n  > 25%, n < 25%), according to
     the percentage of abnormal embryos i n the brood	20

8.    Results from the analysis of the numbers of F2 embryos that
     actually hatched or were estimated to hatch from  the broods
     of the control and radiation-exposed f] mated pairs.  The
     broods were assigned to one of four categories  (n > 150,
     150 > n _>  100, 100 > ri  > 50, and n < 50), according to the
     actual or estimated hatch size	22
                                       VI

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9.   Results of the analysis of survival  to hatching of embryos in
     the broods of the control and radiation-exposed F] mated pairs.
     The percent survival was calculated  by dividing the estimated
     hatch size by the brood size, and then the broods were
     assigned to one of four categories (n :> 75%, 75% > n _> 50%,
     50% > n > 25%, and n < 25%). according to the percent of
     survival of the embryos	24

10.  Comparison of the effects of acute and chronic irradiation
     on Neanthes arenaceodentata.  The values are percents of the
     broods in the category indicated	29

11.  Comparison of the effects on reproductive success of exposure
     of Neanthes arenaceodentata to di fferent doses of contaminants	31


A-l. Experimental data from Neanthes arenaceodentata f] mated pairs
     that were not irradiated with an external gamma-radiation
     source (controls).  The number of days from spawn to hatch
     and from hatch to spawn as well as the estimated hatch size
     are provided	A-2

A-2. Experimental data from Neanthes arenaceodentata f-\ mated pairs
     that ware exposed to 0.19 mGy/h from an external gamma-radiation
     source.  The number of days from spawn to hatch and from hatch
     to spawn as well as the estimated hatch size are provided	A-6

A-3. Experimental data from Neanthes arenac&odentata F] mated pairs
     that were exposed to 2.1 snGy/h from an external gamma-radiation
     source.  The number of days from spawn to hatch and from hatch to
     spawn as well as the estimated hatch size are provided	A-9

A-4. Experimental data from Neanthes arenaceodentata f\ mated pairs
     that were exposed to 17 mGy/h from an external gamma-radiation
     source.  The number of days from spawn to hatch and from hatch
     to spawn as well as the estimated hatch size are provided	..A-12
                                      vn

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                                   ABSTRACT

     The effects of lifetime exposure to  chronic  i./adiation on reproductive
success  were  assessed for  laboratory populations of  the polychaete  worm
Neanthes arenaceodentata.   Lifetime exposure was  initiated  upon the spawning
of the  PI female and was terminated upon  spawning of the F]  female.  Groups of
experimental worms received either  no radiation (controls)  or  0.19, 2.1,  or
17 mGy/h.  The  total dose  received  by the  worms  was either background or
approximately 0.55, 6.5, or 54 Gy, respectively.  The broods from the F] mated
pairs were  sacrificed before  hatching occurred, and information was obtained
on brood size, on the number of normal and abnormal  embryos,  and on the number
of embryos that were living, dying,  and dead.

     The mean number of  embryos in the broods from the  FI  females  exposed to
lifetime radiation of 0.19 and 2.1 mGy/h was not  significantly  different  from
the mean  number of embryos  from control  females; however, the  mean number of
embryos was different from those FI  females exposed  to  17 mGy/h.   There was  a
significant reduction in the number of live embryos  in the broods  from  the F]
mated pairs that were exposed  to  the  lowest dose rate given, 0.19  mGy/h,  as
well as  those  exposed to  2.1  and 17  mGy/h.   Also,  increased percentages  of
abnormal  embryos were determined  in the  broods of all  the radiation-exposed
groups.

     Our  results  on embryo  abnormalities  and  mortalities  indicate  that
dominant-lethal mutations,  and  possibly  recessive-lethal  mutations, were  most
likely  induced  in  the germ cells and that  these mutations  had an adverse
effect  on  reproductive success  by affecting  the survival  ef  early  life
stages.   Except for  those  mated pairs  exposed  to 17  mGy/h, there was no
evidence  of gamete killing,  nor was  there  evidence  of  reduced  fertilization
success  because the number of developing  embryos  in the  broods  did  not
decrease  with   increased dose.   From  our  data  on estimated hatch size and
actual  hatch size,  we concluded that  doses as  low as  0.19 mGy/h can  reduce
significantly the  size of hatches when lifetime doses are given.

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1.   INTRODUCTION

     One of the problems facing managers  and scientists concerned with  the
impact of contaminants on aquatic  environments is assessment of the effects of
chronic exposure to sublethal  levels  of  potentially  toxic  materials.  One
special concern is the  response of  aquatic  organisms  to long-term  exposure to
direct- and indirect-acting  mutagens;  exposure  to  mutagens can result  in
alterations in genetic  material  in both  somatic and germ  cells  (UNSCEAR,
1986).  Important detrimental effects of  mutagens  in  somatic cells  are  the
induction of tumors and  cancer.   Important  detrimental  effects on germ cells
are the induction of dominant- and  recessive-lethal mutations,  cell  killing,
and  the  development  of  abnormalities  in  early life-history stages,  all of
which are factors that affect reproductive  success.   Because preservation  of
the  health  of aquatic  environments requires  insuring  the  maintenance of
indigenous populations as well as  the  survival of individuals, managers  of
aquatic  resources  are  concerned  about  the  impacts  of contaminants  on
reproductive success.

     A direct-acting mutagen  for which  there is  considerable data  is ionizing
radiation (NRC 1980; UNSCEAR  1977,  1982,  1986).   Ionizing   radiation  is  a
genotoxic agent  for  which the  dose to aquatic  animals can  be determined
accurately  without  parallel   studies  on  chemical  metabolism.   Ionizing
radiation is an  ideal  model  mutagen because  the nature of  the damage and  the
processes that modify the lesions  are  well characterized.  Data on  the effects
of radiation on  aquatic  organisms  have been  reviewed extensively  (Polikarpov
1966;  Templeton  et  al.  1971;  Templeton  1976; Chipman  1972;  Ophel   1976;
Blaylock and Trabalka  1978;  Egami  and  Ijiri 1979;  Woodhead  1984;  Anderson  and
Harrison 1986).  However, the great preponderance of the data is on  acute
rather than chronic effects.

     The extensive data on the effects of acute  radiation on mortality rates
in aquatic  animals appear to  indicate that  the radiosensitivity increases  with
biological  complexity,  i.e.,  that  the  higher the phylogenetic position,  the
lower  the  LDso (Templeton  1976;  Blaylock and  Trabalka  1978;  Noodhead 1984).
However, the  limited  data  on effects  of acute radiation at  the cellular level
indicate that  this  conclusion may not be valid.  Induction  of  chromosomal
aberrations  and  sister  chromatid  exchanges  by  acute  radiation  in  the
polychaete  Neanthes arenaceodentata occurred at doses  that did  not differ
greatly  from doses  inducing  such  responses  in some mammals (Harrison et al.
1986; Anderson et al. 1987).   Furthermore,  some  fishes  and   invertebrates  are
as sensitive  to  radiation as  some mammals (Rackham and  Woodhead 1984;  Harrison
and  Anderson   1988;  UNSCEAR  1986),  although  the data  on  the effects  of
radiation  on  reproductive  success  indicate  that  there is  considerable
variation among species  (see  reviews of Woodhead 1984;  Anderson and  Harrison
1986).

      The  impact  of radiation  on  the  reproductive  success   of  an aquatic
organism may  be  related not  only  to the sensitivity of  its  gametes but  also to
its  reproductive strategy.  In a highly fecund species,  the survival of early
life stages may  be  less than  171,  and the  loss of abnormal enbryos  induced  from
radiation  exposure  may  be   masked completely   by   those   lost   from
density-dependent factors,  such as  food  limitation  and  predation.   It might be
expected that  the impact of radiation exposure to  a species of low  fecundity
may  be considerable because  recruitment is  more  closely  related to parent
stock  size.  The limited data available on  the use  of  sealed  sources  for  the

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chronic exposure of fish are not sufficient to allow conclusions to  be  drawn.
Noodhead (1977) found reduced fecundity in the guppy (a low-fecundity spe'cies;
from a  lifetime exposure  to about 1.7 mCy/h,  while Welander et al.  (1948)
noted some long-term deleterious  effects  in  salmon (a high-fecundity species)
at about 2.1  mGy/h.

     The objective of this  study was to obtain information on the effects of
chronic radiation  on the  reproductive success of a  relatively  low-fecundity
invertebrate  marine  animal.    The   species  selected  was  Neanthes
arenaceodentata, which  is a polychaete worm that is available commercially,  is
easily maintained  in the  laboratory,  and for which  considerable information  is
available on effects from acute radiation  (Harrison  et  al.  1986;  Anderson et
al.  "987;  Harrison and  Anderson  '988) and from  toxic inorganic and organic
contaminants (Rossi and Anderson  .978;  Oshida et al. 1981; Oshida  and  Ward,
1982).  The data  obtained from this  study on  effects  of  chronic  exposure to
the direct-acting  mutagen,  radiation, should be useful in  evaluating ocean
disposal of radioactive materials as  well  as other  mutagens.  Also,  comparison
of data for worms exposed chronically to data  for  worms exposed acutely  will
provide information on  the  importance of total dose and dose rate on response
to radiation.

2.  MATERIALS AND METHODS

2.1  Experimental Approach

     The effect of chronic  lifetime  radiation on  the reproductive  success of
N.  arenaceodentata was  determined  by making  observations  on  control  and
radiation-exposed  worms.   Data were  obtained on  the  parental  (Pi), first
filial  (FI),  and  second  filial  (p) generations.   Lifetime exposure  to
radiation  was  initiated  upon the spawning of  the  PI female.   At that  time,
these embryos,  which were being  cared for  by the male,  were placed  in front  of
a radiation source.  The  lifetime exposure was terminated upon the  spawning  of
the FI  female  (Fig.  1).   The number  of gravid  females  (Pi)  used as  sources  for
embryos for the control  group was 6,  for  those receiving 0.19 and  2.1  mGy/h
was 7, and for  those  receiving 17 mGy/h was  3.   The total  number of broods
analyzed for the  control  group was 94, for the group receiving 0.19 mGy/h was
84, for the group  receiving 2.1 mGy/h was  80,  and  for the group receiving  17
mGy/h  was  59.    Numbers  of  offspring of  the  PI  and FI  generations were
determined as  well as  the  times  of  spawning,  hatching, and  exiting of  larvae
from the parental  tube.   In addition, for both control and  radiation-exposed
FI mated pairs, the embryos  in the  brood  were examined for abnormalities and
subjected  to a  dye-exclusion test to determine the number  that were living,
dying,  and dead.   Data accumulated on  the brood  from each F^ mated pair are
provided in the Appendix.

AnimalSources. Culture Conditions, and Irradiation

     Worms  used in the experiment were obtained  either from Dr.  Donald Reish
(California State  University, Long  Beach,  CA)  or from  Brezina  and  As  ociates
(Dillon Beach,  CA).   After the adult worms  were  received from the suppliers,
they were  held  in  80-L aquaria for several weeks.   Once the female  worms began
to  develop oocytes,  they were removed  from  the aquaria,  mated with vigorous
males from the  same supplier, and cultured  according to  procedures by  Reish
(1974).  Oocytes  in the  coelom of  N. arenaceodentata are clearly discernable
b'ecause the  cuticle is translucent.   Each mated  pair (P]) was placed  in a

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IRRADIATION
                   ADULT  MATED  PAIRS
(spawning)
               EMBRYOS AND BROODING MALE (F|)
                         I
                      (hatching)
                      LARVAE (18 to 21 seg.)(F2)
                     (leave tube)
                      (growth)


                     JUVENILES
                      (growth)

                   YOUNG  ADULTS (> 64 seg.)


                    MATED PAIRS

                      (spawning)


                      EMBRYOS (Fa)

                         I
                   SACRIFICE BROOD
                              TIME IN DAYS
                                                       9 to 15
                                   I
                                                       7 to12
                                                      40 to  50
                                60  to 80
                                 4 to 6
Figure  1.  Summary of the life-history stages  and of the steps  in  the
procedure followed to determine  the effects  of  radiation on reproductive
success of Neanthes arenaceodentata.

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large plastic  petri  dish  (120-mm  diameter x 20-mm depth)  containing  about
80 mL of filtered (1.0-nm pore size) seawater; tube formation occurred  within
the next 24  h.   Seawater used in the experiments was pumped from the Pacific
Ocean and passed through sand filters at the  University  of California Bodega
Marine Laboratory before it was transported to the Lawrence Livermore National
Laboratory; the seawater was stored before  use in an  underground540,000-L tank.

     During the  acclimation  period,  observations of  the  mated  pairs  were made
::'"  ieekly.   At these times,  most of the seawater in  the  dishes  was
decanted,  the  tubes were carefully trimmed,  excess mucus  and  fecal  material
were removed by wiping out  the  dish  except in the tube  area,  newly  filtered
seawater was  added,  and fresh  food was supplied (rehydrated  frteze-dried
Enteromorpha sp.).  When the  female stopped  eating,  which  occurred  when her
coelom was  filled with  oocytes, the mated  pair was transferred to the control
area of the radiation facility and was observed daily to determine the  day of
spawning.

     Irradiation  of  the embryos  was  initiated  immediately  after spawning
occurred.   The date  of  spawning  was recorded,  the  female, who dies after
spawning, was removed from the petri dish  (if she had not  been  eaten  by the
male), and  the petri  dish  containing the  brooding male  and the embryos  was
placed randomly  in standard commercial  petri-dish racks that  held  18 petri
dishes (two  stacks each of 9 oetri dishes).   The radiation delivered was from
a 60Co source (about 2.5 x 1010 Bq; 0.7 Ci).   The racks were located in one of
following  four areas  in the radiation facility: behind the radiation source in
a  lead-shielded  site (control  area)  or at one  of three sites increasingly
distant  from  the radiation source (irradiation  areas) (Fig. 2).   The  three
distances  from the  source were chosen  in  advance so that  the  worms  in the
petri dishes would be dosed  at  a  rate of  either  approximately  0.21,  2.1, or
21 mGy/h (about  0.5,  5.0,  or 50 rad/d).  However, actual dose rates delivered
were 0.19  + 0.03, 2.1  0.4, and 17+1.1 mGy/h.  Because the area in front of
the  source from which a dose rate of 17 mGy/h could be delivered was limited,
the number of broods exposed at this dose  rate  was smaller than those at  the
two  lower  dose rates.  The temperature in the exposure facility was 20 + 2C,
and  the  light  level  was low during the day,  except  during the maintenance
periods.

     Doses  delivered to the worms were monitored using  thermoluminescent
dosimeters.  These were  sealed  in plastic  and placed in  the  seawater in the
petri dishes  at positions  similar to those  occupied  by  the worms.  Sets  of
dosimeters were  used at  each of the three  distances  from the source and  were
added  at different  times  during  the  experiment.  From the knowledge of the
radiation  exposure obtained  from  the dosimeters, of  the  number of days  each
worm was  exposed to  the source, and of  the total time the  radiation  source was
down during  maintenance and feeding of  the  worms,  the  total  lifetime  dose
received by each  worm was  calculated.

     The broods  were observed twice weekly, and  care was taken  to  minimize any
disturbance  of the  brood;  the  seawater  was  not changed  unless  it appeared to
be becoming  stagnant.   The amount of food  given  was  reduced and was placed at
the  opening  of the  tube.   The  date  of  hatching of the larvae,  which occurred
generally  between 12  and 15 d after the spawning, was  noted as  well  as the
date  that  the larvae left  the  tube,  which occurred  between 7  and 12 d  after
the  time of  hatching.

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                                                      Control  Area
                                                       17  mGy/h
                                                    Radiation  Source
                                                        2.1 mGy/h
                                                       0.19  mGy/h
Figure  2.   Schematic diagram of the radiation-exposure  facility.   The 60Co
source and the control zone were shielded heavily with lead.

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     When most of the larvae In the brood  left the tube (larvae had  from_40 i"
70 segments),  the  larvae were  removed  from  the large petri dish and  three
larvae,  each from the  same  brood,  were  placed  in a  small  petri  dish (60-mm
diameter x 20-mm depth)  containing 10 to 15  ml of  filtered seawater.   Each
small petri  dish, containing three  larvae,  was placed in  a standard  commercial
petri-dish rack  in the same experimental  area as the large one from which  the
larvae were obtained.   Each  rack  held 64 petri  dishes  (8 stacks  each   of  8
petri dishes).  Seawater  in  the small petri   dishes was changed twice weekly,
and the juveniles were fed dry, ground  alfalfa  that  was  less  than  0.5  mm  in
diameter.  The juveniles  were  fed  immediately after the water  exchange or more
frequently  if they  required  additional  food.    Considerable  cannibalism
occurred among  the  three juveniles  in  the  same petri dish.   In  most  petri
dishes,  only one worm survived to the juvenile stage.

     When most of the  juveniles had grown into young  adults,  their sex was
determined and the females  paired  to  vigorous males  from  the  same  brood,  if
sufficient males were  available.   If  sufficient males were  not available, they
were paired with other males from the  same  dose-rate-exposure group.   Next,
the  mated  pair (first  filial  generation,  F])  was  transferred to  a large
(120-mm diameter x 20-mm depth) petri dish.   The petri  dish with  the  mated
pair was placed in a  petri-dish rack at the  same distance from the  radiation
source as that  of  the juveniles and  parent  worms  (P]) from which  they were
derived.  To  reduce  differences in dosimetry,  the petri  dish  containing  the
mated pair was always rotated  so that their  tube was always at the front  of
the rack (closest to  the radiation source).

     Again,  the mated pairs (Fp were observed and cared  for as described  for
their parents  (P]).   The date  of  spawning of the F|  female was  noted,  the
brood was removed from in front of the source and placed  in  the control area,
and  then  the  brood was sacrificed  about 4 to 6  d after the spawning date.  The
brood was sacrificed  at this time because the nurturing male consumes the dead
embryos  as  part of  taking  care of the brood.   Therefore, to obtain  an
indication of total  number of embryos in the brood,  the brood was  sacrificed
before the male  had  time to consume a significant number of dead  embryos.   In
those cases when  large numbers of embryos died early  in development (before
about 6 d),  the  gut  of the male was yellow  from yolk.  When this  occurred, it
was recorded so that an indication could be obtained of those broods where the
number spawned  was greater than the  number  that was recorded  present at the
time the brood was sacrificed.  The total duration of the experiment was about
8 months.

2.3  Brood Analysis

     The analysis of the brood consisted of (1) enumeration and  examination
of the embryos and (2)  a  trypan-blue-exclusion  test  (Table 1).   The analysis
of the  brood  was performed  by  one or two persons.   For  the first part  of  the
analysis,  the  embryos  were  removed   from  the  tube  and  transferred
quantitatively from their petri dish  to a counting chamber, which was a petri
dish  bottom (60-mm  diameter x 20-mm depth) that had  been divided into
quadrants.  The  counting chamber containing  the  embryos  was  placed on graph
paper, and  then  the  total number  of  embryos in the  spawn  was determined  by
systematically  counting the  embryos  in each quadrant; 6X  magnification was
used.  Next,  the number of abnormal and normal  embryos was  evaluated at 12X

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Table 1.   Steps in the procedure used to harvest the broods from  the  F]  matec
pairs.  The harvest was performed 4 to 6 d after spawning.


Part I.  Enumeration and Examination.
                                                                x

     1.  Removal  of developing embryos from tube to counting chamber.

     2.  Counting of embryos to determine brood size.

     3.  Determination of  the stage  of development of the embryos  and  the
         number of normal and abnormal embryos.

Part II.  Trypan-Blue-Exclusion Test

     1.  Treatment of  brood  with trypan blue  to  identify  living,' dying,  and
         dead embryos.

     2.  Preservation of embryos.

     3.  Calculation of estimated hatch size.
magnification.  The  two  types  of abnormal embryos  identified were  those  that
were aberrant  morphologically  and those that had  delayed development.   The
morphologically abnormal  embryos had atypical  cleavage  patterns  and/or void
regions (Fig. 3); the delayed-development embryos were zygotes or at the  2- or
4-cell  stage  when  the brood was harvested.  In the case where the embryos had
both types  of abnormalities,  this  fact was  noted.   The stages  that  were
quantified  were  the unfertilized egg,  zygote,  2-cell,  4-cell,  prehatch,  and
hatchling stages; these stages were identifiable with  a  minimum of ambiguity.
The  few unfertilized eggs detected were  found  in  broods that were scattered
throughout  the tube.

     The  second part of the brood  analysis  was a trypan-blue-exclusion  test
that was  developed  in our  laboratory.  After  the  embryos were  counted  and
examined, the  seawater  was decanted and  sufficient  0.4% trypan-blue solution
in seawater to cover  the embryos was added.   The  embryos were exposed  to the
trypan  blue for  5 min,  the excess  trypan-blue  solution  was then decanted, and
the embryos rinsed with filtered seawater until the  excess blue dye was  gone.
The  embryos were examined  under  6X magnification,  and  the number  that were
totally stained blue  (dead), partially  stained  blue  (dying),  and  free  of blue
dye  (live)  were  recorded  (Fig. 4).   Because  of the staining  of  the embryos,  it
could  not be ascertained readily whether  the dead  and  dying embryos  were
normal  or abnormal.   Next, the  seawater was decanted and 4% formalin  added to
preserve  the  embryos.

     For  each brood, the number of embryos  that  should hatch  into larvae was
estimated using the  data on  the  total number of embryos  compared to  the number
of  abnormal embryos  or  the number of  embryos  that were dead or  dying.   In

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Figure 3.   Embryo abnormalities  identified  in sacrificed broods.   Normal
cleavage pattern  (a),  atypical  cleavage pattern  (b),  and  embryos  with void
regions (c) are shown.

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Figure  4.   Broods  subjected  to  a  trypan-blue-exclusion  test  were
differentiated into  embryos  that were  (a)  alive (free of blue  color),
dying (partially  stained blue),  and  (c)  dead (totally stained  blue).
(b)
                                      10

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almost all broods, the  number  of abnormal  embryos  was  greater than  the sum of
the numbers  of  dead   and  dying  embryos.   The  assumption made  for  the-
calculation of the hatch  size  was that the abnormal embryos that were living
would not survive to  hatching but would die  and be consumed  by  the  brooding
male.   The  estimated  hatch  size  (EHS) was  calculated from  the  following
relationship:

  EHS  =      (Total  numbers in brood)  - (Total  number  of abnormals)

For example, if the total  number  of  embryos  in  the brood was  400  and if  75
were abnormal,  then

  EHS        400 - 75
              325  .

In the  few  cases  where  the number of  dead  and  dying was greater  than  the
number of abnormal embryos,  the number of live  embryos in the  brood  (the total
number in brood minus number of dead and dying)  was taken as  the EHS.

     Differences among  control  and  radiation-exposed groups  in brood size, in
percentages  of  living  embryos  in the broods,  in  percentages of  abnormal
embryos  in  the  broods,  and in estimated and  actual  hatch size were analyzed
using  a  Test for  Equal Proportions (Snedecor  and Cochran  1967).    Also,
differences  in brood  size for the control and  radiation-exposure groups were
examined using Analysis  of Variance (ANOVA).

3.  RESULTS

3.1  Total Doses Received

     The approximate  doses  received by the experimental  worms from  the  times
(I) the  eggs were spawned by the PI female  until  the larvae hatched  and (II)
the eggs were  spawned by the P]  female  to  the  spawning  of  the  F|   female
(normal  lifespan for females) were determined (Table 2); the  approximate total
dose  is  the product  of  the mean  duration of  exposure  and the mean dose  rate.
Because  the  experimental worms were shielded  from  the  radiation  source during
their  maintenance,  their mean  duration of exposure was  shorter than their
lifetime. The developing PI embryos were  exposed to radiation for an average
of from  10 to 12 d and  received total doses that ranged  from  0.055  to 4.9 Gy.
The life-span dose received by the f] female worms ranged from 0.55  to 54  Gy.

3.2  PI  Hatch Size


     We  determined the  number of embryos that  hatched from each of  the  broods
of  the P]  females (Table 3).   The  mean  number  of  larvae that hatched  from
embryos  exposed to each of  the different dose rates  was  similar  to  the  number
of  control   embryos  that  hatched.  Exposure  to radiation did not appear  to
affect  the  number of  larvae  that  hatched from the  PI broods.
                                       11

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Table  2.   Approximate  total  radiation  doses  received  by  worms in  each
radiation-exposure group.   The mean duration of the exposure is  in parentheses,


Dose rate and duration                                     Total  doses (Gy)


I.  P] spawn to FI hatch

     1.     0.19 mGy/h (12 d)                                     0.055
     2.     2.1 mGy/h (10 d)                                      0.50
     3.    17 mGy/h (12 d)                                        4.9

II. F] life-span dose

     1.     0.19 mGy/h (120 d)                                    0.55
     2.     2.1 mGy/h (128 d)                                     6.5
     3.    17 mGy/h (132 d)                                      54
3.3  F] Brood Size


     The numbers  of  FI  mated pairs that were  placed  in front of  the  source
initially were  sometimes  greater than the numbers  for  which  information was
obtained;  some  worms  were  lost or  killed  accidentally  during  routine
maintenance  (Table  3).   Information  about the  broods  was obtained for  only
about  half  the  F] mated pairs exposed  to  the  highest dose rate  (17  mGy/h)
because some females resorbed their oocytes and then died (see the Appendix).

     The mean Fi  brood  size was always  larger  than the number  that hatched
from  the  PI female  (Table 3).   Because the brood  from the F^  females  was
sacrificed  before hatching occurred  and the  brood from the PI  female  was
allowed to  proceed  to hatching, it would  be  expected that the  total  number
determined  for  the  FI  brood would be  larger  than  the  total  number  of
hatchlings  from the  PI  female.   However,  there  were  some broods  from FJ
females as  small  as  the number of larvae that hatched from the brood of  the  P]
female.

     We determined  the  mean size of  the  broods from the F^ mated pairs that
were  obtained   from  each  parental  brood.  In  the  control  group, brood  size
ranged from 6  to 637 and  had   a  normal   distribution.   Each  brood  was
distributed into  one of four categories (n > 150, 150 > n j> 100,  100 > n _> 50,
and n  < 50), according  to the number of embryos  in the brood (Table  4).   A
Test  for Equal   Proportions  was used  to determine which  radiation-exposed
groups had  brood-size distributions  that  were  significantly different  from
controls.   The  brood-size distribution was  different only for  the group  of
worms  irradiated  at  a rate of 17 mGy/h; the proportion of  broods in  the n  ;>
150  category was lower than that of  controls  (p < 0.001).  The  overall mean
brood  size  of the 0.19 and  2.1  mGy/h  radiation-exposed groups did  not differ
                                       12

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Table 3.   Number of embryos  from parental  (?])  and  first  filial  (Pp
generations in control  and  radiation-exposed  groups.   The brood from the
generation was sacrificed before hatching  occurred.
P] brood
ID
A. Control
8-2
15-4
16-1
17-5
22-7
24-3
x  SD
B. 0.19 mGy/h
1-2
5-1
25-3
25-4
27-5
29-7
31-8
x t SD
C. 2.1 mGy/h
11-4
16-2
20-1
21-3
23-5
24-6
27-8
x t SD
D. 17 mGy/ha
4-1
10-4
11-2
x t SD
Parental
hatch size

189
95
69
170
180
211
152  58

192
48
150
126
72
81
100
110  50

11)
120
93
81
43
150
180
112  45

111
120
120
117  5
Breeding
procedure

Intrabrood
Intrabrood
Intrabrood
Intrabrood
Intrabrood
Intrabrood


Interbrood
Interbrood
Interbrood
Interbrood
Interbrood
Interbrood
Interbrood


Intrabrood
Intrabrood
Intrabrood
Interbrood
Intrabrood
Interbrood



Interbrood
Intrabrood
Intrabrood

Mated pairs
Initial

26
18
9
16
11
18


7
6
18
17
9
21
12


14
14
6
14
8
13
12


23
22
16

Final

26
17
9
16
8
18


7
6
18
17
9
17
10


14
14
5
14
8
13
12


22
21
16

Filial brood size
( x  SD )

188
279
209
281
252
241
238

216
226
169
210
215
244
211
211

201
218
215
249
238
222
253
227

78
133
177
124

102
119
70
138
126
117
118

61
104
64
90
96
93
92
92

97
68
70
85
95
55
123
88

114
148
158
142
a  Females  that  resorbed  their eggs  and  then
compilation as having a brood size of zero.
died were  included in  the
                                       13

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Table 4.  Number of embryos in broods  from  the control and radiation-exposed
mated pairs.   The broods were sacrificed before  hatching occurred and were
assigned to one of four categories (n >_ 150, 150 > n >_ 100, 100 > n _> 50,
and n < 50), according to the number of embryos in the brood.
Experimental
group
Categories of numbers of embryos in broods
n > 150 150 > n. > 100 100 > n > 50 n < 50d
Total
broods
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
73
64
70
23
77.
76.
87.
39.0
                       A.   Number of broods  in  category
10
12
 6
 6
9
6
3
4
                       B.  Percent of broods in category
10.6
14.3
 7.5
10.2
9.6
7.1
3.8
6.8
 2
 2
 1
26
 2.1
 2.4
 1.2
44.1
 94
 84
 80
 59
317
 94
 84
 80
 80
317
a  Females  that resorbed  their eggs  and  then
compilation as having a brood  size of zero.
                              died  were included  in  the
significantly  from  that of the control group,  but that of the  17  mGy/h group
did (one way ANOVA F =  15.04, p < 0.0001).   The group receiving 17  mGy/h  was
significantly  different  from  the controls  because 25  of the  59  females
resorbed their eggs and then died at  approximately the time of spawning,  and
these  females  were included  in the  compilation  as having a brood  size of
zero.  These data indicate that these  levels of radiation, which were  received
over  the  lifetime of  the  female worms and ranged  from about 0.6 to 6.5 Gy, did
not result in a reduced number of F  embryos in the brood.

3.4   Living Embryos in  F] Broods
     For each  brood  from a F] mated  pair,  the percentage of  the  ^2  embryos
 that were  living (as evidenced by  the  exclusion of trypan  blue from  their
 cells)  was calculated  for the group of  control  worms and  for  each  of the
 groups  of  worms  that  were exposed  to  one  of the  three dose  rates  of
 radiation.  The  percentages,  which were distributed  into  four  categories
 (n _> 757.,  757.  >  n  >  507.,  50%  > n _> 257., and  n  <  257.),  were related  to the  dose
 rate received.   For  the control  group,  almost all the developing F2 embryos in
 the  broods were living.  Of  the 90 control  broods,  78 of these  broods  were in
 the  n  > 75% category; stated  as a percentage,  86.7%  of the control  broods  wero
 in  the n   > 75%  category  (Table 5).   In contrast,  the  percentage of the broccis
 in which n >_ 75% of  the  embryos were  living  in the 0.19 mGy/h group  was 62.1;
 in the 2.1 mGy/h group was  49.3; and  in the  17 mGy/h group was 3.4.
                                       14

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Table 5.  Results  from  the  trypan-blue-exclusion test of  the  living,  dying,
and dead F embryos in the broods  from  the  f]  mated pairs.  The  broods  were
sacrificed before  hatching  occurred  and  were  assigned  to  one  of  four
percentage categories (n _>  757.,  75% > n  >  507.,  507. > n _> 257., and n < 257.),
according to the percentage  of living embryos in the brood.
Experimental
   group
Categories of percentages of living embryos in broods     Total
 n > 75       757. > n > 507.   501 > n > 251     n < 257.*  broods
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
  78
  36
  34
   2
  86,
  62.
  49,
   3.4
                       A.  Number of broods in category
 5
13
16
 5
 1
 5
12
 5
                       B.   Percent of broods in category
 5.6
22.4
23.2
 8.5
 1.1
 8.6
17.4
 8.5
 6
 4
 7
47
 6.7
 6.9
10.1
79.6
 90
 58
 69
 59
276
 90
 58
 69
 59
276
a  Females that  resorbed  their eggs  and  then died  were  included  in  the
compilation as broods with n = 01 living embryos.
     The  results  from the  trypan-blue-exclusion  test  indicate  that  with
increased dose  rate  there is a decreased  percentage  of living  embryos  in  the
brood (Fig. 5).  Using the Test for Equal  Proportions,  we determined that the
number of  broods  in  the n _>  751  category  for the group  of  worms exposed  to
0.19 mGy/h was  significantly different from the number in that category for
the  group  of control worms;  x2  12.06, p < 0.001.   The proportion of the
broods that  was in the n >_  75% category for  each of  the other  more  intensely
radiated groups was  also significantly different from that of  the  control
group (p  <  0.001).  These results indicate that, for this species, a lifetime
dose rate  as  low as  0.19 mGy/h or a  total dose of about 0.6 Gy (60 rad)
reduces significantly the percentage of  living embryos  in  the brood.

     The  brooding  males  are  effective at  removing  dead  embryos from the
brood.   This  is evident  from the data acquired on the broods  in which the
embryos hatched into larvae before they were  analyzed  (see comment section of
brood data in  the Appendix).  When  hatching did  occur,  the percentage  of
living embryos  almost always  approached  100.   If  large numbers  of early-stage
embryos are  eaten  by the male,  his gut is  yellow  from the yolk,  consumed;  these
                                       15

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          100
     o
     0)
     o
     o
     o
                                                                                 n<25%
Figure 5.  The percent of broods from the Fj mated pairs in each of four categories (n > 751,  75% > n >

50X, 501 > n > 251,  and n < 25X) of percent living embryos in the brood.

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males are referred to as cannibals.   A few males, even in the control  group,
cannibalized the brood, but at the higher dose rates this was a common
occurrence, presumably because there were more dead embryos present.   The
percentage of the males that were cannibals was 17 in the control  group, 27 in
the 0.19 mGy/h group, 24 in the 2.1  mGy/h group, and 83 in the 17 mGy/h group.

3.5  Abnormal Embryos in FI Broods


     In most broods, some embryos were classified as abnormal because of their
morphology or because  their development was  delayed  severely.  The broods  were
placed into four categories (n >. 150, 150 > n _> 100, 100 > n > 50, and
n  <  50),  according  to the number of  abnormal  embryos in the brood (Table 6,
Fig. 6).  The percent of the broods in the  n < 50 category  was  80,2  for the
control group and was  60.7, 35.3, and 5.1  for  the  groups  exposed  to 0.19,  2.1,
and  17  mGy/h,  respectively.   Ne also calculated  the percentages  of abnormal
embryos that were present, and these were distributed into four categories
(n > 751, 75% > n > 50%, 50% > n >_  251,  and n < 25%)(Table  7,  Fig.  7).  The
percent that  was  in the n _>  75% category was 1 for the control group and 7,
16, and 91 for the  groups  exposed  to 0.19,  2.1,  and 17  mGy/h, respectively.
Incidence  of abnormal embryos  appears  to  be  dose  related.  A  significant
difference from  the control  group was detected  in all  the  radiation-exposed
groups.  For the group exposed to 0.19 mGy/h, x2 =, 6.66,  p  < 0.005.

3.6  Reduced Survival of FT Embryos


     The numbers  of embryos that were estimated  to hatch  or the  actual  numbers
that hatched were grouped  into four categories: (n ^ 150, 150  > n _> 100, 100  >
n   50, and  n  < 50)  (Table  8).   The hatch size  was  related  to dose  rate
(Fig. 8).  The  percent of the broods  that  had  or were  estimated  to  have
hatchlings >  150  in number was 68.1 for the control group and was 50.0, 36.3,
and  0  for  the radiation-exposed groups receiving  0.19,  2.1,  and  17 mGy/h,
respectively.  Also,  the estimated  size of  the  hatch from the F]  mated pairs
exposed to  radiation was significantly different from that of  controls  for all
the  lifetime dose rates  delivered to the worms.

     The effects  of radiation were apparent  also  in the percentage of broods
in which the EHS was zero.  The percentage  was  1.2 for the  control  group  and
was  5.4,  7.7, and  42.3  for the  groups  exposed to 0.19,  2.1,  and 17 mGy/h,
respectively.  An EHS of zero resulted because the female resorbed the  eggs  or
because the embryos  in the brood were either abnormal, dead, or dying.

     The effects  of radiation on the potential  for  embryos to  survive  to
hatching  was assessed.   The  percent of  the embryos that should survive  to
hatching for each brood  was calculated by  dividing the EHS  by the  brood  size
and  multiplying  the fraction  by 100.  Then, the broods were assigned to one of
four categories  (n  ^ 75%, 75%  >  n  > 50%,  50% >  n  >_ 25%,  and  n  <  25%),
according  to the percentages  of survival  (Table  9,  Fig.  9).   The Test for
Equal  Proportions  was   used  to determine  which  radiation  groups  had
distributions  of percentages  that  were  significantly different from that of
the  controls.   All  groups exposed  to radiation  were significantly different
from controls; the  p values were <  0.001.
                                       17

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Table 6.  Results  from  the  analysis  of the normal  and abnormal  embryos in the
broods frofii the control  and  radiation-exposed FI  mated pairs.   The broods were
sacrificed before  hatching  occurred  and  were  assigned  to  one  of  four
categories (n  >  150,  150 >  n >  100,  100  > n _> 50, and n < 50), according to
the number of abnormal  embryos in the brood.
Experimental Categories of numbers of abnormal embryos in broods Total
group
n > 150 150 > n >_ 100 100 > n > 50 n < 50a broods
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
                  4
                  5
                 17
                 47
                  4.4
                  8.9
                 25.0
                 79.6
                       A.  Number of broods  in category
 7
 6
10
 4
17
 5
                       B.   Percent of broods in category
 7.
10.
14.
 6.8
 7.7
19.6
25.0
 8.5
73
34
24
 3
80.2
60.7
35.3
 5.1
 91
 56
 68
 59
274
 91
 56
 68
 59
274
a  Females  that resorbed  their  eggs  and  then died  were
compilation as broods in the n >_ 150 category.
                                                           included  in the
     An analysis was  performed  to determine the  relationship  between chronic
radiation dose  and  embryo  survival.   The  mean  percent survival  for the control
group and for  each  radiation-exposed group was  determined.   For the  control
group, a  value of  82   181 was obtained, and  for the groups  exposed  to
radiation, the  values were 61   28%  for  the  group  exposed  to  0.19  mGy/h,
51  31% for the group exposed  to 2.1  mGy/h,  and 5  13% for the group exposed
to  17  mGy/h.    The  mean percentage  for each  radiation-exposed  group  was
expressed also  as   a  percentage of  the control   group.   A semi  log plot of
percentages versus dose resulted  in  a straight line;  an  LQ$Q  of about 10 Gy
and an LDgg of  about  100 Gy was obtained  (Fig. 10).

     Other parameters  that were examined  for the  experimental animals  were (1)
the  time  from  spawning  of  the  PI  brood to the  hatching  of the F]  larvae
(spawn-to-hatch time)  and  (2) the time  from  the hatching of the  larvae (Fj)
until the spawning  of the adult  females  (F^)  (hatch-to-spawn time).   The  mean
spawn-to-hatch  time  for all  the P]  broods was  11.7 + 1.8 d,  and the irradiated
groups did  not  differ significantly  from  controls.   These data indicate that

-------
    o
    o
    2
    CO
                     02150
150 > n 2 100
100 > n 2 50
n<50
Figure 6.  The percent of broods from Fj mated pairs in each of four categories  (n  >  150,  150 >

100, 100 > n > 50, and n < 50) of numbers of abnormal embryos in the brood.
                                                          n  >

-------
Table 7.   Results  from the analysis  of normal  and abnormal embryos  in  the
broods from the  control and  radiation-exposed mated pairs  The  broods were
sacrificed before hatching occurred,  the number of normal and abnormal embryos
determined, the  percent of abnormal  embryos calculated, and then  the broods
were assigned to one  of  four  categories (n _> 757., 75%  >  n  _> 50%, 50% > n ^
25%, n < 25%), according  to the percent of abnormal  embryos  in  tfte brood.
Experimental
   group
Categories of percentages of living embryos in broods     Total
 n > 75%      75% > n > 50%   50% > n > 25%     n < 25%a  broods
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
   1
   4
  11
  54
   1.1
   7.0
  16.2
  91.5
                       A.  Number of broods  in category
 2
15
19
 4
 9
15
16
 1
                       8.   Percent of broods in category
 2.2
26.3
27.9
 6.8
10.1
26.3
23.5
 1.7
77
23'
22
 0
86.6
40.4
32.4
 0
 89
 57
 68
 59
273
 89
 57
 68
 59
273
a Females  that  resorbed  their eggs and  died  were  included  in  the  compilation
as broods with 100% abnormal embryos.
when worms were irradiated with  doses as high as  17  mGy/h and were given  a
total  dose  of 4.9 Gy  during the spawn-to-hatch time,  the time required to
develop  from fertilized  eggs to  larvae  was not  affected.   The  mean
spawn-to-harvest  time  for all  the  f]  females  was  127    18  d,  and  there  were no
significant  differences among  experimental  groups.  These data  indicate  that
radiation at doses as  high as  17 mGy/h and mean  total  doses of about  54 Gy
also did not affect the life span of  the females.                      1

4.  DISCUSSION

     Living  organisms  are  exposed  to radiation  from  natural  sources and  from
anthropogenic sources,  including nuclear explosions, routine and accidental
releases  from nuclear  power facilities, and  nuclear  waste disposal (UNSCEAR
1977,  1982).  The dose rates  to marine organisms  from natural  background
radiation, global  fallout, and waste  radionuclides were calculated by Woodhead
(1984) and provide a perspective within which  the  possible harmful effects of
increased radiation  exposure can be  considered.   The dose rates in  the marine
environment  due  to radionuclide  inputs arising  from  human activities  range
from  less  than the  natural  background  exposure for typical  nuclear  power
stations in  routine  operations up to a  few tenths of  mGy/h  for  the rather
exceptional  case  of the  Windscale discharge  into the  northeast  Irish Sea
(Woodhead 1984).
                                       20

-------
             100
     o
     co



     o


     c
     o
     o
     o

     at
                                                    CONTROLS



                                                    0.19mGy/h



                                                    2.1 mGy/h



                                                E3  17 mGy/h
                                         75% > n 2 50%
50% > n ;> 25%
n < 25%
Figure 7.  The percent of  broods  in each of four categories  (n  >  75%,  75% >  n > 50X,  50% > n  >  251,

and n c 25%) of percent  abnormal  embryos in the brood.

-------
Table 8.  Results from the analysis of the numbers of F embryos that actually
hatched  or  were estimated  to  hatch  from  the broods of  the control  and
radiation-exposed FI mated pairs.   The  broods were assigned to one  of four
categories (n >_ 150, 150 > n ^ 100, 100  >  '  >_ 50,  and n < 50), according  to
the actual or estimated hatch size.
Experimental
   group
Categories of numbers estimated or actually in hatches    Total
n > 150      150 > n > 100    100 > n > 50     n <  50    broods
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
  64
  42
  29
   0
  68.1
  50.0
  36.3
   0
                       A.  Number of broods in category
12
18
20
 1
10
14
14
 3
                       B.   Percent of broods  in  category
12
21
25
10
16
17
 1.6
 4.9
 8
10
17
57
 8.5
11.9
21.3
93.4
 94
 84
 80
 61
319
 94
 84
 80
 61
319
    It  is  well  documented that radiation  induces  biological  effects  through
the deposition of energy in  the cells  of the irradiated individuals (UNSCEAR
1982).   If  the  effects are produced in  the  somatic cells, they must become
apparent, by definition, within the  life of  the irradiated organism.   If the
effects  are  produced  in the germ cells, whose function is to transmit genet'c
information to new individuals, the  effects may be detected in the descendants
of the irradiated individual in the  first or subsequent generations.

    Most of  the  information available on  radiation  effects  on  reproductive
success  in  aquatic  animals  is  on  the effects of acute  radiation.   Effects were
determined by irradiating early life stages  and adults (see reviews by  Egami
and  Ijiri  1979;  Woodhead 1984; Anderson and Harrison  1986).   The effects of
acute  radiation  on  processes  affecting  reproductive   success  in aquatic
invertebrates were  reported for doses that  range  over at  least  two orders of
magnitude  (Cervini  and  Giavelli  1965;  Ravera  1967; Hoppenheit  1973;
Greenberger  et  al.  1986; Anderson et  al.  1987).   Causes  for  this  broad  range
seem to  be not only actual  species-specific differences in gamete  sensitivity,
but  also  differences  in  the  gamete  stage   irradiated  and  in tha
cell-repopulation capacity of  different organisms.

-------
           100
     o
     at
     a
     O

     2
     m
                                                Controls



                                                0.19mGy/h



                                                2.1 mGy/h



                                                17 mGy/h
                                                                                   n < 50
Figure 8.  The percent of  broods  from the Fj mated pairs in each  of  four  categories (n > 150, 150  >

100, 100 > n ^ 50, and n <  50)  of actual  and estimated numbers of hatchlings.
n >

-------
Table 9.   Results of the analysis of  survival  to hatching of embryos  in  the
broods of  the  control and  radiation-exposed F] mated  pairs.   The percent
survival  was calculated  by dividing  the  estimated  hatch size by  the  brood
size, and then  the broods  were assigned to  one  of  four categories  (n  _>  751,
757.  > n  >  50%,  50% > n >_  25%,  and n  < 25%), according to the percent  survival
of the embryos.
Experimental
   group
Categories of percent survival  of embryos to hatching
 n 2 75%    75% > n > 50%    50% > n > 25%,    n < 25%a
                                                        Total
                                                        broods
                       A.  Number of broods in category
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
Control
0.19 mGy/h
2.1 mGy/h
17 mGy/h
68
20
21
 0
84.1
36.4
32.3
 0
                  9
                 17
                 13
 2
13
18
 3
                       8.   Percent of broods  in  category
                 11.0
                 30.9
                 20.0
                  2.0
 2.5
23.6
27.7
 5.9
 2
 5
13
47
 2.5
 9.1
20.0
92.1
 81
 55
 65
 51
252
 81
 55
 68
 51
252
a  Data  from broods  that hatched or that were  harvested  before day 3  were
excluded.
    Studies  were  conducted  to  assess  the effects  of  chronic  low-level
radiation on  reproduction  in fishes and invertebrates,  and a  number  of these
were conducted over a  full  life cycle.  However, most of  the  experiments to
assess the  effects  of chronic radiation were performed using radionuclides in
the water and the doses delivered were uncertain  (Woodhead  1984;  Anderson and
Harrison 1986).

    Information on the effects of chronic  radiation on reproductive success  in
fishes and  aquatic invertebrates  is  available  from  studies  in  which  the
effects of  relatively low dose rates  were investigated.   Trabalka and  Allen
(1977) compared  populations  of  the mosquitofish Gambusia  affinis from  the
radionuclide-contaminated White Oak Lake at the  Oak  Ridge  National Laboratory
to  those  from a  matched control site.   They  found  no decrease in fecundity,
but an increase  in  embryo mortality of  the  fish from Hhite Oak Lake;  these
fish  received about 0.25 mGy/h.  These  results  were confounded by the fact
that  contaminants  other  than radionuclides were  present in  Nhite Oak  Lake.
Cooley   (1973)   examined     the   reproductive   biology   of   pond
                                       24

-------
ro
en

                                                                   50% > n * 25%
n<25
      Figure  9.   The percent of broods in each  of  four  categories  (n  > 75%, 75% > n >_ 50%, 50% > n 2 25%,  and
      n <  25%) of  percent  survival to  hatching of the embryos in the brood.

-------
      100-1
e
a
       10-
*
o
o
a.
                10
20
30
40
50
60
70
80
90
                                     Dos* (Gy)
  Figure  10.   Mean  percent  survival  of embryos (expressed as percentage of  the
  survival  fraction of the controls)  as  a  function of chronic  dose.   Data  from
  broods  that hatched  or that were harvested before  day  3 were  excluded.
                                        26

-------
snails from White Oak Lake:  these were exposed  to about  the same dose  rate  as
the mosqultofish.   He found that  frequency of  egg-capsule  production was
reduced;  however,  an  Increased  number  of eggs  per capsule  was  also
documented.  It  is interesting to note that a prior laboratory study by Cooley
and Miller (1971) documented a  clear cut reproductive decline at  240 rad/d
(100 mGy/h)  but  not  at  24  rad/d.   Irradiation  was initiated on  45-d-old
snails, and  laboratory effects might have been observed at  lower  levels  if
irradiation had been extended over the entire  lifetime of the organism.

     One of the most rigorous studies involving chronic  exposure to  radiation
was that  of Woodhead  (1977), who examined fecundity of the  guppy Poecilia
reticulata receiving  4.08,  9.6,  and 30.5 rad/d (1.7, 4.0,  and 12.7 mGy/h).
Total   fecundity  was  significantly  reduced at  all dose rates.  Reductions  in
fecundity were probably due  to both  reproductive  effects (damage  to gametes)
and the induction of  dominant-lethal mutations in gametes.  Effects on gonadal
cells were reported also for Gambusia affinis  (Cosgrove  and Blaylock 1973)  and
for aryzias  latipes  (Hyodo-Taguchi and  Egami  1977; Hyodo-Taguchi  1980).
Hyodo-Taguchi (1980) observed an increased percentage of unfertilized Oryzias
latipes eggs after  males  used  to inseminate the  eggs received approximately
6.9 rad/d  (2.9 mGy/h)  for 60 d.   No statistically  significant  effects were
observed at  2.9  rad/d,  the  next lower dose rate  used.   Bonham and Donaldson
(1972) exposed Chinook salmon Oncorhynchus tschawytscha  embryos for  the  first
80 d  of  life to 0.19 to 17  R/d  (about 0.08 to 6.8 mGy/h).   Approximately 4 wk
after  the  irradiations were completed,  gonadal development  was  observed in
smolts.  They  found  that  gonadal development was retarded in  those receiving
at least 10 R/d.

     In a  more recent study,  Rackham and Hoodhead (1984) examined the effects
of chronic gamma irradiation on  the  gonads of  the adult  fish Ameca splenden.
The dose  rate used  was  7.3 mGy/h;  after an accumulated  dose of  0.95 Gy,
spermatogenesis was disrupted,  and after an accumulated   dose of 9.7  Gy,  there
was no further production of sperm.

     It is apparent  from the  data  available  that direct  comparisons of
sensitivity  among species irradiated chronically are often  not valid  because
the duration of  the radiation  differed  from  partial  to several  lifetimes.
Research on  effects  of  chronic  radiation on  the gonads  is of particular
interest,  however, because  the results show effects levels comparable  to those
observed in  mammals.   Dose  rates between 0.2  and 4 mGy/h appear to define a
critical range in which  detrimental effects  on  processes contributing to
reproductive success  are first observed in a variety of  sensitive organisms.

      In our  study, the effects of lifetime radiation on reproductive  success
of  a  relatively low  fecundity  species  were  evaluated.  Information was
obtained on  the  effects  of  chronic  radiation  on  total  number of  developing
embryos in the brood, on the numbers  of normal  and abnormal  embryos  in the
brood, on  the  numbers of embryos that were living, dying, and dead, and on  the
estimated  number of  hatchlings.   Comparisons  were made of  the  data from
control worms  and from worms that were exposed  to radiation immediately after
fertilization  occurred until  they  released  their  gametes  and  the  next
generation of  zygotes were  formed.  Thus,  germ cells were  irradiated from
their  time  of origin (primordial  germ cells)  until mature gametes  were
released.
                                       27

-------
     An important effect of  lifetime  irradiation  of N. arenaceodentata  with
low dose rate (0.19  and  2.1  mGy/h)  was  increased  mortality  of  the  embryos  (F2
generation).   There  was  no  evidence  for F]  gamete  death or  for  reduced
fertilization success because the number of developing embryos in the broods
did not decrease.  However,  at  the  highest dose  rate  used,  17 mGy/h,  brood
size  was  affected and was  related  to  the resorption of oocytes  in  the
females.  Also,  at all  three  dose rates   used, there  was  no  detectable effect
on the  time  required  for the fertilized eggs to develop  into larvae  or on  the
life span of the female F|  worm.

     Increased mortality of  the  F  embryos was  indicated because both  the
number of dead and dying  embryos  and the number of  abnormal  embryos  found  in
the brood  after 4 to  6  days of development  increased with increased dose
rate.   Both of these factors  contributed to a decreased  number of actual  or
estimated number of  hatchlings  in  the broods and occurred at  the lowest rate
used,  0.19 mGy/h.  The increased  mortality was most  likely  from the  induction
of lethal mutations  in the  germ cells during gametogenesis.   Because both  the
males and females were given  lifetime  irradiation and  because  little  is  known
about  the  comparative sensitivity  of  cells in  ths different stages  of
oogenesis and sperraatogenesis in N.  arenaceodentata,  it  is  not known whether
the lethal mutations occurred primarily during oogenesis, spermatogenesis, or
relatively equally during both of these  processes.

     Effects  of  acute radiation on reproduction  of N. arenaceodentata  were
examined in a companion study (Harrison  and Anderson 1988),  and comparisons of
the effects  on  reproduction  of total  doses  received from acute and chronic
radiation were made  (Table  10).   For  the  parameters  compared, the  control
group  for  the worms  irradiated acutely  appeared  to  be less  vigorous than  for
those  irradiated chronically; there was  a greater proportion of  small  broods,
fewer  living  embryos,  etc.   The differences  between  the  two  control groups  may
have  been  due to  differences in their maintenance conditions.   For  the
experiment in which  the  mated pairs were  irradiated chronically,  the broods
were from females that were raised in  our  laboratory under  uniform conditions
of food availability and temperature  whereas  for  those irradiated acutely,  the
females were from multiple sources and may not  have  been  raised  under similar
conditions.

     Effects on brood size, which may be due to oocyte killing, were seen  when
a  total  dose of 50 Gy was given  over the lifetime  of the female and when  an
acute  dose of 10 or 50 Gy was  giver* at  the time  oocytes  were  visible in the
coelom.  Information  available  from the mouse  indicates  that  the  target for
cell killing and that for genetic effects  are different  and distinct  in this
species;  the lethality target in immature  oocytes  appears  to be the  plasma
membrane and the sensitivity of this target differs  almost two  orders  of
magnitude with  stage in  the mouse  life  cycle (Straume et al.  1987; Straume et
al. 1988).   For N. arenaceodentata, we  do  not have  sufficient radiobiological
information  to  evaluate  the effect of  developmental  stage  on  oocyte
radiosensitivity.

     Comparison  of the  values  (except   brood size)  that were corrected for
controls shows that  for  those broods  from  females  receiving a total dose  of
about  0.5  or 5  Gy,  the  effects  were  similar or  greater  for those irradiated
acutely (Table  10).  However, the differences between  the effects elicited  by
                                       28

-------
        Table 10.  Comparison of the effects of  acute  and chronic irradiation on  Neanthes  arenaceodentata.   The
ro
vo
values are percents of the broods in the category indicated.
Effects
Brood sizea
(n < 50 category)
Living embryos''
(n > 75% category)
Abnormal embryos4
 751 category)
Estimated hatch sizea
(n < 50 category)
Survival to hatching3
(n < 251 category)
Survival of embryos^
(mean percent)
Acute Chronic Acute
Control Control 0.5 Gy
10 2 15
(5)
57 87 31
(54)
18 1 25
(7)
23 8 38
(15)
20 2 29
(9)
60 82 48
(80)
Chronic
0.55 Gy
2
(0)
62
(71)
7
(6)
12
(4)
9
(7)
61
(74)
Acute
5.0 Gy
26
(16)
22
(39)
38
(20)
50
(27)
40
(20)
39
(65)
Chronic
6.5 Gy
1
(0)
49
(56)
16
(15)
21
(13)
20
(18)
52
(63)
Acute
50 Gy
56
(46)
14
(25)
71
(53)
82
(59)
73
(53)
20
(33)
Chronic
54 Gy
44
(42)
3
(3)
91
(90)
93
(85)
92
(90)
5
(&)
        a  Values  in  parentheses  are  minus  the  control  values  or  are  expressed  as  percents of  the  control  value.

        b  Values  in  parentheses  are  expressed  as  percents  of  the control  value.

-------
Table 11.   Comparison of the  effects  on reproductive success of exposure of
Neanthes arenaceodentata to different  doses  of contaminants.
Ionizing
radiation
(Gy)
Hexavalent
chromium*
(uq/D
Number 2
x fuel oilb
(%WSF)
Response
Sterility
Reduction in number
Acute
50C
10
Chronic
90C
54
Chronic
100
	
Chronic

2.5
of embryos

Reduction in number        0.5        0.55         16-38                2.5
of hatchlings
  a Oshida and coworkers (1981, 1982).
  b Rossi and Anderson (1978); WSF is the water-soluble fraction.
  c Effective sterility is defined here as 1% survival  of embryos to hatching
    as compared to controls.
radiation  given acutely  and  that  given  chronically  was  less  than was
expected.  These results indicate that  there  was  most  likely accumulation of
radiation damage in  nondividing cells and, then,  this damage became apparent
after fertilization  when  the cells  started to  divide.   This finding  is  of
special   interest because  such damage accumulation may occur not only with the
direct-acting  mutagen,  radiation,   but also  with  other  direct-  and
indirect-acting organic mutagens  that may be present in ecosystems.  Although
we have  no direct evidence for  such, the damage accumulation may be related  to
differences  in  DNA-repair  ability of cells  in  different stages  in
gametogenesis.

     Comparison of  the values (corrected  for  controls) for  females  receiving a
total dose of about  50 Gy  acutely and  chronically indicates that the  effects
appear  to be more  severe in  those irradiated chronically.  There are two
plausible explanations for this response.  First, all gametogenic stages are
irradiated  during   chronic  exposures  and  a  particular  stage  of  oocyte
development  may be  sensitive to high  dose  rates.   This  could be  relatively
short hypersensitive stage that is only  "hit"  by chronic radiation.   Second,
an  unknown  radiation-induced stress may have  been  induced  at the high dose
rate, and this  stress  may  have caused resorption of  the oocytes  prior  to
spawning.  The  overall effect would be  reduced  fecundity.

     Evidence that  the oocytes are the limiting cell system was obtained from
a comparison of the  data from the  preliminary and final  experiments on acute
effects  of  radiation.   In the  preliminary  experiment, only the females were
irradiated (Anderson et al.  1987),  while in the final  experiment, mated pairs


                                       30

-------
          female)  were irradiated (Harrison and Anderson  1988).   The  results
of both the preliminary and the final experiments were similar; this  indicates
that the oocytes were  most  likely the cells in  which  radiation damage  was
accumulated.
     Information available on  mammals  indicates  that  in some  s"pecies  the
oocytes are very  sensitive to radiation (UNSCEAR  1986;  Dobson  et  al.  1984;
Dob'son et al.  1986).   Sensitive  species  include mice and some  primates.   In
th/e mouse,  the  1050  for immature-oocyte killing with ^Co gamma rays is 1.75
Gy in the prenatal mouse and  range from about 0.05 to 0.15 Gy in the juvenile
mouse.  The value of 0.05  Gy for juvenile mice  reflects about a 30 to 50 times
greater sensitivity than found in most other  cells  studied.   In the squirrel
monkey, the 1050  for  radiation from administering tritiated  water  was  0.07 Gy
from prenatal  exposure  and 2.25  Gy from adult  exposure.  It is apparent that
there  are  considerable differences  in  sensitivity with  species  and  life
stage.  In  N. a re n a c eocjgr. ta ta. we know little about differences in sensitivity
with defined life stages.   However,  from the  results we obtained,  it appears
that a dose at  Ie5.st .J^-.i4s higher is required to affect  cell killing in N.
arenaceodentata than in  least  sensitive stage  of  the  mouse, but  that the
sensitivity of N.  arenaceodentata is in the range of most other cells  studied.

    V.ittle is  known  abjwt>  the  effects of  factors that may   modify  the
resporfSj^i^f  aquatic "d^ySlN^  to radiation.   Factors that may  play an
importantii^^^re ONA repair, tissue oxygen concentrations,  and environmental
conditions.^HMjfc^ "".mperature, salinity,  and water  quality  (Anderson and
Harrison 1986^WKine of these  factors  are known to modify  the responses  of
vertebrates to  radiation and require elucidation before conclusions are drawn
about  regulatory  limits on  the  quantities  of  mutagens  released  in  the
environment.

     Information  is  available for  N.   arenaceodentata  on  the effect  on
reproductive  success   of   contaminants  other than  radiation (Table  11).
Considerable data are  available  on the effects of chromium  (Oshida  et al.
1981;  Oshida and  Hard  1982).  Concentrations of  chromium as low as 16 ng/L
reduced  the numbers of  hatchlings.   The  concentration of  chromium  that
resulted in sterility  was  100 jig/L.   However,  sterility occurred  not  because
of effects  on  gametes  but because  of  a behavioral  response of the  adult
worms.  According to  these investigators,  the  worms were jerking and twisting
to such an extent that the prolonged contact required for reproduction did  not
occur.

     The water-soluble  fraction  (WSF)  of  Number 2  fuel  oil  also impacted on
       ction in N.  arenaceodentata (Rossi  and  Anderson 1978).  Effects on the
     f of larvae  that  hatched  occurred at concentrations as  low as  2.57. NSF
(Table  II).  No information  was available on the HSF concentrations resulting
in sterility, but growth was inhibited at 5 and 101 WSF.

-------
     The studies  of  the  effects  of both  chromium and  fuel  oil  were
multigeneration and  provided  evidence  that  there was  accommodation  to  the
contaminants in the  $2  anc'  ^3 generations.  Because our  study  of radiation
effects was only for a  single generation,  no conclusions  can be  drawn as  to
possible accommodation  by  subsequent generations  or  to the response of
populations to continuous exposure to low levels of radiation.

     There are  few data on chronic radiation effects on invertebrates that  can
be compared to  those reported here on N.  arenaceodentata.   However,  it  is
apparent from  the data  available on  fish and  invertebrates  that  the overall
effects on  reproductive success  are dependent upon  a number  of factors.
Important among these are  reproductive  strategy and sensitivity  of  stages  in
gametogenesis  and in  early  development.   It would be  expected  that  species
most vulnerable to  chronic  exposures to low levels of  mutagenic  contaminants
are those that have a low fecundity and  have highly sensitive stages.  Because
the results from  our study indicate  that  in some  invertebrates the range of
sensitivity may overlap with  that  for fish and even for  mammals  and because
the data base  on  effects of chronic  low-level  exposures is limited,  it may  not
be  overly  conservative  to adopt  limits  for   the  chronic   exposure of
low-fecundity aquatic animals based  on  the extensive  data base available on
the responses  of mammals.
                                ACKNONLEDGMENTS

     Special thanks  are given  to  Roger Martinelli,  Marie Kalinowski,  and
Sue Tehensky who  assisted in  the  laboratory.   Ne  also wish  to thank our
project officer Marilyn Varela and others who provided critical reviews  of the
manuscript.

     This work  was  supported by the  U.S.  Environmental  Protection Agency,
Office of  Radiation  Programs (DOE-EPA Interagency Agreement DN 89930414-01-1)
and was  performed  under the auspices of the  U.S. Department  of Energy  by
Lawrence Livermore National  Laboratory (Contract H-7405-ENG-48).
                                       32

-------
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                                       34

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                                       35

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                   APPENDIX

Data Base from the Experiment to Determine the
 Effects of Chronic Radiation on Reproductive
     Success of Neanthes arenaceodentata
                      A-l

-------
               Table A-l.  Experimental data  from Neanthes arenaceoUentata mated pairs that were not irradiated with an external gamma-
               radiation  source  (controls).   The number of days from spawn to hatch and from hatch to spawn as well as the estimated
               hatch number are  provided.
ro
Pj Spawn
to
F) hatch
(days)
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
F1 Hatch
to 10
F] spawn
(days)
119
125
129
129
128
119
134
128
134
122
121
121
152
119
149
136
128
121
134
125
119
125
124
120
125
120
8-2-1
8-2-2
8-2-3
8-2-6
8-2-10
8-2-10b
8-2-16
8-2-32
8-2-36
8-2-37
8-2-42
8-2-49
8-2-52
8-2-53
8-2-55
8-2-56
8-2-60
8-2-63
8-2-66
8-2-68
8-2-74a
8-2-74b
8-2-75
8-2-77a
8-2-80
8-2-88
Spawn
number
73
226
452
103
307
91
361
202
202
96
230
115
38
88
327
121
223
165
306
196
218
239
209
111
96
89
Live
number
57
213
434
101
304
91
357
200
202
93
205
112
34
74
325
121
203
157
305
185
217
229
205
98
83
72
Live
(X)
78.1
94.2
96.0
98.1
99.0
100
98-9
99.0
100
96.9
89.1
97.4
89.5
84.1
99.4
100
91.0
95.2
99.7
94.4
99.5
95.8
98.1
88.3
86.5
80.9
Dying
number
16
11
11
2
1
0
2
0
0
2
20
3
2
14
1
0
17
2
0
5
1
8
3
12
13
17
Dead
number
0
2
7
0
2
0
2
2
0
1
5
0
2
0
1
0
3
6
1
6
0
2
1
1
0
0
Dead
(X)
0
0
1
0
0
0
0
1
0
1
2
0
5
0
0
0
1
3
0
3
0
0,
0.
0.
0
0

.9
.6

.7

.6
.0

.0
.2

.3

.3

.4
.6
.3
.1

.8
,5
9


Abnormal
number
5
60
2
15
62
1
21
15
18
3
24
10
0
11
22
0
40
16
25
52
18
47
21
21
16
0
Est.
hatch Comments
number
57 Cana
166 Can
434 Can
88
245
90 Hatchb
340
187
184
93
205
105
34 Can
74 Can
305
121 Scat
183
149
281
144
200
192
188
90
80
72

-------
            Table A-l  (cont.)
co
PJ Spawn
to
Fj hatch
(days)
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
F, Hatch
to ID
F) spawn
(days)
126
141
125
124
125
138
125
142
119
125
121
123
137
119
124
117
133
117
143
126
113
134
125
125
129
117
15-4-3
15-4-4
15-4-5
15-4-6
15-4-7
15-4-13
15-4-22
15-4-23
15-4-25
15-4-32
15-4-32a
15-4-37
15-4-40
15-4-50
15-4-53
15-4-54
15-4-55
16- -1
16- -2
16- -3
16- -4
16- -8
16- -9
16- -18b
16- -19
16-1-20
Spawn Live Live
number number (X)
311
318
303
257
220
506
325
389
160
325
167
225
533
125
253
106
221
163
158
208
116
200
255
311
311
161
302
228
288
250
208
457
316
336
152
316
159
221
512
26
251
98
218
149
150
203
115
197
251
300
276
122
97.1
71.7
95.0
97.3
94.6
90.3
97.2
86.4
95.0
97.2
95.2
98.2
96.1
20.8
99.2
92.5
98.6
91.4
94.9
97.6
99.1
98.5
98.4
96.5
88.8
75.8
Dying
number
2
21
15
6
9
36
2
37
5
2
6
3
15
81
1
8
1
14
6
4
1
0
3
7
32
39
Dead
number
7
69
0
1
3
13
7
16
3
7
2
1
6
18
1
0
2
0
2
1
0
3
1
4
3
0
Dead Abnormal
{%) number
2.2
21.7
0
0.4
1.4
2.6
0.2
4.1
1.9
2.2
1.2
0.4
1.1
14.4
0.4
0
0.9
0
1.3
0.5
0
1.5
0.4
1.3
1.0
0
7
122
108
63
42
121
7
84
13
0
29
28
166
3
5
6
32
6
20
48
6
3
5
40
115
17
Est.
hatch Comments
numbe r
302
196
195
194
178
385
316
305
147
316
138
197
367
26
248
98
189
149
138
160
110
197
250
271
196
122

Aband











Scat



Can
Can

Can






-------
Table A-l  (cont.)
Pj Spawn
to
F] hatch
(days)
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
11







F1 Hatch
to 10
F| spawn
(days)
129
125
140
121
134
136
123
135
123
147
L
137
139
126
123
165
167
155
172
172
172
154
165
17-5-1
17-5-6
17-5-8
17-5-14a
17-5-14b
17-5-15
17-5-17
17-5-33
17-5-34
17-5-37
17-5-38
17-5-41
17-5-44
17-5-45
17-5-49
22-7-5
22-7-10a
22-7-14
22-7-15
22-7-17
22-7-41
22-7-43
22-7-54
Spawn Live Live
number number (%)
322
216
637
123
429
298
288
297
209
178
125
495
173
265
227
282
209
6
399
229
338
189
368
312
214
607
121
426
294
152
297
i39
154
0
474
171
259
208
253
35
0
368
217
75
180
273
96.9
99.1
95.3
98.4
99.3
98.7
52.8
100
90.4
86.5
0
95.8
98.8
97.7
91.6
89.7
16.8
0
92.2
94.8
22.2
95.2
74.2
Dying
number
8
1
23
1
2
3
32
0
20
20
0
16
2
6
13
21
143
4
28
12
175
5
67
Dead Dead Abnormal
number (%) number
2
1
7
1
1
1
104
0
0
4
125
5
0
0
6
8
31
2
3
0
88
4
28
0.6
0.5
1.1
0.8
0.2
0.3
36.1
0
0
2.2
100
1.0
0
0
2.6
2.8
14.8
33.3
0.8
0
26
2.1
7.6
29
9
18
15
22
26
112
10
36
19
0
113
9
33
53
33
184
1
3
0
338
21
203
Est.
hatch Comments
number
293
207
607
108
407
272
152
287
173
154
0
382
164
232
174
249
25
0
368
217
0
168
165,






Aban

Can
Aban







Can
Hatch
Hatch
Scat
Can


-------
              Table A-l  (cont.)
en
P] Spawn
to
FI hatch
(days)
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
f] Hatch
to 10
F] spawn
(days)
118
119
L
116
112
125
125
121
113
120
120
110
118
119
111
103
103
103
24-3-3a
24-3-4a
24-3-4b
24-3-14
24-3-18
24-3-23a
24-3-23b
24-3-25
24-3-30
24-3-36
24-3-43
24-3-45
24-3-46
24-3-50
24-3-54
24-3-56a
24-3-56b
24-3-57
Spawn Live Live
number number (X)
445
367
309
430
77
279
158
131
304
168
309
192
309
298
253
67
152
82
419
360
248
396
77
278
157
129
275
167
308
181
248
294
241
35
152
38
94.
98.
80.
92.
100
99.
99.
98.
90.
99.
99.
94.
80.
98.
95.
52.
100
46.
2
1
3
1

6
4
5
5
4
7
3
3
7
3
2

3
Dying
number
26
4
35
25
0
1
1
0
26
1
1
11
35
1
10
11
0
20
Dead Dead Abnormal
number (%) number
0
3
26
9
0
0
0
2
3
0
0
0
26
3
2
21
0
24
0
0.8
8.4
2.1
0
0
0
1.5
1.0
0
0
0
8.4
1.0
0.8
31.3
0
29.3
103
30
7
39
3
1
1
16
C8
10
7
0
53
32
31
28
0
12
Est.
hatch Comments
number
342
337
248 Can
391
74 Can, scat
278
157
115
216
158
302 Can
181
248
266
222 Can
35 Can
152 Hatch
38 Aban
                a Can, male eating developing embryos.
                b Hatch, embryos hatched into larvae.
                c Scat, brood scattered throughout tube.
                d Aban, male abandoned the brood.
                e L, original data sheet lost.

-------
               Table A-2.   Experimental data  from Neanthes  arenacgodentata mated pairs  that were exposed  to 0.19 mGy/h from an external gamma-
               radiation  source.   The number  of days  from spawn  to hatch and  from hatch to spawn as well  as the estimated hatch number
               are provided.
O>
P] Spawn
to
F] hatch
(days)
5
5
5
5
5
5
5
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
Fj Hatch
to ID
F] spawn
(days)
126
159
146
129
146
136
140
143
109
122
144
114
137
108
121
108
no
108
108
108
110
108
103
108
108
131
110
108
1-2-15
1-2-18
1-2-35
1-2-39
1-2-40
1-2-46
1-2-50
5-1-2
5-l-3a
5-1-4
5-1-11
5-1-12
5-1-16
25-3-1
25-3-2
25-3-4
25-3-6
25-3-11
25-3-19
25-3-23
25-3-24
25-3-26
25-3-29
25-3-34a
25-3-39
25-3-42a
25-3-42b
25-3-43
Spawn Live Live
number number (%)
139
294
286
227
182
231
154
209
241
58
373
281
193
132
133
209
0
163
148
121
208
169
322
162
160
139
169
174
139
170
192
209
143
169
77
161
239
32
270
276
163
129
133
209
0
163
148
110
206
154
319
159
158
118
167
174
100
57.8
67.1
92.1
78.6
73.2
50.0
77.0
99.2
55.2
72.4
98.2
84.5
97.7
100
100
0
100
100
90.9
99.0
91.1
99.1
98.1
98.8
84.9
98.8
100
Dying
number
0
48
47
11
28
33
43
20
2
15
68
2
22
3
0
0
0
0
0
6
2
0
0
3
2
14
2
0
Dead Dead Abnormal
number (%) number
0
54
47
7
11
29
34
28
0
11
35
3
8
0
0
0
0
0
0
5
0
15
3
0
0
7
0
0
0
18.4
16.4
3.1
6.0
12.6
22.1
13.4
0
19.0
9.4
1.1
4.2
0
0
0
0
0
0
4.1
0
8.9
0.9
0
0
5.0
0
0
0
94
171
72
109
104
77
121
2
8
177
7
40
6
0
0
0
0
0
7
12
5
6
13
0
46
15
0
Est.
hatch Comments
number
139
192
115
155
73
127
77
88
239
32
196
274
153
126
133
209
0
163
148
110
196
154
316
149
158
93
154
174
Hatch*




Can


Can5
Can

Hatch

Hatch
Hatch
Hatch
Can
Hatch
Hatch
Can




Hatch


Hatch

-------
Table A-2.   (cont.)
Pj Spawn
to
F] hatch
(days)
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
12
12
12
12
12
12
12
12
12
F] Hatch
to ID
Fj spawn
(days)
108
109
132
110
114
119
133
103
129
125
108
125
125
107
112
125
151
128
101
108
123
105
107
105
118
110
102
100
107
25-3-45
25-3-47
25-3-48
26-4-3
26-4-5
26-4-7a
26-4-7b
26-4-10
26-4-13
26-4-18
26-4-20a
26-4-23a
26-4-23b
26-4-27
26-4-30
26-4-33
26-4-34b
26-4-35
26-4-38a
26-4-40
27-5-1
27-5-3
27-5-4
27-5-7
27-5-9
27-5-13
27-5-14
27-5-16
27-5-2C
Spawn Live Live
number number (%)
194
232
221
231
132
192
178
227
289
315
152
392
83
181
21
271
234
187
304
190
322
203
185
116
405
222
207
182
96
194
209
183
224
127
181
128
227
243
287
107
17
27
180
21
253
75
156
303
190
258
198
185
116
331
210
185
174
96
100
90.1
82.8
97.0
96.2
94.3
71.9
100
84.1
91.1
70.4
4.3
32.5
99.4
100
93.4
32.1
83.4
99.7
100
80.1
97.5
100
100
81.7
94.6
89.4
95.6
100
Dying
number
0
22
28
6
2
6
39
0
24
23
45
169
12
0
0
16
72
15
0
0
52
2
0
0
52
10
22
7
0
Dead Dead Abnormal
number {%) number
0
1
10
1
3
5
11
0
22
5
0
206
44
1
0
2
87
16
1
0
12
3
0
0
22
2
0
1
0
0
0.4
4.5
0.4
2.3
2.6
6.2
0
7.6
1.6
0
52.6
53.0
0.6
0
0.7
37.2
8.6
0.3
0
3.7
1.5
0
0
5.4
0.9
0
0.5
0
4
26
138
13
19
57
65
0
77
164
11
256
80
1
0
140
80
2
12
0
88
5
23
0
363
49
5
13
0
Est.
hatch Comments
number
190
206
83
218
113
135
113
227
212
151
107
17
3
180
21
131
75
156
292
190
234
198
162
116
42
173
185
169
96

Can
Can
Can



Hatch


Can


Hatch
Hatch
Can



Hatch

Hatch
Can
Hatch

Can

hatch
Hatch

-------
               Table A-2.   (cont.)
i
00
Pj Spawn
to
F, hatch
(days)
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
F1 Hatch
to ID
F] spawn
(days)
146
112
131
153
122
126
122
126
142
170
139
107
122
112
138
126
138
87
97
132
105
128
131
136
131
133
131
29-7-2b
29-7-3
29-7-5
29-7-6
29-7-7b
29-7-8
29-7-9
29-7-10
29-7-1 la
29-7-12b
29-7-14
29-7-16
29-7-17
29-7-19
29-7-25
29-7-28b
29-7-29
31-8-x
31-8-4a
31-8-5
31-8-7
31-8-9a
31-8-9b
31-8-13a
31-8-14
31-8-15
31-8-27
Spawn Live Live
number number (X)
326
99
197
494
231
309
125
127
226
140
507
227
230
127
256
314
217
272
163
278
201
306
229
71
158
84
344
302
98
163
384
83
309
105
119
122
140
403
226
151
127
215
219
199
272
96
278
109
104
201
A
158
11
341
92.6
99.0
82.7
77.7
35.9
100
84.0
93.7
54.0
100
79.5
99.6
65.7
100
84.0
69.7
91.7
100
58.9
100
54.2
34.0
87.8
5.6
100
13.1
99.1
Dying
number
16
1
11
36
117
0
13
7
82
0
47
0
7)
0
32
69
12
0
51
0
79
101
15
21
0
39
3
Dead Dead Abnormal
number (%) number
8
0
23
74
31
0
7
1
22
0
57
1
8
0
9
26
6
0
16
0
13
101
13
46
0
34
0
2.5
0
11.7
15
13.4
0
5.6
0.8
9.7
0
11.2
0.4
3.5
0
3.5
8.2
2.8
0
9.8
0
6.5
33.0
5.7
64.8
0
40.5
0
97
4
51
115
168
0
82
46
143
0
299
62
165
0
57
223
35
0
1
0
98
306
0
71
0
60
3
Est.
hatch Comments
number
229
95
146
379
63
309
43
81
83
140
208
165
65
127
199
91
182
272
96
278
103
0
201
0
158
3
341

Hatch


Can
Hatch

Can

Hatch


Can
Hatch



Hatch

Hatch
Can

Hatch
Can
Hatch

Hatch
                  Hatch,  embryos  hatched  into  larvae.

                  Can,  male eating  developing  embryos.

-------
            Table A-3.  Exparimental data from Neanthes arenaceodentata mated pairs that were exposed to 2,1 mGy/h from an external gamma-
            radiation source.  The number of days from spawn to hatch and from hatch to spawn as well as the estimated hatch number
            are provided.
vo
P] Spawn
to
F, hatch
(days)
12
12
12
12
12
12
12
12
12
12
12
12
12
12
13
13
13
13
13
13
13
13
13
13
13
13
13
13
F] Hatch
to 10
F] spawn
(days)
115
121
127
119
125
119
123
123
128
151
125
118
137
125
114
123
118
123
125
118
121
113
121
125
120
119
115
115
11-4-1
11-4-5
11-4-8
11-4-11
l)-4-14a
11-4-16
11-4-17
11-4-22
11-4-25
11-4-33
11-4-35
11-4-45
11-4-52
11-4-53
16-2-3
16-2-7
16-2-1 la
16-2-1 Ib
16-2-14
16-2-21
16-2-23
16-2-34
16-2-39
16-2-41
16-2-51
16-2-52
16-2-53
16-2-57
Spawn Live Live
number number (%)
188
116
297
165
113
189
189
201
212
478
62
220
181
202
256
329
248
187
109
132
171
153
227
336
232
219
202
205
171
0
89
153
43
175
58
201
104
319
29
186
100
116
245
206
78
151
62
121
108
134
209
94
208
185
182
198
91
0
30
92
38
92
30
100
49
66
46
84
55
57
95
62
31
80
56
92
63
87
92
.0

.0
.7
.1
.6
.7

.1
.7
.8
.5
.2
.4
.7
.6
.8
.7
.9
.1
.2
.6
.1
28.0
89
84
90
96
.7
.5
.1
.6
Dying
number
17
103
157
2
57
4
96
0
17
109
8
25
34
41
10
120
132
21
18
4
13
18
11
181
7
10
18
7
Dead Dead Abnormal
number ('/.) number
0
13
51
10
13
10
35
0
91
50
25
9
47
45
1
3
38
15
29
7
50
1
7
61
17
24
2
0
0
11.2
17.2
6.1
11.5
5.3
18.5
0
42.9
10.5
40.3
4.1
26.0
22.3
0.4
0.9
15.3
8.0
26.6
5.3
29.2
0.6
3.1
18.2
7.3
11
1
0
20
55
90
48
53
28
78
8
138
177
31
38
110
86
11
165
127
120
96
61
79
0
72
213
20
30
20
13
Est.
hatch Comments
number
168 Cana
0
89 Can
117
43 Can
161
58
193 Hatchb
74
301
29 Can
182
71 Abanc
116 Can
245
164
78
67 Can
13
71
92.
134
155
94
208
185
182
192 Can

-------
           Table  A-3.   (cont.)
I
>'
o
Pj Spawn
to
F) hatch
(days)
12
12
12
12
12
13
13
13
13
13
13
13
13
13
13
13
13
13
13
11
11
11
11
11
11
11
11
F] Hatch
to ID
f] spawn
(days)
151
106
121
151
127
128
113
119
114
125
119
119
124
116
134
116
119
120
142
105
117
111
118
118
120
119
105
20-1-1
20-1-10
20-1-16
20-1-24
20-1-31
21-3-2
21-3-5
21-3-6
21-3-8
21-3-9
21-3-10
21-3-13
21-3-17
21-3-18
21-3-19
21-3-20
21-3-24
21-3-26
21-3-27
23-5-1
23-5-3
23-5-12
23-5-12a
23-5-12b
23-5-13
23-5-19
23-5-23
Spawn Live Live
number number (%)
251
73
210
364
180
159
237
69
278
320
277
357
322
247
374
169
289
221
172
111
251
199
207
177
3S8
335
133
24
72
167
5
96
30
228
31
260
230
161
268
101
247
289
147
256
109
106
108
129
179
98
81
215
300
132
9.6
98.6
79.5
1.4
53.3
18.9
96.2
44.9
93.5
71.9
58.1
75.1
31.4
100
77.3
87.0
88.6
49.3
61.6
97.3
51.4
89.9
47.3
45.8
55.4
89.6
99.2
Dying
number
65
0
19
246
67
49
7
4
14
20
22
39
42
0
64
13
31
106
52
2
25
13'
47
59
106
21
1
Dead Dead Abnormal
number ('/.) number
162
1
24
80
17
80
2
34
4
70
94
50
179
0
21
9
2
6
14
1
97
7
62
37
67
14
0
64.8
1.4
11.4
22
9.4
50.3
0.8
49.3
1.4
21.9
33.9
14.0
55.6
0
5.6
5.3
0.7
2.7
8.1
0.9
38.6
3.5
30
20.9
17.3
4.2
0
192
1
88
217
122
69
24
55
6
287
148
242
304
9
238
30
121
78
103
9
26
61
145
163
259
301 A
6JF
Est.
hatch Comments
number
24
72 Hatch
122
 -5
0 - .
30
213 Can
0 Can
260
0
129
115
0
238 Hatch
135 Can
138
168
109
69
102 Jf
129 ^F
138 ^J^
JMT

jWz9 Can
Y 34
127

-------
Table A-3,  (cent.)
P| Spawn   Fj  Hatch
    to         to       10
F] hatch   Fj  spawn
  (days)       (days)
Spawn    Live  Live
number  number (X)
                                Est.
Dying    Dead  Dead  Abnormal   hatch   Comments
number  number (%)     number   number
12
12
12
12
12
12
12
12
12
12
12
12
12
13
13
13
13
13
13
13
13
13
13
13
13
128
99
108
109
100
101
111
106
114
109
105
101
99
176
168
199
182
174
168
192
180
173
174
173
166
24-6-2
24-6-5
24-6-8
24-6-13
24-6-17
24-6-27
24-6-28a
24-6-35a
24-6-36
24-6-37
24-6-39
24-6-43
24-6-47
27-8-1
27-8-2
27-8-3
<>7-8-4
27-8-6
27-8-7
27-8-20
27-8-21
27-8-22
27-8-23
27-8-24
27-8-25
179
158
259
285
188
268
167
252
302
282
187
221
139
152
122
388
508
282
188
38
315
249
267
249
262
170
153
239
84
155
266
99
229
221
232
168
195
138
150
38
337
114
282
188
20
315
245
245
247
160
95.0
96.8
92.3
29.5
82.4
99.3
59.3
90.9
73.2
82.3
89.9
88.2
99.3
98.7
31.2
86.9
22.4
0
100
52.6
100
98.4
91.8
99.2
61.1
1
5
2
81
33
2
56
13
52
39
8
26
0
2
57
50
308
0
0
14
0
0
17
1
45
8
0
18
120
0
0
12
10
29
11
11
0
1
0
27
1
86
0
0
4
0
4
5
1
57
4.5
0
7
42.1
0
0
7.2
4
9.6
3.9
5.9
0
0.7
0
22.1
0.3
16.9
0
0
10.5
0
1.6
1.9
0.4
21.8
56
0
103
266
17
33
90
31
209
174
45
22
5
1
93
72
271
0
0
34
315
0
7
0
24
123
153
156
19
155
235
77
221
93
108
142
195
134
150
29
316
114
282
188
4
0
245
245
247
160
Hatch

Can

Can



Can

Hatch

Hatch
Hatch


Scatd
Hatch
Hatch
Can
Scat
Hatch
Hatch
Hatch

  d  Can. male eating developing embryos.
  k  Hatch, embryos hatched into larvae.
  c  Aban, male abandoned the brood.
    Scat, brood scattered in the tube.

-------
               Table A-4.  Experimental data  from Neanthes  arenaceodentata mated  pairs  that were exposed  to  17 mGy/h  from an external  gamma-
               radiation  source.  The number  of  days  from spawn  to  hatch  and  from hatch to spawn as well  as  the estimated hatch number
               are provided.
i
>
ro
P) Spawn
to
F| hatch
(days)
9
9
9
9
9
9
9
9
9
9
15
15
15
15
15
15
15
15
15
15
15
15
15
F, Hatch
to ID
F] spawn
(days)
128
139
137
130
127
140
131
137
140
120
125
133
136
142
121
124
135
139
123
116
131
125
125
4-1-8
4-1-14
4-1-15
4-1-16
4-1-17
4-1-21
4-1-22
4-1-23
4-1-33
4-1-35
10-4-2
10-4-3
10-4-4
10-4-7
10-4-12
10-4-14
10-4-18a
10-4-19
10-4-20
10-4-31
10-4-32
10-4-35
10-4-40
Spawn
number
278
190
74
190
397
203
109
74
186
3
107
147
109
132
372
261
261
85
523
161
91
186
349
Live
number
0
98
0
63
87
3
34
2
38
1
82
19
19
0
190
49
7
37
55
138
6
21
48
Live
(*)
0
51.6
0
33.2
9.1
1.5
31.2
2.7
20.4
33.3
76.6
12.9
17.4
0
51.1
18.7
2.7
43.5
10.5
85.7
6.6
11.3
13.8
Dying
number
0
56
6
60
!44
130
28
39
106
1
12
73
52
10
50
209
69
27
215
19
66
73
192
Dead Dead Abnormal
number (%) number
278
36
68
67
166
70
47
33
42
1
13
55
38
122
132
3
185
21
253
4
19
92
109
100
19.0
91.9
35.3
41.8
34.5
43.1
44.6
22.6
33.3
12.2
37.4
34.9
92.4
35.5
1.2
70.9
24.7
48.4
2.5
20.9
49.5
31.2
270
183
74
190
315
203
103
74
186
2
67
147
107
132
281
257
260
85
523
44
91
186
333
Est.
hatch Comments
number
0
7
0
0
82
0
6
0
0
1
40
0
2
0
91
4
1
0
0
117
0
0
16
Cana
Abanb
Can
Can

Scatc
Can
Can
Can
Can
Can
Can
Scat
Can
Can
Scat
Can
Can
Can
Can
Can
Can
Can

-------
              Table A-4  (cont.)
3*
i
Pj Spawn
to
F] hatch
(days)
11
11
11
11
11
11
11
11
11
11
11
f} Hatch
to
F] spawn
(days)
141
159
139
143
137
134
134
145
142
142
147

10


11-2-2
11-2-13
11-2-15
11-2-17
11-2-24
11-2-26
11-2-27
11-2-29
11-2-30
11-2-39
11-2-40

Spawn
number

376
313
230
185
199
182
132
187
308
549
168

Live
number

54
5
19
29
128
88
33
18
171
304
19

Live
(%)

14.4
1.6
8.3
15.7
64.3
48.4
25.0
9.6
55.5
55.4
11.3

Dying
number

237
148
137
122
39
84
82
63
99
81
70

Dead
number

85
160
74
34
32
10
17
106
38
164
79

Dead
(X)

22.6
51.1
32.2
18.4
16.1
5.5
12.9
56.7
12.3
29.9
47

Abnormal
number

210
313
214
175
187
164
90
187
285
547
168
Est.
hatch
number

54
0
16
10
12
18
33
0
23
2
0

Comments


Can
Can
Can
Can
Can
Can
Can
Can
Can
Can

                d Can,  male eating developing  embryos.

                b Aban, male abandoned  the  brood.

                c Scat, brood scattered in  the tube.

-------
Table A-4,  (cont.)

        All of these females resorbed their oocytes and then died.



  10       Comments
4-1-2
4-1-4
4-1-7
4-1-9
4-1-18
4-1-20
4-1-26
4-1-27
4-1-30
4-1-31
4-l-32a
4-1-36

10-4-1
10-4-8
10-4-9
10-4-13
10-4-22
10-4-23
10-4-30
10-4-37

11-2-3
11-2-7
11-2-32
11-2-36
11-2-38

-------