520-1-88-004
UCRL—15988
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 + 2°C,
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
-------
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
-------
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
-------
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
-------
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
-------
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.
-------
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
-------
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
-------
REFERENCES
Anderson, S.L., and F.L. Harrison (1986), "Effects of Radiation on Aquatic
Organisms and Radiobiological Methodologies for Effects Assessment," EPA
520/1-85-016 (U.S. Environmental Protection Agency, Washington, DC), 128 pp.
\
Anderson, S.L., F.L. Harrison, G. Chan, and D.H. Moore II (1987), "Chromosomal
Aberrations, Reproductive Success, Life Span, and Mortality in Irradiated
Neanthes arenaceodentata (Polychaeta)," EPA 520-1-87-007 (U.S. Environmental
Protection Agency, Washington, DC), 55 pp.
Blaylock, B.C., and J.R. Trabalka (1978), "Evaluating the Effects of Ionizing
Radiation on Aquatic Organisms," in Advances in Radiation Biology, Vol. 7,
J.T. Lett and H. Adler, Eds. (Academic Press, New York, NY), pp. 103-152.
Bonham, K., and L.R. Donaldson (1972), "Sex Ratios and Retardation of Gonadal
Development in Chronically Gamma-Irradiated Chinook Salmon Smolts," Trans.
Am. Fish. Soc. 101. 428-434.
Cervini, A., and S. Giavelli (1965), "Radiosensitivity of Different Meiotic
Stages of Oocytes in Parthenogenetic Diploid Artemia salina Leach," Mutat.
Res. 2, 452-456.
Chipman, W.A. (1972), "Ionizing Radiation," in Marine Ecology, Vol. I, Pt. 3,
Ch. 11, 0. Kinne, Ed. (John Wiley/Interscience, New York, NY), pp. 1579-1657.
Cooley, J.L. (1973), "Effects of Chronic Environmental Radiation on a Natural
Population of the Aquatic Snail Physa heterostropha." Radiat. Res. 54, 130-140.
Cooley, J.L., and F.L. Miller, Jr. (1971), "Effects of Chronic Irradiation on
Laboratory Populations of the Aquatic Snail Physa heterostropha," Radiat. Res.
47, 716-724.
Cosgrove, G.E., and B.G. Blaylock (1973), "Acute and Chronic Irradiation
Effects in Mosquitofish at 15 or 25°C," in Radipnuclides in Ecosystems,
Vol. 1, D.J. Nelson, Ed. (U.S. Atomic Energy Commission, Washington, DC), pp.
579-584.
Dobson, R.I,., T.C. Kwan, and T. Straume (1984), "Tritium Effects on Germ Cells
and Fertility," in Proceedings of European Seminar on Risks from Tritium
Exposure. G. Gerber and C. Myttanaere, Eds. (Commission of the European
Communities, Luxenbourg), pp. 285-296.
Dobson, R.L., T.C. Kwan, B.F. Brunchkhorst, and T, Straume (1986), "Diminished
Lifetime Reproductive Capacity in the Female Following Early Radiation
Exposure," in Life-Span Radiation Effects Studies in Animals: What Can They
Tell Us? R.C. Thompson and J.A. Mahaffey, Eds. CONF-830951 (U.S. Department
of Energy, Washington, DC), pp. 281-294.
Egami, N., and K.-I. Ijiri (1979), "Effects of Irradiation on Germ Cells and
Emb-yonic Development in Teleosts," Int. Rev. Cytol. 59, 195-248.
33
-------
Greenberger, J.S., J. Pechenik, A. Lord, L. Gould, E. Naperstek, K. Kase, ana
T.J. FitzGerald (1986), "X-Irradiation Effects on Growth and Metamorphosis of
Gastropod Larvae (Crepidula fornicata): A Model for Environmental Radiation
Teratogenesis," Arch. Environ. Contain. Toxicol. J_5, 227-234.
Harrison, F.L., and S.A. Anderson (1988), "The Effects of Acute"'Radiation on
Reproductive Success of the Polychaete Worm Neanthes arenaceodentata," EPA
520/1-88-003 (U.S. Environmental Protection Agency, Washington, DC), 21 pp.
Harrison, F.L., D.W. Rice, Jr., O.H. Moore, and M. Varela (1986), "Effects of
Radiation on Frequency of Chromosomal Aberrations and Sister Chromatid
Exchange in the Benthic Worm Neanthes arenaceodentata," in Wastes in the
Ocean: Biological Effects of Waste Disposal, Volume 9 (Robert E. Krieger,
Malabar, FL).
Hoppenheit, M. (1973), "Effects of Fecundity and Fertility of Single
Sub-Lethal X-Irradiation of Gammarus duebeni Females," in Proc. Symp.
Radioactive Contamination of the Marine Environment (International Atomic
Energy Agency, Vienna), pp. 479-486.
Hyodo-Taguchi, Y., and N. Egami (1977), "Damage to Spermatogenesis in Fish
Kept in Tritiated Water," Radiat. Res. 71, 641-652.
Hyodo-Taguchi, Y. (1980), "Effects of Chronic Y-lrradiation on
Spermatogenesis in the Fish, Qryzias latipes, with Special Reference to
Regeneration of Testicular Stem Cells," in Radiation Effects on Aquatic
Organisms, N. Egami, Ed. (Japan Scientific Societies Press, Tokyo; University
Park Press, Baltimore, MD), pp. 91-104.
National Research Council, Committee on the Biological Effects of Ionizing
Radiations (1980), The Effects on Populations of Exposure to Low Levels of
Ionizing Radiation: 1980 (National Academy Press, Washington, DC).
Ophel, I.L. (1976), "Effects of Ionizing Radiation on Aquatic Organisms," in
Effects of Ionizing Radiation on Aquatic Organisms and Ecosystems. Technical
Report Series 172 (International Atomic Energy Agency, Vienna), pp. 57-88.
Oshida, P., C.S. Ward, and A. Mearns (1981), "Effects of Hexava1 >nt and
Trivalent Chromium on the Reproduction of Neanthes arenaceodontata
(Polychaeta)," Mar. Environ. Res. 5, 41-49.
Oshida, P., and C.S. Ward (1982), "Bioaccumulation of Chromium and its Effects
on the Reproduction of Neanthes arenaceodentata (Polychaeta)," Mar. Environ.
Res. 7, 167-174.
Polikarpov, G.G. (1966), Radioecology of Aquatic Organisms (Reinhold,
New York, NY), 210 pp.
Rachham, B.D., and D.S. Woodhead (1984), "Effects of Chronic Y-lrradiation on
the Gonads of Adult Arneca splendens (Osteichthyes:Teleostei)," Int. J.
Radiat. Biol. 45, 645-656.
Ravera, 0. (1967), "The Effect of X-Rays on the Demographic Characteristics of
Pjvysa acuta (Gastropoda: Basommatophora)," Malacologia 5, 95-109.
34
-------
Reish, D.J. (1974), "The Establishment of Laboratory Colonies of Polychaetous
Annelids," Thalassia Jugosl. ]0, 181-195.
Rossi, S.S., and J.W. Anderson (1978), "Effects of No. 2 Fuel Oil-Water-
Soluble-Fractions on Growth and Reproduction in Neanthes arenaceodentata
(Polychaeta:annelida)," Water Air Soil Pollut. 9_, 155-170.
Snedecor, G.W., and W.G. Cochran (1967), Statistical Methods (The Iowa State
University Press, Ames, Iowa, 6th Ed.), 593 pp.
Straume, T., R.L. Dobson, and T.C. Kwan (1987), "Neutron RBE and the
Radiosensitive Target for Mouse Immature Oocyte Killing," Radiat. Res. 111.
47-57.
Straume, T., R.L. Dobson, and T.C. Kwan (1988), "Size of Lethality Target in
Mouse Immature Oocytes Determined with Accelerated Heavy Ions. Radiat. Env.
Biophys. (in press).
Templeton, W.L. (1976), "Effects of Ionizing Radiation on Aquatic Populations
and Ecosystems," Technical Report Series 172 (International Atomic Energy
Agency, Vienna), pp. 89-119.
Templeton, W.L., R.E. Nakatani, and E.E. Held (1971), "Radiation Effects," in
Radioactivity in the Marine Environment (National Academy of Sciences,
Washington, DC), pp. 223-239.
Trabalka, J.R., and C. P. Allen (1977), "Aspects of Fitness of a Mosquitofish
Gambusia affinis Population Exposed to Chronic Low-Level Environmental
Radiation," Radiat. Res. 70, 198-211.
United Nations Scientific Committee on the Effects of Atomic Radiation (1977),
Sources and Effects of Ionizing Radiation, E.77.IX.1 (United Nations
Publications, New York, NY).
United Nations Scientific Committee on the Effects of Atomic Radiation (1982),
Ionizing Radiation: Sources and Biological Effects, E.82.IX.8 (United Nations
Publications, New York, NY).
United Nations Scientific Committee on the Effects of Atomic Radiation (1986),
Genetic and Somatic Effects of Ionizing Radiation. E.86.IX.9 (United Nations
Publications, New York, NY).
Welander, A.D., L.R. Donaldson, R.F. Foster, K. Bonham, and A.M. Seymour
(1948), "The Effects of Roentgen Rays on the Embryos and Larvae of the Chinook
Salmon," Growth 12, 203-242.
Woodhead, D.S. (1977), "The Effects of Chronic Irradiation on the Breeding
Performance of the Guppy, Poecilia reticulata (Osteichthyes: Teleostei)," Int.
J. Radiat. Biol. 32, 1.
Woodhead, D.W. (1984), "Contamination Due to Radioactive Materials," in Marine
Ecology. Vol. V, Part 3, 0. Kinne, Ed. (John Wiley and Sons, Ltd., Chichester,
UK), pp. 1287-1618.
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
------- |