United States Office of EPA/520/1-88-003
Environmental Protection Radiation Programs December 1988
Agency Washington, DC 20460
Radiation
The Effects of
Acute Radiation on
Reproductive Success
of the Polychaete Worm
Neanthes arenaceodentata
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THE EFFECTS OF ACUTE RADIATION ON REPRODUCTIVE SUCCESS
OF THE POLYCHAETE NORM NEANTHES ARENACEODENTATA
Florence L. Harrison and Susan L. Anderson
Environmental Sciences Division
Lawrence Livermore National Laboratory
Livermore, CA 94550
Marilyn E. Varela
Project Officer
Report prepared for the
Office of Radiation Programs
U.S. Environmental Protection Agency
Nashington, DC 20460
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-52n/i-a8-nrn
2.
3. RECIPIENT'S ACCESSION NP.
4. TITLE AND SUBTITLE
The Effects of Acute Radiation on Reproductive Success
of the Polychaete Worm Neanthes arenaceodentata
5. REPORT DATE
December, 1988
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
F.L. Harrison and S.L, Anderson
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Sciences Division
Lawrence Livermore National Laboratory
Livermore, CA 94550
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. EPA
Office of Radiation Programs
401 M Street, SW
Ua a Vi i T-LCT 1- nn - DP. ?DZl.fifl
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
15.'S~UPPLEttfrENTA'RY NOTES
16. ABSTRACT
Laboratory populations of the polychaete worm Neanthes arenaceodentata were exposed
to acute doses of external gamma radiation to determine effects on reproduction.
Groups of mated pairs received either no radiation (controls) or 0.5, 1.0, 2.0, 5.0,
10 or 50 Gy. The doses were delivered at the time when oocytes were visible in
the females and at a rate of 5 Gy/min. The broods from the mated pairs were^
sacrificed before hatching occurred, and information was obtained on brood size,
on the number of normal and abnormal embryos, and the number of embryos that
were living, dying, and dead.
An important effect of acute irradiation was increase mortality of the embryos.
Except for those mated pairs that received 10 or 50 Gy, there was no evidence for
gamete loss or for reduced fertilization success; the number of embryos in the
brood did not decrease x*ith increase dose. The results on embryo abnormality and
mortality indicate that lethal mutations were most likely induced in the germ
cells and that these affected survival of the early life stages.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
COS AT I Field/Group
Radiation
Reproduction
Worms
Embryos
Dosage
518. DISTRIBUTION STATEMENT
Release Unlimited
j 19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
S3
\20. SECURITY CLASS (This page)
22. PRICE
EPA Form 2220-1 (Rev. 4—77} PREVIOUS EDITION is OBSOLETE
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FOREWORD
In response to the mandate of Public Law 92-532, the
Marine Protection, Research, and Sanctuaries Act (MPRSA) of
1972, as amended, the Environmental Protection Agency (EPA)
developed a program to promulgate regulations and criteria to
control the ocean disposal of 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 of 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 materials into the deep-sea
environment may occur when a package deteriorates. Therefore,
methods for assessing 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 material 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 (ORP)
has had an ongoing program to examine sublethal responses to
radiation at the cellular level, using cytogenetic endpoints.
The results presented in this report demonstrate the effects
that acute ionizing radiation has on the reproductive success of
a low-fecund marine invertebrate species, the polychaete
Neanthes arenaceodentata. Data were obtained on brood size,
abnormal development, and the number of living, dead, or dying
embryos within the brood.
The results of radionuclide effects 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 in comparison of the risks of several
contaminant classes.
The Agency invites all readers of this report to send any
comments or suggestions to Mr. David E. Janes, Director,
Analysis and Support Division, Office of Radiation Programs
(ANR-461), U.S. Environmental Protection Agency, Washington, DC
20460.
Richard J. Gkaimond, Director
Office of Radiation Programs
111
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TABLE OF CONTENTS
Foreword Ill
List of Figures v
List of Tables vi
Abstract 1
1. Introduction 2
2. Material s and Methods 2
2.1 Experimental Approach 2
2.2 Animal Sources, Culture Conditions, and Irradiation 4
2.3 Brood Analysis 5
3. Results 8
3.1 Brood Size 8
3.2 Living Embryos in Broods 11
3.3 Abnormal Embryos i n Broods 11
3.4 Reduced Survival of Embryos 17
4. Discussion 17
Acknowl edgments 25
References 26
Appendix A. Data Base from the Experiments to Determine
the Effect of Acute Radiation on Reproductive Success
of Neanthes arenaceodentata A-l
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LIST OF FIGURES
Embryo abnormalities Identified in sacrified broods.
Normal cleavage pattern (a), atypical cleavage pattern
(b), and embryos with void regions (c) are shown ,
2. Broods subjected to trypan-blue-exclusion test were
differentiated into embryos that were (a) alive
(free of blue color), (b) dying (partially stained blue),
and (c) dead (stained totally blue)
3. The percent of broods in each of four categories
(n > 150, 150 > n _> 100, 100 > n _> 50, and n < 50)
of numbers of embryos i n the broods
4. The percent of broods in each of the four categories
(n > 75%, 75% > n > 50%, 50% > n > 25%, n < 25%) of
percentages of live embryos i n the brood 13
5. The percent of broods in each of the four categories
(n > 75%, 75% > n > 50%, 50% > n > 25%, n < 25%) of
percentages of abnormal embryos i n the brood 16
6. The percent of broods in each of the four categories
(n > 150, 150 > n > 100, 100 > n 2 50, n < 50) of numbers
of actual and estimated hatchlings in the brood 19
7. The percent of broods in each of the four categories
(n > 75%, 75% > n > 50%, 50% > n >_ 25%, n < 25%) of percent
of survival to hatching of the embryos in the broods 21
8. Mean survival of embryos (expressed as percentage
of the survival fraction of the controls) as a function
of acute radiation dose. The survival curve appears to be
biphasic. Data from broods that hatched or that were
harvested before day 3 were excluded 23
VI
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LIST OF TABLES
1. Number of developing embryos in broods from control and
radiation-exposed mated pairs 3
2. Steps in the procedure used to harvest the broods from the mated
pairs. The harvest was performed from about 4 to 6 d after
spawni ng 8
3. 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, n < 50), according to the number of embryos in the
brood 9
4. Results from the analysis of the living, dying, and dead embryos
in the 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 > 75%, 75% > n _> 50%, 50% > n _>
25%, n < 25%), according to the percentage of living embryos
i n the brood 12
5. Results from the analysis of the normal and abnormal embryos in
the 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, n < 50), according to the numbers of abnormal
embryos in the brood 14
6. Results from the analysis of the 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 percents were
assigned to one of four following categories (n > 75%, 75% > n _> 50%,
50% > n > 25%, n < 25%), according to the percentage of abnormal
embryos i n the brood 15
7. Results from the analysis of the numbers of embryos that actually
hatched or were estimated to hatch from the broods of the control
and radiation-exposed mated pairs. The broods were distributed
to one of four categories (n .> 150, 150 > n >_ 100, 100 > n _> 50,
n < 50), according to the number of actual or estimated hatchlings
i n the brood 18
8. Results from the analysis of survival to hatching of embryos
on the broods of the control and radiation-exposed mated pairs.
The percent survival was calculated by dividing the estimated hatch
size by the brood size, and then the percent survival was assigned
to one of four of the following categories (n _> 75%, 75% > n > 50%,
50% > n 2 25%, n < 25%), according to the percentage of survival
of the embryos 20
Vll
-------
A-l. Experimental data from Neanthes arenaceodentata mated pairs that
were not irradiated with an external gamma-radiation source
(controls). The number of days from the date of radiation of the
irradiated mated pairs to spawn (R to S) and from spawn
to sacrifice (S to S) as well as the estimated hatch size (EHS)
are provided A-2
A-2. Experimental data from Neanthes arenaceodentata mated pairs
that received an acute dose of 0.5 Gy from an external gamma-radiation
source. The number of days from radiation to spawn (R to S)
and from spawn to sacrifice (S to S) as well as the estimated
hatch size (EHS) are provided A-6
A-3. Experimental data from Neanthes arenaceodentata mated pairs
that received an acute dose of 1.0 Gy from an external gamma-radiation
source. The number of days from radiation to spawn (R to S)
and from spawn to sacrifice (S to S) as well as the estimated
hatch size (EHS) are provided A-8
A-4. Experimental data from Neanthes arenaceodentata mated pairs
that received an acute dose of 2.0 Gy from an external gamma-radiation
source. The number of days from radiation to
spawn (R to S) and from spawn to sacrifice (S to S) as well
as the estimated hatch size (EHS) are provided A-ll
A-5. Experimental data from Neanthes arenaceodentata mated pairs
that received an acute dose of 5.0 Gy from an external gamma-radiation
source. The number of days from radiation to spawn (R to S)
and from spawn to sacrifice (S to S) as well as the estimated
hatch size (EHS) are provided A-13
A-6. Experimental data from Neanthes arenaceodentata mated pairs
that received an acute dose of 10 Gy from an external gamma-radiation
source. The number of days from radiation to spawn (R to S)
and from spawn to sacrifice (S to S) as well as the estimated
hatch size (EHS) are provided A-l6
A-7. Experimental data from Neanthes arenaceodentata mated pairs
that received an acute dose of 50 Gy from an external gamma-radiation
source. The number of days from radiation to spawn (R to S)
and from spawn to sacrifice (S to S) as well as the estimated
hatch size (EHS) are provided A-l9
VI11
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ABSTRACT
Laboratory populations of the polychaete worm Neanthes arenaceodentata
were exposed to acute doses of external gamma radiation to determine effects
on reproduction. Groups of mated pairs received either no radiation
(controls) or 0.5, 1.0, 2.0, 5.0, 10, or 50 Gy. The doses were delivered at
the time when oocytes were visible in the females and at a rate of
5 Gy/min. The broods from the 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 numbers of embryos in the broods from the mated pairs exposed to
radiation were not significantly different from the mean number from control
mated pairs, except for the groups receiving 10 or 50 Gy. However, there was
a significant reduction in the percentage of live embryos in the broods from
mated pairs that received doses as low as 0.5 Gy. Also, increased percentages
of abnormal embryos were found for the mated pairs exposed to 2.0, 5.0, 10, or
50 Gy. From data on estimated hatch size and actual hatch size, we determined
that only the groups receiving doses as low as 0.5 Gy were significantly
different from controls.
An important effect of acute irradiation on mated pairs of
N. arenaceodentata was increased mortality of the embryos. Except for those
mated pairs that received 10 or 50 Gy, there was no evidence for gamete loss
or for reduced fertilization success; the number of embryos in the brood did
not decrease with increased dose. The results on embryo abnormality and
mortality indicate that lethal mutations were most likely induced in the germ
cells and that these affected survival of the early life stages.
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1. INTRODUCTION
Considerable information is available on the effects of acute radiation
on responses of aquatic organisms (see reviews of Polikarpov 1966; Templeton
et al. 1971; Templeton 1976; Chipman 1972; Ophel 1976; Blaylock and Trabalka
1978; Egami and Ijiri 1979; Noodhead 1984; Anderson and Harrison 1986). Most
of the data on marine invertebrates is on increased mortality rates or on the
induction of histopathological changes. Managers and scientists concerned
with preserving the health of marine ecosystems are concerned with the levels
of radiation that affect reproductive success as well as those that cause
mortality and histopathological changes. Unfortunately, the data available on
effects on reproduction and development, which impacts on reproductive success
of marine invertebrates and may occur at lower doses, are limited to effects
on gonads and fecundity of Artemia salina (Cervini and Giavelli 1965; Squire
1970; Holton et al. 1973), effects on growth and metamorphosis of larvae of
Crepidula fornicata (Greenberger et al. 1986), and some preliminary
information indicating that in Neanthes arenaceodentata effects on chromosomes
and reproductive success occurred in the same dose range (Anderson et al.
1987).
Our preliminary studies on effects of acute radiation on fecundity in
N. arenaceodentata (Anderson et al. 1987) indicated that this species was a
good model animal for studies of reproductive success. This species is
available commercially, is easily maintained in the laboratory, and was
proposed previously as a model animal for genetic toxicology studies (Pesch
and Pesch 1980; Pesch et al. 1981). It is a relatively low-fecundity species,
a terminal spawner, and information is available about the effects of
inorganic and organic contaminants on its reproductive success (Rossi and
Anderson 1978; Oshida et al. 1981; Oshida and Ward 1982).
The objective of this study was to obtain information on the effects of
acute radiation on the reproductive success of a relatively low-fecundity
invertebrate marine animal. N. arenaceodentata were irradiated with a sealed
external source and their broods sacrificed before hatching occurred to obtain
information on brood size, on number of normal and abnormal embryos, and on
the number of living, dying, and dead embryos. The data obtained from this
study are useful in evaluating the effects of acute radiation on marine
organisms and are applicable in assessments of ocean disposal of radioactive
materials.
2. MATERIALS AND METHODS
2.1 Experimental Approach
Four replicate experiments, in which groups of 70 to 200 mated pairs were
subjected to radiation or held as controls, were performed. For each
replicate experiment except the fourth, the total doses delivered to the worms
were either 0.5, 1.0, 5.0, 10, or 50 Gy; for the fourth, a dose of 2.0 Gy was
included (Table 1). For both control and irradiated mated pairs, the
developing embryos in the brood were enumerated, were examined for
abnormalities, and were subjected to a dye-exclusion test to determine the
number that were living, dying, and dead.
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Table 1. Number of developing embryos
radiation-exposed mated pairs.
in broods from control and
Experiment
number
1
2
3
4
1
2
3
4
1
2
3
4
4
1
2
3
4
1
2
3
4
1
2
3
4
Total
broods
25
13
14
38
90
8
9
12
26
55
9
14
13
26
62
35
35
7
12
10
26
55
8
12
12
25
57
7
14
10
25
56
Brood size
(x ± SD)
a. Control
263 ± 129
182 ± 122
204 ± 163
224 i 115
225 ± 129
b. 0.5 Gy
170 ± 77
174 ± 138
168 ± 196
232 ± 127
199 ± 141
c. 1 .0 Gy
264 ± 75
204 ± 119
157 ± 200
249 ± 135
222 ± 172
d. 2.0 Gy
230 ± 172
230 ± 172
e. 5.0 Gy
186 ± 48
152 ± 180
140 ± 34
163 ± 119
162 ± 120
f. 10 Gy
186 ± 95
180 ± 105
86 ± 88
131 ± 115
139 ± 109
g. 50 Gy
128 ± 85
28 ± 43
127 ± 118
65 ± 92
74 ± 93
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The total number of mated pairs used in the experiments was 410. Ninety
control pairs were not exposed to the radiation source, 55 pairs were exposed
to 0.5 Gy, 62 to 1.0 Gy, 35 to 2.0 Gy, 55 to 5.0 Gy, 57 to 10 Gy, and 56 to
50 Gy.
2.2 Animal Sources, Culture Conditions, and Irradiation
Worms used in the experiment were obtained either from Dr. Donald Reish
(California State University, Long Beach, CA), from Brezina and Associates
(Dillon Beach, CA), or raised in our laboratory from parents from those
sources. 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 mated with vigorous males from the same supplier.
Oocytes in the coelom of N. arenaceodentata are clearly discernable because
the cuticle is translucent. Each mated pair was placed in a plastic petri
dish (120-mm diameter x 20-mm depth) containing about 80 ml of filtered
(1.0 vim) 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 underground 40,000-L tank.
Observations of the mated pairs were made twice weekly. 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
supplied (rehydrated freeze-dried Enteromorpha sp.).
Mated pairs were transferred to the radiation facility for irradiation
when oocytes were visible in the female. They were exposed to gamma radiation
in a 5,000-curie 137rjs irradiator (Mark I, Model 68A); doses were delivered at
5 Gy/min.
After the radiation exposure was completed, the mated pairs were returned
to the laboratory and were observed daily to determine the day of spawning.
When spawning occurred, the date was recorded, the female was removed from the
petri dish (if she had not been eaten by the male), and the brood set aside
for about 4 to 6 d before it was analyzed. The brood was sacrificed at this
time because as part of taking care of the brood, the nurturing male consumes
the dead embryos. 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 that had been present in the embryos consumed.
When this occurred, it was recorded (see Appendix A) 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.
Doses delivered to the worms were monitored using thermoluminescent
dosimeters. These were waterproofed and placed in seawater filled petri
dishes at positions similar to those occupied by the worms.
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2.3 Brood Analysis
Analyses of the broods consisted of (1) enumeration and examination of
the embryos and (2) a trypan-blue-exclusion test and were performed by an
analyst who had no knowledge of the dose received by the mated pair. 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 petri dish 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
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. 1); 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 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 Q.47<, trypan-blue solution to cover the
embryos was added. The embryos were exposed to the trypan blue for 5 min, the
excess trypan-blue solution was then discarded, and the embryos rinsed with
filtered seawater until the excess blue dye was removed. The embryos were
examined under 6X magnification, and the number that were totally blue (dead),
partially blue (dying), and free of blue dye (live) were recorded (Fig. 2).
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. This
procedure is summarized in Table 2. The data that were accumulated on each
brood are provided in Appendix A.
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
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 number 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 .
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Figure 1. 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 2. Broods subjected to 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).
-------
Table 2. Steps in the procedure used to harvest the brood from the mated
pairs. The harvest was performed about 4 to 6 d after spawning.
Part I. Enumeration and Examination.
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.
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 (total
number in brood minus number of dead and dying) was taken as the EHS.
Differences between 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 actual or estimated hatch sizes were analyzed
using a Test for Equal Proportions (Snedecor and Cochran 1967). Also,
differences in brood size for the control and radiation-exposed mated pairs
were examined using Analysis of Variance (ANOVA).
3. RESULTS
3.1 Brood Size
The numbers of embryos present in the broods that were sacrificed before
hatching were determined (Table 1). In the control group, brood size ranged
from 19 to 534 and had a normal distribution. Each brood was distributed into
one of four categories (n _> 150, 150 > n >_ 100, 100 > n _> 50, n < 50),
according to the number of embryos in the brood (Table 3, Fig. 3). A Test for
Equal Proportions was used to determine which radiation-exposed groups had
brood-size distributions that were significantly different from controls.
Only groups of mated pairs that received 10 or 50 Gy had brood-size
distributions that were significantly different from those of controls; the
proportion of broods in the n :> 150 category was lower than that in the
control group (p = 0.006 and < 0.001, respectively). Differences in brood
size among treatment groups were tested also using one way ANOVA; again, only
the 10-Gy and 50-Gy groups differed significantly from controls at the 95%
confidence level (Sheffe. F-test). These data indicate that acute radiation
in
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Table 3. Number of embryos in broods from the control and radiation-exposed mated pairs.
sacrificed before hatching occurred and were assigned to one of four categories (n 2 150,
100 > n _> 50, n < 50), according to the number of embryos in the brood.
The broods were
150 > n > 100,
Experimental
group
Categories of numbers of embryos in broods Total
n ^ 150 150 > n > 100 100 > n _> 50 n < 50 broodsa
Control
Radiation exposed (Gy)
0.
1.
2.
5.
10
50
Control
60
32
46
23
31
26
12
a. Number of broods in category
12 9
8
2
2
5
7
3
7
4
1
5
9
10
67
b. Percent of broods in category
13 10
8
10
9
14
15
31
10
90
55
62
35
55
57
56
410
90
Radiation exposed (Gy)
0.5
1.0
2.0
5.0
10
50
58
74
66
56
46
21
13
3
6
9
12
5
13
7
3
9
16
18
15
16
25
26
26
56
55
62
35
55
57
56
410
a Deaths and hatches included in the compilation.
-------
100
o
o
0>
**
re
o
150
150 > n > 100 100 > n > 50
n < 50
Figure 3. The percent of broods in each of four categories (n > 150, 150 > n > 100, 100 > n
n < 50) of numbers of embryos in the broods.
50, and
-------
doses ranging from 0.5 to 5.0 Gy did not reduce the number of embryos in the
brood when the mated pairs were irradiated at the time that the developing
oocytes were discernable through the body wall of the female.
3.2 Living Embryos in Broods
For each brood, the percentage of the embryos that were living was
calculated for the group of control worms and for each of the groups of worms
that received radiation. Then, these percentages were distributed into 4
categories (n > 75%, 75% > n > 50%, 50% > n ^ 25%, n < 25%) (Table 4). For
the control group, most of the developing embryos in the broods were living,
as evidenced by the exclusion of trypan blue from their cells. For this
group, the percentage of the broods in the n > 75% category was 59; in the
radiation-exposed groups the percentages were 31, 40, 49, 22, 23, and 15 in
the groups receiving 0.5, 1.0, 2.0, 5.0, 10, and 50 Gy, respectively. It is
apparent that radiation affects the percentage of living embryos in the brood
(Fig. 4). A Test for Equal Proportions was used to determine which
radiation-exposed groups were significantly different from the controls. The
proportion of broods that was in the n _> 75% category for each of the radiated
groups was significantly different from that of the control group for all
radiation doses (p < 0.01) except that of the 2.0-Gy group, which had the
smallest number of mated pairs; in this case the x2 was 1.16 and p = 0.14.
These data indicate that acute radiation as low as 0.5 Gy (about 50 rad)
caused significant decreases in the percentages of live 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 columns of
tables in Appendix A). When hatching did occur, the percentage of living
embryos almost always approached 100. A few males, even in the control group,
cannibalized the brood; but at the higher doses, this was a common
occurrence. The percentage of the males that cannibalized the brood was 7 in
the control group and 19 and 44 for the 10 and 50 Gy groups, respectively.
3.3 Abnormal Embryos in Broods
Most broods included embryos that were classified as abnormal because of
their morphology or because their development was delayed severely. The
broods were placed into four categories according to the number of abnormal
embryos that were present (n > 150, 150 > n > 100, 100 > n > 50, n < 50), and
the number and percentage of the broods that were in those categories were
determined (Table 5). We also calculated the percentages of abnormal embryos
that were present, and these were distributed into four categories (n >_ 75%,
75% > n > 50%, 50% > n > 25%, n < 25%) (Table 6). The percentage of the
broods that was in the n > 75% category was 18 for the control group and 25,
24, 37, 38, 43, and 71 for the groups exposed to 0.5, 1.0, 2.0, 5.0, 10, and
50 Gy, respectively. Percent of abnormal embryos increased with dose
(Fig. 5). The proportions of broods in the n >_ 75% category of the groups
exposed to 0.5 or 1.0 Gy were not significantly different from that of
controls (p = 0.16 and 0.19, respectively). However, for the groups exposed
to 2.0, 5.0, 10, and 50 Gy, the proportions were significantly different; p =
0.014, 0.004, < 0.001, and < 0.001, respectively.
-------
Table 4. Results from the analysis of the living, dying, and dead embryos in the 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 _> 75%, 75% > n >_ 50%, 50% > n > 25%, n < 25%), according to the percentage of
living embryos in the brood.
Experimental
group
Categories of percentages of living embryos in broods Total
n > 75% 75% > n >. 50% 50% > n > 25% n < 25% broodsa
Control
Radiation exposed (Gy)
0.5
1.0
2.0
5.0
10
50
Control
51
17
25
17
12
14
8
57
a. Number of broods in category
13 8
14
19
4
21
11
4
10
5
5
9
11
8
b. Percent of broods in category
14 9
18
14
13
9
13
21
36
20
90
55
62
35
55
57
56
410
90
Radiation exposed (Gy)
0.5
1.0
2.0
5.0
10
50
31
40
49
22
25
14
25
31
11
38
19
7
18
8
14
16
19
14
25
21
26
24
37
63
55
62
35
55
57
56
410
a Deaths and hatches included in the compilation.
-------
100
o
D>
0)
*-
(0
o
V)
•O
O
O
^
m
40-
20-
n > 75%
75% > n > 50%
50% > n > 25%
n < 25%
Figure 4. The percent of broods in each of the four categories (n > 75%, 75% > n _> 50%, 50% > n _> 25%,
n < 25%) of percentages of live embryos in the brood.
-------
Table 5. Results from the analysis of the normal and abnormal embryos in the 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 _> TOO, 100 > n ^ 50, n < 50), according to the numbers of abnormal
embryos in the brood.
Experimental
group
Categories of numbers of abnormal embryos in broods
n > 150 150 > n > 100 100 > n > 50 n < 50
Total
broodsa
Control
Radiation exposed (Gy)
0.5
1.0
2.0
5.0
10
50
a. Number of broods in category
10 9 23
6
7
4
9
9
4
11
9
2
12
6
3
13
18
9
7
15
13
40
23
23
13
22
24
25
82
53
57
28
50
54
45
369
Control
12
b. Percent of broods in category
11 28
49
82
Radiation exposed (Gy)
0.5
1.0
2.0
5.0
10
50
11
12
14
18
17
9
21
16
7
24
11
7
25
32
32
14
28
29
43
40
47
44
44
55
53
57
28
50
54
45
369
a No deaths or hatches included in the compilation.
-------
Table 6. Results from the analysis of the 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 larvae calculated, and then the percents were
assigned to one of four following categories (n _> 75%, 75% > n >_ 50%, 50% > n _> 25%, n < 25%), according to
the percentage of abnormal embryos in the brood.
Experimental
group
Categories of percentages of abnormal embryos in broods
n > 75% 75% > n > 50% 50% > n > 25% n < 25%
Total
broodsa
Control
Radiation exposed (Gy)
0.5
1.0
2.0
5.0
10
50
Control
16
14
15
13
21
24
40
a. Number of broods in category
10 22
13
6
2
16
14
6
11
18
4
9
9
4
b.
Percent of broods in category
12 25
39
17
23
16
9
9
6
45
87
55
62
35
55
56
56
406
87
Radiation exposed (Gy)
0.5
1.0
2.0
5.0
10
50
25
24
37
38
43
71
24
10
6
29
25
11
20
29
11
17
16
7
31
37
46
16
16
11
55
62
35
55
56
56
406
a No hatches included in the compilation.
-------
100
o
U)
o
03
O
-o
o
o
m
80 -
60 -
n > 75%
75% > n > 50%
50% > n > 25%
n < 25%
Figure 5. The percent of broods in each of the four categories (n _> 75%, 75% > n >_ 50%, 50% > n _> 25%,
n < 25%) of percentages of abnormal embryos in the brood.
-------
3.4 Reduced Survival of Embryos
The estimated or actual number of hatchlings was grouped into four
categories: n > 150, 150 > n _> 100, TOO > n > 50, n < 50. The estimated
number of hatchlings decreased with dose, but there was scatter in the data
(Table 7, Fig. 6). The Test for Equal Proportions was used to determine which
doses resulted in a significant difference from the control group in the
proportion of estimated hatchlings in the n _> 150 category. The percentage of
the broods that had or were estimated to have hatchlings equal to or greater
than 150 in number was 48 for the control group and 27, 52, 54, 20, 14, and 8
for the groups receiving 0.5, 1.0, 2.0, 5.0, 10, and 50 Gy, respectively.
Only the groups receiving 5.0, 10, or 50 Gy were significantly different from
the control group; p = 0.004, < 0.001, and < 0.001, respectively.
An important effect of radiation appears to be an increase in the percent
of broods in which the EHS was zero. This resulted bacause the female
resorbed the eggs or because all embryos in the brood were either abnormal,
dead, or dying. The percentages of broods with EHS of zero were 4 for the
control group and 7, 16, 37, 16, 22, and 54 for the groups that received 0.5,
1.0, 2.0, 5.0, 10, and 50 Gy, respectively. Consequently, significant
differences were determined at lower doses when the category of n < 50 instead
of the category n .> 150 was tested. The percentage of broods that had or were
estimated to have hatchlings less than 50 in number was 23 for the control and
38, 31, 37, 50, 56, and 81 for the groups receiving 0.5, 1.0, 2.0, 5.0, 10,
and 50 Gy, respectively. The p values obtained were 0.02, 0.13, 0.05,
< 0.001, < 0.001, and < 0.001 for the groups receiving 0.5, 1.0, 2.0, 5.0, 10,
and 50 Gy, respectively.
The effects of radiation on embryo survival 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 percent survival was assigned to one of four categories
(n > 75%, 75% > n _> 50%, 50% > n _> 25%, n < 25%), according to the percentages
of survival (Table 8, Fig. 7). The Test for Equal Proportions was used to
determine which radiation groups were different from controls; the proportion
of broods that was in the n < 25% category was compared to that in the
controls. For the groups receiving 0.5, 1.0, 2.0, 5.0, 10, or 50 Gy, the p
values were 0.09, 0.20, 0.02, 0.004, < 0.001, and < 0.001, respectively. The
results indicate that effects on survival are occurring at relatively low
doses.
An analysis was performed to determine the relationship between acute
radiation dose and embryo survival. The mean percent survival for the control
group and for each radiation-exposed group was determined. A normal
distribution of percent survival in each category was assumed even though the
distribution was not normal for all groups (the distribution in the 10- and
50-Gy groups was skewed because of the large number of zero percent
survival). For the control group, a value of 60 + 31% was obtained, and for
the groups exposed to 0.5, 1.0, 2.0, 5.0, 10, or 50 Gy, the values were 48 +
30%, 54 ± 32%, 50 + 41%, 39 + 28%, 35 + 32%, and 20 + 32%. The mean
percentage for each radiation-exposed group was expressed also as a percentage
17
-------
Table 7. Results from the analysis of the numbers of embryos that actually hatched or were estimated to
hatch from the broods of the control and radiation-exposed mated pairs. The broods were distributed to one
of four categories (n > 150, 150 > n _> 100, 100 > n > 50, n < 50), according to the number of actual or
estimated hatchlings in the brood.
Experimental
group
Categories of numbers of actual or estimated hatchlings
n > 150 150 > n > 100 100 > n > 50 n < 50
Total
broodsa
CD
Control
Radiation exposed (Gy)
0.5
1.0
2.0
5.0
10
50
41
15
32
19
11
7
4
a. Number of broods in category
12 14
7
5
0
8
7
3
12
6
3
8
10
3
20
21
19
13
28
32
46
87
55
62
35
55
56
56
406
Control
Radiation exposed (Gy)
47
b. Percent of broods in category
14 16
23
87
0.5
1.0
2.0
5.0
10
50
27
52
54
20
13
7
13
8
0
15
13
5
22
10
9
15
18
5
38
31
37
50
57
82
55
62
35
55
56
56
406
a No hatches included in the compilation.
-------
O
O)
0
ro
o
W
•o
o
o
m
n > 150
150 > n > 100
100 > n > 50
n <50
Figure 6. The percent of broods in each of the four categories (n :> 150, 150 > n 2 100, 100 > n _> 50,
n < 50) of numbers of actual and estimated hatchlings in the brood.
-------
Table 8. Results from the analysis of the survival to hatching of embryos in the broods of the control and
radiation-exposed mated pairs. The percent survival was calculated by dividing the estimated hatch size by
the brood size, and then the percent survival was assigned to one of four of the following categories (n _>
75%, 75% > n 2 50%, 50% > n _> 25%, n < 25%), according to the percentage of survival of the embryos.
Experimental
group
Categories of percentages of survival of embryos to hatching
n > 75% 75% > n > 50% 50% > n > 25% n < 25%
Total
broodsa
Control
Radiation exposed (Gy)
0.5
1.0
2.0
5.0
10
50
Control
Radiation exposed (Gy)
36
13
21
16
9
9
7
41
a. Number of broods in category
23 11
12
19
4
11
9
6
14
7
2
13
10
2
b. Percent of broods in category
26 13
17
16
15
13
22
28
41
20
87
55
62
35
55
56
56
410
87
0.5
1.0
2.0
5.0
10
50
24
34
46
16
16
12
22
31
11
20
16
4
25
11
6
24
18
11
29
24
37
40
50
73
55
62
35
55
56
56
410
a Broods that hatched were excluded.
-------
100
o
O)
0)
•l-i
CO
o
TO
O
O
m
n > 75%
75% > n > 50%
50% > n > 25%
n > 25%
Figure 7. The percent of broods in each of the four categories (n > 75%, 75% > n >_ 50%, 50% > n >_ 25%,
< 25%) of percent of survival to hatching of the embryos in the broods.
-------
of that of the control group. A semi log plot of percentages versus dose
indicated a biphasic survival curve (Fig. 8). The more sensitive target
includes about 30% of the cell population and has an 1059 of roughly 5 Gy,
while the less sensitive target includes about 70% of the cell population and
has an 1059 of roughly 30 Gy.
4. DISCUSSION
The effects of ionizing radiation on reproductive success in mammals,
fishes, and aquatic invertebrates have been observed to occur at lower doses
than those causing mortality or typical histopathological lesions, excluding
tumor development in mammals (Anderson and Harrison 1986). Reproductive
success is the result of a combination of processes, including successful
gametogenesis, fertilization, and development. Important factors that can
affect reproductive success are gamete death, reduced fertilization success,
and impaired development. In many studies of aquatic animals, these effects
cannot be separated (Anderson and Harrison 1986). For example, if an organism
is irradiated and reductions in embryo survival are noted, they may result
from gamete death or dominant-lethal mutations expressed in early development.
Most of the information available on radiation effects on reproductive
success in aquatic animals is on the effects of acute radiation in teleost
fish. Effects have been determined by irradiating gametes, early life stages,
and adults (see reviews by Egami and Ijiri 1979; Hoodhead 1984; Anderson and
Harrison 1986). Research on the effects of acute radiation on processes
affecting reproductive success in aquatic invertebrates have been limited
primarily to those on freshwater organisms and have been 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).
This broad range of radiation doses that cause detrimental effects is not
necessarily explained by actual species-specific differences in sensitivity to
radiation, but may result from differences in the gamete stage irradiated and
in the eel1-repopulation capacity of different organisms.
In experiments by Ravera (1967), adults of the freshwater snail
/sa acuta received 2000 to 220,000 R (about 19 to 2100 Gy) from an x-ray
Exposure to 2000 R reduced fertility and embryo viability. However,
recovery from the damage to germ tissue was evident in snails receiving 2000
and 10,000 R. At 10,000 R, the production of egg capsules and the number of
eggs per capsule were reduced. Moreover, all embryos produced during the
first 50 d after irradiation from snails receiving 10,000 R were not viable.
A greater percentage of the embryos produced after that time was viable.
Ravera (1967) proposed that repopulation of the gonad by undamaged germ cells
could explain this effect. Reproductive activity was completely disrupted by
100,000 R.
Differential radiosensitivity between meiotic stages was studied in
prophase and metaphase oocytes of the brine shrimp Artemia salina. Cervini
22
-------
20
30
40
Dose (Gy)
Figure 8. Mean survival of embryos (expressed as percentage of the survival
fraction of the controls) as a function of acute radiation dose. The survival
curve appears to be biphasic. Data from broods that hatched or that were
harvested before day 3 were excluded.
23
-------
and Giavelli (1965) showed that radiosensitivity of oocytes declined as
prophase progressed. Metaphase oocytes were also less sensitive than prophase
oocytes.
Hoppenheit (1973) observed reduced egg-production rates in adults of the
amphipod Gammarus duebeni receiving as low as 220 R (about 2.1 Gy). However,
this was offset by higher survival of adult females and increased brood size.
The effects of radiation on development of invertebrates have been
observed in only a few studies in which dosimetry was well documented (animals
exposed to an external source rather than radionuclides in the water).
Studies on freshwater animals include those on the calanoid copepod
Diaptomus clavipes (Gehrs et al. 1975) and on the snail Physa acuta (Ravera
1966, 1967). In the copepod, a significant decrease in the percent hatching
was noted at the lowest dose received, 1000 rad (10 Gy). In the snail, a
decrease in percent hatching was seen at exposures as low as 2000 R. In
Artemia salina, at a very high dose (360,000 rad), there was approximately a
25% decrease in hatchability of irradiated cysts. Greenberger et al. (1986)
irradiated larvae of the slipper limpet Crepidula fornicata with x rays at a
dose rate of 2 Gy/min; 5 to 200 Gy were given in a single dose. A significant
increase in larval mortality was detected at doses above 20 Gy. Also, their
results indicated that there was a significant decrease in shell length in
larvae receiving greater than 100 Gy.
Effects on development were demonstrated at lower doses for fish than for
invertebrates. The lowest acute exposure eliciting an effect on fish was 16 R
(about 0.15 Gy). When the one-cell stage of silver salmon embryos
(Oncorhynchus kisutch) was irradiated, an 1050 was observed 150 d after
fertilization (Bonham and Helander 1963). The lowest chronic exposure
eliciting an effect was 0.5 R/d (about 0.2 mGy/h). Increased developmental
abnormalities were documented by Donaldson and Bonham (1964), who performed
their 80-d experiments starting with one-cell Chinook salmon embryos.
Comparison of our results to those available on other invertebrates
indicates that relatively low radiation exposure affects reproduction and
development in N. arenaceodentata. Our results on the response of N.
arenaceodentata to radiation confirm those that were obtained in an earlier
study in which a larger range of doses was given (1.0, 4.0, 8.4, 46, 102, 500,
and 1000 Gy), but fewer mated pairs were tested (Anderson et al. 1987). In
the earlier experiment, only the females were irradiated, and a significant
reduction in fecundity occurred at 4.0 Gy (p < 0.05) for worms irradiated as
adults. At 1.0 Gy, a potentially significant difference was noted (p < 0.1).
Reduction in reproductive success in N. arenaceodentata may result from
oocyte killing, spermatocyte killing, reduced fertilization success, and
induction of lethal mutations. We concluded that oocyte killing was not a
significant factor, except at doses of 10 or 50 Gy, because brood size
distribution of the other radiation-exposed groups were not significantly
different from the controls. The female spawns only once and then dies
(terminal spawner), and oocyte killing would have been reflected in the number
of oocytes spawned, and in turn, in the number of embryos in the brood. There
was little indication that spermatocyte killing or reduced fertilization
success was important at the lower doses because the number of unfertilized
24
-------
eggs found was very small and was not related to dose. However, the number of
viable sperm may have been reduced, but to establish this would have required
a direct determination, which was not performed.
The increased embryo abnormality and mortality observed in this study
were most likely from the induction of lethal mutations in the germ cells
during gametogenesis. This is not unexpected because results of earlier
studies in our laboratory indicated that significant increases in induction of
chromosomal aberrations in larvae and juveniles occurred at about 2 Gy
(Harrison et al. 1984, 1987; Anderson et al. 1987). Because little is known
about cell-cycle time in oogenesis and spermatogenesis in N. arenaceodentata,
it cannot be established whether the effects occurred primarily during
oogenesis or spermatogenesis. In our experiments, both the male and female
worms were irradiated, and effects impacting survival of early life history
stages could have been induced in both male and female germ cells. Increased
incidence of mutations or chromosomal aberrations, e.g., deletions, was
indicated because there were larger numbers of abnormal larvae with increased
dose. These visible manifestations of mutations, however, do not provide an
indication of frequency of mutations that resulted in metabolic errors, which
can cause increased mortality, also.
In addition to the possible induction of dominant-lethal mutations,
recessive-lethal mutations probably occurred and could have been expressed if
they were present in both the egg and sperm forming the zygote. Because we
did not irradiate males and females separately and then ascertain the effects
on reproductive success, it is not possible to determine whether the effects
were primarily on spermatogenesis or on oogenesis. Also, because we did not
perform multigeneration experiments, it is not possible to evaluate the
incidences of recessive-lethal mutations.
The biphasic survival curve that was obtained is indicative of
differences in sensitivity to radiation. Differences in sensitivity may
result from the presence of different kinds of cells in the same population,
different stages of cells in the same population, or of different critical
subcellular targets. Insufficient information is available currently to allow
conclusions to be made about the factors that determine the shape of the
survival curve.
ACKNOWLEDGMENTS
Special thanks are given to Roger Martinelli, Marie Kalinowski, and
Sue Tehensky who assisted in the laboratory. We 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 DW 89930414-01-1)
and was performed under the auspices of the U.S. Department of Energy
(Contract W-7405-ENG-48).
25
-------
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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.
Oshida, P., C.S. Ward, and A. Mearns (1981), "Effects of Hexavalent and
Trivalent Chromium on the Reproduction of Neanthes arenaceodentata
(Polychaeta)," Mar. Environ. Res. 5, 41-49.
Pesch, G.G., and C.E. Pesch (1980), "Neanthes arenaceodentata (Polychaeta:
Annelida) A Proposed Cytogenetic Model For Marine Genetic Toxicology,"
Can. 3. Fish. Aquat. Sci. 37, 1225-1228.
Pesch, G.G., C.E. Pesch, and A.R. Malcolm (1981), "Neanthes arenaceodentata, A
Cytogentic Model For Marine Genetic Toxicology," Aquat. Toxicol. ]_,
301-311.
Polikarpov, G.G. (1966), Radioecology of Aquatic Organisms (Reinhold, New
York, NY), 210 pp.
Ravera, 0. (1966), "Effects of X-Irradiation on Various Stages of the Life
Cycle of Physa aorta, Draparnaud, a Fresh-Water Gastropod," in Proc.
Symp. Disposal of Radioactive Wastes into Seas, Oceans, and Surface
Waters (International Atomic Energy Agency, Vienna), pp. 799-808.
Ravera, 0. (1967), "The Effect of X-Rays on the Demographic Characteristics of
Physa acuta (Gastropoda: Basommatophora)," Malacologia 5, 95-109.
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.
27
-------
Snedecor, G.N., and W.G. Cochran (1967), Statistical Methods. (The Iowa State
University Press, Ames, Iowa, 6th Ed.), 593 pp.
Squire, R.D. (1970), "The Effects of Acute Gamma Irradiation on the Brine
Shrimp, Artemia. II. Female Reproductive Performance," Biol. Bui 1. 139,
375-385.
Templeton, N.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.
Templeton, N.L. (1976), "Effects of Ionizing Radiation on Aquatic Populations
and Ecosystems," Technical Report Series 172 (International Atomic Energy
Agency, Vienna), pp. 89-119.
Woodhead, D.W. (1984), "Contamination Due to Radioactive Materials," in -,
Marine Ecology, Vol. V, Part 3, 0. Kinne, Ed. (John Niley and Sons, Ltd.,
Chichester, UK), pp. 1287-1618.
28
-------
APPENDIX A
DATA BASE FROM THE EXPERIMENT
TO DETERMINE THE EFFECT OF ACUTE
RADIATION ON REPRODUCTIVE SUCCESS
OF Neanthes arenaceodentata
A-l
-------
Table A-l. Experimental data from Neanthes arenaceodentata mated pairs that were not irradiated with an
I
ro
external gamma-radiation source (controls). The number of days from radiation to spawn (R to S) and from
spawn to sacrifice (S to S) as well as the estimated hatch size (EHS) are provided.
Abnormal
Exp.
#
1
1
1
1
1
1
1
1
1
]
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
ID
420
420
413
411
415
421
422
422
423
xxl
1010
444
xx2
1212
xx3
114
100
115
131
666
180
xx4
xx5
xx6
xx8
Brood
size
99
97
198
389
84
77
320
393
404
269
323
141
256
307
534
246
233
180
471
266
127
295
215
457
130
Li
#
99
97
78
295
20
75
292
360
347
250
194
139
240
256
456
47
88
151
452
123
3
253
108
428
116
ve
%
100
100
39
76
24
97
91
92
86
93
60
99
94
83
85
19
38
84
96
46
1
86
50
94
89
#
0
0
4
11
0
0
10
8
0
0
1
0
0
0
2
26
19
0
1
15
42
2
0
0
3
Dead
%
0
0
2.0
3.7
0
0
3.1
2.0
0
0
0.3
0
0
0
0.4
10.6
8.2
0
0.2
5.6
33.1
0.7
0
0
2.3
Dying
#
0
0
116
83
64
2
18
25
57
19
128
2
16
51
76
173
126
29
18
128
82
40
107
29
11
Morph.
#
0
0
130
200
70
2
9
1
152
27
226
22
55
130
71
214
97
38
242
144
119
53
174
40
23
Time
#
0
0
3
8
0
0
1
1
2
0
17
1
27
2
7
31
40
0
18
20
0
4
0
2
2
Total
#
0
0
130
200
70
2
9
1
152
27
237
23
72
132
76
223
124
38
253
144
119
53
174
40
23
Total
%
0
0
65.6
51.4
83.3
2.6
2.8
0.3
37.6
10
73.3
16.3
28.1
43.0
14.2
90.6
53.2
21.1
53.8
54.1
93.7
18
80.7
8.8
17.7
R to S
days
0
0
0
1
1
1
1
1
1
3
6
7
9
10
12
12
13
13
13
13
15
20
20
20
20
S to S
days
5
5
6
5
5
5
4
4
5
4
4
5
3
5
3
4
2
2
4
3
0
5
2
5
0
EHS
99
97
68
189
14
75
292
360
252
242
86
118
184
175
456
23
88
142
218
122
3
242
41
417
107
Comments
Hatchb
Hatchb
Scata
Scata
Hatchb
Placec
Cand
Abane
Place0
Placec
Placec
Scata
Placec
Scata
-------
Table A-l. (continued)
CO
Abnormal
Exp.
#
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
ID
66
80
37
68
73
40
24
45
60
38
39
59
65
169
125
161
173
176
180
160
116
127
115
102
152
164
183
Brood
size
218
444
188
277
182
239
96
230
261
157
70
Femal e
Female
19
272
463
352
44
467
31
317
258
174
Female
124
55
286
Live
#
120
352
121
248
27
22
96
146
210
99
30
died,
died,
6
59
303
264
16
392
27
240
234
170
died,
99
51
197
%
55
79
64
90
15
9
100
64
80
63
43
Dead
#
8
9
4
5
8
2
0
8
7
7
1
7.
3.7
2.0
2.1
1.8
4.4
0.8
0
3.5
2.7
4.5
1 .4
Dying
#
90
83
63
24
147
215
0
76
44
51
39
Morph
#
90
115
45
58
110
206
7
81
64
14
46
. Time
#
0
1
0
18
1
0
0
0
8
5
0
Total Total
# 1.
90
115
45
63
no
206
7
80
69
16
46
10/26/86.
12/1
32
22
65
75
36
84
87
76
91
98
12/1
80
94
69
6/86.
7
24
59
6
13
7
0
30
4
0
1/86.
1
0
11
37.0
8.8
12.7
1.7
29.5
1.5
0
9.5
1.6
0
0.8
0
3.9
6
189
101
82
15
68
4
47
20
4
24
4
78
10
177
79
36
13
87
9
85
18
18.
45
16
102
4
226
6
3
0
6
5
18
7
0
2
1
7
14
258
82
38
13
92
10
94
21
18.
45
16
102
41.3
25.9
23.9
22.7
60.4
86.2
7.3
34.8
26.4
10.2
65.7
100 '
100
73.7
94.9
17.7
10.8
29.5
19.7
32.3
29.6
8.1
10.3
100
36.3
29.1
35.7
R to S
days
13
13
16
16
19
20
22
33
33
36
42
2
2
3
4
5
7
10
10
14
26
38
39
55
S to S
days
6
6
5
5
3
1
6
3
5
6
0
5
5
5
5
2
6
5
5
6
6
5
6
6
EHS Comments
120 Abane
329
121
214
27
22 Scata
89
146 Abane
192
99
24 Placec
0
0
5
14 Abane
303
264
16 Cand
375
21
223
234
156
0
79
39
184 Cand
-------
Table A-l. (continued)
Abnormal
Exp.
#
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
ID
5
13
17
19
46
47
49
54
58
69
76
87
93
95
134
136
137
140
142
144
146
160
165
170
171
174
175
177
185
188
193
Brood
size
227
33
255
202
239
106
312
26
225
133
239
53
392
105
128
116
290
370
229
229
127
335
142
337
332
316
390
400
299
273
82
Li
#
179
14
247
150
194
91
297
15
192
97
213
12
305
24
126
106
283
148
170
199
96
312
107
287
286
307
346
398
243
179
43
ve
%
78.9
42.4
96.9
74.3
81.2
85.8
95.2
57.7
85.3
72.9
89.1
22.6
77.8
22.9
98.4
91.4
97.6
40
74.2
86.9
75.6
93.1
75.4
85.2
86.2
97.2
88.7
99.5
81.3
65.6
52.4
#
10
11
2
8
6
8
1
2
7
3
6
9
11
17
0
0
0
22
1
5
2
2
4
10
10
1
5
0
14
6
3
Dead
I
4.4
33.3
0.8
4.0
2.5
7.6
0.3
7.7
3.1
2.3
2.5
17.0
2.8
16.2
0
0
0
6.0
0.4
2.2
1.6
0.6
2.8
3.0
3.0
0.3
1.3
0
4.7
2.2
3.7
Dying
#
38
8
6
44
39
7
14
9
26
33
20
32
76
64
2
10
7
200
58
25
29
21
31
40
36
8
39
2
42
88
36
Morph.
#
54
22
11
66
43
16
29
16
30
37
24
45
120
85
6
21
14
229
73
41
37
36
36
50
47
29
53
7
60
95
76
Time
#
2
25
5
19
0
9
6
14
28
0
16
6
3
85
5
0
7
10
8
12
24
3
6
14
24
7
8
0
8
20
53
Total
#
56
31
13
66
43
20
30
20
40
37
28
45
120
85
7
21
14
231
76
41
39
36
36
52
47
29
53
7
60
97
79
Total
24.7
93.9
5.1
32.7
18.0
18.9
9.6
76.9
17.8
27.8
11.7
84.9
30.6
81.0
5.5
18-1
4.8
62.4
33.2
17.9
30.7
10.8
25.4
15.4
14.2
9.2
13.6
1.8
20.1
35.5
96.3
R to S
days
5
26
5
10
8
22
10
18
13
8
10
23
19
10
13
15
12
8
15
26
43
12
10
7
7
16
8
20
7
9
17
S to S
days
6
6
6
6
6
6
6
6
6
1
6
6
6
6
6
6
7
6
6
6
5
6
6
6
6
6
6
6
6
6
6
EHS Comments
171
2 Cand
242 Scata
136
194
86
282
6 Cand
185
96 Scat3
211
8 Placec
272
20
121
95
276
139
153
188
88
299
106
285
285
287
337
393
239
176
3 Cand
-------
Table A-1. (continued)
Abnormal
Exp
#
4
4
4
4
4
4
4
a
b
c
d
e
Brood Live Dead Dyjng
ID size # % # 7o #
207 336 314 93.4 2 0.6 20
212 Female died, 5/10/87.
214 370 346 93.5 1 0.3 23
50 294 231 78.6 8 2.7 55
145 228 188 82.5 1 0.4 39
202 285 49 17.2 7 2.5 229
210 72 55 76.4 0 0 17
Scat, brood scattered throughout tube.
Hatch, embryos hatched into larvae.
Place, poor placement of brood for proper i
Cannibal, male eating developing embryos.
Aban, male abandonned the brood.
Morph.
#
52
31
72
78
252
26
ncubati
Time
#
4
6
16
6
0
4
on by
Total
#
53
32
72
79
252
26
male.
Total
7o
15.8
100
8.6
24.5
34.7
88.4
36.1
R to S
days
4
6
55
51
8
5
S to S
days
6
6
6
6
1
6
EHS
283
0
338
222
149
33
46
Comments
Scata
-------
Table A-2. Experimental data from Neanthes arenaceodentata mated pairs that received an acute dose of
0.5 Gy from an external
from spawn to sacrifice
gamma-radiation
(S to S) as well
source.
as the
The number of days from radiation to spawn
estimated hatch size (EHS) are provided.
(R to
S) and
Abnormal
Exp.
#
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
ID
138
102
168
730
211
1313
1818
101
18
20
14
15
67
43
75
13
41
111
113
126
143
182
141
110
121
177
163
146
138
Brood
size
116
82
120
214
243
101
160
305
41
139
35
140
277
198
470
66
199
203
393
649
11
80
25
100
167
299
90
Female
Female
Li
#
32
59
84
28
209
49
153
69
8
47
11
99
231
142
87
63
95
133
342
545
5
46
1
0
153
68
38
died,
died,
ve
%
23
72
70
13
86
49
96
23
20
34
31
71
83
72
18
95
48
66
87
84
46
58
4
0
92
23
42
1/1
Dead
#
43
1
0
11
1
12
2
9
0
3
2
4
9
7
0
0
4
18
8
9
2
11
19
5
0
87
46
2/87.
%
31.4
1.2
0
5.1
0.4
11.9
1.3
3.0
0
2.2
5.7
2.9
3.2
3.5
0
0
2.0
8.9
2.0
1.4
18.2
13.8
75.0
5.0
0
29.1
51.1
Dying
#
41
22
36
175
33
40
5
227
33
89
22
37
37
49
383
3
100
52
43
95
4
23
5
95
14
144
6
Morph.
#
30
53
21
145
150
57
48
268
34
108
22
40
49
44
422
6
110
50
34
74
9
31
19
70
10
112
46
Time
#
0
22
0
0
8
4
16
0
4
0
4
0
2
2
19
0
1
4
25
10
4
0
0
12
9
20
19
Total
#
30
65
21
145
153
57
58
268
34
108
22
40
49
46
122
6
110
53
53
79
9
31
19
76
19
18
50
12/21/86.
Total
%
21 .9
79.2
17.5
67.8
63
56.4
36.2
87.9
77.7
82.9
68.8
28.6
17.7
23.2
89.8
9.1
53.3
26.1
13.5
12.2
81.8
38.8
76
76
11.4
39.5
56
100
100
R to S
days
4
8
9
9
13
13
13
19
12
13
13
15
16
21
23
24
33
1
2
6
8
12
13
14
21
21
26
S to S
days
0
4
2
1
5
4
5
0
5
6
5
4
5
5
5
5
5
4
6
6
4
3
1
5
5
5
6
EHS
32
17
84
28
90
44
102
37
7
31
11
99
228
142
87
60
89
133
340
545
2
46
1
0
148
68
38
0
0
Comments
Scata
Cand
Cand
Abane
Cand
Scata
Cand
Cand
Cand
Cand
-------
Table A-2. (continued)
Abnormal
Exp
#
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
ID
2
4
12
14
23
31
33
36
39
51
52
53
56
61
65
99
124
129
148
157
9
44
77
128
133
141
Scat,
Hatch,
Place,
Brood Live Dead
size # % # %
288 180 62.5 37 12.9
338 145 42.9 23 6.8
13 004 30.8
366 224 61.2 15 4.1
93 86 92.5 0 0
210 97 46.2 3 1.4
147 73 52.6 27 17.3
156 150 96.1 0 0
76 40 52.6 5 6.6
400 344 86.0 6 1.5
333 312 93.7 4 1.2
213 156 73.2 8 3.8
135 110 81.5 5 3.7
23 4 17.4 5 21.7
209 68 32.5 10 4.8
84 44 52.4 7 8.3
165 29 17.6 28 17.0
454 353 77.8 21 4.6
347 233 67.6 10 2.8
202 97 48.0 7 3.5
299 191 63.9 14 4.7
239 212 88.7 3 1.3
229 189 82.5 2 0.9
511 491 96.1 2 0.4
259 198 76.5 11 4.2
237 45 19.0 16 6.8
brood scattered throughout tube
embryos hatched into larvae.
Dying
#
71
170
9
127
7
no
47
6
31
50
17
49
20
14
131
33
108
80
104
98
94
24
38
18
50
176
poor placement of brood for proper i
Morph.
#
123
199
13
155
15
121
60
11
39
62
39
39
27
19
153
52
131
115
129
99
119
39
50
35
78
203
Time
#
18
39
5
120
9
43
8
4
0
6
10
4
3
5
32
32
30
41
7
28
61
7
5
4
8
237
ncubation by
Total
#
125
202
13
202
17
133
60
11
39
63
39
40
27
19
153
59
132
116
131
101
122
40
51
35
78
237
male.
Total
1,
43.
59.
100
55.
18.
63.
40.
7.
51.
15.
11.
18.
20.
82.
73.
70.
80.
25.
37.
50.
40.
16.
22.
6.
30.
100
>
4
8
2
3
3
8
1
3
8
7
8
0
6
2
2
0
6
8
0
8
7
3
9
1
R to S
days
25
17
18
15
10
14
10
12
33
8
10
8
5
17
22
22
19
10
8
22
38
37
40
50
39
52
S to S
days
6
6
6
6
6
5
6
6
6
6
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
EHS Comments
163
136
0 Cand
164
76
77 Cand
73
145 Cand
37
337
294
156
108
4 Scata
56
25
29
338
216 Placec
97
177
199
178 Placec
476
181
0
Can, male eating developing embryos.
Aban,
male abandonned the brood.
-------
Table A-3. Experimental data from Neanthes arenaceodentata mated pairs that received an acute dose of
1.0 Gy from an external gamma-radiation
from spawn to sacrifice (S to S) as well
source.
as the
The number of days from radiation to spawn
estimated hatch size (EHS) are provided.
(R to
S) and
Abnormal
Exp.
#
1
1
1
1
1
1
1
1
1
i
°° 2
2
2
2
2
2
2
2
2
2
2
2
2
2
ID
162
339
184
106
1755
1919
317
117
60
52
83
87
51
54
90
55
88
85
71
50
89
84
49
Brood
size
278
284
218
133
291
361
314
326
170
117
151
35
160
54
353
280
370
325
261
204
277
256
Female
Li
#
228
272
145
120
281
329
287
244
113
41
15
23
26
38
207
209
249
188
227
174
38
132
died,
ve
%
82
96
67
90
97
91
91
75
66
33
10
66
16
70
59
75
67
58
87
85
14
52
Dead
#
0
0
1
2
0
3
5
1
56
1
3
3
13
0
12
4
8
48
6
0
69
3
%
0
0
0.5
1.5
0
0.8
1.6
0.3
32.9
0.8
2.0
8.6
8.1
0
3.4
1.4
2.2
14.8
2.3
0
25.0
1.2
Dyi ng
#
50
12
72
11
10
29
22
81
1
75
133
9
121
16
134
67
113
89
28
30
170
121
Morph.
#
78
36
78
31
103
67
29
129
91
43
134
21
149
8
69
68
93
184
27
25
222
86
Time
#
2
0
0
7
5
25
29
12
6
0
0
4
1
0
0
4
14
32
41
0
5
13
Total
#
80
36
78
34
105
83
52
134
91
43
134
22
149
8
69
71
97
199
51
25
224
91
10/26/86.
Total
y.
28.8
12.7
35.8
25.6
36.1
23
16.6
41.1
53.5
34.4
88.7
62.9
93.1
14.8
19.6
25.4
26.2
61 .2
19.5
12.3
80.9
35.6
100
R to S
days
7
7
9
11
13
14
16
25
27
9
11
15
19
19
19
20
24
26
29
29
33
35
S to S
days
5
5
3
5
5
3
3
5
5
3
0
2
3
3
3
6
5
3
4
4
5
5
EHS
198
248
140
99
186
278
262
192
79
41
15
13
11
38
207
209
249
126
210
174
38
132
0
Comments
Scata
Abane
Abane
Abane
Scata
Cand
Scata
-------
Table A-3. (continued)
3>
I
Abnormal
Exp.
#
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
ID
130
153
155
172
140
170
123
151
133
139
149
114
157
6
8
16
20
21
24
45
48
57
59
63
70
72
73
Brood
size
617
0
203
0
473
0
72
263
220
Female
Female
174
17
243
305
187
234
334
230
211
165
365
196
218
Female
0
354
Live Dead
# % #
572 91 1
Total cannibal ;
186 92 1
Total cannibal ;
453 96 2
21 29 9
161 61 14
50 23 1
died, 12/5/86.
dead, 11/20/86.
170 98 0
11 65 2
223 91.8 0
252 83.4 2
116 62.0 9
217 92.7 6
234 70.1 17
140 60.9 3
204 96.7 1
129 78.2 1
351 96.2 4
45 23.0 60
161 73.8 1
died, 3/31/87.
Total cannibal;
303 85.6 10
%
0.2
brood
0.5
brood
0.4
12.5
5.3
0.5
0
12
0
0.7
4.8
2.6
5.1
1.3
0.5
0.6
1.1
30.6
0.5
brood
2.8
Dying
#
44
gone.
16
gone.
18
42
88
169
4
4
20
51
62
11
83
87
6
35
10
91
56
gone.
41
Morph. Time
it II
TT IT
61
11
23
51
94
122
18
6
24
56
88
49
98
94
16
38
17
174
64
54
4
0
8
15
33
216
0
0
3
4
22
3
32
31
4
1
13
196
15
27
Total Total
# 1,
64
11
28
56
107
219
18
6
24
56
91
49
104
96
17
38
18
196
65
65
10.4
100
5.4
100
5.9
100
77.8
40.7
99.5
100
100
11
35.3
9.9
18.5
48.7
20.9
31.1
41 .7
8.1
23.0
4.9
100
29.8
100
100
18.4
R to S
days
2
3
6
7
13
14
21
21
28
26
51
14
10
10
12
12
21
14
14
12
12
14
12
10
S to S
days
6
6
6
4
5
5
5
6
2
5
6
6
7
6
6
5
5
7
6
5
5
EHS Comments
553
0 Cand
186
0 Cand
445
0 Abane
16
156 Placec
1
0
0
156
1 1 Abane
219
249
96
185
230
134
194
127
347
0
153
0
0 Cand
289
-------
Table A-3. (continued)
>
o
Abnormal
Exp.
#
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
Brood Live Dead
ID size # 1, # 7.
100 408 185 45.3 24 5.9
101 497 260 52.3 38 7.7
104 231 164 71.0 15 6.5
126 274 266 97.1 1 0.4
131 231 87 37.7 83 35.9
139 437 306 70.0 32 7.3
151 153 128 83.7 0 0
152 535 330 61.7 21 3.9
154 87 75 86.2 2 2.3
35 311 301 96.8 0 0
10 68 31 45.6 9 13.2
158 196 103 52.6 8 4.1
Dying Morph. Time
#
199
199
52
7
61
99
25
184
10
10
28
85
#
226
221
38
34
98
122
37
213
12
12
40
106
#
42
30
10
2
5
31
0
27
11
6
68
8
Total
#
226
221
46
34
100
124
37
213
15
12
68
106
Total
55
44
19
12
43
28
24
39
17
3
100
54
%
.4
.5
.9
.4
.3
.4
.2
.8
.2
.9
.1
R to S
days
19
17
8
5
8
12
12
17
14
61
46
50
S to S
days
6
6
5
6
5
6
5
6
5
6
6
6
EHS
182
260
164
240
87
306
116
322
72
299
0
90
Comments
Cand
Scat, brood scattered throughout tube.
Hatch, embryos hatched into larvae.
Place, poor placement of brood for
Can, male eating developing embryos
Aban, male abandonned the brood.
proper
t
incubation
by
mal e.
-------
Table A-4. Experimental data from Neanthes arenaceodentata mated pairs that received an acute dose of
2.0 Gy from an external gamma-radiation source. The
spawn to sacrifice (S to S) as well as the estimated
number of days from radiation
hatch size (EHS) are provided.
to spawn (R to S) and from
Abnormal
Exp.
#
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
ID
161
162
164
168
169
173
178
179
180
181
182
183
186
187
190
191
194
195
196
197
198
199
200
201
204
205
Brood
size
500
228
2
Female
Female
384
323
163
343
Female
112
301
430
3
334
421
324
Femal e
Femal e
436
131
Female
154
448
Female
429
Live Dead
#
454
227
1
died,
died,
381
240
61
276
died,
34
291
393
0
315
413
297
died,
died,
434
61
died,
62
370
died,
378
7. #
90.8 3
99.6 0
50.0 0
5/15/87.
5/11/87.
99.2 1
74.3 9
37.4 2
80.5 3
4/29/87.
30.4 5
96.7 0
91.4 0
0 1
94.3 0
98.1 1
91.7 5
4/29/87.
5/18/87.
99.5 0
46.6 0
5/15/87.
40.3 3
82.6 14
5/9/87.
88.1 3
%
0.6
0
0
0.3
2.8
1.2
0.9
4.5
0
0
33.3
0
0.2
1.5
0
0
2.0
3.2
0.7
Dying
#
43
1
1
2
74
100
64
73
10
37
2
19
7
22
2
70
89
64
48
Morph
#
62
36
1
7
85
106
87
85
26
42
3
19
64
29
18
78
96
94
69
. Time
#
6
5
2
0
36
14
31
112
3
2
3
4
12
7
2
73
154
14
8
Total
#
62
36
2
7
93
106
91
112
38
42
3
19
67
29
19
78
154
94
69
Total
I
12.4
15.8
100
100
100
1.8
28.8
65.0
26.5
100
100
9.3
9.8
100
5.7
15.9
9.0
100
100
4.4
59.5
100
100
21 .0
100
16.1
R to S
days
10
19
4
14
16
15
18
7
15
4
23
11
20
8
17
6
6
8
11
S to S
days
6
6
6
6
6
6
6
6
6
6
4
6
6
6
6
6
6
6
6
EHS Comments
438
192
0 Cand
0
0
377
230
57
252
0
0
263
388
0 Placec
315
354
295
0
0
417
53
0
0
354
0
360
-------
Table A-4. (continued)
ro
Abnormal
Exp,
#
4
4
4
4
4
4
4
4
4
a
b
c
d
e
ID
209
211
213
163
166
176
206
208
189
Scat,
Hatch
Place
Can,
Aban,
Brood Live Dead
size # 1 # %
392 362 92.4 1 0.3
355 295 83.1 2 0.6
387 362 93.5 3 0.8
361 171 47.4 8 2.2
195 178 91.3 4 2.0
81 67 82.7 1 1.2
256 176 68.8 6 2.3
414 248 59.9 6 1.4
159 28 17.6 86 54.1
Dying
#
29
58
22
182
13
13
74
160
45
Morph.
#
46
71
28
236
33
16
89
184
143
Time
#
6
5
4
355
2
13
42
12
159
Total
#
47
72
28
361
33
20
95
186
159
Total
%
12
20
7
100
16
24
37
44
100
.0
.3
.2
.9
.7
.1
.9
R to S
days
4
4
6
5
3
3
3
5
28
S to S
days
6
6
6
6
6
6
6
6
6
EHS Comments
345
283
359
0
162
61
161
228
0
brood scattered throughout tube.
, embryos hatched into larvae.
, poor placement of brood for
male eating developing embryos
male abandonned the brood.
proper i
.
ncubation by
male.
-------
Table A-5. Experimental data from Neanthes arenaceodentata mated pairs that received an acute dose of
>
i
CO
5.0 Gy from an external
from spawn to sacrifice
gamma-radiation
(S to S) as well
source. The number of days from radiation to spawn
as the estimated hatch size (EHS) are provided.
(R to
S) and
Abnormal
Exp.
#
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
ID
301
719
50
1717
107
2020
103
28
46
9
44
10
12
77
53
36
72
81
17
171
165
154
100
105
Brood
size
187
149
203
149
141
281
188
211
284
11
551
222
23
176
352
Female
Female
Female
Female
84
154
193
250
66
Live Dead
#
139
102
101
40
88
160
26
207
176
6
475
47
13
36
224
died,
died,
died,
died,
40
125
68
206
36
% #
74 13
68 6
50 28
27 34
61 7
50 20
14 5
98 0
62 10
55 1
86 3
21 16
56 0
20 43
64 29
10/29/86.
11/13/86.
11/1/86.
10/27/86.
48 20
81 0
35 37
82 11
55 3
%
7.0
4.0
13.8
22.8
4.9
7.1
2.7
0
3.5
9.1
0.5
7.2
0
24.4
8.2
24.0
0
19.2
4.4
4.6
Dying
#
35
41
74
75
46
101
157
4
98
4
73
159
10
97
99
24
29
88
33
27
Morph. Time
#
150
115
139
116
102
158
162
116
106
9
30
143
11
60
122
32
7
117
21
31
#
8
1
25
7
20
0
8
2
2
0
0
4
0
110
7
10
5
10
1
10
Total
#
53
115
139
119
106
158
164
117
106
9
30
144
11
143
126
38
11
117
22
37
Total
7.
81.8
77.2
73.4
79.9
75.2
56.2
87.2
55.4
37.3
81.8
5.4
64.9
47.8
81 .2
35.8
100
100
100
100
45.2
7.1
62.7
8.8
56.1
R to S
days
6
7
13
16
17
17
33
2
12
13
16
20
22
24
32
2
7
8
9
10
S to S
days
4
4
3
3
5
0
4
5
5
4
5
6
0
5
5
5
5
6
5
5
EHS
134
34
64
30
35
123
24
94
176
2
475
47
12
33
224
0
0
0
0
40
125
68
206
29
Comments
Scata
Abane
Scata
Abane
Scata
Scata
Cand
-------
Table A-5. (continued)
:>
i
Abnormal
Exp.
#
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
ID
175
103
107
135
184
11
22
28
29
30
42
64
68
74
78
92
106
108
112
113
114
122
125
135
138
150
90
Brood
size
91
47
254
214
189
21
292
217
211
136
0
414
40
290
44
257
160
157
237
111
59
71
8
182
175
Female
32
Live
#
19
26
227
27
129
16
173
174
68
124
Total
143
19
153
25
89
0
105
43
67
53
43
7
129
120
died,
18
%
21
55
89
13
68
76.2
59.3
80.2
32.2
91 .2
canni
34.5
47.5
52.8
56.8
34.6
0
66.9
18.1
60.4
89.8
60.6
87.5
70.9
68.6
4/6/87
56.2
Dead
#
38
1
4
24
1
2
27
7
24
1
bal;
135
7
29
2
7
160
6
9
8
0
8
1
19
7
.
7
*
41 .8
2.1
1.6
11.2
0.5
9.5
9.2
3.2
11.4
0.7
brood
32.6
17.5
10.0
4.6
2.7
100
3.8
3.8
7.2
0
11.3
12.5
10.4
4.0
21.9
Dying
#
34
20
23
163
59
3
92
36
119
11
gone.
136
14
108
17
161
0
46
185
36
6
20
0
34
48
7
Morph
#
66
29
34
199
97
6
132
52
170
21
185
26
123
22
167
160
62
206
53
8
31
7
44
85
10
. Time
#
0
0
1
16
8
13
30
8
39
6
279
14
45
14
92
0
1
219
13
4
16
3
7
49
6
Total Total
#
-------
Table A-5. (continued)
Abnormal
Exp
#
4
4
4
4
a
b
c
d
e
Brood Live Dead Dyinc
ID size # % # 1 #
149 209 53 25.4 48 23.0 108
67 283 125 44.2 2 0.7 156
40 268 205 76.5 5 1.9 58
156 371 346 93.3 2 0.5 23
Scat, brood scattered throughout tube.
Hatch, embryos hatched into larvae.
Place, poor placement of brood for proper
Can, male eating developing embryos.
Aban, male abandonned the brood.
I Morph.
#
162
167
65
33
incubati
Time
#
209
283
16
4
on by
Total
#
209
283
65
33
male.
Total
*
100
100
24.3
8.9
R to S
days
36
59
46
50
S to S
days
6
6
6
6
EHS Comments
0
0
203
338
I
en
-------
Table A-6. Experimental data from Neanthes arenaceodentata mated pairs that received an acute dose of
10 Gy from
from spawn
an external gamma-radiation source.
to sacrifice (S to S) as well as the
The number of days from radiation to spawn (R to S) and
estimated hatch size (EHS) are provided.
Abnormal
Exp.
#
1
1
1
1
1
1
1
1
f 2
w 2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
ID
274
139
222
129
109
118
120
350
78
1
8
47
64
3
5
76
79
63
42
70
179
159
118
120
Brood
size
15
205
78
162
288
237
273
237
95
320
305
200
52
229
157
51
30
299
167
253
106
96
149
274
Li
#
15
171
1
5
246
166
65
34
73
313
117
171
48
42
142
19
18
143
85
244
80
93
25
227
ve
%
100
83
1
3
87
71
28
14
77
98
38
86
92
18
90
37
60
48
51
96
76
97
17
83
Dead
#
0
0
59
5
1
2
15
1
0
1
34
3
0
62
0
4
7
73
3
1
7
0
29
0
•L
0
0
84.3
3.1
0.4
0.9
5.5
0.4
0
0.3
11.2
1.5
0
27.0
0
7.8
23.3
24.4
1.8
0.4
6.6
0
19.5
0
Dying
#
0
34
18
152
39
65
193
202
22
6
154
26
4
125
15
28
5
83
79
8
19
3
95
47
Morph.
#
0
0
64
100
45
80
223
213
13
12
230
22
4
178
28
44
16
180
'91
35
30
3
48
62
Time
#
0
0
0
0
12
11
0
0
0
3
14
4
1
25
1
2
4
38
0
5
6
5
42
0
Total
#
0
0
64
100
52
85
223
213
13
13
232
23
5
184
28
44
18
197
91
40
33
7
71
62
Total
%
0
0
82.1
61.7
18.2
37.3
81.7
89.9
18.1
4.1
76.1
11.5
9.6
80.4
17.8
86.2
60.0
65.9
54.5
15.8
31.3
7.3
47.7
22.6
R to S
days
4
6
8
12
13
13
23
25
12
16
16
16
19
21
22
23
24
28
42
97
1
2
4
8
S to S
days
2
0
1
0
5
5
0
0
5
5
5
5
7
5
6
5
5
5
1
6
5
5
5
0
EHS
15
171
1
5
236
152
50
24
73
307
73
171
47
42
129
7
12
102
76
213
73
89
25
212
Comments
Hatchb
Scata
Cand
Scata
Scata
Scata
Cand
Scata
Cand
Cand
Placec
Scat4
-------
Table A-6. (continued)
3=-
i
Abnormal
Exp.
#
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
ID
119
158
104
147
166
178
106
185
1
3
7
15
26
27
82
84
88
89
102
103
105
107
109
111
116
121
123
Brood
size
58
198
12
119
14
10
Female
Female
0
70
116
9
230
170
207
37
213
61
271
140
108
3
336
59
142
0
21
Live Dead
#
3
120
2
1
7
9
died,
died,
Total
39
26
5
112
82
115
8
31
31
180
27
70
0
46
27
42
Total
5
% #
5 1
61 20
17 3
1 6
50 1
90 1
11/20/86.
12/19/86.
cannibal ;
55.7 6
22.4 24
55.6 3
48.7 45
48.2 63
55.6 1
21.6 12
14.6 25
50.8 4
66.4 14
19.3 30
64.8 9
0 0
13.7 11
45.8 2
29.6 7
cannibal ;
23.8 5
%
1.7
10.1
25.0
5.0
7.1
10.0
brood
8.6
20.7
33.3
19.6
37.1
0.5
32.4
11.7
6.6
5.2
21.4
8.3
0
3.3
3.4
4.9
brood
23.8
Dying
#
54
58
7
112
6
0
gone.
25
66
1
73
25
91
17
157
26
77
83
29
3
279
30
93
gone.
11
Morph
#
33
63
8
85
7
2
38
68
5
119
73
101
22
168
47
95
107
45
3
317
51
107
15
. Time
#
0
43
8
17
0
1
8
32
8
34
7
40
19
213
22
0
75
28
0
27
54
142
18
Total Total
JJ OJ
33
85
12
92
7
3
42
72
9
122
74
111
27
213
50
95
107
51
3
318
59
142
21
56.9
42.9
100
77.3
50
30
100
100
TOO
60.0
62.1
100
53.0
43.5
53.6
73.0
100
82.0
35.1
76.4
47.2
100
94.6
100
100
100
100
R to S
days
10
13
14
14
14
16
14
13
17
30
13
17
13
5
16
21
8
19
27
30
20
11
18
12
10
S to S
days
2
6
3
5
1
3
6
6
4
6
6
6
4
6
6
1
6
6
4
6
6
6
6
EHS
3
113
0
1
7
7
0
0
0
28
26
0
108
82
96
8
0
11
176
27
57
0
18
0
0
0
0
Comments
Cand
Cand
Abane
Cand
Scata
Scata
Cand
Scata
Cand
-------
Table A-6. (continued)
I
CD
Abnormal
Exp
#
4
4
4
4
4
4
a
b
c
d
e
Brood Live Dead Dying
ID size # % # ?„ #
130 0 Total cannibal; brood gone.
153 305 116 38.0 10 3.3 179
120 354 145 41.0 48 13.6 161
37 156 61 39.1 42 26.9 53
91 258 159 61.6 15 5.8 84
115 0 Total cannibal; brood gone.
Scat, brood scattered throughout tube.
Hatch, embryos hatched into larvae.
Place, poor placement of brood for proper
Can, male eating developing embryos.
Aban, male abandonned the brood.
Morph. Time
it ii
192 80
219 25
100 37
109 10
incubation by
Total
#
192
220
101
84
male.
Total
%
100
63.0
62.2
64.7
43.4
100
R to S
days
13
19
36
45
40
42
S to S
days
6
6
6
6
EHS
0
113
134
55
159
0
Comments
Cand
Cand
-------
Table A-7. Experimental data from Neanthes arenaceodentata mated pairs that received an acute dose of
50 Gy from an external gamma-radiation source. The number of days from radiation to spawn (R to S) and
from spawn to sacrifice (S to S) as well as the estimated hatch size (EHS) are provided.
Abnormal
Exp.
#
1
1
1
1
1
1
1
f 2
^> 2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
Brood Live Dead
ID
539
335
444
110
108
130
333
31
30
11
29
32
34
57
62
22
4
35
61
74
2
174
150
128
size
280
75
192
57
150
96
43
33
0
32
66
0
86
5
20
143
0
Male
Femal
Femal
Femal
48
67
57
#
200
16
135
19
143
38
24
9
Total
8
0
Total
79
1
5
31
Total
% #
71 0
21 3
70 0
33 0
95 0
40 58
56 5
27 2
cannibal ;
25 10
0 40
cannibal ;
92 0
20 0
25 13
22 0
cannibal ;
I
0
4.0
0
0
0
60
11.6
6.1
brood
31.3
60.6
brood
0
0
65.0
0
brood
Dying
#
80
56
57
38
7
0
14
22
gone.
14
26
gone.
7
4
2
112
gone.
Morph
#
66
56
59
53
31
59
24
30
16
63
18
4
13
127
. Time
#
0
0
0
0
11
0
0
16
0
3
0
5
0
0
Total Total
#OJ
/o
66
56
59
53
37
59
24
33
16
63
18
5
13
127
died, 12/1/86.
e died,
e died,
e died,
48
9
57
10/26/86.
10/31/86.
10/24/86.
100 0
13 32
100 0
0
48.0
0
0
26
0
6
60
0
0
2
0
6
60
0
23.4
74.7
30.7
93
24.7
61.2
55.8
100
100
50
95.5
100
20.9
100
65
88.8
100
100
100
100
100
12.5
89.6
0
R to S
days
7
8
12
20
26
29
35
2
5
12
16
23
24
31
33
56
34
2
5
7
S to S
days
0
1
0
2
3
3
0
5
2
3
2
4
2
3
3
2
5
EHS
200
16
133
4
113
37
19
0
0
8
0
0
68
0
5
16
0
0
0
0
0
42
7
57
Comments
Scat*
Cand
Scata
Cand
Cand
Female
resorbing
Cand
Culture
overfed
Cand
Cand
Cand
Cand
Cand
Abane
Cand
Cand
-------
Table A-7 (continued)
I
rv>
O
Abnormal
Exp.
#
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
ID
134
101
186
117
181
124
138
18
25
34
38
43
60
62
79
80
83
85
86
98
110
118
119
132
147
155
32
Brood
size
178
259
0
215
90
357
Femal
147
179
3
0
198
43
1
241
284
0
Femal
113
66
Femal
Male
52
0
6
Male
229
Live Dead
# 1 #
50 28 47
9 4 136
Total cannibal
211 98 0
74 82 3
275 77 7
e died, 12/21/86;
6 4.1 15
9 5.0 38
00 2
Total cannibal
119 60.1 12
16 37.2 4
00 0
227 94.2 0
96 33.8 5
Total cannibal
e died, 3/21/87.
6 5.3 34
14 21.2 5
e died, 4/3/87.
died, 3/22/87.
3 5.8 42
Total cannibal
2 33.3 4
died, 3/22/87.
11 4.8 29
1
26.4
52.5
; brood
0
3.3
2.0
male di
10.2
21.2
66.7
; brood
6.1
9.3
0
0
1.8
; brood
30.1
7.6
80.8
; brood
66.7
12.7
Dying Morph,
it it
81
114
gone
4
13
75
ed 1
126
132
1
gone
67
23
1
14
183
gone
73
47
7
gone
0
189
88
168
19
43
89
2/22/86.
141
170
2
t
28
27
1
4
247
109
66
47
4
212
. Time
#
9
0
0
0
74
22
4
84
2
81
43
1
0
276
98
54
45
6
0
Total Total
93
168
0
19
87
94
141
175
3
98
43
1
4
282
112
66
52
6
212
52.3
64.9
100
8.8
96.7
26.3
100
95.9
97.8
100
100
49.5
100
100
1.7
99.3
100
100
99.1
100
100
100
100
100
100
100
92.6
R to S
days
8
10
24
29
47
50
26
31
30
15
8
11
22
8
19
14
31
26
8
10
18
43
S to S
days
4
2
3
6
5
6
6
4
5
6
6
1
6
6
6
6
6
1
EHS
50
9
0
196
3
263
0
6
4
0
0
100
0
0
227
2
0
0
1
0
0
0
0
0
0
0
11
Comments
Cand
Cand
Cand, Abane
Cand
Cand
Cand
Cand
Scata
Cand
Scata
Cand
Scata
Cand
Cand
Cand
-------
Table A-7 (continued)
Abnormal
Exp.
#
4
4
4
4
4
ID
81
96
66
143
97
Brood
size
19
0
16
Female
26
Live
#
0
Total
0
died,
10
•L
0
canni
0
5/9/87
38.5
Dead
#
4
bal;
10
7
I
21 .1
brood
62.5
26.9
Dying
#
15
gone.
6
9
Morph,
#
19
16
21
, Time
#
19
14
26
Total
#
19
16
26
Total
%
100
100
100
100
100
R to S
days
39
44
71
46
S to S
days
4
6
6
EHS
0
0
0
0
0
Comments
Cand
Cand
Cand
I
ro
a Scat, brood scattered throughout tube.
b Hatch, embryos hatched into larvae.
c Place, poor placement of brood for proper incubation by male,
d Can, male eating developing embryos.
e Aban, male abandonned the brood.
------- |