',.
..,,-
.'
.......
This paper not to be cited without prior reference to the author
International Council for the
Exploration of the Sea·
C.M.1983/E:55
Marine Environmental Quality Cttee
Ref. Demersal and Pelagic Fish Cttees
FREQUENCIES OF MICRONUCLEI IN MATURE AND IMMATURE ERYTHROCYTES OF FISH
AS AN ESTIMATE OF CHROMOSOME MUTATION RATES RESULTS OF FIELD SURVEYS ON WINDOWPANE FLOUNDER,
WINTER FLOUNDER AND ATLANTIC MACKEREL
A. Crosby Longwell, D. Perry, J. B. Hughes
and A. Hebert
National Marine Fisheries Service
Northeast Fisheries Center
Milford Laboratory
Milford, Connecticut 06460
ABSTRACT
Using a micronucleus test modified for the red blood cells of fish, and after
-,
,
training
ourselves~to
recognizethe RBC series in larvae.and adults, chromosome
mutation'frequencies were calculated for mature and .immatureerythrocytes inthe
blood and nematopoietic cells of several'hundred.resource fish.
flounder, windowpane flounderand Atlantic mackerel.
These were winter
Experimentalresultsoncsalmon
larvae.and Fundulus adults affirm .thatthisis alegitimate app1ication:of the test.
Fish·taken. in coasta1.mid-Atlantic waters about the'U~S. havestatistica11yhigher
mutation frequencies .than those from more offshore waters.
...
1t has not been'possib1e
to show a significant relationship of these frequencies to any natural .variables
which might influence mutation rates.
Emp10yant un test "micronucleus modifie pour 1es ce1lules du sang rouge du
ll
poisson, et apres
nous nous:etions instrui en la recconaissance de la serie ("RBC")
,
·en .1es larves et·les.adultes, nous calculions 1es frequences de la mutation chromosome
I
pour 1es.erythrocytes mures et celles qui niest pas
mur.dans·le.s~nt
et,les cellules
"hematopoietic" des plusiers;centaines.de poisson de ressources.
Ces p~issons etaient.
"winter flounder
Les resultats
ll
,
','windowpane flounder"' et "At1antic mackerel".
experimentaux des larvesdesaumon et des adults du Fundulus affirmentque ce11e-ci
est;une app1ication legitime du test.
Lespoissonspris des eaux cotieres au,milieu
de :l l Ocean At1antique,pres des Etas-Unis, ont desfrequences de mutation, statistiqument,
que ceux des.eaux plus loin de 1a cote.
11 n'y avait'pas ,ete possib1e:a montrer un
rapport ·important entre ces frequences a aucunes variables naturelles qu~ peuvent
influencer .les vitesses.des·mutations.
. .'
•
. r
INTRODUCTION
In
~ecent
years
muc~
effort has,gone into the development of sensitive,
though rapid and simple tests of mutagenicity, mammalian as well as bacterial.
As
vertebr~tes,
fish should be good candidates for application of some of the
mammalian tests.
Inasmuch as many of these tests are based on general, well-
known aspects of chromosome form, function and behavior common, to all eukaryotes,
those assays that are cytogenetic in nature are further,applicable to shellfish
and to other, invertebrates. This area was recently reviewed by Longwell (1981).
Though developed as bioassays, several of these tests can be applied to epidemiologie or field studies.
Many environmental contaminants are well-recognized mutagens and carcinogens.
It is inevitable then that their likely,influenceon natural ,mutation rates
should be of interest from several different aspects (Beardmore et
~.,
1980;
Longwell, 1981; Longwell and Hughes,1980, 1982; Neel, 1983).
This report is a summary of chrpmosome mutation frequencies as measured on
.
.. .
.
~
3 resource fish species sampled on Northeast'Fisheries Center Assessment and
Ocean Pulse cruises, and cruises of the Milford Laboratory Shang Wheeler, and.
the analyses of these for significant differences by area of catch.
'~
Frequencies
were determined in mature circulating erythrocytes and in immature erythrocytes
of the fore-ki,dney using a specially modified micronucleus, test •. Winter flounder,
windowpane flounder and Atlantic mackerel were the subjects of these studies.
Some Atlantic cod and larval red hake frequencies have also been,obtained.
Methods' for Calculatin Mutation Micronuclear) Fre'uencies; Laborator Ex eriments
Demonstratlng Suitabi ity of the Micronucleus Test as Applie to Fis
Mutation incidences were calculated on adult fish using a modification of the
mouse micronucleus
test~
and both mature and immature erythrocytes. Mature.erythro-
cytes are prepared in the field in standard blood smears subsequently post-treated
- 3 -
j
,
1
,.
and stained'for microscope examination in the laboratory.
..
•
Immature erythrocytes
are from samples of the hematopoietic fore-kidney trypsinized in the field prior
to fixation.
One thousand tri 5000 cells are scored per fish.
Mutation in larval fish is calculated on the larval"blood cells using again
the modified micronucleus test.
Larval fish are collected and fixed ,in,whole
plankton samples taken with either bongo or neuston nets.
The blood-filled larval
heart, which is itself hematopoietic at this stage, is dissected out of the fish
either free-hand or with a micromanipulator, depending on size of the larva. 'In
the case of very small larvae', microscopic pr~parations consist of the 'entire heart
with its content of blood cells, or of only the blood contents of the heart in
the case of larger larvae.
The suitability of the modified test'formeasuring mutation in the blood cells
"
of adult and larval fish was demonstrated in two laboratory experiments using /
X-rays as the mutating agent.
another on adult Fundulus.
One experiment was conducted on larval salmon, and
In both, a dose-response curve was established. These
experiments support the use of the modified micronucleus test in field surveys of'
chromosome
m~tation
in larval blood, and in the mature and immature erythrocytes
of juvenile and adult fish (manuscripts in preparation, Longwell, Hughes, Perry
and Hebert).
An effort was made to sample enough fish within an area, and to score sufficient cells within a fish to detect an approximate doubling of themutation rate.
At the initiation of these studies the rate seemed to ra~ge from 0 tö 3 in the
f
mature circulating erythrocytes. At first up to 5000 mature cells were scored
per fish, but this was later standardized at 1000 for both mature and immature
erythrocytes.
Winter Flounder Mutation (Micronuclear) Freguencies ,in Different GeneraltWater Masses
The 224 winter flounder (randomly sampled) which could be
processed.~
for,
mutation (micronuclear) frequencies were taken at 27 different sites .in 4 'general
- 4 -
•
'..
.
11,.,
,
water masses. All·but
~
few fish were sampled from October to early December
of 182. The other few came from the same season in 180.' Table 1 identifies
stations within water masses and gives the number of fish sampled, the mean mutation frequency for mature erythrocytesof fish taken at the station and its
variance. Table 2 gives similar information for combined station data for the 4
general water masses. Also given are overall mean mutation frequencies and
variances·for fish caught in different subdivisions of theCoastal Mid-Atlantic.
These subdivisions include the New York Bight apex, Long Island"Sound, and the
•
remainder of the Coastal Mid-Atlantic.
plottedat respective sample sites.
In Figure 1 mean frequencies are shown'
Higher frequency values are clearly clustered
in portions of the Coastal Mid-Atlantic. .
A goodness-of-fit test indicated that the distribution of these counts was
,not Poisson. Therefore, these data were not transformed'for ANOVA.
In the analysis
'of variance, mutation frequencies of fish from the different water masses differed
at the 99% confidence level. The Coastal Mid-Atlantic values were higher than
others,- butthere were no significant differences between any of the other water
units (Scheffe test). There were no significant differences in values for males
and for females.
~
Also, males and females tested alone still showed significant
differe~ces in mutation frequencies i~ the different water ~asses, again at the
99% confidence level.
Both the New York Bight apex and Long Island Sound differed
-,
significantly from the remainder of the Coastal Mid-Atlantic in their mean mutation
frequencies, with Long Island Sound being significantly higher than the New York
Bight (ANOVA and Scheffe tests). With the Bight apex and Long Island Sound
. '. fish
removed, the remainder of the fish in the Coastal Mid-Atlantic did not differ
significantly from those in any of the
othe~
water masses as sampled. Station
differences also proved significant statistically.
When fish with outlier counts were identified as those with frequencies
outside'2standard deviations; they were found to account for 5% of ,the 224
- 5 -
.. '."
winter flounder analyzed.
Long Island Sound.
,
•
I
•.
.'
These occurred only in the New York Bight apex and
Figure 2is a graphie display of the probabilities offish
with different counts occurring in the different waterareas •.
Data on age of the fish from scale readings were available for a subset of
about 75 fish from
7 stations in the Coastal. Mid-Atlantic, Georges Bank . and the
.
"
,
Gulf of Haine. State,of maturation was known for roughly half of all fish analyzed,
from 15 stations in all 4 general water masses. ,Age offish did not differ significantly between water sourees, but maturation did. There was no significant
statistical correlation between mutation frequency and age. Multiple
re~ression
4It
analysis showed no significant relationship of mutation freq4ency with weight, .
length, sex (data for these were available on almost all fish), or with maturation
!
state. Maturation though was more closely related than the other factor~. Analyses
of the subset of data with maturation data again revealedthat fish in the New York
Bight apex and,Long Island Sound had significantly higher mutation frequencies than
flounder sampled elsewhere in this study.
Windowpane Flounder Mutation (Micronuclear) Freguencies in 'Fish Sampled at·Three
Long Island Sound Sites Over Months
,
,
Windowpane flounder for mutation studies were sampled (randomly) at 8 sites
in 180- 181;1 near Georges Bank, 2 in the New York Bight, 2 off New Jersey, 'a~~ 3 in
Long island Sound.
,
However, most data were on fish coliected at the ~ L~ng ,Island
I
Sound sites, which were sampled repeatedly over aperiod of a year.
The'structure
of the data sets for mature erythrocytes and for immature erythrocytes is given
in Tables 3 and 4. Overall, there were 441 adult fish in the study, 8 sample
.
stations, and5 general sample areas.
.~
.
The mean mutation frequencies at the rela,
"
tively most contaminated sample site, Hempstead Bay, were generally 2-3 times
higher than at thecleanest Long Island Sound site for both mature and immature
, .,
erythrocytes. See Figure 3.
- 6 -
•
t.
•
.
t
•
.
Statistical analyses were performed on
~hese
data following a standardization
of the micronucleus counts and normalizing transformations.
In most cases the
square root transformations brought into the normal distribution range outlier
mutation
fr~quencies
of some fish allowing a conventional,analysis of variance.
In every case,analyses were conducted on both the standardized count and on the
transformed data.
Windowpane flounder samp1ed once at the 2 southern Jersey sites in November
'80 did not differ si9~ificant1y in their mutation incidences. Those from the 2
New York Bight sites also sampled in November '80 differed on1y at the level of
borderline statistical significance.
Counts for these 4 sites were all made
on,the mature circulating erythrocytes.
An. analysis was performed for all 3 Long Island Sound stations to test for
time (month of sampling) and water mas~ differences on mutation frequencies of.
mature erythrocytes with stations nested in water mass. Results of the test
showed highly significant statistica1 differences «1%) for time and water mass.
\
Mutation incidences in immature erythrocytes samp1ed 1 month (see Table 4)
at the 3 Long Island Sound stations differed significantly at about the 1% level
•
when tested for station, and also when tested for station and water mass.
Further, the corre1ations between mutation frequencies in mature and immature
erythrocytes differed across stations.
Additional data were co11ected in '82 on mutation frequency in the immature
erythrocytes of 15 juvenile windowpane flounder from Hempstead Bay, and of 12
f10under from off Shoreham, Long Island. These fish were approximate1y 1 year
of age.· For both normal and transformed counts, the differences between frequencies at the two stations were highly significant •. Mature erythrocytes in the
juvenile fish could not be examined in any of the fishfrom Hempstead Bay because
of their extreme fragility.
By contrast, only 2 fish from the Shoreham site
exhibited,a comparable degree of cel1 fragility.
- 7 -
. .'
..
Atlantic Mackerel Mutation (Micronuclear) Frequencies in Fish Sampledin Long
Island Sound and in Three Areas Near the Edge of the Continental Shelf
Atlantic mackerel sampled for mutation frequencies were from 3
gene~al
groups near the edge of the continental shelf, February-April 1982, and from
Hempstead Bay, Lang Island, June 1982.
Blood and kidney samples were taken from
a random sample of fish caught in a large number of offshore tows, anrl f~om all
fish caught at Hempstead Bay. See Figure 4 for location of sample areas~
Table 5 .
provides information on sampling, number of fish analyzed, and mean mutation'
(micronuclear) frequency as measured in both mature and immature erythrocytes of
fish from the 4 sample areas.
As in the case of the windowpane flounder, frequen-
.
4It
cies counted in immature cells are higher than in mature cells.
Statistical analyses were dane using both normal and transformed data.
Using
normal data,the most northerly of the 3 offshore groupings had, likeHempstead
.
~
Bay, significantly higher mutation frequencies.
Thelower number of immature
counts (total of 80 as compared to 352 mature cell counts) available at
~h~
time
.
of the statistical study showed only borderline significance among groups,iwith
.
.
.
I
.
~
' .
the much higher values for Hempstead Bay fish being largely responsible for the
significance in ANOVA.
Other parameters measured on the sampled fish that could conceivably influence
the mutation frequencies were similarly treated statistically. Aside from the
mackerel caught in Hempstead Bay, Long Island, the 4 groups of fish did not differ
significantly in length.
HempsteadBay fish were all among the largest in the
study. State of maturation (based on an integer scale 0-9) did differ significantly
among treated groups. The ripest fish were sampled in June in Hempstead.Bay, and
the least ripe in the Middle Atlantic February 3-12. There was no significant
difference in maturation between fish from the Hudson Canyon shelf border and the
shelf border.south of Hudson Canyon off Cape May.·
Furtheranalyses of the mature and immature mutation frequencies of fish from
the 4 groups were performed using again the standard analysis of variance, fitting
only water mass effects, but w1th
transformed frequencies for the mature and
- 8 -
•
..., \..
immature erythrocytes.
(Since many fish have 0 frequency values, started logs
were used to restare syffimetry to the distributions.) Mature ancl immature counts
were treated as' independent variables. Although sample location does not account
for,much of· the variability in absolute terms, it 1s highly significant statistically. Means of the Middle Atlantic and Hempstead Bay fish are higher than
those of the most southerly group from the shelf border'south'of Hudson Canyon
off Cape
See Table 6.
~ay.
, Since each mutation count on immature cells is paired with a count on mature
•
cells of the same fish, it may not be entirely correct to treat freque~cies counted
in mature and immature erythrocytes independently. At the time the statistical
" study was done mutation frequencies were calculated on 'the immature erythrocytes
of.onlya portion 'of the fish for which mature counts were already available. To
account for any pairing effect, and also to use all dataavailable'at that point,
mature and immature red'blood cell frequencies were considered as jointly distributed
,
,
as bivariate normal random variables within each group of fish analyzed (model and
equations'detailed by Carlstein, 1983)*. Results of this analysis'substantiate the
conclusion reached by the linear model approach with transformed data, and with
the analysis on non-transformed data as'just described above.
...
Further, results
suggest that the ordering across groupsof the mutation frequencies of ~ature vs
immature erythrocytes iswell-determined although the magnitude of the initial
estimates is inflated.
, ' Further statistical treatment of the data in an examination of the relationshipof the auxiliary variables measured on the fish to mutation frequencies of
their mature and immature red blood cells failed to reveal any consistent overall
effect once the group effect was removed. These auxiliary variables included'
* These
latter statistical analyses{and those given in Table 6) were performed as
part of a graduate technical work project by E. Carlstein with data provided
Prof. F. Anscombe, Yale University Statistics Department.
- 9 -
..
~.
length of the fish, length o! gonad, mäturation state and sex. Also included
were water temperature, date of the sample and tow-site within group.
Inthe,
Hempstead Bay fish there may be a sex effect on mutation rates of both'mature and
immature cells though this could be confounded w1th a maturation
effect.~
(As
noted above, Hempstead Bay fish sampled later than others were all more,mature.)
Also, of all the auxiliary variables measured, maturation seems to correlate ,
most closely (though not significantly)with the mutation rates.
~nalyses
Statistical,
of the remaining immature cell data on the 3 offshore groups may provide
some additional information in this regard.
Discussion
One very recent application of the micronucleus test was in a multi-technique
screening of Chicago municipal sewage sludge for mutagenic activity (Hopke et
•
~.,
1982). This'application employed the plant Tradescantia (spiderwort).widely used
in various other genetic studies for decades, and scored micronuclei as mutations
in the pollen mother cells. The usefulness of the micronucleus test as applied
to circulating blood of fish was
(1982).
al~o
recently recognized by Hooftman and de Raat
As reported by us for the immature erythrocytesof Fundulus, the latter
workers obtained a characteristic dose-response in the freshwater mudminnow for
mature erythrocytes using an adaptation of .the micronucleus test.
They recom-
mended development of methods for use of the test on fish kidneys, and the survey
of a variety of fish.
That there should be differences in mutation frequencies of mature and immat~re
erythrocytes of the fish reported in this study is not unexpected.
Immature erythrocytes with severe mutations should not all be capable
of
maturing
and entering, the circulation. Also, some micronuclei are expectedto fuse with
the major nucleus during differentiation of the erythrocyte. The result should be
a tendency for higher incidences to occur in immature cells as found in the
flounders and mackerel.
- 10 -
~
'.
. .
".
However, aside from this, differences in counts between the two cell types
"
"
are expected when the mutagenic burden reaching the blood-forming tissue changes.
This difference depends on the cell kinetics of erythropoiesis, and on the longevity
of the mature circulating erythrocyte.
Published literature places the life span
of the fish erythrocyte at about 150 days with same erythrocytes pos"sibly living
longer. According to one study on an experimental fish~ maturation of the red
blood cell takes about 7 days.
When temperature was varied 5 degrees there was
a 3- to 5-fold increase in the rate of hematopoiesis.
In the experimental study
on Fundulus referred to in the methods section of this paper, a mutagenic response
was measured in the immature erythrocytes already at 3 days post-treatment using
the modified micronucleus test.
I~respective
of the particular kinetics for any given species under any
given envi~onmental conditions, it is 'certain that increased or decreased mutation
frequencies in hematopoiesis is measurable in the circulating erythrocytes only
some time after "ttleir"altered freqLiericy is counted in the immature cells.
(The
micronuclei are formed in the mitosis preceding formation of the immature erythrocytes.)
•
Consequently, frequency data on the immature erythrocytes are more easily
interpreted relevant to environmental conditions in the sampling area of migratory
fish than would be frequency data from the mature erythrocytes. When the fish is
relatively non-migratory, mutation frequencies of the mature cells should better
reflect and integrate the quality of the habitat over aperiod of some time •.
Temporal. metabolie states of the fish at the time it is sampled cf the"sort that
could influence mutation ought always be better reflected in the immature cell
counts.
Windowpane flounder seem to have limited migrations inshore and offshore
related
to spawning, but do migrate considerably greater distances
than do winter
.
.
"
flounder, at least in Long Island Sound (as based on tagging, electrophoretic and
other studies conducted by state and private groups in Connecticut). " The temporal
- 11 -
.. '
variation in mutation frequencies found for the
maturee~ythrocytes
of the
windowpane flounder ,in Long Island Sound could be influenced by migratory
patterns of these fish.
However, variable environmental conditions at·the
3 sites resampled over aperiod of months cannot bediscounted.Thegenerally
clean sample site showed less variation in counts than did the other2.known
to be variously polluted.
Ma
et~.
(1973) reported a seasonal variation in chromatid aberrations in
the spiderwort Tradescantia with enhanced aberration rates also,occurring at times
of peak background rates which may be'related to other contaminants. Otherthan
this report, the matter of seasonal variation in mutation seems not to have been
considered much at all.
In windowpane flounder the generally higher mature cell frequencies in fish
from the Hempstead site, the significantly
higher immature cell counts at. thesite,
.
and the highly significantly greater immature counts of.young fish all ,support
.
the contention that habitat quality contributes to the overall temporal.variability.
In the winter flounder analyzed in this study differences in sex, maturation
and age of the groups cannot account for.the significant variation in mutation
frequencies measured in mature e:ythrocytes in the different water masses sampled.
The clustering of high incidences in the New York Bight apex and thewestern part
of Long Island Sound again suggests a link with environmental po11ution,and.
agrees with the findings of increased incidences of mutation in windowpane flounder
caught at Hempstead Bay.
Non-migratoryspecies as winter flounder'tend to form
distinct sub-populations, and it might be considered that these would
ferent intrinsic rates of mutation.
have:dif~
However, here again there is little precedent
for supposing this would be the case in non-inbred natural populations (Generoso
and Russell, 1969; Nee1 and Rothman, 1981).
Mackerel sampled in the 3 offshore areas near the edge of thecontinenta1
shelf are believed to have overwintered somewhere near the area'in which,they
- 12--
•
".
~.
were caught.
groups
h~d
Even so, it was somewhat surprising to have found that one of these
a significantly higher mutation frequency in both its mature and im-
mature erythrocytes.
(The initial plan called for comparing'frequencies of
mackerel inshore and offshore.) . This group,with the higher frequencies was the
one
cl~sest
to thepolluted Bight apex. The'even higher frequencies of mutations
in.immature cells of mackerel that migrated into western Long Island Sound to
Hempstead Bay support findings of elevated mutation frequencies of windowpane and
winter flounder caught there.
•
Although considerable attention was paid in this study to age-size, sex and
maturation as variables that might confound efforts to examine links between
polluted habitats and mutation frequency, there is little evidence in the literaturethat these can exert a significant influence (Gundy and Varga,·1983;· Ivanov
et~.,
1978; Todorov, 1979). However,in polluted environments and with uptake,
of contaminants, influences ofthese factors on mutation rate may be seen, for
example, when lipid reserves may be mobilized for oogenesis. The result could
be,seasonal flux of mutation frequencies as perhaps seen in the windowpane flounder.
Upon completion of the counts on the immature erythrocytesof the Atlantic mackerel
in the forthcoming month, relationships between their maturation and other auxiliary
variables will be further explored within each of·the offshore sample groups.
" , Prior reports linking aquatic pollution to increased mutation concerned a
population of fern (Qsmunda regalis)·growing in a Massachusetts river heavily
poll uted with industria1 waste. Higher frequencies of chromosome mutation were
reported in developing germ cells than in populations from nearby non-polluted
areas (Klekowski and Berger, 1976; Klekowski, 1978). 'Mudminnows, Umbra pygmaea,
held in water from the river Rhine showed higher incidences of chromosome mutations
1978).
in gill .cells than did fish held in untreated ground water (Prein et.~.,
.
Fish exposed to Rhine water 3 and 11 days showed an increased sister-chromatid
- 13 _.
...
exchange rate in gill and gonad cells two and three times that of fish exposed
to ground water cf drinking quality (Alink et
~.,
1980).
In a test of pooled'
gametes of 5 males and 5 females froma contaminated'dock and from a relatively
uncontaminated area, there were significantly more-aneuploid cells in embryos:
from the contaminated site (Dixon, 1982). Stromberg
et~.
(1981) reported
increased sister-chromatid exchange rates in kidney cells of English sole collected from a polluted river over those found in a relatively unpolluted site
in Puget Sound.
Longwell and Hughes (1980) in a preliminary report of' a study
only now nearing completion found a statistical association between mitotic'
t
abnormalities in planktonic Atlantic mackerel eggs and levels of indicator
•
contaminantsin the New York Bight.
The study reported here is probablj the largest one of its sort conducted'
to date that suggests an influence of coastal contamination, however ephemeral, asfar offshore as near the edge of the'U.S. continental shelf. Though'significant:both statistically and in terms of the 1ikely extent of marine pollution' ':". the'
rates in near coastal waters are generally on the order of'double,or'triple'what.
is assumed to be the background, rate, an increase no greater than that
sidered about the maximum tolerable one for man
from'radiation:hazards~,
once:con~
As
measured in mackerel near the edge of the·continental shelf, themutation:frequency;,
of the northernmost group is less than double that of the most offshore:souther.ly'
group.
Consequences of any increased rates of chromosome mutation. are' expected:
to be a higher incidence of fish with tumors and, when the higher'rates extendto
germ-line tissues, a decrease of reproductive efficiency as mutations'which are
dominant lethals for eggs and larvae occur more frequently.
- 14-
•
..
.
.
Tab1e.l
Mutation Frequency in Micronucleus Test Per
1000 Nature Erythrocytes in Hinter F10under
, •.- By Station HithinWater '~1ass .
.r
.
.
.;'
.
.
locati on 1-1 i thi Tl General
No. of
Hater Hass
Fish'
Nean
5
4
5
9
2tl9
·
2.25
1.76
·.
.2. 18·
Ne\'/ York Bight Apex
Coastal Hid-Atlantic
.·
...
- . ,..... :·
. ··
.
.. :..:
...
,General Water Hass
".
.. Northeast of Bight Apex
C0asta1 l1i d-Ne\·/ Jersey
Albennarle Sound
Coastal long lsland
Narragansett ßay
Hi cl-Atl anti c.
Offshore Ne\'! Jersey
Georges Bank
Mid-Georges Dank
Western Georges Bank
COustal Gulf of Maine
Scotian Shelf
2
.
"
10
1.89'
5.59
. '5
·:1•
2
4
1.5
1.25
10
3
. 9
9
2.7
.71
1~39
1.66
.88
1.88
6 .
7
.5 ':
.42
·5
.2
.'~
.47
1.06
.25
.18
.84
2.88
.32
.. 98
.25
.24
.16
1.2
. 89
1.15 .
1.14
.• 72
.6
.43
13
1.15
..
·.
2.49·
15.3
3.81
.44
20
10
·3
2
2
2
."
2 .
3.43.
1
~89
.-
.'
10
10
10
•8
_:.-.:-
. Variance
2.16 · .
15
20
10
.7
.
. 2.88 .
6
'.
long Is1and Sound
Gu1f of Haine
.
.. 2
.
0
1.66
. ·
..
. .
...
Table"2
..
"
Mutation Frequency in Micronucleus Test Per
1000 t1ature Erythrocytes in Hinter Flounder
- By General and Subdivided Water Mass -
Coastal Mid-Atlantic.
Subdivided
•
New York Bight Apex
39
2.33
2.34
Long Island Sound
35
3.94
10r79
Remainder of Coasta1 Mid-At1antic
54
1.14
1.01
",
.
.
. '.
,
.
.
-:
-'
~."
.. '"
.~.
:
..
..
.
.
.
,.-
"..
'.
.. ': . r•.
"
.
..'
.. - ...
. ..
'
'
:, ' .
.
.
.
:-."
.
..'
~
C
100
>
Figure 1.
Hean chromosome
mutation fr:equency (per
loaa.mature RBC) to the
closest a.5~ for winter
flounder at 27 sampling
sites.
o'
...
1.00,..;:-----~--------'--------------.
.90
Confidence Interval
98%
95%
90%
80%
.80
.70
~
.60
,,
r---l
~
"
rr
(])
\...
,,
,,
,
.50
l.---I
\...
a..
\
.40-
\
'i
\
\
\<"0
\~
.30
,~
\{~
\~
e
\:
,(5)0
\,~
,ö'
.20
,,
\
.10
O-J.-
o
,,
---\----t-----t-----=T----+---:::::=::...f
1
2
3
4
5
6
7
FREQUENCY K
Figul'e 2.
Statistical probability of ,...inter flounder, collected fram 6 major ,...ater
mdsSeS t having 1 to 7 or more chromosomal mutations/lOaD mature RBC, as
measured by the micronucleus test. Note how closely all lines lie except'
for the New York ßight Apex and Lang Island Sound.
All fish with
statistically identified outlier frequencies of mutation occurred in
these two regions.
TABLE 3
Data Structure for Analyses of Mutation Frequencies
in t1ature Erythrocytes of Hindowpane Flounder
'. ...
'
..,.
•..
'
TAßLE 4
Data Structure for Analyses of Mutation Frequencies.
in Immature Erythrocytes of Windowpane Flounder
Adults
General Water Mass
Sample Site and Number of Fish
Sample Time
Long Is1and Sound,
Station 90/14
(off Shoreham, L.I:)
'June '81*
Long Is1and Sound
Station 54/12
(off Milford, eT)
June 181*
Station 9/15
(Hempstead.i' Bay)
June: 181*
. Hempstead Bay
Juveniles
. General Water Mass
Samp1e Site and Number of Fish
Long Island Sound
Station 90/12
(off Shoreham, L.I.)
Hempstead Bay
Station 9/15
(Hempstead Bay)
* Mini Pulse station numbers.
Samp1e Time
. June '82
June 182
•
Shang Wheeler cruises.
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30! "
,
,,
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Long
~ei'1
Isl~nd
Ycrk
.
The frequency of chromosame mutation in wi ndO\'Jpane flounder taken at' the mouth· of Hempstead' Bay,
,Lang Island, is significantly higher than in windowpane flounder sampled in Lang Island Sauna
off Milford, CT, an~ ~hoteham, LI. Mutation'freq~encies of these same fish also varied significantly over time.
',
Mean counts for the mature cells ranged usually from 0-:3. with more 0 counts than in winter
. flounder and subsequently seen in Atlantic mackerel.
,
"
,
'
Figure 3
...... ,
,
.
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"
.
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,
.
,
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ft
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oe
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.'
.........
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I
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Arrm'ls"indicate probable
..general .migratory pattern~
. of the mackerel
.. ' " '
,.- .
I:
I :
'.
::
: KILOMCTE~
o
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iFigure 4
.. ....:. .:-. ....
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Table 5
Data Structure and Mean Mutation (Micronuclear) Frequencies
of Atlantic Mackerel Samples Analyzed as of Spring 183
General Grouping
of the Tows from
which Fish were
Taken in 1982
Middle Atlantic
~dson Canyon
No. of Tows
from which
Fish were
Taken
11
No. of
Time period Dates
Mean Micronuclear
over which Fish
Total No. Count per 1000
Tows were
were
of Fish
Mature ErythroDone
Sampled Analyzed cytes
Feb.
3-12
7
79
4.62
Mean Micronuclear
Count per 1000
Immature Erythrocytes *
9.90
helf Border
.16
Feb .. 14::'25
April 8-12
15
115
3.06
8.07
Shelf Border
South of Hudson Canyon
off Cape May
18
March 4April 13
17
145
3.00
6.17
Hempstead Bay
Long Island
1
1
13
4.23
13.92
May 25
* As based on a total of only 80 fish. Analyses of the immature erythrocytes of the
remaining 272 fish are nearing completion.
based on the entire 352 fish sampled.
Mean counts for the mature cells are
Table 6
Analysis of Variance on the Transformed
Mature and Immature Micronuclear,
Frequencies Fitting Only Water Mass Effects
Dependent
Variable
Transformed
,
Immature
Frequencies
Sample D.F.
5.5.
Size Fit
(Corrected
(Beyond for Mean)
Mean) ,
5.5.
(Res i dua 1)
F
0(
(Attained)
Estimates
A*
80
3
50.75
40.86
6~13
J.q= 2.15
.164·
C2= 2.15
.198
G« .005
A
J.l3= 1.66
A
J.l4= 2.64
Transformed
~1ature
Frequencies
A.
352
3
127.69
' 121.43
5.98
Cl(
=.0007
Standard
Error'
' , .• 128
.203
J.ll = 1.65
.066
A
.055
J.l2= 1.35,
A
J.l3= 1.32,
A
114=' 1.49
* Subscripts 1, 2,3,4, stand respectively for the following'sample groups
so designated in Table 5: f1iddle Atlantic; Hudson Canyon Shelf Border;
Shelf Bordersouth of Hudson Canyon off Cape f1ay; Hempstead Bay, Lang Island.
.049
.154
.
,,
..
REFERENCES
Alink, G.~., E.M.H. Frederix-Wolt~rs~,M.A. van ~er G~ag,J.F.J~ van ~e Kerkhoff
·and C.L.M. Poels. 1980. Induction of sister-chromatid exchanges in fish
. exposed to Rhine water. Mut. Res. 78: 369-374.
Beardmore, J.A., C.J. Barker, B. Battaglia, R.J. Berry, A.Crosby Longwell,
J~F. Payne and A. Rosenfield~
1980. The use ofgenetical apRroaches
to monitoring biological effects of pollution. Rapp. P.-v. Reun. Cons.
Int. Explor. Mer, l7~: 2~9-305.
Dixon, D.R •. 1982. Aneuploidy in mussel embryos (Mytilus edulis L.) originating
from a polluted dock. Mar. Biol. Letters 3: 155.
Generoso, W.M. and W~L~ Russell. 1969. Strain and sex variations.~n the
sensitivity of mice tri dominant-lethal induction with ethyl methanesulfonate. Mut. Res. 8: 589-598.
Gundy, S. and L.P. Varga~ 1983. Chromosomal
Mut. Res. 120: 187-191.
ab~rrations
in healthy persons.
Hooftman, R.N. and W.K. de Raat. 1982. Induction of nuclear anomalies
(micronuclei) in the peripheral blood erythrocytes of the eastern mudminnow Umbra pygmaea by ethyl methanesulphonate. Mut. Res. 104: 147-152
Hopke, P.K., M.J. Plewa, J.B. Johnston, D. Weaver, S.G. Wood, R.A •. Larson, and
T. Hinesly. 1982. Multitechnique screening of Chicago municipal sewage.
sludge for mutagenic activity. Environm. Sci. Technol. 16: 140~
•
Ivanov, B., L. Praskova, M. Mileva, M~ Bulanova and I. Georgieva. 1978.
Spontaneous chromosomal aberration levels in human peripheral lymphocytes. Mut. Res. 52: 421-426.
.
Klekowski, E.J., Jr. 1978. Detection of mutation damage in fern populations:
An in situ bioassay for mutagens in aquatic ecosystems~ In: Chemical
mutagens:-principles and methods for their detection. Vol: 5. Eds.
A~ Hollander and F.J. de Serres.
Plenum Press, New York.
Klekowski, E.J., Jr., and B.B. Berger. 1976.Chromosome mutations in a fern
population growing in a polluted environment: A bioassay for mutagens in
aquatic environments. Amer. J. Bot. 63: 239.
Longwell, A. Crosby. ·1981. Cytologic-cytogenetic perspectives on petroleum
effects in the marine environment - chromosome aberrations. Background
paper for the National AcademY of Sciences' updated review of Petroleum
in the Marine Environment .. Submitted July 1981, reviewed, accepted and
being used. To be re-organized slightly for publication in the open
literature - 45 pp, 2 referenced tables, 285 references.
Longwell, A. Crosby, and J.B. Hughes.
1980. Cytologic, cytogenetic, and
developmental state of Atlantic mackerel eggs from sea surface waters of
the New York Bight, and prospects for biological effects monitoring with
ichthyoplankton. Rapp. P.-v. Reun. Cons. Int~ Explor. Mer 179: 275-291.
~
.
•
l
Longwell, A. Crosby, and J.B. Hughes. 1982. Cytologic, cytogenetic, and
embryologie state of Atlantic mackerel eggs from surface waters of the
New York Bight in relation to pollution, p. 381-388. In section on
IIContributed papers on the effects of pollutants on plankton and neuston. 1I
~: Ecological Stress and the New.York Bight: Science and Management.
G.F. Mayer, editor.
.
Ma, Te-Hsiu, D. Isbandi, S.H. Khan and Ya-Shiu Tseng. 1973. Low level of
S02 enhanced chromatid aberrations in Tradescantia pollen tubes and
seasonal variations in aberration rates. Mut. Res. 21:.93.
Nee1, J.V. and E. Rothman. 1981. Is there adifference among human populations~ .
in the rate with which mutation produces electrophoretic variants? Proc.
Natl. Acad. Sei. USA 78: 3108-3112.
Prein, A.E., G.M. Thie, G.M. Alink, C.L.M. Poels and J.H. Koeman. 1978.
Cytogenetic changes in fish exposed to water of the river Rhine. Sei.
Tot. Environm. 9: 287-291.
Stromberg, P.T., M.L. Landolt and R.M. Kocan. 1981. Alterations in the
frequency of sister chromatid exchanges in flatfish from Puget Sound,
Washington, following experimental and natural exposure to mutagenic
chemieals. NOAA Technical Memorandum OMPA-10, 43 pp.
Todorov, S.L. 1979. Natural variations of human chromosome anomalies and
some data obtained from high background areas of natural radiation.
TEC-DOC-224, IAEA, Vienna, pp. 11-20.
Zimmermann~
F.K. 1979. Workshop on systems to detect induction of aneuploidy
by environmenta1 mutagens. Mut. Res. 64: 279-285.
•