&EPA
EPA 910/9-86-140
United States
Environmental Protection
Agency
Region 10
1200 Sixth Avenue
Seattle WA
Alaska
Idaho
Oregon
Washington
Environmental Services Division
June 1986
A Comparison Between
English Sole Diseases in
Commencement Bay and in
Selected Nonurban
Embayments
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EPA-910/9-86-140
June 1986
A COMPARISON BETWEEN ENGLISH SOLE DISEASES IN
COMMENCEMENT BAY AND IN SELECTED NONURBAN EMBAYMENTS
by
Bruce B. McCain, Linda D. Rhodes, Mark S. Myers, and
Donald C. Mai ins
Environmental Conservation Division
Northwest and Alaska Fisheries Center
2725 Montlake Boulevard East
Seattle, Washington 98112
ENVIRONMENTAL SERVICES DIVISION
REGION 10
U.S. ENVIRONMENTAL PROTECTION AGENCY
SEATTLE, WASHINGTON 98101
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SUBMITTED TO:
Environmental Services Division
Region 10
U.S. Environmental Protection Agency
Seattle, Washington 98101
In Partial Fulfillment of
USEPA Interagency Agreement DW13930536-01-1
NOTICE
This report has been reviewed by Region 10, U.S. Environmental Protection
Agency and approved for publication. Mention of trade names or
commercial products does not constitute endorsement or recommendation
for use.
11
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CONTENTS
Acknowledgements iv
Introduction 1
Methods and Materials 2
Results and Discussion 6
References 13
111
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ACKNOWLEDGEMENTS
We wish to thank the following for their participation in this program:
W.D. Gronlund, P.K. Emry, W.E. Ames, P.O. Plesha, J.T. Landahl,
C.M. Stehr, P.O. Olson, D.R. Craddock, and J.R. Hughes.
iv
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INTRODUCTION
Several studies conducted in Commencement Bay since 1979
(Malins et al. 1980, 1982, 1984) identified a number of pollution-
associated diseases in English sole (Parophrys vetulus), rock sole
(Lepidopsetta bilineata) and Pacific staghorn sculpin (Leptocottus
armatus). The organs of fish containing the greatest numbers of such
lesion types were the liver, kidney, and gills. These lesions were not
associated with parasites or microorganisms, and were defined as idiopathic
(i.e. cause not known). Neoplasms constituted a major type of idiopathic
liver lesion. Other major types of idiopathic liver lesions were
hyperplasia/foci of cellular alteration [which have been reported to be
"preneoplastic" in laboratory rodents (Frith and Ward 1980)], specific
degeneration/necrosis, and storage disorders (McCain et al. 1982). Fish
with hepatic neoplasms were primarily from the waterways of Commencement
Bay (i.e., Hylebos and Sitcum Waterways). No tumor-bearing fish (156
examined) were found in nonurban waters (Case Inlet, Port Madison, Port
Susan, and Discovery Bay) during 1979 to 1982; however, samplings conducted
in 1983 found tumor-bearing English sole (6.7$, 2 of 30 fish) in Port
Madison.
High concentrations of a great variety of chemicals were found in
sediments from Commencement Bay (Malins et al. 1982, 1984). For example,
the mean concentration of polychlorinated biphenyls (PCBs) in sediment
was more than 100 times greater in the Hylebos Waterway than in sediment
from the nonurban area of Port Madison. Mean concentrations of aromatic
hydrocarbons (AHs) were as much as 40 times greater in parts of the
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Hylebos Waterway, compared to nonurban areas. Generally, mean concentrations
of sediment-associated metals (e.g., lead) were higher in Commencement
Bay than in the nonurban areas. The mean concentrations of cadmium,
however, were similar in Commencement Bay and nonurban sediments.
The purpose of the present study was to compare the types and
prevalences of idiopathic lesions of the liver previously observed in
English sole from Commencement Bay with those from selected nonurban
embayments. English sole was selected as the target species because of
the high prevalences of pollution-related lesions observed in this species,
and because this species is widely distributed throughout Puget Sound
and adjacent waters.
METHODS AND MATERIALS
English sole were collected from the RV HAROLD W. STREETER with an
otter trawl having a 7.5 m opening, a 10.8 m total length, 3.8 cm mesh in
the body of the net, and a 0.64 cm mesh liner in the cod end. Individual
trawls were for 5 minutes and covered a distance of approximately 0.2
nautical miles. The results of histopathological examinations of English
sole are reported for sole captured near Bellingham Bay, just southwest
of Eliza Island; Case Inlet, near Stretch and Reach Islands; and
Commencement Bay, several sites including Sitcum and Hylebos Waterways
(Figure 1 and Table 1). At each sampling station, up to 30 adult individuals
of each target species were collected and placed in holding tanks containing
fresh seawater. Only individuals greater than 150 mm in total length
(of as broad a size distribution as possible) were selected for necropsy.
Fish to be necropsied were measured for total length (mm), weighed
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£7 Commencement
Bay
Figure 1. Map showing locations of sampling sites near Eliza Island,
in Case Inlet, and in Commencement Bay.
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Table 1. Sampling sites, sampling dates and latitude and longitude of the trawl
midpoints included in this study.
Eliza Island
(4/84)
Case Inlet
(4/84)
Case Inlet
(4/79)
Sitcum and Hylebos
Waterways
(4/79)
Deepwater Commencement Bay
(4/79)
48°39.1'N
47°18.8'N
47°19.5'N
47°20.8'N
47°21.2'N
47°18.8'N
47°16.2'N
47°16.7'N
47°16.2'N
47°17.1'N
47°17.5'N
47°17.3'N
47°18.5'N
47°18.7'N
47°16.7'N
47°16.2'N
122°33.3'W
122°50.3'W
122°47.9'W
122°48.7'W
122°47.0'W
122°49.5'W
122°22.3'W
122°23.8'W
122°25.0'W
122°25.0'W
122°25.2'W
122°28.7'W
122°30.2'W
122°26.1'W
122°27.5'W
122°26.9'W
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(g) and assigned an individual field number. The gender was determined
and tissues were excised from the liver, kidney, and gill. Any grossly
visible lesions were noted, recorded, and tissue specimens were collected.
Tissues were placed in labeled cassettes and immersed immediately in
Dietrich's fixative (Malins et al. 1980).
Tissue specimens preserved in fixative for a minimum of 48 hours
were dehydrated and paraffin-infiltrated with an automated tissue processor,
embedded in paraffin and sectioned at a thickness of 5-6 urn. Sections
were routinely stained with Mayer's hematoxylin and eosin-phloxine
(AFIP 1968). All slides were examined by light microscopy employing a
"blind" system, in which the examining histopathologist had information
only on the species, length, weight, sex and description of grossly
visible lesions, with no knowledge of the location from which the fish
was collected. Diagnoses were then performed using the lesion categories
described in Malins et al. (1982). The diagnostic information was coded
on National Ocean Data Center (NODC) File type 13, and placed on computer
disk along with information on other biological characteristics of each
fish. Data management and lesion prevalence analyses were performed
utilizing Minitab version 81.1 (Ryan et al. 1981) and SPSS version
9.1 (Nie et al. 1975) on a Burroughs 7800 computer.
For comparisons of lesion prevalences, the specimens were first
classified into 5 size classes (adjusted for gender) roughly
corresponding to age classes 1+, 2+, 3+, 4+ and _>5+. The size class limits
were determined from size distributions of each age-class by gender
of English sole from a variety of sites in Puget Sound (n=1004).
In order to minimize the influence of seasonal differences in liver,
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kidney and gill lesion prevalences, only lesion prevalence data for fish
collected in the spring were compared. Lesion prevalence data was then
computed for 5 sites (sampling month and year in parentheses): (1) Eliza
Island (March 1984); (2) Case Inlet (March 1984); (3) Case Inlet (April
1979); (4) combined Sitcum and Hylebos Waterways (April 1979);
and (5) combined deeper water Commencement Bay sites (April 1979).
Comparisons were made by computation of the G-value from the natural
logarithms of the lesion frequencies and comparison with appropriate
chi-square values. Since 4 multiple tests were required for each organ
(i.e., 4 major lesion categories were considered for each organ), the
critical p-value was adjusted to 0.0125 for all G-tests to reduce Type I
error.
RESULTS AND DISCUSSION
Although the original design of this study called for collecting
English sole from four nonurban embayments in or near Puget Sound, (Sequim Bay,
Dabob Bay, Samish Bay and Case Inlet), sole were not collected from three
of these areas (Sequim Bay, Samish Bay and Dabob Bay) due to a scarcity
or absence of adult English sole. Malins et al. (1982) demonstrated that
English sole less than 2 years old had significantly (p<0.05) lower
prevalences of idiopathic liver diseases than fish over 2 years old. For
example, liver neoplasms were not detected in males less than 2 years old
or in females less than 1 year old. Consequently, our protocol called
for sampling English sole >150 mm, and fish from these year classes were
absent in these areas. In order to partially compensate for this problem,
an additional reference station near Eliza Island (west of Bellingham
Bay, Station No. 04015) was sampled, where a sufficient number of adult
English sole were collected.
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Ten stations in Commencement Bay (Figure 1) were sampled for diseased
English sole in April of 1979 (Maiins et al. 1982, 1984). Seven of these
stations were located around the periphery of the bay at depths between 20
and 150 m. Three of these stations, two in the Hylebos Waterway and the
other in the Sitcum Waterway, were located between 10 and 15 m in depth.
Comparisons in this report were made between the prevalence data
for the major types of idiopathic liver, kidney, and gill lesions observed in
the nonurban embayments sampled in the present study and the prevalence
data for those lesions found in Commencement Bay since they were the only
organs which displayed significant idiopathic lesions. Data comparisons
by site were made for each size class, as well as for all size classes
combined.
The only type of hepatic lesions found in sole from near Eliza Island
were storage disorders (Table 2). This condition was found in sole from
Case Inlet and the Commencement Bay sites, and was previously found in
several bottom fish species from both urban and nonurban areas of Puget
Sound (Malins, et al. 1984). Storage disorders can be caused by a variety
of factors, many of which are unrelated to pollution (e.g. diet, reproductive
stage).
The prevalences of hepatic neoplasms (3.3$) and foci of cellular
alteration FCA (3.32) in sole from Case Inlet (Table 2) were not
significantly different from the prevalences of neoplasms (3.6$) and FCA
(14.3$) found in sole from Sitcum and Hylebos Waterways (3.6$).
However, only one individual from Case Inlet had both the liver neoplasm and
the FCA. None of these liver lesions were observed in sole from
Case Inlet in either April of 1979 (Table 1) or during other sampling
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Table 2. Hepatic lesion prevalences and numbers of examined livers by size
class and by site for English sole collected in March and April
of 1979 and 1984. Size classes 1 through 5 approximately correspond
to age classes 1+, 2+, 3+, 4+, and >5+, respectively. The G-test
was used to determine differences in lesion prevalences among the
sites.
SIZE CLASS
Number of livers examined
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
Neoplasms
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
Foci of cellular alteration
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterwats (1979)
Deep Commencement Bay (1979)
Specific degeneration/necrosis
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
Storage disorders
Eliza Island (1984) 12
Case Inlet (1984) 16
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
1
8
6
0
2
2
0
0
0
0
0
0
0
0
0
0
0
0
.5
.7
0
0
2
19
15
1
2
5
0
0
0
0
0 .
0
0
0
0
0
0
0
0
0
0
5.3
0
0
0
0
3
1
3
0
5
7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
0
2
0
6
14
0
16.7
0
0
33.3
0
0
33.3
0
0
16.7
0
5
0
4
8
13
22
25.0
0
0
0
25.0
0
15.4
0
0
0
23.1
4.6
25.0
0
7.7
0
All size classes
combined
28
30
9
28
50
0
3.3
0
3.6
0
0
3.3
0
14.3
0
0
0
0
17. 9H
2.0
7.1
6.7
0
7.1
0
"H" indicates significantly higher lesion prevalence as determined by G-test,
critical p=0.0125
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periods of 1979 (n=34). It is difficult to evaluate temporal changes
in lesion prevalences in Case Inlet fish, due to the low prevalence
values and the relatively small sample sizes.
The absence of specific degenerative lesions of the liver in sole
from Case Inlet and near Eliza Island contrasted with the significantly higher
prevalence (17.92) in sole from Sitcum and Hylebos Waterways (Table 2). In
previous Puget Sound studies, this lesion type was detected almost exclusively
in bottomfish from urban embayments (Mai ins et al. 1984). Lesions
morphologically similar to this lesion type have been induced in mammals
and fish exposed to a variety of toxic chemicals under laboratory conditions.
Renal depositional disorders were not found among the fish examined in
this study, although these conditions have been found in English sole
in other studies (Maiins et al. 1982). Degenerative and necrotic kidney
lesions and renal inflammations were found at both non-urban and
Commencement Bay sites at statistically uniform prevalences (Table 3).
Renal proliferative disorders were characterized by high prevalences at
the non-urban sites sampled in 1984, and low prevalences at the 1979 sampling
of the Commencement Bay sites and Case Inlet. The significance of this
result is not clear and such a pattern has not yet been previously
reported (Maiins et al. 1980, 1982). Possibly a seasonal factor
may have a role in the etiology of this lesion since Rhodes et al. (In
Preparation) have observed higher prevalences of this condition
in sole captured in the summer and fall than sole captured in winter
and spring.
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10
Table 3. Renal lesion prevalences and numbers of examined kidneys by size class
and by site for English sole collected in March and April of 1979 and 1984.
Size classes 1 through 5 approximately correspond to age classes 1+, 2+,
3+. 4+. and ^5+, respectively. The G-test was used to determine differences
in lesion prevalences among the sites.
SIZE CLASS
Number of kidneys examined
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
Depositional disorders
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
Oegenerati on/necrosi s
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
Proliferative disorders
Eliza Island (1984) 25
Case Inlet (1984) 33
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
Inflammations
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
1
8
6
0
2
2
0
0
0
0
0
0
0
0
.0
.3
0
0
0
0
0
0
2
17
15
1
2
5
0
0
0
0
0
5.9
13.3
0
0
0
35.3
20.0
0
0
0
0
0
0
0
0
3
1
3
0
5
7
0
0
0
0
0 '
0
20.0
0
0
0
0
0
0
0
0
0
4
0
2
0
6
11
0
0
0
0
33.3
0
0
0
0
0
0
0
5
0
4
7
12
20
0
0
0
0
0
0
8.3
15.0
25.0
14.3
0
0
0
0
0
5.0
All size classes
combined
26
30
8
27
45
0
0
0
0
0
3.8
6.7
0
14.8
6.7
30. 8H
20.0
0
0
OL
0
0
0
2.2
"H" indicates significantly higher lesion prevalence as determined by G-test,
critical p=0.0125
"L" indicates significantly lower lesion prevalence as determined by G-test,
critical p=0.0125
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11
With the exception of idopathic respiratory inflammations,
prevalences of gill lesions were statistically uniform among the sites
(Table 4). The prevalence of inflammatory conditions were found to be
significantly higher among sole from the non-urban sites and lower among
sole from Commencement Bay (p <0.0125). Again, a possible role of season
may be involved in lesion prevalence variations.
No statistically significant differences in the prevalences of any
of the liver, kidney or gill lesions were found among the size classes
for any of the sampling sites; however, no fish in the first 3 size
classes had hepatic neoplasm, FCA, or specific degeneration/necrosis.
This suggests that differences in prevalence among the sites are not
attributable to differential lesion prevalences among the size
classes of sole examined.
In conclusion, the number and prevalences of different types of
idiopathic liver lesions were generally greater in English sole from
Commencement Bay compared to the nonurban areas sampled in this study.
Nevertheless, the prevalences of these lesions in Commencement Bay sole
are relatively low compared to other polluted areas ("hot spots") in
Puget Sound [e.g., the Duwamish Waterway (Mai ins et al. 1984), Eagle
Harbor (Malins et al. 1985), and Mukilteo (Malins et al. 1986)]. In
contrast, the prevalences of a renal and a respiratory
lesion were greater at the non-urban sites. These types of results
underscore the necessity of clarifying lesion etiology if lesions are
to be used as indicators of envionmental quality.
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12
Table 4. Respiratory lesion prevalences and numbers of examined gills by size
class and by site for English sole collected in March and April of 1979
and 1984. Size classes 1 through 5 approximately correspond to age
classes 1+, 2+, 3+, 4+, and ^5+, respectively. The G-test was used
to determine differences in lesion prevalences among the sites.
Number of gills examined
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
Degeneration/necrosis
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
Proliferative disorders
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
Vascular disorders
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
Inflammations
Eliza Island (1984)
Case Inlet (1984)
Case Inlet (1979)
Sitcum-Hylebos Waterways (1979)
Deep Commencement Bay (1979)
1
8
6
0
1
1
0
0
0
0
12.5
0
100
0
12.5
16.7
0
0
75.0
66.7
0
0
2
17
15
1
2
3
0
0
0
0
0
29.4
6.7
0
0
0
23.5
0
0
0
0
58.8
60.0
0
0
0
SIZE
3
1
3
0
4
6
0
0
0
0
0
0
0
0
0
0
0
0
0
33.3
0
0
CLASS
4
0
2
0
4
12
0
0
0
0
25.0
8.3
0
0
0
50.0
0
0
5
0
4
6
10
17
0
0
0
0
0
50.0
60.0
17.6
25.0
0
0
0
50.0
0
0
0
All size classes
combined
26
30
7
21
39
0
0
0
0
0
23.1
3.3
42.9
38.1
10.3
19.2
6.7
0
0
0
61. 5H
56. 7"
0
OL
QL
"H" indicates significantly higher lesion prevalence as determined by G-test,
critical p=0.0125
"L" indicates significantly lower lesion prevalence as determined by G-test,
critical p=0.0125
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13
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Armed Forces Institute of Pathology (AFIP). 1968. Manual of Histplogic
Staining Methods, Third Edition, (L.G. Luna, ed.) McGraw-Hill,
New York, 258 p.
Frith, C.H. and Ward, J.M. (1980). A morphological classification of
profliferative and neoplastic hepatic lesions in mice. J_. Environ.
Pathol. Toxicol., _3:329-351.
Malins, D.C., McCain, B.B, Brown, D.W., Sparks, A.K. and Hodgins, H.O.
1980. Chemical contaminants and biological abnormalities in central
and southern Puget Sound. NOAA Technical Memorandum OMPA-2. 295 p.
Malins, D.C., McCain, B.B., Brown, O.W., Sparks, A.K., Hodgins, H.O. and
Chan,CS-L. 1982. Chemical contaminants and abnormalities in
fish and invertebrates from Puget Sound. NOAA Technical Memorandum
OMPA-19. 168 p.
Malins, D.C., McCain, B.B., Brown, D.W., Chan, S-L, Myers, M.S., Landahl , J.T.,
Prohaska, P.G., Friedman, A.J., Rhodes, L.D., Burrows, O.G., Gronlund, W.D.
and Hodgins, H.O. 1984. Chemical pollutants in sediments and
diseases in bottom-dwelling fish in Puget Sound, Washington.
Environ. Sci. Techno! . JL8^709-713.
Malins, D.C., Krahn, M.M., Myers, M.S., Rhodes, L.D., Brown, D.W., Krone,
C.A., McCain, B.B., Chan, S-L. (1985a), Toxic chemicals in sediments
and biota from a creosote-polluted harbor: relationships with hepatic
neoplasms and other hepatic lesions in English sole (Parophrys
vetulus), Carcinogenesis 6(10), 1463-1469.
Malins, D.C., Krahn, M.M., Brown, D.W., Rhodes, L.D., Myers, M.S.,
McCain, B.B. and Chan, S-L. (1985b). Toxic chemicals in marine
sediment and biota from Mukilteo, Washington: relationships with
hepatic neoplasms and other hepatic lesions in English sole
Parophrys vetulus), _J. Natl. Cancer Inst., 74:487-494.
Nie, N.H., Hull, C.H., Jenkins, J.G., Steinbrenner, K. and Bent, D.H.
1975. Statistical Package for the Social Sciences. McGraw-Hill,
NY, 675 p.
Ryan, T.A., Joiner, B.L. and Ryan, B.F. 1981. Minitab reference manual;
Duxbury Press, Boston, 154 p.
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