&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)




4839.1'N

4718.8'N
4719.5'N
4720.8'N
4721.2'N
4718.8'N
4716.2'N
4716.7'N
4716.2'N
4717.1'N
4717.5'N
4717.3'N
4718.5'N
4718.7'N
4716.7'N
4716.2'N
12233.3'W

12250.3'W
12247.9'W
12248.7'W
12247.0'W
12249.5'W
12222.3'W
12223.8'W
12225.0'W
12225.0'W
12225.2'W
12228.7'W
12230.2'W
12226.1'W
12227.5'W
12226.9'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,
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Frith, C.H. and Ward, J.M. (1980).  A morphological classification  of
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Malins, D.C., McCain, B.B, Brown, D.W.,  Sparks, A.K.  and Hodgins, H.O.
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Malins, D.C., McCain, B.B., Brown, O.W., Sparks,  A.K., Hodgins, H.O.  and
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Malins, D.C., McCain, B.B., Brown, D.W., Chan,  S-L, Myers,  M.S., Landahl ,  J.T.,
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Malins, D.C., Krahn,  M.M., Myers, M.S.,  Rhodes, L.D., Brown, D.W.,  Krone,
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Malins, D.C., Krahn,  M.M., Brown, D.W.,  Rhodes, L.D., Myers, M.S.,
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     sediment and biota from Mukilteo, Washington: relationships with
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     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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