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CAUFDRNIA
FBH-GAME



VOLUME 76


SUMMER 1990


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California Fish and Game is a journal devoted to the conservation and
understanding of fish and wildlife. If its contents are reproduced elsewhere,
the authors and the California Department of Fish and Game would appreciate
being acknowledged.

Subscriptions may be obtained at the rate of $10 per year by placing an
order with the California Department of Fish and Game, 2201 Garden Road,
Monterey, CA 93940. Money orders or checks in U.S. currency should be made
out to California Fish and Game. Inquiries regarding paid subscriptions should
be directed to the Editor.

Complimentary subscriptions are granted on an exchange basis.

Please direct correspondence to:

Robert N. Lea, Ph.D., Editor-in-Chief
California Fish and Game
2201 Garden Road
Monterey, CA 93940



\i





VOLUME 76



SUMMER 1990



NUMBER 3




Published Quarterly by

STATE OF CALIFORNIA

THE RESOURCES AGENCY

DEPARTMENT OF FISH AND GAME

—LDA—



STATE OF CALIFORNIA
GEORGE DEUKMEJIAN, Governor



THE RESOURCES AGENCY
GORDON VAN VLECK, Secretary for Resources



FISH AND GAME COMMISSION

ROBERT A. BRYANT. Presider)t
Yuba City

JOHN A. MURDY III, Vice President E. M. McCRACKEN, JR., Member
Newport Beach Carmichael

ALBERT C. TAUCHER, Member BENJAMIN F. BIAGGINI, Member
Long Beach San Francisco

DEPARTMENT OF FISH AND GAME

PETE BONTADELLI, Director

1416 9th Street

Sacramento 98514



CALIFORNIA FISH AND GAME

Editorial Staff

Editorial staff for this issue:

Editor-in-Chief Robert N. Lea, Ph.D.

Editorial Assistant Lisa L. Smith

Inland Fisheries L. B. Boydstun

and Arthur C. Knutson, Jr.

Marine Resources Peter L. Haaker

and Paul N. Reilly

Sturgeon Donald E. Stevens

Wildlife Management Bruce E. Deuel

and Kent A. Smith



131
CONTENTS

Page

Blood Lead Concentrations in Mallards from Delevan and Colusa

National Wildlife Refuges David M. Mauser, Tonie E. Rocke,

John G. Mensik, and Christopher j. Brand 132

Virulence of Four Isolates of Infectious Hematopoietic Necrosis Virus
in Salmonid Fishes and Comparative Replication in Salmonid Fish

Cell Lines Martin F. Chen, Candace M. Aikens,

J. L. Fryer, and J. S. Rohovec 137

The Effects of Different Otolith Ageing Techniques on Estimates of

Growth and Mortality for the Splitnose Rockfish, Sebastes diploproa,

and Canary Rockfish, 5. pinniger Christopher D. Wilson and

George W. Boehlert 146

Crov^'th and Longevity of Golden Trout, Oncorhynchus aguabonita, in

their Native Streams Roland A. Knapp and

Tom L. Dudley 161

Comparison of Efficiency and Selectivity of Three Gears Used to
Sample White Sturgeon in a Columbia River

Reservoir John C. Elliott and

Raymond C. Beamesderfer 174

NOTES

Giant Bluefin Tuna off Southern California, with a New California Size

Record Terry J. Foreman and

Yoshio Ishizuka 181

Bobcat Electrocutions on Powerlines Richard D. Williams 187

BOOK REVIEWS 190

MISCELLANEA 192



132 CALIFORNIA FISH AND CAME

Calif. Fish and Came 76(3) : 1 32-1 36 1990

BLOOD LEAD CONCENTRATIONS IN MALLARDS FROM
DELEVAN AND COLUSA NATIONAL WILDLIFE REFUGES'

DAVID M. MAUSER^

Sacramento National Wildlife Refuge,

Rt. 1 Box 311,

Willows, California 95988

TONIE E. ROCKE

National Wildlife Health Research Center,

6006 Schroeder Rd.,

Madison, Wisconsin 53711

JOHN G. MENSIK

Sacramento National Wildlife Refuge

Rt. 1 Box 311,

Willows, California 95988

and

CHRISTOPHER J. BRAND

National Wildlife Health Research Center,

6006 Schroeder Rd.

Madison, Wisconsin 53711

Blood samples were taken from 181 (108 adult drakes and 73 individuals of mixed
age and sex) mallards. Anas platyrhynchos, from Colusa and Delevan National
Wildlife Refuges during late winter and summer of 1987. The percentage of birds
with elevated lead concentrations was 28.7 for late winter and 16.4 for late summer.
For summer trapped birds, a significantly greater proportion of males than females
contained elevated lead levels. These findings indicate that lead poisoning may be
a year-round event in certain areas of the Sacramento Valley.

INTRODUCTION

Lead poisoning in waterfowl, resulting from ingestion of lead shot, is an issue
that has polarized both biologists and the hunting public for many years. It is
one of the major diseases of wild waterfowl (Friend 1985) with annual losses
estimated to be as high as 2-3 percent of the continent's population (Bellrose
1959). Although large outbreaks have been documented, it is believed that most
losses occur as isolated cases (Sanderson and Bellrose 1986; NWHRC, unpubl.
data). Lead poisoning is usually a chronic, debilitating disease, requiring 3
weeks or longer from ingestion of lead shot to death. Epizootics become
apparent when local predators cannot consume sick and dead birds fast enough
to prevent a visible accumulation of sick or dead birds.

Most studies on lead pellet ingestion and lead poisoning mortality are
conducted during or just after the waterfowl hunting season (Bellrose 1959;
NWHRC, unpubl. data). Few studies have been conducted during the summer
or early fall. We provide information on blood lead concentrations in wild
mallards. Anas platyrhynchos, in California's Sacramento Valley during late
winter and summer.



' Accepted for publication March 1990.

■'Current address: Department of Fisheries and Wildlife, Oregon State University, Corvallis, Oregon 97331



LEAD CONCENTRATIONS IN MALLARDS 133

STUDY AREA

Samples were collected at Delevan and Colusa National Wildlife Refuges
(NWRs), in Glenn and Colusa counties of California. Both refuges are part of
the Sacramento NWR complex and are administered by the U.S. Fish and
Wildlife Service. Delevan and Colusa NWRs are comprised of 2280 and 1635
ha, respectively, of seasonally-flooded marsh (60%), watergrass fields,
Echinochloa cruzgalli, (10%), permanent ponds (5-7%), rice (<2%) and
uplands (20%). Each area is managed primarily for fall migrant and wintering
waterfowl. Approximately 40% of each area is open to waterfowl hunting.
Non-toxic (steel) shot has been required since the fall of 1986.

METHODS

All birds used in the study were captured opportunistically and thus were not
a random sample of mallards from the study area. In addition, most birds
sampled were captured on previously hunted areas of the refuge (hunted since
1962).

During February 1987, 108 drake mallards were captured with baited funnel
traps. These birds were captured for use as sentinels in a study of botulism on
Sacramento NWR. Birds were bled within 24 hours of capture and placed in
holding pens where they were fed a combination of rice and scratch grains.
Pens were checked daily for sick and dead birds, and cause of death was
determined by necropsy and supporting diagnostic laboratory studies at the
U.S. Fish and Wildlife Service, National Wildlife Health Research Center
(NWHRC) Madison, Wisconsin.

Blood samples were similarly collected from 73 mallards of both sexes
trapped from July through early September, 1987. These birds were banded with
U.S. Fish and Wildlife Service leg bands, to insure that recaptured birds would
not be bled a second time and were immediately released.

Using 21 gauge needles and plastic syringes, blood samples (1.0-2.0 ml)
were drawn from the jugular vein of all birds, placed in heparinized glass tubes
and frozen for later analysis at NWHRC. Blood lead concentrations were
determined with a Perkin-Elmer HGA-400 graphite furnace coupled to a
Perkin-Elmer Model 2380 atomic absorption spectrophotometer set at a
wavelength of 283.3 nm (Fernandes and Hilligoss 1982). Blood lead concen-
trations of 0.2-0.5 ppm were considered to be elevated and > 0.5 ppm were
considered within the range known to be toxic to waterfowl (Friend 1985).
Lead poisoning diagnoses in sick and dead birds was based on pathology and
a toxic concentration of lead in liver ( > 8.0 ppm, wet weight) (Friend 1985).

For summer trapped birds, Chi square analysis was used to test for differences
in the numbers of males and females with elevated blood lead concentrations
( >0.2 ppm; age groupings were consolidated to yield cell expected values of
at least 5).

RESULTS

Of the 108 wild drake mallards captured in February, 15 (14%) had blood
lead concentrations greater than 0.5 ppm, 16(1 5% ) had concentrations ranging
from 0.2 to 0.5 ppm, and 77 (71%) were below 0.2 ppm (background
concentrations) . Within 20 days of capture, 1 2 birds had died, including 8 of 1 5



134 CALIFORNIA FISH AND CAME

birds (53%) that had blood lead concentrations exceeding 0.5 ppm when
initially captured; 2 of 16 birds (12%) with concentrations ranging fronn 0.2 to
0.5 ppm; and 2 of 71 birds (3%) below 0.2 ppm. Lead poisoning was diagnosed
as the cause of death in all 12 birds. Ten of the 12 lead poisoned birds had lead
pellets in their gizzards at the time of death ( ranging from 1 to 52 pellets ) . Two
of the 12 also had lesions associated with avian cholera, and Pasteurella
multocida was isolated from their livers. One additional death occurred from
among the 77 birds with background lead concentrations. Cause of death was
diagnosed as emaciation suspected as a result of parasitism by Echinuria
uncinata.

Of the 73 birds trapped during the summer, 12 (16%) had elevated or toxic
blood lead concentrations. Elevated concentrations were detected in 15% (2 of
13) of birds trapped in July, 11% (5 of 44) in August and 31% (5 of 16) in
September (Table 1 ). A significantly greater proportion of males than females
contained elevated concentrations of lead (X^ = 6.62, p <0.05) (Table 2).

TABLE 1. Distribution of Blood Lead Concentrations of 73 Mallards from Colusa and Delevan
National Wildlife Refuges Captured During July, August, and September 1987.

Percenitige of Totdl

Blood Lead Concentration July August September

Background

(<0.2ppm) 85(11) 89(39) 69(11)

Elevated

(0.2-0.5 ppm) 15(2) 9(4) 12(2)

Toxic

(>0.5ppm) 2(1) 19(3)

TABLE 2. Blood Lead Concentrations by Sex for 71 Mallards Captured During July, August and Sep-
tember 1987 on Colusa and Delevan NWRs

Percentage of Total



Blood Lead Concentration Males Females

Elevated

( > 0.2 ppm) 26 (8) 7(3)

Background

( < 0.2 ppm) 70 (19) 93 (41)

A significantly greater proportion of males had elevated lead concentrations than females
(X- = 6.62, p < 0.05).

DISCUSSION

The most sensitive method of determining lead exposure in live waterfowl Is
the measurement of blood lead concentrations (Anderson and Havera 1985).
Dieter (1979) demonstrated that signs of lead poisoning in canvasbacks, Aythya
valisineria, appeared at blood lead concentrations of 0.2 ppm. At lead
concentrations above 0.5 ppm, 12% of canvasbacks exhibited reduced activity
of delta-aminolevulinic acid dehydratase (ALAD), a key enzyme in the
hemoglobin biosynthetic pathway. Reduced ALAD activity in the brain causes
severe biochemical lesions and cerebellar damage (Dieter and Finley 1979).
The resulting motor disfunction coupled with other pathologic effects of lead
poisoning such as anemia, impaction, and tissue degeneration can eventually
lead to death.

Two male mallards trapped in late winter with background lead concentra-
tions ( <0.2 ppm) that later died of lead poisoning, could have ingested lead
pellets shortly before or on the day of capture. One lead pellet was found in the
gizzard of each bird at necropsy, and thus the lead was not yet detectable in the



LEAD CONCENTRATIONS IN MALLARDS I35

blood. The mortality rates of the birds held in captivity do not necessarily reflect
rates of lead poisoning mortally in free-ranging populations. Nonetheless, the
high lead exposure rate is indicative that lead poisoning is a problem to
waterfowl in these areas.

The percentage of adult male mallards trapped in September with elevated or
toxic blood lead concentrations (27%) was nearly as high as that of males
trapped in February (29%). This was unexpected because most cases of lead
poisoning are reported during the winter (Bellrose 1959; NWHRC, unpubl.
data). The availability of lead shot is thought to be correlated with the amount
of shot deposited on an area during the fall hunting season. However, the heavy
clay soils of the Sacramento NWR complex prevent lead pellets from settling
into the sediments. Thus, pellets are available to birds on these wetlands
year-round. High lead shot ingestion rates have been reported prior to hunting
season in other studies. Zwank et al. (1985) found that lead ingestion rates of
mallards and northern pintails. Anas acuta, were higher before, rather than after,
the hunting season on 2 waterfowl wintering areas in Louisiana. Lead pellet
ingestion and lead poisoning mortality rates were also higher in the fall rather
than in the winter in a study conducted at the Sacramento NWR using sentinel
mallards confined to a heavily hunted wetland (NWHRC, unpubl. data).
Moreover, lead poisoning in the summer and fall may not be easily observed
because summer resident populations are sparse, and sick birds or carcasses do
not persist for long in the environment.

The difference in proportions of males and females with elevated blood lead
concentrations could be due to differences in feeding habits. Male and female
mallards are known to molt at different times (Bellrose 1976). During August
and September, males have generally finished molting flight feathers, whereas
females are just entering the molt (adults only). These differences in physio-
logic condition associated with molt might affect their diet (Heitmeyer 1985).
Diets high in calcium and protein have been found to mitigate the effects of lead
shot ingestion (Sanderson and Bellrose 1986). In addition, females have been
shown to be less susceptible to lead poisoning during the breeding season.
Increased mobilization of calcium from bones for egg laying and a high
metabolic rate apparently decrease the absorption of lead (Finley and Dieter
1978).

Recent conversion to non-toxic shot will not result in immediate major
reductions of lead poisoning in certain habitats. The heavy clay soils of the
Sacramento Valley apparently reduce settling of lead shot into sediment beyond
the reach of feeding waterfowl. Surveys for pellets in wetland sediments at the
Sacramento NWR in 1987 revealed densities of up to 900,000 pellets per acre
in the top 10 cm (NWHRC, unpubl. data). Although the use of steel shot has
been enforced on the National Wildlife Refuges of the Sacramento Valley since
the fall of 1986, lead poisoning in waterfowl continues to be documented
(NWHRC, unpubl. data).

ACKNOWLEDGMENTS

Sincere thanks are extended to M. Smith, National Wildlife Health Research
Center for lead analysis, and E. Collins and P. O'Halloran for permission to
collect samples. F. Weekley, S. Shaeffer, C. Franson, L. Locke, and M. Friend
provided helpful editorial comments and H. Berg provided statistical advice.



136 CALIFORNIA FISH AND CAME

LITERATURE CITED

Anderson, W. L., and S. P. Havera. 1985. Blood lead, protoporphyrin, and ingested shot for detecting lead

poisoning in waterfowl. Wildl. Soc. Bull., 13:26-31.
Bellrose, F. C. 1959. Lead poisoning as a mortality factor in waterfowl populations. Illinois Nat. Hist. Surv. Bull.,

27:235-288.

. 1976. Ducks, geese and swans of North America. Stackpole Books. Harrisburg, Pa. 544 p.

Dieter, M. P. 1979. Blood delta-aminolevulinic acid dehydratase (ALAD) to monitor lead contamination in

canvasback ducks (Aythyd valisineria). Pages 177-191 in F. W. Nielson, G. Migaki, and D. C. Scarpelli, eds.

Animals as monitors of environmental pollutants. Natl. Acad. Sci. Washington, D.C., 421 p
and M. T. Finley. 1979. Delta-aminolevulinic acid dehydratase enzyme activity in blood, brain, and

liver of lead-dosed ducks. Environ. Res., 19:127-135.
Fernandes, F. )., and D. FHilligoss. 1982. An improved graphite furnace method for the determination of lead in

blood using matnx modification and the LVOV platform. Atomic Spectroscopy, 3:130-131.
Friend, M. 1985. Interpretation of criteria commonly used to determine lead poisoning problem areas. U.S. Fish

and Wildl. Serv., Fish and Wildl Leaflet no. 2, Washington, D.C. 4 p.
Sanderson, C. C, and F. C. Bellrose. 1986. A review of the problem of lead poisoning in waterfowl. Illinois Natural

FHistory Survey, Special Publication No. 4. 34 p.
Zwank, P. |., V. L. Wright, P. M. Shealy, and ). D. Newsom. 1985. Lead toxicosis in waterfowl on two major

wintering areas in Louisiana. Wildl. Soc. Bull., 13:17-26.



VIRUS IN SALMONID FISHES I37

Calif. Fish and Came 7b{T,): 137-145 1 990

VIRULENCE OF FOUR ISOLATES OF INFECTIOUS

HEMATOPOIETIC NECROSIS VIRUS IN SALMONID FISHES

AND COMPARATIVE REPLICATION IN

SALMONID FISH CELL LINES'

MARTIN F. CHEN 2

CANDACE M. AIKENS '

). L. FRYER"

AND

J. S. ROHOVEC

Department of Microbiology

Oregon State University, Corvallis, OR 97331-3804

The virulence of low-passage isolates of infectious hematopoietic necrosis virus
(IHNV) obtained from diverse geographic locations was compared in juvenile
Chinook, Oncorhynchus tshawytscha, sockeye (kokanee), O. nerka, and coho, O.
kisutch, salmon, and rainbow (steelhead) trout, O. mykiss. All isolates tested were
pathogenic for sockeye salmon and trout, but two isolates were comparatively
avirulent for chinook salmon. Hybrids of IHNV-resistant coho salmon and suscep-
tible trout appeared to be resistant; however, infection of coho salmon with IHNV
was demonstrated. The infectivity and replication of the IHNV isolates were
compared in cell lines derived from chinook, coho, and sockeye salmon. Infective
dose assays, growth curves, and efficiency of plaquing comparisons showed that the
host preference of IHNV isolates could be demonstrated at the cellular level.

INTRODUCTION

Infectious hematopoietic necrosis virus (IHNV) is a highly destructive
pathogen of juvenile salmonid fishes, while adult fish act as asymptomatic
carriers (Wingfield and Chan 1970). The virus affects different host species
throughout its range on the Pacific Coast of North America. In Alaska,
hatchery-reared sockeye salmon, Oncorhynchus nerka, have experienced
severe outbreaks (Grischowsky and Amend 1976). In California, chinook
salmon, O. tshawytscha, have been killed by the virus. (Wingfield and Chan
1970). Resident and anadromous (steelhead) rainbow trout, O. mykiss, are also
susceptible to IHNV, but coho salmon, O. kisutch, are considered resistant
(Pilcher and Fryer 1980).

in Oregon, epizootics of IHNV have occurred in juvenile steelhead trout at
the Round Butte Hatchery on the Deschutes River, juvenile chinook salmon
reared at the same facility have not been affected. However, outbreaks of IHNV
have occurred in juvenile chinook salmon reared at the Elk River Hatchery on
the Oregon coast (Mulcahy et al. 1980). We compared the virulence of virus
isolates from the Round Butte and the Elk River hatcheries, Oregon, and one
each from an Alaska and California hatchery to determine if the Elk River and
California isolates were unique in their ability to kill juvenile chinook salmon.



' Accepted for publication April 1990.

' Present address: California Departnnent of Fish and Came, Mohave River Hatchery, P.O. Box 938, Victorville,

CA 92393.
' Present address: Cascade Valley Hospital, 330 S. Stillaguamish St., Arlington, WA 98223.
'' To whom correspondence should be addressed.



138 CALIFORNIA FISH AND GAME

We also wished to determine if Round Butte chinook salmon were resistant to
IHNV from the four sample locations. The virulence of the four isolates was also
compared in sockeye salmon and rainbow trout. We found the difference in
virus epizootics at the Elk River and the Round Butte hatcheries was shown to
result from a difference in host preference of the two virus isolates found at
these two hatcheries. Round Butte chinook salmon were not more resistant to
the virus than the other chinook salmon stocks. Rainbow trout and sockeye
salmon were susceptible to all virus isolates tested. Juvenile coho salmon,
although demonstrating resistance, could be experimentally infected by IHNV.
Resistance to IHNV was also shown in F, hybrids of coho salmon X rainbow
trout.

Differences have been found in the infectivity of IHNV isolates for salmonid
cell lines (Phillipon-Fried 1980, Fendrick, Groberg, and Leong 1982). To
develop in vitro models of host-specific virulence, the replication of the four
IHNV isolates was compared in cell lines derived from chinook, coho, and
sockeye salmon. Differences among the isolates shown by the in vivo
experiments were also demonstrated in vitro by infective dose assays, growth
curves, and efficiency of plaquing.

MATERIALS AND METHODS
Cell Lines

Chinook, sockeye, and coho salmon embryo cells (CHSE-214, SSE-5, and
CSE-119, respectively, Lannan, Winton, and Fryer 1984) were used in this study.
Cells were grown in Eagle's minimum essential medium (MEM) buffered with
10 mM NaHC03, pH 7.8, supplemented with penicillin (100 lU/ml), strepto-
mycin (100 jLLg/ml) , and 5% fetal bovine serum (MEM-5). Growth of cells and
all incubations of infected cells were carried out at 16°C.

Viral Infectivity Assays

Endpoint dilution assays were performed as previously described (Hedrick,
Leong, and Fryer 1978). Titers were read after 14 d and expressed as the
number of tissue culture infective doses sufficient to infect 50% of inoculated
cultures per ml (TCID5o/ml).

Plaque assays were performed using methylcellulose overlays as previously
described (Hedrick and Fryer 1981 ). Titers were expressed as the average of
triplicate titrations.

Growth Curves

Duplicate, 2-d-old monolayers of CHSE-214 and CSE-119 cells in 25 cm^
flasks were infected with 10"* TCID50 virus in 0.2 ml. After absorption for 1 h,
unattached virus was removed with three washes of MEM, and 6 ml of MEM-5
was added to each flask. At various intervals, 0.1 ml of fluid was removed from
each flask, diluted in 1 ml MEM, centrifuged at 2000^ for 10 min and frozen
at — 70°C. Finally, samples were simultaneously titered by endpoint dilution
assay in CHSE-214 cells.

Viruses

Viruses were received as primary isolates. The Trinity River (TR), California,
chinook salmon isolate was obtained from W. H. Wingfield, California



VIRUS IN SALMONID FISHES 139

Department of Fish and Game, Rancho Cordova, CA. The Elk River (ER)
Chinook salmon isolate and the Round Butte (RB) steelhead trout isolate were
provided by W. J. Groberg, Jr., Oregon Department of Fish and Game and
Wildlife (ODFW), Corvallis, OR. An isolate from adult chinook salmon at
Mendenhall Ponds (MP), Alaska, was received from R. Saft, Alaska Department
of Fish and Game, Anchorage, AK. Second-passage virus produced in CHSE-214
cells were used to infect fish. Cell line infectivity assays and growth curves were
performed using third-passage virus stocks grown in CHSE-214 cells. Virus
harvests were centrifuged at 2000^, pooled, aliquoted and frozen at — 70°C
until used. Each pool was titered after thawing by endpoint dilution assay in
ChHSE-214 cells. Low-passage viruses were used in this study because attenua-
tion and host range adaptation of IHNV has occurred after prolonged serial
passage in cell culture (Fryer et al. 1976, Nims et al. 1970).

Fish

Juvenile fish, eggs, and semen of the four study species of salmonid fishes
were provided by the ODFW: chinook salmon from the Elk, Trask and Round
Butte hatcheries, sockeye salmon from the Wizard Falls Hatchery, steelhead
trout from the Round Butte Hatchery, coho salmon from the Sandy River
Hatchery, and rainbow trout from the Oak Springs Hatchery. The juvenile fish,
eggs, and semen were from adults found to be negative for IHNV at time of
spawning. Juvenile fish were obtained from large lots present in hatching
troughs or ponds. Fish were tested immediately after resorbtion of the yolk-sac,
because susceptibility to IHNV under experimental conditions declines rapidly
with age (Wingfield and Chan 1970). Coho salmon X rainbow trout hybrids
were produced by fertilizing eggs from a single rainbow trout with pooled coho
salmon sperm collected from 10 fish. Eggs from the same rainbow trout were
fertilized with pooled rainbow trout sperm from 10 fish and the pooled coho
salmon sperm was used with coho salmon eggs pooled from three fish to
produce normal rainbow trout and coho salmon, respectively. Production of
true coho salmon X rainbow trout hybrids was verified by liver isoenzyme


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