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Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
THE USE OF SERUM PROTEINS AS BIOLOGICAL MARKERS OF CONTAMINATION OF
GENTOO
Pygoscelis papua
AND ADELIE
P. adeliae
PENGUINS
Roberto NAJLE
1
, Hugo D. SOLANA
1
, Daniela BOTTINO
2
, Mariana A. JUÁRES
2
,
Melina MAUAD
2
and Diego MONTALTI*
2, 3
1
Laboratorio de Biología Celular y Molecular, Departamento de Ciencias Biológicas, Facultad de
Ciencias
Veterinarias, Universidad Nacional del Centro, B7000-Tandil, Argentina
2
Sección Ornitología, Museo de La Plata, Paseo del Bosque, B1900FWA-La Plata, Argentina
3
Departamento Biología Aves, Instituto Antártico Argentino, Cerrito 1248, C1010AAZ-Buenos Aires, Argentina.
Tel: +54 11 42757523. Fax: +54 11 48128169. *E-mail: dmontalti@arnet.com.ar
(Recibido junio 2006, aceptado octubre 2006)
Key words: biological markers, serum proteins, penguins, Antarctica,
Pygoscelis adeliae
,
Pygoscelis papua
ABSTRACT
Two common and widely used liquid fuels with hepatotoxic activity, and trimethyltin, a
compound with neurotoxic activity which is used in several industrial processes, were
tested for their toxicity in two species of
Pygoscelis
penguins. Gentoo penguins
Pygoscelis
papua
were dosed with trimethyltin (TMT)(15 mg/kg) and Adelie penguins
P. adeliae
were dosed with fuel for air (JP1)(0.20 ml/kg) and land transport (Polar Diesel, PD)(0.20
ml/kg). We use the serum proteins as markers of contamination on Gentoo and Adelie
penguins. The following hematological parameters were measured: hemoglobin, hemato-
crit, glucose, total lipids, total proteins and enzymes (aspartate transaminase and alanine
transaminase). Normal hematological values obtained on all penguins are in agreement
with previously published data. All tested birds showed signifcant toxic changes being
seen clinically and by blood tests. No mortality was observed during the experiment.
ß globulin increased in Gentoo penguins treated with TMT, showing highly signifcant
diFFerences. α 1 and
α 2 globulin was Fusioned. There was a marked drop in total proteins
in Adelie penguins dosed with JP1. ß and γ globulin decreased signifcantly in Adelie
penguins exposed to Polar Diesel. This study show that serum proteins and enzymes
levels can be used as biological markers of contamination on penguins.
Palabras clave: marcadores biológicos, proteínas del suero, pingüinos, Antártida,
Pygoscelis adeliae
,
Pygoscelis papua
RESUMEN
Dos combustibles líquidos comunes y ampliamente usados con actividad hepatotóxica y
trimetiltin, un compuesto con actividad neurotóxica, que son usados en varios procesos
industriales, fueron utilizados para probar su toxicidad en dos especies de pingüinos
pigoscélidos. Se administró a pingüinos papúa
Pygoscelis papua
Trimetiltin (TMT)(15
mg/kg) y a pingüinos adelia
P. adeliae
combustible de aeronaves (JP1)(0.20 ml/kg) y
combustible diesel (Polar Diesel, PD)(0.20 ml/kg). Se usaron proteínas séricas como
Rev. Int. Contam. Ambient. 22 (3) 107-112, 2006
R. Najle
et al.
108
marcadores de contaminación en pingüinos papúa y adelia. Los siguientes parámetros
hematológicos fueron medidos: hemoglobina, hematocrito, glucosa, lípidos totales,
proteínas totales y enzimas (AST, aspartato amino transferasa y ALT, alanina amino
transferasa). Los valores hematológicos normales obtenidos en todos los pingüinos
corresponden con los datos publicados previamente. Todas las aves tratadas mostraron
cambios signifcativos clínicos y/o hematológicos. No se observó mortandad durante el
experimento. Los valores de ß globulina aumentaron en los pingüinos papúa tratados
con TMT mostrando diFerencias muy signifcativas, mientras α 1 y α 2 globulinas se
fusionaron. Hubo un marcado descenso en las proteínas totales de los pingüinos Adelia
tratados con JP1. En los pingüinos Adelia expuestos a PD, ß y γ globulinas disminuyeron
signifcativamente. Este estudio muestra que los niveles séricos de proteínas y enzimas
pueden ser utilizados como marcadores biológicos de contaminación en pingüinos.
INTRODUCTION
More than 100 years of human occupation of
the Antarctic continent has inevitably led to anthro-
pogenic contamination in the environment, par-
ticularly in the ice-free areas. Such contamination
is concentrated around occupied and historic bases
and stations, as well as feld camps, where soils
are often visibly contaminated by fuel residues and
solid wastes, or stained by domestic waste water.
Internationally agreed protocols now prohibit the
discharge of any substance onto ice-free areas and
soils in Antarctica. However, prior to the implementa-
tion of the these protocols, there were less stringent
controls on the use, storage and disposal of potential
contaminants and less appreciation of the risk posed
to the environment by inappropriate use and disposal
of these substances. Even now, accidental spills,
particularly of fuel, continue to provide a source of
potential contamination and are, to some degree, an
inevitable consequence of human activity (Webster
et al
. 2003).
The Antarctic Continent constitutes an exceptional
environment in which communities of marine life are
becoming exposed to anthropogenic disturbances,
which are most serious in the areas of greatest human
activity (Harris 1991). In these regions, several birds
requiring rehabilitation have increased dramatically,
the commonest problems being related to oil spills
and starvation (Camphuysen and van Franeker 1992).
Antarctic birds, particularly penguins, are the most
important members of the Antarctic ecosystem, in
terms of total biomass and of interaction with the
environment (Woehler 1993).
Birds that are emplaced in higer trophic level of
the food chain show high levels of xenobiotics and
can be considered as bioindicators for monitoring the
environmental pollution (Thompson
et al.
1990), they
are relatively large and easily identifed. Baseline
surveys of concentration of pollutants in selected bird
species have been conducted in Antarctica (Tatton
and Ruzicka 1967, Szefer
et al.
1993, van Den Brink
and De Ruiter-Dijkman 1997).
Oil by-products, such as fuel for air and land trans-
port (JP1 and Polar Diesel), as well as organometal-
lic compounds (e.g. Trimethyltin: TMT, antifouling
paint), are used in Antarctica. Liquid fuels are used
in large quantities, usually stored in outdoor depots,
which may leak and contaminate the nearby sea and
ice. Trymethyltin has been characterized as a pow-
erful neurotoxin that also affects other organs and
tissues (feathers, muscles, liver, kidneys) (Kannan
et al.
1998) showing a high toxic effect to aquatic
life. One criterion for the persistence of organotins
in the environment is their lipophile character. The
accumulation of organometallic compounds by
higher trophic aquatic organisms proceeds through
either uptake from solution alone or of a combina-
tion with diet ingestion. Due to the extensive use in
numerous areas of human activity, large amounts
of organotin compounds have been introduced to
various ecosystems. Thus, signifcant concentrations
fo these pollutants and their metabolites have been
detected in all compartments mainly of the aquatic
environment: waters, suspended matters, sediment,
and biomass. Despite the high conentrations of toxic
organotin compounds found in aquatic invertebrates,
little is known about the accumulation and toxic ef-
fects in higher trophic vertebrate predators, which
may be exposed to these pollutants via food ingestion
(Hoch 2001).
The impact of a contaminating agent on an organ-
ism is re±ected through changes in physiological,
biochemical and cellular balances. The effects of
toxic substances can be measured as disturbances at
different levels of functional complexity, depending
on the toxin (Larsson
et al.
1990).
A variety of technical approaches have been ap-
BIOLOGICAL MARKERS OF CONTAMINATION OF PENGUINS
109
plied in pollution biomonitoring programs in order to
estimate the bioavailable fraction of toxic substances,
which could be used as baseline levels to monitory
changes in the Antarctic ecosystem.
Considering that the levels of pollutants in birds
exposed to a polluted aquatic medium is the result
of both its accumulation from food and from water,
the aim of this study was to use the serum proteins
as biological indicators of environmental contamina-
tion. For this purpose Gentoo and Adelie penguins
were used.
MATERIALS AND METHODS
Hematological parameters were measured in
samples from adult Gentoo
Pygoscelis papua
and
Adelie
P. adeliae
penguins. Blood samples were
collected from Gentoo (n= 6, mean body weight
5215±134 g) and Adelie (n= 9, mean body weight
4956±123 g) penguins at Potter peninsula (62°14’S
58°38’W) King George Island, South Shetland Is-
lands, Antarctica, at the end of austral summer 1993,
on which there is an active reproductive colony of
penguins: Adelie (number of pairs NP= 14,554),
Gentoo (NP= 2,325) and Chinstrap (
P. antarctica
,
NP= 265)(Aguirre 1995).
Doses of potentially toxic substances, which are
used and stored at Antarctic stations, were given to
Gentoo and Adelie penguins to assess hematologi-
cal changes. The penguins were kept in individual
cages, in good weather conditions and with ad libi-
tum availability of fresh water (snow). They were
distributed in fve groups oF 3 penguins each: two
intact control (Gentoo n= 3, Adelie n= 3) and three
groups of individuals of which were given pollutants
(Gentoo with TMT n= 3, Adelie with JP1 n= 3, and
Adelie with Polar Diesel n= 3). The body weight of
the penguins was recorded.
The experiment was conducted under ethical
conditions. The protocol was approved by Instituto
Antártico Argentino ethics committee. All birds were
adult and were found in the beach of Potter Cove.
Sample collections were done after 12 hours without
food. They were handled very gently and as soon as
the sampling was fnished they were set Free. All the
penguins used in the experiments were banded and
released in good condition in the same place where
they were captured.
We measured proteins and enzymes activity.
Electrophoresis was done on polyacrylamide 5 %
native gels stained with Coomassie Blue and scanned
with a densitometer to determine concentrations of
the different protein fractions. Total proteins were
calculated with Rapid Lowry’s technique.
Serum enzymes, aspartate transaminase (AST)
and alanine transaminase (ALT) were quantifed by
kinetic technique. The enzymatic activity was cor-
rected by mg of protein determined by the Rapid
Lowry technique. Additionally, the following tests
were undertaken: hemoglobin, hematocrit, glucose
and total lipids. The hemoglobin level was deter-
mined by the cyanmethaemoglobin method and the
hematocrit value by the micromethod. All determina-
tions were done in triplicate. Results were expressed
as means ± S.D. Data were evaluated statistically by
one-way analysis of variance and Tukey test to assess
the differences of the means, which were accepted
as signifcant at p < 0.05.
Three Gentoo penguins were given a single dose
of 15 mg/kg body mass of trimethyltin (TMT). The
toxic substances were given orally via an sterile
plastic tube. Samples of blood were taken at 0, 17,
24 and 48 h from the commencement of the experi-
ment, and birds were examined every three hours.
Kerosene-based aviation fuel (JP1) was given orally
to three Adelie penguins (0.20 ml/kg), and Polar Diesel
(PD)(0.20 ml/kg) to the other three via an sterile plas-
tic tube. Blood samples were taken at 0, 24 and 48 h
(Szubartuwska and Gromysz-Kalkowska 1992).
RESULTS
The normal hematological values of two species
of penguins were calculated to determine the good
health of birds (
Table I
). Values obtained on all pen-
guins are in agreement with previously published data
(Milsom et al. 1973, Kostelecka-Myrcha and Myrcha
1980, Rosa
et al.
1993). No mortality was observed
during the experiment. The toxic substances used did
not affect the penguins body weight.
Gentoo Penguins dosed with TMT showed symp-
toms within 24 hours, mainly neurological ones
(diFfculty with balance and walking). Comparison
between the intoxicated animals and control birds 48 h
after the intoxication, revealed differences in several
estimates. In our experimental conditions serum pro-
teins values obtained on the 0 time are in agreement
with unpublished data from adults birds taken in the
same season (D. Montalti, unpublish data). There was
a signifcant increase in total protein From time 17 h
to 48 h, due mainly to a rise in betaglobulin; also the
α 1 globulin and 2 Fractions coalesced, due to that
reason, none of them were focus separated on the
next three samples. Prealbumin, albumin and gamma
R. Najle
et al.
110
globulin didn’t show signifcant diFFerences aFter the
intoxication. ß globulin increased during the experi-
ment showing highly signifcant diFFerences. α 1 and
α 2 globulin was amalgamated, there was no variation
in their percentage from the sample taking at the 17
h, until the end of the experiment (
Table II
).
Thus, on comparing control specimens with
intoxicated Gentoo penguins with TMT, highly sig-
nifcant diFFerences were Found in the serum enzymes
levels after 48 h (
Table II
).
Adelie penguins treated with JP1 remained in
apparently good health with no discernible signs or
symptoms. There was a marked drop in total pro-
teins, showing highly signifcant diFFerences aFter the
experiment. Prealbumin-albumin and alfa globulin
increased showing highly signifcant diFFerences at
24 h while there was no difference at 48 h, showing
the same concentration. On the other hand, ß and
γ globulin, Fell to halF their normal values in the
samples taken at 24 h, showing highly signifcant
difference and keeping their normal values in the
samples taken at 48 h (
Table III
).
No symptoms were observed in Adelie penguins
exposed to Polar Diesel. The biochemical changes
were similar to those seen with JP1, but were not so
marked. Total proteins had a signifcant decreased
at 24 h as well as 48 h. Prealbumin and albumin
showed no signifcant diFFerences aFter 48 h From
the intoxication. Alfa globulin increased and highly
signifcant diFFerences were Found. ß and γ globulin
decreased signifcantly in the two sampled periods
(
Table IV
).
DISCUSSION
The normal values quoted in this study are mostly
within the ranges published
for several penguin spe-
cies (Allison and Feeney 1968, Ghebremeskel
et al.
1991, Aguilera
et al.
1993, Rosa
et al.
1993, Ferrer
et al.
1994). Between Gentoo and Adelie penguins
no signifcant diFFerences were Found in the normal
values of hematological parameters, except in some
of them (e.g. concentration of glucose in Adelie
Hemoglobin
Hematocrit
Glucose
Total lipids
Total proteins
AST
ALT
(g/100 mL)
%
(g/L)
(g/L)
(g/L)
(UI/L)
(UI/L)
Gentoo Penguin
(n=6)
17.9 ± 1.2
49.7 ± 5.3
2.7 ± 0.2
8.3 ±1.1
59.3 ± 5.2
2.3 ± 0.4 4.1 ± 1.1
Adelie Penguin
(n=9)
15.4 ± 1.8
45.9 ± 7.7
1.8 ± 0.1
6.9 ± 1.3
57.9 ± 3.2
2.0 ± 0.2 2.9 ± 0.4
Values expressed as mean ± S.D.
TABLE I.
NORMAL VALUES OF HEMATOLOGICAL PARAMETERS OF GENTOO
Pygoscelis papua
AND ADELIE
P. adeliae
PENGUINS FROM KING GEORGE ISLAND, ANTARCTICA
0 hours
17 hours
24 hours
48 hours
Total proteins
(g/L)
59.3 ± 5.2
66.1* ± 3.3
68.8* ± 4.1
68.9* ± 2.7
Prealbumin
7.6 ± 0.2
7.8 ± 0.3
7.8 ± 0.4
7.9
± 0.3
Albumin
46.9 ± 1.3
46.8 ± 1.3
45.4 ± 1.4
46.7
± 2.1
a
1
globulin
18.8 ± 0.7
a
2
globulin
14.7 ± 0.4
a
1
a
2
globulin
29.9 ± 1.3
28.4 ± 0.9
28.7
± 1.6
b
globulin
12.0 ± 0.5
14.6* ± 1.2
16.4* ± 0.8
16.7* ± 1.3
AST (UI/L)
2.3 ± 0.4
27.4** ± 2.7
ALT (UI/L)
4.3 ± 0.5
7.9** ± 0.3
Values express as mean ±S.D. of percentages of serum fractions ob-
tained by electrophoresis at different times post-dose. Values in the 0
h column are those obtained at 0 h of the experimental group (n= 3)
and those of the control group (n= 3) (each sample run was tripli-
cated).* Signifcantly diFFerent From control (p < 0.05). **Highly sig
-
nifcant diFFerences (p < 0.001).
TABLE II.
EFFECTS OF DOSING WITH TMT ON SERUM PRO-
TEINS AND ENZYMES OF GENTOO PENGUIN
Py-
goscelis papua
BIOLOGICAL MARKERS OF CONTAMINATION OF PENGUINS
111
penguins was signifcantly lower than in Gentoo
penguin). The glycogenic mechanisms are likely to
be important, because of differences in their feeding
habits (Aguilera
et al.
1993). Gentoo penguins feed
inshore and are deep divers, and average body mass
is greater than that of the Adelie penguin (Trivelpiece
et al.
1987). According to Smith and Bush (1978)
total protein estimation is a valuable indicator of the
general nutritional state, with low protein suggesting
either malnutrition, bacterial or chemical toxaemia.
In our experiment, the protein levels increased in
Gentoo penguins treated with TMT (
Table II
), while
in the Adelie penguins dosed with JP1 and PD, the
serum proteins decreased showing highly signifcant
differences (
Tables III
and
IV
).
Gentoo penguins dosed with TMT showed a
signifcant increase in protein levels in their blood,
due mainly to an increase in betaglobulin. One of the
functions of this fraction is excretion of toxins.
The Fusion oF α 1 and α 2 globulins suggests
hepatic shock as a result of ingestion of a potent
hepatotoxin. AST increase more than one order of
magnitud, while ALT rise around two times. This
variation, would be due to the hepatic alterations
produced by the dosed TMT.
The electrophoretic pattern of serum samples
from JP1-dosed penguins showed low levels of beta
and gamma globulin. In spite of this, the penguins
did not show clinical evidence of intoxication. How-
ever, these low globulin levels may lead to greater
susceptibility to concomitant diseases. Variations in
the normal pattern serum proteins, are indicative of
toxic provoked alterations.
The observed serum proteins and enzymes chang-
es would be indicative of hepatotoxic substances. In
our study, the hematological values changed due to
the pollutants administrated to the penguins.
A further posible explanation for the lack of cor-
relation between the degree of contamination and he-
matological values could be the bird’s capacity to carry
out a complete or partial regulation of pollutants.
Our fndings show that serum proteins and en
-
zymes levels can be used as biological markers of
contamination on several penguin species.
ACKNOWLEDGEMENTS
The study was supported by the Instituto Antártico
Argentino. We thank Lucas Marti for improving the
English text and Alfredo Salibián for comments of
the frst draFt oF the manuscript.
Time post-dose
Total proteins
Prealbumin
a
globulin
b
and
g
(hours)
(g/L)
albumin %
%
globulin %
0
59.2 ± 4.1
36.9
± 1.2
7.4 ± 0.4
55.7 ± 3.8
24
40.9* ± 2.0
59.9* ± 2.7
13.7* ± 0.8
26.4* ± 2.4
48
37.5* ± 1.3
60.6* ± 3.7
13.4* ± 0.5
26.0* ± 2.1
TABLE III.
EFFECTS OF DOSING WITH JP1 ON SERUM PROTEINS OF THE ADE-
LIE PENGUIN
Pygoscelis adeliae
Values expressed as mean ±S.D. of percentages of serum fractions obtained by electro-
phoresis at 0 time (n= 3) and times post-dose (n= 3) (each sample run was triplicated).
* Signifcantly diFFerent From control (p < 0.05).
Time post-dose
Total proteins Prealbumin
a
globulin
b
and
g
(hours)
(g/L)
albumin %
%
globulin %
0
58.3 ± 2.4
40.3 ± 2.7
9.4 ± 1.8 50.3 ± 0.8
24
53.3* ± 1.9
41.2 ± 4.8 13.4* ± 1.3 45.4* ± 2.1
48
47.8* ± 0.7
43.6 ± 3.1 18.1* ± 1.0 38.3* ± 1.5
Values expressed as mean ±S.D. of percentages of serum fractions ob-
tained by electrophoresis at 0 time (n= 3) and times post-dose (n= 3)
(each sample run was triplicated). * Signifcantly diFFerent From control
(p < 0.05)
TABLE IV.
EFFECTS OF DOSING WITH POLAR DIESEL (PD)
ON SERUM PROTEINS OF THE ADELIE PENGUIN
Pygoscelis adeliae
R. Najle
et al.
112
REFERENCES
Aguilera E., Moreno J. and Ferrer M. (1993). Blood chem-
istry values in three Pygoscelis penguins. Compar.
Biochem. Physiol. 105A, 471-473.
Aguirre C.A. (1995). Distribution and abundance of birds
at Potter Peninsula, 25 de Mayo (King George) Island,
South Shetland Islands, Antarctica. Marine Ornithol.
23, 23-31.
Allison R.G. and Feeney R.E. (1968). Penguin blood serum
proteins. Archiv. Biochem. Biophys. 124, 548-555.
Camphuysen C.J. and van Franeker J.A. (1992). The value
of beached bird surveys in monitoring marine oil pol-
lution. Technisch Rapport Vogelbescherming 10, pp.
191. Zeist, Netherlands.
Ferrer M., Amat J.A. and Viñuela J. (1994). Daily
variations of blood chemistry values in the Chinstrap
Penguin (
Pygoscelis antarctica
) during the Antarctic
summer. Compar. Biochem. Physiol. 107A, 81-84.
Ghebremeskel K., Williams T.D., Williams G., Gardner
D.A. and Crawford M.A. (1991). Plasma metabolites in
Macaroni Penguins (
Eudyptes chrysolophus
) arriving
on land for breeding and moulting. Compar. Biochem.
Physiol. 99A, 245-250.
Harris C.M. (1991). Environmental effects of human ac-
tivities on King George Island, South Shetland Islands,
Antarctica. Polar Record 27, 193-204.
Hoch M. 2001. Organotin compounds in the environment
– an overview. Appl. Geochem. 16, 719-743.
Kannan K., Senthilkumar K., Elliot J.E., Feik L.A. and
Giesy J.P. (1998). Occurrence of butyltin compounds
in tissues of water birds and seaducks from the United
States and Canada. Archiv. Environ. Contam. Toxicol.
35, 64-69.
Kostelecka-Myrcha A. and Myrcha A. (1980). Hemato-
logical studies on antarctic birds II. Changes of the
hematological indices during the development of the
pygoscelid penguins. Polish Polar Res. 1, 175-181.
Larsson P., Okla L. and Woin P. (1990). Atmospheric
transport of persistent pollutants governs uptake by
Holarctic terrestrial biota. Environ. Sci. Technol. 24,
1599-1601.
Milsom W.K., Johansen K. and Millard R.W. (1973).
Blood respiratory properties in some antarctic birds.
Condor 75, 472-474.
Rosa C.D., Rosa R., Rodríguez E. and Bacila M. (1993).
Blood constituents and electrophoretic patterns in Ant-
arctic birds: penguins and skuas. Compar. Biochem.
Physiol. 104A, 117-123.
Smith E.E. and Bush M. (1978). Haematological param-
eters on various species of Strigiformes and Falconi-
formes. J. Wildlife Disease 14, 447-450.
Szefer P., Czarnowski W., Pempkowiak J. and Holm E.
(1993). Mercury and major essential elements in seals,
penguins, and other representative fauna of the Antarc-
tic. Arch. Environ. Contam. Toxicol. 25, 422-427.
Szubartowska E. and Gromysz-Kalkowska K. (1992).
Blood morphology in quails after poisoning with feni-
trothion. Compar. Biochem. Physiol. 101C, 263-267.
Van Den Brink N.W. and De Ruiter-Dijkman E.M.
(1997). Trans-nonachlor, octachlorostyrene, mirex
and photomirex in Antarctic seabirds. Antarctic. Sci.
9, 414-417.
Tatton J.O.G. and Ruzicka J.H.A. (1967). Organochlorine
pesticides in Antarctica. Nature 215, 346-348.
Thompson D.R., Stewart F.M. and Furness R.W. (1990).
Using seabirds to monitor mercury in marine environ-
ments – the validity of conversion ratios for tissue
comparison. Marine Pollut. Bull. 21, 339-342.
Trivelpiece W.Z., Trivelpiece S.G. and Volkman N.J.
(1987). Ecological segregation of Adélie, gentoo, and
chinstrap penguins at King George Island, Antarctica
Ecol. 68, 351-361.
Webster J., Webster K., Nelson P. and Waterhouse E.
(2003). The behaviour of residual contaminants at a
former station site, Antarctica. Environ. Pollut. 123,
163-179.
Woehler E.J. (1993). The distribution and abundance of
Antarctic and Subantarctic penguins. Scientifc Com
-
mittee on Antarctic Research, Cambridge, U.K.
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