Vorgabe neu

Journal of Applied Botany and Food Quality 81, 45 - 48 (2007)

Leibniz Institut für Gewässerökologie und Binnenfischerei, Berlin

Reduction in germination rate and elevation of peroxidase activity in Zea mays seedlings
due to exposure to different microcystin analogues and toxic cell free cyanobacterial crude extract

Stephan Pflugmacher
(Received February 23, 2007)

Summary

Agricultural crop plants may come into contact with cyanobacterial
toxins via spray irrigation with water contaminated with cyanobacteria/
cyanobacterial toxins. Many of the bloom forming cyanobacteria are
known to produce toxins amongst those the group of the microcystins,
cyclic heptapeptides are the best known once. In this study the
germination of Zea mays under exposure to different microcystins
and cell free cyanobacterial crude extract containing microcystin-LR
was investigated. The concentration used for all microcystins in this
study was 5.0 µg L-1 which is well in the environmental range. The
inhibition of germination was shown as well as the inhibition of root
and shoot length by toxin exposure. As a sign for the generation of
oxidative stress promoted by the toxins taken up, guajacol peroxidase
was measured showing in most toxin exposures an elevation of
peroxidase activity. This study showed that there is a potential concern
a reduction in crop yield and also to human health if agriculturally
important crop plants were exposed to cyanobacterial toxins via spray
irrigation.

Introduction

Modern agriculture is dependent on spray irrigation of the crops to
ensure the crop yield necessary. In many countries, especially in semi-
arid and arid regions this water is taken from water bodies in which
bloom-forming cyanobacteria might be present. From many of these
cyanobacteria it is well known that they can produce a variety of
different secondary metabolites, so called cyanobacterial toxins
(cyanotoxins) and it was estimated that 25 - 75 % of these cyano-
bacterial blooms are toxic (LAWTON and CODD, 1991). Under these
toxins, hepatotoxins, neurotoxins and cytotoxins can be found. The
microcystins, cyclic heptapeptides of five relatively invariant D-amino
acids and two most variable L-amino acids, are the most well known
group from which about 60 analogues were known (SIVONEN and
JONES, 1999). Spray irrigation with water contaminated with cyano-
bacteria respectively their toxins showed to have negative effects on
terrestrial plants. In most cases this studies were performed in
hydroponic culture using the most common hepatotoxin, microcystin-
LR. Effects monitored were decrease in root length (KOS et al., 1995;
MCELHINEY et al., 2001; KURKI-HELASMO and MERILUOTO, 1998),
inhibition of photosynthesis (ABE et al., 1996), inhibition of sucrose
transport (SIEGL et al., 1990), inhibition of protein phosphatases
(MCKINTOSH et al., 1990), indicating the uptake of toxin via the root
system. A previous study showed the uptake of two microcystins (MC-
LR and MC-LF) in different agricultural important crop plants,
amongst those corn (Zea mays) by PEUTHERT et al. (in press) giving
toxin values in roots of Z. mays of 12.7 (MC-LR), 14.5 (MC-LF) and
shoots of Z. mays of 26.5 (MC-LR), 18.0 (MC-LF) after 24 h exposure
time.
From cyanobacterial toxins it is known that they can generate oxidative
stress after being taken up by plant cells (GEHRINGER et al., 2003;
PFLUGMACHER, 2004; PFLUGMACHER et al., 2006). Oxidative stress
is always generated from a rapid and transient production of high
quantities of reactive oxygen species (ROS). Unless the production of

ROS in plants is a natural phenomenon via the photosynthesis in the
chloroplasts, excessive ROS formation could also be triggered by quite
a number of external factors amongst those exposures to xenobiotics
or cyanobacterial toxins (COSSU et al., 1997; PFLUGMACHER et al.,
2006). Oxidative damage has been found in DNA, proteins, carbo-
hydrates and lipids (CADENAS, 1995). To prevent oxidative damage
from ROS a protective system has been evolved based on small
molecular antioxidants such as glutathione (GSH), ascorbate or
tocopherols and antioxidative enzymes such as superoxide dismutase,
catalase, peroxidase, glutathione S-transferase and glutathione re-
ductase.
Aim of this study was to examine the response of corn seedlings when
exposed to different microcystin variants and furthermore to cell-free
cyanobacterial crude extract containing microcystin-LR.

Materials and methods

Plant material and exposure experiments

Corn (Zea mays L. cv Badischer Gelber) seeds were purchased from
Kiepenkerls (Norken, Germany). Seeds were soaked in running tap
water overnight. The seeds were germinated non-aseptically on the
surface of wet filter papers (Whatman No. 1, Roth, Karlsruhe, Ger-
many) in the dark at 13 °C in plastic plates with 24 wells. In each well
one seed was placed in exposure medium (1 ml). For each toxin treat-
ment five plastic plates were used which are 100 seeds per treatment.
Concentration of the different microcystins in the exposure medium
was 5.0 µg L-1 and in the used cyanobacterial cell-free crude extract
was equivalent to a microcystin-LR concentration of 5.0 µg L-1.

Microcystin variant and cyanobacterial crude extract

The different microcystin analogues (Tab. 1) were purchased from
Axxora (Lörach, Germany) and in case of MCHCyR was a gift from
Prof. Dr. G.A. Codd (University of Dundee). The cyanobacterial bloom
material (mainly Microcystis aeruginosa and Aphanezomenon flos-
aqua) was collected in June 2000 from the shore of Lake Müggelsee,
Berlin (Germany). To obtain the cell-free crude extract, 20 g dry weight
of bloom material was suspended in 500 mL Milli-Q water and stirred
on ice for 15 min. After ultrasonication on ice, centrifugation of the
resulting slurry was done at 22,000 x g for 15 min. Supernatant was
collected and stored on ice. The pellet was reprocessed in the same
manner as described above five times and the extracts were combined
thereafter. The extract was stored in the deep freezer (-80°C) before
use. Toxin analysis of the crude extract revealed the presence of
microcystin-LR (MC-LR) and not quantifiable traces of microcystin-
RR (MC-RR). The extract was diluted to a final concentration of
5.0 µg L-1 MC-LR.

Determination of cyanobacterial secondary metabolites

Analyses were performed as described in (PFLUGMACHER et al., 2006)
using a Waters HPLC system (Waters, Eschborn, Germany) with

photodiode array detector (waters 2996) detection. Separation was
carried out on a Symmetry 5 µm C18 column (3.9 x 150 mm). The
mobile phase consisted of solvent A: Milli-Q water and solvent B:
acetonitrile (Rathburn, Walkerburn, UK) both containing 0.1 % (v/v)
trifluoracetic acid. Solvent B was linearly increased from 30 % to
45 % over 10 min at a flow rate of 1 mL min-1. Column temperature
was maintained at 40 °C and the injection volume was set to 80 µl.

Enzyme preparation

Enzyme preparation from Z. mays seedlings roots and shoots was
carried out according to (PFLUGMACHER et al., 2006). Shoots and roots
were cut of with a scalpel and were frozen in liquid nitrogen, ground
to a powder with mortar and pestle and suspended in ice-cold 0.1 M
sodium phosphate buffer pH 6.5 containing 14 mM dithioerythritol
and 1 mM ethylenediaminetetraacetic acid. After removing cell debris
at 5000 x g, the supernatant was centrifuged at 100,000 x g for 60 min
to collect the microsomal fraction. The supernatant (soluble fraction)
was precipitated twice with solid ammonium sulphate (0-30 % and
30-80 % saturation). The pellet from the last precipitation step was
suspended in 20 mM sodium phosphate buffer (pH 7.0) and the
resulting extract (soluble fraction) was desalted using NAP-10 columns
(Amersham Pharmacia, Uppsala, Sweden).

Enzymatic measurements

Photometric determination of peroxidase (POD, E.C. 1.11.1.9) activity
was done according to (PUTTER, 1965) using guajacol as a substrate.
The assay contained 01.mM sodium phosphate buffer pH 6.0, 0.3 mM
guajacol, 0.12 mM H

2
O

2
, 40 µL of protein extract to a total volume of

1200 µL and increase in absorption was measured at 436 nm. Enzyme
activities are given in nkat mg-1 protein (SI unit: katal = conversion
rate of one mol substrate per second). Protein content of the samples

was determined according to (BRADFORD, 1976) using bovine serum
albumin fraction V as calibration standard at 595 nm.

Analysis of Data

The statistical significance was calculated by one-way analysis of
variance (ANOVA) followed by Newman-Keuls test, p < 0.05 (SPSS
9.0 for Windows, Chicago, USA).

Results

Seeds from Z. mays were exposed to different microcystin analogues
in a concentration of 0.5 µg L-1 for 7 days. Tab. 2 showed the
germination rate of Z. mays seeds. The highest reduction in germination
rate was seen using the cell free cyanobacterial crude extract (90 %)
and MC-LA (88 %). For all other microcystin variants the inhibition

Tab. 1: Microcystin variants used in this study and their structural differences concerning the amino acid composition and the molecular weight.

Analogue Structure Molecular weight Reference

Microcystin-LR Cyclo (-D-Ala-L-Leu-D-MeAsp-L-Arg-Adda-D-Glu-Mdha-) 994 Botes et al. (1985)

Microcystin-RR Cyclo (-D-Ala-L-Arg-D-MeAsp-L-Arg-Adda-D-Glu-Mdha-) 1037 Kusumi et al. (1987)

Microcystin-YR Cyclo (-D-Ala-L-Tyr-D-MeAsp-L-Arg-Adda-D-Glu-Mdha-) 1044 Botes et al. (1985)

Microcystin-LF Cyclo (-D-Ala-L-Leu-D-MeAsp-L-Phe-Adda-D-Glu-Mdha-) 985 Azevedo et al. (1994)

Microcystin-LA Cyclo (-D-Ala-L-Leu-D-MeAsp-L-Ala-Adda-D-Glu-Mdha-) 909 Botes et al. (1982)

Microcystin-LW Cyclo (-D-Ala-L-Leu-D-MeAsp-L-Trp-Adda-D-Glu-Mdha-) 1024 Lawton et al. (1994)

[D-Asp3, (Z)-Dhb7]microcystin-HtyR Cyclo (-D-Ala-L-Hty-D-Asp-L-Arg-Adda-D-Glu-(Z)-Dhb-) 1044 Sano et al. (1998)

Tab. 2: Germination rate of Z. mays seeds exposed to different microcystin
variants.

treatment germinated seeds reduction in germination
(n = 100) [%]

Control 100 0

MC-LR 68 32

MC-RR 93 17

MC-YR 73 27

MC-LF 74 26

MC-LA 12 88

MC-LW 89 11

MCHCyR 72 28

Crude extract (MC-LR) 10 90

46 Stephan Pflugmacher

Fig. 1: Z. mays seeds exposed to different microcystin variants A) control, B) MC-LR, C) MC-RR, D) MC-YR, E)MC-LF, F) MC-LA, G) MC-LW, H) Htyr-MC,
I) crude extract

of germination was between 17 - 32 % compared to control seeds.
The exposure to microcystins and to cell free cyanobacterial crude
extract had significant overall effects on primary root length and shoot
length (Fig. 1). The shoot length of the Z. mays seeds were significant-
ly reduced in all exposures but highest reduction was detected in
exposures using the MC-LR containing crude extract (Fig. 2A). A
similar picture was obtained looking at the root length. Also here all
exposures reduced the primary root length significantly from control
up to a factor of 18 (crude extract containing MC-LR) compared to
controls (Fig. 2B).
Activity of the POD in the shoots showed, that in most seeds exposed
to microcystin analogues a significant elevation of enzyme activity
was measured, which was highest in exposures using MC-LF, MC-
LW or MC-LR. The elevation of POD activity was not significant in
exposures using MC-RR. In two exposures (MC-LA and CE) the
activity of the POD was decreased under control levels (Fig. 3A).
A similar activity pattern was measured in the roots with the exception
of MC-RR exposures all POD activity were significantly different from
control. In most cases an elevation was seen also in the shoots. In
exposures using MC-LA and CE a decrease of POD activity was
determined (Fig. 3B).

Discussion

During spray irrigation events, water contaminated with cyanobacterial
toxins could be transferred to the field and the crop plants growing
there.
In this experiment germination rate of the Z. mays seeds were inhibi-
ted by exposure to the different microcystin analogues. The highest

inhibition was detected using the MC-LA one of the more lipophilic
microcystin analogues and cyanobacterial cell-free crude extract
containing MC-LR. Comparing the crude extract with the pure toxin
a big difference in the potency to reduce germination is visible. Where-
as the crude extract inhibits the germination more or less completely
(90 %) the purified toxin gives only a reduction of 32 % from control.
This led to the hypothesis that more bioactive compounds are present
in the crude extract leading to such an effect as also suggested by
PIETSCH et al. (2001).
Inhibition of germination by cyanobacterial toxins was also shown by
METCALF et al. (2004) in pollen of Nicotiana tabacum exposed to
cylindrospermopsin, another cyanobacterial toxin produced by cyano-
bacteria such as Cylindrospermopsis raciborskii, Umezakia natans,
Aphanizomenon ovalisporum or Rhaphidiopsis curvata. OBERMEYER
et al. (1998) correlated the inhibition of pollen tube growth in Lilium
longiorum with the inhibition of protein phosphatases by okadaic acid.
Also microcystins are known to inhibit the activity of protein phos-
phatases (MCKINTOSH et al., 1990) and therefore interfere with plant
growth.
Previous work showed, that the growth and lateral root develop-
ment of Sinapis alba was inhibited by exposure to the cyanobacterial
toxin MC-LR in a concentration of 3.0 µg L-1. A complete inhibition
of root formation was reported by MCKINTOSH et al. (1990) in S. alba
exposed to 5 mg L-1 MC-LR. Also in Medicago sativa a significant
reduction in root growth was monitored when plants were exposed to
two different microcystins (MC-LR, MC-LW) and to cyanobacterial
crude extract containing MC-LR in a concentration of 5.0 µg L-1

(PFLUGMACHER et al., 2006). In this experiment the growth inhibition
was more effective in the shoots of Z. mays than in the roots. Highest

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

co
nt

ro
l

M
C-

M
C
-R

M
C-

M
C
-L

M
C-

M
CH

Cy
R

CE
(M

C-
LR

)

p
e
ro

xi
d

a
se

a
ct

iv
ity

[µ
ka

t
m

g
p
ro

te
in

]
-1

Fig. 3: Peroxidase activity in shoots (A) and roots (B) of Z. mays seedlings
after exposure to different microcystin variants.

Fig. 2: Shoot length (A) and root length (B) of Z. mays seedlings after exposure
to different microcystin variants.

0.0

50.0

100.0

150.0

200.0

250.0

300.0

350.0

0.0

25.0

50.0

75.0

100.0

125.0

150.0

175.0

200.0

co
nt

ro
l

M
C-

M
CH

Cy
R

CE
(M

C-
LR

)

ro
o
t
le

n
g

th
[m

m
]

sh
o
o

t
le

n
g

th
[m

m
]

Effects of microcystins in Zea mays 47

inhibition was achieved using the cell free crude extract containing
MC-LR again pointing out, that there might be more compounds in-
cluded counting for the measured effect.
The generation was oxidative stress by cyanobacterial toxins was
shown in Lepidium sativum and Medicago sativa yielding to an
elevation of antioxidative enzymes such as glutathione peroxidase,
catalase or glutathione S-transferase (GEHRINGER et al., 2003;
PFLUGMACHER et al., 2006). Activity determination of the guajacol
peroxidase in Z. mays root and shoots showed also an elevation of
this enzyme by exposure to the different microcystin analogues. With
MC-LA and the crude extract containing MC-LR a decrease of POD
activity was measured. A possible explanations would be; an inhibition
of the protein (enzyme) by an excessive amount of reactive oxygen
species as known for protein phosphatases (HUBER, 2007).
These experiments have shown that Z. mays seedlings are sensitive to
exposure of cyanobacterial toxins such as microcystins. The contact
of Z. mays to these toxins might come via spray irrigation. After uptake
of the toxin in the seeds germination, root and shoot development
were negatively affected. This might lead to a decrease in crop yield
in two ways: first by the reduced germination of the seeds and second
by morphological damages leading to e. g. smaller plants. Because of
the uptake of toxin in the plants (JÄRVENPÄÄ et al., 2007; PEUTHERT
et al., in press) this could pose a possible risk for human consumption
after such practices too.

References

ABE, T., LAWSON, T., WEYERS, J.D.B., CODD, G.A., 1996: Microcystin-LR
inhibits photosynthesis of Phaseolus vulgaris primary leaves: implications
for current spray irrigation practice. New Phytol. 133, 651-658.

BRADFORD, M.M., 1976: A rapid and sensitive method for the quantification of
microgram quantities of proteins utilizing the principle of protein-dye
binding. Anal. Biochem. 72, 248-254.

CADENAS, E., 1995: Mechanisms of oxygen activation and reactive oxygen
species detoxification. In: Ahmad, S. (ed.), Oxidative stress and antioxidant
defences in biology, 1-61. Chapman & Hall, London.

COSSU, C., DOYOTTE, A., JACQUIN, M.C., BABUT, M., EXINGER, A., VASSEUR,
P., 1997: Glutathione reductase, selenium-dependent glutathione peroxi-
dase, glutathione levels and lipid peroxidation in freshwater bivalves Unio
tumidus as biomarkers of aquatic contamination in field studies. Ecotoxicol.
Environ. Safety 38, 122-131.

GEHRINGER, M.M., KEWADA, V., COATES, N., DOWNING, T.G., 2003: The use
of Lepidium sativum in a plant bioassay system for the detection of micro-
cystin-LR. Toxicon 41, 871-876.

HUBER, S., 2007: Exploring the role of protein phosphorylation in plants: from
signalling to metabolism. Biochem. Soc. Transact. 35, 28-32.

JÄRVENPÄÄ, S., LUNDBERG-NIINISTÖ, C., SPOOF, L., SJÖVALL, O., TYYSTJÄRVI,

E., MERILUOTO, J., 2007: Effects of microcystins on broccoli and mustard,
and analysis of accumulated toxin by liquid chromatography-mass
spectrometry. Toxicon (in press).

KOS, P., GORZO, G., SURANYI, G., BORBELY, G., 1995: Simple and efficient
method for isolation and measurement of cyanobacterial hepatotoxins by
plant tests (Sinapis alba L.). Anal. Biochem. 225, 49-53.

KURKI-HELASMO, K., MERILUOTO, J., 1998: Microcystin uptake inhibits growth
and protein phosphatase activity in mustard (Sinapis alba L.) seedlings.
Toxicon 36, 1921-1926.

LAWTON, L., CODD, G.A., 1991: Cyanobacterial (blue-green algal) toxins and
their significance in UK and Europeanwaters. J. J.W.E.M. 5, 460-465.

MACKINTOSH, C., BEATTIE, K.A., KLUMPP, S., COHEN, P., CODD, G.A., 1990:
Cyanobacterial microcystin-LR is a potent and specific inhi-bitor of protein
phosphatases 1 and 2A from both mammals and higher plants. FEBS
Letters 264, 187-192.

MCELHINEY, J., LAWTON, L.A., LEIFERT, C., 2001: Investigations into the
inhibitory effects of microcystins on plant growth, and the toxicity of plant
tissues following exposure. Toxicon 39, 1411-1420.

OBERMEYER, G., KLAUSHOFER, H., NAGL, M., HÖFTBERGER, M., BENTRUP, F.-
W., 1998: In vitro germination and growth of lily pollen tubes is affected
by protein phosphatase inhibitors. Planta 207, 303-312.

PEUTHERT, A., CHAKRABARTI, S., PFLUGMACHER, S., 2007: Uptake of micro-
cystins-LR and -LF (cyanobacterial toxins) in seedlings of several important
agricultural plant species and the correlation with cellular damage (lipid
peroxidation). Environ. Toxicol. (in press).

PFLUGMACHER, S., 2004: Promotion of oxidative stress in C. demersum due to
exposure to cyanobacterial toxin. Aquatic Tox. 3, 169-178.

PFLUGMACHER, S., JUNG, K., LUNDVALL, L., NEUMANN, S., PEUTHERT, A., 2006:
Effects of cyanobacterial toxins and cyanobacterial cell-free crude extract
on germination of Alfalfa (Medicago sativa) and induction of oxidative
stress. Environ. Toxicol. Chem. 25, 2381-2387.

PIETSCH, C., WIEGAND, C., AME, M.V., NICKLISCH, A., WUNDERLIN, D.,
PFLUGMACHER, S., 2001: The effects of a cyanobacterial crude extract on
different aquatic organisms: Evidence for cyanobacterial toxin modulating
factors. Environ. Toxicol. 16, 535-542.

PUTTER, J., 1965: Peroxidases. In: Bergmeyer, H.U. (ed.), Methoden der enzy-
matischen Analyse, Vol. 2, 685-690. Academic Press, New York.

SIEGL, G., MACKINTOSH, C., STITT, M., 1990: Sucrose-phosphate synthetase
is dephosphorylated by protein phosphatase 2A in spinach leaves. FEBS
Lett. 270, 198-202.

SIVONEN, K., JONES, G., 1999: Cyanobacterial toxins. In: Chorus, I., Bartram,
J. (ed.), Toxic Cyanobacteria in Water. A guide to their public health
consequences, monitoring and management, 41-111. E. & FN. Spon on
behalf of WHO, London.

Address of the author:
PD Dr. Stephan Pflugmacher, Leibniz Institute of Freshwater Ecology and Inland
Fisheries, AG Biochemical Regulation, Müggelseedamm 301, D-12587 Berlin
email: pflugmacher@IGB-Berlin.de

48 Stephan Pflugmacher

<<
/ASCII85EncodePages false
/AllowTransparency false
/AutoPositionEPSFiles true
/AutoRotatePages /All
/Binding /Left
/CalGrayProfile (Dot Gain 20%)
/CalRGBProfile (sRGB IEC61966-2.1)
/CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2)
/sRGBProfile (sRGB IEC61966-2.1)
/CannotEmbedFontPolicy /Warning
/CompatibilityLevel 1.4
/CompressObjects /Tags
/CompressPages true
/ConvertImagesToIndexed true
/PassThroughJPEGImages true
/CreateJDFFile false
/CreateJobTicket false
/DefaultRenderingIntent /Default
/DetectBlends true
/ColorConversionStrategy /LeaveColorUnchanged
/DoThumbnails false
/EmbedAllFonts true
/EmbedJobOptions true
/DSCReportingLevel 0
/EmitDSCWarnings false
/EndPage -1
/ImageMemory 1048576
/LockDistillerParams false
/MaxSubsetPct 100
/Optimize true
/OPM 1
/ParseDSCComments true
/ParseDSCCommentsForDocInfo true
/PreserveCopyPage true
/PreserveEPSInfo true
/PreserveHalftoneInfo false
/PreserveOPIComments false
/PreserveOverprintSettings true
/StartPage 1
/SubsetFonts true
/TransferFunctionInfo /Apply
/UCRandBGInfo /Preserve
/UsePrologue false
/ColorSettingsFile ()
/AlwaysEmbed [ true
]
/NeverEmbed [ true
]
/AntiAliasColorImages false
/DownsampleColorImages true
/ColorImageDownsampleType /Bicubic
/ColorImageResolution 300
/ColorImageDepth -1
/ColorImageDownsampleThreshold 1.50000
/EncodeColorImages true
/ColorImageFilter /DCTEncode
/AutoFilterColorImages true
/ColorImageAutoFilterStrategy /JPEG
/ColorACSImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/ColorImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/JPEG2000ColorACSImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/JPEG2000ColorImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/AntiAliasGrayImages false
/DownsampleGrayImages true
/GrayImageDownsampleType /Bicubic
/GrayImageResolution 300
/GrayImageDepth -1
/GrayImageDownsampleThreshold 1.50000
/EncodeGrayImages true
/GrayImageFilter /DCTEncode
/AutoFilterGrayImages true
/GrayImageAutoFilterStrategy /JPEG
/GrayACSImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/GrayImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/JPEG2000GrayACSImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/JPEG2000GrayImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/AntiAliasMonoImages false
/DownsampleMonoImages true
/MonoImageDownsampleType /Bicubic
/MonoImageResolution 1200
/MonoImageDepth -1
/MonoImageDownsampleThreshold 1.50000
/EncodeMonoImages true
/MonoImageFilter /CCITTFaxEncode
/MonoImageDict <<
/K -1
>>
/AllowPSXObjects false
/PDFX1aCheck false
/PDFX3Check false
/PDFXCompliantPDFOnly false
/PDFXNoTrimBoxError true
/PDFXTrimBoxToMediaBoxOffset [
0.00000
0.00000
0.00000
0.00000
]
/PDFXSetBleedBoxToMediaBox true
/PDFXBleedBoxToTrimBoxOffset [
0.00000
0.00000
0.00000
0.00000
]
/PDFXOutputIntentProfile ()
/PDFXOutputCondition ()
/PDFXRegistryName (http://www.color.org)
/PDFXTrapped /Unknown

/Description <<
/FRA <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>
/ENU (Use these settings to create PDF documents with higher image resolution for improved printing quality. The PDF documents can be opened with Acrobat and Reader 5.0 and later.)
/JPN <FEFF3053306e8a2d5b9a306f30019ad889e350cf5ea6753b50cf3092542b308000200050004400460020658766f830924f5c62103059308b3068304d306b4f7f75283057307e30593002537052376642306e753b8cea3092670059279650306b4fdd306430533068304c3067304d307e305930023053306e8a2d5b9a30674f5c62103057305f00200050004400460020658766f8306f0020004100630072006f0062006100740020304a30883073002000520065006100640065007200200035002e003000204ee5964d30678868793a3067304d307e30593002>
/DEU <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>
/PTB <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>
/DAN <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>
/NLD <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>
/ESP <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>
/SUO <FEFF004e00e4006900640065006e002000610073006500740075007300740065006e0020006100760075006c006c006100200076006f0069006400610061006e0020006c0075006f006400610020005000440046002d0061007300690061006b00690072006a006f006a0061002c0020006a006f006900640065006e002000740075006c006f0073007400750073006c00610061007400750020006f006e0020006b006f0072006b006500610020006a00610020006b007500760061006e0020007400610072006b006b007500750073002000730075007500720069002e0020005000440046002d0061007300690061006b00690072006a0061007400200076006f0069006400610061006e0020006100760061007400610020004100630072006f006200610074002d0020006a00610020004100630072006f006200610074002000520065006100640065007200200035002e00300020002d006f0068006a0065006c006d0061006c006c0061002000740061006900200075007500640065006d006d0061006c006c0061002000760065007200730069006f006c006c0061002e>
/ITA <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>
/NOR <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>
/SVE <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>
>>
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice