In vitro and field studies on the contact and fumigant toxicity of a neem-product (Mite-Stop) against the developmental stages of the poultry red mite Dermanyssus gallinae.

Abstract

The acaricidal activity of the neem product MiteStop was investigated for its potential use as a botanical acaricide for the control of the poultry red mite Dermanyssus gallinae. This neem product is a special formulation of an extract of the seeds of the neem tree Azadirachta indica A. Juss. The efficacy was tested under laboratory conditions as well as in poultry houses. Four different methods of application were used in a filter paper bioassay to evaluate contact and vapour phase toxicity tests. The neem product proved to be already active in very small doses. In order to investigate the efficacy under field conditions, a poultry house was sprayed twice within a 7-day period using 1:33 and 1:50 diluted MiteStop. Cardboard traps were used to assess the mite population before, during and after the treatment. The mite population could be reduced by 89%. In a second poultry house, the spraying of defined areas with a 1:30, 1:33 or 1:50 dilution of the acaricide proved to be highly efficacious against all mite stages. Three other field trials proved that MiteStop is highly active against the red poultry mite. The most efficient dilution is 1:33 with tap water and spraying two or three times at intervals of 7 days.

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ORIGINAL PAPER
In vitro and field studies on the contact and fumigant toxicity
of a neem-product (Mite-Stop®) against the developmental
stages of the poultry red mite Dermanyssus gallinae
Nina Locher & Khaled A. S. Al-Rasheid &
Fathy Abdel-Ghaffar & Heinz Mehlhorn
Received: 17 March 2010 /Accepted: 8 April 2010 /Published online: 28 April 2010
# Springer-Verlag 2010
Abstract The acaricidal activity of the neem product
MiteStop® was investigated for its potential use as a botanical
acaricide for the control of the poultry red mite Dermanyssus
gallinae. This neem product is a special formulation of an
extract of the seeds of the neem tree Azadirachta indica A.
Juss. The efficacy was tested under laboratory conditions as
well as in poultry houses. Four different methods of
application were used in a filter paper bioassay to evaluate
contact and vapour phase toxicity tests. The neem product
proved to be already active in very small doses. In order to
investigate the efficacy under field conditions, a poultry
house was sprayed twice within a 7-day period using 1:33
and 1:50 diluted MiteStop®. Cardboard traps were used to
assess the mite population before, during and after the
treatment. The mite population could be reduced by 89%. In
a second poultry house, the spraying of defined areas with a
1:30, 1:33 or 1:50 dilution of the acaricide proved to be
highly efficacious against all mite stages. Three other field
trials proved that MiteStop® is highly active against the red
poultry mite. The most efficient dilution is 1:33 with tap
water and spraying two or three times at intervals of 7 days.
Introduction
Parasites are a problem wherever poultry are raised and can
lead to significant economic losses (Ruff 1999, Mul et al.
2009). The poultry red mite Dermanyssus gallinae (De
Geer 1778) is the most important ectoparasite of farmed
birds in Europe (Chauve 1998). This haematophagous mite
is a nocturnal feeder that spends the day hidden in cracks
and crevices of the chicken house. An infestation can be
very stressful for the birds resulting in irritation, restless-
ness, feather pecking, and anaemia. Heavy infestations can
decrease egg production, egg quality, weight gain in young
birds, and it can even cause death (Chauve 1998, Kirkwood
1967, Pospischil 2001). It was found that mite-infested
chicken need daily 15-20% more food than uninfested ones
(Mul et al. 2009). Thus, the economic losses are not to be
underestimated. Control and production losses in Europe
have been estimated at €130 million per annum (van Emous
2006). Furthermore, D. gallinae can act as a vector and
reservoir for several bacterial and viral pathogens such as
Salmonella spp., Erysipelothrix rhusiopathiae, Pasteurella
multocida, Borrelia anserina, Coxiella burnetii, chicken
pox virus, Newcastle Disease virus and St. Louis encephalitis
virus (Chirico et al. 2003, Lundh et al. 2005, Moro et al.
2007, Smith et al. 1945). Occasionally it also feeds on
mammals including humans and can cause dermatitis (Auger
Parts of this publication are included in the PhD-thesis of Doctor Nina
Locher. This thesis has been accepted at the Free University of Berlin
(Germany) to obtain the Doctor in Veterinary Medicine.
N. Locher:H. Mehlhorn (*)
Department of Parasitology, Heinrich Heine University,
D-40225 Düsseldorf, Germany
e-mail: mehlhorn@uni-duesseldorf.de
K. A. S. Al-Rasheid
Department of Zoology, Center of Excellence,
College of Science, King Saud University,
Riyadh, Saudi Arabia
F. Abdel-Ghaffar
Department of Zoology, Cairo University,
Giza, Egypt
H. Mehlhorn
Kaiser-Otto-Str. 44,
49393 Lohne, Germany
Present Address:
N. Locher
49393 Lohne, Germany
Parasitol Res (2010) 107:417–423
DOI 10.1007/s00436-010-1882-2

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et al. 1979, Beck and Pfister 2006, Mignon and Losson
2007, Pritchard and Kruse 1982).
Controlofthepoultryredmitehasbeendifficult,especially
for food-producing poultry because there is currently no
registered compound available on the German market.
The European legislation will ban traditional cage
systems for poultry by 2012. Due to the peculiar life cycle
of D. gallinae, less intensive farming systems like barns,
free range, and organic farming show higher prevalence
rates. This and the removal of acaricides from national
markets due to the increase in acaricide resistance and
welfare concerns will probably increase the problems
caused by the poultry red mite D. gallinae (Sparangano et
al. 2009). Therefore, the present situation calls for studies
on the efficacy of alternative control methods such as the
use of, i.e. plant-derived acaricides. The most important
pesticides appear to be those derived from the seeds of the
neem tree Azadirachta indica A. Juss. Several neem
products proved to be efficacious against viruses, bacteria,
endoparasites, and arthropods (Schmutterer 2002). More-
over, the complex mixture of several different, bioactive
analogues in neem is thought to avoid the development of
resistance (Mulla and Su 1999).
The aim of this study was to investigate the potential of a
neem product (MiteStop®) as a botanical acaricide for the
controlofthepoultryredmiteD. gallinae in-vitro and in-vivo.
Material and methods
MiteStop®
The acaricide MiteStop® is a product of Alpha-Biocare
GmbH, Düsseldorf (Germany). It is a special, patented
formulation of the extract of the seeds of the neem tree A.
indica A. Juss. It is sold as a concentrated product, which
has to be diluted with tap water prior to use.
Mites
The mites for laboratory investigations were collected in
different infested poultry houses in Germany. All inves-
tigations were carried out within 3 days after collection of
the mites. The field investigations were done inside large
stables in Europe containing egg laying chicken being kept
either in cages or on the floor.
Life cycle parameters
In order to assess the ovicidal activity, it was necessary to
collect some life cycle parameters of D. gallinae under
specific conditions. All experiments were carried out at a
temperature of 20°C (±0.5°C) and at a 40% relative humidity
(RH). Engorged females, protonymphs and deutonymphs
were placed in single wells of a 24-well plate (Nunclon™
surface, nunc™, Roskilde, Denmark). The wells were sealed
with a special foil that allowed gas exchange. The ongoing
development was observed every 24 h.
Filter paper bioassays
Four different methods of application were used to evaluate
contact and vapour phase toxicity tests. The toxicity was
tested on every developmental stage.
Method A (wetting of individual mites) was used to
evaluate the toxicity of very small doses. About ten mites
were placed on a filter paper (Rundfilter MN 615,
Machery-Nagel) in a Petri dish (92×16 mm, Sarstedt).
Each mite was treated with 0.5 μl of undiluted, 1:20, 1:40,
1:60, and 1:80 diluted acaricide. The control group was
treated with water. Method B1 (contact with wet acaricide)
and method B2 (contact with dried acaricide) were used to
evaluate the toxicity of a treated area. An amount of 0.3 ml
of undiluted and diluted (1:20, 1:40, 1:60, 1:80) acaricide
was applied to filter papers. Control filter papers were
treated with water (Fig. 1).
In method B1, the wet filter paper was placed in a Petri
dish and groups of about 20-30 mites were placed on the
filter paper. In method B2, the filter paper was left to dry at
room temperature before putting it in a Petri dish. In order
to prevent any mites from escaping, the Petri dishes were
sealed with Parafilm®.
Method C was conducted to investigate the vapour phase
toxicity. Groups of 20-30 mites were placed into an
Eppendorf cup (2 ml). The lid of the cups had been cut
off to seal the opening with a special foil allowing the
entrance of vapours. The sealed cups were then placed in a
glass container (20 ml). A folded filter paper treated like in
method B1 and B2 was added and the container was sealed
with a plastic lid. The Petri dishes and the glass containers
were stored at room temperature. The number of dead mites
0
20
40
60
80
100
undiluted1:20 1:401:601:80control
Mortality [%]
Method AMethod B1 Method B2Method C
Fig. 1 Results of the filter paper bioassays for the efficacy of
MiteStop®
418Parasitol Res (2010) 107:417–423

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was determined under a dissecting microscope. Mites were
considered to be dead if no movement resulted upon
pinching with a pair of fine tweezers. Every method was
repeated four times.
The ovicidal activity was investigated by using the
same four application methods. Due to the limited
amount of available eggs, only a 1:50 dilution was
tested. The eggs were observed for a period of 168 h.
Treatment inside poultry houses
Experiment 1
A small heavily infested poultry house of layers was
sprayed twice within a 7-day period using 1:50 diluted
MiteStop®. In order to assess the mite population,
cardboard traps were used at distinct places before, during
and after treatment. The traps were made of ordinary
corrugated cardboard consisting of three layers. The outer
layers were carefully separated from the inner layer by
using a fine scalpel. A paper clip kept the layers together
and allowed opening of the trap. The first monitoring
covered 5 days prior to the first spraying. The second or
third monitoring, respectively, started 2 days after each
spraying and lasted 5 days. At the end of each monitoring
period, the cardboard traps were collected, placed individ-
ually in freezer bags, and were put in a freezer at −18°C to
kill the mites. The total number of the population was
determined by calculating the dead mites of each trap.
Experiment 2
The second experiment was carried out in a scientific
poultry house in Sinsteden (Germany). Defined areas were
secured by using strong double-faced adhesive tape in order
to prevent mites from leaving or entering the area. Two
areas were sprayed with 1:30 and 1:50 diluted MiteStop®.
Other areas were used as a control and remained untreated.
Twenty four hours after the spraying all areas—treated or
not—were cleaned thoroughly with a brush. The resulting
material was put in plastic bags and brought to the
laboratory in order to count the dead and the living
mites.
Experiment 3
This experiment was done in a giant layer house with more
than 50,000 hens close to Nancy in France. The chickens
were kept at semi-night conditions in large metal cages
each containing five chicken. The night situation made it
possible that the mites stayed all the time on or close to the
chicken leading to enormous amounts of mites. Spraying
(1:40 dilution) was done four times at intervals of 7 days.
However, the house construction allowed only the spraying
of the front side of the cages.
Experiment 4
This experiment was done in four chicken houses in Egypt
(Nile Valley). The chickens were kept on the floor inside
60-200 m2rooms inside farms. Spraying was done two
times at an interval of 7 days using a MiteStop®-water
dilution of 1:40 or 1:50. Two hours after spraying, soil was
inspected for living and dead mites.
Experiment 5
This experiment was done in a stable close to Vechta
(Germany), where about 7,500 chickens were kept on the
floor. This experiment was planned to compare the
activity of MiteStop® with another (phoxim containing)
acarizide. Since the latter required due to its toxicity as
organophosphorous compound the retraction of the food
and water for 24 h, if it would be sprayed on the whole
floor, the application was done in both cases only on the
perch using a 1:33 dilution of the product with water.
Controls were done in all five experiments at places that
had not been sprayed or had been sprayed with pure
water.
Data analysis
Acaricidal activity was classified as described by Kim et al.
(2007): strong: mortality >80%; moderate: mortality 80-
61%; weak: mortality 60-40%; little or no activity:
mortality <40%. The LD50values were calculated by using
BioStat 2008.
Results
Life cycle parameters
Engorged females started to lay eggs after 24 h after their
blood meal. The average time required for egg laying was
41.6 h. Within 72 h, all mite eggs had hatched. Almost all
larvae moulted without feeding to protonymphs within
48 h. Moulting to deutonymphs occurred from 48 to 72 h.
Most of the engorged deutonymphs moulted to adults
within 72 h (Table 1).
Filter paper bioassays (Fig. 1)
The treatment of individual mites after method A showed a
strong acaricidal activity for the undiluted neem product
with an average mortality of 96.43%. The 1:20 dilution
Parasitol Res (2010) 107:417–423 419

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proved to be of moderate activity. The 1:40 diluted
MiteStop® showed a weak activity. The 1:60 and 1:80
dilutions were of little acaricidal activity. The LD50was
0.017 μl/cm² (0.019 mg/cm²). MiteStop® used after method
B1 (contact with wet acaricide) proved to be highly
efficacious to all developmental stages. It achieved a
mortality of 100% at an undiluted status as well as when
1:20 and 1:40 diluted. The use of a 1:60 dilution showed
also a strong acaricidal activity. The 1:80 dilution proved to
be of moderate efficacy. The LD50 was 0.047 μl/cm²
(0.052 mg/cm²). The lethal activity of dried acaricide
(method B2) was also very strong. All dilutions up to
1:40 showed a mortality of 100%. The 1:60 dilution also
proved to be very strong reaching an average mortality of
94.67%. The 1:80 dilution showed a moderate lethal
activity. The LD50was 0.051 μl/cm² (0.056 mg/cm²).
The vapour phase toxicity of undiluted and 1:20 diluted
MiteStop® proved to be strong. The 1:40 dilution showed a
moderate toxicity. The 1:60 and 1:80 dilution showed a
weak acaricidal effect. The LD50 for method C was
0.117 μl/cm² (0.128 mg/cm²).
Application method A (wetting of individual eggs) and
method C (vapour phase toxicity) had no ovicidal activity.
All treated eggs had hatched within 72 h. The application
methods B1 (contact with wet acaricide) and method B2
(contact with dried acaricide), however, had a strong
ovicidal activity preventing all eggs from hatching.
Treatment of poultry houses
Experiment 1
In the first experiment, the mite population was reduced by
66.86% after the first spraying. After the second spraying,
the total of the mite population had been reduced by 89%.
Apparently, some mites had hatched from eggs and/or came
from hidden unsprayed places (Fig. 2).
Experiment 2
In the second experiment in Sinsteden (Germany), defined
areas infested with poultry red mites were treated with 1:30
or 1:50 diluted MiteStop®. The efficacy was 100% in both
areas. Here, apparently all mites had been reached during
spraying (Table 2).
Experiment 3
The size if this giant stable and the amount of mites in the
chicken cages and their numbers around the channels and
bands transporting food, faeces and eggs made it necessary
to use an automatic spraying engine which was driven
along the walk boards in front of the cages. This method
implicated that only the front sides of the cages and of the
food and water equipment was fully reached by the 1:40
water diluted neem extract. On occasion of the first
inspection on day 0, the cages and the equipment were
covered with layers of mites reaching diameters of several
millimetres. Furthermore, numerous flies were able to
transport mites from any region of the stable to another
(e.g. the lamps at reduced light were covered with many
mites, too). Furthermore, due to the darkness inside the
stable, the mites did not hide anymore and stayed close to
the chickens and were also found constantly on the
chickens.
The four repeated treatment series (at intervals of
7 days) reduced the number of mites extremely. At the
last inspection, the thick layers of mites had disappeared
and mites were found only at hidden places. The
0
200
400
600
800
1000
1200
1400
1600
1800
1st monitoring
2nd monitoring3rd monitoring
Numbers of caught mites
Fig. 2 Numbers of caught mites during the treatment of a poultry
house (first experiment)
Developmental stageTime required (h) Mode Mean
Minimum (%) Maximum (%)h (%)h
Oviposition
Egg
Larva
Protonymph
Deutonymph
24 (26.67)
48 (5.88)
24 (2.94)
48 (80.00)
72 (80.00)
48 (73.33)
72 (94.12)
48 (97.06)
72 (20.00)
120 (6.67)
48 (73.33)
72 (94.12)
48 (97.06)
48 (80.00)
72 (80.00)
41.60
70.59
47.29
52.80
78.40
Table 1 Developmental time of
D. gallinae at 20°C and 40%
relative humidity
h hours
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reduction was calculated to be gone back to a level of
about 10-15% of the initial value. This value has to be
considered before the background that only the front of
the chicken cages was reached by the automatic spraying
machines and that behind the cages other mites had been
hidden.
Experiment 4
The 1:40 or 1:50 water freshly diluted MiteStop® product
was twice applied (on day 0 and day 7) onto the floor of the
stables in Egypt, where the chickens lived. Samples of soil
were taken 2 h after each spraying and studied by light
microscopy. The soil samples were placed onto a sieve and
a lamp was switched on to provide light and heat. Living
mites are hygrotactic, thermotactic and phototactic. This
introduced the movements of survivors downwards—away
from the lamp. Table 3 shows the results obtained after the
two treatments.
This experiment shows that a dose dependent reduction
of the numbers of mites, which, however, appear still rather
well protected in the soil dust. The 1:40 dilution revealed
slightly better results, but the dust protected still some
survivors.
Experiment 5
In the stable with 7,500 hens in Germany, 12 spots were
selected to become sprayed with the 1:33 MiteStop®-water
dilution. Previous to the first spraying, the 12 spots
contained 878 living mites. After 1 week, these places
contained only 102 living mites, i.e. corresponding to a
reduction of more than 88%. Of course these mites could
have immigrated from neighbouring places. After the
second spraying, the reduction of living mites was another
10% so that it was concluded that the product prohibited
reimmigration at a considerable degree. Otherwise, much
more mites would have been expected to come from the
surrounding non-sprayed places.
Discussion
Life cycle parameters
So far, there had been no data available for life cycle
parameter at a temperature of 20°C and a relative humidity
of 40%. Wisseman and Sulkin (1947) observed mites kept
at room temperature and 70% RH. Tucci et al. (2008)
observed mites at 20°C and a relative humidity ranging
from 70% to 85%. Compared to Tucci et al. (2008) the
average time needed for oviposition, larva, and deuto-
nymph development are higher at 40% RH. However, the
average time in which protonymphs moulted to
deutonymphs was shorter. The prolonged developmental
times might be due to the low relative humidity in
our laboratory, while the poultry red mite favours
high humidity.
Table 2 Assessment of caught mites in the second experiment
AreaNumber of living mites after 24hNumber of dead mites after 24hMortality
Total AdultsNymphs LarvaeTotalAdultsNymphs Larvae%
MiteStop® 1:30
MiteStop® 1:50
Control A
Control B
0
0
0
0
0
0
0
0
1,500
1,013
328
674
437
167
23
698
489
134
41
128
87
27
100.00
100.00
16.45
13.65
1,994
469
824
145
912
236
258
88640
Table 3 Example for mean numbers of counts of living mites found in soil of stables (mites per gram soil) 2 h after spraying of dilutions of the
neem seed extract 1:50) on day 0
MitesDay 0Day 7
D. gallinae (number per gram dust)Control1:40 1:50Control 1:401:50
0.300
0.380
0.420
0.510
1.610
0.180
0.228
0.252
0.306
0.966
0.240
0.304
0.336
0.408
1.288
0.350
0.410
0.481
0.390
1.631
0.070
0.082
0.096
0.078
0.326
0.140
0.164
0.192
0.156
0.652Total per 4 g
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Filter paper bioassays
The filter paper bioassays showed that the neem product
is already efficacious against the poultry red mite at a
low dose. Compared to the commonly used pesticides, it
has a stronger acaricidal activity than fipronil, pheno-
thrin, permethrin, alphacypermethrin, furathiocarb, car-
baryl, and fenotrothion which have an LD50of >5.0 mg/
cm². Diazinone (LD50=0.25 mg/cm²), chloropyrifos-
methyl (LD50=0.15 mg/cm²), fenthion (LD50=0.07 mg/
cm²), and propoxur (LD50=0.06 mg/cm²) are less effica-
cious as well. Only dichlorvos (LD50=0.0004 mg/cm²),
profenofos (LD50=0.003 mg/cm²), prothiofos (LD50=
0.055 mg/cm²), and benfuracarb (LD50=0.053 mg/cm²)
reach a lower LD50value for the same acaricidal activity.
Compared to the 10 most toxic plant extracts for D.
gallinae the neem product is more efficacious than
Glycyrrhiza glabra (LD50=0.14 mg/cm²), Foeniculum
vulgare (LD50=0.15 mg/cm²), Illicium verum (LD50=
0.09 mg/cm²), Lysimachia davurica (LD50=0.09 mg/
cm²), Paeonia suffruticosa (LD50=0.11 mg/cm²) and
Schizonepeta tenuifolia (LD50=0.15 mg/cm²). Only Men-
tha arvensis var. piperascens (LD50=0.0072 mg/cm²),
Eugenia caryophyllata (LD50=0.0069 mg/cm²), Cinnamo-
mum camphora (LD50=0.0051 mg/cm²) and Asarum
sieboldii var. seoulense (LD50=0.0063 mg/cm²) reach a
stronger acaricidal efficacy (Kim et al. 2007). The vapour
phase toxicity is less effective than the contact toxicity
reaching an LD50value of 0.117 μl/cm² or 0.128 mg/cm²,
respectively. A comparison of the LD50value with other
acaricides is not possible because there are no data
available. Kim et al. (2007) published an efficacy of
100% for A. sieboldii var. seoulense, Cinnamomorum
camphora, E. caryophyllata, M. arvensis var. piperascens,
and the efficacy of dichlorvos at a dose of 0.28 mg/cm².
MiteStop® yielded an average mortality of 94.64% at a
dose of 0.25 mg/cm². The efficacy of theses acaricides is
therefore comparable.
Ovicidal activity
An ovicidal activity was only obtained when using for
method B1 (contact with wet acaricide) and method B2
(contact with dried acaricide). However, MiteStop® was
only tested in a dilution of 1:50. It might therefore be
possible that a higher concentration would lead to an
ovicidal activity for the application methods A (wetting of
individual mites) and C (vapour phase toxicity). A higher
dosis may be necessary to penetrate the protective egg shell
to kill the developing larva. It might also be possible that
the neem product inhibits the hatching of larvae. A
transmission electron microscope analysis might give some
answers (Locher et al. 2010).
Poultry house experiments
Experiments 1 and 2
The spraying of the neem product in a low-grade
infested poultry house was very successful. Already
the first spraying, reduced the mite population by
66.86%. After the second spraying, the population had
been reduced by 89%. These results are similar to those
of Abdel-Ghaffar et al. (2008), who treated infested
poultry in Egypt. The efficacy of a repeated spraying of
MiteStop® is comparable to the efficacy of metrifonate
(0.15%; Nordenfors and Hoeglund 2000) and ByeMite®
(phoxim) (Meyer-Kühling 2007). The spraying of distinct
areas with MiteStop® in another infested poultry house
proved to be 100% efficacious (Table 2).
In conclusion, these two studies have shown that the
neem-derived acaricide has a high acaricidal effect against
all developmental stages of D. gallinae. Furthermore, it
shows not only an efficacy at direct contact, but it also is of
fumigant toxicity. Therefore, it has a great potential for the
control of the poultry red mite, if it is used correctly.
Experiment 3
This experiment proved that a dilution of 1:40 is in
principle able to kill the different stages of the red mite.
However, from untreated places, amounts of mites will
attack the chickens again (although in lower numbers). The
short developmental cycle of 8-12 days will always deliver
numerous newcomers from mite eggs that had been laid at
untreated places. Thus, it is needed to spray as much mite-
infested sites as possible.
Experiment 4
In this case, 1:40 and 1:50 dilutions were used being
sprayed directly onto the mite-containing dust on the
floors of the chicken houses. This dust protects several
mites from the product, but nevertheless the reduction
of the blood-sucking mites was considerable. Brush
cleaning of the floors will increase the efficacy of the
spraying.
Experiment 5
In this case, a dilution of 1:33 of the product was
sprayed onto the sitting places of the chickens. Although
there was the chance that the mites may migrate from
unsprayed places to sprayed ones, the reduction of mites
was nearly 90% of the number at the beginning of the
experiment. This shows that the 1:33 dilution is
very effective.
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Conclusions
The in vitro and in vivo experiments show that the neem
seed extract diluted at 1:33 with tap water kills all stages of
the blood sucking mite D. gallinae. Lower concentrations
or in cases when mites do not come into direct contact with
the acaricidal plant compound some mites may survive.
Such cases make it necessary, that treatment is repeated at
least twice at an interval of 5-7 days. Since this biological
biocide has also a high activity against other blood-sucking
insects such as bed bugs, fleas, lice or different ticks, the
product MiteStop® has become a very useful remedy
against house inhabiting pests (Semmler et al. 2009,
Schmahl et al. 2010, Abdel-Ghaffar et al. 2010).
Acknowledgements
Center of Excellence of the College of Science at the King Saud
University in Riyadh, Saudi Arabia. We thank Prof. Dr. Hiepe and
Prof. Dr. Schein (both FU Berlin) for their support and their evaluation
of the thesis of Mrs. Locher.
We gratefully acknowledge the support of the
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