Fish Parasites Bioindicate Danube Water Quality

Bioindication capacity of fish parasites for the
assessment of water quality

in the Danube River
Inaugural-Dissertation
zur
Erlangung des Doktorgrades
Dr. rer. nat.
der Fakultt fr
Biologie und Geografie
an der
Universitt Duisburg-Essen
vorgelegt von
Milen Nachev
aus Sofia (Bulgarien)
Februar 2010
Die der vorliegenden Arbeit zugrunde liegenden Experimente wurden in der Abteilung
Angewandte Zoologie/Hydrobiologie der Universitt Duisburg-Essen und am Zoologischen
Institut I der Universitt Karlsruhe (Karlsruhe Institute of Technology).
1. Gutachter:
Prof. Dr. B. Sures
2. Gutachter:
Prof. Dr. H. Taraschewski
3. Gutachter: ______________________________________________
Vorsitzender des Prfungsausschusses: ______________________________
Tag der mndlichen Prfung: 14.06.2010
`` as an ecosystem matures, parasitism naturally tends to evolve into mutualism; parasites

that fail to make that transition end up destroying their host and consequently themselves.
Human society must make the same transition from exploitation of the natural
environment to harmonious interaction with it. The danger of destroying our host, the planet
earth, was new because until recently neither the size of the human population nor the extent
of humans technological manipulation of the environment had been great enough to affect
regional and global balances.``
Eugene Odum
(Craige, 2001)
Acknowledgements
In the first place, I would like to express my sincere thanks to my supervisor Prof. Dr. Bernd
Sures for giving me the opportunity to carry out this thesis, for the numerous advices and
discussions as well as for the constructive critique regarding my work. Thank you also for
introducing me the exciting world of parasites.
Special thanks to Prof. Dr. Horst Taraschewski for the support during my work at the
University of Karlsruhe.
I am very grateful to Dr. Sonja Zimmermann for helping me during the analytical part of my
work and for the various suggestions, which helped me to improve the dissertation.
Thanks are due Dr. Christoph Singer for teaching me how to use and maintain the mass
spectrometer.
Many thanks go to Mr. Jaroslav Slobodnik and Mr. Igor Liska from the JDS2 expert group for
giving me opportunity to obtain fish samples during the second Joint Danube Survey (JDS2).
All known und unknown fishermen in Bulgaria, who helped me to sample the fish material
for the dissertation, are also thanked.
Thanks are also due to Prof. Thierry Rigaud and Christine Dubreuil from University of
Burgundy (Dijon) for their help in parasite molecular identification.
Dr. Frantiek Moravec is thanked for the morphological identification and verification of the
founded nematodes larvae.
Special thanks to Dr. Frankie Thielen and Dr. Marcel Mnderle for the gathered experience
during the various parasitological investigations.
I am grateful to all my colleagues from the Department of Applied Zoology and Hydrobiology
for the nice working atmosphere and numerous nice unforgettable moments together. Special
thanks to Dr. Christian Feld for the statistical advices and to Elisabeth Mller-Peddinghaus,
Daniel Dangel, Sabrina Frank, Michelle Keppel, Kerstin Gei and Nadine Haus for the
carefully corrections and remarks regarding the dissertation script.
I want to thank also Dr. Todor Hikov for the linguistic corrections and comments on the text
despite his non biological qualification, but well experienced in scientific English.
Many thanks go to my family and especially to my Mom for managing and helping me with
the frozen fish material and for the emotional support. I am also deeply indebted to my
brother Viktor Nachev for coordinating locally the sampling activities as well as for the many
nice sampling off-road trips/adventures with ``The Blue Submarine`` (Ford Escort Kombi),
which we made together. Thank you brother!!!
Thanks are due to all my Bulgarian friends and colleagues and mostly to Ilian Nikolov for the
support and pleasant company during the sampling trips as well as to Plamen Pankov and
Stephan Popov.
Last but not least I would like to thank the Landesgraduiertenfrderung Baden-Wrttemberg
for the financial support.
Thank you all. Without your help the completion of my dissertation could not have been
possible.
Table of Contents
Table of Contents
Table of Contents ........................................................................................................................I
List of Figures ......................................................................................................................... III
List of Tables ............................................................................................................................ IV
Glossary ................................................................................................................................... VI
Background ................................................................................................................................ 8
1
The endohelminth fauna of barbel (Barbus barbus) correlates with water quality of the
Danube River in Bulgaria ................................................................................................ 14
1.1
Introduction ................................................................................................................... 14
1.2
Materials and Methods .................................................................................................. 15
1.2.1
Sampling sites .................................................................................................... 15
1.2.2
Fish sampling ..................................................................................................... 16
1.2.3
Determination of helminth community structure and statistical treatment ............ 18
1.2.4
Water quality ...................................................................................................... 18
1.3
1.3.1
Total parasite fauna............................................................................................. 20
1.3.2
Diversity of helminth communities ..................................................................... 22
1.3.3
Water quality classification ................................................................................. 25
1.4
2
Results ........................................................................................................................... 20
Discussion ...................................................................................................................... 25
Is metal accumulation in Pomphorhynchus laevis dependent on parasite sex or

infrapopulation size? ....................................................................................................... 29
2.1
Introduction ................................................................................................................... 29
2.2
2.2.1
Sample collection ............................................................................................... 30
2.2.2
Molecular identification of Pomphorhynchus laevis ............................................ 31
2.2.3
Heavy metal analysis .......................................................................................... 32
2.2.4
Data analyses and statistical treatment ................................................................ 33
2.3
Results ........................................................................................................................... 34
2.3.1
Fish samples ....................................................................................................... 34
2.3.2
Analytical procedure .......................................................................................... 34
2.3.3
Element concentrations in barbel and Pomphorhynchus laevis ............................ 35
2.3.4
Accumulation differences with respect to parasite infra population size and sex .. 37
2.4
Discussion ...................................................................................................................... 40
Table of Contents
3
II
Seasonal differences of metal accumulation in Pomphorhynchus laevis and its definitive

host Barbus barbus ........................................................................................................... 43
3.1
Introduction ................................................................................................................... 43
3.2
3.2.1
Fish samples ....................................................................................................... 44
3.2.2
3.2.3
3.2.4
Element concentrations in the Danube River ....................................................... 45
3.3
3.3.1
3.3.2
Element concentrations in fish tissues and parasite samples ................................ 46
3.3.3
Seasonal differences in acanthocephalans morphology ...................................... 48
3.3.4
Seasonal variation in concentrations of the elements accumulated by P. laevis .... 49
3.4
4
Results ........................................................................................................................... 46
Discussion ...................................................................................................................... 51
Application of acanthocephalan Pomphorhynchus laevis from its host barbel (Barbus

barbus) as metal indicator in the Danube River ............................................................. 57
4.1
Introduction ................................................................................................................... 57
4.2
4.2.1
Fish samples ....................................................................................................... 58
4.2.2
Heavy metal analysis .......................................................................................... 59
4.2.3
4.2.4
Background metal monitoring data ..................................................................... 60
4.3
Results ........................................................................................................................... 62
4.3.1
Element concentrations in the host-parasite system ............................................. 62
4.3.2
Longitudinal profile of element concentrations in the Bulgarian part of the Danube

River in 2006...................................................................................................... 62
4.3.3
Longitudinal profile of element concentrations in the Danube River in 2007 ....... 67
4.3.4
Long term monitoring of element concentrations in the lower Danube ................ 68
4.3.5
Comparisons between element concentrations in parasite with the available

background data for water and SPM ................................................................... 69
4.4
Discussion ...................................................................................................................... 71
Summary, conclusions and future prospects........................................................................... 75

Zusammenfassung ................................................................................................................... 80
References ................................................................................................................................ 91
Appendix ................................................................................................................................ 102
List of Figures
III
List of Figures
Figure 1.1. Location of the sampling sites along the Danube River in Bulgaria.. .................. 16
Figure 1.2. Prevalence of coexistent helminth species of barbel from three sampling sites of
the Danube River. ................................................................................................. 22
Figure 2.1. Molecular identification of acanthocephalan species, according to the size of PCR
product of the partial ITS sequence. ..................................................................... 32
Figure 2.2. Mean element concentrations in organs of barbels and its intestinal parasite
Pomphorhynchus laevis. ....................................................................................... 35
Figure 2.3. Comparisons of the ratios C[P.laevis] / C[organ barbel] obtained for the toxic elements
arsenic, cadmium and lead between heavily and lightly infected barbels. ........... 39
Figure 2.4. Comparisons of the ratios C[P.laevis] / C[organ barbel] obtained for the essential
elements copper and zinc between heavily and lightly infected barbels. ............. 40
Figure 3.1. Seasonal profile of the mean worm weight. .......................................................... 49
Figure 3.2. Seasonal pattern of the element concentrations accumulated by P. laevis. ........... 50
Figure 3.3. Seasonal pattern of the concentrations of the elements As, Cd, Cu, Pb and Zn
fitted according to changes in prevalence of adult P. laevis in fish and cystacanths
in gammarids. ....................................................................................................... 53
Figure 3.4. Model of metal accumulation by P. laevis derived from data obtained from the
thesis and uptake kinetic suggested by Sures (2008b). ........................................ 56
Figure 4.1. Longitudinal profile of elements accumulated by P. laevis obtained for summer
2006 in Bulgarian part of Danube River. ............................................................. 67
Figure 4.2. Danubes longitudinal profile of elements As, Cd and Pb in P. laevis, obtained in
summer 2007. ....................................................................................................... 68
Figure 4.3. Long term monitoring of elements As, Cd, Cu, Pb and Zn in P. laevis at site
Kozloduy (Bulgaria). ............................................................................................ 69
Figure 4.4. Distribution profile of As, Cd, Cu, Ni, Pb and Zn in the SPM along the Danube
River during JDS2. ............................................................................................... 71
List of Tables
IV
List of Tables
Table I.
List of criteria characterizing the ideal sentinel organisms. ..........................................11
Table 1.1. Morphological parameters and characteristics of collected fish material. .................... 17
Table 1.2. Data on selected aqueous nutrient and pollution parameters according to ICPDR
(2008b) for upper and lower sites of the Bulgarian part of Danube River. .................. 19
Table 1.3. Prevalence, mean intensity and mean abundance of the parasites of barbel from three
sampling sites along the Danube River in Bulgaria...................................................... 21
Table 1.4. Average diversity characteristics of the infra community of helminths of barbel from
the Danube River. ......................................................................................................... 23
Table 1.5. Seasonal profile of the diversity characteristics of the infra community. ..................... 23
Table 1.6. Comparison of the average richness and diversity characteristics of the total component
community of helminths of barbel. ............................................................................... 24
Table 1.7. Seasonal profile of the diversity characteristics of the total component community. .. 24
Table 2.1. Trace metal concentrations in Dogfish Muscle Certified Reference Material
(DORM 3), accuracy and detection limits determined by ICP-MS analyses. .............. 34
Table 2.2. Differences between element concentrations in barbel organs and Pomphorhynchus
laevis. ............................................................................................................................ 36
Table 2.3. Bioconcentration factors C[P.laevis] / C[barbel tissue] for Pomphorhynchus laevis calculated
with respect to different host tissues. ............................................................................ 37
Table 2.4. Differences in element concentrations between heavily infected and lightly infected
barbels, as well as between male and female Pomphorhynchus laevis. ....................... 38
Table 3.1. Morphological data of barbel. ....................................................................................... 44
Table 3.2. Element concentrations in water at two different sites from the Danube River in
Bulgaria. (ICPDR, 2009) .............................................................................................. 46
Table 3.3. Seasonal profile of mean element concentrations in different tissues of barbel and in
P. laevis. ........................................................................................................................ 47
Table 3.4. Seasonal profile of bioconcentration factors C[P.laevis] / C[barbel tissue] for P. laevis
calculated with respect to different host tissues............................................................ 48
Table 4.1. JDS2 (second Joint Danube Survey) sampling site description. ................................... 59
Table 4.2. Data on aqueous element concentrations according to TNMN (2009) for upper and
lower sites of the Bulgarian part of Danube River. ...................................................... 61
Table 4.3. Element concentrations in P. laevis and different host tissues obtained in summer 2006
for the Bulgarian part of Danube River. ....................................................................... 63
Table 4.4. Element concentrations in P. laevis and different host tissues obtained in summer 2007
for upper and lower Danube. ........................................................................................ 64
List of Tables
Table 4.5. Element concentrations in P. laevis and in different host tissues measured for the period
summer 2004 - summer 2007 at site Kozloduy. ........................................................... 65
Table 4.6. Bioconcentration factors calculated for summer 2006 at three sampling sites in
Bulgaria. ....................................................................................................................... 66
Glossary
VI
Glossary
Ammonium EDTA ammonium Ethylendiaminetetraacetic acid. EDTA salts are used as a
chelating agent for metal ions.
BCF Bioconcentration factor. Calculated for each analyzed element according to Sures et al.
(1999a) as a ratio between the metal concentration in the parasite and the host tissue
C[P.laevis] / C[host tissue] as well as between the parasite and the concentration in the water
C[P.laevis] / C[water]. It represents an arithmetical approach for expressing the accumulation
capacity of fish acanthocephalans.
bp base pairs. Pair of nucleotides (bases) which are complementary bounded. In the
molecular biology, the number of base pairs is used as an important measure for the size
of a particular gene or for the entire genome.
DNA Deoxyribonucleic acid. The most important feature of the DNA molecule is to store
the genetic information, which is important for functioning and development of the
living organisms.
DORM-3 Fish protein certified reference material for trace metals. The reference material is
used for control and verification of the entire analytical procedure, which was
performed in the thesis.
ICPDR International Commission for the Protection of the Danube River. The Commission
works to ensure the sustainable and equitable use of waters and freshwater resources in
the Danube River Basin. The work of the ICPDR is based on the Danube River
Protection Convention, the major legal instrument for cooperation and transboundary
water management in the Danube River Basin (ICPDR, 1998).
ICP-MS Inductively Coupled Plasma Mass Spectrometry. This is a methodology for
measuring of numerous metals, which includes inductively coupled plasma for
ionization and mass spectrometer for detecting the ions. ICP-MS is a rapid and highly
sensitive technique in the field of analytical chemistry.
ITS Internal Transcribed Spacer is a region of ribosomal DNA (see rDNA). Comparison of
the sequence of ITS regions is a commonly used approach in taxonomical studies due to
their high variation between close related species.
JDS Joint Danube Survey sampling sites. The abbreviation in combination with the numbers
(e.g. 13, 16, 26 and 32) was used in chapter 4 to represent the localities from which the
fish samples during JDS2 were sampled.
JDS1 First Joint Danube Survey. A scientific expedition along Danube River carried out in
Glossary
VII
2001. It delivered various analyses of the water quality and ecological status of the
Danube River and some tributaries (JDS, 2001).
JDS2 Second Joint Danube Survey. The JDS2 is known as the worlds biggest river research
expedition. It was performed in 2007 and delivered profoundly information about water
quality and pollution in the Danube River and some of its tributaries (JDS, 2007).
PCBs Polychlorinated biphenyls. They represent a group of toxic organic compounds used
mainly in the industry as dielectric fluids for transformers and capacitors. Their
molecule is formed by up to ten chlorine atoms attached on biphenyl (two benzene
rings).
PCR Polymerase Chain Reaction. This is a common technique in the field of molecular
biology, applied to amplify/generate from one or few pieces of DNA thousands/millions
of copies of a particular DNA sequence.
rDNA ribosomal DNA. It represents those sequences of the DNA, which include the genes
of the ribosomal Ribonucleic acid.
SPM Suspended Particulate Matter. It represents the suspended sediment fraction in the
water phase. SMP regulates the transport of all types of water pollutants in dissolved
and particulate phases.
TNMN TransNational Monitoring Network, in short ``TNMN`` was established to support
the implementation of the Danube River Protection Convention in the field of
monitoring and assessment. It was formally launched by the ICPDR in 1996. The main
objective of the TNMN is to provide a structured and well-balanced overall view of
pollution and long-term trends in water quality and pollution loads in the major rivers in
the Danube River Basin (TNMN, 1996).
List of used element abriviations arsenic (As), bismuth (Bi), cadmium (Cd), colbalt, (Co),
copper (Cu), iron (Fe), mercury (Hg) manganese (Mn), molybdenum (Mo), nickel (Ni),
lead (Pb), tin (Sn), titanium (Ti), vanadium (V), zinc (Zn).
Background
Background
In recent years aquatic ecosystems suffer from a permanent increase of pollution caused by
the industrialization and urbanization. Simultaneously, the humans continue to extend their
knowledge regarding the problems emerging after and try to study in detail every component
of the ecosystem in order to understand the consequences of such external stress. In general,
ecosystems are complex systems consisting of a number of mutual interacting components.
Observed independently, each part (component) of a given ecosystem represents a piece of a
puzzle. Combining each of the puzzle pieces should deliver an entire picture of the ecosystem
condition. The size of the puzzle varies according to the size and complexity of the
ecosystem. Therefore, for obtaining precise information over its general condition, we need to
explore as much as possible available parts. At this point the ecologists set the concept for
ecosystem health, which is a measure of how every piece of the puzzle match the entire
puzzle and how are they balanced, if we continue thinking abstractly. Costanza and Mageau
(1999) defined the ecosystem health as a '...comprehensive, multiscale dynamic, hierarchical
measure of system resilience, organization and vigor.' The concept comprises the system's
ability to keep its structure (organization) and function (vigor) over time with regard to
external stress (resilience). In simple words a healthy ecosystem is one which comprises a
balance between system components, stability, diversity and complexity, absence of disease
and last but not least homeostasis. All these aspects are summarized in the term `ècosystem
sustainability``, which is actually the overall performance of the system resulted from the
interaction and behavior of its components (Costanza and Mageau, 1999).
In the field of ecological monitoring, researchers are trying to study as many parts of a given
ecosystem as possible in order to detect external stress factors, which mostly occurring in the
form of contamination. The chemical (all external substances, which naturally do not belong
to the system) or physical (thermal, noise, radioactive etc.) contamination itself can induce
changes in the ecosystems functionality and structure, which on the other hand affects its
overall performance. Therefore, ecological monitoring is mostly aimed at studying the
changes that could be assessed after exploring in detail the balance between the system
components. Following the history of hydrobiological monitoring, at the beginning (until the
middle of the 19th century) water quality assessment was based only on some chemical or
physical parameters of water bodies. Kolenati (1848) and Cohn (1853) for the first time
discovered and described that some organisms are showing a relation to the water quality
(summarized by Bock and Scheubel, 1979). At the beginning of the 20th century Kolkwitz and
Background
Marsson (1902, 1908, 1909) found a close relationship between water organisms and
pollution after studying the biological and chemical processes of self-purification running in
lotic ecosystems (mostly in River Rhine). A methodology (the Saprobic System) for
hydrobiological monitoring based on animal communities labeled as bioindicators was
developed and established for first time. Furthermore, the water quality assessment
implemented more and more components over time, after analyzing their relationship with
pollution. This implies macroinvertebrate communities, macrophytes, algae, fungi, fish, even
ciliats have been studied from a bioindicator perspective. Worth noticing is that all these
groups (components) have a basic common characteristic they are an inseparable part of
aquatic ecosystems. But there are still some components less investigated. One of them could
be the group of fish parasites. The presumption, that aquatic parasites have no relation to the
environment conditions prevailed for quite a while, arguing with the parasites specific
biology. Fish parasites were always underrated by field ecologists in aquatic monitoring,
because they lacked in most of the cases ``direct`` connection with the ambient water
medium. They were observed mostly from the perspective of water born diseases or some
breakout infection events in the fish populations, without searching the reasons which in term
laid mostly on the disturbed environment conditions, respectively pollution. In the last couple
of decades, after gathering more detailed information concerning these aspects, many studies
showed that fish parasite communities also react to alterations in conditions. Furthermore
these alterations resemble those of free living organisms. The first evidence was delivered by
impact surveys on some ectoparasitic species of fish, particularly on monogenean trematodes.
They are common fish parasites occurring on gills and skin, therefore they are in permanent
contact with the surrounding environment. By observing monogeneans presence or absence
and diversity characteristics of their communities, it is possible to obtain valuable information
about the alternation in environment factors (summarized by Sures, 2001). Thus, their close
relation to eutrophication processes was demonstrated (Koskivaara, 1992; Valtonen et al.
1997), as well as to other pollution sources like effluents from the industry (e.g. pulp and
paper mills) (Siddall et al. 1997). This relation was mostly expressed by reduced species
richness and unequal distribution of abundances (summarized by Sures, 2001). However, this
parasite group exhibit some features similar to free living organisms, which are also in
permanent contact with the surrounding environment.
However, endoparasitic assemblages, although `èmbodied`` in the host, may also have a
relationship to pollution. Thus, the first step to achieve an understanding for the interaction
between parasites and environmental factors is to get an overview on the parasite
Background
10
transmission. Despite the high variety and complexity in transmission, the larger part of the
endoparasites exhibit stages affected by the environment conditions. The direct effect is
normally expressed by lethal reactions of the free living larval stages (e.g. Metacercaria) or
adults, whereas the indirect impact is addressed on the intermediate or final host the
pollution could drive the suitable intermediate and final hosts to extinction (Sures, 2008a). It
can also affect the host physiology and thus the infected host as well as the parasites may
suffer more from environmental exposure. In both cases the pollution leads to changes in the
diversity and richness of parasite communities and thus parasites can be used as effect
indicators. For that reason the parasite communities are more frequently analyzed in respect
to pollution in the last decades. In summary, the effect indicators deliver information about
the ecosystem health and integrity through changes in diversity and structure of their
communities (Sures, 2001). However, should an ecosystem rich in parasites be considered as
healthy? In the review paper published by Hudson et al. (2006) the position of parasites on
the ecosystem level and their important regulatory role for the entire biodiversity and
production was clearly defined. Therefore, the parasites diversity and richness is as important
as those of the other ecosystem components like producers and consumers, which always
have been in the focus of ecologists.
In addition to the ecological aspects of bioindication, fish parasites could be also an
appropriate tool for detecting and quantifying some toxic substances in aquatic habitats.
Recently, the intensive research on their application as sentinels showed that they are even
more advantageous than the already established organism (Sures et al. 1997a, 1999b). Due to
their enormous accumulation capacity, parasites such as acanthocephalans can concentrate
toxic chemicals (e.g. heavy metals) even though the ambient concentrations are far below the
detection limits this is advantageous especially in some less polluted habitats like the
Antarctic (Sures and Reimann, 2003) or for substances in very low concentration ranges, like
precious metals (Sures et al. 2005). In general, accumulation indicators are organisms,
which are able to accumulate substances (in the most cases toxic) from the surrounding
environment within their bodies and thus deliver information about the bioavailability of the
given substance and its environment contents. Various experimental and field studies
demonstrated and proved parasites sentinel features, whereas the most promising group was
found to be the group of fish acanthocephalans. They are widely spread intestine parasites of
fish, characterized with a relative short life cycle (Kennedy, 2006). The experiments on heavy
metal uptake mechanism showed that the accumulation process start immediately after the
infection of the definitive host, whereas the uptake occurs through gills over the circulatory
Background
11
system and entero-hepatic route of the fish (Sures and Siddall, 1999). Thus, this considerably
fast mechanism of accumulation leads to achievement of steady state concentrations of the
particular metal in parasite after only 4-5 weeks after the first exposure (Sures, 1996), which
makes the acanthocephalans a very sensitive and quick instrument for the detection of metal
pollution.
Regardless, further investigations of fish parasites in respect to their bioindication features are
needed, in order to be applied in the aquatic monitoring. There are still uncertainties regarding
the ideal sentinel organism (summarized by Sures, 2003; Table I); however, if the fish
acanthocephalans are taken as metal indicators these issues should be overcome. The table
listed below shows in summarized form the information which is available or is still missing:
Table I. List of criteria characterizing the ideal sentinel organisms according to Martin and
Coughtrey (1982), Philips and Segar (1986), Phillips and Rainbow (1993) - summarized by
Sures (2003) for acanthocephalans.
Criteria
Rapid equilibrium whith the source
A linear relationship with source over the range
of ambient concentrations
The relationship between the tissue and source
concentrations should be the same at all sites
studied
Abundant species from which large numbers
can be taken without altering the age structure
or having some other significant effect on
population
Easily identified
Large body of knowledge about the species'
physiology, including the effects of age, size,
season and reproduction activity on the
assimilation of the pollutant
Large body- to provide abundant tissue for
analysis
Sedentary or with a well defined home range
Uptake is from a well defined pollution source
Easily aged and long lived - allowing integration
of the pollutant over long periods
1 More information required
Acanthocephala
Yes
Yes
1
Yes
Yes
No
Yes
Yes
Yes
1
According to Table I it seems that acanthocephalans fulfill almost all necessary criteria
regarding their application as sentinels. The lack of knowledge concerns mostly some
uninvestigated aspects of their biology such as effects of the age and the size of the
Background
12
acanthocephalans as well as the effects of the seasonality and reproduction which might
induce oscillations in the accumulation process. As summarized by Sures (2003), the only
disadvantage, which the acanthocephalans probably exhibit, is that they are hard to be aged
and are not long living animals. However, the short life spawn can be put to an advantage, as
acanthocephalans could possibly deliver a more precise chronological view on metal pollution
than other organisms, postulated their life spawn is restricted to an exact timeframe (e.g.
year). Consequently, it could be able to date accurately the pollution sources and events, when
they occur and subsequently manage them. Therefore, some further investigation concerning
the live duration of the parasites is required.
Even if ecologists are able to fill those knowledge lacks, a logical question appears: Do we
need to implement actually new bioindicators in our hydrobiological praxis?
The need of parasites as accessory bioindicators can be also seen as gathering additional
knowledge over their ecological state, and thus we will improve our view on the overall
condition on ecosystem level. They are an additional piece of the puzzle, which we need to
collect if we want to obtain a more detailed picture of ecosystems homeostasis and integrity.
Therefore, it can be concluded that the water quality could be assessed more precisely by
using accessorily the fish parasites as bioindicators, especially in large and complex lotic
systems like Danube River, where the conventional hydrobiological methods exhibit some
intricacies. The implementation of fish as bioindicator during the second monitoring
expedition in 2007 (Joint Danube Survey) was an example that the hydrobiologists need to
extend their monitoring spectrum to achieve and enhance the desired information about the
ecological state of Danube River. The fish parasites, like fishes, are an inseparable part of
aquatic ecosystems, therefore they should also be taken into account by hydrobiological
monitoring.
Scope of the thesis

The objective of the current thesis is to expand the scientific basis concerning employing fish
parasites as bioindicators. Therefore, a field investigation was carried out as a monitoring
survey over four years from summer 2004 to summer 2007. It covers some faunistical and
ecology aspects of fish parasites in relation to environmental conditions (Chapter 1).
Furthermore, the thesis intends to cover the lack of knowledge regarding the application of
fish acanthocephalans as sentinels for metal pollution (Chapter 2 and Chapter 3).
Additionally, it delivers a detailed heavy metal monitoring over the investigation period
(Chapter 4).
Background
13
Chapter 1: The endohelminth fauna of barbel (Barbus barbus) correlates with water quality of
the Danube River in Bulgaria
This chapter gives an overview on the endohelminth fauna of the barbel in the lower Danube
for the period summer 2004 to summer 2007. The composition and diversity of the parasite
communities were studied in seasonal manner at different sampling sites in Bulgaria in order
to express the capacity of fish parasites as effect indicators. The possible variation in the
composition and diversity of their communities was expected to be related to the local
environmental conditions.
Chapter 2: Is metal accumulation in Pomphorhynchus laevis dependent on parasite sex or
infrapopulation size?
The chapter covers some uninvestigated aspects regarding the application of fish
acanthocephalans as accumulation indicators.
Two questions are in the main focus of this chapter:
Is the metal accumulation by P. laevis dependent on the parasite`s sex?
And: Does the size of the infrapopulation influence the metal accumulation in the parasite?
Chapter 3: Seasonal differences of metal accumulation in Pomphorhynchus laevis and its
definitive host Barbus barbus
This chapter presents the effects of seasonality of P. laevis development on metal
accumulation in the host-parasite system. Furthermore, according to the obtained data was
designed a model, which represents the metal uptake process in natural conditions.
Chapter 4: Application of the acanthocephalan Pomphorhynchus laevis from its host barbel
(Barbus barbus) as metal indicator in the Danube River
This chapter delivers a metal monitoring study conducted with the suggested barbel P. laevis
system. The data was supported with background chemical data delivered by the International
Commission for the Protection of the Danube River (ICPDR) in order to express the
bioindication capacity of fish acanthocephalan regarding heavy metals and arsenic.
1 The endohelminth fauna of barbel (Barbus barbus) correlates

with water quality of the Danube River in Bulgaria
1.1 Introduction
In recent years, fish parasites attain increasing interest from an environmental point of view
(Sures, 2006, 2008a). Many studies demonstrate the close relation between parasitism and
ecological conditions in a given environment and describe how parasites can be used to
enlarge knowledge on ecosystem function and integrity (Hudson et al. 2006; Lafferty et al.
2008). Pollution with toxic substances such as metals or polychlorinated biphenyls (PCBs) as
well as an enrichment of nutrients (eutrophication) may affect the occurrence and physiology
of parasites. The effects of toxic pollutants and eutrophication on parasites can be direct (e.g.
by reduction of the number of free living stages or intermediate host) or indirect (e.g. host
immunosuppression) depending on the pollution type and parasite life cycle (Sures, 2008a).
Various studies demonstrate for example that euthrophication reduces the diversity of
heteroxenous parasites, whereas parasites with direct life cycle (monoxenous) are less
affected. The latter are often ectoparasites which are in direct contact to the surrounding water
and are thus adapted to changes in environmental conditions (Valtonen et al. 1997;
MacKenzie, 1999; Perez-del Olmo et al. 2007). Concerning toxic pollutants it emerges that
certain substances such as metals or PCBs cause immunosuppression in the fish host and thus
may increase parasitism by a reduced host defence (Hoole, 1997). The resulting numerical
changes (increase or decrease of abundance and intensity) of aquatic parasites leading to
changes in structure and diversity of parasite communities as a response to different forms of
pollution may be used for bio-indication purposes (MacKenzie et al. 1995). Accordingly, the
occurrence and diversity of parasites stand as a measure of ecosystem health even if the
underlying functional chains are often unknown.
In order to use fish parasites as pollution indicators, the fish host must be widely distributed
and easy to be sampled (Kennedy, 1997). Therefore, the present investigation was focused on
barbel (Barbus barbus) and its parasite communities at different sampling sites along three
lower reaches of the Danube River. The barbel is the second largest native cyprinid fish
species in Europe, being wide spread in major European river systems. Although many
studies on the parasite fauna of B. barbus have been published from selected localities of the
Danube basin, data from east Europe and especially from the Balkan Peninsula and the
Danube delta is scarce. Only few studies on parasites of barbel in the Danube River in
Bulgaria (Kakacheva-Avramova, 1962, 1977, 1983; Margaritov, 1959, 1966; Nedeva et al.
1 The endohelminth fauna of barbel correlates with water quality
15
2003) and in Romania (Roman, 1955) exist, whereas most information on barbel parasites is
delivered from Central Europe (Michalovi, 1954; Moravec and Scholz, 1991; Moravec et al.
1997; Laimgruber et al. 2005). Until now, the complete endohelminth fauna of B. barbus
reported for the Danube drainage system in Central Europe consists of 43 species with 22
trematodes, 9 cestodes, 7 nematodes and 5 acanthocephalans (Moravec et al. 1997). In
contrast, the list of barbel endohelminths in the Bulgarian section of the Danube River
(Kakcheva-Avramova, 1977) includes only 6 species, but there are a few unpublished studies,
which describe up to 11 species.
The aim of the present chapter was to obtain a more complete picture of the endohelminth
fauna of B. barbus and to study the composition and diversity of parasite communities with
respect to the environmental conditions of the habitats. It is expected that the structure and
diversity of parasite communities over consecutive years at sites that differ in their degree of
eutrophication and in their concentration of toxic metals reflect the ecological conditions.
1.2 Materials and Methods

1.2.1 Sampling sites
The study was carried out in a seasonal manner (April, July and October) ranging from
summer 2004 to summer 2007 at three different localities of the Bulgarian part the Danube
River. The sampling sites (see Figure 1.1) were selected on the basis of different degrees of
eutrophication and toxic pollutants, as the main objective of the current research was to check
if parasite communities reflect the environmental conditions of their habitats. The first
sampling site is located near Vidin (river kilometre 834), about 10-15 km away from the
inflow of the river Timok (845 km), which is one of the biggest metal pollution sources
downstream in the Danube. The second sampling site was selected near to town Kozloduy
(685 km), approximately 160 river kilometres downstream from Vidin. The third site was on
the border between Bulgaria and Romania near the town Silistra (375 km) which represents
the last Bulgarian locality in eastward direction of the river. The sampling stretches covered
approximately 5 river kilometres at each sampling site (Figure 1.1).
16
Figure 1.1. Location of the sampling sites along the Danube River in Bulgaria. BG- Bulgaria;
RO- Romania.
1.2.2 Fish sampling

A total of 407 barbels were collected by fishermen, using drift nets. The number of
individuals with a minimum total length of 20 cm varied between 10 and 35 fishes per
sampling site and season (Table 1.1). During the whole sampling period a total of 165 fish
were caught in Vidin and 193 in Kozloduy. Sampling continued for only two years in Silistra,
where 49 barbels were sampled between 2006 and 2007. Additionally, spring sampling at all
sites was performed only in the years 2006 and 2007. After catching, the fish were frozen at
-15C and transported to the laboratory, where total length (TL), standard length (SL), body
weight (BW), sex and age for each fish was determined. The condition factor (K) was
calculated as follows K=100*BW*TL3 (Schperclaus, 1990). The fish were subsequently
dissected and analysed for parasites using standard parasitological techniques. The skin,
scales, fins, gills, eyes, gut, cavities and organs were examined using a stereomicroscope
(magnification x8 to x50). Nematodes were fixed in 70% ethanol and mounted in glycerine
for further identification whilst all other parasites could be indentified directly.
Sampling sites
Sampling time
No. of fishes
Vidin
Spring
Summer
Autumn
Total
Spring
Summer
Autumn
Total
Spring
Summer
Autumn
Total
48
58
59
165
37
86
71
193
10
27
12
49
Kozloduy
Silistra
Weight [g]
mean SD
range
702 ( 577)
108 - 3909
836 ( 604)
207 - 2145
565 ( 486)
81 - 2390
649 ( 509)
81 - 3909
599 ( 280)
120 - 1125
587 ( 371)
125 - 2208
539 ( 370)
140 - 1785
573 ( 355)
120 - 2208
801 ( 216)
400 - 1050
948 ( 367)
410 - 1785
624 ( 168)
420 - 900
838 ( 327)
400 - 1785
Total Length [cm]

mean SD
range
40.9 ( 8.3)
25.5 - 72
43.2 ( 10.4)
28.7 - 63.7
38.5 ( 9.2)
23.4 - 67.2
39.9 ( 8.8)
23.4 - 72
39.6 ( 6.3)
26 - 52.3
38.9 ( 7.6)
24.5 - 60.5
38.5 ( 7.9)
26.2 - 56.9
38.9 ( 7.5)
24.5 - 60.5
43.2 ( 5.2)
34.8 - 50
44.7 ( 5.9)
34.1 - 54.8
39.7 ( 4.4)
34.3 - 47
43.2 ( 5.7)
34.1 - 54.8
Condition factor
mean SD
range
0.88 ( 0.10)
0.65 - 1.05
0.85 ( 0.08)
0.74 - 1.07
0.83 ( 0.15)
0.30 - 1.28
0.87 ( 0.14)
0.30 - 1.80
0.89 ( 0.10)
0.68 - 1.15
0.88 ( 0.09)
0.64 - 1.07
0.86 ( 0.16)
0.38 - 1.49
0.88 ( 0.13)
0.38 - 1.49
0.99 ( 0.15)
0.77 - 1.28
1.01 ( 0.13)
0.80 - 1.31
0.99 ( 0.12)
0.71 - 1.16
1.00 ( 0.13)
0.71 - 1.31
Table 1.1. Morphological parameters and characteristics of collected fish material.
17
18
1.2.3 Determination of helminth community structure and statistical treatment

Parasitological parameters used followed those suggested by Bush et al. (1997) - prevalence
(P, %), intensity range (IR), abundance (A) and mean intensity (MI) of the infection. The
following diversity indices were calculated to describe the richness and diversity of the
parasite communities: Brilliouin index (HB), Shannon-Wiener index (HS), Shannon-Wiener
evenness (E), Simpsons index (D) and Berger-Parker index (d) according to Magurran (1988)
and Sures et al. (1999c).
Correlations between intensity and fish weight were checked using Spearmans rank
correlation coefficient. A one-way ANOVA was employed to determine significant differences
in the diversity characteristics of the intestinal infra-community and to compare the number of
each parasite species between sampling sites. For estimating differences of fish condition
factors between sampling sites, the Mann-Whitney U-test was applied.
1.2.4 Water quality

Water quality data for sites adjacent to our fish sampling sites (see Table 1.2) were obtained
from the technical reports published by the Joint Danube Survey (ICPDR, 2002, 2008a,c) and
annual reports and the database of TNMN (Trans National Monitoring Network, ICPDR,
2004, 2005, 2008b). These research programs and activities are initialised by the International
Commission for Protection of the Danube River (ICPDR). The available data were used as a
basis to interpret the composition and richness of helminth communities at the same localities.
19
Table 1.2. Data on selected aqueous nutrient and pollution parameters according to ICPDR
(2008b) for upper and lower sites of the Bulgarian part of Danube River.
Parameters
Ammonium
[mg/L]
Year
Vidin1
Kozloduy2
Silistra
2003
0.185
0.265
0.079
2004
0.191
0.183
0.075
2005
0.207
0.288
0.078
2007 *
0.016
0
0
Nitrate
2003
1.203
0.661
1.119
[mg/L]
2004
1.446
0.977
1.435
2005
1.41
0.829
1.574
2007 *
1.45
1.44
1.56
Nitrite
2003
0.033
0.022
0.019
[mg/L]
2004
0.025
0.021
0.02
2005
0.032
0.022
0.016
2007 *
0.059
0.064
0.016
Orthophosphate
2003
0.054
0.053
0.064
[mg/L]
2004
0.116
0.061
0.071
2005
0.12
0.068
0.059
2007
0.069
0.043
0.041
Total phosphorus
2003
0.323
0.108
0.119
[mg/L]
2004
0.184
0.103
0.164
2005
0.21
0.130
0.149
2007 *
n/a
n/a
n/a
Cadmium
2003
1
1.000
1
[g/L]
2004
1
1.167
1
2005
1
1.825
1
2007 *
n/a
n/a
n/a
Copper
2003
14.9
9.083
6
[g/L]
2004
18.7
6.417
2.5
2005
17.5
5.158
1
2007 *
n/a
n/a
n/a
Lead
2003
1.8
2.333
2.8
[g/L]
2004
2
2.583
1
2005
1.8
2.767
1
2007 *
n/a
n/a
n/a
1
: Sampling site Novo Selo, 1 km away from Vidin
2
: Sampling site IskarBaikal, 40 km away from Kozloduy
*: Data delivered by 2nd Joint Danube Survey- Onboard results (ICPDR 2008a)
n/a: Data not available
20
1.3 Results
1.3.1 Total parasite fauna
A total of 10 endohelminth parasites species was recovered, including 3 trematodes
(Dipostomum spathaceum (metacercariae) in the eye lens, Posthodiplostomum cuticola
(metacercariae) on the skin, Metagonimus yokogawai (metacercariae) on the scales), 3
acanthocephalans (Pomphorhynchus laevis, Acanthocephalus anguillae, Leptorhynchoides
plagicephalus in the intestine) and 4 nematodes (Rhabdochona hellichi, Pseudocapillaria
tomentosa, Hysterothylacium sp. (larvae) in the intestine and Eustrongylides sp. (larvae) in the
body cavity) (Table 1.3). One acanthocephalan species (L. plagicephalus) and 2 nematodes
(larvae of Eustrongylides sp. and Hysterothylacium sp.) were recorded for the first time for
barbel. Only one fish from the sampling site Vidin was infected with a single adult male of
L. plagicephalus,
which
is
thus
considered
an
accidental
infection.
Larvae
of
Hysterothylacium sp. were found in the gut of one barbel collected at the sampling site
Kozloduy. Eustrongylides sp. occurred at all sampling sites during the entire period. This
nematode together with the nematode R. hellichi was the second most widely distributed
parasite species at the sampling site Vidin (P, 24.2 %; MI, 10.1). Also at the sampling sites
Silistra and Kozloduy it occurred with high prevalence and intensity (Kozloduy P: 17.1%; MI:
9.1; Silistra P: 14.3%; MI: 2.1). The pattern of infection presents a clear correlation between
fish size, prevalence and intensity of infection. The highest prevalence was found in barbels
with a length between 40 to 60 cm. Infection intensity increased significantly (Spearman
correlation, p<0.05) with body size (Vidin: r = 0.32; Kozloduy: r = 0.39; Silistra: r = 0.34).
Total species richness ranged between 9 worms for Vidin and Kozloduy and 7 for Silistra. The
most abundant parasite was the acanthocephalan P. laevis. At the sampling site Vidin 100% of
the fishes were infected with this acanthocephalan and the mean intensity was 124.6 worms
per fish. Only two fishes from Kozlduy (P, 99%; MI, 84.3) and one from Silistra (P, 98%; MI,
117.7) were not infected with P. laevis. The second most frequent species at all sampling sites
was R. hellichi. The number of R. hellichi individuals showed significant differences between
Vidin and Kozloduy (p = 0.029, F = 4.795), and Vidin and Silistra (p = 0.003, F = 8.78),
whereas no differences were detected between Kozloduy and Silistra.
Parasite species
Rhabdochona hellichi
Pseudocapillaria tomentosa
Eustrongylides sp. larv.
Hysterothylacium sp. larv.
Pomphorhynchus laevis
Acanthocephalus anguillae
Leptorhynchoides plagicephalus
Diplostomum spathaceum larv.
Postodiplostomum cuticola larv.
Metagonimus yokogawai larv.
Samppling site
Vidin
Kozloduy
Silistra
Vidin
Kozloduy
Silistra
Vidin
Kozloduy
Silistra
Vidin
Kozloduy
Silistra
Vidin
Kozloduy
Silistra
Vidin
Kozloduy
Silistra
Vidin
Kozloduy
Silistra
Vidin
Kozloduy
Silistra
Vidin
Kozloduy
Silistra
Vidin
Kozloduy
Silistra
Prevalence
Mean Intensity
P [%]
MI ( SD)
24.2
47.7
46.9
4.8
4,1
10.2
24.2
17.1
14.3
0.5
100
99
98
1.2
0.5
0.6
7.3
8.8
6.1
16.4
17.1
38.8
12.7
15.5
10.2
15.9 ( 35.6)
34 ( 99)
72.9 ( 180.7)
1.4 ( 0.7)
2.3 ( 2.0)
2 ( 1.7)
10.1 ( 20.5)
9.1 ( 14.1)
2.1 ( 1.9)
1
124.6 ( 122.5)
84.3 ( 77.7)
117.7 ( 107.5)
1
2 ( 2)
1
-
Intensity range
Abundance
1 - 207
1 - 759
1 - 761
13
17
1-5
1 93
1 - 68
1-6
1
1 874
2 424
4 - 523
1
2
1
-
3.9
16.2
34.2
0.07
0.09
0.2
2.5
1.6
0.3
0.01
124.6
83.4
115.3
0.01
0.01
0.006
-
Table 1.3. Prevalence, mean intensity and mean abundance of the parasites of barbel from three sampling sites along the Danube River in Bulgaria.
21
22
The trematodes were the third group in terms of prevalence. Metacercariae of P. cuticola were
most frequently found, followed by M. yokogawai and D. spathaceum at all sampling sites.
There are no data available concerning the intensity of infection, since only the presence of
metacercariae was recorded. The nematode P. tomentosa was present in all Danube sites
during the whole sampling period. Whilst the prevalence was similar (4.8% and 4.1%) at the
sampling sites Vidin and Kozloduy, it was more than 2 times higher in Silistra.
1.3.2 Diversity of helminth communities

Diversity and dominance indices were calculated without considering trematodes as they were
not counted individually. Diversity characteristics of the infra-community are presented in
Figure 1.2 and Table 1.4 and Table 1.5. Most of the fish were infected with either one or two
parasite species simultaneously (Figure 1.2). At Vidin more than 50% of all fish were infected
with one species only, whereas at Silistra 10% of the barbels were co-infected with 3 species.
A clear increase in average diversity in downstream direction is reflected by the Brillouin
index, which showed the highest value at Silistra. Statistical analyses revealed significant
differences for the Brilliouin index between Vidin and Kozloduy (p = 0.005, F = 8.101) and
Vidin and Silistra (p = 0.038, F = 4.375), whereas no difference was found between the
sampling sites Kozloduy und Silistra (p = 0.853, F = 0.034). Concerning seasonal differences
highest infracommunity diversity was found in spring and autumn for two sites, only
Kozloduy showed a higher Brillouin index in summer than in autumn.
Prevalence [%]
60,0
50,0
Vidin
40,0
Kozloduy
30,0
Silistra
20,0
10,0
0,0
0
Number of species
Figure 1.2. Prevalence of coexistent helminth species of barbel from three sampling sites of
the Danube River.
Sampling sites
No. of barbels
Mean no. of helminth species per barbel SD
Maximum no. of helminth species per barbel
Mean value of Brillouins Index (HB) SD
Maximum value of Brillouins Index (HB)
Vidin
165
1.55 0.61
3
0.10 0.16
Kozloduy
193
1.68 0.66
4
0.15 0.19
Silistra
49
1.69 0.77
4
0.16 0.22
0.76
0.68
0.66
Table 1.5. Seasonal profile of the diversity characteristics of the infra community.
Sampling sites
Vidin
Summer
Autumn
48
1.71
0.62
3
0.14
0.19
58
1.60
0.65
3
0.07
0.12
59
1.37
0.52
3
0.09
0.17
37
1.81
0.66
3
0.19
0.23
86
1.67
0.69
4
0.15
0.20
71
1.62
0.62
3
0.13
0.17
10
1.40
0.84
3
0.16
0.26
0.68
0.76
0.71
0.68
0.66
0.67
0.65
Spring
No. of barbels
Mean no. of helminth species per barbel
SD
Maximum no. of helminth species per barbel
Mean value of Brillouins Index (HB)
SD
Maximum value of Brillouins Index (HB)
Spring
Kozloduy
Summer Autumn
Spring
Silistra
Summer
27
1.74
0.81
4
0.11 0.18
0.66
Autumn
12
1.59
0.71
3
0.23
0.27
Table 1.4. Average diversity characteristics of the infra community of helminths of barbel from the Danube River.
0.65
23
24
Similarly, component community diversity (Table 1.6) was also found to be higher
downstream (Silistra) than upstream (Vidin). This tendency is also reflected by the BergerParker dominance index, for which highest values were found in Vidin and lowest in Silistra.
Kozloduy showed medium values compared to the other sampling sites. Highest seasonal
diversity was found in spring in Vidin and Kozloduy and in summer in Silistra (Table 1.7).
Table 1.6. Comparison of the average richness and diversity characteristics of the total
component community of helminths of barbel.
Sampling sites
Vidin (n=165)
Kozloduy (n=193)
Silistra (n=49)
Shannon-Wiener Index (HS)
0.23
0.52
0.56
Shannon-Wiener Evenness (E)
0.13
0.33
0.40
Simpsons Index (D)
1.10
1.42
1.56
Berger-Parker Index (d)
0.95
0.82
0.77
P. laevis
P. laevis
P. laevis
No. of helminth species
Dominant species
Table 1.7. Seasonal profile of the diversity characteristics of the total component community.
Sampling sites
Spring
Summer
Autumn
Vidin
Kozloduy
Silistra
HS
0.34
0.60
0.32
E
D
0.25
1.21
0.43
1.61
0.22
1.20
0.91
0.75
0.91
HS
0.09
0.59
0.62
E
D
d
0.06
1.03
0.98
0.37
1.58
0.76
0.44
1.68
0.72
HS
0.24
0.22
0.42
E
D
0.17
1.11
0.16
1.10
0.38
1.33
0.95
0.95
0.86
25
1.3.3 Water quality classification

The mean values of nutrient and heavy metal concentrations for the period 2003-2005
adjacent to our sampling sites are summarized in Table 1.2. Nutrients such as ammonium-N,
nitrite-N, ortho-phosphate and total phosphorus were lowest at the downstream site. Similarly,
concentrations of copper (Cu) and lead (Pb) in the upper Danube sites were higher in the
period 20042005 (ICPDR, 2008b), whereas nearly no difference occurred for Cd. Results
obtained from the second Joint Danube Survey (JDS2) performed in autumn 2007 revealed
the same pattern of pollution and eutrophication parameters between the sampling sites
(ICPDR, 2008a). Accordingly, no significant change in nutrient and heavy metal levels
occurred during our sampling period. Although no taxa lists are available for
macrozoobenthos communities all sampling sites were categorised to class II according to the
saprobic index (ICPDR, 2002).
1.4 Discussion
The composition of endoparasite communities at the investigated Danube sites were
principally similar but showed differences which can be attributed to the local ecological
conditions. In general, ten endohelminth species were identified, none of which is a barbel
specialist. Two of three parasite species recorded for the first time for barbel were considered
as cases of accidental infection. Larvae of Hysterothylacium sp. were found in the intestine of
a barbel collected at the sampling site Kozloduy. The fish most likely acquired this infection
while feeding on crustacean, fish intermediate or paratenic hosts. Various small fishes and
invertebrates serve as obligate intermediate or paratenic hosts for the nematodes third stage
larvae (Moravec, 1994). Some authors suggest that large barbels feed also on small fishes like
bullhead or gudgeon (Moravec et al. 1997). A closer look into the digestive system of barbels
during dissection confirmed small fishes as part of the diet, especially gobiid specimens
(Gobidae) were recovered.
The infection with the acanthocephalan L. plagicephalus observed at the sampling site Vidin
was based on a single well developed male, found in the gut of a fish sampled in the summer
of 2007. The definitive hosts of L. plagicephalus are sturgeons (Acipenseridae) and its
distribution is restricted mainly to Ponto-Caspian basins and drainages including the Danube
river basin, where diverse sturgeon species inhabit. Like its host, L. plagicephalus is
euryhaline, however it has a fresh water life cycle (Skryabina, 1974). The latter suggests that
the barbel might have ingested an intermediate host, infested with this particular
26
acanthocephalan.
In contrast to the single findings of Hysterothylacium sp. and L. plagicephalus the nematode
Eustrongylides sp. occurred with high prevalence and intensity at all sites. Highest infection
rates were usually observed in bigger fish, as they feed on small fishes which are used as
second intermediate hosts for Eustrongylides sp. The barbel serves as a paratenic host for
Eustrongylides sp., similar to other species of the family cyprinidae (Moravec, 1994). The
parasites were located in the anterior part of the body cavity, mainly on the serosa of the
intestine and in the liver tissue. In most cases, the larvae were surrounded by a capsule,
forming a spiral granuloma, as described by Mihalca et al. (2007a). Simultaneously, free
moving nematodes were found, which appeared to cause massive histological damage such as
penetrations of the cavity wall and disruptions of inner organs. Infection with nematodes of
the genus Eustrongylides was recorded from water dwelling reptiles (Reptilia) from different
localities in Romania and from the Danube delta region as well. This parasite occurred with
high prevalence and intensity in dice snake (Natrix tessellata), sampled in the period 2002
2006. The grass snake (Natrix natrix) was described as a new host of Eustrongylides excisus
(Mihalca et al. 2007b).
The dominant parasite species at all sampling sites was the acanthocephalan P. laevis. Similar
results were obtained in the upstream part of the Danube River (Moravec et al. 1997;
Schludermann et al. 2003; Laimgruber et al. 2005). However, the parasite list of B. barbus
published by Margaritov (1966) and Kakacheva- Avramova (1977) for the Bulgarian section
of the Danube River differs greatly from the parasite fauna detected in the present study.
During our study period no cestodes were recovered, although Margaritov (1966) and
Kakacheva- Avramova (1977) reported three cestode species (Caryophyllaeus brachycollis,
C. laticeps, C. fennica) for barbel. The absence of cestodes during our sampling period could
be explained with high P. laevis infection levels, which result from the barbels preferred diet
consisting of amphipods and small fishes. The feeding habits of barbel and its diet are
influenced by the available local invertebrate fauna, which itself is determined by the water
quality and habitat composition. A major characteristic of the principal invertebrate fauna in
the Danube River is the high abundance of gammarids, from which some species are known
to be appropriate intermediate hosts for P. laevis (Rumpus and Kennedy, 1974; Marshall,
1976; Moravec and Scholz, 1991; Dezfuli et al. 2000). Preferred feeding of fish on
amphipods results in high abundance of P. laevis, which obviously reduces the diversity of
parasite communities (Kennedy et. al. 1986; Moravec et. al. 1997).
The second most frequent parasite at all Danube localities, R. hellichi, occurred at the
sampling site Vidin with a prevalence of 24.2%. The prevalence was about two times lower
27
compared to the data obtained from the other two sampling sites. According to Moravec and
Scholz (1995) trichopteran larvae from the genus Hydropsyche serve as intermediate hosts for
the transmission of R. hellichi (see e.g. Moravec, 1995). Thus, the lower prevalence at Vidin
can be explained with a lower abundance of the intermediate host, which could be correlated
to a higher eutrophication and pollution level in this part of the river. The larvae of
Hydropsyche sp. are well established indicators which are used to assess the water quality
(Moog, 1995). For example, the saprobic index of trichopteran larvae varies between 2.1 and
2.3 and corresponds to water quality class 2.
Moreover, the prevalence recorded for the nematode Eustrongylides sp. in Vidin was the
highest at all sampling sites. This supports the pollution hypothesis, since the first
intermediate host described for Eustrongylides sp. are aquatic oligochaetes such as
Lumbriculus variegatus (Lumbriciidae), Tubifex tubifex and Limnodrilus sp. (Tubificidae)
(Moravec, 1994). All these oligochaete species indicate disturbed aquatic habitats (saprobic
index over 3, pollution with chemicals) where they are highly abundant.
The results of the present study correspond very well with data of Valtonen et al. (1997) who
also correlated the occurrence of single parasite species in fish with the abundance of
intermediate hosts from lakes with differences in trophic status and degree of pollution. For
example the acanthocephalan Acanthocephalus lucii showed the highest prevalence in perch
(Perca fluviatilis) from a eutrophic and polluted lake. The intermediate host of A. lucii,
Asellus aquaticus, is known as pollution tolerant and is highly abundant under contaminated
conditions (Murphy and Learner, 1982). Not only the occurrence of a single parasite species
can be related to environmental parameters but also the composition and diversity of whole
parasite communities is determined by environmental conditions such as eutrophication,
pollution and changes in substrate composition. These conditions can either directly affect the
parasite (e.g. toxic effects on free-living stages) or indirectly by affecting the abundance and
distribution of the respective intermediate and final hosts (Sures, 2004a). Evidence from the
field revealed the composition of fish helminth communities being largely dependent on the
benthic invertebrate fauna, which itself is directly dependent on water quality and benthic
habitats (Sures and Streit, 2001; Laimgruber et al. 2005; Thielen et al. 2007).
In the present study the lowest value for the Brillouin index and the Shannon-Wiener diversity
was recorded for Vidin. As parasite diversity is considered a measure of ecosystem health
(Hudson et al. 2006), the higher diversity at Silistra gives evidence for better environmental
conditions in the lower river stretch. This is confirmed by hydrochemical data, which indicate
a higher level of pollution and eutrophication at Vidin compared to Silistra. Eutrophication
might favour the occurrence of intermediate hosts known to be tolerant against high nutrient
28
concentrations such as annelids and crustaceans. Additionally, the presence of toxic metals
supports the occurrence of parasites transmitted by anneldids or crustaceans for example by
compromising the immune system of the definitive host. Thus, the combined effects of high
nutrient and pollutant concentrations represent favourable ecological conditions especially for
the dominant occurrence of P. laevis. This dominance also negatively affects infracommunity
and component community diversity as it leads to lower values for the Shannon-Wiener and
Simpson index. Our results therefore give good evidence that aquatic ecosystem health could
be assessed by investigating the composition and diversity of fish parasite communities,
which also due to their position in food webs (Lafferty et al. 2008) represent an
integrative measure of the overall ecological conditions.
2 Is metal accumulation in Pomphorhynchus laevis dependent on

parasite sex or infrapopulation size?
2.1 Introduction
The increasing industrialization and enhancement of human activities leads to rising levels of
contaminants in aquatic habitats. This requires a permanent monitoring of the presence and
effects of pollutants. For detecting chemicals in aquatic ecosystems, analytical methods are
established and different groups of bioindicators such as bivalves are available (Arndt et al.
1987; Reeders et al. 1993; Gunkel, 1994). Besides established free-living sentinel species,
recent studies suggest that fish parasites might also be useful as monitoring organisms for the
detection of chemical pollution (Sures, 2003). Due to their excellent ability to accumulate
different substances, intestinal parasites, especially fish acanthocephalans, have been
suggested as suitable bioindicators for metal pollution (summarized in Sures, 2003, 2004a;
Vidal-Martinez et al. 2010). The accumulation capacity of acanthocephalans has been shown
to exceed even that of established free living sentinel organisms such as the zebra mussel,
Dreissena polymorpha (see Sures et al. 1997a, 1999b). Accordingly, fish-acanthocephalan
systems represent promising monitoring tools for the detection of chemical pollution in
aquatic systems, especially if contaminant levels are low, e.g. in pristine areas such as the
Antarctic (Sures and Reimann, 2003). For practical reasons the fish host must be widely
distributed and easy to be sampled (Kennedy, 1997) in order to use endoparasites and their
hosts as bioindicators. Moreover, the parasites have to be highly abundant and prevalent in
their host species (Sures, 2004a). Promising host-parasite systems for freshwater habitats are
cyprinid fish species such as barbel, Barbus barbus, infected with the palaeacanthocephalan
Pomphorhynchus laevis (see Sures, 2004b). Different benthic crustacean species serve as
intermediate host for P. laevis (Rumpus and Kennedy, 1974). As they form a substantial part
of the diet of benthivore fishes, high infection intensities are commonly described for fish
species such as barbel.
Although several aspects of the mechanism and kinetics of metal uptake in acanthocephalans
have been elucidated by Sures (2001), there are still some open questions concerning the
applicability of fish parasites as sentinels. For example, the effect of the acanthocephalan
infrapopulation size on the process of metal accumulation in fish-parasite systems is not
known. Some authors reported that fish parasites are able to reduce element concentrations in
their host tissues (Sures and Siddall, 1999), which might correlate with the infrapopulation
size (Thielen et al. 2004). Moreover, there are no data available concerning metal
2 Is metal accumulation dependent on parasite sex or infrapopulation size?
30
accumulation in relation to the sex of fish acanthocephalans, which could also play a
considerable role. Indeed, several metabolic pathways are different according to gender in
acanthocephalans (Crompton and Nickol, 1985), and previous studies on experimental
infections of terrestrial mammals with acanthocephalans provided contradictory results on
metal accumulation according to parasite sex (Scheef et al. 2000; Sures et al. 2000a,b).
The field study presented in this chapter was designed to address these aspects using the fresh
water cyprinid Barbus barbus. Barbel is the second largest cyprinid species in Europe and it is
wide spread throughout large river systems. It is well known for its high infection levels with
the acanthocephalan Pomphorhynchus laevis in the Danube River (Kakacheva-Avramova,
1962, 1977; Margaritov, 1959, 1966; Moravec et. al. 1997; Schludermann et al. 2003; Thielen
et al. 2004; Laimgruber et al. 2005; Nachev and Sures, 2009). Accordingly, the model system
B. barbus- P. laevis was taken for studying the differences in accumulation with respect to
infrapopulation size and the sex of the acanthocephalan.

2.2.1 Sample collection
A total of 27 barbels were collected in September 2006 from local fishermen in the area of
Kozloduy, Bulgaria, around river kilometer 685 of the Danube River. After capture, the fish
were frozen and transported to the laboratory, where length, weight, sex and age were
determined for each fish. The specimens were dissected using standard parasitological
techniques. Tissue samples from fish (muscle, intestine and liver) and parasites were taken
using stainless steel dissecting tools, which were previously cleaned with 1% ammoniumEDTA solution and double- distilled water to avoid contamination. The sample material was
rinsed with physiological solution (0.8% NaCl suprapure) and frozen at -20C until metal
analyses. After collection and identification of the acanthocephalans the following
parasitological parameters were calculated according to Bush et al. (1997) - prevalence
(P, %), intensity range (IR), abundance (A) and mean intensity (MI) of the infection. The fresh
weight and sex (distribution of and ) of the collected acanthocephalans was also
recorded.
Fish were divided into groups according to their infection status or sex. The first group
(lightly infected group, LI) comprised barbels (n=9) infected with less then 20
acanthocephalans. For the second group (heavily infected group, HI) fish (n=9) showed a
mean infection intensity exceeding 100 worms. Another group consisted of fish (n= 8) with an
31
infection intensity around the mean value (80.2 worms per fish) obtained for all collected
fishes. This group was used to compare a sex specific metal accumulation.
2.2.2 Molecular identification of Pomphorhynchus laevis

While the parasite species was identified as P. laevis using morphological traits, the
possibility of co-infection with other closely related acanthocephalan species such as
Pomphorhynchus tereticollis persists, due to their similarity in morphology, biology and
transmission (Perrot-Minnot, 2004). Therefore, a molecular method was used for parasite
identification. DNA was extracted from 200 parasites taken randomly from different fishes
following the procedure described by Franceschi et al. (2008). Species identification was
performed using a diagnostic PCR to amplify a portion of the internal transcribed spacer (ITS)
rDNA gene (see Franceschi et al. 2008 for primers and PCR conditions). For each PCR
reaction, one negative (reaction solution without template DNA) and three positive controls
(template DNA from already-identified P. laevis, P. tereticollis and Polymorphus minutus)
were carried out. The sizes of PCR products determined the parasite species (Franceschi et al.
2008): the products for P. laevis, P. tereticollis and P. minutus were 320, 350 and 290 bp,
respectively (Figure 2.1). The analyses revealed that all acanthocephalans were P. laevis,
which confirmed the absence of co-infection by different acanthocephalan species (Figure
2.1).
32
Figure 2.1. Molecular identification of acanthocephalan species, according to the size of PCR
product of the partial ITS sequence. The central line is the molecular weight marker.
P. l. Pomphorhynchus laevis; P. m. Polymorphus minutus; P. t. Pomphorhynchus
tereticollis;
2.2.3 Heavy metal analysis

The fish and parasite samples were prepared for analysis using microwave assisted digestion
following the procedure described by Zimmermann et al. (2001). Up to 300 mg (wet weight)
of sample, previously homogenized, was weighed and placed into 150 ml perfluoralkoxy
(PFA) vessels, into which a mixture of 1.3 ml nitric acid (65% HNO3, suprapure) and 2.5 ml
hydrogen peroxide (30% H2O2, suprapure) was added. Subsequently, the vessels were heated
for 90 min at about 170C using the microwave digestion system MDS-2000 (CEM GmbH,
Kamp-Lintfort, Germany). After digestion the clear sample solution was brought to volume
with doubly distilled water in a 5 ml volumetric glass flask and kept in polypropylene sample
tubes until analysis.
The concentrations of arsenic (As), cadmium (Cd), colbalt, (Co), copper (Cu), iron (Fe),
manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), tin (Sn), vanadium (V) and zinc
(Zn) were analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The
analyses were carried out with a quadrupole ICP-MS system (Perkin Elmer - Elan 5000)
33
operating at 1100 W plasma power, 13.3 L/min plasma gas flow, 0.75 L/min auxiliary gas
flow and 0.95 L/min nebuliser gas flow and an auto sampler system (Perkin Elmer AS-90)
connected with a peristaltic pump with a sample flow of 1 ml/min. To avoid contamination
and memory effects the wash time between measurements was set at 10 seconds (with 1%
HNO3, suprapure). Before analyses, the samples were diluted 1:10 using a solution of 1%
HNO3 (suprapure) with a concentration of 10 ng/L of yttrium (Y) and thulium (Tm) as
internal standards. In order to control the accuracy and stability during measurements a
standard solution (ICP Multielementstandard V solution, Merck, Darmstadt, Germany) was
analyzed after every 10 samples.
The calibration was carried out with a series of 11 dilutions of a standard solution (ICP
Multielementstandard solution, Merck, Darmstadt, Germany). Element concentrations were
calculated as mg L-1 using corresponding regression lines (correlation factor 0.999). To
check the accuracy of the analytical procedure, standard reference material (DORM-3,
National Research Council, Canada) of dogfish (Squalus acanthias) was analyzed and the
values of 10 certified elements were checked. Detection limits for the investigated elements
were calculated as the three fold standard deviation of concentrations found in 12 procedural
blanks.
2.2.4 Data analyses and statistical treatment

Bioconcentration factors were calculated according to Sures et al. (1999a) as follows:
(C[P.laevis] / C[host
tissue]).
If tissue concentrations for one element were below the respective
detection limit, the detection limit was used to calculate the bioconcentration factor.
As our data did not meet conditions for parametric analyses, even after transformation, non
parametric tests were applied. For comparisons of element concentrations in tissues and P.
laevis between heavily infected and lightly infected barbels a Mann-Whitney U-test was used
with a significance level of p 0.05. Wilcoxon matched pair test was applied to determine
differences between element concentrations of females and males as well as between fish
tissues and the parasites. All statistical tests were performed using STATISTICA 6.0.
34
2.3 Results
2.3.1 Fish samples
The mean ( S.D.) weight and size of the barbels was 376 139 g and 34.6 4.9 cm,
respectively. The age varied between 2 and 5 years, whereas most fishes were 3-4 years old.
Only one out of all collected fish was not infected with the acanthocephalan P. laevis. This
fish was not considered in the following analyses. As expected, the parasite occurred with a
high level of infection (P 97.1%, MI 80.2 and A 77.9).
2.3.2 Analytical procedure

Detection limits and mean concentrations of elements in the reference material (DORM-3) are
listed in Table 2.1. For eight metals present in the standard reference material accuracy rates
ranging between 87% to 106% were obtained with the highest accuracy for iron (100%).
Table 2.1. Trace metal concentrations in Dogfish Muscle Certified Reference Material
(DORM 3), accuracy and detection limits determined by ICP-MS analyses.
Element
DORM-3 values
SD (mg/kg)
As
6.88 0.3
Cd
0.29 0.02
Co
n.c.
Cu
15.5 0.63
Fe
347 20
Mn
n.c.
Mo
n.c.
Ni
1.28 0.24
Pb
0.395 0.05
Sn
0.066 0.012
V
n.c.
Zn
51.3 3.1
n.c.: element not certified
DORM-3 measured
SD (mg/kg)
6.30 0.4
0.27 0.02
16.35 0.93
346.95 28.24
1.21 0.15
0.417 0.04
0.0067 0.010
44.4 3.2
Accuracy
(%)
92%
94%
105%
100%
94%
106%
102%
87%
Detection limit (g/L)

0.008
0.01
0.009
0.19
2.76
0.1
0.02
0.47
0.26
0.01
0.01
2.77
35
2.3.3 Element concentrations in barbel and Pomphorhynchus laevis

The concentrations of the five elements As, Cd, Cu, Pb and Zn were found to be significantly
higher in the acanthocephalan P. laevis when compared with the host tissues (Figure 2.2 and
Table 2.2). With the exception of Sn, the levels of all other metals were higher in the parasite
than in the muscle of barbel. The element Sn was below or close to the detection limit in the
parasites, whereas the levels in the host were significantly higher.
Figure 2.2. Mean ( SD) element concentrations (a-c) in organs of barbels and its intestinal
parasite Pomphorhynchus laevis. *Concentrations of Sn in P. laevis samples are not displayed
as they were below the detection limit.
36
Comparisons of metal concentrations among the fish tissues showed only one clear pattern:
the concentrations of all elements were lowest in the muscle (with the exception of Sn). Else,
some elements such as Cu, Mo and Zn, were present at higher concentrations in the liver;
others such as Co, Mn and Pb were present at significantly higher levels in intestinal samples.
Table 2.2. Differences between element concentrations in barbel organs and Pomphorhynchus
laevis.
Element
P.l. M
P.l. I
P.l. L
MI
As
P.l**
P.l**
P.l.**
I**
Cd
P.l.**
P.l.**
P.l.**
I**
Co
P.l.**
n.s.
P.l.**
I**
Cu
P.l.**
P.l.**
P.l.**
I**
Fe
P.l.**
n.s.
L**
I**
Mn
P.l.**
n.s.
P.l.**
I**
Mo
P.l.**
n.s.
L*
I**
Ni
n.s.
I**
n.s.
I**
Pb
P.l.**
P.l.**
P.l.**
I**
Sn
n.t.
n.t
n.t
I*
V
P.l.**
n.s.
n.s.
I**
Zn
P.l.**
P.l.**
P.l.**
I**
M: Muscle; I: intestine; L: liver; P.l.: Pomphorhynchus laevis
ML
L**
L**
L**
L**
L**
L**
L**
n.s.
L**
L**
L**
L**
LI
n.s.
n.s.
I**
L**
n.s.
I**
L**
n.s.
I*
n.s.
n.s.
L**
*: significant at p 0.05 (Wilcoxon matched pair test)

**: significant at p 0.01 (Wilcoxon matched pair test)
n.t.: not tested as concentration in Pomphorhynchus laevis was below the detection limit.
n.s.: not significantly different (Wilcoxon matched pair test)
In case of significant difference, the site for higher concentration is given in each cell.
The mean bioconcentration factors revealed, that 8 elements (As, Cd, Co, Cu, Mn, Pb, V, Zn)
were overall present in higher levels (BCF>1) in the parasites (Table 2.3). The metal
accumulation capacity of P. laevis with respect to host muscle in decreasing order was as
follows: Pb> Cd> Cu> Zn>As> Mn> Co> V> Mo> Ni. Nearly the same pattern was observed
for the intestine and liver, with Pb> Cd> Cu> Zn> As> Mn> Co> V for the intestine and
Pb> Cd> Cu> Zn> As> Mn> Co> Ni> V, for the liver. The remaining elements were detected
only in low concentrations in the parasite samples (see Table 2.3). The mean concentration
factors were found to be up to 1070 times higher for Pb and 195 times higher for Cd
compared to the host tissues. The ratios for As, Cu and Zn showed the same tendency with the
highest mean values of 12, 95 and 32, respectively.

Table 2.3. Bioconcentration factors C[P.laevis] / C[barbel
tissue]
37
for Pomphorhynchus laevis
calculated with respect to different host tissues.

Element
Muscle
Intestine
Liver
C[P.laevis] / C[Muscle] SD
C[P.laevis] / C[Intestine] SD
C[P.laevis] / C[Liver] SD
As
12.0 ( 9.5)
4.5 ( 3.3)
3.8 ( 3.3)
Cd
194.8 ( 142.8)
22.3 ( 11.9)
23.4 ( 18.7)
Co
8.9 ( 4.6)
1.2 ( 0.7)
2.6 ( 0.8)
Cu
94.7 ( 66.2)
17.8 ( 11.0)
9.6 ( 10.6)
Fe
4.9 ( 3.04)
0.7 ( 0.5)
0.7 ( 0.3)
Mn
22.9 ( 16.2)
1.7 ( 1.6)
5.4 ( 2.6)
Mo
4.7 ( 3.1)
0.9 ( 0.5)
0.3 ( 0.4)
Ni
1.9 (2.2)
0.5 ( 0.3)
2.2 ( 2.1)
Pb
1070.5 ( 781.8)
81.7 ( 88.5)
433.4 ( 602.4)
Sn
n.d.
n.d.
n.d.
V
4.8 ( 3.0)
0.7 ( 0.5)
1.2 ( 0.9)
Zn
32.2 ( 34.0)
10.5 ( 10.8)
6.4 ( 6.9)
n.d.: concentrations for Pomphorhynchus laevis below detection limit
2.3.4 Accumulation differences with respect to parasite infra population size and sex
In general, comparisons between the element concentrations of lightly and heavily infected
barbels showed no significant differences (Table 2.4). Concerning the parasites, the only
significant differences were found for V, with higher levels in the heavily infected group. Fish
liver of this group also contained significantly higher vanadium amounts (see Table 2.4). In
contrast, the concentrations of Cd detected in the intestinal tissue were significantly higher in
the lightly infected group (Table 2.4).
Comparisons of bioconcentration factors calculated for the metals present in significantly
higher levels in P. laevis (As, Cd, Cu, Pb, Zn), showed no differences with respect to
infrapopulation size. Lightly infected fishes displayed more variation in the values obtained
for muscle tissue. However, the pattern of distribution, as well as the ratio ranges for other
organs was similar (see Figure 2.3 and Figure 2.4).
When comparing element concentrations with respect to parasite sex, similar values were
found for females and males of P. laevis. Only the essential elements Zn and V were detected
in significantly higher concentrations in the females (see Table 2.4).
38
Table 2.4. Differences in element concentrations between heavily infected (HI) and lightly
infected (LI) barbels, as well as between male and female Pomphorhynchus laevis.
Element P.l.(HI) P.l.(LI)a P.l. P.l.b M(HI) M(LI)a
As
n.s.
n.s.
n.s.
Cd
n.s.
n.s.
n.s.
Co
n.s.
n.s.
n.s.
Cu
n.s.
n.s.
n.s.
Fe
n.s.
n.s.
n.s.
Mn
n.s.
n.s.
n.s.
Mo
n.s.
n.s.
n.s.
Ni
n.s.
n.s.
n.s.
Pb
n.s.
n.s.
n.s.
Sn
n.t.
n.s.
n.s.
V
P.l.(HI)*
P.l.**
n.s.
Zn
n.s.
P.l.*
n.s.
M: muscle; I: intestine; L: liver; P.l.: Pomphorhynchus laevis
*: significant at p 0.05
**: significant at p 0.01
a
: Mann-Whitney U-test
: Wilcoxon matched pair test
n.s.: not significantly different

n.t.: not tested
I(HI) I(LI)a
n.s.
I(LI)*
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
L(HI) L(LI)a
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
L(HI)*
n.s.
39
Figure 2.3. Comparisons of the ratios C[P.laevis] / C[organ barbel] obtained for the toxic elements
arsenic, cadmium and lead between heavily and lightly infected barbels. Dots are medians,
boxes are interquartile ranges and error bars are interdecile ranges.
Figure 2.4. Comparisons of the ratios C[P.laevis] / C[organ
barbel]
40
obtained for the essential
elements copper and zinc between heavily and lightly infected barbels. Dots are medians,
boxes are interquartile ranges and error bars are interdecile ranges.
2.4 Discussion
As expected and previously reported in various studies on metal accumulation in the hostacanthocephalan system (reviewed by Sures, 2003, 2004b), many of the analyzed elements
were found in higher concentrations in the acanthocephalan P. laevis, compared to its hosts
tissues. Some toxic and essential elements such as As, Cd, Cu, Pb and Zn were present at
significantly higher levels in the parasite. The same results were obtained by Schludermann et
al. (2003) for Cd, Pb and Zn from a field study in the Austrian part of Danube River. The
bioconcentration factors were quite similar in both studies, with the exception of Pb, which
was found to be much higher accumulated in P. laevis in the present study. The present lead
concentrations confirmed previous results of an investigation on chub naturally infected with
P. laevis from the River Ruhr in Germany (Sures et al. 1994a). In the latter study mean Pb
41
values were found to be up to 2700 times higher in the parasite compared with muscle and
770 and 280 times higher than liver and intestine, respectively. The mean Pb concentrations in
the fish organs were similar to those obtained during the present investigation, only the
amounts detected in the parasite were higher. This discrepancy could be due to differences in
lead levels of the river Ruhr and Danube. For example, the average concentration of lead in
the water of the river Ruhr was 5 g/L (Umweltbundesamt, 1992) whereas Pb concentrations
in the Danube river near the fish sampling site varied between 2.3 and 2.8 g/L for the period
of 2003-2005 (TNMN, 2009). The mean bioconcentration factors calculated with respect to
fish muscle differ in the same order as the water concentrations. Whereas the levels in host
tissues were comparable, the acanthocephalans showed a higher Pb accumulation. This
supports the use of P. laevis as an accumulation indicator due to its sensitive and linear
response to aqueous metal concentrations, which was also demonstrated in laboratory
exposure studies (Sures, 2004b).
With respect to parasite infrapopulation size, the study revealed that the intensity of infection
usually plays no significant role for metal accumulation in the host-parasite system. Almost
all elements were found in both groups (LI and HI) in similar concentrations in the parasite
and host tissues. Accordingly, in general metal accumulation is not influenced by the parasite
infrapopulation size. The only significant difference was found for the element vanadium in
P. laevis. Recently, laboratory studies demonstrated that infections with acanthocephalans can
reduce heavy metal burdens in the host (Sures and Siddall, 1999; Sures et al. 2003) when
comparing uninfected fish with infected conspecifics. Due to a lack of a sufficient number of
uninfected fish in the present study, these laboratory findings could not be proven under field
conditions.
A further aspect needing clarification in order to use acanthocephalans as accumulation
indicators is the evaluation of possible effects of parasite sex on metal accumulation. Until
now, there are only few studies based on acanthocephalans of terrestrial mammals that
provide data on differences in element accumulation patterns between male and female
worms. Scheef et al. (2000) and Sures et al. (2000b) reported higher concentrations of lead
and cadmium in females of the acanthocephalan Moniliformis moniliformis in experimentally
infected and subsequently metal exposed rats. In contrast, element levels analyzed by Sures et
al. (2000a) in pigs naturally infected with Macracanthorhynchus hirudinaceus showed the
opposite tendency- male worms contained higher concentrations for each element.
Accordingly, the low number of publications and the contradictory results do not allow
general conclusions. Additionally the mechanism of metal uptake in the fish-acanthocephalan
system differs from that of terrestrial mammals. According to previous studies,
42
acanthocephalans in freshwater fish are exposed to bile bound metals in the small intestine
(Sures and Siddall, 1999), after metals were taken up by fish gills, transported into the liver
and then excreted with the bile into the intestine (Hofer and Lackner, 1995; reviewed by
Sures, 2001). In contrast, excretion of metals in mammals occurs mainly through the kidneys
(Merian, 2004). Therefore data from studies on terrestrial mammals might be completely
different from results obtained from fish hosts. Considering the results of the present chapter,
no clear evidence exists for a possible impact of parasite sex on heavy metal uptake. The only
metals found at significantly higher concentrations in females were V (at p < 0.05) and Zn (at
p 0.05), from which at least the latter element can be considered essential and is therefore
regulated by the fish. Concentrations of other elements and especially those of potential
importance in metal monitoring surveys were not significantly different when comparing both
sexes.
The absence of differences for most element concentrations with respect to parasite sex as
well as with respect to infrapopulation size together with the enormous accumulation capacity
underline the possible use of P. laevis as an excellent sentinel for metal pollution. The
importance of detecting metals with toxic effects on biota persists due to their adverse effects
on the functionality of aquatic ecosystems (Merian, 2004). Furthermore, the EC-Water
Framework Directive (WFD, 2000) has the objective of a good chemical quality status for all
European waters. Accordingly, pollutant levels in surface waters have to be monitored to
decide if their concentrations meet environmental quality standards. In order to use hostparasite systems as bioindicators to detect metal pollution, the contribution of all possible
factors, which might affect metal uptake in the system should be elucidated in detail. In this
context the present chapter evaluated possible influences of parasite sex and infrapopulation
size and the results re-verified the acanthocephalan P. laevis as a highly suitable accumulation
indicator, which meets all criteria suggested for ideal sentinels (see Beeby 2001; Sures,
2004a).
3 Seasonal
differences
of
metal
accumulation
in
Pomphorhynchus laevis and its definitive host Barbus barbus

3.1 Introduction
Despite a large number of studies on metal accumulation in different host-parasite systems
(Vidal-Martinez et al. 2010), information about the influence of possible seasonal dynamics
on the metal uptake by parasites is still missing. Usually, authors focused on studying the
kinetics and metabolism of different heavy metals (Sures and Siddall, 1999; Sures et al. 2003)
or concentrated on the parasites accumulation capacity (reviewed by Sures 2001, 2004a).
However, a seasonal variation of metal concentrations in parasites might exist, connected with
the parasites transmission cycle during the year. The development of fish acanthocephalans,
especially of paleacanthocephala, is characterized by typical annual infection patterns of
intermediate and definitive hosts (Kennedy, 2006). These patterns can be related to water
temperature changes and are, therefore, season and climate dependent (Kennedy, 1985).
Moreover, the lifespan of most fish acanthocephalans in the intestine of the definitive host (in
which metal uptake of the parasites occurs) does usually not exceed a period of some months
(Kennedy, 2006). Regarding the life cycle of Pomphorhynchus laevis it is obvious that its
development in the gut of the definitive host is formally separated into two phases, which
belong to different seasons of the year. The first phase begins immediately after the infection
of the fish and is characterized by an enormous growth of the worms. It continues until the
parasite is fully mature. The second phase covers the time from the release of eggs until the
death of the acanthocephalan. Therefore, a seasonal analysis of metal concentrations in a hostparasite system would help us to elucidate key aspects of the metal uptake process. For
example, metal accumulation in host-parasite systems could be affected by the age structure
of the parasite infrapopulation or by seasonal changes of host physiology during the year. The
latter might be a result of differences in fish activity and metabolism during the cold season
(winter). Changes in host activity are expected to have a considerable impact on the
physiology of the parasite. Although only few data are available for longevity, fecundity and
patency periods, it is known that fish acanthocephalans are able to survive seasonal periods of
host starvation in contrast to acanthocephalans from homeothermic hosts such as mammals
(Kennedy 2006).
All above mentioned aspects have to be considered if parasites such as acanthocephalans are
taken for metal monitoring purposes. Additionally, following the seasonal dynamics of heavy
metal concentrations could be also a good approach for rough estimation of lifespan of
3 Seasonal differences of metal accumulation in host-parasite system
44
P. laevis in its definitive host. Such information is still missing as it is often impossible to
examine the fish faeces qualitatively and quantitatively (Kennedy 2006).
The aim of the present chapter was to analyse if a seasonal pattern of metal distribution in a
host-parasite system exists. The fresh water cyprinid Barbus barbus and its intestinal parasite
P. laevis were taken as a model system, since they are already described as an appropriate
system for environmental metal monitoring (Sures 2004b).

3.2.1 Fish samples
Barbels were collected in a seasonal manner (spring, summer, autumn) during the year 2006.
The fish were caught by local fishermen near to the town Kozloduy, which is situated at river
kilometer 685 of the Danube River. After killing, fish were kept frozen until parasitological
examination in the laboratory.
For metal analyses eight barbels from each season were selected with similar morphological
characteristics such as body size and weight (see Table 3.1). Moreover fish with extremely
hight or low levels of infection were excluded in order to reduce the possible effects of
different infrapopulation sizes even though the intensity of infection plays no considerable
role in the metal uptake process (for details see Chapter 2), at least it has no effects on the
element concentrations in worms.
The fish were dissected using standard parasitological techniques, whereas the dissecting
tools were previously cleaned with 1% ammonium-EDTA solution and double-distilled water
to avoid contamination. The acanthocephalans as well as the fish tissues (muscle, intestine
and liver) were placed in plastic tubes and were kept frozen until metal analyses.
Table 3.1. Morphological data of barbel.

Weight SD (g)
Total Lenght SD (cm)
Standard Lenght SD (cm)
Body Height SD (cm)
Condition Factor SD
Spring (n=8)
430.9 ( 86)
36.5 ( 2.7)
29.9 ( 2.2)
7.2 ( 0.2)
0.88( 0.09)
Summer (n=8)
525 ( 243)
37.4 ( 6.3)
31.1 ( 5.3)
7.6 ( 1.2)
0.95 ( 0.09)
Autumn (n=8)
373.8 ( 134.7)
35.5 ( 7.2)
28.4 ( 4.2)
6.9 ( 0.9)
0.86 ( 0.22)
45

Up to 300 mg (wet weight) of fish tissue (muscle, intestine and liver) and parasite samples
were weighed and prepared for metal analysis using a microwave assisted digestion following
the procedure described by Zimmermann et al. (2001). After digestion the clear sample
solutions were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) and
the concentrations of arsenic (As), cadmium (Cd), colbalt, (Co), copper (Cu), iron (Fe),
manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), vanadium (V) and zinc (Zn) were
determined (for details see Chapter 2).

Collected acanthocephalans were counted and the wet weight of each infrapopulation was
recorded. The mean individual weight was calculated after dividing the total infrapopulation
weight by the total number of worms.
For the comparisons of metal concentrations among the host tissues and the parasites
arithmetic means with standard deviations were calculated. Additionally, in order to express
the accumulation capacity of P. laevis, the mean bioconcentration factors (BCF) for the
different organs were obtained as described by Sures et al. (1999a) - C[P.laevis] / C[host tissue]. The
Wilcoxon matched pairs test was used to compare element concentrations in host tissues and
parasites. The element concentrations between the seasons were compared with the MannWhitney U-test and the t-test. Spearman rank correlation coefficients were determined
between the mean individual weight and the number of parasites with metal concentrations.
3.2.4 Element concentrations in the Danube River

The concentrations of the elements As, Cd, Cu, Pb and Zn in the water are shown in Table 3.2
(ICPDR, 2009). The data represent two Danube sites - one (Novo Selo, 845 km) was situated
upstream from our fish sampling locality and the other one (Iskar, 637 km) was located about
40 river kilometers downstream. The mean concentrations for the years 2005 and 2006 at both
sites were similar, which indicates that the concentrations at site Kozloduy might not differ as
well. The data also showed no clear sings for some incidental contamination (hot spots) with
metals in the period of 2005-2006.
46
Table 3.2. Element concentrations in water at two different sites from the Danube River in
Bulgaria. (ICPDR, 2009)
Elements
(g/L)
Novo Selo (845 km)

(160 km upstream of Kozldoduy)
2005
2006
As
2.2
2.275
Cd
0
0
Cu
17.5
23.5
Pb
0*
0
Zn
22.0
20.92
* Data regards the second half of the year
Iskar (637 km)

(40 km downstream of Kozloduy)
2005
2006
2.692
2.382
0*
0
5.15
6.1
2.767
2.364
29.73
20.0
3.3 Results
The detection limits of the analyzed elements as well as the obtained concentrations for
reference material (DORM 3) are listed in Table 2.1. The recovery of elements in dog fish
material ranged from 87% to 106%.
3.3.2 Element concentrations in fish tissues and parasite samples

The mean element concentrations obtained from the analyzed fish material are presented in
Table 3.3. The amounts of As, Cd, Cu, Pb and Zn were found to be significantly higher in
P. laevis than in the host tissues. This trend was also clearly visualized by the calculated mean
bioconcentration factors for each tissue (Table 3.4). As expected, the lowest concentrations
were observed in muscle tissue, which corresponded to the highest calculated values of BCF.
In general, the parasites demonstrated their enormous capacity to accumulate lead. The mean
lead concentrations in P. laevis obtained in spring, for instance, were 1194 times higher
compared to those in muscle tissue and 78 and 211 times higher than in intestine and liver,
respectively. Cadmium showed a similar pattern, but with lower bioconcentration factors (see
Table 3.3 and Table 3.4). Concerning the elements As, Cu and Zn, the order differed slightly,
due to the higher metal contents in the liver compared to the intestine.
Reviewing the other analyzed elements, the acanthocephalans demonstrated overall a higher
accumulation capacity compared to the muscle tissue. For other organs such a clear tendency
was not obvious.
47
Table 3.3. Seasonal profile of mean ( SD) element concentrations in different tissues of
barbel and in P. laevis.
Season
As
Muscle
Intestine
Liver
P. laevis
Cd
Muscle
Intestine
Liver
P. laevis
Co
Muscle
Intestine
Liver
P. laevis
Cu
Muscle
Intestine
Liver
P. laevis
Fe
Muscle
Intestine
Liver
P. laevis
Mn
Muscle
Intestine
Liver
P. laevis
Mo
Muscle
Intestine
Liver
P. laevis
Ni
Muscle
Intestine
Liver
P. laevis
Pb
Muscle
Intestine
Liver
P. laevis
V
Muscle
Intestine
Liver
P. laevis
Zn
Muscle
Intestine
Liver
P. laevis
n.d.: concentrations below detection limit
Spring
0.28 (0.25)
0.52 (0.32)
0.67 (0.42)
1.08 (0.54)
0.02 (0.03)
0.24 (0.31)
0.16 (0.22)
2.40 (2.09)
0.02 (0.01)
0.15 (0.08)
0.04 (0.01)
0.07 (0.02)
1.45 (1.36)
7.37 (4.87)
16.92 (11.59)
75.37 (35.43)
13.29 (3.54)
84.55 (42.22)
71.88 (23.11)
76.62 (69.21)
0.37 (0.15)
4.44 (1.48)
1.26 (0.33)
4.67 (0.85)
0.01 (0.004)
0.06 (0.04)
0.21 (0.11)
0.05 (0.02)
1.26 (1.31)
1.58 (0.74)
0.33 (0.22)
2.73 (1.30)
0.01 (0.01)
0.09 (0.03)
0.03 (0.02)
6.83 (4.87)
0.04 (0.01)
0.15 (0.05)
0.12 (0.05)
0.07 (0.01)
4.71 (1.49)
11.39 (3.86)
18.82 (3.37)
63.91 (36.39)
Summer
0.21 (0.15)
0.35 (0.20)
0.54 (0.49)
1.01 (0.60)
0.01 (0.01)
0.11 (0.07)
0.08 (0.04)
1.34 (0.55)
0.02 (0.01)
0.17 (0.14)
0.04 (0.01)
0.10 (0.07)
1.57 (1.29)
4.79 (2.44)
13.40 (8.11)
53.13 (24.13)
10.21 (4.72)
70.92 (17.87)
81.34 (39.42)
41.27 (23.28)
0.35 (0.11)
7.35 (6.37)
1.49 (0.77)
7.64 (5.87)
n.d.
0.06 (0.02)
0.28 (0.16)
0.10 (0.06)
0.66 (0.61)
2.28 (1.16)
0.21 (0.15)
0.55 (0.28)
0.004 (0.003)
0.12 (0.10)
0.03 (0.02)
5.19 (3.74)
0.04 (0.01)
0.29 (0.36)
0.17 (0.16)
0.10 (0.04)
4.76 (1.51)
10.40 (1.16)
19.23 (5.84)
34.83 (16.66)
Autumn
0.19 (0.10)
0.45 (0.17)
0.56 (0.21)
1.92 (1.23)
0.02 (0.01)
0.14 (0.05)
0.16 (0.10)
2.63 (0.58)
0.02 (0.01)
0.19 (0.16)
0.05 (0.03)
0.13 (0.08)
1.00 (0.39)
6.23 (2.85)
11.76 (5.90)
77.40 (35.84)
9.83 (4.10)
118.19 (89.73)
67.94 (29.24)
37.59 (10.37)
0.45 (0.42)
13.84 (14.64)
1.45 (0.80)
8.94 (6.87)
0.01 (0.01)
0.03 (0.02)
0.13 (0.06)
0.05 (0.06)
0.31 (0.16)
1.60 (0.84)
0.28 (0.15)
0.58 (0.26)
0.01 (0.01)
0.48 (0.53)
0.06 (0.05)
9.81 (4.63)
0.03 (0,012)
0.48 (0.398)
0.14 (0.094)
0.16 (0.069)
3.66 (0.69)
10.14 (1.87)
18.00 (3.96)
91.06 (43.47)
48

Table 3.4. Seasonal profile of bioconcentration factors C[P.laevis] / C[barbel
tissue]
for P. laevis
calculated with respect to different host tissues.

Spring
As
Cd
Co
Cu
Fe
Mn
Mo
Ni
Pb
V
Zn
Muscle
Intestine
Liver
Muscle
Intestine
Liver
Muscle
Intestine
Liver
Muscle
Intestine
Liver
Muscle
Intestine
Liver
Muscle
Intestine
Liver
Muscle
Intestine
Liver
Muscle
Intestine
Liver
Muscle
Intestine
Liver
Muscle
Intestine
Liver
Muscle
Intestine
Liver
Summer
3.8
2.1
1.6
103.3
10.1
15.3
4.4
0.5
1.9
50.7
10.0
4.4
5.8
0.9
1.1
12.8
1.1
3.7
7.0
0.8
0.2
2.2
1.7
8.3
1194.4
78.1
211.3
2.0
0.5
0.6
13.6
5.6
3.4
4.8
2.9
1.9
132.7
11.7
16.0
5.7
0.6
2.3
33.9
11.1
4.0
4.0
0.6
0.5
21.9
1.0
5.1
5.9
1.6
0.4
0.8
0.2
2.6
1250.0
42.2
151.1
2.8
0.3
0.6
7.3
3.3
1.8
Autumn
10.3
4.2
3.4
116.7
18.3
16.8
8.3
0.7
2.8
77.2
12.4
6.6
3.8
0.3
0.6
20.0
0.6
6.2
5.0
1.2
0.3
1.9
0.4
2.1
794.4
20.2
158.1
5.2
0.3
1.1
24.9
9.0
5.1
3.3.3 Seasonal differences in acanthocephalans morphology

The calculated mean individual weight for the parasite infrapopulations demonstrated a clear
annual pattern (Figure 3.1). The mean individual weight obtained in autumn was found to be
significantly lower compared to spring and summer, which indicates that the infrapopulations
in autumn consisted mainly of young preadult individuals. The comparisons between spring
and summer revealed no significant differences, still the acanthocephalan infrapopulations in
summer showed a slightly elevated mean individual weight as expressed by the higher
49
arithmetic mean and median values. Additionally, the correlation analysis between the mean
individual weight and the number of parasites revealed no significant associations.
Figure 3.1. Seasonal profile of the mean acanthocephalan weight. Dots are medians, lines are
means, boxes are interquartile ranges and error bars are interdecile ranges.
3.3.4 Seasonal variation in concentrations of the elements accumulated by P. laevis

All elements (As, Cd, Cu, Pb, Zn) found with significantly higher concentration in the
parasite demonstrated similar seasonal patterns (Figure 3.2). Some of them showed a
negative correlation with the calculated mean individual weight (e.g. Cd and Pb). Following
the annual distribution of these elements, it turned out that they were less concentrated in the
worms collected from the barbels sampled in the summer. On the other hand the highest mean
concentrations were obtained for autumn, whereas the fish in spring shared concentrations in
the range of those obtained for the other seasons (see Table 3.3 and Figure 3.2). Significant
differences were found only for Cd and Pb comparing the levels in summer and autumn. The
slight variation of mean concentrations obtained for the host tissues during the seasons were
also worth mentioning (see Table 3.3).
As mentioned above, the concentration of some elements (e.g. Cd and Pb), which were
accumulated by the acanthocephalans on a higher level, showed a clear relation to the mean
50
individual weight of acanthocephalans. In general, a higher mean individual weight

corresponded to a lower level of Cd (R = -0.466; p < 0.05) and Pb (R = -0.426; p < 0.05).
Such a relationship was not observed for As and the essential elements Cu and Zn.
Figure 3.2. Seasonal pattern of the element concentrations accumulated by P. laevis. Dots are
medians, boxes are interquartile ranges and error bars are interdecile ranges.
51
3.4 Discussion
The obtained results for the element concentrations in the host-parasite system confirmed
similar tendencies described in other metal monitoring studies on acanthocephalans and their
fish hosts. For example in a field study, accomplished by Schludermann et al. (2003) in the
Austrian part of Danube River, the concentrations for Cd, Pb and Zn in P. laevis were
considerably higher compared to the levels in the barbels tissues. Similar results were also
published by Thielen et al. (2004) for fish from the Danube near Budapest after analyzing a
wide number of elements. P. laevis showed an enormous accumulation capacity (e.g. for lead)
in different fish hosts such as chub (Sures and Siddall, 2003). According to all available data,
there is no doubt that the acanthocephalans are very useful in terms of metal indication, due to
their excellent response to the ambient element levels. Hence, the persent results for As, Cd,
Cu, Pb and Zn in P. laevis support again the fact that the acanthocephalans are good sentinel
organisms.
The observed seasonal pattern of heavy metal concentrations in the parasites could not be
related to some incidental contamination (hotspot pollution) in this part of the river. The
concentrations of these elements in the water showed no wide variation in the period of 20052006 (see Table 3.2). In general, the differences in the course of the year could be explained
with the seasonality of the acanthocephalans transmission, more precisely with the stage of
development (maturation) in the gut of the final host. Seasonal aspects in the transmission
were reported for various aquatic parasites, whereas the climate conditions often played the
decisive role. For instance, the oscillations in prevalence of acanthocephalans were assumed
to be a consequence of seasonal fluctuation in the temperature of the habitat (Kennedy, 1985).
According to Kennedy (2006), if there is a possibility of the intermediate host to breed
throughout the year, for instance in some warmer areas like South England, the seasonality of
infection levels in the intermediate and final host may not be clearly expressed, since
gammarids of all sizes are permanently presented. However, he reported that the prevalence
of cystacanths was slightly higher in summer but lower during winter months. The results
published by Hine and Kennedy (1974) for the dace (Leuciscus leuciscus), as a definitive
host, showed no clear seasonal deviation of prevalence, abundance or maturation, while the
fish was able to acquire infection throughout the whole year. In contrast to the dace, the
barbels activity differs strongly in terms of water temperature. Its activity budget decreases
progressively with the decrease of water temperature and decreasing to the thermal limit for
activity (4.0 C) the barbel enters into a dormancy phase (Baras, 1995). The area where the
sampling was conducted, is characterized by a typical continental climate in contrast to South
52
England, whereas the water temperature in winter months (for the period of December to
March) was mostly below or around the barbels thermal limit of activity, the temperature data
was provided by TransNational Monitoring Network database (ICPDR, 2009, Appendix III).
Thus, the reduced fish activity and the correspondingly altered feeding behavior lead probably
to a complete reduction of infection during winter months, while the host stops feeding on
gammarids. The temperature-related alterations in the host behavior are an important factor in
initiating an outbreak of parasites at the beginning of every annual cycle, due to the fact that
the transmission proceeds via a predator-prey relationship (Kennedy, 1985). Furthermore, the
low temperatures affect the feeding behavior and the reproduction activity of the amphipods,
which also may decrease the transmission efficiency of the acanthocephalans. Consequently,
the biology of the intermediate host is another important factor, which plays a considerable
role in the seasonality of infection. For example, in some areas where P. laevis uses
Gammarus duebeni as intermediate host, a clear seasonality in infection levels of its definitive
host Salmo trutta was observed (Molloy et al. 1995). This was caused by the pronounced
seasonal cycle of growth and reproduction of this gammarid (Fitzgerald and Mulcahy, 1983).
Thus, the combination of the available literature data and the chemical data acquired in the
present study shows that the seasonal pattern of metal accumulation corresponds to the
seasonal dynamics of the acanthocephalans transmission (Figure 3.3), even though the given
data for the prevalence of cystacanths and adults represent the conditions from another
geographical region and are related to another final host. In the lower Danube it might be
expected that the acanthocephalans exhibit a better defined seasonality of transmission than
the observed trends published by Kennedy (1985, 2006).
53
Figure 3.3. Seasonal pattern of the concentrations of the elements As, Cd, Cu, Pb and Zn
fitted according to changes in prevalence of (a) adult P. laevis in fish and (b) cystacanths in
gammarids (Hine & Kennedy, 1974). (From Kennedy, 1985; 2006).
It can be concluded that the acanthocephalans infrapopulations were characterized by

different age compositions in the different seasons covered by the present investigation. The
discrepancies obtained for the mean worms weight in the course of the year (see Figure 3.1)
are taken as evidence for this. Simultaneously, the concentration patterns of all accumulated
elements were similar throughout the year, which was an additional sign that metal uptake
was related to the stage of development. Thus, following the development of
acanthocephalans with respect to the accumulation process, it seems that in autumn P. laevis
infrapopulations consisted mainly of young worms, which were suggested to occur in the
growth phase (preadults). Therefore, due to accelerated metabolism, as previously mentioned,
the obtained mean concentrations of the accumulated elements were on the highest level. An
additional evidence for this was the significantly lower mean individual weight obtained for
the parasites infrapopulations in this period compared to the summer. Furthermore, the
negative relationship between the concentrations of the elements Cd and Pb and the mean
individual weight additionally confirmed the relationship between the accumulation process
54
and the stage of development (characterized by the mean individual weight) in the gut. A
similar tendency was described for other organisms established in metal monitoring like the
shellfish, whereas the small individuals are characterized with faster uptake than the larger
ones (Strong and Luoma, 1981). The increased surface-volume ratios of the younger (smaller)
specimens leads to a higher uptake from the solution (water medium) and causes negative
associations between size and concentration. The same statement could be made for the fish
acanthocephalans, as the assimilation of nutrients and heavy metals occurs mainly through the
worms tegument.
For spring, it can be suggested that the slowed barbels metabolism during the cold months
affected the metabolism of the acanthocephalan regarding accumulated elements. This lead to
a decrease of their concentrations measured in spring compared to autumn. Furthermore, in
the period of dormancy, a process of metal elimination might appear. These aspects combined
with the growth factor during the autumn leads to a diminishment of the element levels,
because if the tissue was gained faster than the metal uptake occurred, the concentrations in
the parasite tissue may be diluted, as described by Strong and Luoma (1981) for the free
living sentinels. It can be assumed that in spring the parasite infrapopulations exibited a
highly heterogeneous age structure, which was a result of the non-simultaneous maturation of
the acanthocephalans. The maturation process itself is mostly affected by various factors such
as host activity and physiology, water temperature and localization of acanthocephalans in the
alimentary tract. For example, Dobson (1985) as well as Bates and Kennedy (1990) reported
that the place of attachment in the intestine of the definitive host also played an important role
in survivorship, maturation and fecundity of acanthocephalans. Therefore it could be expected
that the acanthocephalans were not able to reach the reproductive stage at the same time. The
heterogeneity in age composition during the cold periods was also visible due to the missing
significant differences concerning the element concentrations and mean individual weight,
when compared with other seasons (see Figure 3.1 and Figure 3.2).
During the summer the mean individual weight slightly increased, which indicated that almost
all individuals reached the adult stage. Accordingly, the mean measured concentrations in the
parasites were lower, which confirms that the accumulation process completely differed in the
preadult (growth) and adult phase. While the strategy of the juvenile worms is focused on
gaining both weight and growth, the adults are focused on reproduction, after which they die.
It could be assumed that in the adult stage and in the reproduction phase respectively, the
metabolism of P. laevis regarding the heavy metals and As reached the equilibrium (steady
state) level, where the accumulation and elimination processes were evenly involved. The
hypothesis that in spring a part of individuals have already reached the steady state
55
concentrations was drawn due to the missing significant differences between spring and
summer, when the element concentrations were compared. On the other hand, the slightly
reduced concentrations in summer might be a result of elimination through the egg release
during the reproduction period. Sures et al. (2000b) reported that the acanthocephalans are
able to discharge metals via the shells of their eggs. The fact was proven by the higher Pb
concentrations in the eggs in comparison to the worms body and host tissues. This kind of
detoxification mechanism probably appears not only in the archiacanthocephalan
Moniliformis moniliforms, for which it was firstly described. Our study suggests the same for
the group of paleacanthocephalans, to which P. laevis belongs to. It seems that the process
comprises all accumulated by the parasite elements, not only lead, which was obvious from
the similarity of the concentration pattern throughout the year.
Thus, from the obtained data was designed a model, which represents the accumulation
kinetic of heavy metals and As under natural conditions (Figure 3.4). The model comprises
the suggested by Sures (2008b) uptake progress, considering barbels specific biology
regarding the local climate conditions. The slightly blurred picture in comparison to
laboratory studies persists due to the possibility of the barbel to obtain infection throughout all
warm months. This reduces on the other hand the homogeneity of the acanthocephalan
infrapopulations the individuals are not in the same development stage and respectively the
exposure duration is different for each individual. Under laboratory conditions the infection as
well as the exposure are launched simultaneously, which is actually not the case in the nature.
Therefore, there is a shift in accumulation process when the initial concentrations are
compared. The suggested model was fitted over the year and covered approximately the life
spawn of P. laevis, which probably envelops 7-8 months according to the element
concentration data. The model comprises also the essential metals like Cu and Zn. Their
concentrations showed seasonal pattern similar to the toxic elements As, Cd and Pb, although
the essential metals are known to be regulated by the hosts metabolism (Merian, 2004).
56
Figure 3.4. Model of metal accumulation by P. laevis derived from data obtained from the
thesis (a) and uptake kinetic suggested by Sures (2008b) (b).
As summarized by Luoma and Rainbow (2008), it is necessary to study the seasonal effects
on metal concentration in a particular monitor organism in order to make the impact surveys
more precise after ascertaining the best period for sampling. As previously discussed, seasonal
deviations of metal uptake exist even in already established sentinels such as bivalves and are
mostly associated with the reproductive cycle and growth (Luoma and Rainbow, 2008). For
instance, after the release of gametes, the bivalves enter a period of utilization of energy
reserves characterized by accelerated metabolism and metal uptake, respectively. Moreover,
the accumulation process runs differently according to the size of the mussels, whereas the
smaller one showed a higher accumulation activity than bigger specimens as demonstrated by
Wang and Fisher (1997). When drawing parallels between free living sentinels and parasites it
seems that fish acanthocephalans show some advantages if they are taken as metal monitors.
Even the fact that the fish are able to obtain randomly infection during the active periods and
thus to increase the heterogeneity of age composition of their acanthocephalan
infrapopulations, make the parasites a more flexible tool for assessment of metal pollution,
because the concentrations in the bigger part of their lifespan remain similar (for instance in
spring and summer months). For that reason, there is no need the sampling periods to be
exactly considered like for the other sentinels. However, for the realization of long terms
studies it would be useful and more representative if the sampling activities occurred in the
same season, otherwise the life cycle should be taken into account.
4 Application of acanthocephalan Pomphorhynchus laevis from

its host barbel (Barbus barbus) as metal indicator in the
Danube River
4.1 Introduction
The permanent contamination of aquatic habitats caused by human activities has become one
of the major problems in the era of global industrialization and urbanization. However, the
anthropogenic impact is not only expressed in the form of organic pollution (euthrophication),
but contamination with several toxic substances (e.g. heavy metals, PCBs) might also affect
the functionality and integrity of aquatic ecosystems. Mostly, chemical pollution and in
particular contamination with heavy metals is considered to have an anthropogenic source.
However, natural geogenic deposition might be an important factor for heavy metal pollution
as well. Thus, the detection and management of heavy metal loads in the aquatic environment
is very important from the ecological point of view. Moreover, various aquatic organisms are
used for human consumption and for this reason knowledge about metal contamination is
extremely important from the public health view point.
The large river systems are mostly affected by anthropogenic activities, due to their large
drainage area. After the Volga River, the Danube River is the second largest river in Europe
with a total length of 2850 km. Its entire catchment area covers about 801,463 km 2 and is
shared by ten European countries, which have agreed to monitor the environmental quality of
the river and therefore launched Joint Danube Surveys (JDS) and other monitoring programs,
to produce comparable and reliable information on water quality and pollution (JDS 2001,
2007; TNMN, 1996).
Recently, various sentinel organisms such as bivalves (Arndt et al. 1987; Reeders et al. 1993;
Dallinger, 1994; Gunkel, 1994) have been implemented for assessing levels of pollution in
aquatic habitats. However, in the last two decades fish parasites attained increasing attention,
as they appear to be a more precise tool for detecting metal loads in aquatic habitats. Due to
their enormous accumulation capacity, especially for some elements with severe toxic effects
on biota (e.g. cadmium, lead), fish acanthocephalans are good candidates as bioaccumulation
indicators. In a number of studies concentrations of metals were reported to be 102 to 105
times higher in the parasite than in the water column and the sediment (Sures et al. 1994a;
Schludermann et al. 2003; Thielen et al. 2004). Comparative studies between fish
acanthocephalans and established sentinels such as the zebra mussel (Dreissena polymorpha)
demonstrated the advantage of using parasites, as their accumulation capacity highly exceeds
4 Application of P. laevis as metal indicator
58
that of other accumulation indicators (Sures et al. 1999b). Thus, it can be concluded that fish
acanthocephalans are applicable as a sensitive metal indicator for environmental monitoring
procedures (Vidal-Martinez et al. 2010).
In order to use endoparasites as bioindicators some requirements have to be met, as suggested
by Kennedy (1997): most importantly, the fish host should be abundant and easy to be
sampled and secondly, the parasites must be highly abundant and prevalent among the host
population. The fresh water cyprinid Barbus barbus and its parasite Pomphorhynchus laevis
seems to be a promising model for metal monitoring as they fulfil the above mentioned
requirements. The barbel is the second largest native cyprinid species in Europe, is wide
spread in the epipotamal of large rivers such as the Danube River and is known to show high
infection levels with the intestinal acanthocephalan P. laevis (Kakacheva-Avramova, 1962,
1977; Margaritov, 1959, 1966; Moravec et. al. 1997; Schludermann et al. 2003; Thielen et al.
2004; Laimgruber et al. 2005; Nachev and Sures, 2009). This parasite species is already well
investigated in terms of its metal accumulation (summarized by Sures, 2003; 2004b).
The aim of this study was to perform a long term metal monitoring (in the period from
summer 2004 to summer 2007) and to analyse longitudinal patterns of metal distribution in
the Danube using the acanthocephalan P. laevis. Infected barbels were collected from different
sampling sites along the lower Danube in Bulgaria. Additionally, some fish samples delivered
by the second Joint Danube Survey (JDS2) conducted in summer 2007, were also analysed in
order to detect differences between the upper and the lower Danube reaches. The results were
compared and correlated with the available metal monitoring data provided by International
Commission for Protection of Danube River (ICPDR) for water and suspended particulate
matter (SPM) in order to obtain all possible information on the presence and bioavailability of
trace metals in the Danube River.

4.2.1 Fish samples
The study area was mainly restricted to the Bulgarian part of the Danube River. In the study
period from summer 2004 to summer 2007 up to 35 barbels per sampling were collected in a
seasonal manner (April, July and October) from two localities. To represent the upper Danube
reaches on the Bulgarian river site one sampling site was situated at river kilometre 834 near
the town Vidin. Furthermore, it was in a distance of about 10-15 km downstream from the
inflow of the river Timok, which is known as a one of the major sources for heavy metal
59
pollution in this part of the Danube (Literathy et al. 2002; 2009). The second sampling site
was near the town Kozloduy (685 km) - about 150 river kilometres downstream from Vidin.
Additionally, from spring 2006 to summer 2007, barbels were collected from a site near the
town Silistra (375 km), which represents the last Bulgarian locality in eastward direction of
the river (for details see Chapter 1; Figure 1.1). In summer 2007, during the second Joint
Danube Survey (JDS 2), fish samples were obtained also from four localities in the upper
Danube reach (Table 4.1).
Table 4.1. JDS2 (second Joint Danube Survey) sampling site description.
Sampling site code
JDS 13
JDS 16
JDS 26
JDS 32
River kilometre
1930
1869
1707
1648
Location
Vienna downstream
Bratislava upstream
Szob
Budapest downstram
Number of barbels
n=3
n=10
n=4
n=3
In order to evaluate the use of parasites as accumulation indicators, a long term monitoring
during 4 years, a longitudinal profile in the Bulgarian section of the Danube River and a
longitudinal profile of the entire Danube basin were conducted. All barbels taken for long
term metal monitoring were caught in the same season (e.g. summer) at one sampling site
during the entire period of investigation in order to detect changes of metal concentration in
the lower Danube. Therefore, eight medium sized fishes per summer were selected during the
period 2004-2007 from Kozloduy. Comparative studies for all three sampling sites in Bulgaria
were performed during summer 2006 - eight barbels were taken from each locality. The JDS2
fish samples collected during the survey in 2007 (Table 4.1) were compared with our barbels
collected in summer 2007 at site Kozloduy.
4.2.2 Heavy metal analysis

The concentrations of arsenic (As), cadmium (Cd), colbalt, (Co), copper (Cu), iron (Fe),
manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), vanadium (V) and zinc (Zn) were
were analyzed in fish tissue (muscle, intestine and liver) and parasite samples using
inductively coupled plasma mass spectrometry (ICP-MS) (for details see Chapter 2).
The accuracy of the analytical procedure was verified with the help of a standard reference
material (DORM-3, National Research Council, Canada) of dogfish (Squalus acanthias).
After the analysis, the accuracy rates of seven certified elements were checked. Their values
60
ranged between 87% and 106%, whereas the highest accuracy was obtained for iron (100%)
(Chapter 2; Table 2.1).

The Mann-Whitney U-test was applied to test for significant differences between sampling
sites as well as between metal concentrations in the host-parasite system with data published
for the river Danube. In order to express the accumulation capacity of fish acanthocephalans,
the mean bioconcentration factors were calculated according to Sures et al. (1999a) as
follows: (C[P.laevis] / C[host tissue]). Bioconcentration factors were also determined with respect to
concentrations in water - C[P.laevis] / C[water]. In order to test for significant differences of metal
concentrations between host tissues and parasites, the Wilcoxon matched pairs test was
applied.
4.2.4 Background metal monitoring data

Data published by the ICPDR was used to correlate the metal concentrations in the barbel P. laevis system with those in different matrices of the Danube. The data used for the present
study were from the Trans Nation Monitoring Network (Table 4.2; TNMN, 2009) program
for the period 2003-2006 (monthly metal monitoring in water column) as well as from both
Danube expeditions Joint Danube Surveys (JDS1 in 2001 and JDS2 in 2007), which
delivered an overview of concentrations in water and suspended particulate matter (SPM)
(Literathy et al. 2002; 2009). The metal levels close to our fish sampling sites were used for
comparisons with the parasite data. The JDS sampling sites for the Bulgarian river stretch
were River Timok (confluence with the Danube at km 845 approximately 10 km upstream
from our first sampling site Vidin), Rusenski Lom (450 km, 75 river kilometre upstream from
Silistra) and Silistra (375 km; labelled by JDS2 as Upstream Arges). The TNMN monitoring
sites were, respectively, Novo Selo (833 km), approximately 5 river km upstream form the
sampling site Vidin, Iskar (641 km) about 40 river km downstream from Kozloduy and a
monitoring point directly in Silistra (375 km) (see Table 4.2). Data from the Danube Surveys
was used for comparing the longitudinal distribution of heavy metals in the Danube River
basin.
61
Table 4.2. Data on aqueous element concentrations according to TNMN (2009) for upper and
lower sites of the Bulgarian part of Danube River.
Element
Year
Novo Selo (833km)

(5 km upstream of Vidin)
Right bank
As
2003
3.075
2004
3.4
2005
2.2
2006
2.275
Cd
2003
0
2004
0
2005
0
2006
0
Cu
2003
14.9
2004
18.67
2005
17.5
2006
23.5
Mn
2003
0.0178
2004
0.0141
2005
0.0143
2006
0.0198
Ni
2003
1.975
2004
2.417
2005
4.917
2006
9
Pb
2003
1.842
2004
2
2005
1.833
2006
0
Zn
2003
23.42
2004
24.83
2005
22
2006
20.92
n/a: Data not available
Iskar (675 km)

Righ bank
3.345
3.667
2.692
2.382
0
1.167
1.825
0
9.083
6.417
5.158
6.1
0.0167
0.0108
0.0045
0.0091
2.778
0
0
0
2.333
2.583
2.767
2.364
43.83
24.67
29.73
20
Silistra (375 km)

Right bank
2.813
n/a
n/a
0.3239
1
0
0
0
6
2.545
0
0
0.0303
0.0327
0.0519
0.0373
2
2.727
0
0
2.75
0
0
0
27.08
23.27
21.5
19.83
62
4.3 Results
4.3.1 Element concentrations in the host-parasite system
Similar to the results presented in the Chapters 2 and Chapter 3, the acanthocephalan P.
laevis showed significantly higher concentrations of the elements As, Cd, Cu, Zn and Pb than
the host tissues (Table 4.3; Table 4.4; Table 4.5). Generally, P. laevis showed the highest
accumulation capacity in comparison with fish organs for Pb, followed by Cd, Cu, Zn and As
(see Table 4.6). With the exception of As, the concentration of these elements also exceeded
the concentrations of the local aqueous environment. This was additionally proved by the
bioconcentration factors for the water (Table 4.6).
4.3.2 Longitudinal profile of element concentrations in the Bulgarian part of the

Danube River in 2006
The metal monitoring conducted in summer 2006 at the three selected sampling sites in
Bulgaria showed that the concentrations of the elements As and Cd decreased in downstream
direction in the parasites (Table 4.3 and Figure 4.1). The essential elements Cu and Zn were
found in lowest mean concentrations in Kozloduy, whereas the levels in Vidin were 2 times
higher. In contrast, Pb was found in similar concentrations in the parasites at all sampling
sites, with levels ranging between 5.19 g/kg for Kozloduy and 5.93 g/kg for Silistra. The
distribution of some other elements such as manganese (Mn) in P. laevis, revealed a similar
pattern as the elements As and Cd. Its concentration decreased in the lower part of the
Bulgarian Danube stretch with significantly lower concentrations in Silistra than in Vidin.
Manganese was also found in higher quantities in the parasites compared to fish tissues
although P. laevis had lower levels than the water as can be seen from the mean
bioconcentration factors (Table 4.6).
Generally, no clear longitudinal trend was detected for the element concentrations in fish
tissues, as the data showed a high heterogeneity. One exception was Ni, whose mean
concentration in muscle was elevated in upper section of the Danube- Vidin (2.06 mg/kg);
Kozloduy (0.66 mg/kg); Silistra (0.88 mg/kg) (see Table 4.3).
63
Table 4.3. Element concentrations in P. laevis and different host tissues obtained in summer
2006 for the Bulgarian part of Danube River.
Sampling site
Vidin
As
Muscle
0.07 (0.04)
Intestine
0.15 (0.10)
Liver
0.16 (0.07)
P. laevis
1.53 (0.97)
Cd
Muscle
0.01 (0.01)
Intestine
0.08 (0.05)
Liver
0.10 (0.05)
P. laevis
1.89 (1.23)
Co
Muscle
0.02 (0.01)
Intestine
0.11 (0.17)
Liver
0.04 (0.02)
P. laevis
0.10 (0.11)
Cu
Muscle
1.14 (0.75)
Intestine
2.90 (0.99)
Liver
12.09 (6.10)
P. laevis
57.95 (40.28)
Fe
Muscle
20.18 (10.92)
Intestine
68.85 (66.84)
Liver
92.96 (22.06)
P. laevis
36.22 (20.84)
Mn
Muscle
0.56 (0.21)
Intestine
7.38 (9.91)
Liver
1.79 (1.19)
P. laevis
10.55 (10.41)
Mo
Muscle
n.d.
Intestine
0.05 (0.01)
Liver
0.18 (0.08)
P. laevis
0.03 (0.02)
Ni
Muscle
2.06 (1.34)
Intestine
1.45 (0.92)
Liver
0.48 (0.36)
P. laevis
0.37 (0.27)
Pb
Muscle
0.01 (0.01)
Intestine
0.09 (0.07)
Liver
0.05 (0.04)
P. laevis
5.70 (4.77)
V
Muscle
0.04 (0.01)
Intestine
0.11 (0.11)
Liver
0.25 (0.14)
P. laevis
0.07 (0.02)
Zn
Muscle
4.14 (0.75)
Intestine
12.69 (1.77)
Liver
18.31 (4.13)
P. laevis
52.34 (36.22)
Kozloduy
0.21 (0.15)
0.35 (0.20)
0.54 (0.49)
1.01 (0.60)
0.01 (0.01)
0.11 (0.07)
0.08 (0.04)
1.34 (0.55)
0.02 (0.01)
0.17 (0.14)
0.04 (0.01)
0.10 (0.07)
1.57 (1.29)
4.79 (2.44)
13.40 (8.11)
53.13 (24.13)
10.21 (4.72)
70.92 (17.87)
81.34 (39.42)
41.27 (23.28)
0.35 (0.11)
7.35 (6.37)
1.49 (0.77)
7.64 (5.87)
n.d.
0.06 (0.02)
0.28 (0.16)
0.10 (0.06)
0.66 (0.61)
2.28 (1.16)
0.21 (0.15)
0.55 (0.28)
0.004 (0.003)
0.12 (0.10)
0.03 (0.02)
5.19 (3.74)
0.04 (0.01)
0.29 (0.36)
0.17 (0.16)
0.10 (0.04)
4.76 (1.51)
10.40 (1.16)
19.23 (5.84)
34.83 (16.66)
Silistra
0.08 (0.05)
0.09 (0.07)
0.16 (0.13)
0.91 (0.86)
0.01 (0.01)
0.03 (0.01)
0.04 (0.02)
1.21 (0.65)
0.02 (0.01)
0.05 (0.04)
0.03 (0.01)
0.05 (0.01)
0.76 (0.21)
2.50 (0.68)
13.10 (12.34)
96.01 (47.23)
8.36 (3.05)
38.56 (15.23)
65.89 (20.72)
30.21 (8.79)
0.31 (0.13)
2.28 (1.32)
1.10 (0.29)
5.13 (1.26)
0.02 (0.01)
0.07 (0.05)
0.26 (0.19)
0.11 (0.13)
0.88 (0.97)
2.05 (1.19)
0.72 (0.62)
0.17 (0.14)
0.01 (0.01)
0.09 (0.11)
0.02 (0.01)
5.93 ( 3.66)
0.03 (0.01)
0.09 (0.04)
0.14 (0.10)
0.05 (0.01)
4.85 (1.82)
12.88 (4.41)
16.12 (5.14)
109.38 (62.10)
64
Table 4.4. Element concentrations in P. laevis and different host tissues obtained in summer
2007 for upper and lower Danube.
Element
JDS 13
JDS 16
As
Muscle
0.13 ( 0.17)
n.d.
Intestine
0.46 ( 0.19)
0.26 ( 0.25)
Liver
0.77 ( 0.17)
0.30 ( 0.30)
P. laevis
1.17 ( 0.11)
0.83 ( 0.45)
Cd
Muscle
0.01 ( 0.01)
0.004 ( 0.003)
Intestine
0.02 ( 0.01)
0.04 ( 0.05)
Liver
0.02
0. 08 ( 0.08)
P. laevis
0.14 ( 0.05)
0.30 ( 0.34)
Co
Muscle
0.02 ( 0.006)
0.01 ( 0.01)
Intestine
0.18 ( 0.06)
0.20 ( 0.22)
Liver
0.12 ( 0.01)
0.08 ( 0.06)
P. laevis
0.25 ( 0.10)
0.17 ( 0.11)
Cu
Muscle
0. 62 ( 0.17)
0.81 ( 0.27)
Intestine
3.09 ( 0.22)
2.62 ( 1.02)
Liver
11.5 ( 1.33)
10.3 ( 6.56)
P. laevis
22.9 ( 4.53)
32.6 ( 24.0)
Fe
Muscle
10.4 ( 1.20)
13.6 ( 8.85)
Intestine
60.0 ( 25.3)
49.3 ( 19.9)
Liver
92.1 ( 32.6)
80.4 ( 23.7)
P. laevis
58.0 ( 30.4)
36.6 ( 11.1)
Mn
Muscle
0.46 ( 0.12)
0.28 ( 0.10)
Intestine
7.88 ( 4.34)
6.30 ( 6.43)
Liver
3.96 ( 0.17)
1.80 ( 1.20)
P. laevis
7.90 ( 1.84)
9.37 ( 5.38)
Mo
Muscle
0.22 ( 0.02)
0.01 ( 0.01)
Intestine
0.08 ( 0.03 )
0.04 ( 0.01)
Liver
0.15 ( 0.01)
0.21 ( 0.06)
P. laevis
0.04 ( 0.03)
0.02 ( 0.01)
Ni
Muscle
0.38 ( 0.24)
0.78 ( 1.12)
Intestine
3.25 ( 1.17)
1.01 ( 0.93)
Liver
2.65 ( 2.43)
0.77 ( 1.46)
P. laevis
0.27 ( 0.07)
0.61 ( 0.78)
Pb
Muscle
0.01 ( 0.003)
0.02 ( 0.02)
Intestine
0.18 ( 0.12)
0.13 ( 0.14)
Liver
0.06 ( 0.04)
0.05 (0.04)
P. laevis
1.75 ( 1.34)
2.52 ( 1.75)
V
Muscle
0.02 ( 0.001)
0.02 ( 0.01)
Intestine
0.25 ( 0.16)
0.23 ( 0.23)
Liver
0.12 ( 0.02)
0.40 ( 0.31)
P. laevis
0.06 ( 0.01)
0.06 ( 0.02)
Zn
Muscle
3.64 ( 0.66)
3.56 ( 0.50)
Intestine
13.7 ( 0.77)
12.7 ( 6.25)
Liver
19.8 ( 4.07)
21.9 ( 7.83)
P. laevis
20.9 ( 5.28)
28.8 ( 8.95)
Danube sampling sites

JDS 26
n.d.
n.d.
0.33 ( 0.30)
0.22 ( 0.13)
0.005 ( 0.002)
0.03 ( 0.02)
0.14 ( 0.12)
0.30 ( 0.23)
0.01 ( 0.002)
0.03 ( 0.03)
0.03 ( 0.004)
0.05 ( 0.02)
0.78 ( 0.11)
2.03 ( 0.89)
13.2 ( 7.55)
16.6 ( 12.7)
11.7 ( 2.63)
27.7 ( 14.2)
73.1 ( 5.02)
26.7 ( 15.1)
0.22 ( 0.06)
3.84 ( 2.58)
1.06 ( 0.30)
4.85 ( 2.33)
n.d.
0.05 ( 0.01)
0.15 ( 0.01)
0.05 ( 0.04)
0.71 ( 0.57)
1.16 ( 0.49)
0.44 ( 0.53)
0.26 ( 0.19)
0.004 ( 0.003)
0.04 ( 0.02)
0.03 ( 0.01)
2.67 ( 3.45)
0.03 ( 0.01)
0.08 ( 0.05)
0.69 ( 0.67)
0.03 ( 0.01)
3.45 ( 0.21)
12.3 ( 1.27)
18.4 ( 3.72)
83.6 ( 118.6)
JDS 32
n.d.
n.d.
0.16 ( 0.22)
0.37 ( 0.27)
0.002 ( 0.001)
0.01 ( 0.01)
0.08 ( 0.06)
0. 11 ( 0.04)
0.003 ( 0.002)
0.07 ( 0.05)
0.04 ( 0.02)
0.04 ( 0.03)
0.54 ( 0.13)
1.70 ( 0.12)
12.7 ( 2.92)
8.85 ( 5.11)
11.9 ( 4.19)
52.6 ( 28.2)
89.3 ( 40.8)
76.7 ( 47.6)
0.20 ( 0.07)
3.67 ( 2.82)
1.33 ( 0.52)
6.53 ( 3.86)
n.d.
0.05 ( 0.02)
0.23 ( 0.11)
0.02 ( 0.01)
0.91 ( 0.79)
1.93 ( 0.83)
0.74 ( 0.89)
0.25 ( 0.22)
0.003 ( 0.001)
0.07 ( 0.06)
0.03 ( 0.03)
0.82 ( 0.69)
0.03 ( 0.003)
0.18 ( 0.13)
1.21 ( 1.37)
0.06 ( 0.02)
2.93 ( 0.25)
12.0 ( 0.71)
22.4 ( 3.87)
39.8 ( 14.6)
Kozloduy
0.07 (0.03)
0.21 (0.13)
0.24 (0.13)
0.85 (0.64)
0.02 (0.02)
0.19 (0.14)
0.21 (0.18)
1.82 (1.36)
0.01 (0.01)
0.11 (0.05)
0.04 (0.01)
0.07 (0.04)
0.78 (0.40)
4.43 (2.01)
11.65 (6.28)
26.06 (15.27)
16.36 (8.43)
67.71 (41.22)
99.99 (45.51)
31.11 (16.20)
0.49 (0.46)
4.36 (3.63)
1.56 (1.00)
6.54 (2.46)
n.d.
0.06 (0.02)
0.25 (0.04)
0.06 (0.03)
0.55 (0.35)
2.07 (1.24)
0.49 (0.26)
0.35 (0.29)
0.01 (0.01)
0.17 (0.16)
0.04 (0.03)
3.02 (1.71)
0.04 (0.02)
0.18 (0.12)
0.32 (0.33)
0.08 (0.05)
4.11 (1.01)
13.61 (5.64)
20.34 (5.02)
46.45 (14.83)
65
Table 4.5. Element concentrations in P. laevis and in different host tissues measured for the
period summer 2004 - summer 2007 at site Kozloduy.
Year
As
Summer 2004
Summer 2005
Muscle
0.08 (0.04)
0.16 (0.09)
Intestine
0.37 (0.23)
0.27 (0.23)
Liver
0.20 (0.09)
0.45 (0.36)
P. laevis
0.60 (0.24)
0.93 (0.49)
Cd
Muscle
0.01 (0.01)
0.02 (0.01)
Intestine
0.28 (0.21)
0.10 (0.04)
Liver
0.17 (0.08)
0.11 (0.08)
P. laevis
3.92 (2.87)
1.92 (1.09)
Co
Muscle
0.01 (0.01)
0.02 (0.01)
Intestine
0.11 (0.08)
0.09 (0.10)
Liver
0.06 (0.04)
0.04 (0.02)
P. laevis
0.07 (0.03)
0.08 (0.08)
Cu
Muscle
0.58 (0.32)
0.92 (0.32)
Intestine
7.85 (2.56)
4.26 (3.18)
Liver
8.22 (2.83)
6.99 (2.46)
P. laevis
84.63 (42.38)
56.71 (24.75)
Fe
Muscle
15.56 (8.06)
9.17 (3.11)
Intestine
97.56 (33.99)
77.55 (63.73)
Liver
205.10 (79.11)
71.18 (35.34)
P. laevis
39.54 (14.16)
57.48 (25.71)
Mn
Muscle
0.45 (0.34)
0.41 (0.12)
Intestine
5.55 (5.94)
7.79 (9.56)
Liver
2.07 (1.20)
1.64 (0.94)
P. laevis
5.76 (1.82)
6.38 (4.56)
Mo
Muscle
n.d.
n.d.
Intestine
0.11 (0.04)
0.04 (0.01)
Liver
0.09 (0.04)
0.12 (0.07)
P. laevis
0.04 (0.02)
0.05 (0.03)
Ni
Muscle
0.14 (0.23)
0.50 (0.33)
Intestine
3.99 (2.12)
1.25 (0.85)
Liver
0.95 (0.89)
0.43 (0.23)
P. laevis
0.34 (0.20)
0.51 (0.30)
Pb
Muscle
0.02 (0.01)
0.004 (0.004)
Intestine
0.16 (0.11)
0.18 (0.18)
Liver
0.07 (0.03)
0.03 (0.02)
P. laevis
7.32 (2.98)
6.04 (4.04)
V
Muscle
0.03 (0.01)
0.04 (0.01)
Intestine
0.21 (0.16)
0.23 (0.22)
Liver
0.15 (0.07)
0.27 (0.48)
P. laevis
0.08 (0.03)
0.10 (0.08)
Zn
Muscle
4.42 (1.47)
4.21 (0.79)
Intestine
25.09 (5.05)
13.12 (2.49)
Liver
26.89 (11.94)
16.14 (6.68)
P. laevis
100.78 (63.42)
75.42 (57.03)
Summer 2006
0.21 (0.15)
0.35 (0.20)
0.54 (0.49)
1.01 (0.60)
0.01 (0.01)
0.11 (0.07)
0.08 (0.04)
1.34 (0.55)
0.02 (0.01)
0.17 (0.14)
0.04 (0.01)
0.10 (0.07)
1.57 (1.29)
4.79 (2.44)
13.40 (8.11)
53.13 (24.13)
10.21 (4.72)
70.92 (17.87)
81.34 (39.42)
41.27 (23.28)
0.35 (0.11)
7.35 (6.37)
1.49 (0.77)
7.64 (5.87)
n.d.
0.06 (0.02)
0.28 (0.16)
0.10 (0.06)
0.66 (0.61)
2.28 (1.16)
0.21 (0.15)
0.55 (0.28)
0.004 (0.003)
0.12 (0.10)
0.03 (0.02)
5.19 (3.74)
0.04 (0.01)
0.29 (0.36)
0.17 (0.16)
0.10 (0.04)
4.76 (1.51)
10.40 (1.16)
19.23 (5.84)
34.83 (16.66)
Summer 2007
0.07 (0.03)
0.21 (0.13)
0.24 (0.13)
0.85 (0.64)
0.02 (0.02)
0.19 (0.14)
0.21 (0.18)
1.82 (1.36)
0.01 (0.01)
0.11 (0.05)
0.04 (0.01)
0.07 (0.04)
0.78 (0.40)
4.43 (2.01)
11.65 (6.28)
26.06 (15.27)
16.36 (8.43)
67.71 (41.22)
99.99 (45.51)
31.11 (16.20)
0.49 (0.46)
4.36 (3.63)
1.56 (1.00)
6.54 (2.46)
n.d.
0.06 (0.02)
0.25 (0.04)
0.06 (0.03)
0.55 (0.35)
2.07 (1.24)
0.49 (0.26)
0.35 (0.29)
0.01 (0.01)
0.17 (0.16)
0.04 (0.03)
3.02 (1.71)
0.04 (0.02)
0.18 (0.12)
0.32 (0.33)
0.08 (0.05)
4.11 (1.01)
13.61 (5.64)
20.34 (5.02)
55.47 (33.13)
66
Table 4.6. Bioconcentration factors calculated for summer 2006 at three sampling sites in
Bulgaria.
As
Cd
Cu
Fe
Mn
Ni
Pb
Zn
Muscle
Intestine
Liver
Water
Muscle
Intestine
Liver
Water
Muscle
Intestine
Liver
Water
Muscle
Intestine
Liver
Water
Muscle
Intestine
Liver
Water
Muscle
Intestine
Liver
Water
Muscle
Intestine
Liver
Water
Muscle
Intestine
Liver
Water
Vidin
20.53
9.91
9.33
0.67
146.31
22.70
19.01
1.89
50.93
20.01
4.79
2.47
1.80
0.53
0.39
0.14
18.74
1.43
5.90
0.53
0.18
0.26
0.78
0.04
452.03
60.56
107.80
5.70
12.65
4.13
2.86
2.50
Kozloduy
4.84
2.86
1.87
0.42
132.72
11.68
16.04
1.34
33.85
11.10
3.96
8.71
4.04
0.58
0.51
0.47
21.88
1.04
5.14
0.84
0.83
0.24
2.59
0.55
1250.01
42.16
151.08
2.19
7.31
3.35
1.81
1.74
Silistra
11.94
10.07
5.73
2.81
175.68
38.87
27.50
1.21
126.57
38.35
7.33
96.01
3.61
0.78
0.46
0.05
16.34
2.25
4.66
0.14
0.19
0.08
0.23
0.17
737.83
67.50
271.68
5.93
22.57
8.49
6.78
5.52
67
Figure 4.1. Longitudinal profile of elements accumulated by P. laevis obtained for summer
2006 in Bulgarian part of Danube River.
4.3.3 Longitudinal profile of element concentrations in the Danube River in 2007

Four localities situated in the upper Danube were compared with Kozloduy in the lower
Danube in order to obtain a longitudinal metal profile. According to metal concentrations in
the parasites there were clear differences between the upper and lower Danube for the toxic
elements Cd and Pb (Figure 4.2 and Table 4.4). Increased mean values for their
concentrations were detected in the lower part of the river they were up to 16 and 4 times
higher for Cd and Pb, respectively. Concentrations of the other elements in P. laevis such as
As and Cu showed increased mean values at the upper two localities (downstream from
Vienna; upstream from Bratislava) as well as in the lower Danube in Bulgaria (Kozloduy, see
Table 4.4). The levels of Zn, on the other hand, increased also in downstream direction,
although insufficient number of barbels were investigated from sampling sites Szob (site JDS
26) and downstream from Budapest (site JDS 32) (Table 4.4).
68
Figure 4.2. Danubes longitudinal profile of elements As, Cd and Pb in P. laevis, obtained in
summer 2007.
4.3.4 Long term monitoring of element concentrations in the lower Danube

Elements concentrations in the parasites (Cd, Cu, Pb, Zn) showed a decreasing tendency from
summer 2004 to summer 2007 (Table 4.5 and Figure 4.3) at Kozloduy indicating an
improvement of the water quality. Fluctuations in parasite concentrations were observed only
for the element As, with increasing mean concentrations in 2005 and 2006 and decreasing
levels in 2007, thus reaching a concentration similar to the one measured in 2004 (see Table
4.5). In contrast to the parasites, Cd and Zn concentrations in fish tissues and particularly in
fish muscle remained stable during the years. The As concentrations in barbels muscle
followed the pattern in the parasites during the period of investigation. Similar relationships
were not observed for Cu and Pb. The concentrations of the other elements showed no clear
tendency neither in fish organs, nor in the parasite.
69
Figure 4.3. Long term monitoring of elements As, Cd, Cu, Pb and Zn in P. laevis at site
Kozloduy (Bulgaria).
4.3.5 Comparisons between element concentrations in parasite with the available

background data for water and SPM
The highest aqueous Cu concentration for the entire Danube was reported by JDS1 in 2001
and JDS2 in 2007 at the River Timok confluence (Literathy et al. 2002; 2009). The impact of
the tributary was also evident, at the TNMN site Novo Selo situated below the Timok
effluence (see Table 4.2). At this site, during the JDS2 were found also extended
concentrations of element Zn and Ni (Literathy et al. 2009). Additionally, some of the highest
Cu and Zn levels were reported in the water for other tributaries such as Rusenski Lom (75
river kilometers upstream of Silistra) for Cu and Silistra (375 km) for Zn. As expected, the
longitudinal profile of heavy metal concentrations in the parasites followed the pattern of Cu
and Zn in the water. The reported Zn concentration at Timok confluence was 9.3 g/L and
corresponded to a mean concentration in the parasites of 52.34 g/g at Vidin. On the other
hand, the aqueous Zn concentration at site Silistra was almost 2 times higher (16.1 g/L)
compared to Timok River. Consequently, the obtained Zn levels in the acanthocephalans at
site Silistra (109.38 g/g) were in the same quantity higher as the water- about two times
(chemical data from JDS2 technical report by Literathy et al. 2009). A similar longitudinal
profile was also observed for Cu with higher concentrations in the upper and lower Danube
70
section in Bulgaria and lower levels in Kozloduy (Literaty et al. 2009).

As previously mentioned, the longitudinal profile of the other elements found in higher
amounts in P. laevis (As, Cd, Mn and Pb), showed a decreasing tendency along the Danube in
Bulgaria, except for Pb, for which concentrations in the parasite remained similar at all
monitoring points (see Table 4.3 and Figure 4.1). According to water concentrations
available for 2006, the only significant difference was found for As when the upper and the
lower reaches in the Bulgarian section of the River were compared (see Table 4.2): Novo Selo
(2.275 g/L); Silistra (0.3239 g/L). Elevated aqueous concentrations were reported for Pb at
locality Iskur used as a reference for our sampling site Kozloduy, since at the sites Vidin and
Silistra no Pb has been detected. Cadmium, on the other hand, was below the method
detection limits at all sampling sites. (see Table 4.2).
The comparison of metal concentrations in the parasites with levels determined for SPM
during JDS2 showed identical trends (see Figure 4.2 and Figure 4.4), although detailed
correlation analyses could not be performed as the heavy metal raw data gathered by JDS2 are
still not published.
71
Figure 4.4. Distribution profile of As, Cd, Cu, Ni, Pb and Zn in the SPM along the Danube
River during JDS2 (from Literathy et al. 2009).
4.4 Discussion
As demonstrated in Chapter 2 and Chapter 3 as well as in the present chapter, P. laevis
showed a better accumulation capacity for the elements As, Cd, Cu, Pb and Zn than its fish
host. However, As concentrations in the parasites were lower compared to those in the water,
in contrast to the other elements. Therefore, it seems possible that P. laevis can be employed
as a sentinel for the the metals Cd, Cu, Pb and Zn. These elements were also categorized by
ICPDR into priority groups 1 and 2, according to their toxicological importance. Group 1
includes the elements Cd and Pb together with Hg and Ni, while the elements As, Cu and Zn
were categorized in group 2 together with Bi, Co, Cr, Mo in suspended particulate matter and
72
Co, Ti and V in sediments. The elements Fe and Mn, for instance, were placed in group 3 as
metals important for the overall assessment of water quality (Literathy et al. 2009). Thus, the
acanthocephalans can be successfully applied as metal indicators especially for the priority
metals in the Danube River basin.
The selected river stretch of the Danube in Bulgaria, where the metal monitoring survey was
carried out, can be considered as an optimal part of the river for performing this kind of
investigations. The lower Danube reaches suffer from the impact of some major tributaries
such as Tisa and Sava (joining the Danube in Serbia at 1215 and 1170 river kilometre,
respectively), which are known as the biggest pollution sources along the Danube River
(Literathy et al. 2002; 2009). As a result of their inflow, the lower Danube reaches are
polluted downstream of river km 1000. The influence of the tributaries is evident when
analysing the longitudinal profiles of heavy metals and As in the SPM published by JDS2 (see
Figure 4.4; Literathy et al. 2009). Furthermore, the Timok River has been polluted by copper
mining and heavy metal industry in all of its drainage area since the middle of twentieth
century (Antonovi et al. 1974; Boinovi et al. 2005). Consequently, it pollutes the Danube
with various heavy metals such as cadmium, copper and lead, released by ore leaching.
The longitudinal profile of element concentrations in the parasites corresponded to the
contamination profile reported for the Bulgarian river stretch (Litheraty et al. 2002, 2009;
TNMN 2009). At the localities in upper and lower part of the river the concentrations of Cu
and Zn were higher in comparison to concentrations measured at site Kozloduy. This was a
result from the impact of the tributaries, which join the Danube before the sites Vidin and
Silistra. The profile was clearly expressed by the concentration values obtained both for
parasites and water (see Figure 4.1; Litheraty et al. 2009). As mentioned above, the
relationship between the Zn concentrations in the parasite and the source (water) were the
same at both studied sites. This fact fulfils one of the criteria characterizing the ideal sentinel
organism as summarized by Sures (2003). The lower Cu and Zn levels at Kozloduy were
probably due to dilution along the river stretch after the confluence of the River Timok in the
Danube. The decreasing concentration of As, Cd, Mn and Pb in parasites samples obtained in
downstream direction can be also related to the dilution factor along the river stretch after the
influence of the tributaries such as Rivers Sava, Tisa and Timok. Worth noticing is the
reported during the JDS2 highest Ni concentration at the confluence of Timok River. The
uptake of this element was found to be lower for P. laevis. However, its concentrations in
barbels muscles were significantly higher at Vidin, situated close to the inflow of Timok. The
exposure duration of the pollutants can be evaluated as suggested by Sures et al. (1999a) with
the help of ratios between the elements concentrations of host tissues (e.g. muscle) and
73
parasite. Accordingly, in this part of Danube a permanent contamination with nickel appears
to occur, as fish muscle characterizes with slower uptake kinetics. It should be noted that a
continuous Ni pollution over longer time is required for reaching such high saturation in the
hosts tissue.
During the Danube Surveys the main focus was on element concentrations in suspended
particulate matter, to which heavy metals tend to adsorb and which is suggested to be more
reliable for metal indications purposes than aqueous concentrations (Literathy et al. 2002;
2009). Following the profile of the distribution of heavy metals and As in SPM, published in
the JDS2 report, the concentrations of As, Cd and Pb clearly decreased in downstream
direction after km 1000 (see Figure 4.4). As previously mentioned, the As and Cd amounts in
parasites also followed this profile. Unfortunately, the distribution patterns represented the
conditions in 2007, while our data regards to monitoring performed in 2006. However, it
could be suggested that over the period between both Danube surveys the conditions in this
part of the River did not change drastically, since similar tendencies were observed.
Additionally, for more detailed comparisons between contents in SPM and parasites, the raw
data is required, because the published profiles did not deliver the needed resolution and it
was not possible to assess fully the influence of tributaries like Timok and Russenski Lom.
The analysis on the longitudinal profile conducted in 2007 for the entire Danube River
revealed some differences in element concentrations measured in P. laevis for lower Danube
reaches compared to the upper one. In general, the distribution pattern of the accumulated by
the parasite elements followed the longitudinal profile of SPM. It was clearly visualized by
the profile obtained for As. The values measured for SPM in 2007 characterized with two
humps along the river. The first was around km 2000 after the confluence of River Inn and the
second at km 1000 behind the tributaries Tisa, Sava und Velika Morava. Similar results were
obtained for As in parasite and host tissues, whereas increased concentrations were measured
at the locality Vienna downstream (site JDS 13; 1930 km) followed by the Danube site
Kozloduy in lower Danube (Figure 4.2). A decrease in As content, as reported for SPM, was
observed along the river stretch between km 1930 and 1648 (between the site JDS 13 and the
site JDS 32). As expected from the SPM profile, the elements Cd and Pb also showed highest
values in lower Danube - the mean cadmium concentration at site Kozloduy was up to 16
times higher in comparison than those in the upper Danube sites. A parallel with the SPM
profile could not be found for the essential elements, due to large fluctuations in the measured
values. Therefore, for further detailed analyzes, the raw element data for SMP is required.
The long term monitoring (from summer 2004 to summer 2007) revealed a tendency of
decreasing of Cd, Cu, Pb and Zn concentrations in parasites at site Kozloduy. The
74
improvement of water quality during this period was also obvious from the Cu concentrations
at the confluence of Timok River. For example, in 2001 the reported aqueous Cu
concentration was 163 g/L, which was approximately five times higher compared with 2007
(34.5 g/L) (Literathy et al. 2002; 2009). The descent of concentrations was also detectable in
parasite samples from Kozloduy, situated more than 150 river kilometres downstream of the
river Timok (see Figure 4.3). The enhancement of water quality regarding some of these
heavy metals could be followed also in the chemical data published by the TNMN for the
water column. According to the latter, at the monitoring point near to Kozloduy (Iskur), there
was also a slight decrease in the Zn and Cu concentrations for the period 2003-2006 (Table
4.2). Unfortunately, such clear tendency was not found for the elements Cd and Pb. As
mentioned above, the heavy metal monitoring in the water column does not always deliver a
reliable picture of metal pollution in contrast to bioindicators. A reason for this is that the
water phase is a highly dynamic system and the concentrations of diluted substances (metals)
can vary between the sampling activities, although a pollution source exists. The monitoring
performed by TNMN was based on a monthly assessment of element concentrations in the
water and thus cannot be regarded as highly representative for the environment conditions.
Using parasites, it was possible an obvious reduction of concentrations of elements Cd, Cu,
Pb and Zn to be detected, which was suggested to be related to metallurgy industry in the
catchments of the river Timok. The main reason for this was the constriction of mining in the
region, which led to decreasing of pollution levels in the Timok River and its tributaries. The
ore production at the open pit situated in Timoks catchment took place in the period from
1991 to 2002, after which the mining works were stopped. (Paunovi et al. 2008).
Despite the observed deviations, the mean concentrations in P. laevis seemed to reflect the
local conditions, where the fish were sampled. This deviation was considered to be related to
the mobility of the fish, which might cause variation in the measured concentrations.
However, such tendency is expected to occur even if other organisms (e.g. bivalves) are taken
as sentinels. Even in the case of mussels are sampled from the same locality, the
morphological differences of the riverine may lead to high deviation in the measured
concentrations. The size and the age of the taken specimens play also a considerable role in
the accumulation process, as already discussed in Chapter 3. Therefore, having this
background and according to the obtained data, P. laevis can be regarded as a very appropriate
tool in the field of metal monitoring. Its sentinel properties might be very useful for detecting
and quantifying pollution sources from the industry and particulary for some highly toxic
elements like As, Cd and Pb.
Summary, conclusions and future prospects

Summary
The main focus of the current thesis was to extend the knowledge about the use of fish
parasites as bioindicators. On one hand the research emphasis was aimed at the faunistical and
ecological aspects of parasite communities of barbel (Barbus barbus) in relation to
environment conditions - the use of parasites as effect indicators (Chapter 1). On the other
hand the thesis evaluated and reverified the acanthocephalan Pomphorhynchus laevis as
accumulation indicator, while covering all critical aspects (as summarized by Sures, 2003)
concerning its application - the effects of size and sex composition of its infracommunities
(Chapter 2) as well as the seasonality (Chapter 3) on the metal uptake process in the
parasite. Finally, the thesis delivered a detailed metal monitoring survey focused mainly on
the lower Danube (Chapter 4), which was perforemed using the suggested barbel P. laevis
system.
The important results are summarized in the following:
Chapter 1: The endohelminth fauna of barbel correlates with water quality of the Danube
River in Bulgaria.
Infection of barbel with ten species of metazoan parasites including three trematodes,
three acanthocephalans and four nematodes was observed in fish collected from three
localities in the Bulgarian part of the river Danube between summer 2004 and summer
2007.
New host records for three parasitic species the nematode larvae of genus
Eustrongylides sp. and Hysterothylacium sp. as well as the acanthocephalan
Leptorhynchoides plagicephalus were recorded for first time for the host Barbus
barbus.
The most prevalent species was the acanthocephalan Pomphorhynchus laevis, which
was also the dominant species of the intestinal component communities at all sampling
sites.
The second most frequent parasite at all Danube localities was Rhabdochona hellichi,
which occurred in significantly higher numbers at the less polluted sites.
The composition as well as the diversity characteristics of the parasite communities
showed a clear correlation with the composition of the invertebrate fauna and water
76
quality overall, the diversity of helminth communities increased with decreasing

levels of nutrients and pollutants at all sampling sites.
Chapter 2: Is metal accumulation in Pomphorhynchus laevis dependent on parasite sex or
infrapopulation size?
From total twelve analyzed elements (As, Cd, Co, Cu, Fe, Mn, Mo, Ni, Pb, Sn, V, Zn)
five (As, Cd, Cu, Pb and Zn) were detected in significantly higher concentrations in
the acanthocephalan P. laevis compared to its host tissues (muscle, intestine, liver)
According to the calculated mean bioconcentration factors, three more elements (Co,
Mn, V) usually were with higher concentrations in P. laevis.
Comparisons between high and low infected fish revealed significant differences only
for V with higher concentrations for the heavily infected group.
Concerning sex specific metal accumulation V and Zn showed significant differences
(V, at p < 0.05; Zn, at P = 0.05), with higher levels in females of P. laevis each.
Chapter 3: Seasonal differences of metal accumulation in Pomphorhynchus laevis and its
definitive host Barbus barbus.
The concentrations of As, Cd, Cu, Pb and Zn were significantly higher in the parasite
samples presented than in the hosts tissues.
These elements showed also a clear seasonal pattern, while the concentrations in the
fish tissues remained similar in spring, summer and autumn.
Seasonal variation in the mean individual weight of parasite infrapopulations, whereas
the infrapopulations in autumn characterized with significantly lower mean individual
weight than these in spring and summer - sign for predominant young specimens.
Composition pattern of all accumulated elements reflected the pattern of the mean
individual weight over the year - the highest concentrations obtained for As, Cd, Cu,
Pb and Zn in P. laevis were found in autumn, followed by spring and summer.
Concentration of Cd and Pb in P. laevis correlated negatively with the mean worm
weight.
Significant differences for the metals Cd and Pb were found, when the concentration
in worms from summer and autumn were compared.
77
Chapter 4: Application of acanthocephalan Pomphorhynchus laevis from its host barbel

(Barbus barbus) as metal indicator in the Danube River.
The elements As, Cd, Cu, Pb and Zn were found to be significantly higher
accumulated in the parasite compared to its host tissues.
The concentrations of Cd, Cu, Pb and Zn in P. laevis exceeded the concentrations
reported for the water column at the selected sampling sites.
The longitudinal pattern of As, Cd, Cu, Pb and Zn in the parasite samples
corresponded to the background available data (for water and suspended particulate
matter) along the Danube River in Bulgaria.
The comparisons between upper and lower Danube, performed in summer 2007,
demonstrated increased concentrations of As, Cd and Pb in the lower Danube, whereas
As showed also a peak in the upper reaches. Their concentration pattern reflected the
pattern of suspended particular matter obtained during the 2nd Joint Danube Survey
performed also in summer 2007.
The long term monitoring at sampling site Kozloduy (685 km) showed progressive
decrease in the concentrations of Cd, Cu, Pb and Zn for the period summer 2004 to
summer 2007, while the As contents remained similar.
Conclusions and future prospects

The collective data suggests that the fish parasites can be successfully used to characterize the
ecosystems health and integrity. Following the alternations in composition and diversity of
their communities, it was possible to detect differences in the environmental conditions
between investigated sampling sites. Therefore fish parasites can be efficiently applied as
effect indicators in the aquatic monitoring. The last published data on parasite fauna of barbel
for the Bulgarian part of Danube River was from 1960 and 70-ties (Kakacheva-Avramova,
1962, 1977; Margaritov, 1959, 1966). The results obtained from the present investigation
showed that the fauna composition completely differed from the one reported 40-50 years
ago. The disrepancy can be associated with the changed/disturbed ecological conditions in the
investigated river stretch.
Further investigations aimed on other river stretches in middle and upper part of the river and
on other aquatic habitats as well should be performed for the sake of the future
implementation of fish parasites as effect indicators in the Danube River and in the
hydrobiological praxis as well. The faunistical data from the different parts of the Danube
should be compared and subsequently correlated with the local abiotic and biotic data, in
78
order to confirm profoundly the relationship between parasites and environmental conditions.
Furthermore, different host-parasite systems should be also be included in such surveys and
studied from a bioindication perspective.
A major goal of the current thesis was filling in on the lack of knowledge regarding the
application of fish acanthocephalans as accumulation indicatiors. The obtained results
suggested that the size and the sex composition of acanthocephalans infrapopulations play no
considerable role in the metal uptake process. Therefore, in metal monitoring surveys,
especially in those aimed on toxic elements such as As, Cd and Pb, these aspects should not
be taken into account. Worth noticing is that the results regarded the acanthocephalan
P. laevis. If other acanthocephalan species are taken as accumulation indicators, these aspects
should be studied in order to confirm the tendencies obtained in the thesis.
On the other hand the results revealed a seasonal pattern in the metal uptake, which was found
to be dependent on the stage of acanthocephalans development in the final host. Thus, the
seasonality of transmission of P. laevis under the local climate conditions should be
considered in order to make our monitoring surveys more precise. In some geographical
regions, where the seasonality of transmission is not clearly pronounced, the seasonal factor
can be suppressed. Of course, the sentinel features of fish acanthocephalans should also be
investigated under different climate conditions, in order to select the proper sampling periods
for metal monitoring surveys. The same should be done with other acanthocephalan species,
if they are taken as metal indicators.
With the help of the background metal monitoring data delivered by the International
Commission for Protection of Danube River (ICPDR), it was confirmed that the levels of the
elements accumulated in P. laevis corresponded to these in the environment. The pollution
profile in the Danube River basin, obtained during the both Joint Danube Surveys (in 2001
and 2007), was additionally confirmed by the concentrations measured in the parasites.
Desipite the mobility of the fish host, the results of this thesis suggest the fish-parasite system
is a perfect model in the field of ecological monitoring. However, future detailed analysis and
correlations between the raw data from the JDS2 and obtained parasite data are required.
Unfortunately, this data concerning the element concentrations in water, SPM, sediment and
biota is still not available.
Regarding the practical use of a fish-parasite system as sentinel, the first step was made
during the second Joint Danube Survey in 2007, where fish muscle tissue was analyzed.
During the thesis I had the opportunity to contribute to the survey with metal analysis carried
out on barbel P. laevis system. The combinded results suggest that the additional use of fish
acanthocephalans as sentinels represents a more powerful approach in heavy metal
79
monitoring surveys, due to the higher accumulation capacity of acanthocephalans compared

to fish muscle. Consequently, in future monitoring programs the fish parasites should be
accessorily implemented as sentinels, especially in large and complex lotic systems like
Danube River.
Zusammenfassung
Indikationsvermgen von Fischparasiten zur Beurteilung des kologischen
Zustandes aquatischer Habitate
Hintergrund
Das im letzten Jahrzehnt steigende Interesse an Helminthen als potentielle Bioindikatoren fr
die Belastung und Verschmutzung von Gewssern mit Schwermetallen hat zu einer
verstrkten Forschung auf dem Gebiet der kologischen Parasitologie gefhrt. Die
Mglichkeit, Fischparasiten als Indikatoren fr die Beurteilung der Wasserqualitt zu nutzen
wurde in den letzen Jahren intensiv erforscht (MacKenzie et al. 1995; Kennedy, 1997;
Lafferty, 1997; Overstreet, 1997; Sures et al. 1997b; Valtonen et al. 1997; Lafferty und Kuris,
1999; Sures et al. 1999a; Sures, 2001). Parasiten knnen als Effektindikatoren (Valtonen et al.
1997; Sures, 2001), und als Akkumulationsindikatoren (Sures et al. 1999a; Sures, 2001)
benutzt werden. Dabei bertreffen sie sogar die Bioindikationseigenschaften der bislang
bekannten freilebenden Organismen. Bei den freilebenden Bioindikatoren wurden vor allem
die Akkumulationseigenschaften sowie nderungen in der Physiologie und Ethologie
erforscht, die durch Vernderung der Umweltqualitt entstanden sind (Gunkel, 1994).
Eine Mglichkeit fr einen Einsatz von Parasiten in der Effektindikation, liegt in der
Erfassung der Diversitt und der Vernderung von Parasitengemeinschaften. Bei dieser Form
der Effektindikation liefern die Organismen durch ihre An- oder Abwesenheit Informationen
ber den physikalisch-chemischen Zustand der Umwelt (Sures, 2003). Eine Untersuchung der
Diversitt, Struktur und Dynamik der Parasitengemeinschaften hilft den Zustand und die
Vernderlichkeit natrlicher kosysteme zu erfassen. Um die Auswirkungen, die
Umweltkontaminationen auf Parasitengemeinschaften ausben zu erfassen, mssen viele
Aspekte bercksichtigt werden, wie z.B. die Dynamik und Eingliederung des Fischwirtes in
das Nahrungsnetz (Marcogliese und Cone, 1997), die Beziehung und die Wechselwirkung
zwischen den Parasiten (Overstreet, 1997) sowie die Ab- und Anwesenheit der
Zwischenwirte. Auerdem beeinflussen Faktoren wie der pH-Wert (Marcogliese und Cone,
1997) und der Grad der Eutrophierung (Valtonen et al. 1997) direkt oder indirekt die
Abundanz, die Verteilung und die Struktur von Parasitenpopulationen.
Wie bereits erwhnt, knnen Fischparasiten auch als Akkumulationsindikatoren verwendet
werden. Durch ihre Fhigkeit, verschiedene Substanzen in ihrem Gewebe zu akkumulieren,
liefern sie als Akkumulationsindikatoren Informationen ber den chemischen Zustand ihrer
81
Zusammenfassung
Umwelt. Gegenwrtig ist bekannt, dass nicht nur freilebende Organismen, wie z.B. Krebse
und Muscheln, Schwermetalle in ihrem Gewebe akkumulieren knnen, sondern auch
Parasiten. Und zwar in einem Ma, das die Konzentrationen in den Geweben des Wirtes oder
der Umwelt, um ein Vielfaches bersteigt.
Durchgefhrte Untersuchungen an parasitischen Nematoden deuten darauf hin, dass diese
Helminthen nicht als Akkumulationsindikatoren geeignet sind, da die Anreicherung der
Metalle zu niedrig ist (Sures et al. 1994b; Sures et al. 1998; Szefer et al. 1998; Baru et al.
1999a,b). Cestoden dagegen scheinen vielversprechendere Akkumulationsindikatoren zu sein
(Riggs et al. 1987; Turekov und Hanzelov, 1996; Sures et al. 1997c; Tenora et al. 1997;
Baru et al. 2000; Sures et al. 2002). Anhand experimenteller Daten lsst sich ihre
Akkumulationsfhigkeit hher einstufen als die freilebender Organismen. Die hinsichtlich
ihrer Bioakkumulationsfhigkeiten am besten untersuchte Parasitengruppe sind die
Acanthocephalen (vgl. z.B. Sures 2003, 2004a,b). Es gibt nicht nur eine Reihe von
Freilandsstudien (Sures et al. 1994a,b,c; Sures und Taraschewski, 1995; Sures et al. 1997a,
1999b; Sures und Reimann, 2003), sondern auch Laboruntersuchungen (Siddall und Sures,
1998; Sures und Siddall, 1999; Zimmermann et al. 1999; Scheef et al. 2000; Sures et al.
2000b;
Sures
und
Siddall,
2001,
2003;
Sures
et
al.
2003)
zur
Schwermetallakkumulationskapazitt von Acanthocephalen. Die Metallanreicherung bei

adulten Acanthocephalen kann einige tausend Male hher sein als in den Geweben ihres
Endwirtes. Der Akkumulationsprozess fngt dabei unmittelbar nach der Infektion des
Endwirts
an
und
erreicht
in
4-5
Wochen
seine
Gleichgewichts-Konzentration.
Untersuchungen an Larvenstadien weisen darauf hin, dass diese Stadien noch nicht in der
Lage sind, Metalle in hohen Konzentrationen zu akkumulieren (Sures, 2003). Auch im
Vergleich zu etablierten, freilebenden Bioindikatoren ist die Biokonzentration von Cd und Pb
in Acanthocephalen um ein Vielfaches hher, wie der unmittelbare Vergleich der
Metallanreicherung
in
Dreissena polymorpha,
zeigt
Acanthocephalus lucii
(Sures
et
al.
und
1997a,
der
1999b).
Dreikantmuschel,
Trotz
der
enormen
Akkumulationskapazitt von Fischacanthocephalen bestehen diverse unerforschte Aspekte,

die potenziell ihre praktische Anwendung im Bereich des Metallmonitorings kritisch machen
knnten. Wie von Sures (2003) zusammengefasst, sollten noch die Auswirkung des Alters und
der Gre von Parasiten sowie Effekte der Saisonalitt und der Reproduktionsaktivitt auf die
Metallaufnahme
grndlich
erforscht
werden,
Akkumulationsindikatoren einsetzen zu knnen.
um
die
Acanthocephalen
als
82
Zusammenfassung
In der vorliegenden Arbeit wurde versucht, die oben genannten Aspekte bezglich der
Anwendung von Fischparasiten als Indikatoren zu erfassen. Daraus leiten sich die folgenden
Arbeitshypothesen und Schwerpunkte ab:
(1)
Die
Zusammensetzung
der
Fischparasiten-Gemeinschaft
korreliert
mit
dem
Verschmutzungsgrad in den Flussabschnitten, aus welchen die Fische stammen.

Die Fischparasiten reagieren auf die vernderten Umweltbedingungen mit einer nderung in
ihrer Diversitt und Artenzusammensetzung. Die Verschmutzung (chemische oder
physikalische) kann direkt oder indirekt die Fischparasitenpopulationen beeinflussen. Der
direkte Einfluss zeichnet sich durch eine letale Reaktion der Larven- oder Adultstadien aus.
Bei indirektem Einfluss handelt es sich um letale Effekte auf die Zwischen- oder Endwirte,
wobei die Effizienz der Parasitentransmission verhindert wird. Zudem knnte die
Verschmutzung die Wirtsphysiologie insofern beeinflussen, dass Wirt und Parasit zustzlich
unter diesem externen Stress leiden. In beiden Fllen fhrt die Kontamination zu nderungen
in der Artenzusammensetzung und der Diversitt von Parasitenpopulationen.
(2) Die Infrapopulationsgre und geschlechtsspezifische Metallanreicherung in dem
Acanthocephalen Pomphorhynchus laevis und dessen Endwirt Barbus barbus.
Die Fischacanthocephalen sind fhig die Metallgehalte in den Wirtsorganen zu reduzieren,
wobei die Konzentrationen im Wirtsgewebe negativ mit der Anzahl der Wrmer im Darm
korreliert (Sures und Siddal, 1999; Sures et. al. 2003). Fr den Fall, dass
Fischacanthocephalen als Metallindikatoren verwendet werden, sollte untersucht werden, ob
die Infrapopulationsgre bercksichtigt werden muss. Parallel knnte, bedingt durch den
unterschiedlichen Metabolismus, ihre Geschlechterzusammensetzung auch eine Rolle bei der
Metallaufnahme spielen. Zustzlich knnten die rein morphologischen Unterschiede zwischen
den Geschlechtern auch einen Einfluss auf den Akkumulationsprozess aufweisen. Dies knnte
durch unterschiedliche Oberflche- Volumen-Verhltnisse verursacht werden.
(3) Jahreszeitliche Unterschiede bei der Metallaufnahme in Pomphorhynchus laevis und
dessen Endwirt Barbus barbus.
Eine Saisonalitt bei der Metallaufnahme in den derzeit verwendeten freilebenden
Indikatororganismen (wie z.B. Muscheln) ist bereits bekannt. In den meisten Fllen sind die
Unterschiede durch deren Reproduktionszyklen geprgt. Ein Grund dafr ist hauptschlich die
nderung in der Metabolismusaktivitt vor und nach der Gametenfreisetzung (Luoma und
Rainbow, 2008). hnliche Tendenzen sind auch bei den Fischacanthocephalen whrend ihrer
83
Zusammenfassung
Entwicklung im Darm des Endwirtes zu erwarten. Dadurch besteht ein Bedarf, bestimmte
Beprobungszeitrume zu bercksichtigen, damit der Faktor Saisonalitt vermindert
werden kann.
(4) Die Metallkonzentrationen in dem Acanthocephalen Pomphorhynchus laevis spiegeln die
Konzentrationen in der Umwelt wider.
Um einen Akkumulationsindikator effektiv fr Metallmonitoringszwecke verwenden zu
knnen,
mssten
dessen
Gewebekonzentrationen
jene
der
Umwelt
widerspiegeln
(zusammengefasst von Sures, 2001). Darber hinaus sollten die Fischacanthocephalen eine
realistische Information ber den Metallbelastungsgrad in den entsprechenden aquatischen
Habitaten liefern.
Um die Schwerpunkte der Dissertation abdecken zu knnen, wurden die folgenden Methoden
und Materialen angewendet:
Durchfhrung der Arbeit

In dieser Forschungsarbeit wurde eine Freilandstudie zur Zusammensetzung der
Fischparasitengemeinschaft an verschiedenen Standorten im Verlauf der Donau durchgefhrt.
Zwischen 10 und 30 Barben (Barbus barbus) der jeweiligen Probestellen wurden im Zeitraum
Sommer 2004 bis Sommer 2007 auf ihre Parasitoznosen hin untersucht. Die Fische wurden
von Berufsfischern entlang der Donau bezogen, wobei ein Teil (im Sommer 2007) whrend
der zweiten wissenschaftlichen Donau Expedition (JDS2) gefischt wurde. Die Fische wurden
im eingefrorenen Zustand in das Labor gebracht, wo sie einer vollstndigen parasitologischen
Untersuchung unterzogen wurden (vgl. z.B. Sures et al. 1999c). Aus den Daten zur
Befallssituation der Fische, unter spezieller Bercksichtigung von Helminthen, wurden dann
die mittleren Diversitts- und Dominanzindices (vgl. z.B. Magurran, 1988; Sures et al. 1999c)
berechnet, so dass objektive Gren resultierten, die einen Vergleich der Fischparasitoznosen
zwischen den Probestellen sowie zwischen den Jahreszeiten erlauben. Zustzlich wurden die
Resultate der Fischparasitoznose in Korrelation zu den entsprechenden MakrozoobenthosDaten und den physikalisch-chemischen Wasserparametern gesetzt. Diese Hintergrunddaten
wurden von der Datenbank der Internationalen Kommission zum Schutze der Donau (ICPDR)
entnommen.
Zustzlich wurden die Parasiten (insbesondere Acanthocephalen) wie auch verschiedene
Fischgewebe (Muskel, Darm und Leber) im Labor auf ihren Schwermetallgehalt hin
84
Zusammenfassung
untersucht. Dazu wurden die Proben mittels Mikrowellenaufschluss in Lsung gebracht

(Zimmermann et al. 2001). Anschlieend wurden die Metallgehalte mit Hilfe der
Massenspektrometrie (ICP-MS) gemessen. Die Konzentrationen der Elemente As, Cd, Co,
Cu, Fe, Mn, Mo, Ni, Pb, Sn, V und Zn wurden analysiert. Um die Akkumulationskapazitt
von Fischacanthocephalen zu ermitteln, wurden mittlere Biokonzentrationsfaktoren fr die
gemessenen Elemente wie folgt berechnet (nach Sures et al. 1999a): C[P.laevis] / C[Wirtsgewebe];
C[P.laevis] / C[Wasser].
Wichtige Ergebnisse und Erkenntnisse

Die oben genannten Schwerpunkte und Arbeitshypothesen der Dissertation wurden einzeln
jeweils in Unterkapiteln dargestellt und diskutiert.
1)
Die
Zusammensetzung
der
Fischparasiten-Gemeinschaft
korreliert
mit
dem
Verschmutzungsgrad der Flussabschnitte, aus welchen die Fische stammen.

Insgesamt wurden 407 Barben aus dem Zeitraum Sommer 2004 bis Sommer 2007
parasitologisch untersucht. Die Fische stammten aus drei Beprobungsstellen in dem
bulgarischen Abschnitt der Donau und wurden jeweils im April, Juli und Oktober entnommen.
An den Probestellen Vidin (834 km) und Kozloduy (685 km) wurden jeweils 165 bzw. 193
Barben in dem gesamten Zeitraum untersucht und an der Stelle Silistra (375 km) insgesamt 49
Barben im Jahr 2006 und 2007.
Die erhobene Parasitenfauna umfasste 10 Endohelminthenarten - 3 Trematodenarten
(Metacercarie
von
Diplostomum
spathaceum
in
den
Augenlinsen,
Posthodiplostomum cuticola auf der Haut, Metagonimus yokogawai auf den Schuppen), 3
Acanthocephalenarten
(Pomphorhynchus
laevis,
Acanthocephalus
anguillae
und
Leptorhynchoides plagicephalus im Darm) und 4 Nematodenarten (Adulten von

Rhabdochona hellichi, Pseudocapillaria tomentosa und Larven von Hysterothylacium sp. im
Darm und Larven von Eustrogylides sp. in der Leibeshhle). Der Acanthocephale
L. plagicephalus
und
die
Larven
von
den
Nematoden
Eustrongylides
sp.
und
Hysterothylacium sp. wurden zum ersten Mal fr den Wirt Barbe beschrieben.
Die dominante Parasitenart an allen drei Probestellen war der Acanthocephale P. laevis, der
mit eine Befallsrate von fast 100 % vorkam. Der zweithufigste Parasit war der Nematode
R. hellichi dessen Prvalenz und Befallsintensitt an den Stellen im Unterlauf (Kozloduy und
Silistra) mit einer besseren Wasserqualitt anstieg. Im Gegensatz dazu kam der Nematode
Eustrgylides sp. zusammen mit R. hellichi mit gleichen Befallsraten an der Probestelle im
85
Zusammenfassung
Oberlauf (Vidin) vor, wobei im Lauf der Donau seine Befallsrate und Intensitt absanken. Die
Verteilungsmuster
dieser
Parasitenarten
knnen
auf
den
Verschmutzungsgrad
(Eutrophierungsgrad) bezogen werden, wenn davon ausgegangen wird, dass diese Arten
unterschiedliche Zwischenwirte bentigen. Als Zwischenwirt fr R. hellichi dienen
Kcherfliegenlarven (Trichoptera) von der Gattung Hydropsyche (Moravec, 1995), deswegen
war die niedrigste Prvalenz dieser Art an der Stelle Vidin zu finden- auf Grund der niedrigen
Abundanz des Zwischenwirtes. Der Fakt spiegelt den hheren Eutrophierungs- und
Verschmutzungsgrad wider, welcher von der Internationalen Kommission zum Schutz der
Donau fr diesen Flussabschnitt ermittelt wurde. Die Larven von Hydropsyche sp. werden im
Saprobiensystem mit einem Indikationswert zwischen 2.1 und 2.3 eingestuft (Moog, 1995).
Im Gegensatz zu R. hellichi erfolgt die Entwicklung der Nematoden der Gattung
Eustrogylides sp. ber Oligochaeten (Lumriculus variegatus, Tubifex tubifex, Limnodrilus sp.)
als Zwischenwirt (Morevec, 1994), die mit einem Indikationswert von ber 3 organisch- und
chemisch-belastete aquatische Habitate charakterisieren. Es wurde gefolgert, dass das
Vorkommen von diesen Parasitenarten abhngig von der Belastung war.
Die berechneten Diversittsindizes (Brillouin- und Schannon-Wiener- Index) unterstzten
zustzlich die Korrelation mit dem Belastungsgradienten. Die niedrigsten Werte wurden an
der Probestelle Vidin gemessen, whrend die unteren zwei Stellen deutlich hhere Werte
aufwiesen. Das Belastungsprofil im bulgarischen Abschnitt der Donau konnte zudem durch
die chemischen Hintergrunddaten der ICPDR belegt werden (Literathy et al. 2002, 2009;
TNMN, 2009).
Somit konnte gezeigt werden, dass die Zusammensetzung der Parasitenfauna mit den lokalen
Gewsserbedingungen
korreliert.
Die
Diversittsindizes
spiegelten
auch
den
Belastungsgradient entlang der untersuchten Flussstrecke, wobei die Diversitt hier als ein
Ma fr die allgemeine kosystemgte betrachtet wird.
2) Die Infrapopulationsgre und geschlechtsspezifische Metallanreicherung in dem
Acanthocephalen Pomphorhynchus laevis und dessen Endwirt Barbus barbus.
Die Konzentrationen von 12 Elementen (As, Cd, Co, Cu, Fe, Mn, Mo, Ni, Pb, Sn, V, Zn)
wurden mittels Massenspektrometrie mit induktiv gekoppeltem Plasma (ICP-MS) im
Darmparasiten Pomphorhynchus laevis und in den Geweben (Muskel, Darm, Leber) seines
Wirtes
Barbus barbus
analysiert.
Der
Zweck
der
Untersuchung
war,
eventuelle
Anreicherungsunterschiede, sowohl zwischen gering und stark infizierten Fischen, als auch
zwischen beiden Geschlechtern von P. laevis festzustellen. Dafr wurden 30 mittelgroe
Fische im Oktober 2006 von einer Probestelle bei Flusskilometer 685 an der bulgarischen
86
Zusammenfassung
Flussbank
der
Donau
entnommen
und
entsprechend
ihrer
Befallsintensitt
mit
Acanthocephalen eingeteilt. Fische (n=9) mit einer Befallsintensitt weniger als 20 Wrmer
wurden der Gruppe Gering Infiziert zugeteilt. Eine zweite Gruppe (n=9) mit einer
Befallsintensitt von mehr als 100 Parasiten wurde der Gruppe Stark Infiziert zugeordnet.
Anhand dieser beiden Gruppen wurden potenzielle Akkumulationsunterschiede in den
Wirtsgeweben und den Acanthocephalen zwischen gering und stark infizierten Fischen
untersucht. Eine weitere dritte Gruppe (n=8) von Barben mit einer Befallsintensitt zwischen
66 und 89 Wrmern wurde ausselektiert. Die Gruppe wurde fr die Erfassung
geschlechtsspezifischer Unterschiede in der Metallanreicherung verwendet.
Die Elementzusammensetzung im Wirt-Parasit-System wies einen signifikant strkeren (bis
zu 1070 hheren) Anreicherungsgrad von As, Cd, Cu, Pb und Zn im Parasiten im Vergleich zu
den Wirtsgeweben auf. Gem den berechneten Biokonzentrationsfaktoren wurden drei
weitere Elemente (Co, Mn, V) mit einer hheren Konzentration in P. laevis gefunden. Die
Vergleiche zwischen stark und leicht infizierten Fischen zeigten weder in den Wirtsgeweben
noch im Parasiten signifikante Unterschiede. Die einzigen Anreicherungsunterschiede wurden
fr das Element Vanadium in Parasitenproben und Fischleber gefunden, wobei die stark
infizierte Gruppe hhere Gehalte aufwies. Zusammenfassend blieb die Konzentration von den
in P. laevis stark akkumulierten Elementen (As, Cd, Cu, Pb und Zn) unabhngig von der
Befallsintensitt.
Zwischen den beiden Geschlechtern von P. laevis wurden signifikante Unterschiede nur fr
die Elemente V (p < 0.05) und Zn (p 0.05) festgestellt. Wobei die Weibchen jeweils hhere
Gehalte aufwiesen.
Die Ergebnisse deuten darauf hin, dass P. laevis gut geeignet fr Metallmonitoringstudien ist,
da die Infrapopulationsgre und Geschlechterzusammensetzung keinen groen Einfluss auf
den Akkumulationsprozess im Wirt-Parasit-System ausben. Diese Aspekte zusammen mit
der enormen Akkumulationskapazitt, besonders fr toxische Metalle wie Cd and Pb, sttzen
den Vorschlag fr die Verwendung von P. laevis als Akkumulationsindikator.
Die Arbeitshypothese, dass die Metallanreicherung im Wirt-Parasit System von der Gre und
Geschlechterzusammensetzung der Parasiteninfrapopulationen abhngig ist, konnte nicht
vollstndig besttigt werden. Bis auf die Elemente V und Zn, die noch vom
Wirtsmetabolismus als essentielle Metalle (wie z.B. Zn) beeinflusst werden, war die
Elementzusammensetzung in Parasiten hnlich.
87
Zusammenfassung
3) Jahreszeitliche Unterschiede bei der Metallaufnahme in Pomphorhynchus laevis und

dessen Endwirt Barbus barbus.
Um die Wirkung der Saisonalitt auf die Metallaufnahme in den Acanthocephalen zu
erforschen, wurden die Gehalte der Elemente As, Cd, Co, Cu, Fe, Mn, Mo, Ni, Pb, V und Zn
in dem gewhlten Wirt-Parasit System analysiert. Die Untersuchung deckte Frhjahr, Sommer
und Herbst bzw. die Monate April, Juli und Oktober 2006 ab, wobei acht mittelgroe Barben
jeweils pro Saison von einer Probestelle (Kozloduy, 685 km) entnommen wurden. Nach der
parasitologischen Untersuchung wurde die Anzahl der Acanthocephalen und das
Frischgewicht deren Infrapopulationen erhoben. Zustzlich wurde die mittlere individuelle
Masse von P. laevis fr jede Barbe berechnet, als das Infrapopulationsgewicht geteilt durch
die Anzahl der Wrmer. Der Zweck dieser morphologischen Berechnung war Informationen
ber den Entwicklungszustand der Acanthocephalen im Endwirt zu erhalten. Darber hinaus
bringt die individuelle Wurmmasse auch weitere Erkenntnisse bezglich der Oberflchen
Volumen-Verhltnisse von Parasiten, die eine entscheidende Rolle bei der Metallaufnahme
spielen knnen hnlich wie bei freilebenden Metallindikatoren wie z. B. Muscheln (Luoma
und Rainbow, 2008).
Wie erwartet, waren die Konzentrationen der Elemente As, Cd, Cu, Pb und Zn im Parasiten
im Vergleich zum Wirtsgewebe (Muskel, Darm und Leber) signifikant hher. Diese Elemente
zeigten auch einen hnlich ausgeprgten jahreszeitlichen Konzentrationsverlauf. Die hchsten
mittleren Konzentrationen wurden im Herbst und die niedrigsten im Sommer nachgewiesen.
Die Frhjahrswerte lagen im Bereich zwischen den Werten von Herbst und Sommer. Die
Tendenz wurde zustzlich statistisch fr die toxischen Metalle Cd und Blei geprft- deren
Gehalte waren im Herbst signifikant hher. Die Konzentration dieser Metalle korrelierte
negativ mit dem mittleren individuellen Wurmgewicht, und spiegelte zudem das
jahreszeitliche Konzentrationsprofil der akkumulierten Elemente wider. Die berechnete
mittlere Parasitenmasse war im Vergleich zu Frhjahr und Sommer im Herbst signifikant
niedriger. Ein Zeichen dafr, dass die Achanthocephalen-Infrapopulationen im Herbst
berwiegend aus jngeren Individuen besteht. Dies beeinflusste die Wurmgehalte, da sich
jngere Individuen in der Wachstumsphase durch einen entsprechend intensiveren
Metabolismus auszeichnen. Deswegen waren die mittleren Konzentrationen von As, Cd, Cu,
Pb und Zn im Herbst am hchsten. Das hhere Oberflchen Volumen-Verhltnis der
jngeren Acanthocephalen beeinflusste zustzlich die Metallaufnahme, so wie fr freilebende
Metallindikatoren
bereits
Metallaufnahmemechanismus
beschrieben.
der
Dieser
Acanthocephalen
Aspekt
muss
bercksichtig
auch
werden,
bei
da
dem
die
Akkumulation durch die Tegumentoberflche erfolgt. Zustzlich brachte die Untersuchung
88
Zusammenfassung
Erkenntnisse ber die Saisonalitt der Entwicklung von P. laevis unter den spezifischen
klimatischen Bedingungen im Unterlauf der Donau. Das ausgeprgte Kontinentalklima
beeinflusst den Lebenszyklus und die bertragung der Parasiten, wobei ein jhrliches Muster
deutlich
zu
sehen
ist. Von
den
erhobenen
Elementzusammensetzung der Acanthocephalen
Ergebnissen
zur
wurde
grobe
eine
Morphologie
und
Schtzung der
Lebensdauer von P. laevis im Darm des Endwirtes B. barbus erzeugt. Die Lebensdauer
betrgt hchst wahrscheinlich 7-8 Monate.
Zusammenfassend wird deutlich, dass die Metallanreicherung in P. laevis von der Saisonalitt
bzw. von den Entwicklungsstadien der Parasiten abhngig ist.
4) Die Metallkonzentrationen im Acanthocephalen Pomphorhynchus laevis spiegeln die
Konzentrationen in der Umwelt wider.
Die Metallmonitoringstudie mit Hilfe von Fischacanthocephalen wurde berwiegend entlang
des bulgarischen Donauabschnitts durchgefhrt. Zuerst wurde versucht ein Lngsprofil der
gewhlten Flussstrecke zu erzeugen. Aus diesem Grund wurden im Sommer 2006 jeweils acht
Barben von drei Probestellen entlang der Donau (Vidin, 834; Kozloduy, 685; Silistra, 375 km)
untersucht. Zustzlich wurde eine Langzeitmonitoringstudie im Unterlauf der Donau
durchgefhrt, die vier Jahre umfasste. Die Studie wurde an der Probestelle Kozloduy
durchgefhrt, die als Referenzstelle im Unterlauf gewhlt wurde. Von dieser Stelle wurden in
jedem Sommer in dem Zeitraum 2004 bis 2007 acht Fische entnommen. Im Jahr 2007 wurde
noch eine weitere Studie durchgefhrt, welche fr einen Vergleich zwischen dem Ober- und
Unterlauf der Donau diente. Fr diesen Zweck wurden von vier Probestellen in Mitteleuropa
Barben beprobt und mit der Probestelle Kozloduy im Unterlauf verglichen. Die Fische von
Mitteleuropa stammten aus Probestellen in der Nhe von Wien (1930 km), Bratislava
(1869 km), Szob (1707 km) und Budapest (1648 km) und wurden whrend der zweiten
Donau Forschungsexpedition (JDS2) im Sommer 2007 gefangen.
Die Acanthocephalen und die Fischgewebe (Muskel, Darm, Leber) wurden auf den Gehalt
mehrerer Elemente (As, Cd, Co, Cu, Fe, Mn, Mo, Ni, Pb, V, Zn) analysiert und es wurden
Gehaltprofile erstellt. Die Konzentrationen im Parasit Wirt-System wurden sowohl mit den
Wasserdaten im Zeitraum 2004-2007 (monatliches Metallmonitoring des Wasserkrpers im
Rahmen der TransNational Monitoring Network Programm) als auch mit den Metalldaten (zu
Wasser und Schwebstoffen (SPM)) von beiden Donauexpeditionen (JDS1 in 2001 und JDS2
in 2007) verglichen (Literathy et al. 2002, 2009; TNMN, 2009).
Von den analysierten Elementen As, Cd, Cu, Pb und Zn wurden im Vergleich zu den
Wirtsgeweben wieder signifikant hhere Konzentrationen in P. laevis gefunden. Um einen
89
Zusammenfassung
Vergleich zwischen den Konzentrationen im Parasit und im Wasser zu erlauben, wurden die
mittleren Biokonzentrationsfaktoren bezglich des Wassers berechnet. Damit zeigte sich ein
hherer Anreicherungsgrad fr die Schwermetalle Cd, Cu, Pb und Zn in den
Acanthocephalen. Das Lngsprofil dieser Elemente in den Parasiten spiegelte das Lngsprofil
der Metallgehalte des Wassers und der Schwebstoffe wider. hnliche Ergebnisse wurden auch
fr das Element As beobachtet. Generell senken sich die Gehalte von As, Cd, Pb im Lauf der
Donau in Bulgarien ab. Die essenziellen Metalle Cu und Zn wiesen hhere Konzentrationen
an der oberen (Vidin) und an der unteren Probestelle (Sillistra) auf. Der Grund dafr sind zwei
Nebenflsse
(Fluss
Timok
und
Fluss
Russenski
Lom),
die
als
die
grten
Verschmutzungsquellen fr Cu und Zn von der ICPDR bezeichnet werden.

Das durchgefhrte Langzeitmetallmonitoring an der Stelle Kozloduy zeigte eine Verbesserung
der Wasserqualitt bezglich der vom Parasit akkumulierten Metalle. Mit der Ausnahme von
Arsen senkten sich die Gehalte von Cd, Cu, Pb und Zn im Zeitraum vom Sommer 2004 bis
Sommer 2007 ab. Die Tendenz wird zum Teil von den Hintergrunddaten der ICPDR belegt.
Die Vergleiche zwischen Ober- und Unterlauf der Donau zeigen generell eine deutliche
Metallgehalterhhung im Unterlauf an der Probestelle Kozloduy. Besonders deutlich zeigt
sich dies fr die Elemente Cd und Pb. Die hheren Konzentrationen in der bulgarischen
Donaustrecke stehen unter dem Einfluss zweier groer Nebengewsser (Tisa und Sava), die
im Gesamtverlauf der Donau von km 1000 bis zum Donaudelta zu Belastungen durch
Schwermetalle fhren (Literathy et al. 2009). Fr detaillierte Analysen waren noch
zustzliche Rohdaten der zweiten Donauforschungsexpedition erforderlich.
Die Metallgehalte im Parasit spiegeln die Konzentrationen in der Umwelt wider. Die
beobachtete
Tendenz
in
den
Parasitengehalten
wird
von
den
Ergebnissen
der
unterschiedlichen Donaumonitoringprogramme besttigt.
Schlussfolgerungen
Die durchgefhrte faunistische Untersuchung im Rahmen der Dissertation lieferte eine neue
Wirtsmeldung fr drei parasitische Arten. Die Nematoden der Gattung Eustrongylides sp. und
Hysterothylacium sp und der Acanthocephale L. plagicephalus wurden zum ersten Mal fr
den Wirt B. barubs beschrieben, wobei Eustrongylides sp. einer der hufigsten Vertreter im
Unterlauf der Donau war. Die Parasitenfauna der Barbe wies beim Vergleich der derzeitigen
Ergebnisse mit den zuletzt publizierten Daten aus den 1960-er und 70-er Jahren (KakachevaAvramova, 1962, 1977; Margaritov, 1959, 1966) generell groe Unterschiede auf. Ein
mglicher Grund dafr liegt bei den vernderten Bedingungen im Unterlauf der Donau in den
90
Zusammenfassung
letzten 40 Jahren.
Die abgedeckten Aspekte bezglich der Anwendung von Fischparasiten als Indikatoren
untersttzen deren Einsatz im Bereich des aquatischen Monitorings sowohl als
Effektindikatoren
als
auch
als
Akkumulationsindikatoren.
Ihre
Anwendung
als
Effektindikatoren war von der Artenzusammensetzung untersttzt, da das Vorkommen der

Parasiten zum Teil die Umweltbedingungen widerspiegelten. Zustzlich lieferten die
Diversittindizes hnliche Tendenzen, die den Belastungsgradienten in der Donau folgten.
In der Dissertation wurden diverse, bisher nicht untersuchte, Aspekte betrachtet, welche den
Einsatz von Fischacanthocephalen als Akkumulationsindikatoren verhindern knnten (wie
z.B. der Einfluss der Infrapopulationsgre, der Geschlechterzusammensetzung und der
Saisonalitt auf die Metallanreicherung). Aus den erhobenen Ergebnissen knnen folgende
Rckschlsse gezogen werden:
-
Die Infrapopulationsgre bt keinen groen Einfluss auf den Metallgehalt in P. laevis

aus. Darber hinaus sollte die Befallsintensitt bei Metallmonitoringsstudien mit der
Hilfe von Fischacanthocephalen nicht bercksichtig werden.
Die Geschlechterzusammensetzung der Parasiteninfrapopulationen muss nicht in

Betracht gezogen werden, da beide Geschlechter hnliche Tendenzen bei der
Metallanreicherung gezeigt haben.
Der
Metallanreicherungsprozess
ist
von
der
Saisonalitt
bzw.
vom
Parasitenentwicklungsstadium im Endwirt abhngig. Darber hinaus sollten bei den

Beprobungszeiten unter anderem die lokalen klimatischen Bedingungen bercksichtigt
werden.
Die Fischacanthocephalen, konkret P. laevis, scheinen sehr vielversprechend fr
Metallindikationszwecke zu sein. Die enorme Akkumulationskapazitt besonders fr toxische
Elemente wie As, Cd und Pb zeichnet sie als ein perfektes Werkzeug im Bereich des
aquatischen Monitorings aus. Trotz der hheren Mobilitt des Fischwirtes, spiegelten die
Konzentrationen in P. laevis jene der Umwelt wider und lieferten ein anschauliches Bild ber
die Metallbelastung der Donau. Der erste Schritt fr den Einsatz von Fischen als
Metallindikatoren wurde whrend der zweiten Donauforschungsexpedition (JDS2) gemacht
(Literathy et al. 2009). Im Rahmen der Dissertation wurde bewiesen, dass im Gegensatz zur
alleinigen Analyse von Fischgewebe, die zustzliche Anwendung von Fischparasiten als
Metallindikatoren ein vielversprechenderes Verfahren darstellt.
References
Antonovi, G., Nikodijevi, V., Tanasijevi, D., Vojinovi, L.J., Pavievi, N., Aleksi, .,
Filipovi, D. and Jeremi, M. (1974) Zemljita basena Timoka. Centar za
poljoprivrednaistraivanja Beograd, Institut za prouavanje zemljita, Beograd. 1-342.
Arndt, U., Nobel, W. and Schweizer, B. (1987) Bioindikatoren- Mglichkeiten, Grenzen und
Neue Erkenntnisse. Verlag Stuttgart, Germany.
Baras, E., (1995) Seasonal activities of Barbus barbus: effect of temperature on timebudgeting. Journal of Fish Biology 46, 806-818.
Baru, V., Tenora, F. and Krmar, S. (2000) Heavy metal (Pb, Cd) concentrations in adult
tapeworms (Cestoda) parasitizing birds (Aves). Helminthologia 37, 131-136.
Baru, V., Tenora, F., Krmar, S. and Dvoek, J. (1999a) Contents of several inorganic
Substances in European eel infected and uninfected by Anguillicola crassus
(Nematoda). Diseases of Aquatic Organisms 37, 135-137.
Baru, V., Tenora, F., Krmar, S., Proke, M. and Dvoek, J. (1999b) Microelement
contents in males and females of Anguillicola crassus (Nematoda: Dracunculoidea).
Helminthologia 36, 283-285.
Bates, R.M. and Kennedy, C. R. (1990) Interactions between the acanthocephalans
Pomphorhynchus laevis and Acanthocephalus anguillae in rainbow trout: testing an
exclusion hypothesis. Parasitology 100, 435-444.
Beeby, A. (2001) What do sentinels stand for? Environmental Pollution 112, 285-298.
Bock, K.J. and Scheubel, J.B. (1979) Die biologische Messung der Wassergte.
Naturwissenschaften 66, 505-512.
Boinovi, S., Luki, D., Miljkovi, D. and ivkovi S. (2005) Proceedings of 13th
Scientific and professional conference on natural resources and environmental
protection, Borsko jezero, 01-04.06., 454-456.
Bush, A. O., Lafferty, K. D., Lotz, J. M. and Shostak, A. W. (1997) Parasitology meets
ecology on its own terms: Margolis et al. revisited. Journal of Parasitology 83, 575583.
Cohn, F.J. (1953) ber lebendige Organismen im Trinkwasser. Z. klein. Med. 4, 229-237.
Costanza, R. and Mageau, M. (1999) What is a healthy ecosystem? Aquatic Ecology 33,
205-115.
Craige, B.J. (2001) Eugene Odum: Ecosystem Ecologist and Environmentalist. University of
Georgia Press. Athens, Georgia.
92
References
Crompton, W. T. and Nickol, B. B. (1985) Biology of acanthocephala. Cambridge

University Press, Cambridge.
Dallinger, R. (1994) Invertebrate organisms as biological indicators of heavy metal pollution.
Appl. Biochem. Biotechnol. 48, 27-31.
Dezfuli, B. S., Giari L. and Poulin R. (2000) Species associations among larval helminths in
an amphipod intermediate host. International Journal of Parasitology 30, 1143-1146.
Dobson, A.P. (1985) The population dynamics of competition between parasites.
Parasitology 66, 317-347.
Fitzgerald, R.D. and Mulcahy, M. F. (1983) Parasites of salmon Salmo salar L. and trout
Salmo trutta L. in the River Shournagh. Irish Fisheries Investigations Series A 23, 2431.
Franceschi, N., Bauer, A., Bollache, L. and Rigaud, T. (2008) The effects of parasite age
and intensity on variabilityin acanthocephalan-induced behavioural manipulation.
International Journal for Parasitology 38, 1161-1170.
Gunkel, G. (1994) Bioindikation in Aquatischen kosystemen. Verlag Stuttgart, Germany.
Hine, P.M. and Kennedy, C. R. (1974) The population biology of the acanthocephalan
Pomphorhynchus laevis (Mller) in the River Avon. Journal of fish biology 6, 665-679.
Hofer, R. and Lackner, R. (1995) Fischtoxikologie - theorie and praxis. Fisher Verlag, Jena.
Hoole, D. (1997) The effects of pollutants on the immune response of fish: implications for
helminth parasites. Parassitologia 39, 219-225.
Hudson, P.J., Dobson, A.P. and Lafferty, K.D. (2006) Is a healthy ecosystem one that is rich
in parasites? Trends in Ecology and Evolution 21 (7), 381-385.
ICPDR (1998) International Commission for the Protection of the Danube River. Available
at: http://www.icpdr.org/icpdr-pages/about_us.htm, (accessed: 20.01.2010).
ICPDR
(2002)
JDS
Technical
Report,
available
at
http://www.icpdr.org/icpdr-
pages/jds.htm.Last update 28.08.2008, (accessed 05.09.08).

ICPDR
(2004)
TNMN
Yearbook
2004,
available
at
pages/tnmn_yearbooks.htm Last update 28.08.2008, (accessed 05.09.08).

ICPDR
(2005)
TNMN
Yearbook
2005,
available
at
pages/tnmn_yearbooks.htm. Last update 28.08.2008, (accessed 05.09.08).

ICPDR (2008a) Joint Danube Survey 2 On-board Results, available at
http://www.icpdr.org/jds/latest_results Last update 28.08.2008, (accessed 05.09.08).
ICPDR (2008b) TransNational Monitoring Network Database, available at
http://danubis.icpdr.org/pls/danubis/DANUBIS_DB.DYN_NAVIGATOR.show Last
update 28.08.2008, (accessed 05.09.08).
References
93
ICPDR (2008c) Danube Surveys Database, available at

http://danubis.icpdr.org/pls/danubis/DANUBIS_DB.DYN_NAVIGATOR.show Last
update 28.08.2008, (accessed 05.09.08).
ICPDR (2009) Trans National Monitoring Network Database. International Commission for
the Protection of Danube River. Available at:
http://danubis.icpdr.org/pls/danubis/DANUBIS_DB.DYN_NAVIGATOR.show?p_arg_
names=page&p_arg_values=/servlet/page%3F_pageid%3D53%26_dad%3Ddanubis%2
6_schema%3DDANUBIS%26_type%3Dsite%26_fsiteid%3D1%26_fid%3D14485%26
_fnavbarid%3D1%26_fnavbarsiteid%3D1%26_fedit%3D0%26_fmode%3D2%26_fdis
playmode%3D1%26_fcalledfrom%3D1%26_fdisplayurl%3D, (accessed 02.10.2009).
JDS (2001) First Joint Danube Survey. Available at: http://www.icpdr.org/icpdrpages/jds.htm, (accessed 20.01.2009).
JDS (2007) Second Joint Danube Survey. Available at: http://www.icpdr.org/icpdrpages/jds2.htm, (accessed: 20.01.2009).
Kakacheva-Avramova, D. (1962) Helminthological investigations into fishes of the rivers
Struma, Strumeshnitsa and Mesta. In: Natural foci of infections in the Petrich and Gotse
Delchev Districts, pp. 191-217. Bulgarian Academy of Sciences. Sofia.
Kakacheva-Avramova, D. (1977) Studies on helminths of fishes in the Bulgarian section of
the Danube River. Helminthologia 3, 20-45.
Kakacheva-Avramova, D. (1983) Helminths of freshwater fishes in Bulgaria. Sofia,
Publishing House of Bulgarian Academy of Sciences.
Kennedy, C.R. (1985) Regulation and dynamics of acanthocephalan populations. In:
Crompton , D.W.T., Nickol, B.B., (Ed.), Biology of Acanthocephala. Cambridge
University Press, Cambridge, pp 385-417.
Kennedy, C. R. (1997). Freshwater fish parasites and environmental quality, an overview and
caution. Parassitologia 39, 249-254.
Kennedy, C. R. (2006) Ecology of the acanthocephala. Cambridge University Press,
Cambridge.
Kennedy, C. R., Bush, A. O. and Aho, J. M. (1986). Patterns in helminth communities: why
are the birds and fish different? Parasitology 93, 205-215.
Kolenati, F. A. (1848) Genera et species Trichopterorum. 1. Heteropalpoidea, Pragae.
Kolkwitz, R. and Marsson, M. (1902). Grundstze fr die biologische Beurteilung des
Wassers nach seiner Flora and Fauna. Mitt. a. d. kgl. Prfungsanstalt fr Wasservers. u.
Abwasserbeseitigung zu Berlin Bd. 1, 33-72.
References
94
Kolkwitz, R. und Marsson, M. (1908) kologie der pflanzlichen Saprobien. Ber. deutsch.
bot. Gesellschaft 26a, 505-519.
Kolkwitz, R. und Marsson, M. (1909) kologie der tierischen Saprobien. Int. Rev.
Hydrobiol. 2, 126-152.
Koskivaara, M. (1992) Environmental factors affecting monogeneans parasitic on freshwater
fishes. Parasitology Today 8, 339-342.
Lafferty, K. D. (1997). Environmental parasitology: What can parasites tell us about human
impacts on the environment? Parasitology Today 13, 251-255.
Lafferty, K. D. and Kuris, A. M. (1999). How environmental stress affects the impacts of
parasites. Limnology and Oceanography 44, 925-931.
Lafferty, K. D. Allesina, S., Arim, M., Briggs, C. J., De Leo, G., Dobson, A. P., Dunne, J.
A., Johnson, P. T. J., Kuris, A. M., Marcogliese, D. J., Martinez, N. D., Memmott,
J., Marquet, P. A., McLaughlin, J. P., Mordecai, E. A., Pascual, M., Poulin, R. and
Thieltges, D. W. (2008) Parasites in food webs: the ultimate missing links. Ecology
Letters 11, 533-546.
Laimgruber S., Schludermann C., Konecny R. and Chanovec A. (2005) Helminth
communities of barbel Barbus barbus from large river systems in Austria. Journal of
Helminthology 79, 143-149.
Literathy, P., Koller-Kreimel, V. and Liska, I. (2002) Joint Danube survey 1, Technical
report of the International Commission for the Protection of the Danube River (ICPDR).
Available at http://icpdr.org.
Literathy, P., Enache, I., Pavonic, M., Ocenaskova V. and Diemer, J. (2009) Joint Danube
survey 2, Technical report of the International Commission for the Protection of the
Danube River (ICPDR). Available at http://icpdr.org.
Luoma S.N. and Rainbow P.S. (2008) Metal contamination in aquatic environments.
Cambridge University Press, Cambridge, pp 169-202.
MacKenzie, K. (1999) Parasites as pollution indicators in marine ecosystems: a proposed
early warning system. Marine Pollution Bulletin 38, 955-959.
MacKenzie, K., Williams, H.H. and Williams, B., McVicar, A.H. and Siddall, R. (1995)
Parasites as indicators of water quality and the potential use of helminth transmission in
marine pollution studies. Advances in Parasitology 35, 85-144.
Magurran, A.E. (1988) Ecological diversity and its measurement. London, Cambridge
University Press.
Marcogliese, D. J. and Cone, D. K. (1997). Parasite communities as indicators of ecosystem
stress. Parassitologia 39, 227-232.
References
95
Margaritov N. (1959) Parasites of some freshwater fishes. Publishing House NIRRP, Varna.
Margaritov N. (1966) Helminths of the digestive tract and the abdominal cavity of fishes of
the Bulgarian section of Danube River. Bulletin de L'institut de Zoologie et Muse 20,
157-173.
Marshall, J. P. (1976) Observations on the envelope surrounding Pomphorhynchus laevis
(Acanthocephala) in its intermediate host Gammarus pulex. Parasitology 73, R29-R29.
Martin, M.H. and Coughtrey, P.J. (1982) Biological Monitoring of Heavy Metal Pollution:
Land and Air. Applied Science, London.
Merian, E. (2004) Metals and their compounds in the environment. Occurrence, analysis and
biological relevance. (ed. Merian, E., Anke, M., Ihnat, M. and Stoeppler, M.), Wileyvch Verlag GmbH & Co. KGal, Weinheim.
Michalovi, M. (1954) Vsledky prpruzkumu parasit ryb v podunajsk oblasti u Komrna.
Sbor. VZL v Brn, B. 2, 67-74.
Mihalca, A. D., Gherman C., Ghira I. and Cozma V. (2007b) Helminth parasites of reptiles
(Reptilia) in Romania. Parasitology Research 101, 491-492.
Mihalca, A. D., Fictum P., kori, M., Sloboda, M., Krvemo, S., Ghira, I., Carlsson M.
and Modr , D. (2007a) Severe granulomatous lesions in several organs from
Eustrongylides larvae in a free-ranging dice Snake, Natrix tessellata. Veterinary
pathology 44, 103-105.
Molloy, S., Holland, C. and ORegan, M. (1995) Population biology of Pomphorhynchus
laevis in brown trout from two lakes in the west of Irland. Journal of Helminthology 69,
229-235.
Moog, O. (1995) Fauna Aquatica Austriaca. Wasserwirtschaftskataster. Bundesministerium
fr Land- und Forstwirtschaft, Wien.
Moravec, F. (1994) Parasitic nematodes of freshwater Fishes of Europe. Kluwer Academic
Publishers, Dordrecht.
Moravec, F. (1995) Trichopteran larvae (Insecta) as the intermediate host of Rhabdochona
hellichi (Nematoda: Rhabdochonidae), a parasite of Barbus barbus (Pisces).
Parasitology Research 81, 268-270.
Moravec, F. and Scholz, T. (1991) Observations on the biology of Pomphorhynchus laevis
(Zoega in Mller, 1776) (Acanthocephala) in the Rokytn River, Czech and Slovak
Federative Republic. Helminthologia 28, 23-29.
Moravec F. and Scholz T. (1995) Life history of the nematode Rhabdochona hellichi, a
parasite of the barbel in the Jihlava River, Czech Republic. Journal of Helminthology
69, 59-64.
References
96
Moravec, F., Konecny, R., Baska, F., Rydlo, M., Scholz, T., Molnar, K. and Schiemer, F.
(1997) Endohelminth fauna of barbel, Barbus barbus (L.), under ecological conditions
of the Danube basin in Central Europe. Publishing House of the Academy of Sciences
of the Czech Republic. Praha.
Murphy, P.M., and Learner, M.A. (1982) The life history and production of Asellus
aquaticus (Crustacea: Isopoda) in the River Ely, South Wales. Freshwater Biology 12,
435-444.
Nachev, M. and Sures, B. (2009) The endohelminth fauna of barbel (Barbus barbus)
correlates with water quality of the Danube River in Bulgaria. Parasitology 136, 545
552.
Nedeva, I., Atanassov G., Karaivanova E., Cakic P. and Lenghardt M. (2003)
Pomphorhynchus laevis (Mller, 1776) from the River Danube. Experimental Pathology
and Parasitology 6, 14-16.
Overstreet, R. M. (1997) Parasitological data as monitors of environmental health.
Parassitologia 39, 169-175.
Paunovi, M., Vassilev, V., Cheshmenjiev, S. and Simi, V. (2008) Environmental and risk
assessment of the Timok River Basin. Available at:
http://timok.rec.org/download/timok_assessment.pdf
Perez-del Olmo A., Raga J.A., Kostadinova A. and Fernandez M. (2007) Parasite
communities in Boops boops (L.) (Sparidae) after the Prestige oil-spill: Detectable
alterations. Marine Pollution Bulletin 54, 266-276.
Perrot-Minnot, M.J. (2004). Larval morphology, genetic divergence, and contrasting levels
of host manipulation between forms of Pomphorhynchus laevis (Acanthocephala).
International Journal for Parasitology 34, 45-54.
Philips, D.J.H. and Rainbow, P.S. (1993) Biomonitoring of Trace Aquatic Contaminants.
Elsevier, London.
Philips, D.J.H. and Segar, D.A. (1986) Use of bio-monitors in monitoring conservative
contaminants: programme design imperatives. Marine Pollution Bulletin 17, 10-17.
Reeders, H. H., bij de Vaate, A. and Noordhuis, R. (1993) Potential of zebra mussel
(Dreissena polymorpha) for water quality management. In Zebra Mussels: Biology,
Impacts and Control. (ed. Nalepa, T. F. and Schloesser, D. W.), pp. 439-452. CRC
press, Florida.
Riggs, M. R., Lemly, A. D. and Esch, G. W. (1987) The growth, biomass and fecundity of
Bothriocephalus acheilognathi in a Nord Carolina cooling reservoir. Journal of
References
97
Roman, R. (1955) Cercetari asupra parazitofaunei pestilor din Dunare. EA RPR, Bucuresti.
Rumpus, A. E. and Kennedy, C. R. (1974) The effect of the acanthocephalan
Pomphorhynchus laevis upon the respiration of its host, Gammarus pulex. Parasitology
68, 271-284.
Schperclaus, W. (1990) Fischkrankheiten, 5th Edn. Akademie Verlag, Berlin.
Scheef, G., Sures, B. and Taraschewski, H. (2000) Cadmium accumulation in Moniliformis
moniliformis (Acanthocephala) from experimentally infected rats. Parasitology
Research 86, 688-691.
Schludermann, C., Konecny, R., Laimgruber, S., Lewis, J. W., Schiemer, F., Chovanec,
A. and Sures, B. (2003) Fish macroparasites as indicators of heavy metal pollution in
river sites in Austria. Parasitology 126 (Suppl.) S61-S69.
Siddall, R. and Sures, B. (1998) Uptake of lead by Pomphorhynchus laevis cystacanths in
Gammarus pulex und immature worms in chub (Leuciscus cephalus). Parasitology
Research 84, 573-577.
Siddal, R., Koskivaara, M. and Valtonen E. T. (1997) Dactylogyrus (Monogenea)
infections on the gills of roach (Rutilus rutilus L.) experimentally exposed to pulp and
paper mill effluent. Parasitology 114, 439-446.
Skryabina, E.S. (1974) Gel'minty osetrovykh ryb (Acipenseridae, Bonaparte, 1831).
Izdatel'stvo Nauka, Moscow.
Strong, C.R. and Luoma, S.N. (1981) Variations in correlation of body size with
concentrations of Cu and Ag in the bivalve Macoma balthica. Canadian Journal of
Fisheries and Aquatic Sciences 38, 1059-1064.
Sures, B. (1996) Untersuchungen zur Schwermetallakkumulation von Helminten im
Vergleich zu ihren aquatischen Wirten. PhD thesis, Karlsruhe.
Sures, B. (2001) The use of parasites as bioindicators of heavy metals in aquatic ecosystems:
a review. Aquatic Ecology 35, 245-255.
Sures, B. (2003) Accumulation of heavy metals by intestinal helminths in fish: an overview
and perspective. Parasitology 126 (Suppl.) S53-S60.
Sures, B. (2004a) Environmental parasitology: relevancy of parasites in monitoring
environmental pollution. Trends in Parasitology 20, 170-177.
Sures, B. (2004b) Fish acanthocephalans of genus Pomphorhynchus sp. as globally applicable
bioindicators for metal pollution in the aquatic environment? Wiener Klinische
Wochenschrift 116: 19-23.
Sures, B. (2006) How parasitism and pollution affect the physiological homeostasis of aquatic
hosts. Journal of Helminthology 80, 151-157.
98
References
Sures, B. (2008a) Environmental Parasitology. Interactions between parasites and pollutants

in the aquatic environment. Parasite 15, 434-438.
Sures, B. (2008b) Host-parasite interactions in polluted environments. Journal of Fish
Biology 73, 2133-2142.
Sures, B. and Reimann, N. (2003) Analysis of trace metals in the Antarctic host-parasite
system Notothenia coriiceps and Aspersentis megarhynchus (Acanthocephala) caught at
King George Island, South Shetland Islands. Polar Biology 26, 680-686.
Sures, B. and Siddall, R. (1999) Pomphorhynchus laevis: the intestinal acanthocephalan as a
lead sink for its fish host, chub (Leuciscus cephalus). Experimental Parasitology 93, 6672.
Sures,
B.
and
Siddall,
R.
(2001).Comparison
between
lead
accumulation
of
Pomphorhynchus leavis (Palaeacanthocephala) in the intestine of chub (Leuciscus

cephalus) and in the body cavity of goldfish (Carassius auratus auratus). International
Journal for Parasitology 31, 669-673.
Sures, B. and Siddall, R. (2003) Pomphorhynchus laevis (Palaeacanthocaphala) in the
intestine of chub (Leuciscus cephalus) as an indicator of metal pollution. International
Journal for Parasitology 33, 65-70.
Sures, B. and Streit, B. (2001) Eel parasite diversity and intermediate host abundance in the
River Rhine, Germany. Parasitology 123, 185-191.
Sures, B. and Taraschewski, H. (1995) Cadmium concentrations of two adult
acanthocephalans (Pomphorhinchus leavis, Acanthocephalus lucii) compared to their
fish host and cadmium and lead levels in the larvae of A. lucii compared to their
crustacean host. Parasitology Research 81, 494-497.
Sures, B., Dezfuli, B.S. and Krug, H.F. (2003) The intestinal parasite Pomphorhynchus
laevis (Acanthocephala) interferes with the uptake and accumulation of lead (210Pb) in
its fish host chub (Leuciscus cephalus). International Journal for Parasitology 33,
1617-1622.
Sures, B., Franken, M. and Taraschewski, H. (2000a) Element concentrations in the
archiacanthocephalan Macracanthorhynchus hirudinaceus compared with those in the
porcine host from a slaughterhouse in La Paz, Bolivia. International Journal for
Sures, B., Grube, K. and Taraschewski, H. (2002) Experimental studies on the lead
accumulation in the cestode Hymenolepis diminuta und its final host, Rattus norvegicus.
Ecotoxicology 11, 365-368.
Sures, B., Jrges, G. and Taraschewski, H. (1998) Relative concentrations of heavy metels
References
99
in the parasites Ascaris suum (Nematoda) and Fasciola hepatica (Digenea) and their
respective porcine and bovine definitive hosts. International Journal for Parasitology
28, 1173-1178.
Sures, B., Jrges, G. and Taraschewski, H. (2000b) Accumulation and distribution of lead
in the acanthocephalan Moniliformis moniliformis from experimental infected rats.
Sures, B., Siddall, R and Taraschewski, H. (1999a) Parasites as accumulation indicators of
heavy metal pollution. Parasitology Today 15, 16-21.
Sures, B., Taraschewski, H. and Jackwerth, E. (1994a) Lead accumulation in
Pomphorhynchus laevis and its host. Journal of Parasitology 80, 355-357.
Sures, B., Taraschewski, H. and Jackwerth, E. (1994b) Lead content of Paratenuisentis
ambiguus (Acanthocephala), Anguillicola crassus (Nematodes) and their host Anguilla
anguilla. Diseases of Aquatic Organisms 19, 105-107.
Sures, B., Taraschewski, H. and Jackwerth, E. (1994c) Comparative study of lead
accumulation in different organs of perch (Perca fluviatilis) und its intestinal parasite
Acanthocephalus lucii. Bulletin of Environmental Contamination and Toxicology 52,
269-273.
Sures, B., Taraschewski, H. and Rokicki, J. (1997c) Lead und cadmium content of two
cestodes Monobothrium wageneri and Bothriocephalus scorpii, and their fish hosts.
Parasitology Research 83, 618-623.
Sures, B., Taraschewski, H. and Rydlo, M. (1997a) Intestinal fish parasites as heavy metal
bioindicators: a comparison between Acanthocephalus lucii (Palaeacanthocephala) and
the zebra mussel, Dreissena polymorpha. Bulletin of Environmental Contamination and
Toxicology 59, 14-21.
Sures, B., Taraschewski, H. and Siddall, R. (1997b) Heavy metal concentrations in adult
acanthocephalans and cestodes compared to their fish host and to established free-living
bioindicators. Parassitologia 39, 213-218.
Sures, B., Knopf, K., Wrtz, J. and Hirt, J. (1999c) Richness and diversity of parasite
communities in European eels Anguilla anguilla of the River Rhine, Germany, with
special reference to helminth parasites. Parasitology 119, 323-330.
Sures, B., Steiner, W., Rydlo, M. and Taraschewski, H. (1999b) Concentrations of 17
elements in the zebra mussel (Dreissena polymorpha), in different tissues of perch
(Perca fluviatilis), and in perch intestinal parasites (Acanthocephalus lucii) from the
subalpin lake Mondsee (Austria). Environmental Toxicology and Chemistry 18, 25742579.
100
References
Sures, B., Thielen, F., Baska, F., Messerschmidt, J. and von Bohlen, A. (2005) The
intestinal parasite Pomphorhynchus laevis as a sensitive accumulation indicator for the
platinum group metals Pt, Pd and Rh. Environmental Research 98, 83-88.
Szefer, P., Rokicki, J., Frelek, K., Skra, K and Malinga, M. (1998) Bioaccumulation of
selected trace elements in lung nematodes, Pseudalius inflexus, of harbor porpoise
(Phocoena phocoena) in a Polish zone of the Baltic Sea. The Science of the Total
Environment 220, 19-24.
Tenora, F., Krmar, S., Baru, V and Dvoek, J. (1997) Some inorganic substances in
plerocercoids of Ligula intestinalis (Pseudophyllidea). Acta Universitatis Agriculturae
et Silviculturae Mendelianae Brunensis 45, 23-30.
Thielen, F., Mnderle, M., Taraschewski, H. and Sures, B. (2007) Do eel parasites reflect
the local crustacean community? A case study from the Rhine river system. Journal of
Helminthology 81, 179-189.
Thielen, F., Zimmermann, S., Baska, F., Taraschewski, H. and Sures, B. (2004) The
intestinal parasite Pomphorhynchus laevis (Acanthocephala) from barbel as a
bioindicator for metal pollution in the Danube River near Budapest, Hungary.
Environmental Pollution 129, 421-429.
TNMN (1996) TransNational Monitoring Network. Available at: http://www.icpdr.org/icpdrpages/tnmn.htm, (accessed 20.01.2010).
TNMN
(2009)
TransNational
Monitoring
Network
Database
available
http://danubis.icpdr.org/pls/danubis/DANUBIS_DB.DYN_NAVIGATOR.show
at
Last
update 22.07.2009, (accessed 16.09.2009.)

Turekov, L and Hanzelov, V. (1996) Concentrations of heavy metals in the cestode
Protocephalus percae, parasite of perch. Helminthologia 37, 162-163.
Umweltbundesamt (1992) Daten zur Umwelt 1990/91.
Valtonen, E. T., Holmes, J. C. and Koskivaara, M. (1997) Eutrophication, pollution, und
fragmentation : effects on parasite communities in roach (Rutilus rutilus) and perch
(Perca fluviatilis) in four lakes in central Finland. Canadian Journal of Fisheries and
Aquatic Sciences 54, 572-585.
Vidal-Martinez, V.M., Pech, D., Sures, B., Purucker, T. and Poulin, R. (2010) Can
parasites really reveal environmental impact? Trends in Parasitology 26, 44-51.
Wang, W.X. and Fisher, N.S. (1997) Modeling the influence of body size on trace element
accumulation in the mussel Mytilus edulis. Marine Ecology Progress Series 161, 103115.
WFD (Water Framework Directive) (2000) Official Publication of the European
References
101
Community, Brussels
Zimmermann, S., Sures, B and Taraschewski, H. (1999) Experimental studies on lead
accumulation in the eel specific endoparasites Anguillicola crassus (Nematoda) and
Paratenuisentis ambiguus (Acanthocephala) as comared with their host, Anguilla
anguilla. Archives of Environmental Contamination and Toxicology 37, 190-195.
Zimmermann, S., Menzel, C., Berner, Z., Eckhardt, J. D., Stben, D., Alt, F.,
Messerschmidt, J., Taraschewski, H. and Sures, B. (2001) Trace analysis of platinum
in biological samples: a comparison between high resolution inductively coupled
plasma mass spectrometry (HR-ICP-MS) following microwave digestion and adsorptive
cathodic stripping voltammetry (ACSV) after high pressure ashing. Analytica Chimica
Acta 439/2, 203209.
Appendix
Appendices are available on the enclosed CD-ROM
Appendix I
Parasitological data of barbel.
Appendix I-A
Prasitological data of barbel collected from sampling site Vidin.
Appendix I-B
Prasitological data of barbel collected from sampling site Kozloduy.
Appendix I-C
Prasitological data of barbel collected from sampling site Silistra.
Appendix II
Element concentrations in barbel tissues and P. laevis used in Chapter 2.
Appendix II-A
Element concentrations in host tissues and P. laevis of heavily infected

barbels.
Appendix II-B
Element concentrations in host tissues and P. laevis of slightly infected

barbels.
Appendix II-C
Element concentrations in host tissues and females and males of P. laevis.
Appendix III
Water temperature data at two localities of the Danube River for the period
2005-2006.
Appendix IV
Morphological data of P. laevis used in Chapter 3.
Appendix
103
Appendix I. Parasitological data of barbel.

In the Appendix were used the following abbreviations:
N:
Barbels number in the particular sample
S:
Season (1 spring; 2 summer; 3 autumn)
W:
Fish weight (g)
TL:
Total length (cm)
ST:
Standard length (cm)
BH:
Body height (cm)
K:
Condition factor
P.l.:
Pomphorhynchus laevis
n = number of parasites
L.p.:
Leptorhynchoides plagicephalus
A.a.:
Acanthocephalus anguillae
R.h.:
Rhabdochona hellichi
P.t.:
Pseudocapillaria tomentosa
E.sp.: Eustrongylides sp.
H.sp.: Hysterothylacium sp.
P.c.:
Postodiplostomum cuticola
+ = yes, 0 = no
D.s.:
Diplostomum spathaceum
+ = yes, 0 = no
M.y.: Metagonimus yokogawai
+ = yes, 0 = no
S
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
W
417
1350
1335
1515
1503
740
520
1816
1752
443
435
332
336
1164
432
406
629
876
1685
1095
400
575
314
255
300
800
602
1315
TL
37.5
55.6
54.5
55
53.9
43.3
41.3
60.1
56.6
39
37.6
33
33.1
53.8
37.3
35.3
41.5
45.9
54
49.7
37.4
40.4
34.9
30.9
33.4
45.5
42.6
51.3
SL
30.6
46.6
44.3
45.1
45.8
35.4
33.7
49.4
47.3
31.7
30.8
27.3
26.8
44.3
29.9
29.8
33.8
37.6
46.6
42
30.4
33.7
28.5
26.2
28.4
39.5
35.5
42.6
BH
6.5
10
10.7
11.1
11.9
8.8
8.6
11.5
12.7
7.9
7.3
604
6
9.6
6.9
7.4
8.1
9
11.7
9.7
6.9
7.6
6.2
5.9
6.5
8.5
8.3
10.6
K
0.79
0.79
0.82
0.91
0.96
0.91
0.74
0.84
0.97
0.75
0.82
0.92
0.93
0.75
0.83
0.92
0.88
0.91
1.07
0.89
0.76
0.87
0.74
0.86
0.81
0.85
0.78
0.97
P.l.
125
138
168
283
231
4
191
93
54
76
154
128
26
232
47
183
23
24
223
329
312
166
229
64
133
344
37
91
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
R.h.
0
1
0
0
0
1
3
7
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
P.t.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
E.sp. H.sp.
0
0
0
0
0
0
16
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
22
0
0
0
0
0
0
0
0
0
7
0
5
0
1
0
0
0
0
0
1
0
0
0
0
0
1
0
4
0
P.c.
0
0
0
0
0
0
+
0
0
0
0
+
0
0
0
0
0
+
0
0
0
0
0
+
0
0
+
0
D.s.
0
+
+
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
M.y.
0
0
0
0
0
0
+
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
104
N
1
2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1
2
3
4
5
6
7
8
9
10
Appendix
Appendix I-A. Parasitological data of barbel collected from sampling site Vidin.
W
335
1640
2145
645
238
1563
1521
1446
167
81
208
303
155
216
184
392
1193
1605
725
2390
423
454
296
216
267
305
144
108
372
359
TL
35.6
56.8
63.7
42.9
31.2
54.8
55.4
52.9
27.8
23.4
31.7
34.6
28.4
41.5
28.7
37.6
52.3
56.2
48.5
67.2
34.5
35.3
32.7
30.4
30.9
33.8
27.4
25.5
35.1
35.1
SL
29.2
47.7
52.2
35.3
25.8
45.6
45.6
43.4
23
19
25.6
27.8
22.4
25.3
23.5
30
44
47.6
40
55.5
27.6
29.3
26.5
25.3
25.3
27.5
22.4
20.8
29
28.9
BH
5.6
11.7
11.7
8.4
5.6
11.2
10.3
10.6
5.3
4.1
5.9
6.2
5.3
5.9
5.4
6.7
9.8
10.5
8.7
12.2
7.5
7.4
6.3
5.4
6.5
6.6
4.8
4.2
6.3
6.6
K
0.74
0.89
0.83
0.82
0.78
0.95
0.89
0.98
0.78
0.63
0.65
0.73
0.68
0.30
0.78
0.74
0.83
0.90
0.64
0.79
1.03
1.03
0.85
0.77
0.90
0.79
0.70
0.65
0.86
0.83
P.l.
243
216
191
219
54
90
401
91
28
128
27
112
158
30
10
36
116
227
194
874
66
5
36
37
126
162
71
18
14
220
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
R.h.
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
P.t.
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
3
0
0
0
0
0
0
0
0
0
0
0
0
0
E.sp. H.sp.
0
0
2
0
0
0
0
0
0
0
0
0
24
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
91
0
0
0
93
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
P.c.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
D.s.
0
0
0
+
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
M.y.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
105
S
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
Appendix
N
11
12
13
14
15
16
17
18
1
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
9
10
W
442
372
760
282
350
242
444
330
378
385
384
610
1742
3909
752
1685
1095
400
575
314
255
300
800
602
1315
335
2145
645
238
1563
TL
37.8
35.4
43.3
32.4
34
30.8
35.6
36.4
35
36.1
33.4
40.5
56.3
72
44.6
54
49.7
37.4
40.4
34.9
30.9
33.4
45.5
42.6
51.3
35.6
63.7
42.9
31.2
54.8
SL
30.4
28.5
36
26.5
27.5
25
29.2
30.3
27.7
29.4
27.5
32.5
47.2
60.8
37.3
46.6
42
30.4
33.7
28.5
26.2
28.4
39.5
35.5
42.6
29.2
52.2
35.3
25.8
45.6
BH
6.7
7.3
8.2
6.2
6.9
5.9
7.3
6.6
6.6
6.8
6.8
8.2
11.2
14
8.3
11.7
9.7
6.9
7.6
6.2
5.9
6.5
8.5
8.3
10.6
5.6
11.7
8.4
5.6
11.2
K
0.82
0.84
0.94
0.83
0.89
0.83
0.98
0.68
0.88
0.82
1.03
0.92
0.98
1.05
0.85
1.07
0.89
0.76
0.87
0.74
0.86
0.81
0.85
0.78
0.97
0.74
0.83
0.82
0.78
0.95
P.l.
136
18
21
99
17
62
124
445
69
90
197
32
363
273
24
223
329
312
166
229
64
133
344
37
91
243
191
219
54
90
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
A.a.
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
R.h.
0
0
0
0
0
3
0
2
0
7
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
P.t.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
E.sp. H.sp.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
7
0
26
0
1
0
7
0
5
0
1
0
0
0
0
0
1
0
0
0
0
0
1
0
4
0
0
0
0
0
0
0
0
0
0
0
P.c.
0
0
0
+
0
0
0
0
0
+
0
0
0
0
0
+
0
+
0
0
0
+
+
+
0
0
0
0
0
+
D.s.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
M.y.
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
+
0
0
0
0
0
0
+
0
0
+
0
+
0
106
S
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Appendix
N
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
W
1521
1446
1640
2145
645
238
1563
1521
1446
490
415
640
355
260
400
435
270
405
600
295
475
260
305
230
200
370
375
155
355
610
TL
55.4
52.9
56.8
63.7
42.9
31.2
54.8
55.4
52.9
40
37.8
36.9
36.3
31.9
35.6
37.2
31.4
37.6
40.4
34.6
39
31.4
33.5
30.1
30.4
35.7
36.6
28.2
34.5
36.5
SL
45.6
43.4
47.7
52.2
35.3
25.8
45.6
45.6
43.4
33.3
31.1
29.7
29.8
26.3
29.8
30.8
26.4
30.6
33.2
28.2
31.4
25.8
28
25.1
25
29.8
30.4
23.3
29.2
30.5
BH
10.3
10.6
11.7
11.7
8.4
5.6
11.2
10.3
10.6
6.6
7.2
6.9
6.3
6.3
6.7
6.6
5.9
6.7
7.5
6.3
7.7
6
5.9
5.5
5.7
6.4
6.7
4.5
6.7
6.6
K
0.89
0.98
0.89
0.83
0.82
0.78
0.95
0.89
0.98
0.77
0.77
1.27
0.74
0.80
0.89
0.85
0.87
0.76
0.91
0.71
0.80
0.84
0.81
0.84
0.71
0.81
0.76
0.69
0.86
1.25
P.l.
401
91
216
191
219
54
90
401
91
95
71
53
33
1
59
41
4
74
92
54
6
1
14
86
79
95
29
89
9
22
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
R.h.
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
11
0
1
0
0
0
0
0
0
0
0
P.t.
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
E.sp. H.sp.
24
0
0
0
2
0
0
0
0
0
0
0
0
0
24
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
P.c.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
D.s.
0
0
0
0
0
+
0
0
0
0
0
0
0
+
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
M.y.
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
+
0
0
107
S
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Appendix
N
16
17
18
19
20
21
22
23
24
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
W
440
280
390
230
680
530
420
240
250
140
1000
1050
655
470
890
960
655
870
790
535
625
980
940
960
870
865
835
660
1000
635
TL
38.2
30.8
35.1
30.3
37.6
37.7
36.2
31.5
32
26.3
47.6
46.4
42.3
37.7
47.4
48.6
43
45.2
46.9
37.2
42.8
48.3
48.2
46.2
47.5
44.5
43.1
39.8
48.4
41.4
SL
31
26
29.2
24.5
30.9
31.8
30.5
25.5
26.3
21.6
39.8
40
35.6
30.8
39.3
39.4
35.9
37.8
39
30.8
34.8
40.5
38.6
39.3
40.2
36.5
36
32.3
40
34.5
BH
7.1
6.5
6.3
5.2
7.4
7.8
7.5
5.6
5.9
4.9
8.9
9.5
8
7.4
9.5
9.9
8.1
9.4
8.4
7.9
7.3
9.5
9.3
8.5
8.6
8.4
8.9
7.5
8.9
7.6
K
0.79
0.96
0.90
0.83
1.28
0.99
0.89
0.77
0.76
0.77
0.93
1.05
0.87
0.88
0.84
0.84
0.82
0.94
0.77
1.04
0.80
0.87
0.84
0.97
0.81
0.98
1.04
1.05
0.88
0.89
P.l.
15
168
17
44
5
45
29
5
152
37
149
101
110
40
16
313
142
26
61
4
223
66
24
31
575
173
88
230
98
121
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
R.h.
0
0
14
0
1
21
0
0
0
4
7
0
1
4
0
0
0
9
44
52
39
0
74
0
0
207
26
8
0
0
P.t.
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
E.sp. H.sp.
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
5
0
0
0
0
0
0
0
0
0
5
0
P.c.
0
0
0
0
0
0
+
0
0
0
+
+
0
0
0
0
0
+
+
0
0
0
+
0
+
0
0
0
+
0
D.s.
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
M.y.
+
0
+
+
0
+
0
+
0
0
0
+
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
108
S
3
3
3
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Appendix
N
22
23
24
25
26
27
28
29
30
31
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
S
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
W
1035
745
1325
630
275
220
265
245
330
210
405
370
215
215
207
1000
835
TL
49.3
44.9
52
42.8
32.8
30.8
33
31.2
34.5
29.3
36
35.9
28.7
29.2
30
47.7
45.2
SL
40
36.8
42.7
35
27.5
24.8
27.2
25.5
27.7
24.3
30
29.2
24.2
24
24.6
40.4
37.8
BH
9
7.5
10.6
7.9
5.7
5
5.3
5.4
6
5.5
7.1
6.5
6
6.1
5.3
9.6
8.3
K
0.86
0.82
0.94
0.80
0.78
0.75
0.74
0.81
0.80
0.83
0.87
0.80
0.91
0.86
0.77
0.92
0.90
P.l.
175
293
68
2
40
32
52
21
22
67
367
24
130
177
74
84
63
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
R.h.
0
56
1
3
1
0
6
2
0
0
0
0
3
0
3
0
1
P.t.
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
E.sp. H.sp.
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
P.c.
+
0
0
0
0
0
0
0
0
0
0
0
+
0
0
+
+
D.s.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
M.y.
+
0
0
+
0
+
0
0
0
0
0
0
0
0
0
0
0
Appendix
N
21
22
23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
109
6031B
6048B
605B
6082B
609B
61B
613B
6150B
617B
629B
625B
6235B
6218B
6201B
6184B
6270B
6253B
623B
6219B
620B
6185B
618B
615B
6134B
617B
610B
6083B
60B
6049B
6032B
W
153
396
252
256
460
315
227
151
531
540
202
297
2208
1336
390
375
525
390
373
437
586
418
624
360
283
561
549
410
375
195
603B
TL
25.7
35.3
34.1
31.1
39.5
33.6
31.3
26.4
39.3
39.3
30
32
60.5
51
33.8
34.6
41.4
34.9
35.8
37
40.3
36.7
41
33.7
32.5
40.1
42
35.5
34.1
29
605B
6034B
6052B
608B
6084B
609B
6085B
610B
608B
6102B
618B
6103B
619B
6135B
6120B
613B
6152B
6137B
6153B
619B
6154B
6170B
618B
617B
6187B
6203B
618B
6204B
620B
6205B
621B
6237B
6271B
6035B
6051B
607B
6254B
SL
21
28.5
28.3
24.6
32.9
26.9
25.9
21.8
32
31.9
24.4
27.1
51.5
41.2
28.7
28.6
34
28.5
29.2
29.8
33.6
30.2
33.5
28.8
26.3
33.2
34.1
30
27.6
24.2
6238B
62B
627B
6273B
625B
6239B
625B
BH
4.5
6.8
4.8
5.3
6.4
6.6
4.9
4.7
7.8
7.1
5.2
6
12
10.5
7.3
7.2
7.3
7
6.8
7
7.7
7.2
8.1
6.9
6
7.8
7.4
7.2
7
5.2
603B
K
0.90
0.90
0.64
0.85
0.75
0.83
0.74
0.82
0.87
0.89
0.75
0.91
1.00
1.01
1.01
0.91
0.74
0.92
0.81
0.86
0.90
0.85
0.91
0.94
0.82
0.87
0.74
0.92
0.95
0.80
6053B
6037B
6038B
6054B
607B
605B
6071B
6087B
6072B
608B
6104B
6089B
6105B
612B
610B
612B
6138B
6123B
6139B
615B
6140B
615B
6172B
6157B
6173B
6189B
6174B
6190B
620B
619B
6207B
623B
6208B
624B
6240B
625B
6275B
6258B
6241B
6257B
6274B
P.l.
49
182
28
20
41
124
23
101
55
121
40
32
0
4
43
53
40
27
46
59
44
128
148
174
207
32
115
85
107
41
624B
6259B
627B
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
627B
620B
6243B
62B
6209B
6192B
6175B
6158B
614B
6124B
6107B
609B
6073B
605B
6039B
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6278B
621B
624B
627B
6210B
6193B
617B
6159B
6142B
6125B
6108B
6091B
6074B
6057B
604B
R.h.
2
2
1
27
51
1
3
0
9
0
0
5
3
2
1
8
0
6
12
12
1
0
0
0
0
6
0
6
0
2
6041B
6058B
6075B
6092B
6109B
6279B
62B
6245B
628B
621B
6194B
617B
610B
6143B
612B
P.t.
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
1
0
6280B
623B
624B
629B
621B
6195B
6178B
61B
614B
6127B
610B
6093B
607B
6059B
6042B
E.sp. H.sp.
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
602B
6027B
6043B
604B
60B
601B
607B
6078B
6094B
6095B
61B
612B
6128B
6129B
6145B
614B
612B
613B
6179B
6180B
619B
6197B
6213B
6214B
6230B
6231B
6247B
6248B
624B
625B
6281B
628B
P.c.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
+
0
0
0
0
+
0
0
0
0
+
0
0
6028B
6045B
602B
6079B
609B
613B
6130B
6147B
614B
618B
6198B
6215B
623B
6249B
6283B
62B
D.s.
0
0
0
0
+
0
0
0
0
0
0
0
+
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
6029B
604B
603B
608B
6097B
6284B
627B
6250B
623B
621B
619B
6182B
615B
6148B
613B
614B
M.y.
0
0
+
0
0
0
0
+
0
+
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
+
0
0
0
603B
6047B
604B
6081B
6098B
615B
6132B
6149B
61B
6183B
620B
6217B
6234B
6285B
628B
6251B
110
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
S
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Appendix
Appendix I-B. Parasitological data of barbel collected from sampling site Kozloduy.
630B
6320B
637B
6354B
6371B
638B
6405B
642B
6439B
645B
6473B
6490B
6507B
6524B
6541B
658B
657B
6592B
609B
62B
643B
60B
67B
694B
W
546
145
252
384
1309
966
1120
316
276
350
390
463
779
1070
270
483
480
580
536
1203
1310
1290
1135
1280
766
1155
965
1653
575
470
500
545
451
635B
635B
6372B
6389B
6423B
6457B
641B
6475B
6491B
6508B
6542B
654B
657B
650B
6593B
657B
610B
6594B
6578B
659B
627B
61B
628B
64B
629B
61B
645B
62B
678B
63B
695B
679B
69B
6712B
697B
6729B
6713B
6730B
674B
6731B
674B
674B
675B
6781B
6849B
6832B
6815B
6798B
679B
681B
6850B
683B
6851B
6834B
6817B
680B
6783B
67B
6782B
6718B
6734B
6750B
6735B
6751B
67B
6752B
6801B
681B
6835B
6853B
683B
6819B
6802B
6785B
678B
6784B
6852B
6701B
671B
673B
6749B
684B
670B
671B
6732B
6748B
67B
683B
69B
6715B
650B
6B
682B
698B
6714B
63B
649B
65B
681B
61B
632B
648B
64B
680B
659B
615B
631B
647B
6582B
6598B
614B
630B
64B
65B
6581B
6597B
613B
6548B
654B
6580B
659B
612B
6531B
6547B
653B
6579B
6514B
6530B
654B
652B
6497B
6513B
6529B
654B
651B
6480B
649B
6512B
6528B
643B
6479B
6495B
651B
6527B
6543B
659B
684B
6510B
652B
64B
642B
6478B
649B
6429B
645B
641B
647B
6493B
6509B
652B
6831B
647B
6492B
6412B
6428B
64B
640B
6395B
641B
6427B
643B
6459B
6378B
6394B
6410B
642B
642B
6458B
647B
6814B
6425B
631B
637B
639B
6409B
634B
630B
637B
6392B
6408B
642B
640B
679B
6391B
6407B
6327B
634B
6359B
6375B
6310B
632B
6342B
6358B
6374B
6390B
640B
6780B
6357B
637B
6293B
6309B
6325B
6341B
P.l.
154
68
24
103
69
168
142
98
81
173
122
98
119
252
128
45
53
44
170
80
222
36
138
78
106
31
63
257
189
69
68
11
33
629B
6308B
6324B
6340B
K
1.00
0.76
0.83
0.84
0.88
0.85
0.86
1.00
0.99
0.76
0.86
0.96
0.90
0.86
0.84
0.99
0.79
0.87
0.95
0.87
0.86
0.98
0.86
1.07
0.77
0.79
0.80
0.91
0.77
0.38
0.76
0.74
0.70
6291B
6307B
632B
639B
BH
7.4
5.3
5.9
6.4
10.4
9.5
10
6.4
5.7
5.7
7.1
7.9
8.8
9.6
5.8
7.4
6.7
8.2
7.3
9.6
10
9.8
9.4
10.2
8.5
9.4
9.3
10.6
7.6
7.3
7.8
7.6
7.5
6290B
630B
632B
638B
SL
31
22.2
26.6
30.2
44.4
40.8
41.1
27.3
25.4
28.9
29.3
30.4
36.4
41
26.4
31.5
31.7
32.8
32.8
43.2
44.2
43.8
42.8
41.4
37.8
44.8
40.4
49
35.5
32.7
33.4
34.3
33.2
6289B
6305B
6321B
673B
TL
38
26.7
31.2
35.8
52.9
48.4
50.6
31.6
30.3
35.8
35.7
36.4
44.2
49.9
31.8
36.5
39.3
40.6
38.4
51.7
53.4
50.9
50.9
49.2
46.4
52.6
49.5
56.7
42.1
49.9
40.3
42
40
628B
6304B
679B
678B
6803B
6820B
6854B
6837B
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6294B
685B
683B
6821B
6804B
678B
670B
6753B
673B
6719B
6702B
685B
68B
651B
634B
617B
60B
6583B
65B
6549B
6532B
651B
6498B
6481B
64B
647B
6430B
6413B
639B
6379B
632B
6345B
6328B
631B
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
6295B
685B
6839B
682B
6805B
678B
671B
6754B
673B
6720B
6703B
68B
69B
652B
635B
618B
601B
6584B
657B
650B
653B
651B
649B
6482B
645B
648B
6431B
641B
6397B
6380B
63B
634B
6329B
6312B
R.h.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
1
0
0
0
2
629B
631B
630B
6857B
6840B
6823B
680B
6789B
672B
675B
6738B
6721B
6704B
687B
670B
653B
63B
619B
602B
658B
658B
651B
6534B
6517B
650B
6483B
64B
649B
6432B
6415B
6398B
6381B
634B
6347B
P.t.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
6297B
685B
6841B
6824B
6807B
6790B
673B
675B
6739B
672B
6705B
68B
671B
654B
637B
620B
603B
658B
659B
652B
653B
6518B
6501B
648B
647B
6450B
643B
641B
639B
6382B
635B
6348B
631B
6314B
E.sp. H.sp.
7
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
26
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
21
0
0
0
4
0
68
0
0
0
0
0
4
0
16
0
0
0
0
0
6298B
629B
6315B
631B
632B
63B
6349B
6350B
63B
637B
638B
6384B
640B
6401B
6417B
6418B
643B
6435B
6451B
6452B
648B
649B
6485B
648B
6502B
6503B
6519B
6520B
653B
6537B
653B
654B
6570B
6571B
6587B
658B
604B
605B
621B
62B
638B
639B
65B
65B
672B
673B
689B
690B
670B
670B
6723B
6724B
6740B
6741B
675B
6758B
674B
675B
6791B
6792B
680B
6809B
6825B
682B
6842B
6843B
6859B
680B
P.c.
+
0
0
0
0
0
0
0
0
+
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
630B
6317B
634B
6351B
638B
6385B
6402B
6419B
643B
6453B
6470B
6487B
6504B
6521B
6538B
65B
6572B
6589B
60B
623B
640B
657B
674B
691B
6708B
6725B
6742B
6759B
67B
6793B
6810B
6827B
681B
684B
D.s.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6301B
682B
6845B
682B
681B
6794B
67B
670B
6743B
672B
6709B
692B
675B
658B
641B
624B
607B
6590B
6573B
65B
6539B
652B
650B
648B
6471B
645B
6437B
6420B
6403B
638B
639B
6352B
635B
6318B
M.y.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6302B
6319B
63B
635B
6370B
6387B
640B
6421B
6438B
645B
6472B
6489B
650B
6523B
6540B
657B
6574B
6591B
608B
625B
642B
659B
67B
693B
6710B
672B
674B
671B
678B
6795B
6812B
683B
684B
6829B
111
6847B
6830B
6813B
679B
679B
672B
6745B
6728B
671B
S
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
6287B
Appendix
N
17
18
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
1
2
3
4
5
6
7
8
9
628B
681B
689B
6915B
6932B
694B
69B
6983B
70B
701B
7034B
7051B
7068B
7085B
7102B
719B
7136B
7153B
710B
718B
7204B
721B
7238B
725B
72B
7289B
W
362
581
215
1047
1785
1224
1350
1686
1270
1370
332
410
500
235
385
605
315
385
350
385
445
320
210
385
415
465
415
300
650
193
125
805
645
689B
691B
7426B
7409B
7392B
735B
7358B
7341B
7324B
730B
7290B
723B
7256B
7239B
72B
7205B
718B
71B
7154B
713B
7120B
698B
7053B
70B
7054B
705B
701B
708B
702B
708B
7104B
7089B
7105B
712B
7106B
712B
7138B
7123B
7139B
715B
7140B
7156B
712B
715B
713B
7189B
714B
7190B
7206B
719B
720B
723B
7208B
724B
7240B
725B
7241B
725B
724B
7258B
724B
7259B
725B
7291B
726B
729B
7308B
7293B
7309B
7325B
7310B
7326B
7342B
732B
734B
7359B
734B
7360B
736B
7361B
73B
739B
738B
7394B
7410B
742B
7038B
7395B
7428B
741B
7429B
7412B
6905B
692B
693B
695B
695B
6972B
6973B
698B
690B
706B
702B
70B
7023B
7039B
7024B
704B
7056B
7041B
705B
703B
7058B
709B
704B
705B
7091B
710B
7092B
7108B
7124B
7109B
7125B
714B
7126B
7142B
7158B
7143B
7159B
715B
7160B
716B
7192B
71B
7193B
7209B
7194B
7210B
726B
721B
7243B
72B
7294B
731B
7328B
7345B
7362B
739B
7396B
7413B
7431B
741B
739B
7380B
736B
7346B
7329B
7312B
7295B
728B
7261B
724B
72B
7260B
7430B
68B
6938B
705B
7021B
703B
6871B
6921B
698B
704B
702B
7036B
6904B
6971B
6987B
703B
6903B
6954B
6970B
P.l.
8
150
61
140
237
307
33
258
60
3
25
18
157
27
114
82
32
88
70
125
179
151
46
20
98
88
76
99
91
15
9
5
73
6870B
687B
6937B
6953B
69B
K
0.71
0.81
0.67
0.77
1.41
0.86
0.86
0.92
0.76
0.75
0.77
1.04
0.87
0.96
0.79
0.82
0.91
0.88
1.06
0.95
0.92
0.89
1.02
0.90
0.90
0.98
0.93
0.92
0.93
0.96
0.85
0.96
0.86
68B
6920B
693B
6952B
7019B
BH
6.9
7.9
5.7
10.3
11.9
10.3
10.9
11.5
10.2
10.3
6.5
7.2
8
5.4
6.9
7.6
6.2
6.8
7.2
6.9
7.9
7
5.9
6.4
6.8
8.2
7.6
6.5
8.1
4.9
4.4
7.8
7.3
689B
691B
6935B
702B
7018B
7103B
6902B
6918B
6985B
701B
7086B
6901B
698B
6984B
7069B
690B
6951B
697B
7052B
685B
68B
684B
6934B
6950B
SL
30.4
33.5
25.6
42.2
49.9
43.4
44.6
47.2
46
47
28.1
28.2
31.8
23.5
29.6
34.5
26.7
29
27
28.1
29.6
26.7
22.4
28.9
29.3
29.7
29.5
26.5
33.6
22.5
20
36
34.9
687B
683B
6917B
693B
7035B
TL
37.1
41.6
31.7
51.4
50.2
52.3
54
56.9
55.2
56.8
35
34
38.6
29
36.5
42
32.6
35.2
32.1
34.3
36.5
33
27.4
35
35.9
36.2
35.5
32
41.2
27.2
24.5
43.7
42.1
68B
682B
728B
7245B
726B
729B
7296B
731B
730B
734B
7364B
7381B
7398B
7432B
7415B
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6872B
743B
7416B
739B
7382B
7365B
7348B
731B
7314B
729B
7280B
7263B
7246B
729B
721B
7195B
718B
716B
714B
712B
710B
7093B
706B
7059B
7042B
7025B
708B
691B
6974B
6957B
6940B
6923B
690B
689B
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6873B
743B
741B
740B
738B
736B
7349B
732B
7315B
7298B
7281B
7264B
724B
7230B
7213B
7196B
719B
7162B
7145B
7128B
71B
7094B
70B
706B
7043B
7026B
709B
692B
6975B
6958B
6941B
6924B
6907B
6890B
R.h.
5
0
0
0
0
0
2
1
1
0
0
0
1
1
0
0
114
0
0
1
1
0
0
0
0
9
3
3
1
105
136
7
1
6874B
6891B
6908B
6925B
6942B
695B
697B
693B
701B
702B
704B
7061B
708B
7095B
712B
7129B
7146B
7163B
7180B
719B
7214B
7231B
7248B
7265B
728B
729B
7316B
73B
7350B
736B
7384B
7401B
7435B
7418B
P.t.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
6875B
7436B
7419B
7402B
7385B
7368B
7351B
734B
731B
730B
7283B
726B
7249B
723B
7215B
7198B
718B
7164B
714B
7130B
713B
7096B
709B
7062B
7045B
7028B
701B
694B
697B
690B
6943B
692B
690B
6892B
E.sp. H.sp.
0
0
11
0
0
0
11
0
31
0
37
0
1
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
687B
687B
6893B
6894B
6910B
691B
6927B
6928B
694B
6945B
691B
692B
6978B
697B
695B
69B
7012B
7013B
7029B
703B
7046B
704B
7063B
7064B
708B
7081B
709B
7098B
714B
715B
713B
7132B
7148B
7149B
7165B
716B
7182B
7183B
719B
720B
7216B
721B
723B
7234B
7250B
7251B
726B
7268B
7284B
7285B
7301B
7302B
7318B
7319B
735B
736B
7352B
735B
7369B
730B
7386B
738B
7403B
740B
7420B
7421B
743B
7438B
P.c.
0
0
0
0
0
+
0
0
0
0
+
0
0
0
0
+
0
0
0
0
0
+
0
0
0
0
0
0
+
0
0
0
0
687B
6895B
6912B
692B
694B
693B
6980B
697B
7014B
7031B
7048B
7065B
7082B
709B
716B
713B
7150B
716B
7184B
7201B
7218B
7235B
725B
7269B
7286B
730B
7320B
73B
7354B
731B
7439B
742B
7405B
738B
D.s.
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
6879B
689B
6913B
6930B
6947B
694B
6981B
698B
7015B
7032B
7049B
706B
7083B
710B
71B
7134B
715B
7168B
7185B
720B
7219B
7236B
7253B
720B
740B
7423B
7406B
7389B
732B
735B
738B
7321B
7304B
728B
M.y.
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
+
+
0
0
0
0
0
680B
6897B
6914B
6931B
6948B
695B
6982B
69B
7016B
703B
705B
706B
7084B
710B
718B
7135B
7152B
7169B
7186B
7203B
720B
723B
7254B
721B
728B
7305B
732B
739B
7356B
73B
7390B
741B
742B
740B
112
7425B
7408B
7391B
734B
735B
7340B
732B
7306B
S
3
3
3
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
685B
Appendix
N
10
11
12
13
14
15
16
17
18
19
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
1
2
3
4
5
6
7
8
684B
W
710
255
270
1360
855
720
985
685
1000
470
475
440
465
195
580
375
440
395
300
330
625
410
375
360
565
570
385
240
380
405
280
320
455
746B
7493B
7510B
752B
754B
7561B
758B
759B
7612B
7629B
764B
763B
7680B
769B
714B
731B
748B
765B
782B
79B
7816B
783B
7850B
786B
784B
7901B
7918B
803B
7986B
796B
7952B
7935B
74B
749B
804B
798B
790B
7953B
7936B
791B
7902B
785B
786B
7851B
7834B
781B
780B
783B
76B
749B
732B
715B
7698B
7681B
764B
7632B
7648B
765B
76B
7682B
769B
716B
70B
71B
73B
750B
751B
76B
784B
785B
7801B
781B
7835B
7819B
7836B
7852B
7869B
780B
786B
7903B
7904B
7920B
793B
7938B
805B
798B
791B
7954B
792B
806B
807B
7942B
7958B
794B
795B
795B
791B
80B
7908B
7925B
7941B
795B
790B
7891B
7924B
7940B
793B
798B
784B
790B
7923B
7956B
785B
7890B
7906B
793B
795B
7840B
783B
789B
792B
7823B
7856B
782B
7905B
7921B
789B
7806B
7839B
785B
78B
72B
782B
783B
781B
78B
75B
7805B
7821B
7854B
738B
78B
7804B
783B
7853B
721B
71B
78B
7820B
704B
754B
70B
7803B
768B
73B
753B
786B
7802B
760B
720B
736B
769B
7653B
703B
719B
752B
768B
763B
768B
702B
735B
7619B
769B
7685B
718B
734B
7602B
7652B
768B
701B
758B
7635B
7651B
7684B
7568B
7618B
7634B
76B
7683B
751B
7601B
761B
7650B
7534B
7584B
760B
763B
7649B
751B
756B
7583B
761B
750B
750B
756B
759B
7615B
7483B
753B
7549B
7582B
749B
7516B
7532B
756B
P.l.
197
74
55
18
13
55
37
18
9
4
94
18
10
69
174
112
46
109
63
60
235
9
127
69
38
63
6
77
28
43
60
68
120
746B
749B
751B
7548B
7598B
7631B
7482B
7498B
7531B
7581B
7614B
7481B
7514B
7564B
759B
7465B
748B
746B
7480B
754B
K
1.08
0.97
1.00
1.00
0.91
0.83
0.92
1.04
1.05
1.05
0.94
0.96
0.90
0.95
0.89
0.95
0.90
1.13
0.39
0.94
0.95
0.77
0.84
0.89
0.88
0.90
1.04
0.97
0.88
0.91
0.90
0.89
1.06
74B
749B
7530B
7580B
BH
8.5
5.6
5.2
9.2
7.5
8.4
9.1
8.2
8.8
7
7.7
7.2
7.5
5.5
8.3
7.2
7.4
7.6
6.8
6.9
9
6.4
6.9
6.9
8.2
7.6
6.6
5.9
6.6
6.6
5.7
6.3
6.9
746B
7513B
7563B
759B
SL
33.4
24
24.5
43.5
37.8
36.4
40.4
33.8
39
29.2
31
29.5
30
22.1
33.5
27.6
29.5
28.3
27.4
26.8
32.8
31.5
29
27.8
33.5
32
27.8
23.8
28.5
29.8
26
27.4
30.5
7463B
7496B
7546B
7562B
764B
749B
7529B
754B
7630B
748B
7512B
7528B
7613B
7462B
745B
7461B
7495B
751B
7596B
TL
40.3
29.7
30
51.4
45.5
44.3
47.5
40.4
45.7
35.5
37
35.8
37.2
27.4
40.2
34.1
36.6
32.7
42.6
32.7
40.3
37.6
35.5
34.3
40.1
39.8
33.3
29.1
35.1
35.4
31.4
33
35
74B
7460B
796B
809B
792B
801B
793B
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7450B
801B
794B
79B
7960B
7943B
7926B
790B
7892B
785B
785B
7841B
7824B
780B
790B
73B
756B
739B
72B
705B
768B
761B
7654B
763B
7620B
7603B
7586B
7569B
752B
753B
7518B
7501B
748B
746B
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7451B
8012B
795B
798B
7961B
794B
792B
7910B
7893B
786B
7859B
7842B
7825B
780B
791B
74B
75B
740B
723B
706B
7689B
762B
765B
7638B
7621B
7604B
758B
750B
753B
7536B
7519B
7502B
7485B
7468B
R.h.
1
26
132
1
386
5
759
1
21
0
13
2
196
2
1
30
7
5
1
2
1
0
10
9
0
27
0
4
0
0
6
7
0
7452B
7469B
7486B
7503B
7520B
753B
754B
751B
758B
7605B
762B
7639B
765B
763B
7690B
70B
724B
741B
758B
75B
792B
7809B
7826B
7843B
7860B
78B
7894B
8013B
796B
79B
7962B
7945B
7928B
791B
P.t.
0
0
0
7
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7453B
8014B
79B
7980B
7963B
7946B
792B
7912B
7895B
78B
7861B
784B
782B
7810B
793B
76B
759B
742B
725B
708B
7691B
764B
765B
7640B
7623B
760B
7589B
752B
75B
7538B
7521B
7504B
748B
740B
E.sp. H.sp.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
745B
745B
741B
742B
748B
7489B
750B
7506B
752B
7523B
7539B
7540B
756B
75B
753B
754B
7590B
7591B
760B
7608B
7624B
7625B
7641B
7642B
7658B
7659B
765B
76B
7692B
7693B
709B
710B
726B
72B
743B
74B
760B
761B
7B
78B
794B
795B
781B
7812B
782B
7829B
7845B
7846B
7862B
7863B
789B
780B
7896B
789B
7913B
7914B
7930B
7931B
794B
7948B
7964B
7965B
7981B
7982B
798B
79B
8015B
8016B
P.c.
+
0
0
0
0
+
+
0
+
+
+
0
0
0
+
0
0
0
0
0
0
+
0
0
+
0
0
0
0
0
0
0
0
7456B
743B
7490B
750B
7524B
7541B
758B
75B
7592B
7609B
762B
7643B
760B
76B
7694B
71B
728B
745B
762B
79B
796B
7813B
7830B
784B
7864B
781B
8017B
80B
7983B
796B
794B
7932B
7915B
789B
D.s.
0
0
0
+
0
0
0
0
0
0
0
0
0
+
0
0
+
0
0
0
0
+
0
+
0
0
0
0
0
0
+
0
0
745B
74B
7491B
7508B
752B
7542B
759B
756B
7593B
7610B
762B
764B
761B
768B
7695B
712B
729B
746B
763B
780B
79B
7814B
7831B
784B
7865B
782B
789B
7916B
793B
7950B
796B
7984B
801B
801B
M.y.
0
0
+
0
0
0
0
+
0
+
0
0
0
0
+
0
0
0
+
0
0
+
0
0
0
0
0
0
0
0
0
+
0
7458B
745B
7492B
7509B
7526B
7543B
7560B
75B
7594B
761B
7628B
7645B
762B
769B
769B
713B
730B
74B
764B
781B
798B
7815B
7832B
7849B
786B
783B
790B
791B
7934B
7951B
7968B
7985B
802B
8019B
113
S
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
743B
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
1
2
3
4
5
7459B
Appendix
N
742B
W
520
465
450
180
345
325
470
455
180
455
210
140
275
400
275
595
210
300
295
625
625
355
290
250
455
215
680
515
375
870
1090
685
865
8054B
8071B
80B
8105B
812B
8139B
8156B
8173B
8190B
8207B
824B
8241B
825B
8275B
829B
8309B
8326B
834B
8360B
837B
8394B
841B
842B
845B
8462B
8479B
8496B
8513B
851B
8564B
8547B
8530B
805B
8072B
8057B
8175B
819B
820B
8209B
825B
8210B
824B
826B
8243B
8259B
824B
8276B
8260B
8293B
827B
8310B
8294B
827B
8295B
8327B
831B
832B
834B
8329B
8361B
8345B
8362B
837B
836B
8395B
8379B
8412B
8396B
8429B
8413B
8397B
841B
846B
8430B
847B
8463B
84B
840B
846B
841B
8497B
842B
8514B
849B
851B
8531B
8516B
8532B
8549B
853B
856B
850B
854B
85B
856B
851B
8567B
8469B
845B
846B
8502B
8503B
8519B
853B
8520B
8536B
852B
8537B
857B
8570B
853B
8569B
856B
8452B
846B
851B
8534B
8435B
8451B
8501B
8517B
853B
841B
843B
84B
850B
8401B
8417B
8467B
843B
849B
834B
840B
8450B
846B
8367B
83B
843B
849B
8465B
8350B
836B
8416B
8432B
83B
8349B
839B
8415B
8431B
8316B
832B
832B
839B
829B
8315B
8365B
831B
82B
829B
834B
8364B
830B
8265B
821B
831B
8347B
824B
8264B
8314B
830B
8346B
8231B
8247B
8297B
831B
8214B
8230B
820B
8296B
8312B
810B
8197B
8213B
8263B
8279B
8163B
8196B
8246B
826B
8146B
8179B
829B
8245B
8261B
8129B
8162B
821B
82B
812B
8145B
8195B
821B
827B
8095B
812B
817B
8194B
8061B
807B
81B
816B
817B
8027B
8094B
814B
8160B
P.l.
383
89
158
6
2
66
72
24
0
90
5
2
22
14
11
3
249
5
119
74
47
51
65
16
27
85
123
403
117
14
384
176
35
804B
807B
8127B
8143B
8193B
806B
810B
8126B
8176B
8192B
8059B
8093B
8109B
8159B
8043B
8026B
8042B
8076B
8092B
8142B
815B
8174B
8075B
8125B
K
0.78
0.39
0.89
0.84
0.72
0.84
0.85
0.90
0.74
0.80
0.77
0.75
0.82
1.02
0.90
1.00
0.66
0.88
0.84
0.89
0.75
1.18
0.95
0.91
0.91
1.20
1.49
0.88
0.88
0.93
0.89
0.89
0.89
8025B
805B
810B
814B
8157B
BH
7.4
7.2
7.5
5.2
6.4
6.8
7.4
7
5.4
7.6
5.5
4.4
6.2
6.9
6.4
8.2
5.1
6.1
6.1
8.5
7.7
6.7
6.6
6
7.7
6.3
7.6
6.7
6.4
8.7
10.3
8.3
9.4
8024B
8091B
8124B
8140B
SL
33.2
33
31
22.5
29.8
27.8
31.7
29.8
23.8
32
25.2
22
26.4
28.4
25.7
32.9
25.8
27.4
26.4
33.8
36.7
26.6
25.9
25
30.5
21.6
30.1
33
28.7
37
40.7
35.3
36.9
8041B
8074B
8107B
8123B
852B
8056B
809B
8106B
856B
804B
8023B
8039B
8073B
809B
854B
TL
40.5
49.2
37
27.8
36.3
33.8
38.1
37
29
38.5
30.1
26.5
32.3
34
31.3
39
31.7
32.4
32.7
41.3
43.6
31.1
31.2
30.2
36.9
26.2
35.7
38.8
34.9
45.4
49.6
42.5
46
802B
803B
854B
8571B
85B
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
802B
859B
8572B
85B
853B
8521B
8504B
847B
8470B
8453B
8436B
8419B
8402B
835B
836B
8351B
834B
8317B
830B
823B
826B
8249B
823B
8215B
819B
81B
8164B
8147B
8130B
813B
8096B
8079B
8062B
8045B
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8029B
8590B
8573B
856B
8539B
852B
850B
84B
8471B
845B
8437B
8420B
8403B
836B
8369B
8352B
835B
831B
8301B
824B
8267B
8250B
823B
8216B
819B
812B
8165B
814B
813B
814B
8097B
80B
8063B
8046B
R.h.
1
0
0
1
11
0
0
0
0
0
0
1
1
0
0
0
1
0
0
6
0
2
0
1
0
1
0
0
0
0
0
138
0
803B
8047B
8064B
801B
809B
815B
8132B
8149B
816B
813B
820B
8217B
8234B
8251B
826B
825B
8302B
8319B
836B
835B
8370B
837B
840B
8421B
843B
845B
8472B
849B
8506B
8523B
8540B
857B
8574B
8591B
P.t.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8031B
8592B
857B
85B
8541B
8524B
8507B
8490B
8473B
8456B
8439B
842B
8405B
83B
8371B
8354B
837B
8320B
830B
826B
8269B
825B
8235B
821B
8201B
814B
8167B
8150B
813B
816B
809B
802B
8065B
804B
E.sp. H.sp.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9
0
0
0
0
0
8032B
803B
8049B
805B
806B
8067B
803B
804B
810B
810B
817B
81B
8134B
8135B
815B
8152B
816B
8169B
815B
816B
820B
8203B
8219B
820B
8236B
8237B
8253B
8254B
8270B
8271B
827B
82B
8304B
8305B
8321B
832B
83B
839B
835B
8356B
8372B
837B
839B
8390B
8406B
8407B
8423B
842B
840B
841B
8457B
845B
847B
8475B
8491B
8492B
850B
8509B
852B
8526B
8542B
8543B
859B
8560B
8576B
857B
8593B
8594B
P.c.
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
+
0
+
0
0
0
0
0
0
+
0
0
0
0
+
8034B
8051B
806B
805B
8102B
819B
8136B
8153B
8170B
817B
8204B
821B
823B
825B
827B
829B
8306B
832B
8340B
8357B
8374B
8391B
840B
8425B
842B
8459B
8476B
8493B
8510B
8527B
854B
8561B
859B
857B
D.s.
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
8035B
8052B
8069B
806B
8103B
8120B
8137B
8154B
817B
81B
8205B
82B
8239B
8256B
8273B
8290B
8307B
8324B
8341B
835B
8375B
8392B
8596B
8579B
8562B
854B
852B
851B
849B
847B
8460B
843B
8426B
8409B
M.y.
0
0
0
0
0
0
+
+
0
+
0
+
0
0
0
+
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
+
8036B
8053B
807B
807B
8104B
812B
813B
815B
8172B
819B
8206B
823B
8240B
8257B
8274B
8291B
830B
8325B
8342B
8359B
8376B
839B
8410B
8427B
84B
8461B
847B
8495B
8512B
8529B
8546B
8563B
8597B
850B
114
S
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1
1
1
1
8021B
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
1
2
3
4
8037B
Appendix
N
802B
8632B
8649B
86B
863B
870B
871B
8734B
8751B
876B
875B
802B
819B
836B
853B
870B
87B
8904B
8921B
893B
895B
8972B
89B
906B
9023B
904B
9057B
9074B
901B
9108B
W
835
740
1050
1000
965
1105
925
120
220
590
630
1125
505
455
395
545
885
530
460
580
590
430
820
955
1025
320
400
495
800
845
755
863B
8650B
8752B
876B
820B
854B
8B
872B
8906B
892B
8907B
893B
8923B
8940B
8956B
8941B
8973B
8957B
8974B
890B
8975B
907B
891B
908B
90B
9025B
9042B
905B
9043B
906B
9076B
903B
904B
9127B
910B
9128B
9062B
912B
9064B
908B
9081B
913B
914B
907B
913B
9130B
9047B
9063B
906B
912B
903B
9046B
907B
905B
91B
9013B
902B
9045B
9078B
896B
9012B
9028B
9061B
907B
897B
895B
901B
904B
8962B
897B
894B
9027B
8945B
8961B
897B
901B
9026B
892B
894B
8960B
893B
891B
8927B
8943B
8976B
892B
894B
8910B
8926B
895B
87B
893B
890B
8942B
895B
860B
876B
892B
8925B
843B
859B
875B
890B
8924B
826B
842B
85B
891B
809B
825B
841B
874B
890B
8792B
80B
824B
857B
873B
89B
8905B
9126B
856B
875B
875B
8791B
807B
840B
8741B
874B
8790B
823B
839B
85B
871B
910B
82B
83B
8724B
875B
873B
806B
870B
8740B
8756B
879B
805B
821B
837B
902B
87B
804B
8690B
8723B
8739B
872B
8673B
8706B
872B
875B
871B
8639B
865B
869B
8705B
873B
862B
8672B
86B
8721B
P.l.
86
31
133
21
37
153
124
5
69
220
45
134
17
148
15
52
29
67
20
17
93
12
6
48
19
29
41
59
137
424
87
8605B
865B
8671B
8704B
8754B
87B
803B
863B
8654B
867B
873B
870B
8637B
8670B
8720B
8753B
8769B
8621B
8604B
8620B
8653B
8703B
K
0.79
0.89
1.15
1.03
0.92
0.77
0.93
0.68
0.78
0.76
0.85
0.96
0.97
0.83
0.88
1.03
0.81
0.75
0.81
1.02
0.86
0.92
0.70
0.86
0.91
0.69
0.76
0.89
0.71
0.87
0.79
8603B
863B
869B
8736B
BH
9.6
8.6
9.8
8.3
9.8
9.8
8.8
4.4
5.7
7.1
7.4
10
7.8
7.3
6.4
7.5
8.4
7.5
7
7.6
8
7.2
8.2
8.5
9.8
5.1
6
7.3
8.5
8
8.2
8602B
86B
8719B
8735B
SL
39.3
36.5
39
36.9
38.5
42.5
39.5
21.2
24.6
35.5
34.9
40.9
31.4
31.4
29.3
31.3
39.6
33.7
33.5
31.4
33.5
29.1
40.1
38.5
40.4
29.7
31.1
31.4
40.2
38.4
37.3
8619B
8652B
8702B
871B
9075B
8635B
865B
8701B
9058B
8634B
86B
864B
9041B
861B
8601B
8617B
8651B
867B
9024B
TL
47.2
43.6
45
46
47.1
52.3
46.4
26
30.5
42.6
42
48.9
37.3
38
35.6
37.6
47.8
41.3
38.5
38.5
40.9
36
48.9
48
48.3
36
37.5
38.2
48.4
46
45.7
860B
861B
908B
915B
9132B
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
860B
913B
916B
90B
9082B
9065B
9048B
9031B
9014B
897B
890B
8963B
8946B
892B
8912B
895B
87B
861B
84B
827B
810B
8793B
876B
8759B
8742B
8725B
870B
8691B
8674B
8657B
8640B
8623B
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8607B
9134B
917B
910B
9083B
906B
904B
9032B
9015B
89B
891B
8964B
8947B
8930B
8913B
896B
879B
862B
845B
82B
81B
8794B
87B
8760B
8743B
8726B
8709B
8692B
8675B
865B
8641B
8624B
R.h.
0
0
0
189
0
203
2
6
1
1
0
87
0
1
0
253
1
0
0
0
0
0
0
1
1
1
0
0
2
0
0
860B
8625B
8642B
8659B
867B
8693B
8710B
872B
874B
8761B
87B
8795B
812B
829B
846B
863B
80B
9135B
918B
910B
9084B
9067B
905B
903B
9016B
89B
892B
8965B
894B
8931B
8914B
897B
P.t.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8609B
9136B
91B
9102B
9085B
9068B
9051B
9034B
9017B
90B
893B
896B
894B
8932B
8915B
89B
81B
864B
847B
830B
813B
8796B
879B
8762B
8745B
872B
871B
8694B
867B
860B
8643B
862B
E.sp. H.sp.
0
0
0
0
11
0
0
0
1
0
0
0
10
0
0
1
0
0
2
0
2
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
2
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
8610B
861B
8627B
862B
864B
8645B
861B
862B
867B
8679B
8695B
869B
8712B
8713B
8729B
8730B
8746B
874B
8763B
8764B
870B
871B
879B
879B
814B
815B
831B
832B
84B
849B
865B
86B
82B
83B
89B
890B
8916B
8917B
893B
8934B
8950B
8951B
8967B
896B
894B
895B
901B
902B
9018B
901B
9035B
9036B
9052B
9053B
906B
907B
9086B
9087B
9103B
9104B
9120B
912B
9137B
9138B
P.c.
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
+
+
+
0
0
+
0
0
0
0
0
0
+
0
8612B
8629B
864B
863B
860B
8697B
8714B
8731B
874B
8765B
872B
879B
816B
83B
850B
867B
84B
8901B
891B
8935B
8952B
896B
896B
903B
902B
9037B
9054B
9071B
908B
9105B
912B
913B
D.s.
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
+
0
+
0
0
0
0
8613B
8630B
8647B
864B
861B
869B
8715B
8732B
8749B
876B
873B
80B
817B
834B
851B
86B
85B
8902B
891B
8936B
8953B
8970B
897B
904B
9021B
9038B
905B
9072B
9140B
9123B
9106B
908B
M.y.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
+
0
0
0
0
0
+
0
+
0
0
0
0
0
+
8614B
8631B
864B
865B
862B
869B
8716B
873B
8750B
876B
874B
801B
81B
835B
852B
869B
86B
8903B
8920B
8937B
8954B
8971B
89B
905B
902B
903B
9056B
9073B
90B
9107B
914B
9124B
115
9125B
S
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
859B
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1
2
3
4
5
6
7
8
9
10
11
8615B
Appendix
N
859B
N
9142B
W
430
410
660
670
640
535
670
610
550
1640
1395
1065
1785
790
739
600
420
420
530
555
900
890
770
655
475
530
400
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1
2
3
4
5
6
7
8
9
10
11
12
1
915B
9176B
913B
9210B
927B
924B
9261B
9278B
925B
9312B
932B
9346B
936B
9380B
937B
941B
9431B
948B
9465B
9482B
94B
9516B
953B
950B
9567B
9584B
9601B
9160B
928B
9245B
92B
926B
9246B
926B
9280B
930B
9314B
9347B
931B
9602B
958B
9568B
951B
9534B
9517B
950B
9483B
946B
94B
9432B
9415B
9315B
9348B
932B
9365B
934B
936B
9382B
93B
9384B
940B
9416B
9401B
9417B
943B
9418B
943B
9450B
9435B
9451B
9467B
9452B
9468B
948B
946B
9485B
9501B
9486B
9502B
9518B
9503B
951B
953B
9520B
9536B
952B
9537B
953B
956B
954B
9570B
9586B
9603B
9367B
938B
9571B
9604B
9587B
9605B
958B
931B
935B
9351B
936B
9352B
9368B
935B
936B
9385B
9370B
9386B
9402B
9387B
9403B
941B
940B
9420B
9436B
9421B
9437B
9453B
9438B
945B
9470B
945B
9471B
9487B
9472B
948B
9504B
948B
950B
9521B
9506B
95B
9572B
958B
9607B
950B
9573B
956B
953B
952B
9538B
960B
9302B
9318B
934B
9350B
9285B
9301B
9317B
93B
9268B
9284B
930B
9316B
9251B
9267B
9283B
92B
9234B
9250B
926B
928B
928B
9217B
923B
924B
9265B
9281B
927B
931B
938B
9264B
920B
9216B
923B
9248B
9183B
91B
9215B
9231B
9247B
9263B
927B
9381B
9230B
916B
9182B
918B
9214B
914B
9165B
918B
917B
9213B
P.l.
77
112
60
126
131
128
42
208
167
82
236
282
81
523
114
22
57
94
86
88
120
166
282
24
4
4
0
9148B
9164B
9180B
916B
921B
K
1.08
0.99
1.16
0.99
1.21
0.96
1.09
0.99
0.89
1.31
0.96
0.95
1.08
1.08
0.71
0.98
1.04
1.04
0.98
0.98
0.99
1.06
0.83
1.16
0.97
1.09
0.95
9147B
9163B
917B
915B
921B
BH
7.5
6.6
8.6
9.3
9.2
8.1
9
8.9
8.2
12
11.1
10.9
12.5
7.5
9
8.2
7.4
6.8
7.8
8.5
10
8
9.1
8.7
7.5
7.9
7.3
9146B
9162B
9178B
914B
SL
29
29
32.4
34.1
31.2
32.6
33.2
32.1
32.8
41.6
44
39.5
44.5
35
38
32.6
28.5
28.2
31.3
32.2
37.7
37
36.7
32.3
29.9
30
28.5
9145B
916B
917B
9364B
TL
34.1
34.6
38.5
40.8
37.5
38.2
39.5
39.5
39.6
50
52.5
48.3
54.8
41.8
47
39.4
34.3
34.3
37.8
38.4
45
43.8
45.2
38.4
36.6
36.5
34.8
914B
9523B
9540B
957B
9608B
951B
9574B
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9150B
960B
952B
957B
958B
9541B
9524B
9507B
940B
9473B
9456B
943B
942B
9405B
938B
9371B
9354B
937B
9320B
930B
9286B
926B
925B
9235B
9218B
9201B
9184B
9167B
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
915B
9610B
953B
9576B
95B
9542B
952B
9508B
941B
947B
9457B
940B
9423B
9406B
938B
9372B
935B
938B
9321B
9304B
9287B
9270B
9253B
9236B
921B
920B
9185B
9168B
R.h.
5
1
0
0
1
0
1
2
26
0
0
0
1
0
3
1
0
26
46
32
0
0
0
39
12
51
0
9152B
916B
9186B
9203B
920B
9237B
9254B
9271B
928B
9305B
932B
93B
9356B
937B
930B
9407B
942B
941B
9458B
9475B
942B
950B
9526B
9543B
9560B
957B
954B
961B
P.t.
0
0
0
0
2
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9153B
9612B
95B
9578B
9561B
954B
9527B
9510B
943B
9476B
945B
942B
9425B
9408B
931B
9374B
9357B
9340B
932B
9306B
928B
927B
925B
9238B
921B
9204B
9187B
9170B
E.sp. H.sp.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
9154B
915B
917B
9172B
918B
918B
9205B
9206B
92B
923B
923B
9240B
9256B
9257B
9273B
9274B
920B
921B
9307B
9308B
9324B
9325B
9341B
9342B
9358B
935B
9375B
9376B
932B
93B
940B
9410B
9426B
9427B
943B
94B
9460B
9461B
947B
9478B
94B
945B
951B
9512B
9528B
952B
954B
9546B
9562B
9563B
957B
9580B
956B
957B
9613B
9614B
P.c.
0
+
0
+
+
0
+
+
+
0
+
+
0
0
0
0
0
0
0
0
+
0
+
+
0
0
0
9156B
9173B
910B
9207B
924B
9241B
9258B
9275B
92B
930B
9326B
934B
9360B
937B
934B
941B
9428B
945B
9462B
947B
946B
9513B
9530B
9547B
9564B
9615B
958B
9581B
D.s.
0
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
9157B
9174B
91B
9208B
925B
924B
925B
9276B
923B
9310B
9327B
934B
9361B
9378B
935B
9412B
942B
946B
9463B
961B
95B
9582B
956B
9548B
9531B
9514B
947B
9480B
M.y.
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9158B
9175B
912B
920B
926B
9243B
9260B
927B
924B
931B
9328B
9345B
9362B
937B
936B
9413B
9430B
947B
946B
9481B
948B
951B
9532B
954B
956B
9617B
960B
9583B
116
S
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
1
9143B
Appendix
Appendix I-C. Parasitological data of barbel collected from sampling site Silistra.
71B
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
11
12
13
9635B
9652B
96B
968B
9703B
9720B
973B
9754B
971B
978B
9805B
982B
983B
9856B
9873B
980B
907B
924B
941B
92B
975B
958B
S
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
W
735
745
485
815
1050
965
885
995
930
1350
1385
915
875
1070
1175
995
1265
1105
920
560
965
1165
961B
963B
9687B
9704B
968B
9721B
9705B
975B
973B
978B
973B
9806B
970B
9840B
9824B
9874B
985B
982B
90B
983B
926B
910B
943B
927B
94B
960B
94B
97B
95B
978B
96B
948B
98B
981B
964B
980B
97B
931B
947B
963B
97B
914B
930B
946B
962B
987B
913B
92B
945B
961B
980B
986B
912B
928B
9863B
987B
985B
91B
9846B
9862B
987B
984B
982B
9845B
9861B
987B
9812B
982B
984B
9860B
9876B
975B
981B
9827B
9843B
985B
9875B
981B
93B
9842B
978B
974B
9810B
9826B
9761B
97B
973B
980B
9825B
9841B
9857B
976B
980B
974B
9760B
976B
972B
972B
9743B
975B
975B
971B
9807B
9823B
95B
974B
9710B
9726B
9742B
9758B
963B
970B
9725B
9741B
975B
967B
962B
9708B
9724B
9740B
9756B
972B
942B
9723B
965B
9675B
961B
970B
9642B
9658B
9674B
960B
9706B
972B
9738B
925B
968B
9625B
9641B
9657B
9673B
P.l.
263
195
37
104
114
68
13
164
10
5
287
99
39
81
31
45
409
80
66
23
181
19
9624B
9640B
965B
9672B
K
1.07
1.28
1.00
0.89
0.89
0.77
0.84
1.10
1.08
0.90
0.97
0.80
0.99
0.81
1.05
0.95
1.18
1.18
0.88
0.85
1.04
1.06
9623B
963B
965B
9671B
BH
8.4
9.4
7.9
9
10
10
9.1
9.1
9.8
9.3
10.5
8.4
9.1
9.3
9
8.5
11.7
11.1
9.5
8.1
9.3
10.8
962B
9638B
9654B
9670B
SL
34.5
31.7
30
37.5
40.3
40
38
36.9
36.1
44.2
42.3
40.4
36.5
42.8
39.5
38.5
39.8
37
38.7
33
37.3
39.7
9621B
9637B
9653B
908B
TL
41
38.8
36.5
45.1
49
50
47.2
44.9
44.2
53.1
52.3
48.5
44.6
51
48.2
47.2
47.5
45.4
47.1
40.4
45.3
47.9
9620B
965B
982B
9B
L.p.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
962B
10B
983B
96B
94B
932B
915B
98B
981B
9864B
9847B
9830B
9813B
976B
97B
9762B
9745B
9728B
971B
964B
967B
960B
9643B
A.a.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9627B
10B
984B
967B
950B
93B
916B
98B
982B
9865B
984B
9831B
9814B
97B
9780B
9763B
9746B
972B
9712B
965B
9678B
961B
964B
R.h.
0
0
0
66
0
27
2
0
0
761
1
72
0
0
0
492
0
0
0
8
0
0
9628B
9645B
962B
967B
96B
9713B
9730B
974B
9764B
9781B
978B
9815B
9832B
984B
986B
983B
90B
102B
985B
968B
951B
934B
917B
P.t.
0
0
0
0
1
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
962B
103B
986B
96B
952B
935B
918B
901B
984B
9867B
9850B
983B
9816B
97B
9782B
9765B
9748B
9731B
9714B
967B
9680B
963B
964B
E.sp. H.sp.
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
9630B
9631B
9647B
9648B
964B
965B
9681B
9682B
968B
96B
9715B
9716B
9732B
973B
974B
9750B
976B
976B
9783B
9784B
980B
9801B
9817B
981B
9834B
9835B
9851B
9852B
986B
986B
985B
986B
902B
903B
91B
920B
936B
937B
953B
954B
970B
971B
987B
98B
104B
105B
P.c.
+
+
+
+
+
0
0
0
0
0
0
0
0
0
0
+
0
+
0
0
+
0
9632B
964B
96B
9683B
970B
971B
9734B
9751B
9768B
9785B
9802B
981B
9836B
9853B
9870B
987B
904B
921B
938B
95B
972B
98B
106B
D.s.
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
963B
9650B
967B
9684B
9701B
9718B
9735B
107B
90B
973B
956B
93B
92B
905B
98B
9871B
9854B
9837B
9820B
9803B
9786B
976B
9752B
M.y.
0
0
0
0
+
0
0
0
0
0
0
+
0
0
0
0
+
0
0
0
0
+
9634B
9651B
Appendix
N
9618B
968B
9685B
9702B
971B
9736B
9753B
970B
978B
9804B
9821B
983B
985B
9872B
98B
906B
923B
940B
957B
974B
91B
108B
117
Appendix
118
Appendix II: Element concentrations in barbel tissues and P. laevis used in Chapter 2.
109B
Used abbreviations in the appendix:

10B
N:
barbel number
S:
Sample tissue (M muscle; I intestine; L liver; P.l. Pomphorhynchus laevis)
10B
102B
Appendix II-A. Element concentrations in host tissues and P. laevis of heavily infected
72B
barbels.
10532B
1053B
N S As
Cd
Co
Cu
1 M 0.123 0.015 0.009 0.731
2 M 0.238 0.176 0.025 0.964
3 M 0.095 0.016 0.011 1.235
4 M 0.085 0.007 0.027 1.297
5 M 0.043 0.028 0.018 0.691
6 M 0.140 0.006 0.007 1.226
7 M 0.180 0.011 0.011 1.217
8 M 0.406 0.016 0.013 0.428
9 M 0.181 0.012 0.009 1.238
1 I
0.425 0.060 0.030 1.843
2 I
0.469 0.161 0.052 3.816
3 I
0.160 0.058 0.074 1.678
4 I
0.301 0.085 0.088 5.993
5 I
0.217 0.126 0.190 6.918
6 I
0.422 0.050 0.049 4.318
7 I
0.496 0.127 0.239 6.390
8 I
*
0.175 0.142 2.222
9 I
0.495 0.098 0.173 3.733
1 L
0.400 0.160 0.028 5.837
2 L
0.354 0.187 0.035 9.739
3 L
0.229 0.085 0.045 7.243
4 L
0.245 0.204 0.033 178.782
5 L
0.135 0.181 0.129 5.597
6 L
0.343 0.101 0.032 8.677
7 L
0.616 0.111 0.038 25.398
8 L
*
0.232 0.037 10.823
9 L
0.557 0.118 0.033 10.654
1 P.l. 1.731 1.599 0.069 72.826
2 P.l. 1.732 2.616 0.074 91.379
3 P.l. 2.156 2.275 0.102 90.997
4 P.l. 1.505 3.442 0.096 159.436
5 P.l. 2.436 3.583 0.148 130.552
6 P.l. 0.432 1.523 0.056 73.184
7 P.l. 1.594 2.908 0.138 99.337
8 P.l. *
0.915 0.107 54.935
9 P.l. 1.261 1.715 0.099 63.732
*: values not taken
106B
Fe
13.134
15.961
8.269
8.693
13.831
9.347
5.372
7.910
6.389
27.648
37.593
68.852
69.514
143.787
46.778
158.238
90.310
120.122
87.927
82.479
60.049
83.719
762.201
36.935
44.419
129.575
53.948
36.385
63.844
44.627
45.643
75.201
24.229
42.942
152.905
49.728
Mn
0.265
0.804
0.313
0.230
0.329
0.259
0.347
0.282
0.266
1.369
2.975
13.054
4.250
11.371
2.991
18.169
5.119
9.070
0.923
1.122
1.168
0.753
3.352
1.046
1.079
1.782
1.267
3.617
4.382
4.490
4.656
8.671
4.783
5.210
28.407
5.043
Mo
0.006
0.009
0.005
0.007
0.008
0.007
0.005
0.004
0.003
0.023
0.041
0.025
0.030
0.042
0.046
0.018
0.109
0.018
0.087
0.117
0.101
0.243
0.088
0.231
0.142
0.124
0.092
0.182
0.059
0.048
0.035
0.072
0.022
0.028
0.014
0.015
Ni
0.229
1.808
0.163
0.335
0.506
1.188
0.542
0.249
0.221
0.806
0.290
1.370
1.311
1.288
1.536
2.065
0.756
1.237
0.043
0.383
0.375
0.404
0.425
0.267
0.484
1.550
0.058
0.272
0.190
0.251
0.300
0.552
0.234
0.387
0.648
0.435
Pb
0.007
0.027
0.000
0.016
0.021
0.006
n.d.
0.003
0.047
0.040
0.039
0.421
0.126
0.303
0.019
0.482
0.199
0.228
0.027
0.133
0.038
0.034
1.104
0.032
0.016
0.035
0.000
7.708
10.348
4.959
9.222
51.803
6.861
11.747
3.491
5.692
Sn
0.002
0.005
0.003
0.004
0.004
0.003
0.000
0.003
0.004
0.008
0.008
0.008
0.015
0.017
0.010
0.009
0.037
0.009
0.014
0.035
0.010
0.036
0.016
0.043
0.005
0.006
0.007
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
V
0.045
0.046
0.032
0.035
0.050
0.032
0.025
0.025
0.021
0.083
0.104
0.237
0.201
0.439
0.201
0.783
0.244
0.394
0.375
0.272
0.179
0.267
0.331
0.203
0.075
0.295
0.125
0.125
0.277
0.194
0.162
0.255
0.118
0.167
0.317
0.173
104B
105B
107B
108B
109B
102B
102B
102B
1023B
1024B
1025B
1026B
1027B
1028B
1029B
103B
103B
1032B
103B
1034B
1035B
1036B
1037B
1038B
1039B
104B
104B
1042B
1043B
104B
1045B
1046B
105B
1056B
1057B
1058B
1059B
106B
1069B
107B
107B
1072B
1073B
1074B
1083B
1084B
1085B
1086B
1087B
108B
1097B
1098B
109B
10B
10B
102B
10B
102B
103B
104B
105B
106B
1025B
1026B
1027B
1028B
1029B
103B
1039B
104B
104B
1042B
1043B
104B
1053B
1054B
105B
1056B
1057B
1058B
1067B
1068B
1069B
107B
107B
1072B
108B
1082B
1083B
1084B
1085B
1086B
1095B
1096B
1097B
1098B
109B
102B
1029B
102B
102B
102B
1023B
1024B
1023B
1024B
1025B
1026B
1027B
1028B
10237B
10238B
10239B
1024B
1024B
1024B
1025B
1025B
10253B
10254B
1025B
10256B
10265B
1026B
10267B
10268B
10269B
1027B
10279B
1028B
1028B
1028B
10283B
10284B
10293B
10294B
10295B
10296B
10297B
10298B
1037B
1038B
1039B
103B
103B
1032B
1032B
1032B
1032B
10324B
10325B
10326B
1035B
1036B
1037B
1038B
1039B
1034B
10349B
1035B
1035B
10352B
1035B
10354B
1036B
10364B
10365B
1036B
10367B
10368B
1037B
10378B
10379B
1038B
1038B
10382B
1039B
10392B
1039B
10394B
10395B
10396B
1045B
1046B
1047B
1048B
1049B
104B
1049B
1042B
1042B
1042B
10423B
1042B
1043B
1043B
10435B
10436B
10437B
10438B
1047B
1048B
1049B
1045B
1045B
10452B
1046B
10462B
10463B
1046B
10465B
1046B
10475B
10476B
1047B
10478B
10479B
1048B
10489B
1049B
1049B
10492B
10493B
1049B
1053B
1054B
105B
1056B
1057B
1058B
1057B
1058B
1059B
1052B
1052B
1052B
Zn
4.515
4.941
4.069
3.352
4.069
3.986
3.427
2.369
3.498
11.199
10.227
7.558
10.393
21.279
12.703
15.106
14.086
8.823
19.413
22.913
17.080
32.103
16.005
16.826
20.777
25.277
18.855
34.287
128.232
178.334
162.001
208.329
40.123
65.568
61.954
70.784
103B
1047B
1048B
106B
1075B
107B
103B
108B
1045B
1032B
109B
1059B
1046B
103B
1087B
1074B
106B
1025B
102B
1089B
10243B
1023B
1027B
1027B
10258B
10245B
1029B
10286B
10273B
10327B
1034B
103B
1035B
10342B
10329B
1038B
1037B
10357B
104B
10398B
10385B
10439B
10426B
1043B
10467B
1045B
104B
10495B
10482B
10469B
10524B
105B
10497B
1048B
10526B
1052B
1049B
10486B
10528B
1054B
105B
10529B
105B
1053B
1056B
1052B
1048B
1047B
10487B
105B
1053B
10527B
10473B
1046B
1046B
10459B
10472B
10485B
10498B
105B
1052B
1047B
10458B
1045B
10432B
1048B
1043B
104B
10457B
1047B
10483B
10496B
1059B
10456B
1043B
1043B
1047B
104B
1039B
1043B
1046B
10429B
1042B
1045B
10468B
1048B
10428B
1045B
1042B
10389B
10376B
10362B
10375B
1038B
104B
104B
10427B
104B
10453B
104B
10387B
10374B
1036B
10348B
1034B
10347B
1036B
1037B
10386B
1039B
1042B
10425B
10372B
10359B
10346B
103B
1032B
1036B
1039B
1032B
10345B
10358B
1037B
10384B
10397B
1034B
103B
1038B
1035B
1029B
10278B
1029B
1034B
1037B
103B
1034B
10356B
10369B
1036B
103B
1029B
1027B
10264B
1025B
10263B
10276B
10289B
1032B
1035B
10328B
1034B
1028B
10275B
1026B
10249B
10236B
102B
10235B
10248B
1026B
10274B
10287B
103B
103B
1026B
10247B
10234B
102B
1028B
1094B
1027B
102B
1023B
10246B
10259B
1027B
10285B
1023B
1029B
1026B
1093B
108B
106B
1079B
1092B
1025B
1028B
1023B
1024B
10257B
1024B
109B
1078B
1065B
1052B
1038B
105B
1064B
107B
109B
1023B
1026B
1029B
1076B
1063B
105B
1037B
1024B
10B
1023B
1036B
1049B
1062B
1075B
108B
102B
1048B
1035B
102B
109B
1096B
1082B
1095B
108B
102B
1034B
1047B
106B
1073B
102B
107B
1094B
108B
1068B
1054B
1067B
108B
1093B
106B
1053B
106B
1079B
1092B
105B
1052B
1065B
1078B
109B
104B
105B
1064B
107B
109B
103B
105B
1063B
1076B
1089B
10523B
1049B
1062B
Appendix
119
Appendix III-B. Element concentrations in host tissues and P. laevis of slightly infected
73B
barbells.
N S
As
Cd
Co
Cu
1 M 0.035 0.026 0.011
0.960
2 M 0.218 0.025 0.023
1.104
3 M 0.059 0.022 0.013
0.904
4 M 0.049 0.013 0.010
1.071
5 M 0.116 0.002 0.012
0.610
6 M 0.349 0.032 0.008
1.171
7 M 0.060 0.005 0.008
0.243
8 M 0.058 0.018 0.004
1.233
9 M 0.382 0.078 0.016
2.797
1 I
0.166 0.257 0.063
4.482
2 I
0.521 0.217 0.077
9.775
3 I
0.190 0.212 0.042
4.417
4 I
0.211 0.246 0.107
7.013
5 I
0.144 0.019 0.013
1.850
6 I
0.622 0.450 0.187
11.781
7 I
0.332 0.169 0.196
1.944
8 I
0.070 0.435 0.037
4.323
9 I
0.817 0.259 0.168
17.227
1 L
0.177 0.149 0.032
8.870
2 L
0.786 0.188 0.055
6.431
3 L
0.345 0.304 0.039
30.341
4 L
0.195 0.137 0.047
15.912
5 L
0.839 0.052 0.024
10.107
6 L
0.963 0.136 0.041
5.500
7 L
0.173 0.157 0.033
4.574
8 L
0.189 0.195 0.030
17.607
9 L
1.239 0.092 0.033
15.374
1 P.l. 0.985 3.794 0.161
112.572
2 P.l. 1.263 5.257 0.133
387.022
3 P.l. 1.940 13.127 0.089
137.278
4 P.l. 0.387 5.316 0.089
111.940
5 P.l. 0.304 0.528 0.025
20.667
6 P.l. 1.958 14.666 0.076
111.687
7 P.l. *
*
*
*
8 P.l. 0.253 3.891 0.062
42.600
9 P.l. 0.470 0.990 0.089
57.236
*: values not taken
Fe
13.786
15.791
9.656
10.857
5.814
4.023
4.655
7.897
6.202
54.507
55.435
26.020
77.421
9.965
128.525
111.115
29.648
132.828
101.215
61.371
92.301
119.598
84.958
20.414
67.664
88.017
39.185
49.427
65.487
43.133
30.769
15.820
26.227
*
28.013
20.107
Mn
0.545
0.324
0.463
0.739
0.207
0.202
0.368
0.276
0.280
3.183
3.451
3.414
6.377
0.846
15.224
18.227
1.552
13.448
1.902
1.714
2.456
4.492
1.114
1.738
1.528
1.390
0.983
17.030
3.238
17.327
18.891
2.532
4.741
*
7.505
4.305
Mo
0.007
0.008
0.007
0.008
0.000
0.008
0.004
0.009
0.008
0.045
0.044
0.053
0.038
0.029
0.100
0.036
0.053
0.029
0.134
0.086
0.205
0.105
0.093
0.100
0.101
0.150
0.081
0.291
0.061
0.065
n.d.
n.d.
0.035
*
0.009
0.004
Ni
1.364
0.193
0.337
0.506
0.272
0.075
0.128
0.077
1.798
1.252
1.256
0.734
2.288
0.856
1.493
1.661
0.387
1.698
0.532
0.836
0.394
0.456
0.117
0.383
0.064
6.150
3.719
0.597
0.337
0.627
0.382
0.000
0.344
*
0.418
0.286
Pb
0.011
n.d.
0.007
0.038
0.002
0.009
n.d.
0.002
0.003
0.174
0.139
0.131
0.284
0.067
0.366
0.241
0.023
0.355
0.098
0.045
0.046
0.065
0.006
0.005
0.000
0.033
0.016
19.753
9.978
16.308
11.515
2.267
15.109
*
5.307
1.438
Sn
0.003
0.003
0.001
0.004
0.002
0.007
0.000
0.002
0.002
0.008
0.014
0.006
0.010
0.007
0.035
0.009
0.003
0.007
0.012
0.007
0.013
0.004
0.013
n.d.
0.001
0.008
0.007
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
*
n.d.
n.d.
V
0.029
0.055
0.026
0.029
0.020
0.037
0.022
0.032
0.034
0.104
0.178
0.073
0.225
0.054
0.553
0.292
0.041
0.519
0.093
0.233
0.113
0.090
0.290
0.064
0.158
0.131
0.121
0.086
0.124
0.078
0.088
0.050
0.126
*
0.052
0.121
1053B
10536B
10537B
10538B
10539B
1054B
1054B
10542B
10543B
1054B
1054B
10546B
10548B
10549B
105B
105B
1052B
1053B
1054B
105B
1056B
1057B
1058B
1059B
1056B
1056B
10562B
10563B
10564B
1056B
1056B
1057B
10576B
1057B
10578B
10579B
1058B
10589B
1059B
1059B
10592B
10593B
10594B
1063B
1064B
1065B
106B
1067B
1068B
1067B
1068B
1069B
1062B
1062B
1062B
1063B
10632B
1063B
10634B
10635B
1063B
10645B
1064B
10647B
10648B
10649B
1065B
10659B
106B
106B
1062B
1063B
1064B
10673B
10674B
10675B
1067B
1067B
10678B
10687B
1068B
10689B
1069B
1069B
10692B
107B
1072B
1073B
1074B
1075B
1076B
1075B
1076B
107B
1078B
1079B
1072B
10729B
1073B
1073B
10732B
1073B
10734B
10743B
1074B
10745B
10746B
1074B
10748B
1075B
10758B
10759B
1076B
1076B
10762B
107B
1072B
1073B
1074B
1075B
1076B
10785B
10786B
1078B
1078B
10789B
1079B
1079B
108B
108B
1082B
1083B
1084B
1083B
1084B
1085B
1086B
1087B
108B
10827B
1082B
10829B
1083B
1083B
10832B
1084B
10842B
10843B
1084B
10845B
10846B
1085B
10856B
10857B
1085B
10859B
1086B
10869B
1087B
1087B
10872B
10873B
10874B
1083B
1084B
1085B
1086B
1087B
108B
10897B
1089B
1089B
109B
109B
1092B
109B
1092B
1093B
1094B
1095B
1096B
10925B
10926B
10927B
10928B
1092B
1093B
1093B
1094B
1094B
10942B
10943B
1094B
10953B
10954B
1095B
10956B
10957B
10958B
10967B
10968B
1096B
1097B
1097B
10972B
1098B
10982B
10983B
10984B
10985B
10986B
1095B
1096B
1097B
1098B
109B
10B
109B
10B
10B
102B
103B
104B
1023B
1024B
1025B
1026B
1027B
1028B
1037B
1038B
1039B
104B
104B
1042B
1052B
105B
10534B
10547B
Zn
3.645
4.003
3.674
3.969
2.846
4.721
2.915
3.128
3.681
11.295
11.123
11.830
13.525
14.772
14.465
9.217
14.737
12.980
16.971
14.733
27.322
21.549
27.191
13.168
12.784
20.835
17.329
589.719
46.968
360.795
457.576
20.470
228.797
*
43.652
36.901
1053B
10567B
1058B
10568B
1059B
10582B
10637B
1065B
10638B
1065B
10652B
10639B
10679B
106B
10653B
107B
10694B
1068B
10735B
1072B
1079B
10763B
1075B
1073B
1079B
1078B
10765B
1089B
1086B
10793B
10847B
10834B
1082B
10875B
10862B
10849B
1093B
1089B
1087B
1093B
1098B
1095B
1095B
10946B
1093B
10987B
10974B
1096B
105B
102B
1098B
104B
103B
107B
104B
1046B
1032B
109B
106B
1048B
1034B
102B
1049B
1035B
105B
1036B
102B
108B
1094B
107B
102B
103B
1047B
1093B
1098B
1096B
1097B
1092B
105B
108B
103B
1045B
109B
10978B
10965B
10952B
10938B
1095B
10964B
1097B
109B
103B
106B
1029B
10976B
10963B
1095B
10937B
10924B
109B
10923B
10936B
1094B
10962B
10975B
1098B
10B
10948B
10935B
1092B
109B
10896B
1082B
10895B
1098B
1092B
10934B
10947B
1096B
10973B
1092B
1097B
10894B
108B
1086B
10854B
10867B
108B
10893B
1096B
109B
10932B
10945B
10892B
10879B
1086B
10853B
1084B
10826B
10839B
10852B
10865B
1087B
1089B
1094B
1097B
10864B
1085B
1083B
10825B
1082B
10798B
108B
10824B
10837B
1085B
10863B
10876B
1089B
10836B
10823B
108B
1079B
10784B
107B
10783B
10796B
1089B
1082B
10835B
1084B
1086B
108B
10795B
10782B
10769B
10756B
10742B
1075B
10768B
1078B
10794B
1087B
1082B
1083B
1078B
1076B
10754B
1074B
10728B
1074B
1072B
1074B
10753B
1076B
1079B
10792B
1085B
10752B
10739B
10726B
1073B
107B
1068B
1069B
1072B
10725B
10738B
1075B
10764B
107B
10724B
107B
10698B
10685B
10672B
10658B
1067B
10684B
10697B
107B
10723B
10736B
10749B
1069B
10683B
1067B
10657B
1064B
1063B
10643B
1065B
1069B
10682B
10695B
1078B
1072B
1068B
1065B
10642B
10629B
106B
1062B
1065B
10628B
1064B
10654B
1067B
1068B
10693B
1064B
10627B
1064B
106B
1058B
10574B
10587B
106B
1063B
1062B
10573B
10586B
1059B
1062B
10625B
10572B
1058B
10598B
106B
10624B
1057B
10584B
10597B
106B
10623B
1057B
10583B
10596B
1069B
1043B
10569B
120
Appendix
Appendix II-C. Element concentrations in host tissues and females and males of P. laevis.
74B
N
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
S
As
Cd
Co
Cu
Fe
M
0.050 0.007 0.004
0.587
6.139
M
0.196 0.012 0.018
0.856
7.354
M
0.204 0.026 0.009
1.219
9.504
M
0.300 0.025 0.010
1.186
11.635
M
0.128 0.015 0.016
0.595
6.737
M
0.110 0.043 0.035
1.193
13.374
M
0.160 0.042 0.023
1.715
17.603
M
0.342 0.010 0.010
0.674
6.326
I
0.140 0.062 0.035
1.579
22.278
I
0.667 0.217 0.414
8.076
283.876
I
0.390 0.099 0.070
2.883
38.581
I
0.509 0.127 0.053
7.520
36.942
I
0.461 0.115 0.144
5.542
112.767
I
0.558 0.198 0.452
9.707
196.072
I
0.290 0.131 0.154
5.781
143.294
I
0.608 0.198 0.176
8.721
111.702
L
0.488 0.171 0.032 16.792
116.333
L
0.729 0.100 0.072
9.838
51.846
L
0.394 0.293 0.037 18.403
80.057
L
0.805 0.090 0.038
9.739
47.926
L
0.303 0.093 0.026
4.565
76.498
L
0.471 0.320 0.110
20.237
95.855
L
0.440 0.105 0.034
6.196
45.563
L
0.880 0.079 0.020
8.285
29.439
P.l.
0.284 1.266 0.102 25.046
34.251
P.l.
0.820 2.030 0.157 87.794
43.857
P.l.
2.232 2.381 0.074 50.133
36.770
P.l.
2.988 3.551 0.077 64.596
44.711
P.l.
3.861 1.615 0.083 51.662
33.999
P.l.
1.496 2.867 0.295 80.038
54.780
P.l.
2.038 2.437 0.094 40.771
32.482
P.l.
0.752 2.931 0.079 130.318
41.992
P.l.
0.406 2.573 0.090 43.225
25.163
P.l.
0.949 2.580 0.221 122.360
54.722
P.l.
2.836 2.178 0.078 47.181
33.968
P.l.
2.240 3.538 0.080 92.528
33.372
P.l.
4.034 1.659 0.089 42.370
33.810
P.l.
1.463 2.517 0.281 87.445
52.640
P.l.
2.601 3.189 0.104 53.952
33.844
P.l.
0.820 2.772 0.089 130.160
33.196
1053B
1054B
105B
1067B
1057B
1058B
107B
107B
1072B
1073B
1082B
1083B
1084B
1085B
1086B
1087B
1096B
1097B
1098B
109B
10B
10B
10B
1B
12B
13B
14B
124B
125B
126B
127B
128B
140B
14B
142B
143B
157B
1095B
109B
123B
137B
139B
15B
138B
154B
15B
156B
16B
167B
168B
169B
170B
180B
18B
182B
183B
184B
194B
195B
196B
197B
198B
1208B
1209B
120B
12B
12B
123B
12B
123B
124B
125B
126B
127B
1236B
1237B
1239B
1240B
1250B
125B
125B
1253B
1254B
125B
1264B
1265B
126B
1267B
1268B
1269B
1278B
1279B
1280B
128B
128B
129B
1293B
1294B
1295B
1306B
1307B
1308B
1309B
1320B
132B
132B
132B
134B
135B
136B
137B
138B
139B
1348B
1349B
1350B
135B
1352B
135B
1362B
136B
1364B
1376B
137B
1378B
1379B
1380B
138B
1390B
139B
1392B
139B
1394B
1395B
140B
1405B
1406B
1407B
1408B
1409B
148B
149B
1420B
142B
142B
1423B
1432B
143B
143B
1435B
1436B
1437B
146B
147B
148B
149B
1450B
1460B
146B
1462B
1463B
146B
1465B
147B
1475B
1476B
147B
1478B
1479B
148B
1489B
1490B
149B
1492B
1493B
1502B
1503B
1504B
150B
156B
157B
158B
159B
1530B
153B
1532B
153B
154B
154B
1546B
1547B
1548B
1549B
158B
159B
1560B
156B
1562B
1563B
1572B
1573B
1574B
157B
1576B
157B
1586B
1587B
158B
1589B
1590B
159B
160B
160B
1602B
1603B
1604B
1605B
164B
165B
16B
167B
179B
193B
1207B
12B
1235B
1249B
1263B
127B
129B
1305B
139B
13B
1347B
136B
1375B
1389B
1403B
147B
143B
145B
1459B
1473B
1487B
150B
15B
1529B
1543B
157B
157B
158B
159B
1627B
1238B
120B
124B
124B
1270B
136B
1298B
1326B
1354B
1506B
1396B
1534B
142B
169B
14B
1452B
1439B
146B
1453B
140B
1480B
1467B
145B
149B
148B
1468B
1508B
1495B
1482B
152B
1509B
1496B
1536B
1523B
150B
150B
1537B
1524B
158B
1594B
1607B
162B
1567B
1580B
1593B
160B
153B
156B
1579B
1592B
1539B
152B
156B
1578B
152B
1538B
15B
1564B
15B
162B
1608B
159B
1623B
1609B
1430B
143B
14B
1457B
1470B
1458B
147B
148B
1472B
1485B
1498B
1486B
149B
152B
150B
153B
1526B
154B
154B
1568B
1582B
1596B
160B
1625B
16B
1597B
1583B
1569B
15B
154B
1527B
1540B
1624B
146B
1429B
1456B
1497B
1402B
145B
142B
1483B
138B
140B
1428B
1469B
1374B
1387B
14B
145B
1360B
137B
140B
14B
1346B
1359B
1386B
1427B
132B
1345B
1372B
143B
1426B
138B
13B
1358B
139B
142B
1425B
1438B
1620B
1398B
1304B
137B
134B
1385B
1290B
130B
130B
137B
1384B
1397B
140B
153B
168B
138B
1370B
1276B
1289B
136B
1357B
1248B
126B
1275B
1302B
134B
1356B
1369B
1382B
152B
1342B
135B
1368B
1507B
1520B
134B
1234B
126B
128B
1329B
120B
1247B
1274B
135B
1328B
129B
1206B
123B
1260B
130B
134B
1327B
1340B
145B
130B
13B
192B
1205B
1246B
1287B
178B
19B
123B
1273B
1286B
129B
132B
1367B
127B
1285B
164B
17B
128B
1259B
150B
163B
1204B
1245B
1258B
127B
1284B
1325B
1365B
1257B
124B
136B
149B
190B
123B
12B
135B
176B
127B
1230B
1243B
1256B
13B
126B
129B
108B
12B
162B
1203B
1094B
107B
148B
189B
120B
125B
128B
1297B
18B
106B
108B
1093B
134B
175B
Zn
2.822
3.376
3.791
4.039
4.598
3.302
4.526
2.844
8.126
10.602
10.740
8.210
9.483
11.575
8.794
13.584
21.816
16.441
22.953
16.146
16.205
22.998
13.455
13.989
51.338
73.472
33.353
89.820
36.158
43.792
49.566
69.439
152.305
154.508
65.014
70.738
53.999
51.264
63.857
116.758
1065B
120B
16B
174B
V
0.011
0.017
0.038
0.043
0.031
0.039
0.037
0.024
0.044
1.033
0.118
0.105
0.425
1.004
0.719
0.405
0.052
0.055
0.144
0.186
0.227
0.300
0.084
0.049
0.054
0.141
0.102
0.118
0.097
0.225
0.122
0.121
0.061
0.254
0.108
0.121
0.145
0.258
0.139
0.162
1079B
106B
147B
160B
120B
1092B
13B
146B
187B
109B
19B
132B
173B
1078B
1064B
107B
105B
18B
159B
186B
1283B
104B
145B
Sn
0.003
0.001
0.004
0.004
0.004
0.001
0.004
0.002
0.006
0.018
0.010
0.008
0.011
0.010
0.002
0.003
0.015
0.024
0.016
0.061
0.016
0.008
0.004
0.004
0.143
0.020
0.005
n.d.
0.010
0.005
n.d.
0.000
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1063B
109B
13B
172B
Pb
0.014
0.003
0.000
0.017
0.006
0.016
0.026
0.017
0.024
1.379
0.194
0.107
0.266
1.282
0.292
0.335
0.097
0.066
0.086
0.143
0.003
0.073
0.019
0.010
3.335
10.919
10.273
10.865
3.863
16.145
6.402
15.278
5.657
8.956
8.960
11.698
2.997
15.544
8.120
16.576
1062B
17B
158B
124B
Ni
0.112
0.549
0.342
0.227
0.148
2.588
0.325
0.466
0.462
2.096
2.540
0.804
1.027
2.507
1.081
2.322
0.396
0.214
0.113
0.201
0.277
0.326
0.589
0.159
0.559
1.167
0.466
0.408
1.690
1.149
0.361
0.915
0.314
0.805
0.536
0.367
0.412
1.108
0.503
0.634
1076B
103B
14B
19B
1324B
1089B
130B
185B
130B
108B
16B
17B
1296B
1075B
106B
1074B
102B
129B
153B
Mo
0.004
0.005
0.005
0.006
0.005
0.008
0.029
0.004
0.089
0.043
0.023
0.027
0.021
0.039
0.019
0.019
0.161
0.108
0.101
0.209
0.105
0.243
0.077
0.062
0.392
0.058
0.019
0.034
0.023
0.031
0.017
0.013
0.197
0.053
0.015
0.022
0.017
0.035
0.021
0.016
106B
15B
152B
165B
Mn
0.284
0.495
0.246
0.272
0.283
1.455
0.342
0.192
2.923
33.500
2.173
2.587
8.623
39.889
12.036
9.010
2.207
2.117
0.911
1.099
0.885
2.821
0.759
0.826
10.164
14.928
4.588
5.567
4.117
24.787
3.801
3.819
9.768
17.824
4.907
3.785
4.224
21.294
5.084
4.621
1059B
1069B
108B
163B
1056B
1068B
1528B
1542B
156B
1570B
1584B
1598B
162B
162B
121
Appendix
Appendix III: Water temperature data (in C)at two localities of Danube River for the period
1628B
2005-2006 (data from TNMN (2009)).

Month
Novo Selo
(5 km upstream of Vidin)
2005
2006
J
4.9
2.8
F
1.5
6.4
M
2.1
5.3
A
11.5
10.6
M
16.2
15.4
J
17.5
16.8
J
23.9
24.9
A
24.2
25.1
S
15.2
21.1
O
15.5
18.3
N
10.6
11.2
D
7.3
7.2
* value is not available
1629B
1632B
163B
1634B
164B
1645B
164B
1650B
165B
164B
1640B
1649B
1654B
165B
1659B
16B
164B
165B
16B
169B
1670B
167B
1674B
1675B
167B
1679B
1680B
168B
1685B
168B
1690B
170B
1694B
1637B
169B
1643B
1647B
1648B
1652B
1653B
1657B
1658B
162B
163B
167B
168B
1672B
1673B
167B
1678B
1682B
1683B
1687B
169B
1695B
1638B
1642B
165B
160B
1684B
163B
163B
1635B
1639B
1689B
Iskar
2005
2006
8.8
6.9
6.5
6.2
3
5.3
10.4
11.4
15.6
18
21.4
20
24.2
25.1
27.6
*
22.4
24
18.8
22.9
11.2
14.7
8.3
11
1630B
168B
1692B
1697B
1693B
1698B
169B
122
Appendix
Appendix IV. Morphological data of Pomphorhynchus laevis used in Chapter 3.
170B
In the Appendix were used the following abbreviations:

1702B
N:
Fish number
S:
Season (1 spring, 2 summer, 3 autumn)
W:
Weight of P. laevis infracomunity (g)
N P.l.:
Number of individuals
MAW:
Mean acanthocephalan weigh (g)
1703B
1704B
1705B
1706B
170B
N
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1708B
182B
1823B
18B
183B
180B
1803B
1798B
1793B
178B
1783B
178B
173B
1768B
1763B
1758B
1753B
1748B
1743B
1738B
173B
1728B
1723B
178B
173B
S
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
W
0.375
0.421
2.382
2.124
1.025
1.852
1.832
3.784
1.471
1.376
1.868
1.225
0.827
1.213
1.409
3.945
0.441
1.105
0.720
1.247
1.934
0.846
0.641
1.280
1709B
175B
179B
1720B
170B
174B
1724B
1734B
1739B
174B
1749B
1754B
1759B
1764B
1769B
174B
179B
1784B
1789B
1794B
179B
1804B
1809B
184B
189B
1829B
1824B
1830B
1825B
1820B
185B
180B
1805B
180B
1795B
1790B
1785B
1780B
175B
170B
1765B
1760B
175B
1750B
1745B
1740B
1735B
1730B
1725B
1729B
N P.l.
26
18
157
114
82
88
125
179
88
76
99
91
55
55
37
109
61
68
89
66
72
74
65
85
MAW
0.014
0.023
0.015
0.019
0.013
0.021
0.015
0.021
0.017
0.018
0.019
0.013
0.015
0.022
0.038
0.036
0.007
0.016
0.008
0.019
0.027
0.011
0.010
0.015
17B
17B
172B
172B
172B
176B
1726B
172B
173B
1732B
1736B
173B
174B
1742B
1746B
175B
174B
1752B
1756B
175B
176B
1762B
176B
17B
176B
172B
176B
17B
178B
1782B
1786B
178B
179B
1796B
1792B
179B
180B
1802B
1806B
1807B
18B
182B
186B
182B
187B
183B
1832B
1827B
182B
1826B
Lebenslauf Milen Nachev

183B
Hohlweg 24, 45147 Essen
Anschrift
1835B
1834B
21.04.1979
Geburtsdatum
183B
1836B
Sofia, Bulgarien
Geburtsort
1839B
1837B
Ausbildung
1840B
1985-1991
Grundschule in Sofia
184B
1991-1997
1853B
Nationales Gymnasium fr Naturwissenschaften und Mathematik in

Sofia, Profil: Biologie und Biotechnologie
1842B
1997-2002
1854B
Studium an der Universitt Sofia (Fakultt fr Biologie)

Fach: kologie und Umweltschutz; Magister-Abschluss
1843B
185B
1856B
Von Herbst 2002 bis

Sommer 2003
Immatrikulation am Studienkolleg der Universitt Karlsruhe (Zwei

Semester Deutschkurs)
184B
1845B
Im Herbst 2003
1857B
Die DSH (Deutsche Sprachprfung fr den Hochschulzugang

auslndischer Studienbewerber) erfolgreich bestanden
01.2004 05.2005
185B
Projektpraktika am Zoologischen Institut I der Universitt Karlsruhe

(Abt. kologie und Parasitologie)
1846B
Seit Sommer 2005
1859B
Promotionsstudium, Universitt Karlsruhe und Universitt

Duisburg-Essen Probenahme im Rahmen meiner Doktorarbeit
`Ìndikationsvermgen von Fischparasiten zur Beurteilung des
kologischen Zustandes aquatischer Habitate``
1847B
1860B
Sonstiges
184B
Seit 1992
Mitglied in der Bulgarischen Ornithologischen Gesellschaft bei

BAS (Bulgarian Academy of Sciences). Teilnahme an
ornithologischen Untersuchungen in europischen VogelSchutzgebieten
1849B
Vom 17. bis

23. April 2004
186B
1850B
``Workshop & Training course on fish parasites`` an der

biologischen Station Neusiedler See, in Ilmitz, sterreich
185B
Sprachkenntnisse
1852B
Essen, den
10.02.2010
1947B
1862B
Deutsch, Englisch, Russisch

1863B
Unterschrift:
1864B
/Dipl.-kol. Milen Nachev/

1865B
Erklrung:
184B
Hiermit erklre ich, gem. 6 Abs. 2, Nr. 7 der Promotionsordnung der Math.-Nat.186B
Fachbreiche zur Erlangung der Dr. rer. nat., dass ich das Arbeitgebiet, dem das Thema
Bioindication capacity of fish parasites for the assessment of water quality in the
Danube River zuzuordnen ist, in Forschung und Lehre vertrete und den Antrag von Milen
Nachev befrworte.
Essen, den _________________ ____________________________________
185B
Unterschrift eines Mitglieds der Universitt Duisburg-Essen

186B
Erklrung:
187B
Fachbereiche zur Erlangung des Dr. rer. nat., dass ich die vorliegende Dissertation selbstndig
verfasst und mich keiner anderen als der angegebenen Hilfsmittel bedient habe.
Essen, den _________________ ______________________________
189B
Unterschrift des/r Doktoranden/in

1890B
Erklrung:
189B
Fachbereiche zur Erlangung des Dr. rer. nat., dass ich keine anderen Promotionen bzw.
Promotionsversuche in der Vergangenheit durchgefhrt habe und dass diese Arbeit von keiner
anderen Fakultt/Fachbereich abgelehnt worden ist.
Essen, den _________________
1893B
Unterschrift des Doktoranden

1894B
_________________________

Fish Parasites Bioindicate Danube Water Quality

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Fish Parasites Bioindicate Danube Water Quality

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Bioindication capacity of fish parasites for the

assessment of water quality

Prof. Dr. B. Sures

Prof. Dr. H. Taraschewski

Vorsitzender des Prfungsausschusses: ______________________________

Tag der mndlichen Prfung: 14.06.2010

`` as an ecosystem matures, parasitism naturally tends to evolve into mutualism; parasites

Materials and Methods .................................................................................................. 15

Sampling sites .................................................................................................... 15

Fish sampling ..................................................................................................... 16

Determination of helminth community structure and statistical treatment ............ 18

Water quality ...................................................................................................... 18

Total parasite fauna............................................................................................. 20

Diversity of helminth communities ..................................................................... 22

Water quality classification ................................................................................. 25

Is metal accumulation in Pomphorhynchus laevis dependent on parasite sex or

Materials and Methods .................................................................................................. 30

Sample collection ............................................................................................... 30

Molecular identification of Pomphorhynchus laevis ............................................ 31

Heavy metal analysis .......................................................................................... 32

Data analyses and statistical treatment ................................................................ 33

Fish samples ....................................................................................................... 34

Analytical procedure .......................................................................................... 34

Element concentrations in barbel and Pomphorhynchus laevis ............................ 35

Seasonal differences of metal accumulation in Pomphorhynchus laevis and its definitive

Materials and Methods .................................................................................................. 44

Fish samples ....................................................................................................... 44

Analytical procedure .......................................................................................... 45

Data analyses and statistical treatment ................................................................ 45

Element concentrations in the Danube River ....................................................... 45

Analytical procedure .......................................................................................... 46

Element concentrations in fish tissues and parasite samples ................................ 46

Seasonal differences in acanthocephalans morphology ...................................... 48

Seasonal variation in concentrations of the elements accumulated by P. laevis .... 49

Application of acanthocephalan Pomphorhynchus laevis from its host barbel (Barbus

Materials and Methods .................................................................................................. 58

Fish samples ....................................................................................................... 58

Heavy metal analysis .......................................................................................... 59

Data analyses and statistical treatment ................................................................ 60

Background metal monitoring data ..................................................................... 60

Element concentrations in the host-parasite system ............................................. 62

Longitudinal profile of element concentrations in the Bulgarian part of the Danube

Longitudinal profile of element concentrations in the Danube River in 2007 ....... 67

Long term monitoring of element concentrations in the lower Danube ................ 68

Comparisons between element concentrations in parasite with the available

Summary, conclusions and future prospects........................................................................... 75

List of criteria characterizing the ideal sentinel organisms. ..........................................11

Scope of the thesis

1 The endohelminth fauna of barbel (Barbus barbus) correlates

1 The endohelminth fauna of barbel correlates with water quality

1.2 Materials and Methods

1 The endohelminth fauna of barbel correlates with water quality

1.2.2 Fish sampling

Total Length [cm]

1 The endohelminth fauna of barbel correlates with water quality

Table 1.1. Morphological parameters and characteristics of collected fish material.

1 The endohelminth fauna of barbel correlates with water quality

1.2.3 Determination of helminth community structure and statistical treatment

1.2.4 Water quality

1 The endohelminth fauna of barbel correlates with water quality

1 The endohelminth fauna of barbel correlates with water quality

1 The endohelminth fauna of barbel correlates with water quality