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Fitting a new assessment system for rivers in Greece using fish fauna to the results of the MED GIG


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This report documents the development of a new ecological classification method for rivers using the BQE Fish. The approaches followed comply with the procedures specified in CIS Guidance Document no 30 (Wilby et al. 2014). Greece did not intecalibrate existing fish indices in the previous rounds of the intercalibration exercise due to lack of sufficient standardised data and the absence of a state-wide fish index. Although important fish-based bioassessment projects have been worked on in Greece for more than a decade, routine fish data collection began following the establishment of a national bioassessment monitoring programme in 2012. This led to the development of the “Hellenic Fish Index” (HeFI) during 2014-2016.
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Fitting a new
assessment system
for rivers in Greece
using fish fauna to
the results of the
Annex Report submitted to the
Institute of Marine Biological
Resources and Inland Waters -
December 24th 2016
(revised version)
Athens, Greece
Fitting a new assessment system for rivers in Greece using fish
fauna to the results of the MED GIG
Table of contents
1. INTRODUCTION ...................................................................................................................... 3
2. BACKGROUND ........................................................................................................................ 3
3. SURVEY DESIGN, FIELD SAMPLING AND RECORDING PROCEDURES ..................................... 4
4. ACTIVITIES SUPPORTING BIOASSESSMENT RESEARCH .......................................................... 7
5. FISH-BASED BIOASSESSMENT INDICES ................................................................................... 9
6. DEVELOPMENT OF A STATE-WIDE INTERREGIONAL FISH INDEX (HeFI)............................... 11
7. DISCUSSION .......................................................................................................................... 19
8. A WAY AHEAD ....................................................................................................................... 20
APPENDIX ..................................................................................................................................... 22
REFERENCES ................................................................................................................................. 27
This report should be cited as:
Tachos V., Zogaris S., Oikonomou E. & Economou A.N. Dec 2016. Fitting a new assessment system for
rivers in Greece using fish fauna to the results of the MED GIG - ANNEX REPORT. Unpublished Annex
Report submitted to WG ECOSTAT through the Special Secretariat for Water, Hellenic Ministry of
Environment and Energy. Institute of Marine Biological Resources and Inland Waters, HCMR, Athens. 34
This report documents the development of a new ecological classification method for rivers
using the BQE Fish. The approaches followed comply with the procedures specified in CIS
Guidance Document no 30 (Wilby et al. 2014). Greece did not intecalibrate existing fish indices
in the previous rounds of the intercalibration exercise due to lack of sufficient standardised
data and the absence of a state-wide fish index. Although important fish-based bioassessment
projects have been worked on in Greece for more than a decade, routine fish data collection
began following the establishment of a national bioassessment monitoring programme in 2012.
This led to the development of the “Hellenic Fish Index” (HeFI) during 2014-2016. This index is
based on the site-specific approach for defining reference conditions and offers prospects for a
nation-wide application. A number of fish-based indices developed earlier through the type-
specific approach could only have local application due to biogeographic variability,
hydrographic idiosyncrasies and substantial biological heterogeneity in the country
constraining their transferability to other areas. Methods for data collection, for setting
reference conditions and class boundaries, and for selecting and calibrating metrics are
described and are generally compliant with the WFD normative definitions. HeFI has
undergone some preliminary validation using monitoring data. However, its performance and
efficiency in some river systems remains untested.
Before 2012 there was no functional biological monitoring programme utilising the BQE "fish in
the rivers" of Greece. However, since 2003, bioassessment was included in various projects
that pursued various objectives, such as environmental protection. The fish sampling
procedures have generally been following the field protocols produced by the FAME project
(Schmutz et al. 2007a). In the summer of 2009 a single nation-wide fish survey was conducted
within an initial project for river assessment following the WFD (Chatzinikolaou & Economou
2009; for results see: Economou et al. 2016). In 2012 a monitoring system compatible with the
WFD procedural requirements was formally established. The delay in initiating monitoring
affected progress in bioassessment research through limiting the opportunities for the
collection of fish data upon which bioassessment tool development and testing heavily relies.
Additional difficulties arose from biogeographic variability in the country, hydrographic
idiosyncrasies and biological complexities (e.g. data-gaps in endemic fish ecological knowledge,
fish taxonomy revisions, etc). Greece is a hydrologically fragmented country with many
medium and small sized rivers (over 120 autonomous river basins containing fish have been
identified; Economou et al. 2007a; Koutsikos et al. 2012). The freshwater fish fauna is
characterised by a strong biogeographic structure (eight freshwater ecoregions have been
defined; Zogaris et al. 2009a, 2009b). Like in other Mediterranean countries (Logez et al. 2005;
Ferreira et al. 2007a, 2007b; Magalhães et al. 2008; Hermoso et al. 2010; Alonso et al. 2011,
Aparichio et al. 2011; Dallas 2013; Benejam et al. 2015), the rivers Greece are characterised by
fluctuating and seasonally arid hydroclimatic conditions, and they host species-poor, highly
endemic and greatly diversified fish faunas which are dominated by tolerant taxa with wide
environmental tolerances and low functional specialisation (Skoulikidis et al. 2009; Economou
et al. 2016). This combination of conditions limit the direct applicability of bioassessment
approaches that have been developed in central and northern European countries and make
make fish-based bioassessment particularly challenging (Logez et al 2005; Ferreira et al. 2007a;
Pont et al. 2007; Magalhães et al. 2008; Benejam et al. 2015; Hermoso et al. 2010; Dallas
Under the circumstances described above, the strategy for advancing fish-based bioassessment
consisted in making the best use of what resources were available (workforce, equipment,
datasets, bioassessment tools) and covering as many aspects of river assessment as possible.
Initial efforts (before monitoring was implemented) focused on the development of spatially-
based (type-specific) ecological status evaluation systems. These were often the by-product of
small-scale regional projects which were undertaken with other objectives. Although fish
sampling was conducted with WFD-compliant methodologies, the data generated by these
projects were too localised and variable in temporal time scales to serve as a reliable basis for
nation-wide bioassessment analysis. Some fish-based indices were generated, which were
designed for local applications. However, the substantial environmental and biological
heterogeneity across the country constrained the transferability of already established indices
to national or even ecoregional scales. As a result, when the intercalibration exercise was
taking place, a national fish-based bioassessment system was not in place. Because of this,
Greece was only an observer in the "fish BQE for rivers" intercalibration process. However,
researchers maintained close ties with many team members working on bioassessment and
intercalibration in Mediterranean countries, and even worked on joint projects (Pont et al.
In 2009 a state-wide biological sampling programme was established, but the programme was
suspended after only one round of sampling (summer 2009) had been completed. However,
this sampling exercise provided basic insights into planning and procedural aspects of survey
design and field protocol development and guided subsequent research efforts towards
bioassessment tool development. The results of these sampling activities have been reported
by Economou et al. (2016).
Routine and systematic fish sampling over broad spatial and temporal scales with the use of
standardised protocols began in 2012, when monitoring activities were re-initiated. Data
collection through these activities enabled the development of the “Hellenic Fish Index” (HeFI),
which is based on a site-specific approach for predicting reference conditions. The index offers
perspectives for a nation-wide application and has undergone some preliminary validation
using existing data.
The starting point of most bioassessment monitoring methodological tasks and analyses was
work accomplished during the EU-funded research project FAME in which the HCMR
participated from 2002 (Schmutz et al. 2007a). HCMR ichthyological sampling for
bioassessment pioneered something new for Greece: the use of several field protocols
concurrently at each site following a holistic potamological approach (Zogaris et al. 2008,
Zogaris 2009).
Greece's official WFD sampling network, as defined in the Joint Ministerial Decision
140384/2011, includes 449 sampling stations (149 for operational and 300 for surveillance
monitoring), in 14 major River Basin Districts. Operational stations are sampled annually with
two samples per year (spring and summer). Surveillance stations are scheduled to be sampled
on a 3-year rotation basis, again with two samples (spring and summer) taken in the year of
sampling. An increase of the sampling stations to 490 (195 for operational and 294 for
surveillance monitoring) has been decided for the next sampling cycle (beginning in early
The design of the sampling network took into account a preliminary list of river Water Bodies
which was provided by the administration. The positioning of the sampling sites was
determined on the basis of best available knowledge to represent the landscape and macro-
habitat features and the range of prevailing human pressures. Several sites were selected to
correspond with sites sampled in previous fish and macroinvertebrate surveys in order to
ensure the inclusion of interannual biological variability in the datasets. Sites contained in a
pre-existing hydrochemical monitoring programme (the "National Network for Monitoring the
Quality of Surface Waters") were also included in the sampling scheme. The designation of sites
as routine or surveillance was based on available knowledge of important pressures and
assessments of impacts.
The sampling network contained undisturbed or minimally disturbed sites (reference sites) to
serve for the determination of reference conditions. Reference sites were selected on the basis
of pressure “exclusion criteria”, following general guidelines developed during the FAME
project (Economou 2002). Data from sites with low levels of anthropogenic disturbance or
affected relatively little by human pressures (e.g. low levels of pollution from agricultural
activities or water abstraction) were used for the derivation of reference conditions.
Fish sampling was carried out solely with the use of electrofishing and took place at the same
locations as with sampling for macroinvertebrates and other BQEs. At each sampling site
measurements of chemical and physicochemical parameters were made and
hydromorphological analysis was performed with the use of the River Habitat Survey (RHS)
method (Raven et al. 1997); QBR was also used as an index of riparian condition at most sites.
Photographs and video of the sampled localities and specimens caught were taken for
documentation and follow up evaluations.
The official sampling site area was defined as a 500-m long river section covered by the RHS
method within which sampling for the various BQEs was conducted. Within each site, a
representative river reach containing typical fish habitats (i.e. riffle, run, pool, glide) was
selected and electrofished. A single pass was conducted and no stop nets were used. In deep
rivers the electrofishing gear was fitted on a boat. Fish sampling and environmental data
recording followed standardised procedures; key environmental and habitat parameters were
recorded (IMBRIW 2012).
Fish sampling procedures were adapted from the CEN standard BS EN 14011:2003 Water
Quality- Guidance standard on sampling fish with electricity. With regard to the length and
method of the sampled stretch, some flexibility was allowed dependant on river size and
depth, flow velocity, morphological, habitat characteristics and biological conditions (e.g.
number of autochthonous species, abundance of individuals). Except in fishless streams, at
least 15 individual fish were set to be collected (minimum catch; if not the sampling was
continued in most cases until the minimum catch as completed). Generally more sampling
effort was put in sites with higher species diversity and habitat heterogeneity or when fish
density was particularly low. At the end of fish sampling occasion a qualitative assessment of
sampling effort and efficiency was made. Sampling effort was assigned to quality classes A, B,
C, D as described in the Institute’s manual (IMBRIW 2012) and was recorded in the sampling
protocol (see APPENDIX Figure A3). Quality class D indicates highly insufficient and/or non-
representative sampling and the results from this sampling occasion were not used for
ecological status assessments. This was often the case when there were particularly strong flow
conditions, great depths, or morphological obstacles preventing sampling the desired minimum
sampling length. Sampling quality class D is also used for qualitative records of fish. Sampling
quality C is a borderline condition, and in many cases it was also not considered in our
ecological status assessment.
An important parameter is the actual length of river (and the areal cover) sampled. The 20X
empirical rule (fished length at least 20 times the mean wetted stream width) was set for small
wadable streams (less than 6 m wide). This rule was nearly always satisfied, and stream portion
about or over 100 m was typically sampled. The rule was not met in a small number of sites
(less than 10) with highly homogeneous fish fauna, e.g. mono-species or low-species sites such
as in small "mountain barbel type" streams. For larger wadable streams up to 15 m wide a ten-
fold rule was generally assigned. The great majority of sites (80%) satisfied the rule and
between 80 and 400 m were sampled. The sampling area always exceeded 100m2 for small
streams and 200m2 for larger streams. For rivers with a wetted width >15 m also a ten-fold rule
was generally assigned and at least 150 m length was always sampled. Boat sampling was used
in most cases and several hundred meters were usually sampled. Boat sampling always
exceeded 600m2 sampling area, with most samples lying over 1000m2.
Finally, regardless of the sampling method used (wading or boat), all samples, were assigned
specific sampling form categories: “one bank”, “partial whole” and “whole”. “One bank”
category covered all habitats of the shore, “partial whole” covered all habitats of the shore plus
habitats of the mid-channel and “whole” category covered all habitats of the wetted channel
(when "whole" was done with significant space left un-sampled it was distinguished as
"ambient"; i.e. nearly whole but more fragmented and/or dispersed sampling).
Throughout the surveys, two types of electrofishing devices were used: a) Battery-powered
Backpack: Hans-Grassl GmbH (Model IG200-2, DC pulsed, 1,5 KW output power, 35-100 Hz,
max. 850 V) and/or Smith-Root 24L (DC pulsed 1,5 KW, 10-100 Hz, max. 980 V) which were
routinely used to sample fish in streams and small rivers; and, b) a generator powered unit
EFKO Elektrofischereigeräte GmbH, Model FEG 6000 (DC unpulsed, 7,0 KW output power, 600
V), which was used in deeper streams and rivers. When the latter gear was used, a common
practice involved the operator thrusting (and throwing) the anode at a distance ahead to
surprise the fish and limit fish escape. The latter gear was mounted on a small aluminum boat
and used in non-wadable river sections during boat-based sampling in deeper waters.
The fish recording procedures were based on several economic and technical considerations,
such as limitations in the available workforce and the restricted time frame within which the
daily sampling plan and each sampling round had to be completed. Thus, the fish caught were
identified to species level, measured in situ in 5 cm class intervals and returned alive to the
river. Measuring at 5 cm intervals provides rapid measurement documentation and this has
been effectively utilised in some EU member states such as in the FiBS application in Germany
(Düssling 2009). In cases of identification problems (usually juveniles), samples were preserved
in formalin solution for laboratory identification. In some localities young fry occurred in
enormous densities and could not be quantitatively sampled due to large numbers and/or gear
limitations. However, an index of fry abundance was assessed and recorded in the protocol. In
each sampling occasion the wetted surface area sampled was estimated from geometrical
characteristics (fished length and cross-sectional width). Fish densities could not be
quantitatively determined because no stop nets were used (also only one anode was applied).
Bioassessment monitoring is an interdisciplinary operation which includes various steps and
stages and strongly depends on the accomplishment of tasks that provide input to monitoring
tool development (e.g. species inventories, biogeographic regionalisation and ecological trait
classifications). Significant milestones accomplished include the following:
4a. State-wide distributional inventory of fish species
A basin-level ichthyological database of all major catchments of Greece has been
developed, using sampling data and literature information. Initially established using
distribution records for native and introduced freshwater fish species from 105
hydrographic basins (Economou et al. 2007), the database was subsequently expanded with
the addition of new information from monitoring and other research activities (e.g.
Koutsikos et al. 2012). The database now contains data for 127 hydrographic basins. More
than 160 species are accommodated in this distributional compilation. Species lists are
given for each basin area, and information on conservation status and provenance are
provided. This database has supported research for ecoregional delineations, and has
facilitated the creation of an inventory of fish species with documented occurrence in
freshwaters (see below). It has also supported efforts for species conservation.
4b. Biogeographic classification of the southern Balkans based on fish
The WFD's typology is largely based on J. Illies' biogeographical regionalisation and
important discrepancies have been identified between the WFD's proposed ecoregoinal
map and current understanding of major freshwater biogeographical boundaries (Zogaris et
al. 2009a). A freshwater ecoregional delineation in the southern Balkans based on
biogeographic associations of drainage-specific fish communities was developed and refined
(Zogaris 2009, Zogaris et al. 2009b) (see APPENDIX Figure A1.). This work was corroborated
by a classification based on site-specific assemblage data by Economou et al. (2016).
Elaboration of the data obtained by the monitoring activities has revealed that the different
freshwater ecoregions host different species and different assemblage types (e.g. APPENDIX
Table A1). Fish taxonomy and especially our understanding and interpretation of
systematics and phylogeography are also important in defining among-basin
biogeographical affinities (Economou et al. 2007a).
4c. Type-specific reference conditions and fish-based biotic typology classification
The issue of reference conditions is keystone within WFD bioassesment and especially
challenging with fish assemblages in Mediterranean rivers (Economou 2002, Ferreira et al.
2007a). Recently, several projects have researched and identified fish community patterns,
such as longitudinal changes and environmental parameters affecting these assemblages
(Economou et al. 2003, Zogaris et al. 2004, Economou et al. 2007a, Zogaris 2009, Vardakas
et al. 2015). Specific longitudinal distribution patterns under near-natural conditions have
been identified (APPENDIX Figure A2)
4d. Checklist of Greek freshwater fish species and taxonomic clarifications
There has been a serious problem in bioassessment and conservation research and
applications with changing fish taxonomy (Economou et al. 2007a). A special project to
promote national checklist management (called has been recently pursued by
IMBRIW-HCMR and a web-based educational tool is being developed. Greece's most recent
annotated checklist was published during this project (Barbieri et al. 2015).
4e. Sampling standardisation and field protocol development
Sampling procedures and field data collections targeting bioassessment are in accordance
with the FAME (2005) project protocols and largely follow the CEN electrofishing sampling
methodology. Stardardised field forms have been developed since 2003 in Greece (see
APPENDIX Figure A3.) The standard method and protocols are outlined in a detailed manual
(IMBRIW, 2012) and have also been utilised in other countries in the Eastern Mediterranean
(Zogaris et al. 2012; Zogaris & Özeren 2014). Differences from CEN procedures and training
event developments have been outlined in Zogaris et al. (2015).
4f. Species guild classifications
An important need in bioassessment tool development is to assign fish species into guild or
functional trait categories for making fish metrics transferable among different basins or
ecoregional units. We exploited our own natural history knowledge and published
information on species ecologies, biologies and life histories, together with assessments of
habitat requirements derived from our site-specific database to produce guild classifications
of all species normally encountered in the riverine systems of Greece (APPENDIX Table A2.)
4g. Site-specific databases for fish and environmental parameters
Fish data, environmental attributes and human-induced pressure information relevant to
ecological status assessments are accommodated in a site-specific database. These data
were acquired from a number of river basins through the use of fish sampling protocols
specifically designed to serve the environmental demands of the WFD. The databases were
initially fed with data collected during early EU-funded research projects that were
undertaken to support the WFD (FAME, STAR). Since then the databases serves as the
repository of data collected during all subsequent fish surveys. Data from a pre-existing site-
specific fish database (Economou et al. 1999) were evaluated; those assessed as being
pertinent and compatible with the WFD requirements were included in the new database.
Bioassessment tools developed so far are multi-metric and are based on the reference
conditions concept. Two basic approaches have been followed for reference conditions
designation and tool development: the spatially-based (type-specific) approach, and the
model-based (site-specific) approach.
5a. Spatially-based indices
The simplest approach, and the default in the WFD, is the spatially-based approach. This
involves the establishment of a river typology that allows variability of biological conditions
over a broad geographical area to be partitioned into smaller spatial units (river types), for
which type-specific reference conditions are defined. Four indices have been developed
through this method. All were designed for local applications and were developed in the frame
of research projects which were adequately resourced to enable sampling at spatial scales
sufficient for multimetric indices to be constructed.
Procedures followed the guidelines of the FAME project (Schmutz et al. 2007b) and involved
the use of similarity analysis for identifying biologically homogeneous regions (biotic typology),
discriminant analysis for assigning probabilities of class membership to sites, the
characterisation of reference conditions through a combination of approaches (use of data
from reference sites, historical information when available, and expert judgment), the selection
of metrics representing the local taxonomic composition and community structure, metric
testing (redundancy, responsiveness to pressures), and setting class boundaries to metrics.
Metrics were selected to represent all relevant biological parameters defined in the WFD. To
the degree possible, the instructions provided in the Guidance Document no 10 (EU, 2003;
Table in page 48) were followed.
Upland rivers fish index
The index was developed for use in upland areas of the following river basins: Acheloos,
Aliakmon, Alpheios, Arachthos and Aoos (Economou et al. 2007b). This index defined three
major river types and can potentially be applied to other upland rivers of Greece and the
Balkans. Several other indices and a detailed analyses of pressures including riparian habitat
and hydromorphological human-induced degradation were studied concurrently during this
bioassessment development (Zogaris et al. 2009; Chatzinikolaou et al. 2011). Four river types
were defined (see Appendix A.
Evrotas fish index
Evrotas is a hydrologically impacted river basin that hosts a species-depauperate and highly
endemic fish fauna. Much of the main stem of this river is considered artificially intermittent
(Skoulikidis et al. 2012) and this presents a challenge due to significant variablility especially
after droughts. An index was developed based on local conditions and non-perennial reach
idiosynchrasies (Skoulikidis et al. 2008; Vardakas et al. 2009). Three river types were defined.
Aliakmon fish index
This index was developed for use in upper Aliakmon where the construction of a dam expected
to impact the area was scheduled (Economou et al. 2009; Tachos et al. 2013). Three river types
were defined. This index can be potentially be applied to the upper portions of other rivers in
the Macedonia-Thessaly ecoregion. Three main river types were defined.
Sperchios fish Index
This index was recently developed during an in-depth research project (KRIPIS Project Report
2016) in a basin of the northernmost part of the Western Aegean ecoregion. The index closely
follows the WFD demands for uncertainty analysis (Oikonomou et al. 2015) and is available in
recently completed unpublished report (see Sperchios River website:
coastal-and-marine-zone/). Four river types were defined.
5b. Model-based indices
Effort has been invested in building interregional indices that go beyond biogeographical
regions. Since the EFI+ index is not applicable to Greece, it was important to begin working on
model-based indices that rely on species traits and thereby surpass biogeographically-limited
basin conditions. Two indices have been developed through this approach. Both use a
combination of river-landscape descriptors and environmental variables to assist in
“predicting” ichthyological reference conditions at a site, with which the observed fish
attributes in the samples are compared.
Fish-Assessment Tool for Hellenic Rivers - FATHeR
This model-based fish index was developed through the cooperation of Uwe Dussling (German
FAME partner) using limited data from the middle and upper sections of a number of
watersheds within three freshwater ecoregions (Economou et al. 2007b). FATHeR employs
environmental variables to predict the “reference fish community” in a site and then employs
sample data from the site to calculate the deviation of fish community attributes from the
reference attributes.
Hellenic Fish Index (HeFI)
This model-based index was developed at HCMR between 2014 and 2016 and is largely based
on procedures developed in the European Fish Index approach (Schmutz et al. 2007a). It is
based on data from all relevant standardised samplings including the national monitoring
programme of surface waters. The index follows the WFD requirement to utilise fish
composition, abundance and age structure for assessment (although size class length is used as
a proxy of age). The index construction details are provided in the next section of this report.
Here we provide details of the development and a description of the Hellenic Fish Index (HeFI).
This work was developed between 2014 and early 2016 at HCMR with the cooperation of Prof.
Stefan Schmutz (BOKU, Austria) and will soon be submitted for publication (Zogaris et al. in
prep.). This index aims to be a standard national index, applicable in a wide variety of rivers
among different biogeographical regions in the southern Balkans. The index utilises fish
composition, abundance, age (size) structure and disturbance-sensitive taxa for assessment,
and therefore meets all the requirements of the WFD.
A total of 640 samples were considered for the analyses leading to the index; this is the largest
dataset ever used for inland fish assemblage-environmental research in Greece. Sampling
followed the IMBRIW protocol largely applying the CEN procedure; however only one anode
was used despite the width of the river's wetted area (and this creates a semi-quantitative
aspect to sampling, especially in deeper waters). The sampled sites are fairly evenly distributed
across the mainland of Greece and include two major islands (Evia, Lesvos). Several islands are
not included since their ichthyofaunal composition is poorly developed, and the type-specific
reference conditions are especially difficult to establish as is the case throughout the
Mediterranean islands and peninsulas (Zogaris et al. 2012, Skoulikidis et al. 2014). In total, the
analysed dataset contains 248,178 fish of 103 species. A median number of 223 fish (25%
quantile 73.8, 75% quantile 516.0) and 4 species (25% quantile 2, 75% quantile 7) were caught
in each sample. Fish sampling effort was about the same in reference (median 510 m2) and
impacted samples (median 450 m2).
Functional guild definitions were applied to developed potential bioassessment metrics. Six
biological and ecological traits were considered according to previous classifications of
European fish traits with regard to reproduction, trophic position, habitat preference, habitat
alteration and migratory behaviour. Each species was assigned to one of the different
categories of a trait (24 categories). We assigned species to categories based on published
accounts (e.g., Economou et al. 1999; FAME 2005, Logez et al., 2013) and recent field
observations of endemic and range-restricted species whose natural history and ecology is
poorly documented. Since the number of non-native species was comparatively low they were
included and not given special significance as was documented in an initial monitoring review
(Economou et al. 2016). In total, species were classified into 13 categories out of 6 traits. Each
category represents 10 to 68 species (Table 1). Figure 1 shows the proportion of fish species
contributing to the four final metrics.
Direction of response of ecological traits were predefined according to ecological expectations
(i.e positive or negative expected response to human-induced degradation; see Table 1). Due
to the "semi-quantitative" type of sampling only relative density ("dens") and relative number
of species ("rich") were considered. All metrics were additionally calculated for small (<100 mm
or <150 mm total length) and large fish (>=100 mm or >=150 mm total length).
Table 1: Species traits and categories tested and selected: category, code, number of classified
species, acceptable reference model established, significant response to pressures and non
redundant to other metrics, and finally selected metrics used for Index. Of the metrics selected
three had a positive expected response to degradation, the rest negative.
Figure 1: Proportion of fish species contributing to the four final metrics used for the Helenic
Fish Index (HeFI) within the reference samples.
In order to develop the index the following pressure attributes were assessed (following
Schinegger et al 2013) to identify near-reference and degraded sites: channel modification
Non redundant
Feeding habitat
(channelization), Instream habitat modification, embankment, riparian vegetation
modification, barrier upstream, barrier downstream, barrier basin, water abstraction,
hydropeaking, hydrological modification, impoundment, pollution, urbanisation, and irrigation.
Each site was assessed during a distance-based desk-study by a core team of expert field
ichthyologists who understand human-induced pressures on the ichthyofauna (Figure 2). So-
called "Reference sites" were defined as not or minimally impacted sites. For the selection of
responsive metrics two datasets were defined: (1) “No or minimally impacted samples” (REF:
class 1 and 2, n=135 sites), (2) “Strongly impacted samples” (IMPACT: class 4 and 5, n=297
sites) (see Table 2)
Figure 2: Classification of human-induced pressures in sites used to develop the Hellenic Fish
Index (HeFI). The class-categories follow WFD practice (1=reference/high; 5=bad).
Table 2: Scoring of pressures into 5 classes and definition of reference and impacted dataset.
Pressure information
Channel modification
Instream habitat modification
Riparian vegetation modification
Barrier upstream
Barrier downstream
Barrier basin
Water abstraction
Hydrological modification
Reference data: no or
minimally impacted
Strongly impacted
6.a- Metric modeling and selection
Classification and regression trees (CRT), a recursive partitioning method, were used to model
fish metrics as a function of environmental characteristics. Tree methods encompass several
advantages: (1) nonparametric basis, (2) no implicit assumption of linearity, (3) simplicity of
results for interpretation and (4) ability of predictive classification for new observations. Trees
depth level was limited to 3 levels and minimum bucket size to 15 samples in order to avoid
overfitting. Models performance were tested by calculating Pseudo-R2 and by 10-fold cross-
validation using the intern routine of the “rpart” algorithm.
Models were then used to predict metric theoretical values in reference conditions at any site.
Predictions were compared with observations and residuals (residuals = observations
predictions) were calculated. Assuming that most of the natural variability of the metrics was
included in the models, the metric residuals were supposed to vary according to the intensity
of human disturbances and independently of natural environmental variables (Pont et al.,
2006). Metrics were selected regarding model quality (Pseudo-R2 > 0.3, cross-validation
results), metric sensitivity to pressure (Wilcox u-test, p<0.001, metric median deviation > 20%)
and redundancy. Redundancy (Spearman rank correlations |r| >0.7) was considered by
iteratively removing the metric with the highest redundancy with other metrics until
redundancies among metrics were entirely eliminated.
6.b- Index computing and scoring
The index was derived by averaging selected metrics. The index derived from the
untransformed metrics was rescaled to range between 0 and 1. The thresholds of the five
ecological status classes (high, good, moderate, poor, or bad) were defined in agreement with
European intercalibration rules by splitting the index range in five equally spaced classes with
class boundaries at 0.8, 0.6, 0.4 and 0.2.
Fish index performance was tested by Spearman rank correlations comparing the fish index
with the cumulative pressure index and testing the response to pressures in very small (<100
km2), small (>= 100, <250 km2), medium (>=250, <1000 km2) and large (>=1000, <40000 km2)
using bootstrap method (sample size 30, 100 replicates).
6.c- Metrics selection
Reference models were derived for all but habitat traits, however, only feeding habitat,
feeding, migration traits responded to pressures: (1) proportional density of insectivorous
larger than 100 mm (dens.INSV.p.100large), (2) proportional density of omnivorous smaller
than 100 mm (dens.OMNI.p.100small), (3) proportional density of benthic species smaller than
150 mm (dens.BENTH.p.150small) and (4) proportional density of potamodromous
(dens.POTAD.p.all) (Table 1).
6.d - Reference models
Redundant environmental variables were identified by means of PCA and removed from the
further analyses. Finally, altitude, slope, altitude of source, catchment and mean January air
temperature were used to predict reference conditions (Figure XX).
Figure 3. PCAs of environmental parameters of reference samples before (left) and after (right)
removing redundant variables.
Under reference conditions the spatial pattern of the metric proportion of large (>=100 mm)
insectivorous fish is mainly triggered by catchment area and altitude with high proportions in
small rivers and high altitude. The proportion of small (<150 mm) benthic fish show similar
patterns but with very low proportion of benthic fish at very high altitudes (>918 m). For
potamodromous fish lower proportions can be expected in northern ecoregions at lower
altitudes. In contrast to these three metrics, the proportion of small (< 100 mm) omnivorous
fish is generally very low in all environments with the exception of southern rivers with
catchment areas >208 km2 (see Figure 4).
Figure 4. Reference models (decision trees) for the final metrics: (a) proportion of large (>=100
mm) insectivorous fish, (b) proportion of small (<150 mm) benthic species, (c) proportion of
potamodromous species and (d) proportion of small (<100 mm) omnivorous species.
Environmental parameters: Area = catchment area (km2) upstream of sampling site, Altitude =
altitude of sampling site (m), Alt_source = altitude of stream source (m), EcoRegionNS =
southern or northern ecoregions, Temp_Jan = mean monthly January air temperature.
Figure 5 reflects the response of the four metrics to pressure by comparing reference samples
with strongly impacted samples. The metrics show differences in the distributions of samples in
the two categories: insectivorous and benthic fish respond more distinct than the other two
metrics, however, potamodromous species vary less than other metrics under reference
conditions. Potamodromous and omnivorous demonstrate a bimodal distribution under
pressure indicating only partial response to pressures.
Figure 5. Response of individual metrics to site degradation (i.e.pressures).
6.e- Metrics response, index scoring and index performance
The performance of the index was evaluated along a gradient of human degradation (each site
had been pre-classified based on a cumulative pressure index during the earlier stage). Figure
6a shows a clear but non-linear relationship between the cumulative pressure index and the
fish index (Spearman rank correlation -0.537). While a slight increase in pressure is not
reflected by the fish index, a pressure index >18-20 results in a significant decrease of the fish
index. Variation of the fish index in response to pressures is low in case of low and high
pressure but high in case of medium pressure levels. There are no influence of catchment size
on index performance (Figure 6b). Therefore the index functions well both in small and large
Figure 6. A) HeFi index and its responsiveness with the cumulative pressure index of samples
and Β) index response for different catchment size (Spearman rank correlations).
The model-based fish index performed well in discriminating human-induced degradation
classes. The index scores have been preliminarily mapped using the five-scale categories
indicated by the WFD (Figure 7). Based on a screening with other indices and expert knowledge
of the particular sites during the sampling period, the results of the index provide evidence for
a robust bioassessment tool that works across biogeographic regions in a remarkable variety of
stream and river types.
Figure 7. Fish ecological status of sampled sites in Greece based on the Helenic Fish Index
The spatially-based approach is relatively straightforward and can be completed at regional
scales with local datasets. It is a logical option for bioassessment studies that are applied at
fine-scales, particularly in drainages with high endemicity levels and atypical landscape and
hydro-morphological characteristics that do not permit the application of broad-scale
approaches. A disadvantage of the spatially-based approach is that in the Greek situation, the
indices created through this approach have poor generalisability and spatial transferability. No
simple typology can fully capture the spatial diversity of environmental settings and biological
diversity in Greek rivers. Therefore, our knowledge of the environmental factors that influence
the structure and functional organisation of fish communities at regional scales must increase
substantially before spatial typologies that allow to reliably predict reference conditions can be
Another potential disadvantage is that river typologies cannot always control sufficiently
natural variability of biological conditions, unless they are sufficiently fine-scaled to allow
delineation of areas of relatively high biological homogeneity. In the main stem of some river
basins we observed a longitudinal (upstreamdownstream) gradient of change in metric values
pertaining to species richness, assemblage composition and ecological guilds, as is predicted by
the River Continuum Concept (Vannote et al. 1980). This longitudinal pattern has been
observed by other researchers as well (e.g. Oberdorff et al. 2001, 2002; Grenouillet et al. 2004;
Oliveira et al. 2012; Hermoso and Linke 2012) and implies that "broad" typologies cannot
adequately predict reference conditions, because different reference conditions would apply to
sites occupying upstream and downstream positions within a river type. Therefore, it deems
essential, if the spatially-based approach is to be used for defining reference conditions, to
include a model for controlling longitudinal patterns in assemblage attributes.
Model-based bioassessment indices offer a solution to the problem of poor transferability,
which is associated with the spatially-based approach. This is important from a cost-
effectiveness perspective. The Hellenic Fish Index (HeFI) is the most recent development and
seems to be a promising tool for bioassessment in Greek rivers. However, its performance and
efficiency in some river systems remains untested. The real evaluation will become possible in
the years to come with more data from monitoring operations.
Currently an analysis is being conducted of parameters that are likely to influence the HeFI
performance. We found it difficult to assign some fish species to rigid "guild categories" and we
are now examining more closely the functional and life-history attributes of these species using
data from field observations and the literature. We have also undertaken an analysis of all
tasks and methodologies involved in our monitoring operations in order to identify biases and
uncertainties that are likely to be associated with the survey plan and the procedures applied
so far. In this context, we are examining issues of sampling scale and sampling sufficiency,
spatial representativeness of the sampling network, interannual variability of catches in
reference sites, and the degree to which the spatial configuration of the sampling sites ensures
a fair and representative coverage of the officially designated water bodies.
Fish-based bioassessment is a very active research area in the Mediterranean (Ferreira et al.
2007a, Benejam et al. 2015) and our experience has shown that fish are important indicators of
hydromorphological impairment, habitat degradation and connectivity disruptions. Moreover,
their presence and distribution may be a good criterion for identifying "significant waters" and
reforming Greece's river water body delineations (for a general current review for Greece, see
Zogaris et al. 2016). There are also serious indications that fishes will be affected by climate
change as well (Papadaki et al. 2015) and they are especially important as indicators for
modeling environmental flow management and restoration (e.g. for an example from Greece
see Muñoz-Mas et al. 2016).
There is important work in progress and difficult challenges ahead for WFD-relevant
bioassessment using fish in rivers in Greece. The following important steps are seen as
necessary to provide a way forward for fish-based bioassessment in river monitoring in this
1. Further development and further refinement of HeFI in order to provide a baseline
interregional index for reporting and intercallibration.
2. The development of local indices at the ecoregional and basin level should also be
encouraged, since these spatially-based indices may be better honed to local conditions and
special environments (increasing their accuracy and precision). Furthermore, indices for
small streams with depauperate ichthyofaunas should also be attempted and used in insular
and peninsular small basins (e.g. see Segurado et al. 2014).
3. The protocols and field forms for sampling should be further refined in order to streamline
field work among different sampling campaigns, and electrofishing gears. The method
should be used to train all field workers sampling fish communities for bioassessment
purposes in Greece. This rigorous standardization and streamlining of sampling should be
promoted to assure quality control in sampling. Sampling bias is one of the most serious
problems producing unwanted noise and variation since at least three methods of
electrofishing are currently practiced in running waters (i.e. boat-based, bank-based and
back-pack procedures).
4. Research on species assemblages, fish community dynamics, functional traits and other
ecological research is important for refining existing indices.
5. A nationally agreed biotic typology scheme is an important unmet need and should be
addressed urgently. Biogeographic sub-regionalisation of Greece's freshwater ecoregions is
also important for regionally defined index development.
6. Research on temporal variation of samples in different sites is lacking and this is important
for explaining the effects of natural variability on metrics. Work is in progress, using the
available monitoring data, to study seasonal and annual biological variability in reference
7. Anthropogenic pressures on fishes have been poorly studied in Greece and eastern
Mediterranean river basins. Species traits and ichthyological indices should also be utilised
in assessing the amelioration of ecosystem integrity after measures are applied.
Figure A1. Freshwater Ecoregions in the Greek territory (following Zogaris 2009 with GIS
cartography by Y.Chatzinikolaou and N. Koutsikos). Ecoregions by number: 1: Thrace, 2:
Macedonia-Thessaly, 3: Southeastern Adriatic, 4: Western Aegean, 5: Ionian, 6: Crete, 7:
Eastern Aegean, 8:Southern Anatlolia (corresponding only to the Kastellorizo Island cluster).
Note that in the HeFi index the northern ecoregions (1 & 2) which have a widespread
ichthyofauna of Danubian origin are grouped as “Northern” and the other ecoregions are
grouped as “Southern” since they have species-depauperate assemblages dominated by range-
restricted endemics.
Figure A2. Reference conditions along longitudinal pattern of fish assemblages as exemplified
in the Acheloos basin in the Ionian Ecoregion (adapted from Zogaris 2009). In this approach,
hypothetical zones are named in order to broadly indicate the affect of combined physical
attributes such as altitude, distance from river source, catchment area and slope. Of the five
zones; the montane cyprinid also occurs as a mono-species "barbel zone" with small-sized
Barbus sp (e.g. Barbus peloponnesius in the Acheloos river).
Figure A3. The standardised field forms used to record habitat and pressure data (two at L) and
the fish sample data which is in size-class categories (at R). These have been published in the
institute’s manual (see IMBRIW 2012).
Table A1. Summary of data compiled in the HCMR fish database as of June 2016. Row titles
relate to freshwater ecoregional entity (see Map on Fig. A1).
Icthyological Attributes
Sample Number
Samples lacking fish
Total species richness
Sampled individuals
Species per sample
Individuals per sample
Endemic species
%Endemic species
%Endemic individuals
Introduced species
%Introduced species
% Introduced individs.
Table A2. Development of HeFI: Biological traits of the fish species (following EFI+ project
classification, and adjusted to fish communities of Greece).
Species Feeding Migration Repro Hab_Repro Hab_rheo Hab_feed
Alburnoides.bipunctatus INSV ---- LITH RH_LITH RHEO WC
Alburnoides.strymonicus INSV ---- LITH RH_LITH RHEO WC
Alburnus.alburnus OMNI ---- LITH RH_LITH EURY WC
Alburnus.scoranza OMNI ---- LITH RH_LITH EURY WC
Alburnus.sp.volvi OMNI ---- LITH RH_LITH LIMNO WC
Alburnus.thessalicus OMNI ---- LITH RH_LITH EURY WC
Alburnus.vistonicus OMNI LONG LITH RH_LITH EURY WC
Anguilla.anguilla PISC LONG ---- ---- EURY BENTH
Aphanius.fasciatus INSV ---- PHYT ---- LIMNO WC
Atherina.boyeri OMNI LONG PHYT ---- LIMNO WC
Barbatula.barbatula INSV ---- LITH RH_LITH RHEO BENTH
Caspiomyzon.graecus ---- ---- LITH RH_LITH RHEO BENTH
Chelon.labrosus OMNI LONG ---- ---- EURY WC
Chondrostoma.vardarense ---- POTAD LITH RH_LITH RHEO BENTH
Cobitis.arachthosensis INSV ---- PHYT ---- EURY BENTH
Cobitis.hellenica INSV ---- PHYT ---- EURY BENTH
Cobitis.ohridana INSV ---- PHYT ---- LIMNO BENTH
Cobitis.puncticulata INSV ---- PHYT ---- LIMNO BENTH
Cobitis.punctilineata INSV ---- PHYT ---- EURY BENTH
Cobitis.strumicae INSV ---- PHYT ---- EURY BENTH
Cobitis.trichonica INSV ---- PHYT ---- LIMNO BENTH
Cobitis.vardarensis INSV ---- PHYT ---- EURY BENTH
Ctenopharyngodon.idella ---- POTAD ---- ---- EURY WC
Cyprinus.carpio OMNI ---- PHYT ---- EURY BENTH
Dicentrarchus.labrax PISC LONG ---- ---- EURY WC
Dicentrarchus.punctatus PISC LONG ---- ---- EURY WC
Economidichthys.pygmaeus OMNI ---- PHYT ---- EURY BENTH
Economidichthys.trichonis OMNI ---- PHYT ---- LIMNO WC
Esox.lucius PISC POTAD PHYT ---- EURY WC
Gambusia.holbrooki INSV ---- ---- ---- LIMNO WC
Gasterosteus.gymnurus INSV ---- PHYT ---- EURY WC
Gobio.bulgaricus INSV ---- LITH RH_LITH RHEO BENTH
Gobio.feraeensis INSV ---- LITH RH_LITH RHEO BENTH
Gobio.skadarensis INSV ---- LITH RH_LITH RHEO BENTH
Knipowitschia.caucasica INSV ---- PHYT ---- LIMNO BENTH
Knipowitschia.milleri INSV ---- PHYT ---- LIMNO BENTH
Knipowitschia.thessala INSV ---- PHYT ---- LIMNO BENTH
Lepomis.gibbosus INSV ---- PHYT ---- LIMNO WC
Leucaspius.delineatus OMNI ---- PHYT ---- LIMNO WC
Liza.aurata OMNI LONG ---- ---- EURY WC
Liza.ramada OMNI LONG ---- ---- EURY WC
Liza.saliens OMNI LONG ---- ---- EURY WC
Mugil.cephalus OMNI LONG ---- ---- EURY WC
Neogobius.fluviatilis INSV ---- LITH RH_LITH EURY BENTH
Oncorhynchus.kisutch PISC ---- LITH RH_LITH RHEO WC
Oncorhynchus.mykiss INSV ---- LITH RH_LITH RHEO WC
Oxynoemacheilus.bureschi INSV ---- LITH RH_LITH RHEO BENTH
Oxynoemacheilus.pindus INSV ---- LITH RH_LITH RHEO BENTH
Pachychilon.macedonicum OMNI ---- PHYT ---- EURY WC
Pachychilon.pictum OMNI ---- PHYT ---- EURY WC
Pelasgus.laconicus OMNI ---- PHYT ---- LIMNO WC
Pelasgus.marathonicus OMNI ---- PHYT ---- LIMNO WC
Pelasgus.prespensis OMNI ---- PHYT ---- LIMNO WC
Pelasgus.stymphalicus OMNI ---- PHYT ---- LIMNO WC
Pelasgus.thesproticus OMNI ---- PHYT ---- LIMNO WC
Perca.fluviatilis PISC ---- PHYT ---- EURY WC
Petroleuciscus.borysthenicus INSV ---- PHYT ---- LIMNO WC
Phoxinus.strymonicus INSV ---- LITH RH_LITH RHEO WC
Proterorhinus.semilunaris INSV ---- LITH RH_LITH EURY BENTH
Pseudorasbora.parva OMNI ---- PHYT ---- EURY WC
Pungitius.hellenicus INSV ---- PHYT ---- LIMNO WC
Rhodeus.amarus OMNI ---- ---- ---- EURY WC
Rhodeus.meridionalis OMNI ---- ---- ---- EURY WC
Romanogobio.elimeius INSV ---- LITH RH_LITH RHEO BENTH
Rutilus.panosi INSV ---- PHYT ---- EURY WC
Rutilus.rutilus INSV ---- PHYT ---- EURY WC
Rutilus.sp.sperchios INSV ---- PHYT ---- EURY WC
Rutilus.ylikiensis INSV ---- PHYT ---- EURY WC
Sabanejewia.balcanica INSV ---- PHYT ---- RHEO BENTH
Salaria.economidisi INSV ---- LITH RH_LITH LIMNO BENTH
Scardinius.acarnanicus OMNI ---- PHYT ---- LIMNO WC
Scardinius.erythrophthalmus OMNI ---- PHYT ---- LIMNO WC
Silurus.aristotelis PISC ---- PHYT ---- LIMNO BENTH
Squalius.peloponensis OMNI POTAD LITH RH_LITH RHEO WC
Squalius.vardarensis OMNI POTAD LITH RH_LITH RHEO WC
Telestes.pleurobipunctatus OMNI POTAD LITH RH_LITH RHEO WC
Tinca.tinca OMNI ---- PHYT ---- LIMNO BENTH
Tropidophoxinellus.hellenicus OMNI ---- PHYT ---- LIMNO WC
Tropidophoxinellus.spartiaticus OMNI ---- PHYT ---- EURY WC
Valencia.letourneuxi INSV ---- PHYT ---- LIMNO WC
Valencia.robertae INSV ---- PHYT ---- LIMNO WC
Contributors to this report
This report reviews the process and products of several years of work in fish-based index-
building in Greece. Over 10 IMBRIW-HCMR scientists have been involved, the contributor's
names are present in the papers and presentations that have disseminated this work (see
references). We should specifically acknowledge the contributions of the FAME project and
the scientists that worked closely with the IMBRIW-HCMR team: S. Schmutz, U. Dussling, M.T.
Ferreira, W.R.C. Beaumont and P. Segurado. During the building of the HeFi (2014-2016) S.
Schmutz was responsible for guiding the index development; his involvement has been
instrumental in finalizing the proposed national fish-based index. IMBRIW-HCMR ichthyologists
are also grateful to the coordination efforts of N. Skoulikidis who was responsible for the WFD
monitoring project for rivers at HCMR. Finally, part of this work is based on E. Oikonomou’s
doctoral dissertation “Assessing and handling uncertainty associated with WFD bioassessment
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Edited volume of abstracts of a scientific meeting held at the Hellenic Ministry of Environment in Athens on March 16th 2015. 19 presentations are included on various themes related to the EU Water Framework Directive application in Greece and aquatic biodiversity conservation (In Greek).
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