water

Article
Back to Ecology: Reference Conditions as a Basis for
Assessment, Restoration and Sustainable Management of
Large Rivers
Gorazd Urbanič 1,2, *, Zlatko Mihaljević 3 , Vesna Petkovska 4 and Maja Pavlin Urbanič 1

1 URBANZERO Institute for Holistic Environmental Management, Ltd., Selo pri Mirni 17,
8233 Mirna, Slovenia; [email protected]
2 Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
3 Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov Trg 6, 10000 Zagreb, Croatia;
[email protected]
4 Ministry of the Environment and Spatial Planning, Dunajska Cesta 48, 1000 Ljubljana, Slovenia;
[email protected]
* Correspondence: [email protected]

Abstract: Under the EU Water Framework Directive, ecological assessment and management are
based on type-specific reference conditions. In the EU it may be difficult to find sites in large rivers
with at least near-natural conditions, though this is not the case in southeast Europe, where stretches
of large rivers still exist with at least near-natural conditions, meaning that there is little or no
disturbance from hydromorphological alteration, water quality, land use in the catchment and alien



species. We examined benthic invertebrate assemblages in 45 samples collected from near-natural

 sites of several large rivers: Sava, Drava, Mura, Kupa and Una. The near-natural benthic invertebrate
Citation: Urbanič, G.; Mihaljević, Z.; assemblages of large rivers contained several rare or remarkable species, especially among stoneflies,
Petkovska, V.; Pavlin Urbanič, M. e.g., Marthamea vitripennis, Xanthoperla apicalis. We compared benthic invertebrate communities in
Back to Ecology: Reference river sections with fine and coarse substrates and in three eco-hydromorphological (ECO-HM) types
Conditions as a Basis for Assessment, of large rivers, reflecting habitat heterogeneity: lowland-deep, lowland-braided and intermountain.
Restoration and Sustainable Multivariate analysis of variance (PERMANOVA) was used to statistically evaluate similarities
Management of Large Rivers. Water among assemblages. It was found that the composition of benthic invertebrate assemblages varied
2021, 13, 2596. https://doi.org/
by both ECO-HM types and substrate category. Similarity percentage (SIMPER) analysis showed
10.3390/w13182596
that the average dissimilarity of benthic invertebrate assemblages was high between all ECO-HM
type pairs and between fine and coarse substrate. We found that habitat heterogeneity and substrate
Academic Editor: Rui Cortes
independently influenced benthic invertebrate assemblages. To achieve ecological goals in the
Received: 10 August 2021
management of large rivers, in addition to functionality, a holistic view with at least near-natural
Accepted: 18 September 2021 assemblages, including the names of the taxa present, should also be considered.
Published: 20 September 2021
Keywords: large rivers; reference conditions; near-natural conditions; eco-hydromorphological types;
Publisher’s Note: MDPI stays neutral substrate; river-basin management; WFD; southeast Europe
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Large rivers provide a plethora of utilities for humankind, such as a source of water
for domestic, industrial and agricultural purposes, a means of energy production and
Copyright: © 2021 by the authors. waste disposal, routes for navigation, and space for recreational activities [1]. However,
Licensee MDPI, Basel, Switzerland. these human uses are also pressures and, in combination with land use changes in their
This article is an open access article catchments, pose a threat to the function and ecological integrity of aquatic ecosystems.
distributed under the terms and The long history of intensive use of large rivers has led to the degradation of unique
conditions of the Creative Commons ecosystems, and today large rivers are among the most degraded ecosystems on Earth.
Attribution (CC BY) license (https:// Concerns about the ecological quality of aquatic habitats have led to legislation in most
creativecommons.org/licenses/by/
developed countries that sets the objectives (e.g., EU Water Framework Directive 2000, US
4.0/).

Water 2021, 13, 2596. https://doi.org/10.3390/w13182596 https://www.mdpi.com/journal/water

Water 2021, 13, 2596 2 of 19

Clean Water Act of 1972, Australian Water Reform Framework [2]). The European Union
in its Water Framework Directive set 2015 as the year by which good ecological status
should also be achieved for rivers [3], though these aspirations are yet to be achieved, due
primarily to hydromorphological alterations that were identified as the main reason for
water bodies being unable to achieve the environmental objectives of Water Framework
Directive [4]. The European Green Deal was developed and published at the EU level [5]
as a holistic document that aims to improve the wellbeing and health of citizens and future
generations. It contains a proposal for legally binding EU nature conservation targets
for restoring degraded ecosystems in the framework of the EU Biodiversity Strategy by
2030 [6]. The importance of “semi-natural” rivers was recognized through the restoration
of river continuity and reinforced by a target to transform at least 25,000 km of rivers into
free-flowing rivers by 2030, mainly through the removal of obsolete barriers and restoring
floodplains and wetlands. In addition, according to the strategy, Member State authorities
are also required to review water abstraction and impoundment permits with the aim of
restoring ecological flows.
Due to the understanding that altering the hydrology, water chemistry and biology of
rivers has unintended consequences on human lives, an increasing number of restoration
projects were initiated [7–10]. A prevailing paradigm in ecological restoration is that
increasing habitat heterogeneity promotes biodiversity restoration, which is reflected in
stream restoration projects through the common practice of reconfiguring channels by
adding meanders and physical structures. A scientific review of river restoration projects
found that most projects successfully improved physical habitat heterogeneity, though very
few showed a statistically significant increase in biodiversity, increasing their similarity
to reference reaches or sites [11]. Improved ecological conditions are a true measure of
river restoration success, where observed conditions should be compared to reference
conditions. The reference conditions approach, which implies past historical conditions,
is widely used in ecological assessment and current legislation now widely depends on
the establishment of reference standards (e.g., Water Framework Directive). In Europe,
type-specific reference conditions have been established in a variety of ways, often taking
a pragmatic approach [12]. The latter is particularly true when sites with at least near-
natural conditions are lacking, as is the case for most large rivers in the EU. Regardless of
whether reference conditions are based on true reference sites or near-natural sites, their
selection is crucial for the development of useful measures of ecological status [13]. The
characteristics of biota occurring at reference sites are used to develop metrics and indices
that serve as a basis for comparison with impacted sites of the same river-type to assess
their ecological status. Therefore, biota representing reference conditions are a key element
of river ecological assessment, restoration and sustainable river basin management. The
key question is whether reference sites or at least near-natural sites are available for large
rivers and what the differences are in the biota between large river types.
In Europe, river engineering began several centuries ago, and today most European
large rivers are channelized and highly fragmented by dams, with few catchments still
containing free-flowing large rivers [14]. In large rivers, human impact often increases
along the river course, i.e., the lower reaches are the most disturbed [15,16]. The Dinaric
Western Balkan and the Pannonian Lowland (Ecoregions 5 and 11) [17,18] are areas in
Europe where free-flowing large rivers still exist. According to water quality results, the
impact of pollution is relatively low (e.g., [19,20]); thus, near-natural large river stretches
can still be found. The Kupa and Una are in near-natural conditions along their river
courses. The Drava is one of the few rivers in Europe that is not regulated in its middle and
lower sections. The lower Drava in Hungary has been protected as a national park since
1996 [21]. Large parts of the Sava River still show a relatively natural geomorphic structure
and hydrological regime, lined by large, protected wetlands. The mainstream has been
channelized for flood control only in a few, short sections (e.g., in Zagreb); the mainstream
has been channelized for flood control [14]. There are two Ramsar sites and three Important
Bird Areas [22] in the middle reaches of the Sava, and this river is a priority area in the Pan
Water 2021, 13, 2596 3 of 19

European Biodiversity and Landscape Strategy programmes and a key site in the Danube
River Basin programmes. The Sava, the lower Drava, Mura and Kupa also have significant
floodplains. A good example of intact floodplains is the Lonjsko Polje Nature Park on the
Sava [23]. However, these types of habitats are becoming increasingly rare as development
fundamentally changes river courses and floodplain landscapes. Although all these rivers
are located in the same region, certain sections differ in their natural habitat heterogeneity,
which is reflected in the eco-hydromorphological types [19,20].
The aims of this study were (i) to define near-natural benthic invertebrate assemblages
of large rivers, (ii) to examine difference in benthic invertebrate assemblage compositions be-
tween three eco-hydromorphological types of large rivers; lowland-deep, lowland-braided
and intermountain and (iii) to explain some of the key drivers of benthic invertebrate
variation across these river types. We hypothesized that (i) the composition of near-natural
benthic invertebrate assemblages and contribution of taxa vary among large river types,
and (ii) substrate and habitat heterogeneity independently influence the composition of
benthic invertebrate assemblages.

2. Materials and Methods

2.1. Study Area
The study was conducted in two neighbouring countries: Slovenia with a total area
of 20,273 km2 and Croatia with a total area of 56,594 km2 . The rivers in each of these
countries flow into either the Danube Basin or Adriatic Basin, but this study focused only
on the Danube catchment, covering 16,381 km2 (80.8%) of the Slovenian territory and
35,101 km2 (62%) of the Croatian territory. This study was limited to five major rivers
of the Danube Basin: Drava, Mura, Sava, Kupa and Una Rivers. Only stretches with a
catchment area of 5000 to 64,000 km2 at altitudes between 74 and 338 m were considered.
These stretches belong to three eco-hydromorphological types [19,20]: intermountain,
lowland-braided and lowland-deep (Table 1, Figure 1). The Sava and Drava are among
the largest tributaries of the Danube River (first and fourth, respectively) and represent
some of the most preserved rivers in Europe in terms of their biological and landscape
diversity. Nature conservationists and scientists consider the Sava to be one of the “crown
jewels” of European nature [24]. The lower Drava, together with the lower Mura, forms a
380 km long, free-flowing and relatively natural watercourse, representing one of the last
remaining contiguous riverine landscapes in Central Europe [23].

Table 1. Main characteristics of the sampled rivers according to the eco-hydromorphological river type and the number of
sampling sites and samples.

Eco-Hydromorphological Catchment Size Range

River Altitude Range (m a.s.l.) No. Sites (Samples)
Type (km2 )
Sava Intermountain 4946–5203 191–222 3 (7)
Drava Lowland-braided 13,189–31,038 122–236 7 (11)
Mura Lowland-braided 9784–10,930 153–246 6 (12)
Sava Lowland-deep 7151–64,073 74–191 7 (8)
Drava Lowland-deep 33,916–39,982 81–100 3 (3)
Kupa Lowland-deep 9184–9184 92–92 1 (2)
Una Lowland-deep 9368–9368 94–94 1 (2)
Water 2021, 13, 2596 4 of 19

Figure 1. Study area with large river eco-hydromorphological (ECO-HM) types and near-natural sampling sites with
prevailing natural substrate (closed circles).

2.2. Environmental Variables

The 28 sampling sites (Figure 1) selected cover “near-natural conditions”, reflecting
no or slight disturbance levels caused by hydromorphological alteration, water quality,
catchment land-use and alien species (Table 2). Some variables were used as exclusion
criteria for selection of suitable samples, or simply to represent values recorded at “near-
natural” sites. The following criteria related to hydromorphology [19,25], water quality
conditions [19,26–29], Corine land use/land cover [30] and alien species were used to select
these near-natural sites:
(a) Hydromorphological quality and assessment index (HQM), reflecting the combined
influence of habitat quality, habitat modifications and upstream and/or downstream
barriers: HQM > 0.6 (at least class 2).
(b) Hydrological modification index (HLM) reflecting influence of upstream/downstream
barriers (impoundments): HLM > 0.6 (at least class 2).
(c) Saprobic index (minimum normalised value–ecological quality ratio (EQR) for the
Slovenian (SIG3) and Croatian version (SIHR): EQR > 0.6 (at least good ecological
status).
(d) Percentage of CLC natural and semi-natural land use/land cover in the catchment at
least 50% and CLC intensive agriculture + urban land use/land cover in the catchment
<30%, where CLC urban land use/land cover in the catchment <5%.
(e) Alien species could be present but account for <35% of the benthic invertebrate
assemblage.
Water 2021, 13, 2596 5 of 19

Table 2. Median, minimum and maximum values of the selected hydromorphology, water quality, land use variables and
proportion of alien species at near-natural sites.

Variable Median Minimum Maximum

Low annual discharge (m3 /s) 99 7 648
Mean annual discharge (m3 /s) 140 9 998
High annual discharge (m3 /s) 399 90 1530
Hydromorphological quality and modification index (HQM) 0.84 0.71 1
Hydrological modification index (HLM) 0.94 0.68 1
Water temperature—median (◦ C) 12.0 9.8 16.1
pH—median 8.0 7.7 8.3
Conductivity—median (µS/cm) 350 299 480
Total suspended solids—median (mg/L) 7 3 56
Total nitrogen—median (mgN/L) 1.60 0.69 2.34
Ammonium—median (mgN/L) 0.06 0.01 0.30
Nitrate—median (mgN/L) 1.40 0.50 2.01
Orthophosphate—median (mgP/L) 0.05 0.01 0.29
Biochemical oxygen demand (5 days)—median (mg/L) 1.2 0.8 2.4
Chemical oxygen demand (K2 Cr2 O7 )—median (mg/L) 6.15 2.5 19.6
Oxygen concentration—median (mg/L) 9.55 8.55 11.5
Oxygen saturation—median (mg/L) 90 80 104
Saprobic index (EQR) 0.78 0.63 1.00
CLC Natural (%) 71 55 78
CLC Agriculture (%) 25 19 43
CLC Intensive agriculture (%) 12 8 26
CLC Extensive agriculture (%) 12 11 25
CLC Urban (%) 4 1 5
Alien species (%) 0 0 33.3

Twenty-four environmental variables were measured or calculated: five hydromor-

phological, fourteen water quality, and five catchment land cover/land use variables.
Details of how the variables were obtained or calculated are described elsewhere [20]; only
a summary is provided here. Water flow data were obtained from available discharge
data from the hydrological databases of the Slovenian Environment Agency (ARSO) and
Croatian Meteorological and Hydrological Service (DHMZ). The mean daily discharges
were used to calculate the mean annual discharge (MQ), the lowest annual discharge
(daily mean, NQ) and the highest annual discharge (daily mean, HQ) of the sampling year.
The Slovenian hydromorphological (SIHM) assessment method [19,25,31] was applied
to examine habitat quality, habitat modifications and the influence of the main upstream
barriers/impoundments. A river habitat survey [32,33] was conducted once between 2005
and 2012 in the benthic invertebrate sampling year or after sampling for each sampling site
and the data were used to calculate the two morphological indices [25,31]: river habitat
quality index (RHQ), and river habitat modification index (RHM). Normalised values (con-
verted to a common scale of 0–1; RHQnor and RHMnor [19]) were used according to the
eco-hydromorphological type of the considered river stretches according to Urbanič and Ur-
banič et al. [32,33] (Table 1, Figure 1). The data on impoundments recorded in the catchment
of each sampling site in the year of benthic invertebrate sampling were used to calculate the
hydrological modification index (HLM, [19,25]). The combination of the RHQnor, RHMnor
and HLM indices was used to calculate the hydromorphological quality and modification
index (HQM) [19,25,31]. Physical and chemical data were obtained monthly or at least four
times per year (each season) from national surface water monitoring programmes during
the sampling year. Only selected water quality parameters were used that were available
for all selected sites: water temperature, conductivity, pH, oxygen concentration, oxygen
saturation, water temperature, chemical oxygen demand (K2 Cr2 O7 ), biochemical oxygen
demand (BOD5 ), orthophosphate, total nitrogen, ammonium and total suspended solids.
The ecological quality ratio values were calculated based on the saprobic index according
to the national standards [26–29]. The median of data collected for each parameter in the
Water 2021, 13, 2596 6 of 19

benthic invertebrate sampling year is shown in Table 2. In addition to the variables listed,
for each site the naturally predominant substrate of each site was classified into two classes:
fine (psammal) or coarse (lithal). The information on eco-hydromorphological river type
was based on previous studies [19,20]. Land use variables were based on the proportion of
land use categories at the catchment scale, extracted from Corine land cover (CLC) data [30]
using ArcGIS version 10.2.1 (Esri Corp., Redlands, CA, USA). The categories were grouped
into five land use variables: urban land use (CLC class 1), natural and semi-natural land
use (CLC classes 3–5), extensive agricultural land use (CLC categories 2.3.1, 2.4.3, 2.4.4),
and intensive agricultural land use (CLC categories 2.1, 2.2, 2.4.1, 2.4.2).

2.3. Benthic Invertebrates

Biological data were obtained as part of the WFD monitoring and assessment system
development programmes in Slovenia and Croatia between 2005 and 2011. A total of
104 samples were collected at 56 sites [20], and of these 45 samples at 28 sampling sites
met the criteria for “near-natural sites” as used in this study. Most samples (13) were
collected in the lowland-braided sections of the Drava and Mura Rivers, 12 samples in the
lowland-deep sections of the Sava, Drava, Kupa and Una Rivers, and seven samples were
collected in the intermountain sections of the Sava River (Table 1). Some sites were sampled
several times, but not more than once per year. Benthic invertebrates were collected at
low to moderate flows using a multihabitat sampling approach. A detailed description of
benthic invertebrate sampling is provided elsewhere [19,20]. Samples were collected in the
wadeable part of the main channel using a hand net (frame 25 × 25 cm, mesh-size: 500 µm).
Twenty subsamples were collected at each site with a total sampling area of 1.25 m2 along
a 100–250 m river stretch. The sampling procedure in Slovenia followed the standardized
Slovenian river bioassessment protocol and is described in detail elsewhere [19,34,35].
Twenty sampling units were selected in proportion to the coverage of microhabitat types.
Microhabitat types were defined as a combination of substrate and flow type with coverage
of at least 5%. The channel substrates of each sampling site were classified according
to [36], while the flow characteristics were classified according to [19,35,37]. Sampling units
were pooled, preserved in the field with 96% ethanol and transported to the laboratory
for further processing. Each sample was subsampled, and benthic organisms from one
quarter of the total field sample were identified and counted. In Croatia, samples were
collected according to the AQEM sampling strategy [36]. A total of 20 sampling units from
representative substrates (i.e., substrates with a coverage >5% in the sampled reach) were
sampled. In 2009, 10 subsampling units were collected from sampling sites (five sites)
with homogenous substrates (sand and other soft sediments). In these cases, the sample
was collected by pushing the hand net through the top (2–5 cm) layer of the substrate.
Sampling units were pooled, preserved in the field with 96% ethanol and transported
to the laboratory for further processing. In 2006, a more elaborate subsampling design
was used: habitat (substrate)-specific subsampling units were pooled and analysed as
separate samples. At least one-sixth of the sample was sorted in the laboratory until the
target minimum number of 500 (habitat-specific samples) or 700 individuals (multihabitat
samples) was reached. Benthic invertebrates were generally identified to the species and
genus level but Oligochaeta and Diptera were identified to the (sub)family and genus level
(Appendix A).

2.4. Data Analyses

Variation in composition and structure of benthic invertebrate data were analysed
using the Bray–Curtis similarity measure. Assemblage similarities were then visualized
by nonmetric multidimensional scaling (NMS) plots, and statistically evaluated via per-
mutational multivariate analysis of variance (PERMANOVA, [38]). In NMS plots, the
samples vicinity to other samples in portrayable space—typically two dimensions—reflects
their underlying similarity in multivariate space, and accordingly, this is the preferred
method for illustrating relationships among ecological communities [39,40]. PERMANOVA
Water 2021, 13, 2596 7 of 19

is a multivariate, nonparametric permutation test for assessing differences in commu-

nity composition or structure between two or more predefined treatment groups (eco-
hydromorphological river types). We used a two-way PERMANOVA to test for sig-
nificant structural differences between the benthic invertebrate assemblages of (a) the
three eco-hydromorphological river types, and (b) the two substrate categories. When
differences in benthic invertebrate assemblage structure were detected between the eco-
hydromorphological river types and the substrate categories, similarity percentage (SIM-
PER) analyses were conducted to determine the contribution of specific taxa to these
differences. The benthic invertebrate abundances were ln (y + 1) transformed prior to anal-
ysis. All statistical and graphical analyses were performed using the PAST data analysis
package (v 3.14), (Natural History Museum, Oslo, Norway) [41].

3. Results
A total of 268,471 individuals representing 229 benthic invertebrate taxa were collected
in 45 samples (Appendix A). Most of the taxa collected were autochthonous, but nine alien
species were also recorded. Alien species were recorded in 16 benthic samples. Most
alien species were detected in one to six samples (Dugesia tigrina, Branchiura sowerbyi,
Potamopyrgus antipodarum, Sinanodonta woodiana, Corbicula fluminea, Dikerogammarus villosus,
Dikerogammarus haemobaphes and Jaera istri), but Chelicorophium curvispinum was found
in 11 samples. The recorded taxa included several species, e.g., the stonefly Marthamea
vitripennis and Xanthoperla apicalis. Marthamea vitripennis was recorded in the Una River,
while Xanthoperla apicalis was found in the Mura and Drava Rivers. Other large stonefly
species from the Perlidae family were also recorded, e.g., Dinocras cephalotes and Perla
abdominalis (P. burmeisteriana) in the Mura and Una Rivers. Several caddisfly species typical
of large rivers were recorded: Hydropsyche bulgaromanorum in the Drava and Sava rivers, H.
ornatula in the Sava, H. modesta in the Mura, Drava and Sava rivers, H. contubernalis was
present in all five rivers, and Setodes punctatus in the Drava, Kupa and Una rivers.
Site grouping in the NMS showed that the benthic invertebrate assemblages differ
among substrates (Figure 2A). The site grouping of eco-hydromorphological river types
was distinct, but less clear, especially between intermountain and lowland-braided rivers
(Figure 2B). PERMANOVA confirmed significant differences in assemblage structure be-
tween coarse and fine substrates (F = 4.62, R2 = 0.19, p < 0.0001) and among ECO-HM types,
where a slightly higher explanatory power was observed (F = 4.62, R2 = 0.22, p < 0.0001).
Despite the observed overlap of intermountain and braided samples in the NMS diagram,
PERMANOVA revealed differences in assemblage composition between all pairs of ECO-
HM types (F = 3.67 − 7.85, p = <0.0001 − 0.0003). The interaction between ECO-HM types
and substrates was not significant (p > 0.05) (Table 3).
Similarity percentage (SIMPER) analysis indicated that the overall average dissim-
ilarity of benthic invertebrate assemblages of all eco-HM type pairs was >50, with the
highest dissimilarity between lowland-braided and lowland-deep rivers (69.21) and the
lowest dissimilarity between intermountain and lowland-braided rivers (54.59). Gam-
marus fossarum, Nais sp., Orthocladiinae, Stylodrilus heringianus, Lithoglyphus naticoides,
Hydropsyche incognita, Tubificidae (with and without hair chaetae), Heptagenia sulphurea,
Baetis rhodani, Hydropsyche sp.-juv. and Gammarus roeseli were largely responsible for the
observed differences among benthic invertebrate assemblages (Table 4). However, Nais
sp. was the only taxon that was among the ten most influential taxa contributing to the
observed differences between benthic invertebrate assemblages of all pairs of ECO-HM
types. These taxa were also largely responsible for the observed differences between ben-
thic invertebrate assemblages of coarse and fine substrates (Table 5), confirming that some
differences between assemblages of ECO-HM types were also due to substrate differences.
Most taxa with the highest percentage contribution to differences between coarse and fine
substrates were found in higher abundance in rivers with coarse substrates, e.g., Gammarus
fossarum, Orhocladiinae, Stylodrilus heringianus, Nais sp., Hydropsyche sp.-juv, Baetis rhodani,
Psycomyiia pusilla and Hydropsyche incognita. The latter two species were not found in rivers
Water 2021, 13, 2596 8 of 19

with fine substrate. On the other hand, Lithoglyphus naticoides and Tubificidae without hair
chaetae preferred river sections with fine substrate.

Figure 2. Nonmetric multidimensional scaling ordination diagrams of sampling sites. (A) Overlay
indicates eco-hydromorphological types: intermountain (), lowland-braided (+), lowland-deep (o).
(B) Overlay indicates coarse () and fine (+) substrate. Stress (two-dimensional space) = 0.19.

Table 3. PERMANOVA test of eco-hydromorphological types of large rivers and substrates and their
interactions. ECO-HM—eco-hydromorphological river type, Df—degrees of freedom, p—probability
of statistical significance based on 9999 permutations of the data.

Scheme 2. Df Sum of Sqrs Mean Square F R2 p

ECO-HM type 2 2.22 1.11 2.64 0.22 0.0001
Substrate 1 1.94 1.94 4.62 0.19 0.0001
Interaction 2 −10.78 −5.39 −12.83 1
Residual 40 16.81 0.42
Total 45 10.18
Water 2021, 13, 2596 9 of 19

Table 4. Results of SIMPER analyses indicating the contribution of benthic invertebrate taxa to the observed differences between benthic invertebrate assemblages of eco-hydromorphological
(ECO-HM) types. Only taxa found in at least five samples were considered. Abbreviations refer to the ECO-HM types sampled: InterM—intermountain, L-braided—lowland-braided,
L-deep—lowland-deep. For taxon abbreviations, see Appendix A.

Overall Average Ten Most Percent Contibution to Cumulative Percent Average abundance Average Abundance
ECO-HM Type A vs. B
Dissimilarity Influential Taxa Difference Contibution to Difference (log10)—Type A (log10)—Type B
InterM vs. L-braided 54.59 Nai_sp. 3.2 3.2 1.10 1.65
The_dan 2.9 6.1 1.32 0
Bae_f_s 2.7 8.8 1.26 0.30
Hyd_inc 2.7 11.5 1.95 0.97
Gam_roe 2.6 14.1 0 1.25
Hep_sul 2.6 16.7 0 1.18
Sim_sp. 2.5 19.2 1.38 0.78
Hyd_spj 2.5 21.7 1.97 1.17
Eis_tet 2.4 24.1 1.18 0.39
Tubb_dae 2.3 26.4 0.96 1.68
InterM vs. L-deep 66.15 Gam_fos 5.4 5.4 3.20 0.85
Hyd_inc 3.8 9.1 1.95 0.26
Lith_nat 3.2 12.4 0 1.39
Hyd_spj 3.1 15.4 1.97 0.62
Psy_pus 3.1 18.5 1.80 0.54
Orth_nae 2.9 21.4 2.46 1.67
Bae_rho 2.8 24.2 1.57 0.69
Sto_her 2.8 27.0 1.55 0.98
Ant_sp. 2.7 29.7 1.39 0.15
Nai_sp. 2.5 32.2 1.10 0.41
L-braided vs. L-deep 69.21 Gam_fos 5.4 5.4 3.12 0.85
Lith_nat 3.3 8.7 0.03 1.39
Orth_nae 3.3 11.9 2.62 1.67
Nai_sp. 3.2 15.1 1.65 0.41
Sto_her 2.8 17.9 1.63 0.98
Gam_roe 2.4 20.3 1.25 0.13
Tubb_dae 2.4 22.7 1.68 1.77
Hep_sul 2.3 25.1 1.18 0.55
Bae_rho 2.2 27.3 1.02 0.69
Tubz_dae 2.2 29.5 0.75 1.13
Water 2021, 13, 2596 10 of 19

Table 5. Results of SIMPER analyses indicating the contribution of benthic invertebrate taxa to observed differences between benthic invertebrate assemblages of coarse and fine substrate
types. Only taxa found in at least five samples were considered. For taxon abbreviations, see Appendix A.

Overall Average Ten Most Percent Contibution to Cumulative Percent Average Abundance Average Abundance
Substratum
Dissimilarity Influential Taxa Difference Contibution to Difference (log10)—Coarse (log10)—Fine
Coarse vs. fine 74.91 Gam_fos 6.4 6.4 2.88 0.08
Orth_nae 4.6 11.0 2.63 0.68
Lith_nat 4.6 15.6 0.11 2.14
Sto_her 3.3 18.9 1.65 0.22
Nai_sp. 2.8 21.7 1.33 0.36
Psy_pus 2.7 24.4 1.29 0
Tubb_dae 2.5 26.9 1.48 2.19
Hyd_spj 2.5 29.4 1.31 0.18
Bae_rho 2.4 31.8 1.13 0.36
Hyd_inc 2.3 34.1 1.07 0
Water 2021, 13, 2596 11 of 19

4. Discussion
The selection of reference or near-natural sites and description of reference commu-
nities should be based on criteria associated with human disturbances that affect aquatic
assemblages at a level of no or low impact. We chose stressors of different spatial scales
describing different aspects of the aquatic environment (water quality, hydromorphology,
land use in the catchment, alien species) that have been shown to be critical in shaping
benthic invertebrate assemblages in large rivers [19,20]. Usually, the stressors applied in
studies are a simplification of the true stressors to which biota are exposed. In this study, we
performed river habitat surveys (RHS) once, which could bias the results because habitat
condition changes over time. The hydromorphological quality and modification (HQM)
index used in this study is partly based on the RHS conducted once in a given period.
However, it proved to be very robust to natural change as it is based not only on river
habitat survey data, but also on the presence of upstream and downstream impoundments
considered in the year of benthos sampling. Environmental conditions in large rivers gen-
erally deteriorate over time and rarely improve (e.g., through restoration); thus, conducting
RHS surveys after benthos sampling should not be an issue. We dealt with near-natural
sites and all parameters were used as knockout criteria. In the case that habitat condition
deteriorated prior to RHS was conducted, such sites would not be considered.
Reference conditions are not only commonly used for ecological assessment, but
are also important for restoration and river management in general, as improvement in
ecological conditions is a true measure of management success. However, it can be difficult
to find sites with at least near-natural conditions, as is the case with most large rivers in
the EU. Southeast Europe still has stretches of large rivers with near-natural conditions
where some rare benthic invertebrate taxa occur, as confirmed here. Marthamea vitripennis,
rediscovered after 100 years in the Una River [42], once occurred in the rivers of central
and southeast Europe, but is now largely extinct [43]. Xanthoperla apicalis, another stonefly
species that is a potamal species, at least in central Europe, had disappeared for decades
and was only recently rediscovered [43]. Other detected large stonefly species from the
family Perlidae (Dinocras cephalotes and Perla abdominalis in the Mura and Una Rivers,
respectively) confirm that remarkable large insect species can also occur in near-natural
conditions of large rivers, playing an important role in the functioning of large rivers. We
also found some other less endangered but typical potamal species typical for larger rivers,
e.g., the caddisfly Hydropsyche bulgaromanorum, which still occurs in the lower Sava and the
lower Drava. This was a common and dominant Hydropsyche species farther upstream (in
the middle Drava near Maribor, Slovenia), prior to the construction of hydroelectric power
plants, but it no longer occurs today (Urbanič, unpublished data). In addition to rare and
typical potamal species, alien species are becoming increasingly common in near-natural
sections of large rivers [44]. In the present study, we excluded sites dominated by alien
species. However, in the near future, alien species might dominate river stretches with near-
natural conditions, especially due to artificial connections between river catchments [45,46].
Most alien species recorded in this study are Ponto–Caspian elements that have spread
upstream.
In the EU, type-specific reference conditions have often been established in the spirit
of national individuality and with a distinct lack of stringency in defining reference
states [47,48]. It is well known that river degradation leads to the homogenisation of
aquatic communities [49]. Therefore, the use of communities from degraded sites could
lead to a reduced number of taxa in reference communities, lower ecological expectations
and a smaller number of recognised distinct river types. Abiotic factors, particularly hy-
dromorphological characteristics, are often used for river typologies, which may lead to
a lower number of river types [50] compared to the number of ecological river types. In
Slovenia, a much smaller number of hydromorphological types compared to ecological
types were recognised, although habitat diversity was taken into account. This is especially
true for small rivers [25,34] and to a lesser extent for large rivers [19]. In the present
study, the benthic invertebrate community composition differed significantly in all three
Water 2021, 13, 2596 12 of 19

eco-hydromorphological types that were considered for large rivers. This is even more re-
markable because not all taxa were determined to species level. Nevertheless, Nais sp. was
the only taxon that was among the ten most influential taxa contributing to the observed
differences between the benthic invertebrate assemblages of all pairs of ECO-HM types.
Thus, it is evident that not only the taxonomic composition but also the abundance of taxa
differs between ECO-HM types. Habitat heterogeneity is important from the biodiversity
point of view and was considered when delineating the eco-hydromorphological large
river types [19]. However, the eco-hydromorphological types considered differ not only
in habitat heterogeneity, but also in water depth associated with flow type and substrate
size. We identified the latter as one of the key factors defining the benthic invertebrate
communities of large rivers, as in many studies (see [51]). There were several taxa that
preferred river types with coarse substrates, while some, such as the snail Lythoglyphus
naticoides and Tubificidae without hair chaetae, preferred rivers with fine substrates. This
is consistent with the finding that rivers with coarse substrates harbour more diverse
communities compared to rivers with fine substrates [52].
A pragmatic approach has often been taken in setting reference conditions in the
EU [12]. It has often been stated that reference conditions or near-natural conditions are
also lacking in the case of most large river types in the EU. We have shown here that large
rivers with near-natural conditions still exist or existed recently, and provide a description
of the type-specific near-natural benthic invertebrate communities. Therefore, it might
be possible to set ecological conditions that would serve as a true measure of assessment
river restoration success and river management, especially in line with the EU Water
Framework Directive. Following this pragmatic approach, the European Green Deal has
set the restoration targets for rivers at EU level based only on the length of stretches
that have been transformed back into free-flowing rivers by removing mainly obsolete
barriers. Addressing one of the main pressures [4,53] could be a good starting point for
river restoration. Parameters related to barriers have also been shown to be key factors
for benthic invertebrate community composition and ecological status [19], together with
fish composition in large rivers [54]. Fish communities may show a rapid response to dam
removal [55]. However, although restoration is needed as river degradation is ongoing
in Europe, it is evident that large rivers are affected by multiple stressors [4,35,56,57]. For
benthic invertebrate assemblages in southeast European large rivers, water quality and land
use effects are almost equally important as hydromorphological conditions [20], indicating
that considering hydromorphological conditions alone in restoration may be insufficient.
A more holistic view of restoration may be required to achieve ecological goals [58],
particularly where the names of taxa present (reference communities) are important in
addition to functionality.

5. Conclusions
Reference conditions, or at least near-natural conditions, are not only commonly used
for ecological assessment, but are also important for restoration and river management, as
improvement in ecological conditions is a true measure of management success. In most
large rivers in the EU, it can be difficult to find sites with at least near-natural conditions,
though southeast Europe still has stretches of large rivers with near-natural conditions
where some typical potamal and rare benthic invertebrate taxa occur. We defined the
near-natural benthic invertebrate assemblages of large rivers containing several rare and
remarkable species among the stoneflies, e.g., Marthamea vitripennis, Xanthoperla apicalis,
Dinocras cephalotes, Perla abdominalis (P. burmeisteriana). We compared the composition of
benthic invertebrate assemblages among three eco-hydromorphological (ECO-HM) types
of large rivers (lowland-deep, lowland-braided and intermountain) and found that the
composition of near-natural benthic invertebrate assemblages varied among large river
types. Overall, the average dissimilarity in assemblage composition among all ECO-HM
type pairs was high, with the highest dissimilarity between lowland-braided and lowland-
deep rivers, and the lowest dissimilarity between intermountain and lowland-braided
Water 2021, 13, 2596 13 of 19

rivers. ECO-HM large river types were defined based on habitat heterogeneity, but also
differed in water depth associated with flow type and substrate size. We found that
habitat heterogeneity and substrate independently influence the composition of the benthic
invertebrate assemblages. Habitat heterogeneity is often generally considered in river
assessment, restoration projects or river management. However, it is evident that other
factors should also be considered to maintain or restore ecological conditions with relevant
benthic communities. For example, simple restoration goals for rivers, such as the length
of stretches restored to free-flowing rivers, as stated in the European Green Deal, may
not ensure sufficiency to meet all ecological objectives. To achieve ecological goals in
the management of large rivers, a holistic view with at least near-natural assemblages,
including the names of the taxa present, should be considered in addition to functionality.

Author Contributions: Conceptualization, G.U. and Z.M.; methodology, G.U., Z.M., V.P. and M.P.U.;
investigation, G.U., Z.M., V.P. and M.P.U.; validation, G.U. and Z.M.; writing—original draft prepara-
tion, G.U., Z.M., V.P. and M.P.U.; writing—review and editing, G.U., Z.M., V.P. and M.P.U.; visualiza-
tion, G.U.; supervision, G.U. and Z.M.; project administration, G.U. and Z.M.; funding acquisition,
G.U. and Z.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research was conducted within the Slovenian–Croatian bilateral project: Benthic
invertebrate based ecological status assessment of large rivers with management goals focused on
hydromorphological alterations (G.U. and Z.M.). The study was also supported by the Ministry of
the Environment and Spatial Planning of the Republic of Slovenia as a part of the national program
for the implementation of the EU Water Framework Directive conducted at the Institute for Water of
the Republic of Slovenia.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available on special request.
Acknowledgments: Authors gratefully acknowledge the members of the project teams for their as-
sistance both in the field and in the laboratory. The Croatian Meteorological and Hydrological Service
and Croatian Waters and Slovenian Environment Agency are thanked for providing hydrological
and water physicochemical data. The authors would like to thank the anonymous reviewers for their
useful comments to a previous version of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.

Appendix A

Table A1. Benthic invertebrate taxa recorded at near-natural sites of large rivers.

Higher Taxon Taxon Abbreviations

Turbellaria Dendrocoelum album
Turbellaria Dendrocoelum lacteum
Turbellaria Dugesia lugubris/polychroa
Turbellaria Dugesia lugubris
Turbellaria Dugesia tigrina
Turbellaria Phagocata sp.
Turbellaria Planaria torva
Turbellaria Polycelis nigra/tenuis
Nematoda Nematoda Gen. sp.
Oligochaeta Enchytraeidae Gen. sp.
Oligochaeta Haplotaxis gordioides
Oligochaeta Eiseniella tetraedra Eis_tet
Oligochaeta Lumbriculidae Gen. sp.
Oligochaeta Lumbriculus variegatus
Oligochaeta Rhynchelmis sp.
Water 2021, 13, 2596 14 of 19

Table A1. Cont.

Higher Taxon Taxon Abbreviations

Oligochaeta Stylodrilus heringianus Sto_her
Oligochaeta Stylodrilus sp.
Oligochaeta Dero sp.
Oligochaeta Nais sp. Nai_sp.
Oligochaeta Ophidonais serpentina
Oligochaeta Pristina sp.
Oligochaeta Stylaria lacustris
Oligochaeta Uncinais uncinata
Oligochaeta Propappus volki
Oligochaeta Aulodrilus pluriseta
Oligochaeta Branchiura sowerbyi
Oligochaeta Tubificidae—without setae Tubb_dae
Oligochaeta Tubificidae—with setae Tubz_dae
Hirudinea Dina punctata
Hirudinea Erpobdella nigricollis
Hirudinea Erpobdella octoculata
Hirudinea Erpobdella sp.
Hirudinea Erpobdella testacea
Hirudinea Trocheta bykowskii
Hirudinea Glossiphonia complanata
Hirudinea Glossiphonia concolor
Hirudinea Glossiphonia nebulosa
Hirudinea Helobdella stagnalis
Hirudinea Hemiclepsis marginata
Hirudinea Piscicola geometra
Gastropoda Acroloxus lacustris
Gastropoda Ancylus fluviatilis
Gastropoda Bithynia tentaculata
Gastropoda Borysthenia naticina
Gastropoda Lithoglyphus naticoides Lith_nat
Gastropoda Potamopyrgus antipodarum
Gastropoda Sadleriana sp.
Gastropoda Radix auricularia
Gastropoda Radix balthica/labiata
Gastropoda Radix balthica
Gastropoda Esperiana daudebartii acicularis
Gastropoda Esperiana esperi
Gastropoda Holandriana holandrii
Gastropoda Theodoxus danubialis The_dan
Gastropoda Theodoxus transversalis
Gastropoda Physa fontinalis
Gastropoda Physella acuta
Gastropoda Gyraulus albus
Gastropoda Valvata cristata
Gastropoda Valvata piscinalis
Gastropoda Viviparus viviparus
Bivalvia Dreissena polymorpha
Bivalvia Musculium lacustre
Bivalvia Pisidium sp.
Bivalvia Sphaerium corneum
Bivalvia Sphaerium sp.
Bivalvia Sinanodonta woodiana
Bivalvia Unio crassus
Bivalvia Unio pictorum
Bivalvia Corbicula fluminea
Arachnida Hydrachnidia Gen. sp.
Amphipoda Synurella ambulans
Amphipoda Gammarus fossarum Gam_fos
Amphipoda Gammarus roeseli Gam_roe
Water 2021, 13, 2596 15 of 19

Table A1. Cont.

Higher Taxon Taxon Abbreviations

Amphipoda Corophium curvispinum
Amphipoda Dikerogammarus haemobaphes
Amphipoda Dikerogammarus villosus
Isopoda Asellus aquaticus
Isopoda Jaera istri
Ephemeroptera Baetis buceratus
Ephemeroptera Nigrobaetis digitatus
Ephemeroptera Baetis fuscatus/scambus Bae_f_s
Ephemeroptera Baetis lutheri
Ephemeroptera Baetis rhodani Bae_rho
Ephemeroptera Baetis scambus
Ephemeroptera Baetis sp.-juv.
Ephemeroptera Baetis vardarensis
Ephemeroptera Baetis vernus
Ephemeroptera Baetis buceratus/vernus
Ephemeroptera Centroptilum luteolum
Ephemeroptera Centroptilum sp.
Ephemeroptera Cloeon dipterum
Ephemeroptera Caenis sp.
Ephemeroptera Serratella ignita
Ephemeroptera Ephemerella notata
Ephemeroptera Ephemerella mucronata
Ephemeroptera Torleya major
Ephemeroptera Ephemera danica
Ephemeroptera Ephemera sp.
Ephemeroptera Ecdyonurus sp.
Ephemeroptera Epeorus sylvicola
Ephemeroptera Heptagenia sp.
Ephemeroptera Heptagenia sulphurea Hep_sul
Ephemeroptera Rhithrogena sp.
Ephemeroptera Habroleptoides confusa
Ephemeroptera Paraleptophlebia submarginata
Ephemeroptera Oligoneuriella rhenana
Ephemeroptera Potamanthus luteus
Ephemeroptera Siphlonurus sp.
Plecoptera Chloroperla sp.
Plecoptera Xanthoperla apicalis
Plecoptera Leuctra sp.
Plecoptera Nemoura sp.
Plecoptera Nemurella pictetii
Plecoptera Protonemura sp.
Plecoptera Dinocras cephalotes
Plecoptera Perla abdominalis (P. burmeisteriana)
Plecoptera Marthamea vitripennis
Plecoptera Isoperla sp.
Plecoptera Perlodes sp.
Plecoptera Brachyptera sp.
Plecoptera Taeniopteryx nebulosa
Odonata Calopteryx splendens
Odonata Enallagma cyathigerum
Odonata Ischnura elegans
Odonata Coenagrionidae-juv.
Odonata Cordulegaster heros
Odonata Gomphus sp.
Odonata Gomphus vulgatissimus
Odonata Gomphus flavipes
Odonata Onychogomphus forcipatus forcipatus
Odonata Platycnemis pennipes
Heteroptera Aphelocheirus aestivalis
Water 2021, 13, 2596 16 of 19

Table A1. Cont.

Higher Taxon Taxon Abbreviations

Heteroptera Micronecta sp.
Hymenoptera Agriotypus armatus
Coleoptera Bidessus sp. Ad.
Coleoptera Elmis sp. Ad.
Coleoptera Elmis sp. Lv.
Coleoptera Esolus sp. Ad.
Coleoptera Esolus sp. Lv.
Coleoptera Limnius sp. Ad.
Coleoptera Limnius sp. Lv.
Coleoptera Normandia nitens Ad.
Coleoptera Oulimnius sp. Ad.
Coleoptera Oulimnius sp. Lv.
Coleoptera Stenelmis canaliculata Ad.
Coleoptera Orectochilus villosus Lv.
Coleoptera Haliplus sp. Lv.
Coleoptera Helophorus sp. Ad.
Coleoptera Hydraena sp. Ad.
Coleoptera Ochthebius sp. Ad.
Trichoptera Brachycentrus montanus
Trichoptera Brachycentrus subnubilus
Trichoptera Ecnomus tenellus
Trichoptera Agapetus delicatulus/ochripes
Trichoptera Agapetus laniger
Trichoptera Goera pilosa
Trichoptera Silo nigricornis
Trichoptera Silo piceus
Trichoptera Cheumatopsyche lepida
Trichoptera Hydropsyche bulbifera
Trichoptera Hydropsyche bulgaromanorum
Trichoptera Hydropsyche contubernalis contubernalis
Trichoptera Hydropsyche incognita Hyd_inc
Trichoptera Hydropsyche modesta
Trichoptera Hydropsyche ornatula
Trichoptera Hydropsyche pellucidula
Trichoptera Hydropsyche siltalai
Trichoptera Hydropsyche sp.-juv. Hyd_spj
Trichoptera Hydroptila sp.
Trichoptera Orthotrichia sp.
Trichoptera Lepidostoma hirtum
Trichoptera Athripsodes albifrons
Trichoptera Athripsodes sp.
Trichoptera Ceraclea annulicornis
Trichoptera Ceraclea dissimilis
Trichoptera Mystacides azurea
Trichoptera Mystacides nigra
Trichoptera Oecetis lacustris
Trichoptera Oecetis notata
Trichoptera Setodes punctatus
Trichoptera Oecetis sp.
Trichoptera Anabolia furcata
Trichoptera Halesus digitatus
Trichoptera Limnephilinae-juv.
Trichoptera Potamophylax rotundipennis
Trichoptera Philoptamus ludificatus/montanus
Trichoptera Polycentropus flavomaculatus
Trichoptera Lype reducta
Trichoptera Psychomyia pusilla Psy_pus
Trichoptera Rhyacophila sp. s. str.
Trichoptera Sericostoma sp.
Water 2021, 13, 2596 17 of 19

Table A1. Cont.

Higher Taxon Taxon Abbreviations

Diptera Limnophora sp.
Diptera Lispe sp.
Diptera Atherix ibis
Diptera Ibisia marginata
Diptera Liponeura sp.
Diptera Ceratopogoninae Gen. sp.
Diptera Chironomini Gen. sp.
Diptera Chironomus obtusidens-Gr.
Diptera Chironomus thummi-Gr.
Diptera Chironomus sp.
Diptera Corynoneurinae Gen. sp.
Diptera Diamesinae Gen. sp.
Diptera Orthocladiinae Gen. sp. Orth_nae
Diptera Paratendipes sp.
Diptera Potthastia longimana-Gr.
Diptera Procladius sp.
Diptera Prodiamesa olivacea
Diptera Prodiamesa rufovittata
Diptera Tanypodinae Gen. sp.
Diptera Tanytarsini Gen. sp.
Diptera Thienemanniella sp.
Diptera Dolichopodidae Gen. sp.
Diptera Clinocerinae Gen. sp.
Diptera Hemerodromiinae Gen. sp.
Diptera Antocha sp. Ant_sp.
Diptera Hexatoma sp.
Diptera Limnophilinae Gen. sp.
Diptera Limoniinae Gen. sp.
Diptera Dicranota sp.
Diptera Pedicia sp.
Diptera Psychodidae Gen. sp.
Diptera Prosimulium sp.
Diptera Simulium sp. Sim_sp.
Diptera Syrphidae Gen. sp.
Diptera Chrysops sp.
Diptera Tabanus sp.
Diptera Tipula sp.

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