w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

Establishing physico-chemical reference conditions in

Mediterranean streams according to the European Water
Framework Directive

Marı́a del Mar Sánchez-Montoya a,*, Maria Isabel Arce a, Marı́a Rosario Vidal-Abarca a,
Marı́a Luisa Suárez a, Narcı́s Prat b, Rosa Gómez a
a
Department of Ecology and Hydrology, University of Murcia, 30100 Espinardo, Murcia, Spain
b
Department of Ecology, University of Barcelona, Av. Diagonal, 645, 08028 Barcelona, Spain

article info abstract

Article history: Type-specific physico-chemical reference conditions are required for the assessment of
Received 18 July 2011 ecological status in the Water Framework Directive context, similarly to the biological and
Received in revised form hydro-morphological elements. This directive emphasises that natural variability of
23 December 2011 quality elements in high status (reference condition) needs to be quantified. Mediterranean
Accepted 28 January 2012 streams often present a marked seasonal pattern in hydrological, biological and
Available online 6 February 2012 geochemical processes which could affect physico-chemical reference conditions. This
study establishes general physico-chemical reference conditions (oxygenation, nutrient,
Keywords: salinity and acidification conditions) for different Mediterranean stream types. 116
Water quality potential reference sites located in 23 Mediterranean catchments in Spain were sampled in
Physico-chemical reference condi- spring, summer and autumn in 2003. All sites were subjected to a screening method for the
tions selection of reference sites in Mediterranean streams (Mediterranean Reference Criteria)
Mediterranean streams and classified using a pre-established stream typology that establishes five different
Water Framework Directive stream types (temporary streams, evaporite-calcareous at medium altitude, siliceous
headwaters, calcareous headwaters and large watercourses). Reference conditions (refer-
ence value and reference threshold equivalents to high-good class boundary) were calcu-
lated using two different methods according to the availability of reference sites: the
reference site 75th percentile approach of all reference sites and the 25th percentile of the
population approach. The majority of the studied potential reference sites (76 out of 116)
were selected as reference sites. Regarding type-specific reference conditions, only sili-
ceous headwaters could be considered different from the rest of stream types because
lower conductivity and pH. All reference stream types presented seasonal differences as
regards some parameters, except for temporary streams due to the high natural variation
of this stream type. For those parameters which presented seasonal differences in
a specific stream type, the least restrictive values were proposed as reference conditions.
ª 2012 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: þ34 868 884653; fax: þ34 868 883963.
E-mail addresses: marsanch@um.es (M.M. Sánchez-Montoya), marisarce@um.es (M.I. Arce), charyvag@um.es (M.R. Vidal-Abarca),
mlsuarez@um.es (M.L. Suárez), nprat@ub.edu (N. Prat), rgomez@um.es (R. Gómez).
0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2012.01.042
2258 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9
1. Introduction
Increasing water quantity and quality problems in Europe have
led to the development of the Water Framework Directive
(WFD; EU/2000/60/EC-WFD, 2000), an integrated approach for
water management systems that includes all water-related
impacts (Achleitner et al., 2005). One of the main objectives of
this directive is to achieve “good ecological status” in all water
bodies of the European Union until 2015 by applying an inte-
grated approach for water management and avoiding the
deterioration of the present status of water bodies. In this
frame, the ecological status is defined as an expression of the
quality of the structure and functioning of aquatic ecosystems
by integrating biological, physico-chemical and hydro-
morphological components of water bodies. Before the WFD,
water quality monitoring programmes in most 2000 Member
States were mainly based on physico-chemical variables and
only around half the programmes included biological parame-
ters in their assessment and classification of river quality
(Hering et al., 2003).
General physico-chemical quality elements are required for
the determination of high, good and moderate ecological status
classes in the WFD context. For the other two quality classes
(poor and bad), the conditions of the physico-chemical
elements are expected to be consistent with the achievement
of values for biological quality elements. According to the WFD,
the physico-chemical conditions are: thermal, oxygenation and
nutrient conditions, salinity and acidification status.
The Reference Condition Approach (RCA) has been adopted
by the WFD as it requires the evaluation of ecological status,
which may be expressed as a deviation from the near-natural
reference conditions (RC). This condition should be linked to
stream typology, and reference sites should present the full
range of conditions expected to occur naturally in a given
stream type (e.g. Barbour et al., 1996; Reynoldson and Wright,
2000; Stoddard et al., 2006). Therefore, the use of physico-
chemical indicators for ecological assessments entails previ-
ously established physico-chemical reference conditions
(physico-chemical RC), similarly to the biological and hydro-
morphological elements.
Understanding the natural variability of ecological
resources and abiotic conditions associated with this varia-
tion is fundamental to develop management directives
(Hawkins et al., 2010). In this sense, the WFD emphasises that
the natural variability of quality elements in high status water
bodies needs to be quantified (Irvine, 2004).
When physico-chemical RC are established, it must be
considered that the hydrochemical properties of streams in
undisturbed catchments are determined by different factors
such as rainfall, evaporation rate, geology catchments,
bedrock weathering, soils and biological processes
(e.g. Vitousek, 1977; Cooper et al., 2000). Knowledge of the
relationship between hydrochemical parameters and their
influenced on environmental factors is essential to gain
a complete understanding of aquatic biological communities
and processes. In particular, benthic macroinvertebrate
communities undergo considerable changes as a response to
varying environmental factors, such as water physico-
chemical characteristics (Jacobsen et al., 1997), among others.
Therefore, physico-chemical parameters are markedly
affected by both biotic and abiotic variables which, in turn,
may change through space and time. In particular, Mediter-
ranean streams often present a marked seasonal pattern due
mainly to the seasonality of precipitation in Mediterranean
areas (Gasith and Resh, 1999). The Mediterranean climate
brings about a strong seasonal pattern in hydrological,
biological and geochemical processes (e.g. Holloway and
Dahlgree, 2001), which could affect physico-chemical RC.
Establishing RC through the selection of a set of reference
sites in European rivers is expected to be problematic (Bonada
et al., 2002; Nijboer et al., 2004; Sánchez-Montoya et al., 2005),
particularly in lowland rivers with a long history of intense
flow regulation, agricultural development and pollution (Petts,
1989). In this frame, Mediterranean streams in Spain, where
this work was carried out, are no exception (see Sánchez-
Montoya et al., 2009).
Along these lines, this study specifically aimed to: 1) recog-
nise and select reference sites in Mediterranean streams
applying an approach to select undisturbed sites in Mediterra-
nean streams for different stream types defined previously in
the same study area, 2) analyse natural seasonal variation of
physico-chemical parameters in type-specific reference sites to
be taken into account for establishing RC, and 3) establish both
reference value and the reference threshold (equivalents to the
high-good boundary) for water management purposes in each
stream type for the studied physico-chemical parameter.
For these purposes, the data from sites sampled in the
GUADALMED II project (Prat, 2002) were used, providing
a unique opportunity to study the physico-chemical RC in
Mediterranean streams.
2. Materials and methods
2.1. Study area and sampling sites
The study was carried out in 23 catchments located along
a latitudinal, thermal and pluviometric gradient along the
Spanish eastern coast covering a wide range of basis sizes
(Fig. 1). The annual range of temperatures lies between 2  C
and 42  C, and annual precipitation ranges from 280 mm to
1000 mm (MIMAN, 2000). Given its Mediterranean climate, this
area is characterised by hot dry summers and cool wet winters,
and rivers may show high seasonality with annual and inter-
annual variability in discharge regimes, with frequent periods
of flooding and drying (Gasith and Resh, 1999). Limestone and
other sedimentary materials prevail along this coast, conse-
quently most of the studied catchments are predominately
calcareous, although few siliceous catchments are also present.
Sclerophyllous and evergreen trees are the dominant vege-
tation, although deciduous and coniferous forests are present
in some areas. Some temporary streams such as ramblas
present a particular kind of vegetation that has adapted to
conditions of high salinity, marked hydrological fluctuations,
and to severe dry and flood periods (Gómez et al., 2005).
A total of 116 sampling sites were studied as potential
reference sites (Fig. 1). These sites are distributed along the
main rivers and their tributaries. All the sites were sampled in
 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9 2259

Fig. 1 e Distribution of the 116 sampling sites indicating their typology (T1: temporary streams, T2: evaporite-calcareous at
medium altitude, T3: siliceous headwaters at high altitude, T4: calcareous headwaters at medium and high altitudes and
T5: large watercourses).

spring, summer and autumn 2003, except for sites belonging
to T1 (temporary streams) that were sampled at most two
times because they were dry out at least in summer.
2.2. Definition of stream types
All 116 studied sites were classified using the stream typology
proposed by Sánchez-Montoya et al. (2007) for Mediterranean
streams, following the criteria suggested in System B of
the WFD. This classification grouped the 116 studied sites
into five different stream types: temporary streams (T1),
evaporite-calcareous streams at medium altitude (T2),
siliceous headwaters streams at high altitude (T3), calcareous
headwaters streams at medium to high altitude (T4), and large
watercourses (T5). The five stream ecotypes were identified
using a combination of hydrological, geological, climatic and
morphological criteria (top-down approach). Table 1 shows
the main environmental characteristics of these five stream
types.
2.3. Reference site screening method
In order to establish RC, reference sites or near-natural sites
were selected from the total potential reference sites

Table 1 e Average and standard deviation (in parenthesis) values of the main environmental variables which characterise
the five stream types established by Sánchez-Montoya et al. (2007). Only the two predominant percentages of geology of
the three categories (calcareous, evaporite and siliceous) are shown.
Code Definition Hydrologic state % geology Stream order Surface drainage Altitude (m)
drainage area area (Km2)

T1 Temporary streams Intermittent/Ephemeral 69% carbonate 1.1 (0.3) 22 (35) 645 (523)
27% siliceous
T2 Evaporite-calcareous Perennial seasonal 67% carbonate 1.9 (0.8) 200 (290) 541 (188)
at medium altitude streams 17% evaporite
T3 Siliceous headwaters Perennial seasonal 97% siliceous 1.3 (0.6) 47 (118) 720 (225)
at high altitude streams 3% carbonate
T4 Calcareous headwaters Perennial seasonal 88% carbonate 1.7 (0.9) 149 (38) 689 (192)
at medium and high 9% siliceous
altitude streams
T5 Large watercourses Perennial seasonal 70% carbonate 4.3 (0.7) 3490 (1675) 239 (187)
2% siliceous
2260 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9

(n ¼ 116) by applying the Mediterranean Reference Criteria
methodology (MRC) defined by Sánchez-Montoya et al. (2009)
to distinguish undisturbed sites in Mediterranean streams.
This quantitative method includes 20 pressure criteria
related to the riparian vegetation zone, presence of invasive
species, point and diffuse sources of pollution, river
morphology, habitat structure and hydrologic conditions
(Table 2). These 20 criteria were analysed at the 116 potential
reference sites.
To calculate the percentage of different land uses, firstly
a digital terrain model (DTM 30  30 m; Centro Geográfico del
Ejército, Spanish Ministry of Defence, 2005) and the Arc/Info
software (version 9.0, ESRI, Redlands, California, USA, 2005)
were used to delimit and calculate the water drainage area as
a new polygon for all the sampling sites. Secondly, the
percentages of intensive and extensive agriculture, urban
zones, burnt area and natural land use were calculated by
intersecting the Corine Land Cover 2000 Programme (Spain,
Scale 1:100.000, Instituto Geográfico Nacional, Centro Nacio-
nal de Información Geográfica) with the drainage area. Infor-
mation on the remaining criteria was obtained from maps and
available data, some of which originated from the water
agencies of each river basin district. This information was
checked during a preliminary site inspection.
The 116 studied sites were classified into three levels of
disturbance (1e3) determined by the number of MRC criteria
that each fulfilled. Only those that satisfied all 20 criteria were
considered reference sites, which were termed undisturbed
sites or very minor disturbed sites (level 1). The rest, which ful-
filled fewer criteria, were classified as non-reference sites with
two degrees of degradation. If sites fulfilled between 16 and 19
criteria, they were considered minor disturbed sites (level 2).
Finally, sites were considered disturbed sites when they ful-
filled fewer than 16 of the 20 criteria (level 3).
For those stream types where undisturbed sites were
found, only those which fulfilled the 20 pressure criteria were
selected as reference sites (hereafter referred as “reference
sites”). However, in stream types where undisturbed sites are
not available to generate a database of reference sites, we
selected as reference sites those which have received less
intense impacts fulfilling most of the reference criteria (> 13
criteria; hereafter referred as “the least disturbed sites”). This
approach matches with “the Best Attainable Condition”
concept defined by Stoddard et al. (2006).
2.4. Physico-chemical parameters and environmental
variables
Eight physico-chemical parameters were studied, and they
were all referred by the WFD as general physico-chemical
elements supporting biological elements: oxygen saturation
and dissolved oxygen as oxygenation conditions, conductivity
as an indicator of salinity, pH as an indicator of acidification
status, and nitrate (NO3eN), nitrite (NO2eN), ammonium
(NH4eN) and phosphate (PO4eP) concentrations as indicators
of nutrient conditions.
On each sampling occasion (spring, summer and autumn),
we recorded in situ oxygen saturation, dissolved oxygen,
conductivity and pH. We also collected surface water samples
in 250 ml acid-washed polyethylene bottles. We maintained
samples in the dark and at a temperature of 4  C until they
were analysed in the laboratory. Nutrients concentration was
determined in a photometer (LASA 100) or by using standard
protocols following APHA (1998).

Table 2 e Mediterranean reference criteria (MRC; Sánchez-Montoya et al., 2009) applied in this study to select reference
sites in order to establish physico-chemical reference conditions.
Elements Criteria

Riparian vegetation zone 1. Cover and composition appropriate for the type and geographical location of the river.
2. Lateral connectivity between river and riparian corridor is maintained (no cultivation
and significant impervious area in riparian zone).
Introduced species 3. No significant impairment by exotic plant or animal species on autochthonous species.
Point sources of pollution 4. No dumping of urban effluents.
5. No dumping of industrial effluents.
6. No irrigation return channel for floodwater farming.
Diffuse sources of 7. Dry land farming <20% of drainage area (cereal, vineyard and tree crops as olive)
pollution and land uses and not connected to riparian vegetation zone.
8. Intensive irrigated farming <3% in drainage area (rice field, irrigated vineyard
and others irrigated fruit tress) and not connected to riparian vegetation zone.
9. Urban use <0.7% in drained area.
10. Burnt vegetation <7% in drainage area at least seven years ago and not
connected to riparian vegetation zone.
11. No evidence of intensive use of grazing.
12. Natural land uses >80% in drainage area.
River morphology and 13. Representative diversity of substrate materials appropriate for the type.
habitat conditions 14. No canalization (stream bottoms and stream margins must not be fixed).
15. No transversal structures “dams” (no retention of sediments).
16. No sand or gravel extraction.
Hydrological conditions 17. No water diversion for irrigation or other purpose.
and regulation 18. No alterations of the natural hydrograph and discharge regime (reservoirs, hydroelectric.).
19. No effect of inter-basin water transfer.
20. Near-natural level of groundwater (aquifer not affected by over-exploitation).
 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9
2.5. Reference values and boundaries setting procedures
Before establishing the physico-chemical RC, we examined
the effect of seasonal variability of physico-chemical param-
eters for establishing the reference value and setting the high-
good boundary for each physico-chemical parameter in each
stream type for assessment purposes. For those parameters
that did not show significant differences among seasons
within each stream type, the mean physico-chemical
parameter values of the selected reference sites or the least
disturbed sites on the three sampling occasions (spring,
summer and autumn) were used to estimate the reference
value and the high-good boundary. Conversely, for those
parameters which showed seasonal effect, if seasonal values
were similar, then the least restrictive value was proposed for
establishing RC (reference value and reference threshold).
To establish the reference values of the eight physico-
chemical parameters in all the studied stream types, the
median value of each physico-chemical parameter measured
at the selected reference sites or the least disturbed sites was
used as the reference value.
The reference threshold equivalents to high-good class
boundary was calculated in each stream type for each studied
parameter using two different methods proposed by the
USEPA (2000) according to the availability of reference sites: 1)
the reference site 75th percentile approach of reference sites
and 2) the 25th percentile of the population approach.
The reference site 75th percentile approach uses the 75th
percentile value of reference sites (fulfilled all 20 criteria) as
the reference threshold. The EPA considers this percentile of
reference sites because it is associated with minimally
impacted conditions, is protective of designated uses and
provides management flexibility (Herlihly and Sifneos, 2008).
The 25th percentile of the population approach is used as
a surrogate for reference conditions and is proposed if in
a specific stream type do not suffice, in which case all the least
disturbed sites (fulfilled > 13 criteria) from that type are used.
For the studied parameters under oxygen conditions, both
the 25th and 75th percentiles of the reference sites or the least
disturbed sites were used to establish an upper and lower
reference threshold, because eutrophication can lead to over-
saturation in the daylight.
2.6. Data analysis
After selecting reference sites by applying the MRC, and in
order to analyse physico-chemical variability in stream types
in RC, the physico-chemical data at the reference sites were
subjected to a Principal Component Analysis (PCA) on the
mean value of the three sampling occasions using the STA-
TISTICA program (Stat Soft, Inc., 1999). Previously, all eight
variables were transformed to ascertain each variable’s
normal distribution. Spearman’s correlation was used to
identify strongly correlated variables (correlation coefficient
> 0.8), and these variables were removed to avoid including
redundant information in this analysis.
In addition to a PCA, an analysis of similarities (ANOSIM)
was also performed to test for any significant difference
among stream types regarding physico-chemical parameters.
This analysis was done using the mean value of the three
2261
sampling occasions of the eight physico-chemical parameter
at each reference site. Each ANOSIM test produces an
R-statistic, which contrasts the similarities of the sites within
an ecotype with the similarities of those among stream types
(when the R value is close to 1, similarities between sites
within an ecotype are higher than those between sites from
different stream types, whereas values close to 0 indicate no
differences among types). The number of Monte Carlo
permutations was set at 999. We also did a pair-wise ANOSIM
comparison among stream types to distinguish among
possible contrasting effects. ANOSIM analysis was conducted
in PRIMER version 6.0. (Clarke and Warwick, 1994).
Boxplots of the all reference sites in the studied stream
types for the eight physico-chemical parameters were per-
formed to study their variability. The possible differences in
parameter among stream types were detected using a Krus-
kaleWallis non-parametric ANOVA test (Chi-square statistic)
which was carried out using the mean value of the three
sampling occasions for each site.
The seasonal differences in parameters under the RC were
assessed using a non-parametric repeated measure ANOVA
(Friedman test). For temporary streams, only spring and
autumn were compared because all the reference sites had
completely dried in summer. All ANOVA analyses were per-
formed using the SPSS (version 15.0, Chicago).
3. Results
3.1. Selection of reference sites
Most of the sites included in this study (76 out of 116; 65.5%)
fulfilled all 20 MRC, therefore they were considered reference
sites (Table 3). Of the total sites, 28.5% were classified as minor
disturbed and only 6% were classified as disturbed sites. Based
on the number of reference sites, the least disturbed stream
type was siliceous headwaters (T3: 84% of the sites were
undisturbed), followed by temporary streams (T1: 76.5%),
calcareous headwaters (T4: 71.5%) and evaporite-calcareous
at medium altitude (35%). Therefore, reference sites were
found for all stream types, except for T5 (large watercourses).
In general, the most limiting criteria were those relating to
natural land use and to dry land farming, which affected more
than 47% of the non-reference sites. Additionally, distur-
bances caused by the presence of reservoirs, retention of
sediments and irrigated farming affected a considerable
percentage of sites (32.5%).
In particular, the most limiting criteria for T1 and T2 were
those relating mostly to dry land farming and natural use. For
the case of T3, the most restraining criteria were the presence
of reservoirs, the retention of sediments and the riparian
vegetation. T4 and T5 were limited by riparian vegetation and
land use, and reservoirs and retention of sediments,
respectively.
3.2. Physico-chemical reference conditions in different
stream types
Fig. 2 shows the projection of the 76 selected reference sites
belonged to T1, T2, T3 and T4 and seven physico-chemical
2262 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9

Table 3 e Number of sites and proportions in relation to the total (in parenthesis) according to the number of Mediterranean
reference criteria fulfilled for each stream type.
Exposure to stress levels Criteria Steam types
fulfilled
T1 T2 T3 T4 T5 Total

(1) Undisturbed or 20 13 (76.5%) 7 (35.0%) 21 (84.0%) 35 (71.5%) 0 (0.0%) 76 (65.5%)

very minor disturbed sites
(2) Minor disturbed sites 19e16 4 (23.5%) 11 (55.0%) 3 (12.0%) 13 (26.5%) 2 (40.0%) 33 (28.5%)
(3) Disturbed sites 15 0 (0.0%) 2 (10.0%) 1 (4.0%) 1 (2.0%) 3 (60.0%) 7 (6.0%)
Total 17 20 25 49 5 116

parameters in the two axes of the PCA analysis. Dissolved
oxygen was removed of this ordination analysis because
Spearman’s correlation showed that dissolved oxygen and
oxygen saturation were highly correlated (r ¼ 0.827; p < 0.01).
Hence, the seven remaining previously transformed variables
were used to perform the PCA analysis. The two first axes
absorbed a total variance of 53.9%. The first axis, explaining
32.6% of variance, was positively related to nitrate and
ammonium concentrations, conductivity and pH. The second
axis, explaining 21.3% of variance, was positively related to
phosphate concentration and oxygen. The majority of sili-
ceous reference sites (T3) were located to the left of axis 1, and
presented lower conductivity, pH and nitrate concentrations,
and most were located separately to the rest of the type-
specific reference sites. The remaining reference sites
belonged to T1, T2, T4, located to the right of axis 1, showed
a clear overlap among stream types.
The ANOSIM analysis revealed that, although there were
some overall differences in the physico-chemical composition
of the four reference stream types according to the R global
value (Table 4), pair-wise comparisons indicated that only
siliceous headwaters (T3) could be considered physico-
chemically different from the rest. Moreover, the temporary
stream type (T1) showed differences with calcareous head-
waters (T4).
In order to study the variability of the reference sites
belonged to each stream types for the eight studied physico-
chemical parameters, boxplots were performed (Fig. 3). Box-
plots revealed that temporary streams (T1) presented the
highest dispersion of the reference data for dissolved oxygen,
evaporite-calcareous (T2) for conductivity, pH, nitrate and
phosphate, and calcareous headwaters (T4) for oxygen satura-
tion. Conversely, siliceous headwaters (T3) were characterised
by showing the lowest dispersion for conductivity, nitrate and
nitrite, although presented the highest variability for pH.
According to the KruskaleWallis analysis, only conduc-
tivity (F4,78 ¼ 42.4; p < 0.001) and pH (F4,78 ¼ 29.3; p < 0.001) gave
significant differences in RC among the four stream types.

Table 4 e ANOSIM results for global and pair-wise

comparisons among the four Mediterranean stream
types where reference sites were found. This analysis
was based on the reference datasets of the combined
(three seasons) values of the eight studied physico-
chemical parameters.
R P-value

Global test 0.459 0.001

T1eT2 0.091 0.201
T2eT3 0.497 0.001
T1eT4 0.192 0.018
T2eT3 0.614 0.001
Fig. 2 e Projection on the two axes in the PCA of the 76
T2eT4 0.129 0.152
type-specific reference sites and the seven physico- T3eT4 0.794 0.001
chemical parameters.
 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9 2263

Fig. 3 e Boxplots of the eight parameters studied in the four stream types where reference sites were found. The median
values (central line), 25th and 75th percentile values (box) and the maximum and minimum values are shown.

Respect to these two parameters, water conductivity pre- presented the lowest values for this parameter
sented higher mean and standard deviation values in the (104.8  106.4 mS/cm). As regards pH, siliceous headwaters (T3)
temporal stream type (T1: 1285.9  3343.3 mS/cm) than the presented a lower mean and standard deviation values
remaining permanent types, while siliceous headwaters (T3) (T1: 7.1  0.4) than the rest of stream types.
2264 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9

3.3. Seasonal variation in reference physico-chemical

conditions 4. Discussion

The Friedman test indicated that all the parameters showed
a seasonal pattern, except for nitrate concentration (Table 5).
All reference stream types presented seasonal differences in
some of the studied parameters, except for temporary
streams. In the case of evaporite-calcareous streams (T2), only
phosphate concentrations presented seasonal differences.
With siliceous headwaters (T3), seasonal differences were
detected in four parameters (dissolved oxygen and concen-
tration of phosphate, nitrite and ammonium) and finally, for
calcareous headwaters (T4) the parameters showing seasonal
differences were oxygen saturation, conductivity, pH and
nitrite concentration.
3.4. Reference values and boundaries setting results
Table 6 provides reference values and references thresholds
for each season for only those parameters which presented
seasonal differences for each stream type where reference
sites were found (T1, T2, T3 and T4). In all cases, it is generally
noted that although seasonal differences were detected, the
reference values and thresholds for each single season were
similar among them. In this situation, as it was mentioned in
Methods, the least restrictive both reference and threshold
values of the three seasons were proposed for each parameter
in each stream type (see proposed values in Table 6).
Table 7 shows reference values and reference thresholds
using the two approaches according to the availability of
reference sites mentioned in Methods for each parameter in
the five studied stream types. For T1eT4, where reference
sites were found, the 75th percentile value of reference sites
was used as reference threshold. However, for T5 the 25th
percentile value of the least disturbed sites was used as
reference threshold. In general, reference values and refer-
ence thresholds were very similar, with a low variability for all
the stream types, except for conductivity and pH, as it was
showed by the KruskaleWallis analysis. For these two
parameters, T3 had the lowest reference values and reference
thresholds. Also, it must be highlighted that large water-
courses (T5) had highest reference values for all the parame-
ters, except for dissolved oxygen and conductivity, being
these differences more relevant for the case of nutrients.
4.1. Reference sites in Mediterranean streams
Although employing the data from reference sites proves
a useful tool to estimate the RC of the physico-chemical
parameters, there are three major drawbacks in developed
countries to establish boundaries for class quality values: i) real
pristine reference sites are essentially non-existent in most
regions; ii) natural nutrient concentration at reference sites
varies because of variation in any of the (several) basin charac-
teristics, including climate, hydrology, natural vegetation, and
mineral composition of soil and rock; iii) all the near-natural
reference sites are allocated in small catchments because few
large catchments remain undisturbed (Smith et al., 2003).
Evidently, these three problems must be faced in Mediter-
ranean areas. The first is resolved by applying screening
methods to select reference sites. This type of methods allows
the presence of very minor disturbances, which are compatible
with a condition corresponding to a state of very low environ-
mental pressure or degradation. This is the case of the present
study by applying MRC (Sánchez-Montoya et al., 2009). The
second obstacle relating to RC variability should be addressed
by using river typologies as they reduce the natural variability
of basin characteristics. The present work used the Mediterra-
nean streams typology developed in the same study area,
which establishes five environmentally stream ecotypes, which
are homogeneous in terms of the hydrological, geological,
climatic and morphological criteria or characteristics (Sánchez-
Montoya et al., 2007). For obstacle 3, we and others (e.g. Davies,
1994; Hering et al., 2003; Bailey et al., 2004) justify the use of
more permissive approaches as regards the presence of some
pressures at reference sites, thus allowing environmental and
management goals with a view to applying the WFD, similarly
to the middle and lower reaches of large watercourses (T5) in
Mediterranean streams and rivers.
The majority of the studied potential reference sites
(n ¼ 116) fulfilled the 20 MRC established for this study area
thanks to the huge efforts made during the field campaign
searching for the least impacted sites in the studied basins.
The most limiting criteria were those relating to agriculture.
Although all European catchments generally undergo the
same types of human pressure, it seems that an increasing
gradient of intensity to Mediterranean regions exists. Dry and

Table 5 e Results of the Friedman test to check seasonal differences in RC in the four stream types. Only the cases which
presented significant differences are shown. For those parameters, mean values from spring, summer and autumn are
present.
Stream type Parameters N Spring (mean) Summer (mean) Autumn (mean) c2 df p

T2 Phosphate (mg/l) 7 0.049 0.048 0.010 8.361 2 0.039

T3 Dissolved oxygen (mg/l) 21 10.02 8.82 9.80 10.381 2 0.006
Phosphate (mg/l) 21 0.058 0.031 0.033 8.391 2 0.015
Nitrite (mg/l) 21 0.304 0.326 0.345 7.000 2 0.030
Ammonium (mg/l) 21 0.033 0.026 0.019 7.536 2 0.023
T4 Oxygen saturation (%) 33 104.21 111.21 99.49 8.061 2 0.018
Conductivity (mS/cm) 33 447.70 482.20 486.30 10.093 2 0.006
pH 33 8.13 8.16 8.24 11.282 2 0.004
Nitrite (mg/l) 33 0.019 0.015 0.029 0.731 2 0.005
 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9 2265

Table 6 e Reference values (Ref-Val) and reference thresholds (Ref-Thr) in each sampled season for those parameters
showing seasonal differences. The proposed reference values and reference threshold values are also indicated, using the
least restrictive of the three studied seasons.
Spring Summer Autumn Proposed values

Ref-Val Ref-Thr Ref-Val Ref-Thr Ref-Val Ref-Thr Ref-Val Ref-Thr

T2 Phosphate (mg/l) 0.040 0.050 0.040 0.050 0.045 0.114 0.045 0.110
T3 Oxygen saturation (%) 100.6 90.7e115.1 101.4 93.8e109.2 98.0 93.1e100.8 98.0 90.7e115.1
Phosphate (mg/l) 0.035 0.052 0.020 0.049 0.028 0.049 0.035 0.052
Nitrite (mg/l) 0.015 0.015 0.015 0.015 0.015 0.022 0.015 0.022
Ammonium (mg/l) 0.019 0.050 0.017 0.032 0.016 0.019 0.019 0.050
T4 Dissolved oxygen (mg/l) 9.5 8.7e10.5 9.3 8.0e10.5 10.0 9.2e10.6 9.3 8.0e10.6
Conductivity (mS/cm) 400 705 461 743 468 731 468 743
pH 8.1 8.6 8.1 8.6 8.2 8.6 8.1 8.6
Nitrite (mg/l) 0.015 0.040 0.015 0.015 0.015 0.045 0.015 0.045

irrigated farm lands are significantly greater in circum-
Mediterranean countries than in other European countries
(MIMAN, 2000; Prat and Munné, 2000) because agriculture has
traditionally been very important productive sector in the
Mediterranean Europe (Allen, 2001). In fact, 90% of lands irri-
gated in Europe are located in Mediterranean countries, and
this is the most water-demanding activity (MIMAN, 2000). This
activity has led to increasing nutrients, pesticides and herbi-
cides input through point and diffuse sources of pollution,
impact on riparian vegetation and channel morphology, and
also to the alteration of natural discharge in rivers and
streams (Sánchez-Montoya et al., 2009).
Regarding stream type 5 (large watercourses), no reference
sites were found. For this stream type, and in order to overcome
the third obstacle, we considered that those sites receiving less
intense impacts that fulfil most reference criteria (> 13) might
be justifiably used to establish reference physico-chemical
conditions. This approach matches the ‘Best Attainable
Condition’ concept defined by Stoddard et al. (2006), which
indicates that reference sites are selected where the impact of
inevitable land use on biota has been minimised.
4.2. Differences in physico-chemical reference conditions
among Mediterranean stream types
In the present study, only pH and conductivity presented
significant differences among stream types where reference
sites were found.
Regarding oxygen reference concentrations, which depend
mainly on primary production, organic matter decomposition,
altitude and temperature, significant differences were not
detected. In relation to nutrient concentrations, the low
concentrations found in all stream types reflect the absence of
anthropogenic perturbations. However, it must be taken into
account that several authors have reported high natural
nitrate concentrations in streams located in Mediterranean
sedimentary watersheds (Holloway et al., 1998; Dahlgren,
1994; Gómez et al., 2005; Holloway and Smith, 2005). These
authors have demonstrated that bedrock containing consid-
erable concentrations of fixed nitrogen contributes a surpris-
ingly large amount of nitrate to surface waters, which could
have a profound effect on water quality in small basins. In this
study, some temporary streams where high nitrate concen-
trations have been attributed to this geological origin (Gómez
et al., 2005) were not selected as reference sites given the high
percentage of dry land farming in their drainage areas.
However, this geological nitrogen must be considered as
a plausible explanation of high nitrate concentrations in
undisturbed streams when the ecological status is evaluated
according to physico-chemical elements.
On the other hand, only the conductivity and pH parame-
ters differed among stream types, and were lower and
significantly different in siliceous headwater than in the other
stream types. Regarding water conductivity, this parameter is
primarily affected by the geology of the area through which
water flows due to differences in weathering reactions (e.g.

Table 7 e Reference values (Ref-Val) and reference thresholds (Ref-Thr) for the five studied stream types. Numbers in italics
correspond to those variables which showed significant seasonal differences, whose reference value and reference
threshold were proposed in Table 6.
T1 T2 T3 T4 T5

Ref-Val Ref-Thr Ref-Val Ref-Thr Ref-Val Ref-Thr Ref-Val Ref-Thr Ref-Val Ref-Thr

Oxygen saturation (%) 97.0 91.0e103.2 100.7 98.9e104.7 98.0 90.7e115.1 102.0 93.2e110.6 100.7 89.0e116.4
Dissolved oxygen (mg/l) 9.9 8.1e10.8 10.0 9.2e10.6 9.5 8.64e10.4 9.3 8.0e10.6 9.8 9.0e10.4
Conductivity (mS/cm) 337 443 585 660 67 151 468 731 543 463
pH 8.0 8.3 8.1 8.4 7.3 7.6 8.1 8.6 8.2 7.8
Phosphate (mg/l) 0.027 0.041 0.040 0.110 0.035 0.052 0.021 0.038 0.080 0.032
Nitrate (mg/l) 0.232 0.480 0.462 0.672 0.230 0.416 0.298 0.602 1.010 0.404
Nitrite (mg/l) 0.019 0.041 0.015 0.047 0.015 0.022 0.015 0.045 0.020 0.015
Ammonium (mg/l) 0.023 0.031 0.029 0.038 0.019 0.050 0.023 0.030 0.043 0.033
2266 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9
Orive et al., 1989). Therefore, lower conductivity values were
expected in siliceous headwaters (T3) due to the insolubility of
this rock type. Conversely, the high solubility of carbonate
rocks, in addition to the high salt content in sedimentary
evaporite rocks, implies that evaporite-calcareous headwaters
(T2) display the highest value of conductivity under natural
conditions. Our results also highlighted the high variability of
conductivity values (according to its standard deviation value)
present in reference temporary streams, as have been also
previously reported in the same Mediterranean region for this
stream type (Vidal-Abarca et al., 1995; Moreno et al., 1995,
2001). In temporary streams, apart from differences in
geology, variability in surface water conductivity is generated
by the spatial variation in advection and by other hydrological
parameters (water residence time and channel velocity) that
affect processes implicated in salt dissolution (e.g. tempera-
ture, sedimentewater interaction, etc.), as consequence of the
low flow (Gómez et al., 2009).
Respect to pH, the higher acidity in siliceous headwaters
(T3) reflects the relative inert siliceous rock if compared with
more reactive rock types such as carbonate and evaporite
geology (Walling and Webb, 1975) in T1, T2 and T4.
In short, siliceous headwaters was the only stream type
that revealed clear differences if compared with the
other stream types regarding to conductivity and pH.
Moreover, temporary stream was the ecotype showing
greater variation for some parameters, mainly in water
conductivity.
It must be emphasised that the aforementioned differ-
ences in physico-chemical conditions detected in siliceous
headwaters and temporary streams if compared to the
remaining studied ecotypes, completely coincide with differ-
ences detected in reference macroinvertebrate communities
in the same stream types (Sánchez-Montoya et al., 2007). In
this previous work conducted in the same study area, sili-
ceous streams clearly differed from the other three ecotypes,
while the evaporite-calcareous and calcareous headwaters
stream types (T2 and T4) presented no large differences in
macroinvertebrate assemblages. Moreover, temporary
streams formed a very heterogeneous group of reference
communities. Therefore, when physico-chemical and biolog-
ical elements are analysed together we could conclude that: i)
the distinct reference macroinvertebrate community in sili-
ceous headwaters is associated with lower conductivity and
pH values and ii) the diverse group of reference macro-
invertebrate communities found in the temporary stream
type seems to respond to the high variability of water
conductivity values, among other important factors as
hydrological regime. This large variability in the biological and
physico-chemical elements of temporary streams may justify
a further division of this ecotype into subgroups to assure the
proper application of the WFD methodology, as has been
formerly pointed out for the reference macroinvertebrate
community (Sánchez-Montoya et al., 2007).
Besides, similarities found between evaporite-calcareous
at medium altitude (T2) and calcareous headwaters (T4)
stream types with respect to both macroinvertebrate
communities and physico-chemical parameters in RC, lead to
propose that these two stream types must be considered as
a single one for assessment purposes.
4.3. Seasonal differences in Mediterranean stream types
Quantitative ecological assessments ultimately require the
establishment of numerical criteria that describe the
maximum acceptable level of water quality elements
(Hawkins et al., 2010). Along these lines, type-specific refer-
ence conditions must be established which consider their
natural variability (Irvine, 2004).
In this study, all the stream types where references sites
were available presented seasonal differences as regards
some parameters, except for temporary streams (T1).
A special consideration must be taken into account to
understand the lack of seasonal pattern in the temporary
stream type in this study. Reference sites belonging to
temporary stream type were completely dry in the summer of
2003. Therefore, the effect of the high evaporation rate and
system contraction due to the cessation of flow in summer,
have been not detected. In fact, in temporary streams it is
expected that during summer solute concentrations increase
as result of lower water velocities and higher water residence
time, unlike the more hydrologically stable permanent
streams where solute concentrations are less variable (Gómez
et al., 2009). Besides, the high spatial variability registered for
water conductivity in this stream type could hide potential
seasonal differences between spring and autumn. In this
sense, many studies have detected seasonal pattern in
physico-chemical conditions for temporary streams not only
in this work study area (Vidal-Abarca et al., 2000; Gómez et al.,
2005, 2009) but also in other Mediterranean streams (Martin
and Sabater, 1996; Butturini and Sabater, 2002; Bernal et al.,
2003, 2005; Lillebo et al., 2007).
Moreover, permanent and temporal reaches can appear in
the same temporary stream at the same time due to the effect
of surface flow variability and local substrate heterogeneity
(Bonada et al., 2007; Gómez et al., 2009). For this reason to
define the exact location of the sampling site is essential when
reference physico-chemical conditions are studied. It must be
also consider that this intermittency in temporary streams is
expected to increase by changes in the flow and flood regime
induced by climate change and human use (Giorgi and
Lionello, 2008).
Although all parameters showed a seasonal pattern for
some stream types except for nitrate concentration (Table 5),
we believe that the similarity found among the three seasonal
reference values and reference thresholds justify the proposal
of use the least restrictive values in order to simplify the water
quality assessment for management goals.
4.4. Establishment of reference values and reference
thresholds in different Mediterranean stream types
As far as we know, this is the first study from a Mediterranean
area which, apart from nutrient concentration, the most of
physico-chemical parameters demanded by the WFD have
been analysed in order to establish reference values and
reference threshold. In the context of Mediterranean streams
several studies have been developed recently. In Spain,
Moreno and Navarro (2006) and the Spanish Environmental
authorities (IPH, 2008) have defined reference value for some
parameters. In this later case, only reference values for three
 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9 2267

Table 8 e Comparison of range of reference values (Ref-Val) and reference thresholds (Ref-Thr) for physico-chemical
parameters established for different stream types defined in Mediterranean streams in several studies.
Present study Moreno and IPH, 2008 Skoulikidis Erba
Navarro, 2006 et al., 2006 et al., 2009

Ref-Val Ref-Thr Ref-Val Ref-Val Ref-Thr Ref-Thr Ref-Val

Oxygen saturation (%) 97.0e102.0 (89.0e98.9) 98.6

(103.2e116.4)
Dissolved oxygen (mg/l) 9.3e10.5 (8.1e9.2) 8.0e9.8 9e10.2 7.6e8.6
(10.4e10.8)
Conductivity (mS/cm) 67e585 151e660 148.0e1029.9 80e510 300e1000
pH 7.3e8.2 7.6e8.4 7.9e8.2 7.1e9.0
Phosphate (mg/l) 0.021e0.080 0.032e0.082 0.019e0.031 0.070 0.029
Nitrate (mg/l) 0.230e1.010 0.404e0.672 0.903e3.208 0.220 0.740
Nitrite (mg/l) 0.015e0.020 0.015e0.047 0.006e0.014 0.003 0.000
Ammonium (mg/l) 0.023e0.043 0.021e0.038 0.019e0.090 0.024 0.020

physico-chemical parameters have been established,
emphasizing the urge of studies for determining reference
physico-chemical RC in Spanish Mediterranean streams. In
Greece, Skoulikidis et al. (2006) established reference thresh-
olds for nutrient concentrations using 36 reference sites
throughout Greece, and more recently Erba et al. (2009)
showed RC for few parameters, mainly nutrient concentra-
tions, within the context of Mediterranean Geographical
Intercalibration Group.
When comparing the physico-chemical RC established in
those previous studies with the present work (Table 8), simi-
larities and differences are observed in accordance with the
parameters analysed. Reference acidification conditions are
similar in all the studies, but conversely, differences are
detected for salinity, oxygenation and nutrient conditions.
Respect to salinity conditions, RC established in the present
work were more restrictive than in the other studies. The
greater differences are detected in nutrients conditions due to
significant lower reference thresholds for all nutrients, except
for ammonium, reported by Skoulikidis et al. (2006) for Greek
streams. The rest of studies show greater similarities
according to nutrient concentrations, especially between the
present study and that developed by Erba et al. (2009) in the
Mediterranean GIG context.
conductivity. A further division of this ecotype into
subgroups is justified by the results in this stream type.
 Similarities found between evaporite-calcareous at medium
altitude and calcareous headwaters stream types with
respect to both macroinvertebrate communities and
physico-chemical parameters in RC suggest the union of
these two stream types into one ecotype for management
purpose.
 All the studied stream types presented seasonal differences
as regards some parameters, except for temporary streams
due to the high natural variation of this stream type.
Acknowledgements
This study has used of the GUADALMED Project database
(Phase II; Data available at: http://webfe04.ird.ub.es/fem/
index.php/guadalmed). The authors wish to thank all
researchers who have worked in this project. This research
has been supported by the GUADALMED-2 project (REN2001-
3438-C07-01) and partially by the IþD project (CGL2010-21458)
from the Spanish Ministry of Science. Special thanks to
Victoria Garcı́a and Andrés Mellado for their help in the
fieldwork in the Segura Basin, and Joaquı́n Pajarón for his help
with the figures of this article.

5. Conclusions
 The majority of the studied potential reference sites fulfilled
the 20 Mediterranean reference criteria established for this
study area. The least disturbed stream type was siliceous
headwaters, followed by temporary streams, calcareous
headwaters and evaporite-calcareous at medium altitude.
Reference sites were not found for large watercourses. The
most limiting criteria were those relating to agriculture.
 Siliceous headwaters can be considered different from the
rest of studied stream types because they presented lower
conductivity and pH. This finding is completely coincident
with that detected previously when reference macro-
invertebrate communities was analysed.
 Temporary streams was the ecotype that showed greater
variation than permanent streams, mainly water
references
Achleitner, S., Toffol, S., Engelhard, C., Rauch, W., 2005. The
European Water Framework Directive: water quality
classification and implications to engineering planning.
Environmental Management 35 (4), 517e525.
Allen, H.D., 2001. Mediterranean Ecogeography. Harlow Pearson
Education, England.
APHA, 1998. Standard Methods for the Examination of Water and
Waste Water, twentieth ed. American Public Health
Association, Washington, DC.
Bailey, R.C., Norris, H., Reynoldson, B., 2004. Bioassessment of
Freshwater Ecosystems. Using the Reference Condition
Approach. Kluwer Academic Publishers, Dordrecht.
Barbour, M.T., Stribling, J.B., Gerritsen, B.D., 1996. Biological
Criteria: Technical Guidance for Streams and Small Rivers.
2268 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9
EPA/822/B-96/001. US. Environmental Protection Agency,
Washington, DC.
Bernal, S., Butturini, A., Nin, E., Sabater, F., Sabater, S., 2003. Leaf
litter dynamics and nitrous oxide emission in a Mediterranean
riparian forest: implications for soil nitrogen dynamics.
Journal of Environmental Quality 32 (1), 191e197.
Bernal, S., Butturini, A., Sabater, F., 2005. Seasonal variation of
dissolved nitrogen and DOC: DON ratios in an intermittent
Mediterranean stream. Biogeochemistry 74 (2), 351e372.
Bonada, N., Prat, N., Munné, A., Rieradevall, M., Alba-Tercedor, J.,
Álvarez, M., Avilés, J., Casas, J., Jaı́mez-Cuéllar, P., Mellado, A.,
Moyá, G., Pardo, I., Robles, S., Ramón, G., Suárez, M.L.,
Toro, M., Vidal-Abarca, M.R., Vivas, S., Zamora-Muñoz, C.,
2002. Criterios para la selección de condiciones de referencia
en los rı́os mediterráneos. Resultados del proyecto
GUADALMED. Limnetica 21 (3e4), 99e114.
Bonada, N., Rieradevall, M., Prat, N., 2007. Macroinvertebrate
community structure and biological traits related to flow
permanence in a Mediterranean river network. Hydrobiologia
589, 91e106.
Butturini, A., Sabater, F., 2002. Nitrogen concentrations in a small
Mediterranean stream: 1.Nitrate 2. Ammonium. Hydrology
and Earth System Sciences 6 (3), 539e550.
Clarke, K.R., Warwick, R.M., 1994. Changes in Marine
Communities: An Approach to Statistical Analysis and
Interpretation, first ed. Plymouth Marine Laboratory,
Plymouth.
Cooper, D.M., Jenkins, A., Skeffington, R., Gannon, B., 2000.
Catchment-scale simulation of stream water chemistry by
spatial mixing: theory and application. Journal of Hydrology
233 (1e4), 121e137.
Dahlgren, R.A., 1994. Soil acidification and nitrogen saturation
from weathering of ammonium-bearing rock. Nature 368,
838e841.
Davies, P.E., 1994. Monitoring River Health Initiative. River
Bioassessment Manual. National River Process and
Management Program. Freshwater system, Tasmania.
Erba, S., Furse, M.T., Balestrini, R., Christodoulides, A.,
Ofenböck, T., van de Bund, W., Wasson, J.G., Buffagni, A., 2009.
The validation of common European class boundaries for river
benthic macroinvertebrates to facilitate the intercalibration
process of the Water Framework Directive. Hydrobiologia 633,
17e31.
EU/2000/60/EC-WFD, 2000. Directive of the European Parliament
and of the Council establishing a framework for Community
action in the field of water policy (2000/60/EC). October 2000.
Gasith, A., Resh, V.H., 1999. Streams in Mediterranean climate
region: abiotic influences and biotic responses to predictable
seasonal events. Annual Review of Ecology and Systematics
30, 51e81.
Giorgi, F., Lionello, P., 2008. Climate change for the Mediterranean
region. Global and Planetary Change 63, 90e104.
Gómez, R., Hurtado, I., Suárez, M.L., Vidal-Abarca, M.R., 2005.
Ramblas in south-east Spain: threatened and valuable
ecosystems. Aquatic Conservation: Marine and Freshwater
Ecosystems 15, 387e402.
Gómez, R., Garcı́a, V., Vidal-Abarca, M.R., Suárez, M.L., 2009. Effect
of intermittency on N spatial variability in an arid
Mediterranean streams. Journal of the North American
Benthological Society 28 (3), 572e583.
Hawkins, P.C., Olson, J.R., Hill, R.A., 2010. The reference condition:
predicting benchmarks for ecological and water-quality
assessments. Journal of the North American Benthological
Society 29, 312e343.
Hering, D., Buffagni, A., Moog, O., Sandin, L., Sommerhaüser, M.,
Stubauer, I., Feld, C., Johnson, R., Pinto, P., Skoulikidis, N.,
Verdonschot, P., Zahrádková, S., 2003. The development of
a system to assess the ecological quality of streams based on
macroinvertebrates design of the sampling programme within
the AQEM project. International Review of Hydrobiology 88
(3e4), 345e361.
Herlihly, A.T., Sifneos, J.C., 2008. Developing nutrient criteria and
classification schemes for wadeables streams in
conterminous US. Journal of the North American
Benthological Society 27 (4), 932e948.
Holloway, J.M., Dahlgren, R.A., Hansen, B., Casey, W.H., 1998.
Contribution of bedrock nitrogen to high nitrate
concentrations in stream water. Nature 395, 785e788.
Holloway, J.M., Dahlgree, R.A., 2001. Seasonal and event-scale
variations in solute chemistry for four Sierra Nevada
catchments. Journal of Hydrology 1e4 (250), 106e121.
Holloway, J.M., Smith, R.L., 2005. Nitrogen and carbon flow from
rock to water: regulation through soil biogeochemical
processes, Mokelumne River watershed, California, and Grand
Valley, Colorado. Journal of Geophysical Research 110, F01010.
doi:10.1029/2004JF000124.
IPH (Instrucción de Planificación Hidrológica), 2008. Ministerio de
Medio Ambiente, y Medio Ambiente, y Medio Rural y Marino
de España. Orden ARM/ 2656/2008. BOE 229.
Irvine, K., 2004. Classifying ecological status under the European
Water Framework Directive: the need for monitoring to
account for natural variability. Aquatic Conservation: Marine
and Freshwater Ecosystems 14, 107e112.
Jacobsen, D., Schultz, R., Encalada, A., 1997. Structure and
diversity of stream invertebrate assemblages: the influence of
temperature with altitude and latitude. Freshwater Biology 38,
247e261.
Lillebo, A.I., Morais, M., Guilherme, P., Fonseca, R., Serafim, A.,
Neves, R., 2007. Nutrient dynamics in Mediterranean
temporary streams: a case study in Pardiela catchment
(Degebe River, Portugal). Limnologica 37 (4), 337e348.
Martin, E., Sabater, F., 1996. High variability in temporal and
spatial nutrient retention in Mediterranean streams. Ecology
77 (3), 854e869.
MIMAN, 2000. Las aguas continentales en los paı́ses
mediterráneos de la Unión Europea. Ministerio de Medio
Ambiente de España, CEDEX.
Moreno, J.L., Suárez, M.L., Vidal-Abarca, M.R., 1995. Hidroquimica
de las ramblas litorales de la región de Murcia. Variaciones
espacio-temporales. Limnetica 11 (1), 1e13.
Moreno, J.L., Aboal, M., Vidal-Abarca, M.R., Suárez, M.L., 2001.
Macroalgae and submerged macrophytes from fresh and
saline waterbodies of ephemeral streams (‘ramblas’) in
semiarid south-eastern Spain. Marine & Freshwater Research
52 (6), 891e905.
Moreno, J.L., Navarro, C., De las Heras, J., 2006. Abiotic ecotypes in
south-central Spanish rivers: reference conditions and
pollution. Environmental Pollution 143, 388e396.
Nijboer, R.C., Jhonson, R.K., Verdonschot, P.F.M.,
Sommerhaüser, M., Buffagni, A., 2004. Establishing
reference conditions for European streams. Hydrobiologia
516, 91e105.
Orive, E., Basaguren, A., de Bikuña, B., Cacho, M., 1989. A
comparative study of water mineralization and nutrient
status in the main water courses of Biscay (Basque Country).
Water Research 23, 705e710.
Petts, G.E., 1989. Historical Change of Large Alluvial Rivers:
Western Europe. Wiley, Chichester.
Prat, N., Munné, A., 2000. Water use and quality and stream flow
in a Mediterranean stream. Water Research 34 (15),
3876e3881.
Prat, N., 2002. El proyecto GUADALMED sobre el Estado Ecológico
de los rı́os Mediterráneos. Limnética 21 (3e4), 1e3.
Reynoldson, T.B., Wright, J.F., 2000. The reference condition:
problems and solutions. In: Wright, J.F., Sutcliffe, D.W.,
Furse, M.T. (Eds.), Assessing the Biological Quality of Fresh
 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 2 5 7 e2 2 6 9
Waters: RIVPACS and Others Techniques. Freshwater
Biological Association, Ambleside, Cumbia, UK, pp. 293e303.
Sánchez-Montoya, M.M., Suárez, M.L., Vidal-Abarca, M.R., 2005.
Propuesta de criterios para la selección de estaciones de
referencia en rı́os mediterráneos en el contexto de la DMA.
Tecnologı́a del Agua 167, 42e52.
Sánchez-Montoya, M.M., Puntı́, T., Suárez, M.L., Vidal-
Abarca, M.R., Rieradevall, M., Poquet, J.M., Zamora-Muñoz, C.,
Robles, S., Álvarez, M., Alba-Tercedor, J., Toro, M., Pujante, A.,
Munné, A., Prat, N., 2007. Concordance between ecotypes and
macroinvertebrate assemblages in Mediterranean streams.
Freshwater Biology 52, 2240e2255.
Sánchez-Montoya, M.M., Vidal-Abarca, M.R., Puntı́, T.,
Poquet, J.M., Prat, N., Rieradevall, M., Alba-Tercedor, J.,
Zamora-Muñoz, C., Toro, M., Robles, S., Álvarez, M.,
Suárez, M.L., 2009. Defining criteria to select reference sites in
Mediterranean streams. Hydrobiologia 619, 39e54.
Skoulikidis, N.T., Amaxidis, Y., Bertahas, I., Laschou, S.,
Gritzalis, K., 2006. Analysis of factors driving stream water
composition and synthesis of management tools e a case
study on small/medium Greek catchments. Science of the
Total Environment 362, 205e241.
Smith, R.A., Alexander, R.B., Schwarz, G.E., 2003. Natural
Background concentrations of Nutrientes in streams and
2269
rivers of the conterminous United States. Environmental
Science & Technology 37 (14), 3039e3047.
Stoddard, J.L., Larse, D.P., Hawkins, C.P., Johnson, R.K.,
Norris, R.H., 2006. Setting expectations for the ecological
conditions of streams: the concept of reference condition.
Ecological Applications 16 (4), 1267e1276.
USEPA, 2000. Nutrient Criteria Technical Guidance Manual: Rivers
and Streams. EPA-822-B00-002. Office of Water, US
Environmental Protection Agency, Washington, DC.
Vidal-Abarca, M.R., Suárez, M.L., Ramı́rez-Dı́az, L., 1995.
Caracterı́sticas ambientales y recursos hı́dricos de la Cuenca
del Rı́o Segura. In: Senté, M., Cabezas, F. (Eds.), Agua y
Futuro en la Región de Murcia. Asamblea Regional de
Murcia, España.
Vidal-Abarca, M.R., Suárez, M.L., Moreno, J.L., Gómez, R.,
Sánchez, I., 2000. Hidroquı́mica de un rı́o de caracterı́sticas
semiáridas (Rı́o Chı́camo; Murcia): análisis espacio-temporal.
Limnética 18, 57e73.
Vitousek, P.M., 1977. The regulation of element concentrations in
mountain streams in the Northeastern United States.
Ecological Monographs 47 (1), 65e87.
Walling, D.E., Webb, B.W., 1975. Spatial variation of river quality:
a survey of the River Exe. Transactions of the Institute of
British Geographers 65, 155e171.