PRIMARY RESEARCH PAPER

A multimetric diatom index to assess the ecological statusof coastal Galician rivers (NW Spain)

Cristina Delgado • Isabel Pardo • Liliana Garcıa

Received: 3 September 2009 / Revised: 24 February 2010 / Accepted: 1 March 2010 / Published online: 17 March 2010

� Springer Science+Business Media B.V. 2010

Abstract There are many rivers in northwest Spain

as a consequence of the mountainous landscape and

the granitic geology subjected to Atlantic influences.

Water and epilithic diatoms samples were collected

at 72 sites in Galicia flowing into the Atlantic Ocean

and Cantabrian Sea in summer 2002–2003 and

spring 2004. These sites included minimally dis-

turbed sites, defined as reference sites, and impacted

sites which were influenced by different human

pressures. We used the diatom assemblages to

calculate diatom indices using the Omnidia software,

but we also developed new metrics based on the

similarity of species composition in reference sites.

The response of the metrics was tested in relation to

physicochemical variables. We developed a diatom

multimetric index (MDIAT) as a combination of

metric values. The sensitivity of the MDIAT to

organic and nutrient stressors supports the use of this

index to classify the ecological status of Galician

rivers. The MDIAT showed higher correlations with

some variables and nutrients than the individual

metrics. According to the MDIAT, 69% of the sites

sampled in Galician coastal rivers achieve good

ecological status. The MDIAT has been developed

specifically for Galician granitic rivers (NW Spain),

and has been intercalibrated at the European level in

the Central Baltic Rivers GIG. Our study validates

the application of this multimetric index to evaluate

the water quality in coastal Galician rivers.

Keywords Coastal Galician rivers � Ecological

status � Diatoms � Multimetric index � Water

Framework Directive

Introduction

The degradation of freshwater ecosystems has been a

cause of concern for several decades. Since the

implementation of the European Water Framework

Directive (WFD; European Union, 2000) has encour-

aged different applied ecological studies for under-

standing the impact caused to freshwater ecosystems

by anthropogenic pressures (Hering et al., 2006a;

Muxika et al., 2007). The WFD requires that

ecological status assessments of rivers and lakes are

based on evaluations of phytoplankton, macrophytes

and phytobenthos, benthic invertebrates and fish.

Methods to assess the phytobenthos have tended to

focus on diatoms which often form a large part of the

algal diversity in freshwaters (King et al., 2000).

Although, macroinvertebrates and fishes have been

frequently used in the evaluation of the ecological

status in rivers (Oberdorff & Hughes, 1992; Morais

et al., 2004; Hering et al., 2006a), the diatoms are

Handling editor: P. Noges

C. Delgado (&) � I. Pardo � L. Garcıa

Department of Ecology and Animal Biology,

University of Vigo, 36330 Vigo, Spain

e-mail: cdelgado@uvigo.es

123

Hydrobiologia (2010) 644:371–384

DOI 10.1007/s10750-010-0206-y

very sensitive organisms to sudden and minor

changes occurring in water chemistry (Leira &

Sabater, 2005). Benthic diatoms are traditionally

considered to be regulated mainly by local rather than

large scale factors (Pan et al., 1999) although large-

scale spatial factors, such as climate, geology and

vegetation also influence the structure of the diatom

community (Leland, 1995). Benthic diatoms seem not

only to respond to hydromorphological modification

(Hering et al., 2006a) but also have been used

extensively in rivers for assessing nutrient enrichment

(e.g. Coring et al., 1999; Rott et al., 1999). In Latvia,

the relationship between diversity indices and envi-

ronmental variables is stronger in the case of

small*bodied organisms, such as diatoms and macr-

oinvertebrates, compared to macrophytes and fishes

(Springe et al., 2006).

The response of the community to a pressure

gradient can be converted into a continuous variable

using diatom metrics (Kelly et al., 2008). This

simplifies the complicated ecology of rivers in a way

that permits the rapid assessment of the overall

condition in a manner that is easily understood

(Atazadeh et al., 2007). Some studies show that diatom

metrics detect eutrophication effects better than met-

rics calculated using fishes, macroinvertebrates and

macrophytes, and they respond most strongly to land-

use gradients (Hering et al., 2006a; Johnson et al.,

2006). For all these reasons, the use of diatom indices

has undergone an increase in recent years as a tool to

provide a reflection of water quality (Prygiel & Coste,

1993; Kelly, 1998, 2002; Wu, 1999; Gomez & Licursi,

2001; Wu & Kow, 2002). Moreover, the appearance of

software packages, such as Omnidia, which facilitates

the calculation of indices, has intensified its use in

Europe (Kwandrans et al., 1998; Eloranta & Soininen,

2002; Pardo et al., 2005; Garcıa et al., 2008). Mean-

while, in North America, the use of metrics based on

sensitive and tolerant species is more developed (Fore

& Grafe, 2002; Passy & Bode, 2004).

The objectives of this study are (i) to test the

response, in Galician rivers, of different diatom

indices and metrics (some newly developed for the

area) that taken into account the composition and

abundance of reference species assemblages, (ii) to

build a multimetric diatom index by a combination of

different types of response metrics (taxonomic,

organic, trophic, and sensitivity of taxa) to fulfil the

normative definitions of the WFD, and finally (iii) to

assess, using this multimetric, the ecological status of

Galician rivers.

Materials and methods

Study area

The Northwest of the Iberian Peninsula is influenced by

the Atlantic climate, and it is characterized by rainy

weather with mild temperatures throughout the year,

similar to the rest of Western Europe. The region of

Galicia lies in this area of Spain with the Cantabrian

Sea to the north and the Atlantic Ocean to the west

(Fig. 1). The geology is dominated by siliceous rocks:

granite in the west and metamorphic rocks in the east.

The region can be divided into two areas: Inland

Galicia and Coastal Galicia which are separated by a

mountain range known colloquially as ‘Galicia0sbackbone’. Coastal Galicia has mild winters and cool

summers with precipitation exceeding 1,500 mm per

year. The mountainous geomorphology and regular

precipitation influences the occurrence and perma-

nence of many small- and medium-sized rivers with

regular discharge throughout the year. This study

focuses on these systems within the area of Coastal

Galicia that includes all Galician river basins that flow

into the Cantabrian Sea and to the Atlantic Ocean

(Fig. 1).

Sampling design

Initially, we differentiated river types using ‘system A’

of the WFD, which uses geology, altitude and catch-

ment area as descriptors (WFD, Annex II). Galician

coastal rivers have granitic geology and they were thus

divided into three categories according to the catchment

area: small, medium and large at different altitudes

(Table 1). Most of the rivers were small to medium

sized and only the downstream parts of the rivers Ulla,

Umia and Tambre were considered to be large rivers.

These, however, were not included in this study.

Selecting reference and non-reference sites

Candidate reference sites had to satisfy a series of

a priori selection criteria based on the absence of

significant pressures (dams, water treatment plants, fish

farms and percentage of agricultural land\30%). We

372 Hydrobiologia (2010) 644:371–384

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used local information on pressures in the Galician

coastal area and data from CoORdination of INforma-

tion of the Environment (CORINE) Land Cover 1990

(Bossard et al., 2000) to calculate the percentage of land

used for different purposes. The reference condition

criteria used to select the reference sites were those of

the Central/Baltic Geographical Intercalibration Group

(C/B GIG; van de Bund, 2009; Kelly et al., 2009).

Field sampling and laboratory processing

During this study, we sampled a total of 72 small and

medium rivers from 27 river basins in Coastal Galicia.

Sampling was conducted in two seasons: summer

2002–2003 and spring 2004, giving a total of 144

samples. Environmental factors, such as water temper-

ature (�C), pH, dissolved oxygen (mg l-1) and electri-

cal conductivity (lS cm-1) were measured in situ using

portable meters. Temperature and oxygen were

measured with a WTW Oxi 197 oxymeter, conductiv-

ity with an Orion Model 115 corrected for 25�C, and

pH with a Thermo Orion 290?. Water samples for

chemical analyses were collected into polypropylene

bottles and transported chilled to the laboratory.

Standard methods for chemical water analysis were

carried out following American Public Health Asso-

ciation (APHA) (1989): BOD5 with oxitop WTW

after incubation for 5 days at 20�C; alkalinity by the

potentiometric method, nitrates (NO3-), silica (SiO2),

phosphates (PO43-) using an auto-analyzer for nutri-

ents (Auto-Analyzer 3, Bran ? Luebbe, Germany),

ions, such as calcium (Ca2?), iron (Fe2?), magnesium

(Mg2?), potassium (K?), sodium (Na?) with a

spectrophotometer of masses, and chlorides (Cl-)

and sulphates (SO42-) with Inductively Coupled

Plasma-Mass Spectrophotometry (ICP-MS).

Three rocks were selected at random from each site

and their upper surface divided into two halves,

Fig. 1 Localization and

distribution of the 72 sites

sampled in this study

Table 1 Types of rivers found in Galicia coastal area in the system A and B classification and the number of reference sites and total

sites

System A Number of sites System B

Size catchment area Altitude (m) Total References Size catchment area Altitude (m)

Small \200 16 0 Small and Medium (10–1000 km2) \200; 200–800; [800

Small 200–800 31 5

Small [800 4 4

Medium 200–800 11 0

Medium \200 10 0

Large \200 0 0 Large (1000–10000 km2) \200

Hydrobiologia (2010) 644:371–384 373

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providing a total of six replicate samples of approx-

imately 20–60 cm2 each. Samples of periphyton were

taken from the upper surfaces of each rock removed

by brushing with a toothbrush and rinsing with

distilled water. They were stored in ice, kept in

darkness and transported to the laboratory. Three

samples were used for analysis of chlorophyll a (chl a)

and the other three were used to estimate periphyton

biomass as ash-free dry mass (AFDM). The periph-

yton samples were filtered through Watman GF/C

glass fibber-filters, and the chl a concentration was

extracted with acetone (90%) for 48 h at 4�C, kept in

the dark and measured using a Hitachi Model U-2001

UV/Visible Spectrophotometer. Values were cor-

rected for degradation products using the equations

given by Lorenzen (1967). Samples for AFDM were

filtered through pre-ashed and weighed glass-fiber

filters, dried to constant mass at 105�C for 24 h, and

reweighed. Filters were then placed in a muffle

furnace at 505�C for 1.5 h to estimate the AFDM.

Organic mass lost during combustion was determined

as the difference between initial and ash masses

(American Public Health Association (APHA) 1989).

Epilithic diatoms were collected from stones

following the European standard (CEN, 2003) with

a small toothbrush. Immediately after collection,

the diatom samples were fixed with formaldehyde

(4% v). Diatom samples were digested by following

the procedure of Renberg (1990), and permanent

slide mounts were prepared for each sample using the

high-resolution mountant Naphrax�. Diatoms were

observed and identified at the lowest taxonomic level

possible using a light microscope (Olympus BX40);

and a minimum of 400 diatom valves were counted

on each slide. The identification and the nomencla-

ture were based on Krammer & Lange-Bertalot

(1986–1991).

Analysis of the diatom communities

Diatom abundance data in this study was log-

transformed (x ? 1), to give more weight to large

species that are often found at low relative abundance

in benthic diatom communities and which can be

important for defining assemblages (ter Braak &

Verdonschot, 1995; Snoeijs et al., 2002; Tison et al.,

2005). The data were analyzed using the program

Primer 6 (Plymouth Marine Laboratory, UK, 2001).

Two analyses were performed: (i) a SIMPER analysis

(SIMilarity PERcentage) to estimate the degree of

similarity between the reference and non-reference

samples, and (ii) a Non-metric MultiDimensional

Scaling (NMDS), based in the Bray–Curtis similarity

index to examine patterns of community composi-

tion. The SIMPER indicated the individual contribu-

tion and the importance of each taxon to the global

similarity between sites by considering the frequency

and the abundance of each taxon. This analysis

allowed us to identify the reference taxa that char-

acterized the community of reference sites.

Candidate indices and metrics

Calculation

The diatom abundance data was used to calculate 17

biological indices and metrics (Table 2). The soft-

ware OMNIDIA v.3.6 (Lecointe et al., 1993) was

used to calculate the first 13 diatom indices listed in

Table 2. Each index differed in the number of species

that were used and in the constant values (tolerance

values) that have been evaluated for ecological

relevance from the compiled literature information

(Prygiel & Coste, 1993, van Dam et al., 1994).

The other four metrics were calculated for each

sample in an excel spreadsheet: (a) relative abun-

dance (ABSS) and richness (FSS) of reference taxa

and (b) the ratio of reference taxa to the total taxa

expressed as percentages of abundance (PABSS) and

richness (PFSS).

Selection

Diatom indices and metrics representing ecologically

relevant aspects of the assemblage, and responding to

the targeted stressors tested were considered as

potential metrics to combine in a multimetric index.

The selection of indices and metrics followed the

procedure described by Barbour et al. (1999) with

some modifications as follows:

(i) Assessing redundancy: this method identifies

pairs of metrics with significant Spearman rank

correlations. If a pair had a correlation coeffi-

cient greater than [0.7, one of the two metrics

was excluded from further analyses. We used

r = 0.77 as a limit following Ofenbock et al.

(2004).

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(ii) Relation with the physicochemical variables:

correlations were used to establish the relation-

ship of the different indices and metrics with the

physicochemical variables. Those that did not

correlate with the physicochemical variables,

and did not sufficiently discriminate among

sites of different condition were eliminated. The

remaining metrics, which were used in further

analysis, were termed ‘candidate metrics’.

(iii) Estimating discrimination efficiency: as the

percentage of samples with metric values lower

than the P25 of reference values for decreasing

metrics, and higher than the P75 for increasing

metrics, respectively (Ofenbock et al., 2004).

Discriminatory efficiencies were calculated to

evaluate the most suitable indices by visualis-

ing each metric’s distribution between the

reference and the non-reference group.

Results

Typology and reference community

The geology in the Galician coastal area is homoge-

neously siliceous, thus, the a priori ‘system A’

provided six types of rivers based on catchment area

and altitude (Table 1). A total of 51 small rivers and

21 medium rivers were sampled in this study. The

most abundant were the small rivers at mid-altitudes

(Table 1), but only nine sites were designated as

‘reference sites’ according to the criteria established

by CB-GIG. These reference sites were located in

mountainous areas of coastal Galicia area, and

corresponded to small rivers: four of these were

located at an altitude greater than 800 m, and five at

mid-altitudes from 200 to 800 m (Table 1). We could

not find sites that achieved these criteria in medium

rivers and in small rivers at low altitudes.

Diatom samples from these reference sites, along

with sites not influenced by pressures (having less

than 50% of agriculture in their basin), a total of 56

samples, were analyzed for two purposes:

(i) The SIMPER routine compared both groups

‘reference’ and ‘non-reference’ groups. The

within-group percentage of similarity in the

references was 50.14, and in the non-references,

was 35.57%. Indeed, the reference group was

characterized by nine species that contributed

[90% to characterize the diatom reference

assemblage (Tables 3 and 4). This group was

dominated by the genus Eunotia with five taxa:

Eunotia exigua, E. minor, E. subarcuatoides,

E. intermedia and E. paludosa Grunow var.

Table 2 Diatom indices

and metrics tested in this

study

Metrics

CEE Commission for Economical Community metric (Descy & Coste, 1991)

DESCY Descy’s pollution metric (1979)

EPID Pollution metric based on diatoms (Dell’Uomo, 1996)

IBD Biological Diatom Index (Prygiel & Coste, 1999)

IDG Generic Diatom Index (Coste & Ayphassorho, 1991)

IDAP Indice Diatomique Artois Pircardie (Prygiel et al., 1996; Lecointe et al., 2003)

IPS Specific pollution sensitivity index (Cemagref, 1982)

L&M Leclercq & Maquet‘s pollution index (1987)

ROOT Trophic metric (Rott et al., 1999)

SHE Steinber & Schiefele trophic metric (1988)

SLAD Sladecek’s pollution index (Sladecek, 1986)

TDI Trophic Diatom Index (Kelly & Whitton, 1995)

WAT Watanabe et al. pollution metric (Watanabe et al., 1986; Lecointe et al., 2003)

ABSS Abundance of reference taxa (Present study)

PSS Richness of reference taxa (Present study)

PABSS Percentage abundance of reference taxa (Present study)

PFSS Percentage of richness of reference taxa (Present study)

Hydrobiologia (2010) 644:371–384 375

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paludosa, together with Achnanthidium minu-

tissimum, Navicula angusta, Peronia fibula and

Surirella roba. These nine species plus other

nine taxa, more abundant in reference sites than

in non-reference sites (Table 4), were considered

to be the core of the reference assemblage and

their presence and abundance (Table 2) were

used to build the new metrics based on the

reference community. Although, the dissimilar-

ity of two groups had a value of 60.62%, the

nine taxa of the reference community appeared

as well in the non-reference group, but at lower

percentages (Table 3).

(ii) Non-metric MultiDimensional Scaling (NMDS)

ordination plot (stress 0.17), based on the

samples of diatom communities, revealed spa-

tially the similarity between reference samples

in relation with some of the non-reference

samples (Fig. 2).

Metric selection

We compared indices/metrics based on their correla-

tions, discriminatory efficiency and response to the

physicochemical variables: (1) The indices EPID and

CEE were excluded due to their high correlation

coefficient with other indices, such as IPS and SLAD;

(2) IDAP and WAT were excluded because they

were not significantly correlated with the physical–

chemical variables (Table 5); (3) For the rest of indices,

we selected those with discrimination efficiencies higher

than 50%: IDG, IPS, L&M, SHE, SLAD and TDI

(Table 5); (4) From the four metrics based on

reference taxa, PFSS and PABSS were selected due

to their higher discriminatory efficiency, 77.78 and

84.13%, respectively (Table 5).

We mainly considered indices and metrics for the

multimetric construction, but values of Chl a were

also analyzed and related to indices/metrics

(Table 5). The Chl a was inversely related to diatom

multimetric index (MDIAT) indicating a significant

increase in biomass with lower values of MDIAT and

consequently higher in nutrients.

Generating a multimetric index

Finally, these six indices and two metrics were

combined in a multimetric, the MDIAT. All selected

indices, calculated with the Omnidia software, had

values from 0 to 20, while the metrics PFSS and

PABSS ranged from 0 to more than 1 with values

decreasing with increased degradation (Table 5). The

TDI index is an exception, with values 0–100 and

positive correlations in pressure variables, for this

reason, this index had to be inverted. We rescaled each

of the eight metrics, transforming them by dividing

each individual value by the median value of the

reference population, so that each index ranged from

0 to [1. The eight rescaled values were summed to

the MDIAT. Subsequently, the Ecological ratio

(EQR_MDIAT) was calculated by dividing each value

of the multimetric by the MDIAT median value of the

reference data, the expected value without significant

human influence. The ranges of values obtained for the

selected indices/metrics, MDIAT and EQR_MDIAT

in reference sites and the rest of samples from Galician

coastal area are summarized in Table 6. The values of

the MDIAT ranged from 2.804 to 8.819 and EQRs

from 0.349 to 1.096 (Table 6).

Classification system of the ecological status

Diatom multimetric index (MDIAT) had the highest

discriminatory efficiency and highest correlations

with the physical–chemical variables of all indices

and metrics tested (Table 5). Initially, the EQR values

were subdivided into five classifications according to

its status: High, Good, Moderate, Poor and Bad, using

the P25 of the reference values as the limit between

High and Good. The remaining EQR values from the

Fig. 2 Ordination (MDS) of the diatom community of

reference (ref) and some of no reference sites (no ref), all of

them with percentages of agriculture \50%

376 Hydrobiologia (2010) 644:371–384

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P25 EQR to 0 were divided into four equal intervals

corresponding to the remaining classifications

(Table 7). Minor adjustments to these boundary

values were made during the European Intercalibra-

tion Exercise, and the final values were 0.93, 0.70,

0.50 and 0.25, respectively (Table 7). Figure 3 shows

the ranges of the measured physicochemical variables,

percentage of agriculture, MDIAT and EQR_MDIAT

for each of the status classifications (High, Good,

Moderate and Poor) defined by MDIAT.

The relationship between the EQR_MDIAT and

some of its component metrics is shown in Fig. 4.

The other component metrics have similar curves in

relation to the EQR_MDIAT, but the IDG had the

highest correlation with the MDIAT (R2 [ 0.7).

Figure 4 represents the metrics tendency within the

EQR_MDIAT. All metrics tend to decrease with

increasing degradation, except for the TDI that

diminishes with higher values. According to the

shape of the curves, the two metrics built from the

reference community are more sensitive to low levels

of pressures than the other indices with a more

conservative tendency, such as the NIDG. NPFSS

had the same tendency as NPABSS, but only the

Table 3 Percentage of

contribution and

accumulative percentage of

the diatom species that

characterized reference and

no reference groups

(resulted by SIMPER)

Species Reference group No Reference group

% Contribution %A Contribution % Contribution %A Contribution

ADMI 34.38 34.38 25.55 25.55

ESUB 21.24 55.61 14.6 40.14

SRBA 14.71 70.32 9.79 49.93

EUIN 8.87 79.19 6.58 56.51

EEXI 3.28 82.46 2.07

PFIB 2.95 85.41 1.99

NAAN 2.36 87.77 2.44

EPAL 2.09 89.86 1.52

EMIN 1.95 91.81 3.13

Table 4 Codes and names

of the 18 reference taxa for

coastal Galician rivers

* Taxa that characterized

the reference community

until 90%

Code Reference taxa

ADMI *Achnanthidium minutissimum (Kutzing) Czarnecki

BBRE Brachysira brebissonii Ross spp. brebissonii

DLAE Diadesmis laevissima (Cleve) Mann

EAQL Encyonopsis aequalis (W.Smith) Krammer

EBIL Eunotia bilunaris (Ehrenberg) Mills var. bilunaris

EEXI *Eunotia exigua (Brebisson ex Kutzing) Rabenhorst

EINC Eunotia incisa Gregory var. incisa

EMIN *Eunotia minor (Kutzing) Grunow

EPUN Eunotia pectinalis (Kutzing) Rabenhorst var. undulata (Ralfs) Rabenhorst

ESUB *Eunotia subarcuatoides Alles, Norpell & Lange-Bertalot

EUIN *Eunotia intermedia (Krasske ex Hustedt) Norpel & Lange-Bertalot

EUPA *Eunotia paludosa Grunow var. paludosa

GGRA Gomphonema gracile Ehrenberg

NAAN *Navicula angusta Grunow

PFIB *Peronia fibula (Brebisson ex Kutzing) Ross

PCHL Psammothidium chlidanos (Hohn & Hellerman) Lange-Bertalot

SLIN Surirella linearis W. M. Smith

SRBA *Surirella roba Leclercq

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former was represented in the figure. We defined the

centre of good status as the point at which the NPFSS

and TDI crossed, whilst the TDI and NIDG crossed

on the moderate class. The NIDG and NPFSS

crossing near the value of one represent the centre

of the high classification (Fig. 4).

The ecological improvement of the MDIAT

Having established the MDIAT using existing con-

cepts for classification systems, we aimed subse-

quently to develop and simplify this. In particular, an

important variable that we had estimated, Chl a, was

Table 5 Significative Spearman correlations among indices/

metrics and physico-chemical variables and percentage of

discriminatory efficiency and the Spearman correlation

coefficients (n = 144) with some of physicochemical variables

(** significative correlation at the level 0.01; * significative

correlation at the level 0.05)

NO3- SiO2 SO4

2- PO43- BDO5 Chl a AFDM PH Alkalinity % Discriminatory

efficiency

IDAP -0.073 -0.098 -0.153 -0.102 -0.006 -0.173 -0.169 0.095 -0.103 15.38

EPID -0.172* -0.494** -0.203* -0.134 -0.380** -0.235** -0.183* -0.252** -0.103 59.52

ROTT -0.237** -0.469** 0.018 -0.351** -0.148 0.099 -0.014 -0.537 -0.231** 33.33

IBD -0.210* -0.569** -0.326** -0.261** -0.380** -0.207* -0.338** -0.338** -0.190* 47.62

WAT -0.099 0.173* -0.081 -0.023 -0.122 0.013 -0.106 0.293** 0.058 42.86

DES -0.199* -0.516** -0.154 -0.290** -0.353** -0.179* -0.164* -0.472** -0.192* 44.44

CEE -0.265** -0.607** -0.364** -0.365** -0.425** -0.337** -0.380** -0.256** -0.226* 72.73

IDG -0.323** -0.530** -0.170* -0.319** -0.360** -0.362** -0.321** -0.412** -0.252** 78.57

IPS -0.242** -0.515** -0.239** -0.232** -0.461** -0.285** -0.349** -0.237** -0.101 57.94

L&M -0.335** -0.594** -0.263** -0.364** -0.393** -0.195* -0.286** -0.474** -0.281** 59.52

SHE -0.372** -0.551** -0.265** -0.483** -0.288** -0.267** -0.288** -0.288** -0.274** 77.78

SLAD -0.233** -0.684** -0.260** -0.265** -0.466** -0.341** -0.378** -0.459** -0.195* 65.87

TDI 0.381** 0.741** 0.201* 0.440** 0.440** 0.384** 0.413** 0.560** 0.336** 90.48

PABSS -0.388** -0.673** -0.297** -0.447** -0.386** -0.316** -0.390** -0.511** -0.287** 84.13

PFSS -0.413** -0.734** -0.279** -0.481** -0.419** -0.194* -0.373** -0.595** 0.322** 77.78

MDIAT -0.411** -0.722** -0.292** -0.478** -0.474** -0.302** -0.411** -0.527** -0.315** 93.65

Nitrates (NO3-), silicon dioxide (SiO2), sulphates (SO4

2-), phosphates (PO43-), biological demand of oxygen (BDO5), chlorophyll a

(chl a), biomass as ash-free dry mass (AFDM), pH and alkalinity

Table 6 Minimum, maximum, mean and standard error (SE) of the selected metrics, MDIAT and EQR_MDIAT of reference sites

and the rest of samples

Reference sites All sites

Minimum Maximum Mean SE Minimum Maximum Mean SE

MDIAT 7.250 8.600 7.920 0.091 2.804 8.819 5.811 0.110

EQR_MDIAT 0.900 1.070 0.980 0.011 0.349 1.096 0.722 0.010

SHE 16.50 17.20 16.85 0.03 4.20 17.80 11.00 0.22

SLAD 14.90 16.90 15.90 0.14 11.20 18.50 14.85 0.14

IDG 16.10 18.10 17.10 0.12 9.60 19.00 14.30 1.15

TDI 1.90 30.40 16.15 2.29 0.10 96.90 48.50 2.21

IPS 17.70 20.00 18.85 0.15 7.30 20.00 13.65 0.21

L&M 15.10 16.50 15.80 0.10 1.50 17.60 9.55 1.16

PABSS 0.57 1.00 0.78 0.02 0.00 1.00 0.50 0.03

PFSS 0.36 1.00 0.68 0.05 0.00 1.00 0.50 0.02

378 Hydrobiologia (2010) 644:371–384

123

missing from the classification. For this reason, we

decided to build a new, simpler multimetric, MDIATa

that included Chl a along with a trophic index (TDI),

and PABSS, one of the new metrics calculated with

the reference community. We then compared the

results from the MDIAT and the MDIATa. The Chl a

had to be inverted due to its increase with stream

nutrients/organic degradation. The three metrics were

averaged to produce the MDIATa. The interpretation

of the change of the metrics along a degradation

gradient evaluated by the MDIATa can be seen in

Fig. 5. The crossing between metrics is an indicator of

the consistency with the limits already established, 0.7

for the High/Good, and 0.5 for the Good/Moderate. It

is interesting to note that Chl a only increases below

the moderate class boundary. The lineal regression

between the MDIAT_EQR and the MDIATa_EQR

was significant (P \ 0.01) with R2 = 0.90.

Discussion

The main objective of this study was to analyze

different diatom metrics, to check their response to

organic/nutrient pressures, and to develop a classifi-

cation system for the evaluation of ecological status,

based on diatoms, for small and medium rivers. For

these rivers, we identified a spatial network of

reference sites [minimally disturbed according to

Stoddard et al. (2006)], that fulfilled the reference

criteria that has been widely applied across Europe in

the intercalibration exercise (Wallin et al., 2005; van

de Bund, 2009). Defining reference sites is a very

important step in this study (WFD, Annex V). The

reference criteria applied in Galicia has been agreed

to and intercalibrated (Central/Baltic Rivers GIG),

and it has been included in the first phase of

intercalibration (van de Bund, 2009).

The diatom reference assemblages found in small

and medium Galician rivers showed that a high

degree of similarity independent of altitude. The

reference group showed more than 50% similarity,

whilst the non-reference group was more diverse. The

nine taxa that characterized the reference assemblage

also appeared in sites with low levels of disturbance,

but at lower relative abundances. This assemblage

characterizes sites under minimally disturbed condi-

tions, but some species can appear in disturbed sites

in this area. We considered this assemblage to be very

sensitive because they tend to disappear with increas-

ing levels of human disturbance. The reference

community was dominated by five species of the

genus Eunotia: E. exigua, E. minor, E. subarcuato-

ides, E. intermedia and E. paludosa var. paludosa, a

genus that is usually well-represented and character-

istic of acidic waters (van Dam et al., 1994; De

Nicola, 2000; Sala et al., 2002), such as the waters of

Galician rivers.

The existing differences in the water quality of the

studied Galician rivers corresponded to changes in the

diatom assemblages and consequently to the values of

diatom indices and metrics. Diatom indices have been

shown to be one of the most effective tools for

evaluating ecological status in European rivers

(Eloranta & Soininen, 2002; Kelly et al., 2008). The

response to pressures that the diatom indices provided

when applied to small and medium Galician rivers was

very weak and not able to discriminate low enrichment

levels, and for this reason, it was necessary to develop

new metrics, using the conceptual framework pro-

posed by the WFD of characterizing the reference

community. The new metrics based on the diatom

reference community for Galician rivers were more

sensitive indicators than the diatom indices used and

developed for other geographical areas of Europe

(Kelly, 1998; Kelly & Whitton, 1998; Prygiel et al.,

2002). Even though diatoms species are widely

distributed across regions, there is a regional constraint

as local test datasets may not include the whole

spectrum of the taxa’s autoecology that exist across

regions. Our observations are consistent with findings

from studies that show that some diatom indices

developed in certain parts of Europe are not effective

when they are used in other areas of the same continent

(Pipp, 2002; Rott et al., 2003); for this reason, we also

used the new metrics based in the reference commu-

nity. Galician coastal rivers are, in general, systems

Table 7 Boundaries between the different status classes in the

EQR_MDIAT sensu P25 of our values and sensu the Intercal-

ibration European Exercise

Boundaries P25 EQR_MDIAT Intercalibration

European Exercise

High/Good 0.960 0.930

Good/Moderate 0.720 0.700

Moderate/Poor 0.480 0.500

Poor/Bad 0.240 0.250

Hydrobiologia (2010) 644:371–384 379

123

characterized by low levels of nutrients and low

conductivity. This oligotrophic character may be the

reason for the absence of significant responses from

indices derived in other, more nutrient-rich regions.

Multimetric Indices can be easily interpreted, a

fact which is regarded as a main advantage of this

type of classification systems, but European countries

have little experiences with these multimetrics

(Hering et al., 2006b). We demonstrated that the

metrics selected for the multimetric satisfied three

important requirements: (i) They were not strongly

correlated with other metrics (Fore & Grafe, 2002);

(ii) They responded to disturbance in the predicted

ecological direction and magnitude; and (iii) They

Fig. 3 Box and whisker

plots of different

physicochemical variables

and percentages of

agriculture, for the different

class that we found in

Coastal Galician Rivers

380 Hydrobiologia (2010) 644:371–384

123

were significantly associated with water physico-

chemical variables. Therefore, the MDIAT consti-

tutes a good tool to evaluate the ecological status of

Galician coastal rivers. The multimetric values were

better correlated with the physicochemical variables

than with the individual indices, integrating the

effects of the pressures studied (organic pollution

enrichment and eutrophication). The values of MDI-

AT have been converted into EQR values, computed

from the observed and the expected status value

(Kelly et al., 2008) giving a value for each river in

terms of their deviation from the biota expected at

the reference state. We used the limits of the last

intercalibration exercise, and checked that the limits

between the different status classifications that arose

in this exercise corresponded with significant rele-

vant metric crossing and ecological interpretation.

The crossing between the trophic index (TDI) and

the normalized metric that represented the reference

community (NPFSS) indicates that community

changes due to nutrient enrichment decreased the

abundance and richness of sensitive species, the

crossing of their curves representing the middle of

the Good classification. We confirmed that the

crossing between NIDG and TDI was on the

Moderate classification. The evaluation resulting

from our multimetric indicates that none of the study

sites were under the Bad classification, and that 69%

of the studied sites achieved the Good ecological

status according to the diatoms in this area.

The classification provided by the MDIAT in

Galician rivers has been intercalibrated at the Euro-

pean level under the first phase of the exercise of

intercalibration with other countries (Kelly et al.,

2009), showing a good correlation with the intercal-

ibration common metric (composed of IPS and Rott’s

TI). However, the MDIAT has a complex composi-

tion that includes newly developed metrics which are

more sensitive and specific for Galician oligotrophic

waters than the intercalibration indices. The EQR

provided by the MDIATa is highly related to the

MDIAT, indicating a high agreement of classification

results between both multimetrics.

The new MDIATa also includes Chl a, a good

indicator of the trophic status of streams (Dodds,

2006). Granitic rivers are known to be poor in

nutrients, and they usually have developed riparian

areas that shade the channel, conditions that increase

the probability for nutrient and light limitation of

diatom communities (Pardo & Alvarez, 2006). The

fact that we only perceive an increase in Chl a from

values prevailing under reference sites after the

moderate classification seems indicative of a diffi-

culty to build biomass even though nutrient levels

increase.

Previous studies have applied diatom indices to

Galician rivers (Ector, 1992; Penalta & Lopez, 2007)

without an analysis of pressures or suitability to

Galician waters. This study represents the first

extensive research on the application of diatom

indices and composition metrics to Galician rivers.

Fig. 4 Bivariate graphic between normalized IDG (NIDG),

normalized PFSS (NPFSS), inverted TDI (TDI/100) and the

EQR_MDIAT

Fig. 5 Bivariate graphic between standardized Chl a (SChl a),

normalized PABSS (NPABSS), inverted TDI (TDI/100) and

the EQR_MDIATa

Hydrobiologia (2010) 644:371–384 381

123

However, further studies should be done when more

data are available to ascertain whether MDIAT may

be improved and, ideally, further simplified. Our

research has confirmed that the application of the

multimetric diatom indicator system is a valuable tool

to classify the ecological status in Galician rivers

because it integrates the effects of stressors on

different indicators and components of the diatom

community.

Acknowledgements This article complemented some of the

results obtained by a project dealing with the application of

the Water Framework Directive in Galician coastal area. The

financial support for this study has been provided by Augas de

Galicia (Xunta de Galicia, Spain), and this also included the

support of the University of Vigo (Spain). We are grateful to

the editor and the reviewers for their criticism and comments

that improved the final manuscript. We thank M. Kelly for

improving the language and content of the article, M.H. Novais

for their commentaries, M. Dominguez for the help with the

chemical analysis and L.M. Gonzalez for the help with figures.

We also thank C. Veiga, A. Nebra, M. Arndal and Sofia for

their assistance with sample collection and for their friendship.

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