Journal of Applied Phycology (2005) 17: 119–128DOI: 10.1007/s10811-005-4801-7 C© Springer 2005

Response of diatom indices to simulated water quality improvementsin a river

Frederic Rimet1,2, Henry-Michel Cauchie1, Lucien Hoffmann1 & Luc Ector1,∗1Centre de Recherche Public – Gabriel Lippmann, 41 rue du Brill, L-4422 Belvaux, Grand Duchy of Luxembourg;2Present address: Direction Regionale de l’Environnement, 19 avenue Foch, BP 60223, F-57005 Metz, France

∗Author for correspondence: e-mail: ector@lippmann.lu; fax: +352470264

Received 30 September 2004; revised and accepted 31 December 2004

Key words: bioindication, biotic indices, diatoms, epilithon, integration interval, water quality

Abstract

Various diatom indices are routinely used in European countries to monitor water quality in waterways. In orderto assess their sensitivities and their integration interval after a sudden and lasting environmental change, epilithicdiatom biofilms were transferred from several polluted rivers to an unpolluted stream. To monitor the changes ofthe index values, the biofilms were sampled in a first experiment 20 and 40 days after transfer, and in a secondexperiment 30 and 60 days after transfer. Sensitivities of the indices to the water quality improvement were assessedcalculating the differences between the index values of the reference and the transferred assemblages. Some indiceshave intermediate sensitivities (BDI, GDI, ILM, SLA), others higher sensitivities (CEE, EPI, ROT, SPI, TDI). Theintegration interval of these indices was 40–60 days. Some differences were observed between the indices, buttheir results were homogeneous when compared to those obtained with other metrics such as Bray-Curtis or Chorddistances, used to assess the difference between the transferred and the reference diatom assemblages. These othermetrics showed that even after 60 days, the transferred assemblages still differed from the reference. This underlinesthat metrics do not have the same integration intervals and do not assess the same stresses; the choice of the metricused to assess water quality is of prime importance.

Abbreviations: BDI, Biological Diatom Index; CEE, European index; EPI, Eutrophication Pollution Index usingdiatoms; GDI, Generic Diatom Index; ILM, Index of Leclercq & Maquet; ROT, Rott saprobic index; SHE, Schiefele& Schreiner index; SLA, Sladecek index; SPI, Specific Polluosensitivity Index; TDI, Trophic Diatom Index

Introduction

Diatom generation time is one of the quickest amongbioindicators of river water quality (Rott, 1991). Thesealgae divide frequently and can thus indicate rapidlya change in water quality (Stevenson & Pan, 1999).Because of these features, biotic indices based on theautecological requirements of diatoms have been de-veloped. Water managers use benthic diatoms as waterquality assessment tools alongside macroinvertebratesand fish that integrate environmental quality on longertimeframes. A number of diatom indices have been

developed in European countries to assess the biologi-cal quality of running waters. The commonest indicesare: in France, the Specific Polluosensitivity Index –SPI – (Cemagref, 1982), the Generic Diatom Index –GDI – (Rumeau & Coste, 1988) and the standardizedBiological Diatom Index – BDI – (Lenoir & Coste,1996); in Belgium the Leclercq and Maquet Index –ILM – (Leclercq & Maquet, 1987); in UK, the TrophicDiatom Index – TDI – (Kelly & Whitton, 1995); in Ger-many, the SHE index (Schiefele & Schreiner, 1991);in the Czech Republic, the Sladecek index – SLA –(Sladecek, 1986); in Italy, the Eutrophication Pollution

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Index using Diatoms – EPI – (Dell’Uomo, 2004); inAustria, the saprobic index of Rott – ROT – (Rott et al.,1997); and at the European level, the CEE index – CEE– (Descy & Coste, 1991).

Nevertheless, information on the integration intervalof benthic diatom assemblages following an improve-ment or a deterioration of the water quality is ratherunknown (Kelly & Whitton, 1995). The first objectiveof this study was to compare existing diatom indicesand other metrics, such as Bray–Curtis (Bray & Curtis,1957) and Chord (Orloci, 1967) distance measures, byassessing their sensitivity when diatom biofilms wereexposed to a sudden and lasting water quality improve-ment. The second objective was to estimate the timeneeded for different diatom indices and distance mea-sures to integrate a change in water quality. To experi-mentally simulate this water quality improvement, di-atom biofilms were transferred from polluted sites to anunpolluted site. The transferred biofilms were sampledand examined during two experiments; a first experi-ment lasting 40 days, and a second 60 days.

Material and methods

Study area

The study was carried out in Luxembourg (Figure 1),during 2002 and 2003. On the basis of its geology

Figure 1. Location of Luxembourg and of the 7 study sites. Arrowsindicate direction of the biofilm transfers. A1, A2, A3, Alzette River,At, Attert River, C: Chiers River; S, Sure River; R, Rollingerbaachstream.

and landscape, the country can be divided into twodifferent regions: the ‘Oesling’ in the north and the‘Gutland’ in the south. These regions are characterisedby different diatom assemblages (Rimet et al., 2004).In this study, seven river sites with differing water qual-ities were chosen in the ’Gutland’ region. Their phys-ical and chemical characteristics are summarized inTable 1.

Sampling site A1 (49◦42′N, 6◦27′E), an upstreamsampling site on the Alzette river (2 km from the source,4–5 m wide), is characterised by a high organic load. Itis downstream of a wastewater treatment plant. Sam-pling site A2 (49◦34′N, 6◦9′E), 19 km downstream ofA1, is 20–25 m wide. It is characterised by an importantorganic and nutrient load. Its catchment area is essen-tially urban. Sampling site A3 (49◦50′N, 6◦6′E), thefurthest downstream site on the Alzette River (65 kmfrom the source), is 30 m wide. It is characterisedby lower organic and trophic loads than A1 and A2.The Alzette basin is essentially urban and agricultural.Site At, situated on the Attert river (49◦48′N, 6◦5′E)is 35 km from the source and 15 m wide. Its organicand trophic loads are significant but lower than in theAlzette and Chiers rivers. Sampling site C (49◦33′N,5◦50′E), situated on the upper stretch of the Chiersriver, is 5 m wide and 8.5 km from the source. It ischaracterised by high organic and trophic loads. Thisriver’s basin is industrial and urban. Sampling site S(49◦43′N, 6◦30′E) is situated on the Sure river, one ofthe widest rivers in Luxembourg (35 m wide, 70 kmfrom source). Compared with A1, A2, A3, At and C,this sampling site was less polluted. Site R (49◦44′N,6◦7′E) was situated in an unpolluted stream 2–3 m wide(Rollingerbaach stream), 2.5 km far from the sourceand flowing on sandstone. Its basin is forested. It wasconsidered as a reference site for this experiment.

Experimental set-up

The geology of the ‘Gutland’ region is dominated bysandstone. To reproduce the substrate found in therivers, the transfers were performed with sandstoneblocks. Two experiments were carried out, the firstlasted for 40 days, the second for 60 days. In both ex-periments, 35 sandstone blocks (10 cm×10 cm×6 cm)were used. At each of the seven sites (A1, A2, A3, At,C, S and R), five blocks were deposited in order to al-low diatom colonisation. After 20 days, three blocksfrom each site were sampled and fixed with 4% for-mol (AFNOR, 2000). These three blocks were usedto check the homogeneity of the assemblages between

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Table 1. Physical and chemical characteristics of study sites (A1, A2, A3, At, C, S, R).

Experiment Physical andlength chemical parameters A1 A2 A3 At C S R

40-day Temperature (◦C) 14.0 20.6 19.2 17.7 18.6 21.6 14.0

Conductivity (µS cm−1) 753.0 887.7 777.0 629.0 940.3 645.7 454.3

pH 7.7 7.7 7.9 8.0 7.6 8.2 8.2

O2 (%) 18.5 58.9 97.5 87.7 23.0 106.7 106.3

Cl (mg L−1) 57.5 90.5 45.5 22.5 95.0 35.0 8.2

SO42−(mg L−1) 127.5 152.5 108.0 36.0 186.0 87.0 38.2

BOD5 (mg L−1) 18.2 13.0 5.2 2.0 18.0 2.8 3.3

NO3-N (mg L−1) 0.3 6.5 5.5 5.2 5.4 4.4 2.1

NO2-N (mg L−1) 0.114 0.750 0.254 0.110 0.397 0.038 0.005

NH4-N (mg L−1) 3.422 1.120 0.311 8.069 1.812 0.272 0.055

Ortho-P (mg L−1) 0.170 0.21 0.08 0.04 0.16 0.08 0.00

Total P (mg L−1) 0.680 0.77 0.33 0.23 0.70 0.34 0.03

60-day Temperature (◦C) 16.0 19.1 18.4 17.4 18.4 19.8 14.2

Conductivity (µS cm−1) 611.3 806.7 625.7 454.7 775.3 598.3 474.7

pH 7.7 7.6 7.6 7.5 7.5 7.9 8.3

O2 (%) 58.7 49.0 71.7 89.5 56.5 113.7 111.7

Cl (mg L−1) 43.3 78.3 43.7 23.0 84.0 37.0 15.0

SO42− (mg L−1) 104.0 142.7 93.0 34.3 131.7 97.5 38.4

BOD5 (mg L−1) 13.7 11.8 7.6 6.1 16.6 4.6 1.2

NO3-N (mg L−1) 1.2 5.0 4.8 4.5 2.4 4.3 2.0

NO2-N (mg L−1) 0.255 0.556 0.371 0.207 0.259 0.060 0.001

NH4-N (mg L−1) 1.680 1.174 0.772 0.418 2.248 0.120 0.001

Ortho-P (mg L−1) 0.12 0.18 0.09 0.08 0.16 0.06 0.01

Total P (mg L−1) 0.36 0.54 0.27 0.23 0.49 0.23 0.03

Average values during the experiments are given (data for 40-day experiment: 10/07/2002, 30/07/2002 and 19/08/2002; data for 60-dayexperiment: 26/06/2003, 26/07/2003 and 25/08/2003).

blocks on the same site. Biofilms on the upper partof the blocks were sampled with clean brushes. Thetwo other blocks in the polluted sites (A1, A2, A3,At, S and C) were transferred to the unpolluted site R(Figure 1). In the first experiment, the transfers wererealized on 10 July 2002, and the blocks were exposedfor 40 days. One of the two blocks from each pollutedsite and from the reference site (R) was sampled after20 days (30/07/2002) and after 40 days (19/08/2002).For the second experiment that lasted 60 days, the trans-fers were carried out on 26 June 2003 and the diatomcommunities were sampled after 30 days (26/07/2003)and 60 days (25/08/2003).

The diatom valves were cleaned with 40% hydro-gen peroxide to eliminate organic material and with hy-drochloric acid to dissolve carbonate. Clean frustuleswere mounted in a synthetic resin (Naphrax) and up to400 valves were identified and counted in each sample(AFNOR, 2000) by light microscopy with 1000× mag-nification. The floras of Krammer and Lange-Bertalot

(1986–1991) were used as the basis for identificationtogether with more recent bibliography.

Water temperature, dissolved oxygen, conductivityand pH were measured in the field. Water samples wereanalysed in the laboratory (APHA, 1995) for NO3

−,NO2

−, NH4+, total phosphorus, PO4

3−, Cl−, SO42−

and biological oxygen demand (BOD5).

Data analysis

The diatom indices SPI, GDI, BDI, ILM, TDI (pro-portion of resistant taxa was not used in this study),SHE, SLA, EPI, ROT and CEE were calculated usingthe OMNIDIA software (Lecointe et al., 1993). All theindices were transformed to range from 0 (or 1) to 20to be comparable.

Distances can be used to compare an assemblagefrom a reference assemblage as in the Percent ModelAffinity that compares an actual stream communitywith an ideal community (Novak & Bode, 1992).

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Distances between the transferred diatom assemblagesand the diatom assemblage of the unpolluted site Rwere calculated for each sampling date. Bray–Curtisdistance (Index of Bray-Curtis – IBC: Bray & Curtis,1957) and Chord distance (–Chord: Orloci, 1967)were used.

Results

Modifications of diatom assemblages in the biofilms

For both experiments, the dominant diatoms in the ref-erence sites were Achnanthidium biasolettianum, A.minutissimum, Amphora pediculus, and Gomphonemapumilum (Table 2). Whereas the diatom communitycomposition of the reference site R remained quite sta-ble over the duration of the two experiments, the as-semblages of the transferred sites underwent markedchanges. The assemblages of site A1 were initiallydominated by Gomphonema parvulum, E. minima, andNitzschia palea. A. minutissimum already appeared insignificant abundance after 20 days. G. parvulum re-mained at significant levels after 40 days, whereas af-ter 60 days the dominant taxa were the same as inthe reference site R. For the transferred assemblagesof site A2, the pollution-tolerant taxa Eolimna submi-nuscula, Mayamaea atomus var. permitis, and Fistulif-era saprophila were already replaced after 20 days bythe taxa dominant in the reference assemblage, i.e. G.pumilum and A. minutissimum. This was also the casefor the transferred assemblages of site A3, although theinitially dominant M. atomus var. permitis was still thedominant taxon after 40 days. For the transferred Atassemblages, the initially dominant F. saprophila wasstill very abundant after 40 days, but was negligibleafter 60 days. A. biasolettianum and A. minutissimumreplaced the dominant N. palea and G. parvulum in thefirst experiment after 40 days for site C, whereas in thesecond experiment they already were among the mostabundant taxa after 30 days.

Variation of indices during the experiments

Based on the values for the indices, the water qualityof the reference site R can be characterised as good tovery good over the two experimental periods (Table 3).Calculation of GDI, SPI, EPI, TDI and ROT indicestook into account more than 98% of the taxa, BDI andILM about 95%; SHE, CEE and SLA about 88%. Someindices did not take into account some abundant taxa(over 1% in the experiments):

EPI: P. frequentissimumCEE: F. saprophila, P. frequentissimumILM: C. placentula var. lineata, Nitzschia fonticolaSLA: F. saprophilaSHE: C. placentula var. lineata, G. pumilumROT: C. placentula var. lineata

The changes in each index value are summarizedwith box-plots (Figure 2). In order to compare the vari-ation of the different indices during the experiments,the difference between the index values of the trans-ferred assemblage and of the index value of the refer-ence site was calculated for each index at each sam-pling date. These differences are represented on Fig-ure 2a for the 40-day experiment and in Figure 2b forthe 60-day experiment. Each box represents the 25thand 75th percentile and the bar inside the box the me-dian of the calculated differences for a given index andsampling date. Three boxes are given for each index,representing respectively the differences for D0, D20,and D40 (40 day-experiment) or D0, D30 and D60 (60day-experiment). Results of Figure 2 show that a moreimportant overlapping of the box-plots of each indexwas observable for the 40-day experiment. In both ex-periments, the greatest changes in the diatom index val-ues occurred during the first 20, respectively 30 days ofthe experiments. At the end of the 40-day experiment,few indices reached the value obtained for the refer-ence (R). On the other hand, at the end of the 60-dayexperiment, most of the index values of the transferredassemblages reached the value of the reference site R.For sites A1 and C, this was already the case for mostindices after 30 days. The TDI even reached highervalues for the transferred biofilms than for those of thereference site R. The similarity between the transferredand the reference assemblages measured on the basisof Bray–Curtis and Chord distances increased duringthe course of the experiments. The maximum similaritywas observed in the 60-day experiment.

The differences between the indices at the beginningand end of the experiments (Figure 2) were greater inthe 60-day than in the 40-day experiment. Neverthe-less similar results can be seen in both experimentsconcerning the reactivity of the different indices. ThusTDI, SHE, SPI, EPI, ROT and CEE were the indiceswith the highest differences between the end and the be-ginning of the two experiments. SLA, BDI, ILM, GDIhad smaller differences between the beginning and theend of the two respective experiments; the latter indiceswere thus less sensitive to the change in water qualitythan the first group of indices.

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Table 2. Most abundant taxa in the biofilms for the 40-day experiment (upper panel) and 60-day experiment (lower panel).

Days Five most abundant taxa in biofilms (percentage in brackets)

A1 D0 GPAR (61%), EOMI (17%), NPAL (10%), ADMI (5%), FSAP (4%)

D20 EOMI (39%), GPAR (22%), ADMI (22%), NPAL (5%), FSAP (4%)

D40 GPAR (61%), ADMI (19%), ADBI (5%), EOMI (4%), ENMI (3%)

A2 D0 ESBM (25%), MAPE (25%), FSAP (18%), PLFR (8%), CPLI (5%)

D20 GPUM (19%), ADMI (12%), ADBI (10%), GPAR (8%), NTPT (7%)

D40 GPUM (33%), ADBI (14%), SPIN (9%), NTPT (8%), GPAR (7%)

A3 D0 MAPE (49%), FSAP (14%), NPAL (9%), MAAL (8%), NFON (6%)

D20 MAPE (29%), ADMI (25%), GPAR (8%), GPUM (6%), RSIN (4%)

D40 MAPE (33%), ADMI (29%), GPUM (8%), GPAR (5%), ADBI (5%)

At D0 FSAP (87%), MAPE (4%), ADMI (2%), RSIN (1%), NCTE (1%)

D20 FSAP (29%), ADMI (24%), MAPE (24%), NFON (4%), ADBI (3%)

D40 FSAP (27%), ADMI (19%), NTPT (10%), MAPE (9%), ENMI (8%)

C D0 NPAL (35%), GPAR (31%), ESBM (10%), FSAP (7%), NVEN (6%)

D20 GPAR (31%), ESBM (16%), NAMP (12%), NPAL (7%), NVEN (7%)

D40 ADBI (21%), ADMI (15%), GPAR (15%), GPUM (15%), NTPT (7%)

S D0 MAPE (33%), FSAP (13%), NINC (11%), ESBM (9%), CPLI (8%)

D20 ADMI (27%), GPUM (16%), ADBI (13%), ENMI (7%), NCTE (7%)

D40 ADBI (27%), GPUM (23%), ADMI (21%), NTPT (7%), ENMI (6%)

R D0 ADBI (89%), GPUM (3%), NCTE (2%), ADMI (1%), APED (1%)

D20 ADBI (72%), ADMI (16%), GPUM (4%), ENMI (3%), CAFF (1%)

D40 ADBI (68%), ADMI (19%), GPUM (5%), NTPT (2%), ENCM (1%)

A1 D0 GPAR (46%), EOMI (21%), NPAL (13%), NGRE (9%), NSEM (3%)

D30 ADMI (60%), ADBI (24%), GPUM (10%), ENMI (1%), NCTE (1%)

D60 ADBI (58%), ADMI (23%), GPUM (7%), APED (4%), RABB (2%)

A2 D0 FSAP (67%), ESBM (11%), GPAR (7%), MAPE (7%), EOMI (2%)

D30 ADMI (25%), GPUM (17%), MAPE (14%), ESBM (11%), ADBI (10%)D60 ADBI (35%), ADMI (18%), GPUM (13%), NTPT (8%), NCTE (5%)

A3 D0 MAPE (32%), ESBM (19%), FSAP (18%), RSIN (7%), MAAL (5%)

D30 MAPE (32%), ADMI (20%), GPUM (14%), ESBM (8%), FSAP (5%)

D60 ADMI (40%), ADBI (33%), GPUM (8%), ENMI (3%), APED (2%)

At D0 FSAP (37%), MAPE (17%), ADMI (9%), ESBM (7%), RSIN (3%)

D30 ADMI (21%), FSAP (19%), MAPE (18%), GPUM (10%), ESBM (6%)

D60 ADBI (25%), ADMI (23%), GPUM (18%), NCTE (12%), NTPT (6%)

C D0 ESBM (31%), MAPE (26%), GPAR (13%), FSAP (12%), PLFR (5%)

D30 ADMI (43%), GPUM (19%), ADBI (12%), FSAP (6%), ENMI (4%)

D60 ADBI (38%), ADMI (25%), APED (14%), GPUM (8%), RABB (4%)

R D0 ADBI (43%), ADMI (42%), GPUM (3%), ENMI (2%), APED (2%)

D30 GPUM (36%), APED (21%), ADBI (16%), ADMI (9%), ENMI (3%)

D60 ADBI (41%), APED (20%), GPUM (10%), NCTE (7%), ADMI (4%)

Blocks of site S were lost during the 60-day experiment. ADBI: A. biasolettianum (Grunow) Round et Bukhtiy., ADMI: A.minutissimum (Kutz.) Czarn., APED: A. pediculus (Kutz.) Grunow, CAFF: Cymbella affinis Kutz., CPLI: Cocconeis placentulaEhrenb. var. lineata (Ehrenb.) Van Heurck, ENCM: Encyonopsis microcephala (Grunow) Krammer, ENMI: Encyonema minutum(Hilse) D.G. Mann, EOMI: E. minima (Grunow) Lange-Bert., ESBM: E. subminuscula (Manguin) Moser, Lange-Bert. et Metzeltin,FSAP: F. saprophila (Lange-Bert. et Bonik) Lange-Bert., GPAR: G. parvulum (Kutz.) Kutz., GPUM: G. pumilum (Grunow)Reichardt et Lange-Bert., MAAL: M. atomus (Kutz.) Lange-Bert. var. alcimonica (Reichardt) Reichardt, MAPE: M. atomus var.permitis (Hust.) Lange-Bert., NAMP: Nitzschia amphibia Grunow, NCTE: Navicula cryptotenella Lange-Bert., NFON: Nitzschiafonticola Grunow, NGRE: Navicula gregaria Donkin, NINC: Nitzschia inconspicua Grunow, NPAL: N. palea (Kutz.) Smith,NSEM: Navicula seminulum Grunow, NTPT: N. tripunctata (O.F. Muller) Bory, NVEN: N. veneta Kutz., PLFR: Planothidiumfrequentissimum (Lange-Bert.) Lange-Bert., RABB: Rhoicosphenia abbreviata (C. Agardh) Lange-Bert., RSIN: Reimeria sinuata(Gregory) Kociolek et Stoermer, SPIN: Staurosira pinnata Ehrenb.

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Figure 2. Box-plots of changes in values for indices and distances after the transfer. For each index, the difference between the index valuesof the transferred assemblage and the index value of the reference site was calculated at each sampling date. These differences are representedfor the 40-day and 60-day experiments. Three boxes are given for each index: (1) corresponds to differences for D0; (2) D-20; (3) D-40. Boxesrepresent the 25th and 75th percentile and the bar in the box the median. The diatom indices are ranked by decreasing difference between thebeginning and end of the experiment for transferred assemblages.

Discussion

Sensitivity of diatom indices to water qualityimprovement

In this study, the diatom communities were exposedto a sudden and lasting improvement of the waterquality. Although all indices indicated an improve-ment of the water quality and thus tended towardsthe values obtained for the unpolluted reference site,the differences of the index values observed betweenthe transferred blocks and the reference blocks at theend of the experiments varied markedly from oneindex to another. These differences can be interpreted

as a sensitivity of the indices to a water qualitychange. Thus, indices that showed small differencesbetween the beginning and end of the experiment canbe considered as less sensitive to the water qualitychange than those showing larger differences. Someindices, such as SPI, ROT, SHE, EPI, and CEE showedrelatively large differences and thus well reflected theimprovement in water quality.

SPI and ROT were developed on large databases(respectively, 1332 and 450 samples) and include animportant number of taxa in their calculation; thus, allthe taxa of the study are integrated in the index calcula-tion (except one for ROT). SPI and ROT have been usedsuccessfully in numerous European countries (Prygiel

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Table 3. Values for indices at the beginning of the experiment, 20and 40 (D40) days for the 40-day experiment (upper panel) and after30 and 60 days for the 60-day experiment (lower panel).

Day SPI BDI CEE TDI ROT EPI SHE SLA ILM GDI

A1 0 5.4 9.4 5.0 0.8 8.2 7.8 2.6 10.9 7.1 11.2

20 8.7 12.9 7.5 4.4 12.1 8.6 6.4 12.3 9.8 11.0

40 10.5 12.5 9.9 3.0 15.1 12.5 6.1 12.6 9.0 13.4

A2 0 8.4 11.4 8.6 1.5 5.0 4.8 3.5 7.4 6.9 11.9

20 16.4 14.9 15.1 7.3 14.3 13.6 12.1 13.7 13.0 14.3

40 17.6 15.4 16.2 8.5 15.5 15.5 14.0 14.6 13.9 14.6

A3 0 7.3 12.1 6.9 1.3 6.0 5.6 3.2 7.3 6.4 9.3

20 13.8 15.0 12.8 6.8 10.3 10.9 8.9 10.8 9.4 12.5

40 14.2 15.4 12.8 7.8 10.3 10.9 9.2 10.8 9.3 13.0

At 0 6.8 12.8 12.2 0.9 3.9 4.1 2.3 10.3 6.3 6.8

20 12.6 15.2 13.4 7.1 8.4 9.0 8.0 11.1 8.2 10.7

40 15.1 15.3 15.6 7.4 10.7 10.5 10.2 13.5 10.1 12.8

C 0 2.7 7.1 3.3 1.2 4.5 5.4 2.3 7.5 5.7 7.3

20 6.5 8.3 6.1 1.1 8.7 7.0 5.4 8.9 7.5 10.3

40 15.5 15.6 13.9 7.4 14.4 13.9 11.8 14.1 12.7 13.6

S 0 10.0 12.3 8.2 2.9 7.5 6.3 5.4 9.2 7.9 10.2

20 17.7 16.3 16.0 9.8 14.5 14.8 14.9 14.6 13.3 13.5

40 19.1 17.6 17.5 11.8 15.7 16.7 16.5 15.6 14.6 15.4

R 0 19.8 20.0 18.3 14.2 17.1 17.5 16.8 17.8 15.1 17.2

20 19.8 20.0 17.9 14.9 17.0 17.5 16.8 17.5 15.2 17.3

40 19.6 20.0 17.5 14.4 16.9 17.4 16.5 17.2 15.0 16.9

A1 0 5.2 9.7 6.1 0.8 9.6 6.9 3.2 10.5 7.5 10.4

30 19.8 19.0 17.9 14.4 16.2 17.4 16.5 16.0 15.1 16.3

60 19.5 20.0 17.9 14.3 16.7 17.3 16.5 17.0 15.1 16.6

A2 0 6.0 11.5 7.3 0.2 3.8 3.9 2.0 7.3 6.0 8.6

30 15.3 15.9 14.7 8.6 12.0 11.6 10.2 12.2 10.8 13.5

60 18.7 18.2 17.5 11.6 15.7 16.5 15.9 16.0 14.4 15.1

A3 0 7.9 11.8 7.9 1.7 5.4 5.3 3.8 7.4 6.8 10.7

30 13.4 15.1 13.5 7.0 9.8 9.5 7.7 10.5 9.3 12.5

60 19.2 19.0 17.7 13.3 15.8 16.9 16.2 16.1 14.5 15.8

At 0 10.4 13.2 11.4 3.3 7.1 6.8 6.1 10.0 8.0 9.8

30 13.5 14.7 13.4 7.2 9.7 9.4 8.3 11.5 9.5 11.5

60 18.9 17.5 17.7 10.8 15.8 16.6 16.5 15.8 14.8 15.2

C 0 6.8 10.3 6.6 0.8 4.0 4.7 3.0 7.1 6.4 11.9

30 17.9 17.3 17.2 12.3 14.3 15.0 14.3 14.8 13.4 14.1

60 19.0 18.3 17.3 11.5 15.7 17.3 15.9 16.1 14.8 14.7

R 0 19.5 20.0 17.5 14.2 16.4 17.3 16.6 16.6 15.0 16.6

30 18.6 16.0 17.3 7.7 15.4 16.4 15.6 15.3 15.1 13.0

60 18.4 17.7 17.2 9.0 15.7 16.5 15.6 16.3 15.0 14.3

To compare the indices the values were transformed to range from 0to 20. Blocks of site S were lost during the 60-day experiment.

et al., 1999) and showed high correlations with the pa-rameters characterising pollution level.

The new EPI version proposed by Dell’Uomo(2004), taking into account 492 worldwide distributedtaxa, was very sensitive to the change in water quality.

This was also the case for CEE, already tested in Lux-embourg and other European countries (Prygiel et al.,1999). Its values calculated on the basis of a two-entrytable are based on typological levels encountered alonga hypothetical ecosystem. It may be difficult to applythis index to some rivers, but this was not so in ourstudy, since most taxa were included in the CEE cal-culation.

SHE showed a significant increase for the trans-ferred blocks during the experiment. This method hassuccessfully been used outside Germany, for instancein the Danube river (Dokulil et al., 1997). Neverthelessin our study, SHE probably underestimates the qualityof the polluted rivers, because its values for the pollutedsites were very often the lowest of all. Abundant taxa,present in the unpolluted site, were not included in theSHE calculation.

TDI showed the largest differences between the be-ginning and end of the 60-day experiment. The indexvalues of the transferred assemblages were even higherthan those of the reference assemblages after 60 days.This was because of the development in the transferredbiofilms of A. minutissimum, considered as a good wa-ter quality indicator in TDI, whereas it remained rarein the reference biofilms coming from the Rollinger-baach stream. The disappearance of pollution-resistanttaxa when the blocks were transferred to the referencesite released free space on the substrate, and this mayexplain the increase of A. minutissimum, which is alsoconsidered as a coloniser that quickly occupies freespace (Sabater, 2000).

On the other hand, a second group of indices (SLA,GDI, ILM, BDI) showed smaller differences betweenthe beginning and end of the experiment and thus wereless sensitive to the water quality improvement thanthe previous group of indices. For SLA, taxa presentin the polluted sites of the experiment (E. subminus-cula, M. atomus var. permitis, Nitzschia palea) haverelatively low pollution sensitivity values. To reachthe highest sensitivity values, taxa should present eco-logical preferendums corresponding to BOD5 con-centrations of 50 mg O2 L−1 (Sladecek, 1986). Suchconcentrations are never seen in Luxembourg rivers;therefore this method is probably not adapted to ourstudy area. SLA values of the polluted sites (A1,A2, A3, At and C) were more optimistic than theother indices, as already noticed by Coste (1989) andLeclercq and Maquet (1987). ILM developed fromthe SLA and suffers the same problems as the lat-ter index; 76% of the taxa have low pollution sen-sitivity values, corresponding to moderate to weak

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pollution levels. Therefore ILM values were oftenintermediate and never approached extreme values.This probably explains the low sensitivity of these twoindices.

GDI also showed a reduced variation between thebeginning and end of the experiments. As stated bythe developers of this index considering the taxa at thegenus level, the results given cannot be highly pre-cise (Rumeau & Coste, 1988). For instance, the cos-mopolitan taxa G. pumilum and G. parvulum havethe same sensitivity values and weights in GDI, al-though the first taxon is common only in oligosaprobicand oligotrophic waters and the second can be foundwith mass developments in alpha-mesosaprobic andmoderately polysaprobic waters (Krammer & Lange-Bertalot, 1986–1991). This explains the relatively re-duced sensitivity of GDI to the water quality improve-ment. Nevertheless, studies comparing performancesof several indices showed good correlations of thisgeneric index with phosphorus concentrations, biolog-ical oxygen demand and total inorganic nitrogen (Kellyet al., 1995).

BDI also showed a relatively small variation rangein our study. In this index, some morphologically sim-ilar taxa are regrouped, thus reducing the indicationaccuracy. Goma et al. (2004) noted that BDI tendedto overestimate water quality in medium and high-polluted waterways, mainly because it overvalues thequality of small Naviculaceae, such as F. saprophilaand M. atomus var. permitis. This explains the overesti-mation of the quality of moved blocks at the beginningof the experiments, when dominated by these smalltaxa.

Integration interval of diatom assemblages and bioticindices

Colonization of new substrates and establishment ofmature communities in equilibrium with the environ-ment can take from two weeks in a reservoir (USA:Hoagland et al., 1982), to one month in temperaterivers (France: Eulin & Le Cohu, 1998; USA: Oemke &Burton, 1986). Colonization rate depends among oth-ers on the nature of the surface of the substrate; it canbe slower on smooth surfaces than on rougher surfaces(Antoine & Benson-Evans, 1985). Processes that takeplace when a well-established diatom biofilm faces achange in water quality are partly different from thoseoccurring during the colonization of new substrates.Among these processes, the competition betweenspecies for already occupied space and the effects

of changing abiotic parameters on the growth rates ofthe different taxa play a crucial role.

Transfer experiments studying the changes of di-atom assemblages due to changes in water qualityare scarce. In a rather short experiment, Tolcach andGomez (2002) and Ivorra (2000) showed that after 2weeks, the assemblages of biofilms transferred froma polluted to unpolluted sites were still different. Ex-periments over a longer period (45 days) have cometo the same conclusion (Iserentant & Blancke, 1986).Transfer experiments from unpolluted to polluted sitesshowed quicker changes of the biofilms (Ivorra, 2000;Iserentant & Blancke, 1986). Our results are compara-ble with those of Iserentant and Blancke (1986), sinceafter 40 days, most of the diatom indices of the trans-ferred biofilms from most of the polluted sites were stilldifferent from those of the reference site. Only after 60days, most of the diatom index values of the transferredbiofilms approached the values of the unpolluted ref-erence site.

Our study showed that 40 to 60 days were neces-sary for the diatom indices calculated with the trans-ferred assemblages to indicate similar water qualitythan the indices calculated with the reference assem-blages. These results give an indication of the timeneeded for diatom assemblages to adapt to new wa-ter quality conditions, and thus of the time intervalnecessary for diatom indices to integrate water qualityimprovement. Even if some differences were observedbetween the different diatom indices, these results werehomogeneous when compared to results obtained usingother metrics such as Chord and Bray–Curtis distances.Bray–Curtis or Chord distances, used to assess the dif-ference between an assemblage and a reference com-munity (Novak & Bode, 1992; Barton, 1996), showedthat the transferred assemblages were still quite differ-ent after 40 and even 60 days. This was mainly becausesome taxa pointing to pollution still remained in thebiofilms with low quantities. This underlines that thesemetrics do not have the same integration interval. Thechoice of the metric to use for water quality assessmentis thus of crucial importance.

Some differences in the observed sensitivity ofthe indices depend largely on the aims for whichthey were developed, such as the assessment of over-all pollution, saprobic load or trophic level. Fur-thermore, as stated by Rott et al. (2003), the useof diatom indication methods in regions differentfrom their region of creation, without any checkof their applicability, should be avoided. The WaterFramework Directive (European Parliament & The

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Council of the European Union, 2000) strengthensthis idea by requesting the development of newbioindication tools adapted to each ecoregional andtypological river type.

Acknowledgments

The financial support of Luxembourg’s National Re-search Fund in the framework of the MODELECOTOXproject is gratefully acknowledged. Christophe Bouil-lon, Loıc Tudesque and Olivier Monnier are thankedfor their help. The anonymous reviewers are thankedfor their constructive criticism.

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