241

J. Phycol.

38,

241–248 (2002)

EFFECT OF COPPER ON ALGAL COMMUNITIES FROM OLIGOTROPHIC CALCAREOUS STREAMS

1

Helena Guasch,

2

Maria Paulsson,

3

and Sergi Sabater

Departament d’Ecologia, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, Barcelona E-08028, Spain

Two sets of experiments were done to quantify theeffects of chronic copper exposure on natural peri-phyton in a nonpolluted calcareous river. The resultsof short-term (up to 6 h exposure) experiments cor-roborated the significance of pH on copper toxicity.Copper toxicity increased when pH was reducedfrom 8.6 to 7.7, and this was related to the effect ofpH on copper speciation (free copper concentrationincreased from 0.2% to 2.3% of total copper). Longerterm experiments demonstrated that periphyton com-munities exposed to copper under pH variation

(8.2–8.6) were already affected at 10

�

g

�

L

�

1

(20–80ng

�

L

�

1

Cu

2

�

) after 12 days of exposure. Copper ex-posure caused stronger effects on structural (algalbiomass and community structure) than on func-tional (photosynthetic efficiency) parameters of peri-phyton. Changes in community composition included

the enhancement of some taxa (

Gomphonema gracile

),the inhibition of others (

Fragilaria capucina

and

Phor-midium

sp.), and the appearance of filament malfor-mations (

Mougeotia

sp.). The results of our studydemonstrated that several weeks of exposure to cop-per (10–20

�

g

�

L

�

1

) were sufficient to cause chronicchanges in the periphyton of oligotrophic calcareousrivers. This degree of copper pollution can be com-monly found in the Mediterranean region as a resultof agricultural practices and farming activities.

Key index words:

algae; calcareous; channel; copper;Mediterranean; periphyton; pH; speciation; stream;toxicity

Abbreviations:

Cu, copper; EC

50

, effective concentra-tion producing 50% inhibition; F

o

, constant fluores-cence; TOC, total organic carbon

Copper is a phytotoxic heavy metal (Rai et al.1981) that inhibits algal cultures at very low concen-trations (Garvey et al. 1991). However, the bioavail-ability of copper is conditioned by the organic and in-organic chemistry of the water body (Genter 1996).Copper toxicity is expected to be relatively low in alka-line waters because the concentration of Cu

2

�

is low-ered at high pH (Stumm and Morgan 1981). In addi-

tion, high alkalinity is usually correlated with calciumconcentration, and calcium ions offer protection againstCu

2

�

toxicity (Folson et al. 1986, Welsh et al. 2000).On the other hand, low phosphate and organic mat-ter concentrations may enhance copper toxicity (e.g.Twiss and Nalewajko 1992, Verma et al. 1993). Thiscomplex scenario points out the need for field studiesto elucidate the effects of copper under natural con-ditions.

Field studies on copper toxicity are scarce, andthey have been done in acidic or poorly mineralizedlotic systems in temperate zones (Leland and Carter1984, 1985). Studies performed in calcareous zones

are even more rare (Weber and McFarland 1981), andnone of them has evaluated the effects of low copperlevels.

Nonpolluted calcareous rivers in the Mediterra-nean area have diverse and peculiar flora and fauna(Gasith and Resh 1999, Sabater et al. 2000). Thesesites are usually affected by non-point sources of pol-lution from agricultural and recreational activities intheir catchments. Agricultural crops in this area,mainly olive trees and vineyards, are still treated with

copper sulfate or copper oxychloride (15–20 Kg

�

ha

�

1

,Departament d’Agricultura, Generalitat de Catalunya).Dissolved copper may thus enter surface water viarunoff, and low copper levels (5–15

�

g

�

L

�

1

) are oftendetected.

Here we study the effect of chronic copper expo-sure on natural periphyton communities in a nonpol-luted calcareous river. We posed three specific ques-tions: (1) What is the lowest concentration that affectsthe community? (2) How long does it take to detectthe first effects? and (3) Are the structural or func-tional parameters more sensitive to chronic copperexposure?

materials and methods

Study site.

Based on data provided by the Agància Catalanade l’Aigua, copper concentration in the study area (NE Spain)ranged from

�

5–15

�

g

�

L

�

1

in agricultural catchments to 10–50

�

g

�

L

�

1

in industrial zones (unpublished data). The study wascarried out in the Brugent, a calcareous stream tributary of theFrancolí river, a Mediterranean river in Tarragona (NE Spain).

The Brugent is a typical limestone stream with low nutrient con-tent (Table 1).

The microbenthic community is similar to that found in un-disturbed calcareous Mediterranean streams (Guasch and Sa-bater 1994). It is dominated by diatoms (several species of

Cym-bella

,

Navicula

,

Synedra

,

Gomphonema

, and

Melosira

), green algae(mainly

Mougeotia

but also

Closterium

and

Cosmarium

), and colo-

nies of cyanobacteria, especially

Rivularia biasolettiana.

This com-munity is characterized by high potential phosphatase activity,especially on the colonies of

Rivularia

(Romaní 2000).

1

Received 14 June 2001. Accepted 10 December 2001.

2

Author for correspondence: e-mail hguasch@porthos.bio.ub.es.

3

Present address: Golder Associate AB, Anders Perssongatan 12, SE410 64 Göteborg, Sweden.

242

HELENA GUASCH ET AL.

The influence of natural variations in pH and alkalinity oncopper toxicity was first examined by short-term tests of coppertoxicity on periphyton communities from the Brugent river. Thesame community was thereafter used to follow long-term effectsof various copper concentrations. Chemical characterization ofthe site was carried out by the Agància Catalana de l’Aigua fol-lowing standard methods (APHA 1991). Nitrates were mea-sured spectrophotometrically, phosphates by the ammoniummolybdate method, total organic carbon by catalytic combus-tion, calcium concentration by EDTA titration, and total cop-per by inductivity coupling plasma (optic emission spectros-copy). Periphyton communities used in the first experimentwere allowed to colonize the surface of etched glass squares(1.4 cm

2

) in the Brugent throughout spring 1996. Two series ofsubstrata were left in the field for 2 consecutive weeks, and sam-pling was performed after colonization (6 weeks) over 2 consec-utive weeks. For each experiment, 60–80 L of stream water and100–150 colonized glass squares were sampled and transportedto the laboratory in cool boxes filled with stream water. The ex-periments were initiated

�

2 h after sample collection. For eachexperiment, 10–15 colonized substrata were placed in six chan-nels (170 cm long

�

10 cm wide). Water was recirculated froma container by means of a pump at a flow rate of 1.5 L

�

min

�

1

.Water depth in the channels ranged between 0.2 and 0.8 cm.All containers were placed in a water bath for temperature con-trol (maintained within the range of water temperature measuredin the field). Light was provided by halogen lamps giving a photonflux density of 200

�

mol photons

�

m

�

2

�

s

�

1

at the water surface.The effect of copper was assessed at pH 8.6–8.8 (high pH

treatment), pH 8.1–8.2 (intermediate pH treatment), and pH7.6–7.8 (low pH treatment). pH was reduced by continuous ad-dition of diluted H

2

SO

4

. The addition of diluted sulfuric acidensured constant pH (c.v.

�

1%) during the incubation. Thisaddition increased total sulfur by 18%–16% and 38% in the in-termediate and low pH treatments, respectively. Alkalinity de-creased by 10%–28% in the intermediate pH treatment and52% in the low pH treatment (Table 2). Copper was added ascopper chloride (copper titrisol, Merck, Darmstadt, Germany)to achieve nominal concentrations of 0, 30, 60, 120, 300, and600

�

g

�

L

�

1

. The effect of pH without copper addition was eval-uated using recirculating channels: two channels at pH 7.6–7.8,

two at pH 8.1–8.2, and two without acid. For each channel, pHand water temperature were measured every 10 min in the re-spective container and two water samples (one at time 0 andthe other after 6 h) were taken for the analysis of nutrients(phosphate, nitrate, and ammonium), alkalinity, cations andanions, and copper concentration. Copper analyses were per-formed using inductivity coupling plasma (optic emission spec-troscopy).

For each experiment, photon yield was measured after 1, 2,4, and 6 h of exposure. On three occasions (treatments 1, 2-a,and 3), the uptake of radiolabeled

14

C-HCO

3

was measured af-ter 5 h exposure for comparison. For each measurement, fivecolonized glass squares were taken per channel. At the end ofthe incubation, two colonized substrata from the control andtwo from the highest copper concentration were frozen in liq-uid nitrogen and analyzed for pigment composition with HPLCfollowing Guasch and Sabater (1995).

Photon yield of periphyton communities was measured witha mini-pulse modulation chl fluorometer (Walz Mess. und Regel-technik, Effeltrich, Germany) as described in Ivorra et al. (2000).Incorporation of

14

C was achieved following Guasch and Sabater(1998). From the concentration–effect curves generated, coppertoxicity was estimated as effective concentration producing 50%inhibition (EC

50

) of the photosynthetic activity. EC

50

values werequantified by log-linear interpolation, giving the photosyntheticactivity in copper-treated samples as a percentage of the averageactivity of controls, which was set at 100%.

For each pH treatment, estimates of copper speciation werebased on Cu-inorganic complexation. The influence of organicligands was considered negligible. This assumption was basedon the low copper complexing capacity characteristic of hardwater rivers with low dissolved organic carbon and fulvic acidproportions (Breault et al. 1996).

Estimates of the proportion of total copper that was Cu

2

�

was based on Eq. 1 and the stability constants for inorganiccomplexation (Morel 1994):

(1)

TOTCu Cu2�( ) 1 β1 OH�( ) β2 OH�( )2

β1 CO32�( ) β2 CO3

2�( )2

β1 SO42�( ) β1 Cl�( )

+ +

+ + +

+=

Table

1. Chemical characteristics of monthly sampling during 1997–1998 in the Brugent River.

Conductivity (

�

S

�

cm

�

1

)Alkalinity

(mEq

�

L

�

1

) pHTemperature

(

�

C)O

2

(% sat)

NO

3

�

(

�

mol

�

L

�

1

)PO

43

�

(

�

mol

�

L

�

1)TOC

(

�

mol

�

L

�

1

)Cu

(

�

g

�

L

�

1

)

Average 533 3.6 8.28 15.6 103 17 1.4 62 4Minimum 446 3.1 7.70 6.0 67 2.4 0.03 21 0Maximum 593 4.1 8.80 23.9 174 65 5.2 108 15c.v. (%) 7.1 14 3.5 31 26 85 121 55 101

Data provided by the Agència Catalana de l’Aigua.

Table

2. Water chemistry in the recirculating channels at high pH (8.6–8.8) treatment, intermediate pH (8.1–8.2) treatment, and

low pH (7.6–7.8) treatment.

Treatment

Alkalinity(mEq

�

L

�

1

)Ca

2

�

(mg

�

L

�

1

)S

(mg

�

L

�

1

)SRP

(

�

mol

�

L�1)

Initial Final Initial Final Initial Final Initial Final

High pH 3.65 3.62 77.0 76.5 42.8 44.0 0.07 0.07(0.05) (0.06) (2.2) (2.9) (0.8) (1.2) (0.08) (0.03)

Intermediate pH 3.29 2.95 67.1 69.2 43.8 51.7 0.07 0.065(0.19) (0.7) (0.14) (0.45) (0.05) (0.027)

Intermediate pH 2.93 2.40 66.8 65.9 47.4 55.9 0.025 0.040(0.04) (0.03) (1.0) (1.3) (0.6) (0.9) (0.024) (0.032)

Low pH 3.29 1.59 67.4 67.5 43.6 71.4 0.07 0.12(0.11) (1.6) (0.95) (0.8) (1.71) (0.07) (0.07)

Average and standard deviation (in parentheses) of alkalinity, calcium, sulfur, and soluble reactive phosphorus (SRP) at thebeginning and the end of the incubations are given. Data are the average of six samples, one from each channel.

243COPPER TOXICITY ON STREAM ALGAE

where TOTCu is the total copper.Etched glass substrata colonized in the Brugent for 6 weeks

were also used to monitor the effects of longer (16 days) cop-per exposure during autumn 1998. After colonization, 300–400substrata were randomly distributed in 12 once-through Per-spex channels as described above. The channels were locatedindoors near the study site. The water flowing through thechannels came from the stream, and water temperature andpH followed the same pattern of variation as in the field. Watertemperature ranged between 14 and 18� C and pH between 8.2and 8.7. River water was distributed to the channels using irri-gation pipes that gave a homogeneous flow of 1.5 L�min�1. Ve-locity in the channels was kept at 30 cm�s�1. Light was providedby halogen lamps (200 �mol photons�m�2�s�1) with a 12:12-hlight:dark cycle. Copper addition started after 7 days of adapta-tion for the periphyton. Copper was continuously added bymeans of peristaltic pumps (2.5 mL�min�1). The experimentfollowed an exponential design with three controls and ninecopper concentrations (0; Cu1 � 5; Cu2� 7.5; Cu3 � 10; Cu4 �15; Cu5 � 20; Cu6 � 30; Cu7 � 40; Cu8 � 60; Cu9 � 80 �g�L�1

Cu). Every 2–4 days, samples were taken for analyses. After sam-pling, the toxicant flows were adjusted to nominal concentra-tions with an error � 5%.

On days 0, 3, 6, 9, 12, and 16, five substrata from each chan-nel were randomly sampled for fluorescence measurementsand then returned to the channels. A mini-pulse modulationchl fluorometer was used to measure constant fluorescence(Fo) (in vivo fluorescence of dark-adapted cells) and photonyield of illuminated cells. Fo values were used as relative mea-surements of algal biomass. Photon yield was measured follow-ing the procedure described above. On days 0, 1, 3, 6, and 12,one glass substratum per channel was sampled and frozen foranalysis of total chl a that was used for comparison with Fo val-ues. Fo and chl a concentration showed a linear relationship foreach sampling date, but the correlation was nonlinear when alldata were plotted together (Fo � 71 � 77 � ln chl a, r 2 � 0.72,n � 50). Fo is proportional to algal biomass for a given commu-nity, but these relationships are different among algal groups.Taking into account that Fo could underestimate the effects ofcopper on algal biomass, we used temporal changes in Fo toevaluate copper toxicity because it is a fast and nondestructivemethod. Complementary biomass-related measurements wereperformed at the end of the experiment. Sampling on day 16included five substrata for the study of community composition(microscopic identification and enumeration) and pigmentcomposition (HPLC analysis). Community composition wasstudied following Sabater et al. (1998) and used to calculate rel-ative abundances and total biovolume. On each sampling occa-sion, EC50 was obtained from plots of each measured endpoints(Fo, photon yield, chl a, biovolume, or relative abundance) ver-sus measured copper concentrations.

resultsInfluence of water pH and alkalinity on short-term copper

toxicity. The addition of diluted sulfuric acid used toensure constant pH (c.v. �1%) during the incubationdid not alter photon yield of periphyton. Photon yieldincreased 16%–20% at pH 8.1–8.2 and 5%–17% at

pH 7.6–7.8. These changes were within the range ofvariation of photosynthesis in control channels (18%).HPLC analysis did not reveal any effect of short-termexposure to copper on pigment composition.

For each range of pH, EC50 values obtained with14C-uptake were similar to those obtained with photonyield. When all data were plotted together (Fig. 1), asignificant (r 2 � 0.99, n � 6) exponential correlationbetween EC50 values and pH was obtained. EC50 in-creased with pH, showing the maximum slope (EC50more than 10-fold for every 0.5 pH unit) at the highestpH (between 8.2 and 8.6).

Equilibrium constants showed that the percentageof free copper was influenced by pH (Table 3). Freecopper was 0.2% of total copper at the high pH treat-ment, 0.6%–0.8% at the intermediate pH treatment,and 2.3% at the low pH treatment. Therefore, changesin pH slightly modified copper toxicity when EC50 val-ues were converted to free copper (Fig. 1).

Long-term effects of copper on algal communities. Con-tinuous addition of copper to the nine copper treat-ments resulted in copper concentrations of 20%within nominal concentration in most treatments(Cu1 � 6; Cu2 � 9.7; Cu3 � 11.3; Cu4 � 15; Cu5 � 17;Cu6 � 28; Cu7 � 36; Cu8 � 55; Cu9 � 94 �g�L�1). Incontrol channels, traces of copper were detected insome cases (4.5 �g�L�1 on average), close to Cu1 nom-inal concentration.

The first effects of copper on periphyton (mea-sured as photon yield and Fo) were detected after 3days of exposure, with Fo being affected more strongly.The EC50 for Fo was lower than EC50 for photon yield(Table 4). The effects of copper on Fo increased untilday 12 and remained constant until day 16. The EC50based on photon yield progressively decreased untilthe end of the experiment. EC50 based on chl a analy-sis showed the same pattern as Fo but was generallylower (Table 4).

After 16 days of Cu exposure, clear effects on thecommunity composition were observed. Forty-four taxawere identified. The community was dominated by di-atoms, but there were also green algae (seven taxa)and cyanobacteria (three taxa). Copper did not affectdiversity (calculated as the index of Shannon-Wiener)or the number of species (Table 5). Copper exposureaffected the density of cells and thus their biovolume,which was significantly reduced (Tables 4 and 5). Thetotal number of cells of most taxa was reduced, buttheir relative abundance showed various patterns of

Table 3. Inorganic speciation of copper in the Brugent River at the minimum, average, and maximum pH values recorded duringthe study period.

pHCO3

(mM) [CuOH] [CuOH2] [CuCO3] [Cu(CO3)2] %Cu2� %CuOH %CuOH2 %CuCO3 %Cu(CO3)2

7.99 0.020 100.49 100.22 102.00 100.80 0.91 2.81 0.55 90.9 5.748.28 0.039 100.78 100.36 102.29 101.37 0.44 2.68 1.02 85.9 10.58.57 0.078 101.07 100.94 102.59 101.99 0.20 2.31 1.71 77.0 19.0

Concentration of the largest complexes and the percentage of the different copper species. Free ligand concentrations werecalculated from each pH value and total alkalinity (3.6 mEq�L�1) following the equations of the carbon-carbonate equilibrium.

244 HELENA GUASCH ET AL.

variation. The relative abundance of some species re-mained constant (Achnanthes minutissima, Cymbella mi-crocephala, and Navicula cryptocephala; Table 5); a fewtaxa increased, and Fragilaria capucina and Phormidiumsp. showed a marked reduction (Fig. 3). The relativeabundance of Mougeotia showed a two-phase pattern.It increased at intermediate copper treatments (15–30 �g�L�1) and decreased at higher copper exposure.Filaments of Mougeotia under high (60 �g�L�1) cop-per exposure differed in their morphology from thosedeveloping under lower copper concentrations. Morethan 80% of the cells and filaments were bent (Fig. 2).

Pigment composition of periphyton in controls wasdominated by chl a (53%–61%); the proportion of to-tal carotenoids ranged between 33% and 39%, chl bbetween 2% and 3%, chl c between 1% and 2%, andpheopigments were scarce (1%–5%). The percentageof chl a and pheopigments remained constant in

treatments �40 �g�L�1. At the highest copper con-centrations, the percentage of chl a decreased andthe percentage of pheopigments increased (Fig. 4).No change in chl b was observed between 0 and 10 �gCu�L�1, whereas an increase was observed at a copperexposure concentration of 20–40 �g�L�1 and a de-crease at 40–80 �g�L�1. The percentage of chl cshowed the opposite pattern of variation (Fig. 4).

discussionInfluence of water pH and alkalinity on short-term copper

toxicity. Our results highlight the effect of pH onshort-term copper toxicity. The effects of the reduc-tion of about one pH unit and 50% of total alkalinityon copper toxicity can be explained by copper specia-tion, because the amount of Cu2� increases at lowerpH. In fact, this manipulation caused an exponentialreduction in toxicity (increase in EC50) but also a 10-fold increase in the amount of Cu2�. Similar resultshave been reported for phytoplankton cultures at thesame range of pH (Starodub et al. 1987).

In addition to speciation, pH may also modify thebiological sensitivity of cellular surfaces (Starodub et al.1987). An increase in pH moderately decreased thetoxicity of Cu2�, whereas other studies show that pHenhances the toxicity of free copper (Campbell andStokes 1985). These authors suggest that pH reduc-tion attenuates copper toxicity due to H� competitionfor binding sites when the metal is not stronglybound. If the metal is strongly bound at the biologicalinterface (i.e. H� ineffective in competing for bindingsites), pH reduction does not affect copper toxicity.This was probably the case in our investigation. Per-iphyton communities in calcareous streams are em-bedded in a matrix of polysaccharide and calcium car-bonate that can bind copper more efficiently thansome phytoplankton cultures.

Decreased copper toxicity due to the high water al-kalinity of the Brugent River is expected, but short-term decreases in alkalinity concomitant with pH re-duction are not likely to influence copper toxicitymarkedly. The maximum change in alkalinity oc-curred between high and intermediate pH treat-ments, and these treatments showed similar coppertoxicity. In addition, pH treatments did not influence

Fig. 1. Effect of water pH on the toxicity of copper on periph-yton communities after 6 h of exposure. EC50 values based on theinhibition of 14C-HCO3 uptake (�) and photon yield (∆) andgiven as total copper (top) or free copper (bottom) concentration.

Table 4. Temporal changes in EC50 values (in �g Cu�L�1) ofphotosynthetic efficiency (photon yield), biomass (Fo, chl a, andbiovolume), and species composition (in relative abundance ofdifferent taxa) of periphyton.

Day Photon yield Fo Chl a BiovolumeFragilariacapucina

Phormidiumsp.

1 n.s. n.s. n.s. n.m. n.m. n.m.3 935 45 20 n.m. n.m. n.m.6 336 20 12 n.m. n.m. n.m.9 319 21 n.m. n.m. n.m. n.m.12 97 17 9.7 n.m. n.m. n.m.16 64 18 n.m. 16 21 6.4

n.s., not significant; n.m., not measured.

245COPPER TOXICITY ON STREAM ALGAE

calcium concentration. High alkalinity reduces toxic-ity of a variety of metals by preventing them from pass-ing through the membrane (Rai et al. 1981). This ef-fect has been observed when the concentration ofcations is changed before exposure (Garnham et al.1993) but not after exposure (Stauber and Florence1985).

Long-term effect of copper on algal communities. The ef-fects of copper were fast and occurred at low concen-tration levels. Changes in algal biomass were clear atintermediate treatments after 3 days of exposure(EC50 � 40–60 �g Cu�L�1) and were maximal (EC50 �10–17 �g Cu�L�1) after 12 days. At the end of the ex-

periment, three biomass-related parameters, Fo, chl acontent, and total biovolume, showed similar results(i.e. EC50s between 10 and 18 �g Cu�L�1). Taking intoaccount diurnal changes in pH, it may be concludedthat 12 days of exposure to 20–80 ng�L�1 Cu2� causessignificant effects on the biomass of periphyton.

Copper exposure for 16 days under variation in pHsimilar to that observed in the stream (i.e. diurnalchanges from 8.2 to 8.6) affected the structural (algalbiomass and community structure) more than the func-tional (photosynthetic efficiency) parameters of thestream algal community. Long-term exposure to inor-ganic chemical stress, such as that produced by cop-

Table 5. Community composition (relative abundance) after 16 days of exposure to various copper concentrations.

Nominal copperconcentration

(�g�L�1) 0 0 0Cu1

5Cu2

7Cu310

Cu415

Cu520

Cu630

Cu7 40

Cu860

Cu980

CyanobacteriaCalothrix sp. 1.3 3.8 8.9 3.1 6.1 7.4 5.4Chroococcus sp. 0.5 1.9 0.8 0.3 0.5 0.6 1.5Oscillatoria sp. 0.8Phormidium sp. 7.1 2.2 2.2 0.5DiatomsAchnanthes flexella 0.4 0.2 0.7Achnanthes lanceolata 0.5Achnanthes minutissima 27.6 19.8 21.2 32.8 32.4 17.2 28.9 31.2 27.4 22.7 21.3 23.5Amphora pediculus 0.2Cymbella microcephala 19.8 21.7 22.1 32.6 28.0 13.2 23.2 18.8 24.4 25.3 22.2 28.6Cymbella minuta 0.5 0.6 0.2 0.3 0.3 0.2Cymbella affinis 0.8 1.7 3.0 1.1 0.4 1.3 0.4 1.9 2.5 0.6 2.5Cymbella amphicephala 0.5Cocconeis pediculus 0.1Cocconeis placentula 0.6 0.7 0.7 2.1 1.0 2.0 0.4 1.5 2.2 3.0 3.1 1.0Cyclotella comta 0.5Denticula tenuis 0.6 0.2 0.5 0.3 0.2Diatoma vulgare 0.2Diatoma hiemale 0.3 0.2 0.4 0.1 0.8 0.8 0.6 0.2Diatoma elongatum 0.2Epithemia turgida 0.1Eunotia arcus 0.2 0.2 0.1 0.5 1.0 1.2 0.2 4.0 0.6 1.0Fragilaria capucina 17.0 11.3 17.8 8.0 5.8 21.3 7.7 3.5 7.2 2.5 2.2 4.7Fragilaria construens 0.1 0.3 1.5Fragilaria pinnata 0.1 0.2 0.4 0.3Fragilaria dilatata 0.2 0.1Gomphonema gracile 6.3 4.8 7.4 4.1 6.6 4.3 4.4 6.2 7.0 12.1 15.7 10.4Gomphonema angustatum 3.3 4.6 0.8 0.5 0.6Gomphonema angustum 0.1 2.4 1.1 1.0 1.5 1.1 1.2 0.5 0.6 0.5Gomphonema minutum 0.1 0.4 0.7 2.8 0.8 1.9 0.7 1.0 0.6 0.5Navicula cryptocephala 5.4 5.5 9.8 4.5 5.9 6.8 10.5 6.2 1.5 14.6 6.8 4.9Navicula contenta 0.3 0.5 0.7 1.0 0.3 0.5 0.2 0.2 0.2Navicula radiosa 0.2 0.2Nitzschia fonticola 0.1 0.2 0.2 0.2Nitzschia sinuata delognei 0.2 0.4 0.3Neidium iridis 0.2Rhoicosphenia abbreviata 0.1 0.2 0.1 0.2 0.5Synedra ulna 0.4 5.3 4.5 1.5 2.0 5.1 2.3 1.7 0.3 0.5Synedra acus 2.0 0.2 0.4 0.7 0.4 0.8 0.7 0.5 0.3 5.4

Green algaeCosmarium cf vexatum 0.4 1.9 2.8 1.0 1.6 3.5 1.8 4.2 1.5 3.0 2.5 3.7Mastoigloia smithii 0.1Mougeotia sp. 6.3 8.9 5.4 2.3 2.0 5.6 14.5 12.3 21.1 5.1 9.6 4.4Oedogonium sp. 3.7 7.0 0.7 1.3 3.5 0.8 1.5Phacus sp. 0.4Staurastrum sp. 0.1 0.2 0.6 0.2

Number of taxa 24 30 18 29 25 23 16 20 18 12 22 21Diversitya 2.91 3.56 3.14 2.92 3.02 3.21 2.85 3.26 2.72 2.96 3.27 3.03Biovolumeb 6.2 6.3 7.3 3.1 3.7 3 2.3 2.7 4.3 6.7 1.4 1.1

a Shannon-Wiener index.b 108 � �m3·cm�2.

246 HELENA GUASCH ET AL.

per, cause a replacement of metal-sensitive species bymetal-tolerant ones (Foster 1982, Deniseger et al. 1986,Lindstrom and Rorslett 1991). Therefore, an early re-duction of overall biomass should be followed by bio-mass recovery due to growth of pollution-tolerant spe-cies (Foster 1982, Leland and Carter 1984, Genter andLehman 2000). During the experiment, we detectedchanges in species composition and an increase in rela-tive abundance of tolerant species, but it was not longenough to allow biomass recovery. Because the physiol-ogy of tolerant species does not necessarily differ fromthat of sensitive species, changes in species compositionmay be associated with decreases in functional variables.In agreement with our results, Balczon and Pratt (1994)reported that the effects of copper on periphyton com-munity structure are about one order of magnitudehigher than the effects on community processes. In con-trast, Leland and Carter (1984, 1985) found that slightincreases in Cu stress significantly affected communitycomposition and community functional variables (i.e.autotrophic and heterotrophic production) during a1-year exposure in a natural stream.

Our results show that several weeks of exposure tolow copper concentration (10–20 �g�L�1) cause chronicchanges in the periphyton of oligotrophic calcareousrivers because they modify the composition of the com-munity. Algal community composition was affected bycopper, especially in terms of relative abundance ofcertain taxa but not in terms of percentages of groupsor species diversity. Negative effects of metal exposureon periphyton diversity are common but are usuallyrelated to high metal pollution (Foster 1982, Deni-seger et al. 1986, Sabater 2000). Although some spe-cies were stimulated, others were clearly inhibited bycopper. Phormidium disappeared completely at copperconcentrations above 7.5 �g�L�1, and so it can be re-garded as highly sensitive to copper. The disappear-ance of Phormidium coincided with a peak of Calothrixthat increased their relative abundance under somecopper exposure concentrations. Lindstrom and Rors-lett (1991) found Phormidium in sites with intermedi-ate copper and zinc concentrations (5–44 �g�L�1 Cu,5–102 �g�L�1 Zn) but not in more polluted sites (80–150 �g�L�1 Cu). Calothrix is considered a tolerant spe-

Fig. 2. Filaments of Mougeotia in the control (a) and Cu8 treatment (b–d) after 16 days of exposure. Bar, 10 �m.

247COPPER TOXICITY ON STREAM ALGAE

cies (Leland and Carter 1984). Analogously to cyano-bacteria, copper addition affected diatom species.The effects of metal pollution described by Lindstromand Rorslett (1991), Deniseger et al. (1986), and Fos-ter (1982) are in agreement with our results, althoughGenter and Lehman (2000) observed an overall re-duction of diatoms due to metal pollution. Fragilariacapucina was sensitive, whereas Gomphonema gracile in-creased under high copper (40–80 �g�L�1) exposure.Fragilaria construens is sensitive to copper according toLeland and Carter (1984), but there are no reportsabout the copper sensitivity of F. capucina. The varietyof responses to copper for the genus Gomphonemafound in this study (whereas Gomphonema gracile wasenhanced by copper, G. angustum and G. minutum wereunaffected) disagrees with reports on copper sensitiv-ity of Gomphonema spp. (Leland and Carter 1984) andGomphonema olivacea (Deniseger et al. 1986). On theother hand, a metal-tolerant species like Achnanthesminutissima (Leland and Carter 1984, Deniseger et al.1986, Genter and Lehman 2000) was not enhanced inour study. There is controversy on the effect of cop-per on the composition of some taxa of algae and cy-anobacteria, but this is not surprising. Metal adapta-tion differs between strains of the same species (Ivorra2000); therefore, descriptions of metal tolerance pro-files based on the species composition should be takenwith caution.

Copper exposure modified the algal communitystructure (in terms of relative abundance of taxa) andits morphology. Here, copper exposure at the highestconcentrations (60–80 �g�L�1) resulted in a marked

malformation of the filaments of Mougeotia sp., themost abundant green algae in the algal community ofthe Brugent. The pattern of variation of Mougeotia wasalso reflected in the analysis of pigments. Chl b was max-imal at intermediate copper exposure (15–30 �g�L�1).At high copper exposure, there were less chl b and morepheopigments, and Mougeotia showed highly curved fila-ments. The dominance of green algae in many metal-polluted rivers points to their metal resistance (Whit-ton 1970, Foster 1982, Leland and Carter 1984, Lind-strom and Rorslett 1991). However, species differ intolerance. Mougeotia parvula was found under strongmetal pollution, whereas other species of Mougeotiadominated in reference sites (Foster 1982). Mougeotiaat alkaline pH is highly tolerant to zinc, aluminum,and copper (Whitton 1970). However, these malforma-tions have not been recorded at these copper concen-trations. Therefore, they may be typical of Mougeotiastrains developing in calcareous streams.

Ecological implications. Low levels of copper (5–15�g�L�1) are commonly found in the Mediterraneanregion as a result of agricultural and farming prac-tices. Even though high pH and alkalinity decreasecopper toxicity, alkalinity does not exclude coppertoxicity, and fluctuations in pH (after natural shifts)are sufficient to render copper toxic to algal commu-nities. In addition, organic complexation is almost sup-pressed by the alkaline water chemistry. Our resultshighlight the significance of copper toxicity in non-polluted calcareous rivers. Recommended safety val-ues should therefore be adapted and taken into ac-count for the conservation of these aquatic ecosystems.In the case the nonpolluted calcareous rivers from theMediterranean region, the use of copper in agricul-tural practices or any activity (e.g. farming activities)that could release copper in their watershed shouldbe restricted to preserve their ecological integrity.

Fig. 3. Effect of copper on the relative abundance of Gom-phonema gracile, Fragilaria capucina, Calothrix, and Phormid-ium after 16 days of exposure.

Fig. 4. Effect of copper on pigment composition after 16days of exposure. Changes in relative abundance (%) or totalcarotenes (car), chl a, chl b, chl c, and pheopigments (pheop).

248 HELENA GUASCH ET AL.

Supported by the European Community project (ENV4-CT96-0298). The paper factory Gomà-Camps, located near the Bru-gent, provided its facilities for the operation and purification ofeffluents of the 12 once-through channels used for long-termcopper exposure experiments. The Serveis Científico-tècnicsde la Universitat de Barcelona offered their facilities and tech-nical help in the HPLC analysis of pigments and inductivitycoupling plasma copper analysis. The “Agència Catalana del’Aigua” supplied chemical data (including copper concentra-tion in surface water) corresponding to their monitoring area(NE Spain). We also thank E. Navarro for assistance during theexperiments.

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