Algal growth responses to waters of contrasting tributaries of the River Dee, north-east Scotland

PII: S0043-1354(97)00450-8

Wat. Res. Vol. 32, No. 8, pp. 2471±2479, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $19.00 + 0.00

ALGAL GROWTH RESPONSES TO WATERS OF CONTRASTING TRIBUTARIES OF THE RIVER DEE, NORTH-EAST SCOTLAND M M M H. TWIST12** , A. C. EDWARDS1* and G. A. CODD2*

Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB15 8QH, Scotland, U.K. and 2 University of Dundee, Dundee DD1 4HN, Scotland, U.K.

1

(First received January 1997; accepted in revised form November 1997) AbstractÐIncreasing legislative emphasis is being placed on the use of biological indices in water quality assessments. The majority of current techniques have been developed for use in standing waters. Similar techniques are needed which provide in situ biological assessments of ¯owing waters, which should intrinsically include all of the time-integrated factors controlling algal growth. This paper presents a biomonitor which uses 3 alginate-immobilised algal species, and shows its practical use by measuring growth responses in two contrasting tributaries of the R. Dee. Growth was quanti®ed using a non-destructive, automated absorbance scan (650 nm) across the surface of nylon-supported, thin (01 mm) ®lms of alginate-immobilised algal cells. Signi®cant di€erences (P < 0.001) occurred between growth responses at the two sites, and between the responses of individual algal species. These data are compared with the results obtained from a laboratory-based, free-cell bioassay, which suggested that growth was not being limited by either nitrogen or phosphorus added at 10 and 1 mg cmÿ3 respectively. The two methods provided consistent assessments of the water quality, although for di€erent reasons; the eutrophic status of one river meant further N and P additions were not signi®cant, and the chemical properties of a second river, such as pH, may have been the growth-limiting factors. This preliminary ®eld assessment shows the alginate ®lm method is sensitive, tough and durable, and allows continual, site-speci®c, assessment of water quality. We also discuss the adaptability and potential standardisation of this bioassessment method. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐbioassay, biomonitor, immobilised, algae, Scenedesmus subspicatus

INTRODUCTION

Algae are well suited to monitor changes in the trophic status of water systems since they occur almost universally in waterbodies and are sensitive to small changes in water quality (McCormick and Cairns, 1994; Skulberg, 1995). At present, bioassessment methods for both ¯owing and standing waters commonly use either diatom community structures (Kelly et al., 1996) or colonisation of nutrient-diffusing arti®cial substrates for taxonomically-based indices (Fairchild et al., 1985). Other uses of algae as bioindicators for ¯owing waters are rare; keeping algal cells in place has been the major problem for turbulent systems such as rivers. Faafeng et al. (1994) have shown the potential of algal cells immobilised in alginate beads to monitor water quality, but the beads became unstable and cell losses became signi®cant. However, responses of immobilised algae to nutrient levels in water are rapid (photosynthetic responses can occur within minutes), sensitive and potentially simple to measure and standardise. *Author to whom all correspondence should be addressed. [E-mail: [email protected]].

European legislation (The Urban Waste Water Treatment Directive, EEC, 1991) and several federal U.S. regulations (The Clean Water Act, see Lewis, 1995, for a review) imply that in addition to commonly used animal/plant-based assessments of water quality, algal bioassays will also become required for ``sensitive'' areas. New techniques based on in situ measurements need to be developed in order to achieve this. The majority of mandates e.g. EEC (1991), quote either single concentrations for individual chemicals which can cause damage, or use poorly de®ned terms such as ``eutrophication'' in their assessment of water quality. This creates problems for the long-term management of water bodies. It is dicult to de®ne at which point a site is at risk, or has become, eutrophic. Eutrophication guidelines for lakes and reservoirs give concentration ranges of <10 and >20 mg dmÿ3 total P, and <200 and >500 mg dmÿ3 total N for oligotrophic and eutrophic status respectively (INDITE, 1994). In situ biomonitoring using algae could provide a predictive estimate of eutrophication using one of its most obvious features, algal growth. In fact, it has been reported that in the U.S.A. 12 000 lakes and 61% of surface drinking

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water supplies had already been adversely a€ected by algae by the mid-1980s (Welch and Lindell, 1992). Existing laboratory-based algal bioassays usually use ``exotic'' species, primarily Scenedesmus subspicatus Chodat, incubated under strictly controlled, optimised, conditions which are at variance with the ¯uctuating conditions usually encountered in natural environments. Therefore, the data obtained from such laboratory-based bioassays may not realistically describe responses under natural conditions (Boutin et al., 1995). Diculties also arise because the information is based on a single sampling and cannot, therefore, take account of episodic/periodic nature of individual pollution inputs, nutrient spiralling (Mulholland et al., 1984), or rapid alterations in physical and chemical properties. The present paper describes a bioassessment technique which provides growth data which include these temporally-integrated environmental parameters, and thus avoids the inherent limitations associated with laboratory-based bioassays. We also compare and contrast the growth responses of freeliving and immobilised algae to widely di€erent trophic conditions.

MATERIALS AND METHODS

Field sites Two sub-catchments, the Water of Dye (5780' N, 2833' W) and Leuchar Burn (5788' N, 2823' W), of the River Dee, north-east Scotland (Fig. 1), were selected on the basis of their contrasting chemical and physical properties (NERPB, 1993). The Water of Dye is an upland catchment, originating in the eastern Cairngorm mountains, having a land use of predominantly heather moorland and rough grazing. The Leuchar Burn drains from the Loch of Skene, which has a land use dominated by agriculture (mixed arable and livestock). Both catchments are approximately 40 km2 and drain directly into the main river channel. The soils at both sites are derived from granites and belong to the Countesswells Association (Glentworth and Muir, 1963). Free-cell cultivation and starvation Three algal species were used; the reference isolate Scenedesmus subspicatus NIVA-CHL.55 (Skulberg and Skulberg, 1990) obtained from the Norwegian Institute for Water Research, Oslo, and two native strains, originally isolated from the River Dee at a point close to where the two tributaries enter the main river. These were held in the culture collection at the University of Dundee for approximately one year prior to their use in these experiments. These were preliminarily identi®ed as Scenedesmus strain UD16, and a unicellular cyanobacterium strain UD32. Cultures were maintained in 2 dm3 of half-strength

Fig. 1. Part of the River Dee catchment areas showing the location of the ®eld sites.

Laboratory and in situ algal bioassessments modi®ed Z8 medium (Kotai, 1972), sparged with approximately 6 dm3 of sterile air per minute, with an incident photon ¯ux density of 40 mmol mÿ2 sÿ1 (white ¯uorescent light) and incubated at room temperature (238C). Prior uptake of P, and possible consequent storage, made a period of P-starvation necessary prior to experimental use. P-limited cells were obtained from the inoculation of a washed cell pellet, harvested by centrifugation (20 000  g, 15 min) from a week-old exponentially growing culture into half-strength Z8 minus P. P-limited cells, washed free of N, were used in all experiments. Laboratory-based algal bioassays Un®ltered river water, sampled mid-way through the experimental period at d 7, was used to inoculate laboratorybased bioassays of the growth responses of free cells of all 3 strains under standard conditions of 23 238C and an irradiance of 30 mmol mÿ2 sÿ1 photon density incident on the growth ¯ask surfaces, on an orbital shaker at 60 rpm. Triplicate water samples (4.8 cm3) were incubated with free-cell algal suspension (0.1 cm3) from the same batch used to produce the alginate ®lms. Z8 nutrient solution (0.1 cm3, minus N and P), was also added to provide adequate amounts of trace elements and iron. N and/or P, supplementary to that already present, was added at 10 or 1 mg cmÿ3 respectively, where speci®ed. Absorbance of the cell suspensions, using river water as a blank, was determined at 663 nm at time zero and again after ®ve days incubation. Multivariate one way analysis of variance (ANOVA) of the data was carried out using Genstat 5 (1993). Immobilisation technique A technique was developed for the immobilisation of cells which gave thin (01 mm) tough and easily handled ®lms of alginate-immobilised algae (Twist et al., 1997). Cells were suspended in aqueous sodium alginate (Sigma, derived from Macrocystis pyrifera) at room temperature to a ®nal alginate concentration of 2% (w/v) after a 1:1 dilution with cell suspension. Cultures for immobilisation were diluted until their absorbance at 650 nm in the immobilised ®lms was approximately 0.2 units to standardise biomass at zero time. The alginate and cell mixture was thinly spread onto nylon sheets (mesh size 500  500 mm, 47% open area, G. Bopp, Derbyshire), allowed to settle under gravity, and the gel hardened by ¯ooding with 5% (w/v) calcium chloride. Films identical to those described above, but without cells, were used as controls.

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N/P determinations Nitrate concentrations in the river samples were determined colorimetrically using a Technicon TRAACS autoanalyser system based upon reduction to nitrate using hydrazine. Inorganic P was determined by the colorimetric molybdenum blue method (Murphy and Riley, 1962). Growth determination of algae immobilised in ®lms Microplate reader. The spatial average absorbance across the surface of the ®lms was determined at 650 nm using an EMAX microstation plate reader (Molecular Devices, Crawley, U.K.) connected to a PC, and the data were collected using Softmax2 software. This system enabled 96 simultaneous readings from a single ®lm (11  7.5 cm) placed directly onto a 96-well plastic plate. Single factor ANOVA's were used for statistical analysis of the triplicate absorbance values for blank or cell-containing ®lms. Extraction of chlorophyll a. Approximately 1 cm2 triplicate samples were randomly cut from ®lms and stored in the dark at ÿ208C until extraction. For chlorophyll a extractions, samples were air-dried (3 h, in the dark at room temperature) and weighed. The calcium alginate gel was dissolved in 0.2 cm3 5% (w/v) sodium hexametaphosphate, the cell suspension diluted with 1.3 cm3 Z8 nutrient solution minus P, and the free cells pelleted (10 000  g, 1 min). Chlorophyll a was extracted from the pellet in 1 cm3 methanol (24 h) in the dark at room temperature, and the absorbance of the supernatant read at 663 nm (CE 594 spectrophotometer). Chlorophyll a concentrations were calculated according to MacKinney (1941) per unit weight of ®lm, and single factor ANOVA's performed on the triplicate data sets. E€ect on the growth of Scenedesmus NIVA-CHL.55 of cellulose dialysis membrane protection Six portions (11  7 cm) of alginate-immobilised, P-limited, Scenedesmus NIVA-CHL.55 ®lm were cut from one newly formed sheet. Triplicate ®lms were either enclosed in cellulose dialysis membrane (mw cut-o€; 12 to 14 kDa) or left exposed. Absorbance scans (650 nm) were made pre- and post-incubation in Z8 medium (2 dm3 for ®ve days at 258C and 100 mmol photons mÿ2 sÿ1), and single factor analysis used to compare the di€erences in the increase in absorbance between treatments.

RESULTS

Field experiments

Site characteristics

The ®eld apparatus consisted of weighted plastic baskets, with perforated sides to allow the free ¯ow of water, to which the alginate-immobilised algal ®lms contained in either nylon (as above) or dialysis membrane bags were ®xed. Two baskets were placed approximately 10 m apart per site, with water ¯owing parallel to the length of the ®lms. Triplicate ®lms of each algal strain, plus blanks, were placed in cellulose dialysis bags, mw cut-o€ 12 to 14 kDa. An additional series of blank and ScenedesmusNIVA-CHL.55-containing ®lms were placed in nylon bags. The alginate ®lms were re-hardened at each sampling time by soaking in 5% (w/v) CaCl2. The ®lms were incubated in the ®eld for two weeks at each site, during which they were collected after 2, 7 and 14 d, analysed for absorbance and chlorophyll a, and then returned to the ®eld (after around 3 h). River samples were collected simultaneously and analysed for N and P concentration. The temperature and pH were recorded at the time of sampling for each site. Continuous river discharge was recorded for the Water of Dye, and rainfall data for both sites, provided by the Scottish Environmental Protection Agency (SEPA).

The two sites contrasted over a range of chemical and physical properties. The Water of Dye was oligotrophic, acidic and had an overall higher water quality index. The Leuchar Burn drains a eutrophic loch and generally has higher pH and nutrient availability (Table 1). River ¯ow properties also differed sharply between the two sites, with the Water of Dye being ¯ashy in nature and typical of an upland river. Two spate ¯ows at the start and end of the experimental incubation were recorded, the average ¯ow over the two week period was 1.95 m3 sÿ1 and ranged between a minimum of 0.416 m3 sÿ1 and a maximum of 5.836 m3 sÿ1. The Leuchar Burn remained at a relatively constant ¯ow throughout the experimental period, due to the regulatory e€ect of the Loch of Skene. During the two week incubation period the total rainfall

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Table 1. Physical and chemical characteristics of the two sample sites during the incubation period and over the long-term (14 y period) Water of Dye Incubation period pH PAR (mmol mÿ2 sÿ1)d Water temperature (8C) ÿ3 NOÿ 3 -N (mg cm ) ÿ3 PO3ÿ 4 -P (mg cm ) WQIe BOD (mg cmÿ3)

5.6 20.1 5.3 24.0 8.3 21.8 0.19 20.05 0.09 20.08 ND ND

a

Leuchar Burn

Long-term average 6.7 (5.3±7.6) ND 8.2 (ÿ2.0±20.5) 0.31 (lod±2.60) 0.0083 (lod±0.01) 96.3 (83±100) 0.76 (0±1.6)

b

Incubation perioda

Long-term averagec

6.5 20.4 36.3 25.6 9.2 21.8 1.46 20.10 0.06 20.01 ND ND

7.9 (6.1±10) ND 9.5 (2.0±23.0) 2.8 (0.01±9) 0.056 (lod±0.036) 67 (27±89) 3.9 (0.1±40)

ND: Not determined. lod: limits of detection. a 15/09/96±30/09/96 (n = 4). b 1980±1994 (n = 82). (Source: SEPA, Aberdeen, U.K.). c 1980±1994 (n = 145). (Source: SEPA, Aberdeen, U.K.). d Photosynthetically active radiation (at depth of ®lms). e Water Quality Index % (NERPB, 1993, maximum quality = 100).

amounts were 67.8 and 42.4 mm at Water of Dye and Leuchar Burn, respectively. Laboratory-based algal bioassays Growth of all three algal strains was always greater in water from Leuchar Burn compared to that from the Water of Dye (P < 0.001, (Fig. 2)). Growth responses between algal strains were also signi®cantly di€erent (P < 0.001) at each site, although the general trend was similar. Growth changes increased in the order cyanobacterium UD32 (where decreases in absorbance implied cell death); native Scenedesmus UD16; and NIVACHL.55. Water sampled on day seven was used for the incubations and contained: 0.144/1.55 mg cmÿ3 ÿ3 NOÿ PO3ÿ 3 -N, and 0.012/0.051 mg cm 4 -P, and was at pH 5.75 and 6.1 for Water of Dye and Leuchar Burn respectively. Additional N and/or P had no in¯uence on the growth responses of free-living cells in water from either site, despite increasing the P and N concentrations for the oligotrophic Water of Dye by approximately 10 and 50-fold respectively. Table 2 shows the relative increases in the absorbance of both Scenedesmus strains incubated in Water of Dye compared to that of Leuchar Burn. These two strains reacted in a very similar manner during ®eld incubations with immobilised cells, both increased their absorbancies by around 20%. However, for the free-cell laboratory incubations, a large di€erence in the response between the two strains was evident. The native Scenedesmus strain produced an approximately 1% increase in absorbance, whereas the absorbance of the NIVACHL.55 cultures increased by nearly 60%. Growth responses of ®eld-incubated immobilised algae A highly signi®cant increase in the absorbance for each species occurred with increasing length of incubation time of the ®lms of immobilised algae placed in the Leuchar Burn (Fig. 3). The growth trend was di€erent for the Water of Dye, where absorbance values only signi®cantly increased over the total incubation time for the two Scenedesmus

strains (P = 0.021 and 0.0002 for Scenedesmus NIVA-CHL.55 and native Scenedesmus UD16, respectively), whilst there was a small, but signi®cant (P = 0.007) decrease in the absorbance values for the cyanobacterium UD32. The rate of growth response also varied with di€erences between sites becoming signi®cant at 14 (cyanobacterium strain UD 32), 7 (Scenedesmus strain UD16) and 2 d (Scenedesmus NIVA-CHL.55). The overall signi®cance factor associated with the increase in absorbance as an indicator of growth of each strain at Leuchar Burn compared to Water of Dye was P < 0.001. There was also a highly signi®cant di€erence (P < 0.001) in response between the strains placed in the Leuchar Burn. This method gave low standard errors due to the accurate estimation of the true mean absorbance value of the ®lms from 96 simultaneous readings determined using the plate reader. The coecient of determination was 3.38 2 1.13% (n = 24). These incubations were all carried out with ®lms placed in dialysis bags which were then closed. (Preliminary trials resulted in the colonisation of alginate ®lms by water snails which then grazed on the ®lms. After placing the ®lms in dialysis bags no further colonisation by grazers occurred.) All ®lms sealed in dialysis membrane bags remained intact during the 14 d ®eld exposures. Growth responses of all strains, indicated by changes in the concentration of extracted chlorophyll a (Fig. 4) were similar to those indicated by changes in absorbance at 650 nm of the intact alginate ®lms (Fig. 3). The correlation between chlorophyll a and absorbance was highly signi®cant (R2=0.87) for the Leuchar Burn and is shown in Fig. 5. There were, however, di€erences which arose at d 14, with the native Scenedesmus UD16 and Scenedesmus NIVA-CHL.55 strains incubated in the Water of Dye; consequently the R2 value was lower at 0.41 (Fig. 5), and the two outlying data points are obvious. A comparison of the relative changes in absorbance at 650 nm of free and immobilised Scenedesmus strains is shown in Table 2. Clear

Laboratory and in situ algal bioassessments

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Fig. 2. Change in absorbance at 663 nm of a laboratory-based, free-cell batch bioassay of: (A) Scenedesmus strain UD16; (B) cyanobacterium strain UD32; (C) Scenedesmus NIVA-CHL.55, in river water collected on d 7. Incubations were under standard conditions for 5 d, and spiked with N (10 mg cmÿ3) and/or P (1 mg cmÿ3). (Error bars represent standard errors of n = 3 determinations.) Table 2. Relative growth (absorbance) of free-cell laboratory and immobilised cell ®eld incubations of Scenedesmus strains using the oligotrophic Water of Dye, as a percentage of each ones respective growth under the eutrophic conditions of Leuchar Burn Growth of algae (estimated from absorbance 650 nm) incubated in the Water of Dye as a percentage of that found in the Leuchar Burn Laboratory incubations Site Scenedesmus NIVA-CHL.55 Scenedesmus UD16

Immobilised ®eld incubations

Leuchar Burn (absorbance)

Water of Dye (absorbance)

Relative increase (%)

Leuchar Burn (absorbance)

Water of Dye absorbance)

Relative increase (%)

0.137

0.080

58.4%

0.558

0.089

15.9%

0.108

0.001

1.0%

0.234

0.039

16.7%

Absorbance values are the mean of n = 3 determinations.

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Burn, means that the algal ®lms need to be protected, especially for long exposures (weeks). Cellulose dialysis membrane (pore size 12±14 kDa) and nylon bags, were tested. No signi®cant di€erences in absorbance between the two protective covers were found during the incubation for the Water of Dye, but for the Leuchar Burn, at day seven only, nylon-incubated ®lms had a signi®cantly greater absorbance than dialysis membrane incubated ®lms (P = 0.017). However, when the e€ect of dialysis membrane protection on growth was tested under laboratory conditions, in Z8 medium, the increase in absorbance (650 nm) of NIVA-

Fig. 3. Change in intact ®lm absorbance (650 nm) of: (A) Scenedesmus strain UD16; (B) cyanobacterium strain UD32; (C) Scenedesmus NIVA-CHL.55 during in situ river exposures of algae immobilised in alginate ®lms. Stars indicate signi®cance levels, *r 5%, ** r1% (single factor ANOVA). (Error bars represent standard errors of n = 3 determinations, apart from 14 d where n = 2.)

di€erences between the laboratory and ®eld responses occurred, and was particularly noticeable between the two grown in laboratory culture. In contrast, despite the di€erences in the rate of change noted between species under ®eld conditions (Fig. 3), both algae showed a very similar proportional growth depression at the Water of Dye. E€ect of dialysis membrane vs nylon bag isolation Grazing pressures by aquatic invertebrates, particularly in slower ¯owing rivers like the Leuchar

Fig. 4. Chlorophyll a levels extracted from ®lms of immobilised algae and cyanobacteria: (A) Scenedesmus strain UD16; (B) cyanobacterium strain UD32; (C) Scenedesmus NIVA-CHL.55, after in situ river exposures. Chlorophyll a was not detected in alginate-only ®lms. Stars indicate signi®cance levels, *r5%, ** r1% (single factor ANOVA). (Error bars represent standard errors of n = 3 determinations.)

Laboratory and in situ algal bioassessments

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Fig. 5. Correlation of absorbance (650 nm) of intact alginate ®lms of immobilised cells with levels of chlorophyll a extracted from ®lms for: (A) Leuchar Burn; (B) Water of Dye: Q, Scenedesmus strain UD16; W, cyanobacterium strain UD32; ., Scenedesmus NIVA-CHL.55.

CHL.55 was not signi®cantly a€ected by the presence or absence of the membrane (P = 0.218).

DISCUSSION

Three strains of freshwater phytoplankton were immobilised in thin alginate ®lms which were subsequently immersed in ¯owing waters of two streams of di€erent trophic status over a 14 d period. The rational for these trials was to determine whether microalgae and cyanobacteria, immobilised in ®lms, can be used as in situ biomonitors of water quality. With the further aim of measuring growth responses by a straightforward and non-destructive method, enabling the biomonitors to be returned to the aquatic environment for continual use, we have assessed the potential of measuring the absorbance of the ®lms using a standard microplate reader. This included a ®lter with a wavelength transmission of 650 nm, i.e. close to the absorbance maximum of chlorophyll a (663 nm in methanol). The in situ biomonitors were durable over the 14 d ®eld trials, although further work is needed before the longer-term life expectancy can be determined. The signi®cant di€erences in growth response of the immobilised algae at the two ®eld sites provides an indication of how native and exotic strains may respond to oligotrophic and eutrophic systems. These di€erences were attributed in part to the integrated e€ects of current velocity, light intensity at incubation depth, pH, temperature, and concentrations of N and P. The e€ects on algal metabolism of such complex interactions are not well understood. Film-immobilised cells gener-

ally gave patterns of growth similar to those obtained in the laboratory-based free-cell bioassays of the point- sampled waters, and similar responses to traditional growth determination methods such as chlorophyll a extractions. Di€erences between laboratory and ®eld growth responses, e.g. the cyanobacterial isolate in Leuchar Burn, probably re¯ect the ¯ow-dependent ¯ux of the available nutrient supply. The signi®cantly di€erent growth responses between each site according to the laboratory-based and in situ methods correlated with that which would be predicted from the averaged long-term chemical analysis data. Leuchar Burn, which arises from a eutrophic source, promoted a much larger growth response than did the oligotrophic Water of Dye. Surprisingly, in free-cell bioassays, additions of N and/or P did not increase growth in comparison to that supported by river water alone. Consequently the results obtained are a re¯ection of the algal growth potential, as opposed to the nutrient status, of particular water bodies. One characteristic of many upland rivers is the high organic carbon content, particularly during periods of high discharge. This could have two e€ects upon algal growth potential, ®rstly due to light penetration and secondly due to the direct toxicity of certain humic acids (Freeman et al., 1990). This demonstrates the need for in situ exposure trials in addition to chemical analysis for the assessment of water quality. It is generally assumed that P is the most likely limiting factor controlling algal growth in the aquatic environment (Foy and Withers, 1995), unusually this was not the case in these samples.

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Standard methods for the cultivation and P-limitation of phytoplankton growth, combined with the fact that alginate is a suitable matrix for the longterm (years) storage of viable algal cells (Day et al., 1987), makes possible the production and standardisation of large batches of pre-formed ®lms containing phytoplankton cells. Immobilisation combined with appropriate storage conditions also reduces the problems of expense, mutation and selection encountered in free-cell cultivation. Work has recently been carried out on Scenedesmus cell distribution within alginate ®lms, and the e€ect of immobilisation on growth responses (Twist et al., 1997). The inclusion of native algal strains isolated directly from the water system under investigation would incorporate the existing pre-adaptation of species to localised conditions into the bioassay itself, giving site-speci®c algal growth potentials. Such data could be used as the basis for a water quality index which takes into account the existing habitat conditions. The signi®cant di€erences found between the exotic and native Scenedesmus strains in the present study highlight the importance of the choice of indicator species used in the biomonitor. Individual species and strains of algae are known to vary considerably in their sensitivity to common pollutants (Genter et al., 1987). Protection of the ®lms, particularly from grazing pressures is an important consideration. Dialysis membrane in the form of broad tubing with a molecular cut-o€ of 12 to 14 kDa was used in this study, and this may have in¯uenced the availability of particulate-associated forms of phosphorus. It did not, however, a€ect growth increases under laboratory conditions in Z8 medium. Algal growth in ®lms protected by dialysis membranes have been compared to that in ®lms placed in nylon bags, with a relatively large mesh size (500  500 mm). The nylon mesh appeared to initially allow marginally better growth in the eutrophic site, however, grazing through the mesh occurred between 7 and 14 d after exposure. Dialysis membranes are available in a range of pore sizes and further trials are merited to evaluate alternative materials for the protection of ®lms from grazers, whilst allowing the passage of bioavailable compounds and particles to the ®lms and immobilised cells. The selection of particular membranes with de®ned pore sizes may also permit the selective monitoring of groups of bioavailable molecules based on size. Microscopic examination of the membrane bags, or the ®lms minus algal cells, in these experiments did not reveal any signi®cant colonisation by epiphytes. At present a limited number of freshwater algal species, such as Scenedesmus subspicatus NIVACHL.55 used in this paper, provide the standard test strains used in the majority of laboratory-based algal growth potential/inhibition (ISO, 1989). S. subspicatus NIVA-CHL.55 is used because it grows well in a wide range of type and concentration of

chemical species (Whitton and Kelly, 1995), and it was this strain which grew best in both the ®eld and in situ experiments in this study. The use of algae immobilised in ®lms as presented here allows a biomonitor to be designed for a particular site by the inclusion of native algal strains. The procedure is also based on the response to cumulative growthcontrolling factors. The authors consider these to be two of the most important factors which need to be included in the design of e€ective biomonitors. This biomonitor ful®ls the criteria set out by McCormick and Cairns (1994) in that it is ecologically relevant, broadly interpretable and is capable of continuity through space and time. Such developments are required as it appears water quality assessment in the future will have both a legal and scienti®c basis for an increased emphasis on biological monitoring techniques. AcknowledgementsÐThis work was funded by the Scottish Oce (Agriculture Environment and Fisheries Department) and by the Natural Environmental Research Council (grant # GST/02/1200). The authors would like to thank Dr R. Owen, for suggesting ®eld sites and SEPA for supplying water ¯ow and quality data. Our thanks also to Mrs Y. Cook and Mrs E. Fisher for N and P analyses. REFERENCES

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