Hydrobiologia (2006) 572:103114 DOI 10.

1007/s10750-006-0371-1

BIOASSESSMENT METHODS

Development of diatom-based tools for assessing stream water quality in south-eastern Australia: assessment of environmental transfer functions
A. Philibert P. Gell P. Newall B. Chessman N. Bate

Received: 11 July 2005 / Revised: 7 June 2006 / Accepted: 27 July 2006 Springer Science+Business Media B.V. 2006

Abstract Models were developed of relationships among water quality, geospatial and specieslevel diatom data for 465 samples collected from stream sites across south-eastern Australia. Transfer functions were derived from weighted averaging and articial neural network
Guest editors: R. H. Norris, R. Marchant and A. Milligan Evaluation and Application of Methods for Biological Assessment of Streams Handling editor: R. Norriss

Electronic Supplementary Material Supplementary material is available in the online version of this article at http:// dx.doi.org/10.1007/s10750-006-0371-1 and is accessible for authorized users.
A. Philibert P. Gell (&) Diatoma, Geographical & Environmental Studies, The University of Adelaide, 5005 Adelaide, South Australia, Australia e-mail: peter.gell@adelaide.edu.au P. Newall N. Bate Environment Protection Authority, 40 City Road, Southbank, Victoria 3006, Australia B. Chessman P. Newall Co-operative Research Centre for Freshwater Ecology, University of Canberra, Canberra, ACT 2601, Australia B. Chessman Department of Infrastructure, Planning and Natural Resources, P.O. Box 3720, Parramatta, NSW 2124, Australia

approaches. Analysis of spatial variations in species assemblages was used to divide the sites into two groups according to site elevation. The strongest predictive models for the upland group associated diatom assemblages with conductivity, longitude, altitude, and to a lesser extent pH, NOx and TKN. The strongest predictors for the lowland group were longitude and conductivity, but articial neural network models performed well for NOx and temperature. The importance of the geospatial variables suggests that there may be a capacity to develop diatom sub-regions within which robust models for other water quality variables important to management can be generated. Keywords Bioassessment Diatoms Salinity Pollution Transfer function Articial neural networks

Introduction Biological assessment is used or advocated increasingly in the management of aquatic ecosystems (e.g. USEPA, 1990; Yoder & Rankin, 1998; Metzeling et al., this volume). Concomitantly, biological objectives are being included in legislation drafted to protect the aquatic environment (e.g. Government of Victoria, 1999; 2003; Federal Water Pollution Control Act, 2002). Although aquatic macroinvertebrates (Rosenberg

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& Resh, 1993; Norris & Norris, 1995) and sh (Karr, 1981; Harris, 1995) are commonly used in biological assessment, diatoms are particularly suitable for the assessment of water quality in lakes and in running waters (Gasse, 1987; Smol, 1992; Prygiel & Coste, 1993; Reid et al., 1995; Moser et al., 1996; Stoermer & Smol, 1999; Gell et al., 2002). Diatoms are widely distributed and form an important component of aquatic food webs (Chessman, 1986). Diatom assemblages can provide a simultaneous indication of several water quality variables because they respond to a range of physical and chemical characteristics (Smol, 1992; Moser et al., 1996). Benthic diatom assemblages, being sessile, are exposed to water quality at a site over a period antecedent to sampling. They therefore indicate recent as well as current water quality (Breen, 1998). Transfer functions are common among the techniques for diatom-based assessments of aquatic environments. Transfer functions are formulae that relate species assemblages to specic environmental measures, whereby empirical data on species ranges and optima for a given indicator (e.g. pH, salinity) can be used to predict that indicator at a new site, based on the species assemblage. Transfer functions therefore allow quantitative reconstruction of specic variables. Mathematical tools used to derive transfer functions include weighted averaging (WA) (ter Braak & van Dam, 1989; Birks et al., 1990), weighted averaging partial least squares (WA-PLS) (ter Braak & Juggins, 1993), and linear methods of PLS (Wold et al., 1984). Articial neural networks (ANN) have recently been added (Racca et al., 2001; Gevrey et al., 2004). Initially, diatom-based transfer functions were used primarily in lake studies, particularly for the reconstruction of historical and pre-historical environments (Battarbee & Charles, 1987; Dixit et al., 1993; Gell, 1998; Battarbee et al., 2001; Philibert & Prairie, 2002a, b). Recently, however, transfer functions have been used successfully in streams to infer a series of environmental measures e.g. pH, TP, conductivity, from diatom communities (Reavie & Smol, 1997; Winter & Duthie, 2000; Belore et al., 2002; Prygiel et al., 2002; Schonfelder et al., 2002; Soininen & Niemela, 2002; Potapova & Charles, 2003; Tibby et al., 2003).

Recent studies have suggested that diatombased indices and transfer functions that were developed within one region are inaccurate in other regions (Philibert & Prairie, 1999; Pipp 2001). Reasons for this may include regional differences in water quality (Pipp, 2001), polymorphism, force-tting species to keys (Tyler, 1998), endemism (Potapova & Charles, 2002) and local history (Philibert & Prairie, 1999). These problems can be overcome if the condition of aquatic ecosystems is assessed with local or regional indices and models. The broad aim of this study was to produce regional transfer functions for diatoms in south-eastern Australia. Additional objectives included: (i) the assessment of within-region variation of diatom-water quality relations; and (ii) the use of geospatial measures to determine whether diatom regions need to be delineated at a smaller spatial scale than that used in this study. Methods Site and sample selection This study used diatom and environmental data, mostly unpublished, from several programs and studies commissioned in three states in southeastern Australia: South Australia, Victoria and New South Wales (one site from the South Australian eld sampling program was located just inside Queensland). These data were readily available, were collected in a largely consistent fashion, and were from a restricted area within Australia. The geographic range of the sample sites is approximately 1000 km (northsouth) by 1500 km (eastwest) (Fig. 1). This area is mostly of low relief relative to other continents, even though it includes uplands in south-eastern Australia. Even within this part of the continent, environments range from hot deserts in South Australia, where summertime temperatures can exceed 45C, through to alpine herbelds in Victoria and New South Wales, where snow can occur any time and typically is present for six or more months of the year. Sites were not restricted

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Hydrobiologia (2006) 572:103114 Fig. 1 Location of sites sampled for diatoms and water quality

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to particular environments, because the intent was to include a broad array of conditions across the states. Most samples were collected from sites between sea level and 600 m ASL. Collection and processing of the diatom samples Periphytic diatom assemblages were sampled from owing waters and stream pools. All samples were collected, prepared and counted in accordance with a draft protocol produced by Chessman et al. (1999a). In summary, this consisted of either submerged substrata being removed from the stream and scraped with sharpened wooden spatulas or a thin veneer of sediment being removed from mud surfaces using a spoon or syringe. At each site samples were taken from several surfaces, aggregated and preserved in the eld in ethanol and the frustules cleared in the laboratory. The cleared samples were then mounted in Naphrax and at least 200 valves were normally counted per sample, as described by Bate and Newall (2002). Fifteen samples contained so few valves that only 100 were counted. Seventy-one samples with fewer than 100 valves were omitted from further analyses. The diatoms were identied to the lowest practical taxon (usually species) with standard international (Krammer & Lange-Bertalot, 1986, 1988, 1991a,b; Lange-Bertalot & Metzelin, 1996; Reichardt, 1999; Krammer, 2000;

Witkowski et al., 2000; Lange-Bertalot, 2001) and Australian keys (Gell et al., 1999; Sonneman et al., 2000). Biological data preparation The data set comprised 393 samples from 326 sites (Fig. 1) after the exclusion of samples with fewer than 100 valves. A total of 641 taxa was identied from these samples. Rare taxa were deleted on the following criteria: each retained taxon had to occur in at least ve samples, each had to reach 1% relative abundance in more than three samples, and each had to reach 3% relative abundance in at least one sample. These criteria resulted in the omission of 396 taxa and left 245 taxa in the nal data set. In order to partition variance in the data arising from the wide altitudinal range (41650 m ASL), a non-hierarchical clustering program (ALOC) was applied to the diatom data in the PATN package (Belbin, 1993) and this separated the sites into two species-based groups. This analysis supported an uplandlowland division at 180 m ASL. This resulted in a lowland (L) site group containing 175 sites with an altitude below 180 m and an upland (U) site group with 218 sites at 180 m or higher. Environmental data preparation The independent origin of the data sets resulted in inconsistencies among the suites of environ-

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mental data collected with each set. All three states had data for electrical conductivity (EC) at 25C (lS/cm), water temperature (C) and pH. Nutrient measures, however, varied among the states, with South Australia sampling for total phosphorus (TP) and total nitrogen (TN), Victoria sampling for TP, TN, total Kjeldahl nitrogen (TKN) and oxides of nitrogen (NOx), and New South Wales sampling for NOx and lterable phosphorus (FP). The geospatial measures used to assess the potential for subregional delineations comprised latitude, longitude and altitude (m ASL). These three measures were available for all States. Predictive models for variables were developed on the subset of samples with data for the variable. Most environmental variables were logarithmically transformed to approximate a normal distribution. The co-efcient of variation (V = St. Dev./mean) was used to compare the relative variability of the environmental variables. Numerical analyses The numerical analyses involved a 3-step process: (i) A canonical correspondence analysis (CCA) was used to explore the relationships between the diatom assemblages and the environmental variables (ter Braak, 1986, 1987). Forward selection, based on Monte Carlo tests with 999 permutations (P << 0.05), was then used to identify the variables most likely to produce reliable models. Caution must be applied when interpreting outcomes from forward selection because signicant variables that are highly correlated with others may be omitted from the model. This occurs because their contribution to the total level of explanation is reduced after covariables are selected. An examination of the outcomes of the rst CCA may reveal variables worthy of closer scrutiny for model formulation. A nal CCA was executed on the forward-selected variables to calculate the total variance explained by these variables. This was undertaken using the CANOCO program version 4.0 for Windows (ter Braak & Smilauer, 1998). (ii) Detrended CCA ordinations or DCCA (ter Braak & Prentice, 1988) were then used to establish the signicance of selected variables in the explanation of diatom distributions and to aid

the design of prediction models. A series of DCCAs, constrained to each environmental variable, was performed to determine the species gradient lengths with respect to the selected variables. This was done to see whether unimodal or linear models were the most appropriate (Birks, 1985, 1998). This was also achieved with the CANOCO program version 4.0 for Windows (ter Braak & Smilauer, 1998). (iii) Transfer functions were then developed for the prediction of satisfactorily robust variables from diatom assemblages. Commonly used methods of Weighted Averaging (WA) (ter Braak & Looman, 1986; Birks et al., 1990) were compared with and without tolerance weighting and/or deshrinking. The Weighted Averaging Partial Least Square (WA-PLS) technique was also tried (ter Braak & Juggins, 1993). This last method was used because it takes into account both the unimodal response of diatoms along environmental gradients and the information contained in the residuals, thereby diminishing bias (ter Braak & Juggins, 1993). The analyses were carried out with the program C2 version 1.3 (Juggins, 2003). Transfer functions were also developed using articial neural network (ANN) techniques (Racca et al., 2001; Gevrey et al., 2004). The ANN technique is designed to quantify the relationships between diatom species (inputs) and environmental variables (outputs). Non-linearly inter-connected neurons or networks are used and work in parallel without requiring any necessary assumptions of relationships (linear or unimodal) between diatom taxa and environmental variables. Relationships are quantied by the back-propagation algorithm (descending gradient algorithm), which continuously adjusts the weights of the network connections until optimization is reached (i.e., the difference between observed and predicted values of the variable to be reconstructed is minimal). More details of the back-propagation procedure and the algorithm are given in Racca et al. (2001). In all, six models for deriving transfer functions were compared: classical WA, classical WA/TOL (downweighted by the tolerance factor), inverse WA, inverse WA/TOL, WA-PLS and ANN. The predictive ability of the calibration models was assessed with the apparent coefcient of deter-

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mination (r2) between measured (observed) and the diatom-inferred (predicted) values, and with the associated apparent root mean squared error (RMSE) whose unit depends on that of the variable to be predicted. Estimates based on jackknife resampling, r2jackknife and RMSEjackknife, also called RMSEp, were also used as they represent more realistic measures of the predictive power than the apparent r2 or RMSE (Birks et al., 1990). Arbitrary thresholds, commensurate with those published elsewhere, were selected to accept or reject variables. Models with an r2jackknife greater than 0.35, and an r2apparent greater than 0.6, were accepted. Models with r2jackknife less than 0.35, but an r2apparent greater than 0.6, were allocated a low utility ranking and models with r2jackknife less than 0.35, and an r2apparent less than 0.6, were classed as failing. Optimally, the RMSE and the RMSEp should be lower than 10% of the range of the variable of interest. Models were derived separately for the upland and lowland site groups, and for both groups combined.

frequentissimum Lange-Bertalot and Tabellaria occulosa Kutzing). Few taxa (4.2%) were found exclusively in upland streams (e.g. Achnanthes imperfecta Schimanski, Actinella eunotioides, Eunotia bilunaris var. mucophila Lange-Bertalot & Norpel and Pinnularia divergentissima Cleve) and even fewer (3.4%) were conned to samples in lowlands (e.g. Amphora coffeaeformis var. borealis, Amphora holsatica Hustedt, Aulacoseira subarctica form subborealis, Gyrosigma obscurum Grifth & Henfrey and Navicula cincta f. minuta Grunow). General water quality results Several measures of water quality had wide ranges across the sites (Table 1). The streams generally had low conductivity (median EC at 25C of 404 lS/cm). A few saline sites exceeded 10,000 lS/cm and elevated the mean EC at 25C to 1117 lS/cm. By contrast the ion-poor, alpine sites had conductivities (at 25C) as low as 6 lS/ cm. Temperatures centred on 15C with extreme values of 3 and 28 C. Most sites were circumneutral to alkaline with extreme pH values of 4.4 and 9.8. All nutrients were detected in nearly all samples with upper values well into eutrophic levels (e.g. TKN: 24 mg/l; NOx: 11 mg/l; TP: 9 mg/l). Low median values for nutrients relative to means show that high-nutrient sites were in the minority. The uplands occur in the southern and eastern parts of the study area, with the lowlands extending north and west. Mean conductivities (at 25C) were greater in the lowland samples but those in the upland data set were more variable (CV-upland: 179%; V-lowland: 129%). Lowland temperatures were also higher but less variable (CV-upland: 30%; V-lowland: 26%). Upland sites encompassed a large pH gradient from acidic to alkaline waters, while most lowland sites were circumneutral to alkaline. The mean nitrogen values for the lowland sites were usually greater than their upland counterparts, but the NOx (CVupland = 461%) values for the upland sites were highly variable. This variability and the low median values show that while some hypereutrophic sites were elevating the means, most sites were relatively low in nutrients. This can also be said for TP for upland sites

Results General oristic description The taxa that occurred most frequently in the full data set were Achnanthidium minutissimum Kutzing (304 samples), Gomphonema parvulum Kutzing (234), Tryblionella apiculata Gregory ` (232), Fragilaria capucina Desmazieres (169) and Synedra ulna Ehrenberg (162). The occurrence and effective occurrence (Hills N2) of the taxa found in the upland and lowland subsets are provided in the supplementary material. Most taxa were in both the upland and lowland data sets (e.g. Achnanthidium minutissimum, Cocconeis placentula Ehrenberg, Gomphonema parvulum and Tryblionella apiculata), although several were relatively more abundant in low land streams (e.g. Cyclotella meneghiniana Kutzing, Navicula viridula Ehrenberg and varieties, Tryblionella hungarica D.G. Mann and Trybionella levidensis W. Smith) and others in upland streams (e.g. Gomphonema angustum Agardh, Navicula schroeteri Meister, Planothidium

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Table 1 Minima, maxima, mean, median and standard deviations of environmental variables for the upland sites, lowland sites and whole data set for sites sampled Min Max value value Latitude (degrees) Whole data set 27.60 39.12 Group L 27.60 39.12 Group U 30.57 39.12 Longitude (degrees) Whole data set 136.61 151.37 Group L 136.61 151.37 Group U 138.50 150.93 Altitude (m) Whole data set 4 1650 Group L 4 175 Group U 180 1650 EC at 25 (uS/cm) Whole data set 6 14030 Group L 43 12410 Group U 6 14030 EC ambient (uS/cm) Whole data set 4 10610 Group L 47 10130 Group U 4 10610 Water temperature (C) Whole data set 2.6 28.0 Group L 7.6 25.4 Group U 2.6 28 Mean Median St. Dev 36.58 36.55 36.60 No. samples Min Max Mean Median St. No. value value Dev samples pH (units) Whole data set 4.42 Group L 6.15 Group U 4.42 NO2 + NO3 (mg/l) Whole data set 0.002 Group L 0.002 Group U 0 TKN (mg/l) Whole data set 0.04 Group L 0.06 Group U 0.04 TN (mg/l) Whole data set 0.02 Group L 0.06 Group U 0.02 TP (mg/l) Whole data set 0.003 Group L 0.003 Group U 0.003 9.83 7.56 9.80 7.54 8.75 7.57 11.40 0.27 5.15 0.25 11.40 0.28 23.60 0.75 23.60 0.95 12.30 0.59 5.60 0.54 5.60 0.86 1.64 0.35 9.48 0.16 5.86 0.11 9.48 0.19 7.60 7.52 7.70 0 0.02 0 0.34 0.52 0.20 0.38 0.60 0.22 0.02 0.03 0.01 0.59 388 0.54 174 0.63 214 1.10 249 0.49 107 1.29 142 1.98 257 2.26 117 1.70 140 0.64 174 0.89 64 0.29 110 0.93 250 0.56 110 1.14 140

35.75 36.06 35.49

2.17 393 1.96 175 2.29 218 4.13 393 4.74 175 3.52 218 308.4 393 51.2 175 327.4 218 1715.0 393 1939.4 175 1442.7 218 1605.0 172 1903.2 62 1290.0 110 4.2 259 4.1 116 4.2 143

145.29 146.40 144.81 144.70 145.67 146.72 288.1 80.6 454.6 1117.3 1507.3 804.3 365 85 345 404 747 172

809.2 137 1437.2 2033 455.2 62 14.5 15.5 13.6 13.8 15.5 13.0

in which there was even greater variability (Vupland = 604%; Vlowland = 509%). Multivariate analyses Forward selection revealed that conductivity provided the best explanation of the variance in the diatom data for both upland and lowland sites (Fig. 2a, b). In addition, the selection of NOx and water temperature from stepwise techniques of a CCA undertaken on the lowland sites, suggested that useful inference models could be developed for these latter variables also. From the upland sites TKN and TP were selected after the stepwise procedure in the CCA as potentially useful predictive variables for explaining the variance in assemblages at these sites. Finally, the geospatial variables altitude and longitude were also identied as likely to produce reliable inference models of diatom communities in both upland and lowland sites. The total variance explained by the CCA technique was 8.4 and 8.7% for the lower

and upper altitudinal subgroups respectively. Such low values are possibly because of noise and are not unusual for large data sets across broad geographic ranges (e.g. Potopova & Charles, 2002). There was however a strong relationship between the species and the CCA axes with 72% of species variation explained in the rst three axes. In each of the upland and lowland groups all the selected variables from the previous stepwisebased CCAs were associated with the diatom assemblages in a unimodal fashion as the DCCAs yielded species gradient lengths greater than 2 standard deviations for each. In addition, all these selected variables were nally used in the model generation because the correlations between the canonical axes and the species were all signicant. Lowland regression models In lowland sites (group L altitude < 180 m), conductivity was well predicted from all inference

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NOx

(a)

Axis two

5 4 3 2

Upland regression models The conductivity of upland sites was well inferred by all tested inference models (Table 2). The signicance of pH in explaining variation in diatom communities was hidden after the stepwise selection in the rst CCA because pH was highly correlated with conductivity and temperature. However, inference models were generated for pH because it is ecologically important, despite its elimination in forward selection. Most inference functions for the prediction of pH from diatom taxa from upland sites were very strong (Fig. 3b). Several models performed very well in inferring TKN and NOx. The geospatial variables altitude and longitude were reconstructed well by most models. Full data set regression models

temperature Longitude
-4 -2 1 0

altitude
-2

-1

2 Axis one 4

EC

Forward CCA on the group L (< 180 meters)

samples longitude altitude

EC NOX o T

(b)
altitude

15
Axis two

EC

10 5 0 -15 -10 -5 -5 -10 0 5

Axis one

TKN

longitude

TP

Forward CCA on the group U (> 180 meters)

samples longitude altitude

EC TKN TP

Fig. 2 Canonical Correspondence Analysis (CCA) of lowland sites after forward selection

Water quality inference models were derived only for conductivity, and to a limited extent NOx, across the two sets of streams (L & U). Species optima Within each of the lowland and upland subgroups output from the weighted averaging models were used to derive optima (see supplementary material) for each taxon and for each variable of ecological interest even if some failed to perform well in the inference models (Table 2).

models (Table 2, Fig. 3a), as was the geospatial variable longitude. None of the WA models tested provided reliable inferences of NOx, and the ANN techniques provided only weak models for water temperature and NOx.

Table 2 Performance of models based on diatom assemblages for the selected variables in each upland and lowland group Lowland Group Long WA classical WA/TOL classical WA inverse WA/TOL inverse WA-PLS ANN EC NOx TEMP Upland Group Long Low Low Alt EC NOx Low Low Low TKN Low Low pH Low

Low

Low

* indicates models with high predictive

capacity(r2jack

>> 0.35 and r2apparent >> 0.6)

** Low indicates models with a weak predictive capacity (r2jack is < 0.35 but r2apparent >> 0.6) *** Empty cells indicate that the model failed (r2jack is < 0.1) LONG: longitude (degrees); EC: conductivity at 25C (lS/cm); NOx: nitric oxides (NO2 + NO3) (mg/l); TEMP: temperature (C); ALT: altitude (m); TKN: total Kjeldahl nitrogen (mg/l); pH (units)

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(a)
4.5

Plot of regression between observed and inferred EC values in lowlands

4 3.5 3 2.5 2 1.5 1.5

2.5

3.5

4.5

inferred log EC values

(b)
9

Plot of regression between observed and inferred pH values in uplands

8.5 8 7.5 7 6.5 6 5.5 5.5 6 6.5 7 7.5 8 8.5 9

inferred pH values

Fig. 3 Correspondence between observed and inferred values of log EC from an inverse Weighted Averaging (WA) model

Discussion The support from the species-based classication for the initial division of samples above and below 180 m demonstrated a clear altitudinal split between upland and lowland diatom assemblages. Despite this, 96% of the taxa identied from upland samples were also found in the lowlands, and 97% vice versa. That the assemblages were distinct suggests that the uplandlowland division was based principally on the relative abundance of taxa rather than presence and absence. The performance analysis of the models also demonstrated that altitude remained an inuential variable even within the upland subsetperhaps because the range and variability in elevation were greater in the upland set. Another geospatial variable, longitude, produced reliable diatom-based inference models for both upland and lowland site groups. The chemically dilute streams are predominantly in the

eastern uplands and coastal lowlands, whereas most solute-rich streams are in the western lowlands. Despite the association of longitude with conductivity, both variables remained important in explaining the diatom data after forward selection revealing variation in conductivity beyond the eastwest correlation. The independent importance of altitude and longitude suggest that geospatial descriptors potentially can predict diatom assemblages. Such prediction can be used in bioassessment methods whereby data from unpolluted reference sites are used to generate expected diatom assemblages at sites of unknown water quality status (Chessman et al., 1999b). Anthropogenic impacts on stream water quality can then be detected as mis-matches between expected and observed diatom assemblages. The high performance of the geospatial models also suggests that a ner scale sub-regional delineation than simply uplands and lowlands is warranted in the development of transfer functions. The construction of functions for individual sub-regions is likely to remove spatial variance that cannot be explained by spatial patterns in water quality, and so enhance the capacity for prediction of water chemistry. Studies with macroinvertebrates in Victoria (e.g. Newall and Wells, 2000; Wells et al., 2002) divided that State into four and ve or more sub-regions respectively, indicating substantial opportunity for subregionalisation across the three states. Of the non-spatial variables that were measured, conductivity appeared to be most inuential on diatom assemblages. This is not surprising given the evidence from diatom-salinity studies of lakes and wetlands within the study area (Gell, 1997; Blinn & Bailey, 2001). It is likely that, above a particular salinity threshold, salinity is the dominant water quality inuence on diatom assemblages. This phenomenon is possibly accentuated in the Australian landscape where shallow, saline groundwaters are impacting widely on running waters (Macumber, 1991). Other chemical variables may be more inuential in less saline sites. The high predictive capacity of pH in the upland site group but not the lowland is probably because the pH in the upland group ranged across

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thresholds (i.e. acidneutralalkaline) that have been demonstrated to inuence the diatom ora (Birks et al., 1990), whereas the lowland sites were predominantly alkaline. There are few species (e.g. Fragilaria nanana Lange-Bertalot, Eunotia paludosa Grunow, Eunotia exigua Rabenhorst) that appear to be largely conned to acidic waters, and these were most abundant in the upland sites. The non-selection of pH during forward selection probably attested to its co-variation with conductivity, longitude (as conductivity relates to longitude) and altitude. Similarly, the weak relationships between diatom assemblages and nutrient variables such as TN, TKN and TP, may have been because of dominance of geospatially related inuences as well as conductivity and pH. Nitrogen variables performed better in upland sites where the variability in concentrations was greater. The weakness of the nutrient models, therefore, may have arisen also because the range of nutrient values was uneven, with most sites having lower concentrations. The paucity of medium- and high-nutrient data may have caused the diatom nutrient relationship to be statistically weak even though the diatom assemblages were responding to nutrients at the extremes. Also, the narrower range of phosphorus variables may explain why diatoms appear to be more responsive to nitrogen variables. This could be tested through supplementation of the data set with a greater range of samples from enriched sites. The possibility remains, however, that diatoms may be robust nutrient bioindicators only in waters of lower conductivity and within a limited pH range. The lower performance of some models in terms of r2 and associated errors (RMSE) also may have been caused by the uneven spread of values across the ranges of environmental variables, with the exception of pH. Additional sampling to ll gaps in the environmental data is likely to strengthen the predictive capacity of the models. In addition, some of the apparent lack of strength in the models may have arisen because the diatom assemblages were a reection of a uctuating regime of water chemistry prior to sampling, rather than the chemical conditions at the time of sampling. Intensive, localized analysis of the response times of diatoms would provide

evidence to direct the modication of sampling protocols to limit this inuence on model robustness.

Conclusion This data set can be used to infer stream conductivity and pH using diatoms. In much of southeastern Australia, these environmental variables reect some of the most substantial human impacts on stream systems. Given the importance of altitude and longitude, it is likely that sub-regional transfer functions can be developed that, with conductivity and pH variation constrained, can be used to infer nutrient regimes. The utility of this approach may be conned to the lowsalinity, upland areas. The capacity for use of diatoms to infer nutrients may increase through extension of the data set by sampling of more sites with low and high concentrations of both salts and nutrients. The uses of diatoms to infer water quality may be more cost-effective than direct physical and chemical analysis if several water quality properties can be inferred from a single diatom sample, or if diatoms can be used to deduce an average of water quality conditions over an antecedent period. Diatom transfer functions developed for rivers also are likely to prove useful in inference of long-term water quality histories from diatom stratigraphy of deposits in wetlands on riverine oodplains (Gell et al. 2002, 2005). This may provide a means of understanding the past condition of streams that were impacted well before the development of monitoring programs.
Acknowledgements Peter Goonan authorized the use of samples and data collected within South Australia. Numerous eld technicians collected diatoms and water samples. Diatom samples were analysed by Sophie Bickford, Greg Smith, Craig McVeigh, Angus MacGregor and Cameron Barr. John Tibby provided valuable comments on the manuscript.

References
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