Biological indicators track differential responses of pelagic and littoral areas to nutrient load reductions in German lakes

External nutrient loading was reduced over the past decades as a measure for improving the water quality of eutrophic lakes in western Europe, and has since been accelerated by the adoption of the European Water Framework Directive (WFD) in 2000 (EC, 2000). A variety of eutrophication-related metrics have indicated that the response of biological communities to this decreased nutrient loading has been diverse. Phytoplankton, a major component of the pelagic community, often responded rapidly, whereas a significant delay was observed for submerged macrophytes colonizing littoral areas. In this study we tested whether assessment methods developed for phytoplankton and macrophytes in lakes during Germany's implementation of the WFD reflect this differential response. An assessment of 263 German lakes confirmed that a lower ecological state was recorded when based on the biological quality element (BQE) for macrophytes than the BQE for phytoplankton during the investigated period (2003–2012). On average, lakes had a moderate ecological status for both phytoplankton and macrophyte BQEs, but differences of up to three classes were observed in single cases. Long-term data were available for five lowland lakes subject to strong reductions in phosphorus loading. Their phytoplankton-based assessments indicated a constant improvement of the ecological status in parallel to decreasing water phosphorus concentrations. In contrast, macrophyte-based assessments indicated a 10–20 year delay in their ecological recovery following nutrient load reduction. This delay was confirmed by detailed data on the temporal development of macrophyte species diversity and maximum colonization depths of two lakes after nutrient load reduction. We conclude that the available WFD assessment methods for phytoplankton and macrophyte BQEs are suitable to track the differential response of pelagic and littoral areas to nutrient load reductions in German lakes.     

Quantifying uncertainties in biologically-based water quality assessment: A pan-European analysis of lake phytoplankton community metrics

Lake phytoplankton are adopted world-wide as a sensitive indicator of water quality. European environmental legislation, the EU Water Framework Directive (WFD), formalises this, requiring the use of phytoplankton to assess the ecological status of lakes and coastal waters. Here we provide a rigorous assessment of a number of proposed phytoplankton metrics for assessing the ecological quality of European lakes, specifically in response to nutrient enrichment, or eutrophication, the most widespread pressure affecting lakes. To be useful indicators, metrics must have a small measurement error relative to the eutrophication signal we want them to represent among lakes of different nutrient status. An understanding of variability in metric scores among different locations around a lake, or due to sampling and analytical variability can also identify how best this measurement error is minimised.To quantify metric variability, we analyse data from a multi-scale field campaign of 32 European lakes, resolving the extent to which seven phytoplankton metrics (including chlorophyll a, the most widely used metric of lake quality) vary among lakes, among sampling locations within a lake and through sample replication and processing. We also relate these metrics to environmental variables, including total phosphorus concentration as an indicator of eutrophication.For all seven metrics, 65–96% of the variance in metric scores was among lakes, much higher than variability occurring due to sampling/sample processing. Using multi-model inference, there was strong support for relationships between among-lake variation in three metrics and differences in total phosphorus concentrations. Three of the metrics were also related to mean lake depth. Variability among locations within a lake was minimal (<4%), with sub-samples and analysts accounting for much of the within-lake metric variance. This indicates that a single sampling location is representative and suggests that sub-sample replication and standardisation of analyst procedures should result in increased precision of ecological assessments based upon these metrics.For three phytoplankton metrics being used in the WFD: chlorophyll a concentration, the Phytoplankton Trophic Index (PTI) and cyanobacterial biovolume, >85% of the variance in metric scores was among-lakes and total phosphorus concentration was well supported as a predictor of this variation. Based upon this study, we can recommend that these three proposed metrics can be considered sufficiently robust for the ecological status assessment of European lakes in WFD monitoring schemes.     

Use of marker pigments and functional groups for assessing the status of phytoplankton assemblages in lakes

Phytoplankton is a key biological quality element for the establishment of the Water Framework Directive (WFD) ecological status in reservoirs and lakes. In freshwaters, inverted microscope examination is the traditional standard method for estimating phytoplankton and assessing taxonomic composition. Based on the enumeration of algal units and measurements for biovolume calculation, this technique is cumbersome and time-consuming. In large monitoring programmes, such as the application of the WFD in lakes and reservoirs, chemotaxonomy (HPLC pigment analysis and CHEMTAX treatment) is ideally suited as an alternative method because it allows the rapid processing of large numbers of samples from numerous locations and depths, thereby providing ideal temporal and spatial resolution. The low taxonomical detail obtained by HPLC and CHEMTAX (phytoplankton classes or phyla) can easily be overcome by a rapid inverted microscope screening with identification of the dominant species. Combining HPLC and microscopy provides a useful method for monitoring phytoplankton assemblages, which can be used to implement the WFD with respect to phytoplankton. Here, we present the application of a method combining marker pigments and microscopy to phytoplankton samples from 12 Belgian reservoirs. This method substantially reduced the workload and enabled us to assess the status of the phytoplankton assemblage in these lakes. The method complies with the WFD, as it takes into account taxonomic composition, assesses abundance and biomass of the phytoplankton taxa, and easily detects blooms. Additionally, a set of templates of probability of occurrence of phytoplankton functional groups at the maximal ecological potential for reservoirs from the Central/Baltic region is presented, based on reference conditions defined for natural lakes from other regions.     

Ecological threshold responses in European lakes and their applicability for the Water Framework Directive (WFD) implementation: synthesis of lakes results from the REBECCA project

The objective of this synthesis is to present the key messages and draw the main conclusions from the work on lakes in the REBECCA project, pointing out their links to theoretical ecology and their applicability for the WFD implementation. Type-specific results were obtained from analyses of large pan-European datasets for phytoplankton, macrophytes, macroinvertebrates and fish, and indicators and relationships showing the impact of eutrophication or acidification on these biological elements were constructed. The thresholds identified in many of the response curves are well suited for setting ecological status class boundaries and can be applied in the intercalibration of classification systems. Good indicators for phytoplankton (chrysophytes, cyanobacteria) and macrophytes (isoetids and charaphytes) responses to eutrophication were identified, and the level of eutrophication pressure needed to reach the thresholds for these indicators was quantified. Several existing metrics developed for macrophytes had low comparability and need further harmonisation to be useful for intercalibration of classification systems. For macroinvertebrates, a number of metrics developed for rivers turned out to be less useful to describe lake responses to eutrophication and acidification, whereas other species based indicators were more promising. All the biological elements showed different responses in different lake types according to alkalinity and humic substances, and also partly according to depth. Better harmonisation of monitoring methods is needed to achieve better precision in the dose–response curves. Future research should include impacts of hydromorphological pressures and climate change, as well as predictions of timelags involved in responses to reduction of pressures.     

Integrated assessment of ecological status and misclassification of lakes: The role of uncertainty and index combination rules

The European Water Framework Directive (WFD) requires that the ecological status of waterbodies is assessed using multiple biological quality elements (BQEs) that are combined into a single status class. The recommended combination rule (the “one-out, all-out” rule; OOAO) has been criticised for being unreasonably conservative and for being sensitive to uncertainty. In this study, the objective was to compare the sensitivity to uncertainty of four different combination rules: (1) OOAO, (2) OOAO with exclusion of one element, (3) average and (4) weighted average. Index values for 5 BQEs (phytoplankton, phytobenthos, macrophytes, macroinvertebrates and fish) sampled from 10 lakes in the Wel River catchment in Poland were used to classify the lakes according to the OOAO and the three alternative combination rules. Based on the mean and (where possible) standard deviation of these index values, we modelled the risk of misclassification by simulating 10,000 resamples for each BQEs in each lake, classifying each resample and calculating the proportion of misclassified resamples under each combination rule. For individual BQEs, the risk of misclassification increased both with higher uncertainty (standard deviation) and with the proximity of the index value to a class boundary. Under the OOAO rule, the risk of misclassification was more biased towards worse status (“underclassification”) than towards better status. Furthermore, risk of underclassification was more affected by uncertainty under the OOAO rule compared with the alternative combination rules. This analysis has demonstrated the weaknesses associated with the OOAO rule for integration of BQEs for lake classification. However, the alternative combination rules are associated with other shortcomings, such as the need for subjective judgement, and involve a higher risk of not protecting the most sensitive BQE and thus the whole ecosystem. We recommend that future versions of instructions for WFD implementation consider alternatives to the OOAO combination rule, and provide guidelines for weighting of individual BQEs.     

A fish-based index for the assessment of the ecological quality of temperate lakes

A fish – based index for the assessment of the ecological quality of natural temperate lakes was developed, in accordance to the requirements of the Water Framework Directive (WFD) 2000/60/EC. As a case study, 11 natural lakes located at northern and western Greece were selected. Fish surveys were conducted during mid summer to mid autumn in 2010, 2011 and 2012 using Nordic gillnets and electrofishing. Environmental parameters and anthropogenic pressures were assessed for each lake. Fish species richness, abundance, trophic, reproductive and habitat functional guilds were used for extracting a set of 107 metrics, meeting the requirements of the WFD. All metrics were initially tested as candidates for the index development. A stepwise linear regression of each metric against environmental parameters (lake area, altitude, maximum depth, alkalinity) and anthropogenic pressures (drainage area covered by non-natural land uses – NNLC, water total phosphorus concentrations – TP, Lake Habitat Modification Score – LHMS) was initially conducted for ensuring pressure-response relationships. Reference conditions for each lake were estimated by the hindcasting procedure and the ecological quality for each lake was expressed as the ecological quality ratio (EQR) by a value ranging from 0 (poor quality) to 1 (excellent quality). Two fish fauna metrics, the relative numerical abundance of introduced species (Introduced_a) and the relative biomass of omnivorous species (OMNI_b) were finally extracted as the most significant, responding to LHMS and TP, respectively. The final index was expressed as the mean values of the EQRs of these two metrics. The multimetric fish index presented herein could serve as a tool for assessing the ecological quality of natural lakes at broad geographical scale and generally, in the Mediterranean temperate lakes with similar hydromorphological characteristics.     

Combination of multiple biological quality elements into waterbody assessment of surface waters

The Water Framework Directive (WFD) requires EU Member States to classify the ecological status of surface waters by using multiple biological quality elements (BQEs). According to the WFD Classification Guidance, a ‘one-out-all-out’ (OOAO) rule should be applied when integrating multiple BQEs into an overall biological status of a waterbody, i.e. classification is determined by the lowest status BQE. Using both simulated and monitoring datasets, we analyzed the effects of different combination rules in classification outcome and classification reliability. The OOAO represented the strictest combination rule in terms of increased probabilities of waterbodies being in moderate or worse status in comparison to other rules. The OOAO approach gave acceptable results when different BQEs were complementary, showing the effects of different pressures, and when level of uncertainty in the metrics used in the assessment was not high. Increasing the number of BQEs used in the assessment affected the classification outcome when using the OOAO approach; this was especially problematic if all BQEs address the same pressure. Our study showed that grouping of metrics and metrics uncertainty has a large influence on classification outcomes and that this should be carefully considered to ensure that final classification adequately reflects ecological status.     

A phytoplankton trophic index to assess the status of lakes for the Water Framework Directive

Despite improvements in wastewater treatment systems, the impact of anthropogenic nutrient sources remains a key issue for the management of European lakes. The Water Framework Directive (WFD) provides a mechanism through which progress can be made on this issue. The Directive requires a classification of the ecological status of phytoplankton, which includes an assessment of taxonomic composition. In this paper, we present a composition metric, the plankton trophic index, that was developed in the WISER EU FP7 project and demonstrate how it has been used to compare national phytoplankton classification systems in Northern and Central Europe. The metric was derived from summer phytoplankton data summarised by genus from 1,795 lakes, covering 20 European countries. We show that it is significantly related to total phosphorus concentrations, but that it is also sensitive to alkalinity, lake size and climatic variables. Through the use of country-specific reference values for the index, we demonstrate that it is significantly related to other national phytoplankton assessment systems and illustrate for a single European (intercalibration) lake type how it was used to intercalibrate WFD boundaries from different countries.     

Transitional and coastal waters ecological status assessment: advances and challenges resulting from implementing the European Water Framework Directive

The derivation, performance, sensitivity and inherent uncertainty of ecological quality indicators have become major topics in developing tools for the management of marine, transitional and coastal waters. In reviewing the advances in these waters, related to an ecological status assessment, we show the future challenges to be addressed within the European Water Framework Directive (WFD). Using new analyses carried out under the research project ‘Water Bodies in Europe: Integrative Systems to Assess Ecological status and Recovery’, we provide a complete set of assessments for the biological quality elements (BQEs) (phytoplankton, macroalgae-seagrasses, macroinvertebrates and fish) to be assessed, as well as the validation of existing indicators and multimetric indices and, in some cases, the development of new assessment indices. We show that these indices respond differently to different human pressures and they each have challenges in defining reference conditions against which future changes are judged. In investigating good ecological potential, as the response to heavily modified water bodies, we show that there are flaws in the Directive, not least in its definitions. Our analyses have also focussed on uncertainty in using the indices and we emphasise the problems of defining ecological class boundaries based on indices which themselves may be combined indices (multimetrics). The analysis shows that some of those multimetrics are redundant and/or are inter-correlated and thus may reduce the sensitivity in defining ecological class boundaries. If this is related to the drivers-pressures-state change-impacts-response approach then there are lessons for management measures aimed at achieving good ecological status and even the potential for legal challenges to decisions based on uncertain indices under the WFD. Hence, we conclude the continued need for advances in assessing pressures and gradients, and defining reference conditions for state change, index development, impact assessment and the validation of indices for each BQE.     

天津市典型湖库湿地生态完整性评价

对湖库湿地进行生态健康评价,可为湖库湿地生态系统修复和管理提供决策依据。本文以天津市为例,选取20个典型的湖库湿地采样点,基于2018年8—9月期间调查获取的物理、化学和生物群落指标(浮游动物、浮游植物、底栖动物、鱼类、水生大型植物、河岸带植物)数据,构建包含物理完整性、化学完整性和生物完整性在内的生态完整性指数(IEI)对采样点生态健康状况进行评价。根据栖息地环境质量(QHEI)、水质状况和人类活动干扰3方面选取参照点,采用标准化方法筛选候选指标,应用层次分析法计算各指标权重,最终得出天津市典型湖库湿地生态完整性评价结果。结果表明: 1)所有样点中,“健康”点位占5.0%,“较好”点位占20.0%,“一般”点位占35.0%,“较差”点位占30.0%,“差”点位占10.0%。天津市典型湖库湿地生态健康状况整体处于一般水平,呈现出西部优于东部的趋势,空间差异显著;2)基于栖息地评分、水质状况与人类活动干扰相结合选取参照点是可行的,依据水质指标可降低选择参照点时的主观性;3)适用性验证结果表明,IEI可较好表征各点位的健康状况,区分效率明显,适用于评价研究区湖库湿地生态健康状况。     

Assessing ecological quality of shallow lakes: Does knowledge of transparency suffice?

The European Water Framework Directive (WFD) requires that all aquatic ecosystems in their member states should reach ‘good’ ecological quality by 2015. To assess ecological quality, the WFD requires the definition of reference conditions using biological, physical and chemical indicators and the assignment of each water body to one of five quality classes using these indicators. Elaborate assessment schemes using large sets of variables are now being developed. Here we address the question whether all this is really needed and what the simplest assessment approach would be for the case of shallow lakes. We explore the relationships between the quality class assigned to a lake by experts in shallow lake ecology and a rich set of biological, physical, and chemical data. Multinomial logistic regression analyses were carried out based on data from 86 shallow lakes throughout Europe that were sampled in 2000 and/or 2001. Ecological quality of shallow lakes judged by experts was strongly correlated to physical and chemical variables associated with light regime and nutrients and much less to biological variables.Our regression model showed that ecological quality of this set of shallow lakes judged by experts could be predicted quite well from water transparency expressed as Secchi depth and that other variables did not contribute to it significantly. According to the WFD, lakes should at least have a ‘good’ ecological quality. Quality judged by experts and predicted quality were similar for 78% of the lakes with respect to meeting this standard. As a cautionary note we stress that Secchi depth alone will be a less useful indicator if effects of stressors other than eutrophication (e.g. lake acidification and toxic pollution) are to be considered.     

Mining lakes in a disturbed landscape: Application of the EC Water Framework Directive and future management strategies

Some of the several hundred Lusatian mining lakes originating from lignite mining will be among the largest and deepest lakes in Germany. A special problem of many of these lakes is severe acidification. According to the EC Water Framework Directive (WFD), artificial lakes larger than 0.5 km² have to be monitored and managed in order to establish or maintain a state of so-called good ecological water quality. Therefore the WFD demands the definition of type-specific reference conditions as those of the closest comparable surface water body type in nature, such as natural acidic volcanic crater lakes or oligo- to mesotrophic neutral hard water lakes. We propose a classification scheme for mining lakes applying acidity and planktonic colonization as criteria. Five types of lakes are distinguished ranging from extremely acidic to neutral hard water lakes. Phytoplankton and especially metazoan zooplankton are introduced as intriguing and simple indicators for different states of ecological quality in highly acidic mining lakes. We recommend that extremely and very acidic lakes should remain in the acidic state to protect these as valuable ecosystems, whereas moderately and weakly acidic lakes can be managed by external flooding, chemical or ecotechnological measures to achieve neutral water quality conditions necessary for recreation or fishery use.     

Cascading effects of generalist fish introduction in oligotrophic lakes

Introduction of strictly planktivorous fish to lakes can alter plankton communities via cascading interactions in food webs. Less is known about the large-scale and long-term effects resulting from the introduction of fish with more generalist feeding habits, and the extent to which these effects depend on lake trophic status. Paleolimnological records of three oligotrophic lakes in Maine, USA were used to analyze the response of plankton communities to the introduction of white perch (Morone americana), a fish that often numerically dominates fish assemblages and switches from strict planktivory to a more generalist diet during ontogeny. After white perch introduction, cladoceran ephippia size increased up to 50 %, suggesting that the most important role of this generalist fish, with respect to water quality, is as a piscivorous trophic link. Algal standing crop declined by a quarter to over a half of pre-introduction levels, suggesting that top-down effects of white perch reduced phytoplankton biomass. In comparing these oligotrophic lakes to prior work in a eutrophic system, white perch introduction had similar effects on zooplankton body size; however, cascading effects to phytoplankton were only observed in low productivity lakes.     

Phytoplankton and ecological status of forest lakes in the Omsk Priirtyshye

According to the materials of multiyear studies, the author estimates the ecological status of forest lakes in the Omsk Priirtyshye with respect to the phytoplankton development indicators. The species composition, structure, and floristic similarity degree of the phytoplankton of polytypic forest lakes are described. The author also determines the trophic status and water quality of these lakes. It is found that excessive recreation has a negative impact on the ecological status of the lakes.     

Introducing a zooplanktonic index for assessing water quality of natural lakes in the Mediterranean region

In the pelagic food web of lakes, zooplankton offers the linkage between phytoplankton and fish, greatly affecting but also mirroring the functionality and stability of the ecosystem. Despite the increased interest on the development of water quality indices, incorporating zooplankton data on metrics used for the assessment of natural lakes remains a challenge. Here, we used information of natural lakes hosted in Eastern Mediterranean on zooplankton abundance, biomass, body weight and cladocerans' ratio to develop a novel zooplanktonic index (Zoo-IQ). Α 5-grade classification scheme was generated (Bad ≤6, 6 < Poor ≤10, 10 < Moderate ≤14, 14 < Good ≤18, 18 < High) for water quality assessment. The Zoo-IQ was originally parametrized based on data collected from 13 natural lakes, located in Greece and successfully classified hypertrophic lakes with poor quality and deep lakes with good quality, while it identified lakes subjected to restoration actions. Using independent data from five lakes hosted in Greece, Turkey and Croatia, Zoo-IQ successfully assessed their water quality. Specifically, from the 25 sampling cases, used for the development and the evaluation of the index, Zoo-IQ identified 5 as good, 8 as moderate, 11 as poor and 1 as bad. Our results demonstrate the potential of using Zoo-IQ for the assessment of lakes water quality in the Mediterranean. The proposed index could serve as a first step towards the development of similar indices in other climatic zones.     

Ecological status assessment of European lakes: a comparison of metrics for phytoplankton, macrophytes, benthic invertebrates and fish

Data on phytoplankton, macrophytes, benthic invertebrates and fish from more than 2000 lakes in 22 European countries were used to develop and test metrics for assessing the ecological status of European lakes as required by the Water Framework Directive. The strongest and most sensitive of the 11 metrics responding to eutrophication pressure were phytoplankton chlorophyll a, a taxonomic composition trophic index and a functional traits index, the macrophyte intercalibration taxonomic composition metric and a Nordic lake fish index. Intermediate response was found for a cyanobacterial bloom intensity index (Cyano), the Ellenberg macrophyte index and a multimetric index for benthic invertebrates. The latter also responded to hydromorphological pressure. The metrics provide information on primary and secondary impacts of eutrophication in the pelagic and the littoral zone of lakes. Several of these metrics were used as common metrics in the intercalibration of national assessment systems or have been incorporated directly into the national systems. New biological metrics have been developed to assess hydromorphological pressures, based on aquatic macrophyte responses to water level fluctuations, and on macroinvertebrate responses to morphological modifications of lake shorelines. These metrics thus enable the quantification of biological impacts of hydromorphological pressures in lakes.     

Planktonic indices in the evaluation of the ecological status and the trophic state of the longest lake in Poland

Due to the intensive mixing polymictic lakes should be homogenous. However, morphometric diversity and high water dynamics contribute to the differentiation of many parameters in various areas of the lakes. This study analyzes both phytoplankton and zooplankton to assess differences in water quality along the north–south axis of the longest lake in Poland. New phytoplankton indicators were applied for determining the lake's ecological status: the Q index based on functional groups and the PMPL (Phytoplankton Metric for Polish Lakes) index based on phytoplankton biomass. TSI_ROT index (Rotifer Trophic State Index), which comprises the percentage of species indicating a high trophic state in the indicatory group and the percentage of bacteriovorus in the Rotifera population, was used for zooplankton analysis.TP content was different at different sites – we observed its gradual increase from the south to the north. Spatial variation of phosphorus did not considerably affect plankton diversity. The phytoplankton was dominated by Oscillatoriales, typical of shallow, well-mixed eutrophic lakes. The ecological status of the lake based on the EQR (Ecological Quality Ratio) was poor or moderate. The zooplankton was dominated by rotifers (at almost all sites), which indicates a eutrophic state of the lake. The values of phytoplankton indices at the studied sites did not differ considerably; the differences resulted more from local conditions such as the contaminant inflow and the macrophyte development than water dynamics.We have demonstrated that in the lake dominated by filamentous Cyanobacteria the ecological status should be determined according to the PMPL index or other indices dependent on the dominant Cyanobacteria species. Since the Q index does not include the functional group S1, the results can lead to the false conclusion that water quality improves with an increased amount of phytoplankton. The high abundance of Cyanobacteria in the lake may have contributed to the poor growth of rotifers.     

Decoupling of pelagic and littoral food webs in oligotrophic Canadian Shield lakes

The importance of top-down effects of piscivorous fish on phytoplankton in natural oligotrophic lakes is still debated. In this study, we analyzed patterns in phytoplankton and zooplankton abundance in 37 oligotrophic Canadian Shield lakes in relation to variations in both piscivorous fish predation and resources (total phosphorus; TP). Zooplankton community structure (but not total biomass) was partially affected by the variation in fish predation while the phytoplankton community structure and total biomass showed no response. Carbon isotope analyses revealed that the lack of top-down effects is due to the uncoupling of the littoral and the pelagic food webs. We found that the fish community depends mostly on benthic resources, suggesting that only low planktivory occurred in our study lakes. Due to the absence of specialized zooplanktivorous fish, zooplankton is poorly exploited in these lakes and thus able to control phytoplankton by grazing. A comparison of our data with published studies on the TP–chlorophyll a relationships in both natural and manipulated systems shows that the phytoplankton biomass per unit of TP is relatively low in Canadian Shield lakes.     

Site-specific chlorophyll reference conditions for lakes in Northern and Western Europe

The Water Framework Directive (WFD) requires EU Member States to assess the “ecological status” of surface waters. As a component of ecological status, many European countries are developing a classification scheme for chlorophyll concentrations as a measure of phytoplankton biomass. The chlorophyll classification must be based on the degree of divergence of a water body from an appropriate baseline or ‘reference condition’. This article describes the development of a series of regression models for predicting reference chlorophyll concentrations on a site-specific basis. For model development, a large dataset of European lakes considered to be in reference condition, 466 lakes in total, was assembled. Data were included from 12 European countries, but lakes from Northern and Western Europe dominated and made up 92% of all reference lakes. Data have been collated on chlorophyll concentration, altitude, mean depth, alkalinity, humic type, surface area and geographical region. Regression models were developed for estimating site-specific reference chlorophyll concentrations from significant predictor ‘typology’ variables. Reference chlorophyll concentrations were found to vary along a number of environmental gradients. Concentrations increased with colour and alkalinity and decreased with lake depth and altitude. Forward selection was used to identify independent explanatory variables in regression models for predicting site-specific reference chlorophyll concentrations. Depth was selected as an explanatory variable in all models. Alkalinity was included in models for low colour and humic lakes and altitude was included in models for low colour and very humic lakes. Uncertainty in the models was quite high and arises from errors in the data used to develop the models (including natural temporal and spatial variability in data) and also from additional explanatory variables not considered in the models, particularly nutrient concentrations, retention time and grazing. Despite these uncertainties, site-specific reference conditions are still recommended in preference to type-specific reference conditions, as they use the individual characteristics of a site known to influence phytoplankton biomass, rather than adopt standards set to generally represent a large population of lakes of a particular type. For this reason, site-specific reference conditions should result in reduced error in ecological status classifications, particularly for lakes close to typology boundaries.