Environmental Pollution 157 (2009) 18411848

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

SPEAR indicates pesticide effects in streams Comparative use of species- and family-level biomonitoring data
fer a, C.A. Schriever a, A. Sacchi b, E. Capri b, J. Biggs c, C. Wells d, M. Liess a M.A. Beketov a, *, K. Foit a, R.B. Scha
a

UFZ Helmholtz Centre for Environmental Research, Department System Ecotoxicology, Permoserstrasse 15, D-04318 Leipzig, Germany ` Cattolica del Sacro Cuore, Istituto di Chimica Agraria ed Ambientale, Piacenza, Italy Universita c Pond Conservation, c/o Oxford Brookes University, Headington, UK d Environment Agency of England and Wales, Science Department, Bristol, UK
b

We show that SPEARpesticides can be based on family-level biomonitoring data and is applicable for large-scale monitoring programmes to detect and quantify pesticide contamination.

a r t i c l e i n f o
Article history: Received 18 September 2008 Received in revised form 22 January 2009 Accepted 25 January 2009 Keywords: Bioassessment Large-scale Macroinvertebrates Pesticides Streams SPEAR Traits

a b s t r a c t
To detect effects of pesticides on non-target freshwater organisms the Species at risk (SPEARpesticides) bioindicator based on biological traits was previously developed and successfully validated over different biogeographical regions of Europe using species-level data on stream invertebrates. Since many freshwater biomonitoring programmes have family-level taxonomic resolution we tested the applicability of SPEARpesticides with family-level biomonitoring data to indicate pesticide effects in streams (i.e. insecticide toxicity of pesticides). The study showed that the explanatory power of the family-level SPEAR(fm)pesticides is not signicantly lower than the species-level index. The results suggest that the family-level SPEAR(fm)pesticides is a sensitive, cost-effective, and potentially European-wide bioindicator of pesticide contamination in owing waters. Class boundaries for SPEARpesticides according to EU Water Framework Directive are dened to contribute to the assessment of ecological status of water bodies. 2009 Elsevier Ltd. All rights reserved.

1. Introduction Efcient management of freshwater resources, including conservation of aquatic ecosystem services and biodiversity, is one of the major challenges to modern society. Such management requires tools to detect and quantify effects of various anthropogenic stressors. Among the stressors affecting freshwater ecosystems, pesticides are particularly important, as they can inuence structure and functional parameters of biological communities fer et al., 2007; Liess et al., (Liess and Von der Ohe, 2005; Scha 2008a), have wide-spread exposure (Schriever and Liess, 2007), and can affect all taxonomic groups of aquatic organisms including microorganisms (DeLorenzo et al., 2001), invertebrates (Hata fers et al., 2006), plants (Frankart keyama and Yokoyama, 1997; Scha et al., 2003), and sh (Grande et al., 1994). However, the effects of pesticides in freshwater ecosystems remain elusive, as detection and evaluation of their effects require specic methods (Liess et al., 2005, 2008a).

* Corresponding author. Tel.: 49 341 235 1498; fax: 49 341 235 1785. E-mail address: mikhail.beketov@ufz.de (M.A. Beketov). 0269-7491/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.01.021

The difculties in detection and evaluation of pesticide effects on aquatic ecosystems are two-fold. First, specic exposure patterns hinder chemical monitoring of pesticide concentrations pesticide pollution is mostly diffuse and transient and often occurs at low concentrations (Guo et al., 2004; Leu et al., 2004; Richards and Baker, 1993; Comoretto et al., 2008). Second, variability in biological communities there is a high diversity in communities inhabiting freshwaters, and these communities are affected by numerous factors including natural and anthropogenic stressors that may confound effects of pesticides (Beketov and Liess, 2008a). All this makes attributing any observed changes in biological communities to pesticide contamination complicated (Liess and Schulz, 1999; Hose et al., 2003; Liess et al., 2005; Anderson et al., fers et al., 2006). 2006; Scha To tackle these difculties, the pesticide-specic bioindicator system SPEAR (SPEcies At Risk) was developed (Liess et al., 2001a; Liess and Von der Ohe, 2005) and successfully employed to link pesticide exposure and effects (namely insecticide toxicity of fer et al., 2007; Liess et al., 2008a). The main pesticides) (Scha advantage of the SPEAR system is that this system is based on biological traits of stream invertebrates, and not on taxonomic composition or abundance parameters like many conventional

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bioassessment indices (e.g. EPT, Lenat, 1988). Therefore, it is relatively independent from confounding factors, and application of this system is not constrained by geographical and geomorphological factors and associated differences in biological communities. Furthermore, missing information on biological traits of species can be extrapolated from closest phylogenetic relatives for which such information is available. The SPEAR bioindicator is based on biological traits responsive to the effects of pesticides (i.e. insecticide toxicity of pesticides physiological sensitivity, spatio-temporal co-occurrence of organisms and toxicants) and post-contamination recovery (generation time, migration ability). Validation of the SPEAR system has shown that it is (i) exceptionally sensitive to pesticide contamination (ii) relatively independent of abiotic environmental factors other than pesticides, and (iii) applicable across different biogeographical regions in Europe (Liess and Von fer et al., 2007; Schriever et al., 2007; Von der der Ohe, 2005; Scha Ohe et al., 2007; Liess et al., 2008a). The capability of the currently applied bioassessment methods to detect effects of pesticides is very low, as they were not designed hmer et al., 2004). to detect effects of this specic stressor (e.g. Bo Recent analysis of available large-scale pesticide- and biomonitoring data has shown that such conventional bioassessment indices as BMWP (Armitage et al., 1983), Saprobic Index (Friedrich, 1990), EPT (Lenat, 1988), and Species Number do not correlate well with pesticide contamination and vary signicantly with environmental variables such as pH, current velocity, and temperature (Liess et al., 2008a). However, there is a clear and acute need for pesticidespecic methods in routine biomonitoring programmes in Europe as well as other continents, because pesticides are shown to be a widespread and relevant stressor (Castillo et al., 2006; Schriever and Liess, 2007; Liess et al., 2008a). In recent decades, environmental policies increasingly focused on sustaining and improving water quality across large geographical areas. In Europe, the EU Water Framework Directive stipulated protection of European river basins in its totality and aimed to achieve at least a good ecological status for all water bodies (European Commission, 2000). Hence, indicators applicable across national borders are more desirable than a diverse set of local indicators, which require inter-calibration (Sandin and Hering, 2004; Birk and Hering, 2006). The SPEAR bioindicator is a promising candidate for inclusion in biomonitoring programmes under to the EU Water Framework Directive, because it is sensitive to pesticide contamination and applicable across different biogeo fer et al., 2007; graphical regions (Liess and Von der Ohe, 2005; Scha Von der Ohe et al., 2007; Beketov et al., 2008a; for petrochemicals and surfactants see: Beketov and Liess, 2008a). The SPEAR system has been developed and validated using data based on species level of taxonomic identications. However, in many countries routine bioassessment of surface waters utilise family level of taxonomic resolution (Alba-Tercedor and SanchezOrtega, 1988; Barbour et al., 1999; Smith et al., 1999; Environment Agency, 2008), because of cost efciency, lack of time and human resources, and lack of taxonomic expertise (Marshall et al., 2006; Chessman et al., 2007). Hence, the question arises can the SPEAR system be effectively used with family-level data? Such a familylevel bioindicator would be a cost-effective and widely applicable bioassessment tool focused on detecting and quantifying pesticide contamination over large territories. The aim of the present study was to test applicability of the pesticide-specic SPEAR index based on the family level of taxonomic resolution for detection of pesticide effects in European streams. In particular, we aimed to compare the effectiveness of the indices based on species and family levels. In addition, to contribute to the assessment of the ecological status of water bodies in Europe and to allow for the inclusion of the SPEAR indicator into

monitoring programmes according to EU Water Framework Directive we aimed to dene SPEAR boundaries for ve ecological status classes (referring to pesticide pollution) according to the EU Water Framework Directive (European Commission, 2000).
2. Materials and methods 2.1. SPEAR bioindicator system: terminology update and classication SPEAR (SPEcies At Risk) is a stressor-specic bioindicator system designed by combining biological traits responsive to the effects of particular stressors and associated with recovery. Currently the SPEAR system includes two types of indicators designed for two different types of contaminants (particularly for two different exposure scenarios of organic toxicants): pesticide-specic SPEAR designed for agricultural pesticides occurring in water in short-term pulses (Liess and Von der Ohe, 2005) and (ii) SPEARorganic that is specic for organic toxicants with a relatively constant exposure regime (e.g. synthetic surfactants, petrochemicals) (Beketov and Liess, 2008a). To identify the bioindicators according to the stressor type, we introduce here the title SPEARpesticides for the pesticide-specic SPEAR (similarly to SPEARorganic, Beketov and Liess, 2008a) instead of the index name %SPEAR(abundance) and related indices used previously (Liess and Von der Ohe, fer et al., 2007; Schriever et al., 2007; Von der Ohe et al., 2007). Therefore, 2005; Scha the SPEAR bioindicator system includes the SPEARpesticides and SPEARorganic indices focused on respective stressors (see also Beketov et al., 2008a). To differentiate the SPEARpesticides indices based on different levels of taxonomic resolution we introduce the terms SPEAR(sp)pesticides and SPEAR(fm)pesticides for the indices based on species (or other lowest possible level) and family levels of taxonomic identication respectively. 2.2. Data set In order to compare the species and family-level indices SPEAR(sp)pesticides and SPEAR(fm)pesticides, we used available biomonitoring data sets for small streams in three different biogeographical regions of Europe (Liess and Von der Ohe, 2005; fer et al., 2007) (Fig. 1). These are the data sets from the Fennoscandian shield Scha region (Finland, Fig. 1a), Western plains region (France, Fig. 1b), and Central plains (Germany, Fig. 1c) (classication of biogeographical regions according to Illies, 1978). The data sets include the results of extensive pesticide measurements (with shortterm peak concentrations measured), information on macroinvertebrate community composition (abundance of species), relevant landscape characteristics (presence of undisturbed upstream reaches), and sets of basic water quality parameters (pH 6.458.6, ammonium 01.75 mg/L, nitrate 047.5 mg/L, nitrite 00.8 mg/L, orthophosphate 03.5 mg/L, min. max.). In total, the data sets comprise information on 48 sampling sites (Fig. 1). The samples were collected during the periods of intensive pesticide usage two times (Finland July and August, France April and May, Germany May and June) with a Surber sampler (area 0.062 m2, four replicate samples per each site/date). The streams investigated were characterised by the following: current velocity ranging between 0.1 and 0.5 m/s, maximum stream depth of 0.8 m, no drying up in summer, no dredging in the present or past year, presence of adjacent elds with vegetable, corn, sugar beet, or oil-seed crops. The sites were checked in eld survey and with maps to have no waste-water treatment plants, industrial facilities or mining drainage upstream. Thus, pollution other than from agricultural sources was unlikely (for details see Liess and Von der Ohe, 2005; fer et al., 2007). Scha 2.3. Computation of SPEARpesticides index The SPEARpesticides is based on dichotomic classication of taxa into species at risk and species not at risk (can be any taxonomic category, e.g. genus, family etc.) according to the following biological traits: physiological sensitivity to organic toxicant (Sorganic, developed by Wogram and Liess, 2001, revised by Von der Ohe and Liess, 2004), generation time, presence of aquatic stages in water during the maximum pesticide usage period, and migration abilities. These traits are used for dening species at risk according to criteria dened in Liess and Von der Ohe (2005) with the only exception that the parameter presence of aquatic stages during maximum pesticide exposure is now dened not only according to emergence time of merolimnic insects, but also by considering duration of the adult lifespans. A taxon is regarded as a species at risk only if it has: (i) Sorganic value >0.36 (ii) generation time 0.5 year (iii) aquatic stages which are unable to avoid exposure during the periods of intensive pesticide usage, and (iv) low migration abilities (for details see Liess and Von der Ohe, 2005). The taxon-specic biological traits are compiled in the freely available SPEAR database and are regionally differentiated according to potential differences in the life-cycle traits (Liess et al., 2008b). Currently the database comprises information for the following regions: Central Europe, Finland, UK, and Western Siberia. For computation of the SPEARpesticides indices for Germany and France the information from Central Europe was used, and respectively, such information from Finland was used to derive the indices for

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Fig. 1. Maps of the sampling sites in Finland (A), France (B), and Germany (C). Large rivers and some of the big cities are shown only. Sampling streams are not displayed due to scale.

sampling sites in this region. Classication of species at risk and species not at risk is performed automatically by the algorithm included in the database. In order to adapt the SPEAR database for the family level of taxonomic resolution, the family-level SPEAR denitions (whether family at risk or not at risk) were derived for each species included in the database. These denitions were calculated according to the majority of the species comprising the family (50%). Hence, all species from a particular family have the same family-level SPEAR denition for a particular region. SPEAR denitions at the family level for different regions are collected in the database. As for species, dening of the families at risk is performed automatically by the algorithm included in the SPEAR database (Liess et al., 2008b). After dening the species and families at risk the SPEARpesticides index was computed as relative abundance of these taxa for each site and date as follows: Pn i 1 logxi 1$y $100 SPEARpesticides P n i 1 logxi 1 where n is the number of taxa, xi is the abundance of the taxon i and y is 1 if taxon i is classied as at risk, otherwise 0. These calculations were performed for the lowest possible identied taxonomic levels (down to species level) to dene SPEAR(sp)pesticides and for the families to dene SPEAR(fm)pesticides. The ranges of SPEAR(sp)pesticides and SPEAR(fm)pesticides were 2.1469.41 and 4.8669.71 respectively. Calculations of the indices were made using the program SPEAR Calculator (UFZ, Leipzig, Germany) that is freely available on the internet (http://www.systemecology.eu/SPEAR/Start.html), and was recently developed to automate computation of the SPEARpesticides indices. This program derives site- and date-specic values of both the species- and family-level indices automatically using information on all the relevant traits accumulated in the SPEAR database (Liess et al., 2008b). 2.4. Expression of pesticide-related water toxicity To compare the toxicity associated with the pesticide concentrations measured in the sampling sites, toxic units (TU) were computed from the maximum peak water concentrations measured at each site according to Liess and Von der Ohe (2005): TUDaphnia magna maxn i1 logCi =LC 50i

LC50s of D. magna were used for toxicity assessment (for discussions on relations between laboratory toxicity values and eld effect see Hose et al., 2003; Hose and van den Brink, 2004; Kefford et al., 2006; Beketov et al., 2008b), and (ii) only the maximum toxic unit was considered instead of the sum toxicity of the pesticides detected at the respective site. Obviously, this method is only an estimation of water toxicity, as it does not consider possibility of shorter peak and longer low concentrations. However, we assume that this method estimates toxicity well, as post-peak low concentrations are not relevant when high peaks are present. Higher toxicity due to delayed effects (Beketov and Liess, 2008b; Beketov et al., 2008b) and higher sensitivity due to environmental stressors and biological interactions (Liess et al., 2001b; Duquesne and Liess, 2003; Beketov and Liess, 2006) are expected to be reected by SPEARpesticides, as effects at concentration levels much below the 48-h LC50s D. magna have been detected with this indicator previously (Liess and Von der fer et al., 2007). Ohe, 2005; Scha 2.5. Data analysis Prior to the analysis, the average values for the two sampling dates were calculated for all variables that were measured twice at each site, in order to avoid temporal pseudoreplication. The data set for the streams having upstream undisturbed reaches (recovery areas) was analysed separately from the set for the streams without undisturbed reaches, as it had previously been shown that the presence of such reaches signicantly inuenced the correlation between pesticide exposure fer et al., 2007; Schriever and observed effect (Liess and Von der Ohe, 2005; Scha et al., 2007). The relationship between the SPEARpesticides indices and water toxicity was analysed by linear regression. Analysis of covariance (ANCOVA) was applied to check for signicant differences in slope and intercept between the regression models for SPEAR(sp)pesticides and SPEAR(fm)pesticides. To compare actual values of these two indices a paired t-test was used, with the data points paired for each observation site. To dene water toxicity classes of the sampling sites and to compare the SPEAR indices across the studied countries, the sites were grouped according to their TU as: uncontaminated (reference) (TU < 4) slightly contaminated (4 < TU < 2) highly contaminated (TU > 2) The class boundaries (4 and 2) were selected to roughly classify the sites fer et al., according to pesticide contamination levels (slightly modied after Scha 2007). The limit of 4 TU was chosen for dening uncontaminated sites as biological effects of pesticides are highly unlikely at such negligible concentration level (effects have been rarely shown in literature, Liess, 2002; Beketov and Liess, 2005). The use fer et al., 2007) yielded the of different class boundaries (3.5 and 2.25, as in Scha

where TU(Daphnia magna) is the maximum number of toxic units of the n pesticides detected at the considered site, Ci is the concentration (mg/L) of pesticide i and LC50i is the 48-h LC50 of pesticide i for Daphnia magna (mg/L) as given in Tomlin (2001). Although peak water concentrations could be underestimated due to a delayed response of the sampling equipment, we assume that the computed TU is an estimation of pesticide toxicity since (i) pesticide concentrations usually decrease strongly within 24 h during runoff (Richards and Baker, 1993) and therefore 48-h

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same results. To compare values of SPEAR(fm)pesticides for the study sites in Finland, France, and Germany, two-way analyses of variance (ANOVA) were applied with the factors region and TU. In this analysis sites with and without recovery areas were analysed together due to limited amount of uncontaminated and heavily contaminated sites without recovery areas in France. Levenes test was used to test for homogeneity of variances. As variances were not homogeneous in all cases, Games-Howell post-hoc test (robust with respect to the potential deviations from normality or variance homogeneity) was used to compare the site groups with different contamination levels. Denition of boundaries for ve ecological status classes according to EU Water Framework Directive (bad, poor, moderate, good, and high, European Commission, 2000) was begun by establishing the boundary between good and moderate classes. This boundary was dened as the minimum observed value of SPEAR(fm)pesticides derived for uncontaminated sites (dened as sites with TU < 4, for rationale to take this limit see above). The rationale of this conservative approach was to prevent false positive detections of contamination (e.g. to classify uncontaminated sites as contaminated). Values of SPEAR(sp)pesticides and SPEAR(fm)pesticides indices were ranked in increasing order to inspect for the presence of distinct groups. As no groups were found, and the gradients of values were considered even, the entire ranges of values were equidistantly divided into 3 and 2 classes below and above the border between good and moderate classes established previously. Use of the same interval between the classes resulted in relatively bigger size of the class high that was considered realistic, as uncontaminated sites are characterised by high variability of the SPEARpesticides values. Zero value taken as a lowest possible limit indicates that species or families at risk are absent. However, this does not exclude that respective biological communities are taxonomically rich and abundant but consist of taxa not at risk only. The analyses were performed using programs STATISTICA 7.1 for Windows (StatSoft, Tulsa, OK, USA), SPSS 11.0 for Macintosh (SPSS Inc., Chicago, Illinois), and Prism 4.0c for Macintosh (GraphPad Software, San Diego, USA).

3. Results All the linear regression models between the SPEAR indices and water toxicity were statistically signicant (p < 0.001) with higher values of the determination coefcient r2 for the sites without upstream recovery areas (Fig. 2). Variation in both the species-level
80

Recovery area absent

species level family level

60

40

20

r2 = 0.73 r2 = 0.77

0 -6 80 -5 -4 -3 -2 -1 0

Recovery area present

60

40 r2 = 0.48 20 r2 = 0.49

0 -6 -5 -4 -3 -2 -1 0

Toxic Units(Daphnia magna)

Fig. 2. Linear regressions for the SPEARpesticides indices based on family and species levels of taxonomic resolution and water toxicity expressed as Toxic Units (Daphnia magna). Sites with and without upstream recovery areas are analysed separately (n 27 and 21 respectively). The slopes and intercepts for the species- and family-level indices are not signicantly different (p > 0.05, ANCOVA) in both the sites with and without the recovery areas.

index SPEAR(sp)pesticides and the family-level index SPEAR(fm) pesticides was explained well by the pesticide toxicity (Fig. 2). There were no signicant differences in slopes or intercepts of the linear regressions for SPEAR(sp)pesticides and SPEAR(fm)pesticides for both the sites with and without upstream recovery areas (p > 0.05, ANCOVA, n 27 and 21 respectively). Comparison of actual values of SPEAR(sp)pesticides and SPEAR(fm)pesticides by paired t-test showed signicant differences between them for both the streams with and without upstream recovery areas (p < 0.01, n 27 and 21 respectively), with SPEAR(fm)pesticides values being higher for the same sample. To reveal the sources of differences between species- and family-level indices, the basic SPEAR database (Liess et al., 2008b) used for computation of the indices (not the eld-collected data set) was checked for the number of discrepancies between the species- and family-level SPEAR denitions (i.e. number of species at risk in families classied as not at risk and vice versa). This analysis has shown that for regional groups Central Europe and Finland included in the database the species/family discrepancies comprise a very small part of the entire amount of taxa entries (0.4 1.2%, Table 1). To understand which particular species were responsible for the differences between species- and family-level indices in the present study, the entire data set analysed here (not the SPEAR database) was checked for the discrepancies. This analysis revealed that only ve species had SPEAR denitions different from their family-level denitions (Table 2). These ve species comprise 1.8% of the overall number of species present in the analysed data set (281 species), which is comparable to the proportion of exceptional species in the SPEAR database (Table 1, Liess et al., 2008b). In all cases, the species were classied as not at risk due to specic species-level traits, although they belong to families classied at risk (Table 2). Notably high abundance and occurrence in the samples were found for two of these species: caddisy Anabolia nervosa and mayy Baetis vernus (Table 2). These species are classied as not at risk because of the good in-stream migration abilities (avoidance movement and drift, Liess and Von der Ohe, 2005) and short generation time in central Europe (3 generations per year, Faasch, 1999) respectively. Comparison of the family-level SPEAR(fm)pesticides values for the study sites in Finland, France, and Germany (Fig. 3) performed with two-way ANOVA showed that effect of the factor region is insignicant (p > 0.05, n 48). In contrast, effect of the factor TU was statistically signicant (p < 0.001, n 48). Interaction between the factors was insignicant (p > 0.05, n 48). The post-hoc Games-Howell test has shown signicant differences between the uncontaminated sites and both slightly and highly contaminated sites (p < 0.004, Fig. 3). However, no signicant difference was found between the slightly and highly contaminated sites (p > 0.05, Fig. 3). In order to evaluate the effect of upstream recovery areas on the species- and family-level SPEAR indices the linear regressions derived for these indices and water toxicity (Fig. 2) were compared between the sites with and without recovery areas. For both SPEAR(fm)pesticides and SPEAR(sp)pesticides, intercepts at sites with upstream recovery area were signicantly higher than at sites without (p < 0.05, ANCOVA), but the slopes were not signicantly different (p > 0.05, ANCOVA) between the sites with and without recovery areas. The boundaries of the ve classes of ecological status according to the EU Water Framework Directive were dened as follows: bad < 11  poor < 22  moderate < 33  good < 44  high (Fig. 4). As the ranges of the species- and family-level SPEAR indices were similar, the same boundaries have been dened for both of these indices (Fig. 4).

SPEARpesticides

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Table 1 Analysis of the SPEAR database: numbers of families classied as at risk and not at risk, their percentage of total number of families, and species/family discrepancies in SPEAR denitions in these families for regional groups Central Europe and Finland. Central Europe Number of families (% of total) Families at risk Families not at risk 59 (39.3) 91 (60.7) Number of species/family discrepancies (% of total) 13 (1.2) 4 (0.4) Finland Number of families (% of total) 59 (39.3) 91 (60.7) Number of species/family discrepancies (% of total) 12 (1.1) 4 (0.4)

4. Discussion In bioassessment of freshwaters the question of the required level of taxonomic identications is repeatedly and intensively discussed. Use of species-level information is, in general, expected to yield more precise results as compared with data based on higher taxonomic levels (Resh and McElravy, 1993; Hawkins and Norris, 2000; Schmidt-Kloiber and Nijboer, 2004; Waite et al., 2004). However, data on higher taxonomic levels, such as family level, are frequently used because of cost efciency, lack of time and human resources, and lack of taxonomic expertise (Marshall et al., 2006; Chessman et al., 2007). Indeed, a number of studies have shown that for many indices family-level data are sensitive enough to detect and quantify environmental impacts (Bournaud et al., 1996; Bowman and Baily, 1997; Chessman et al., 2007), and can yield even better results than species-level information (Reynoldson et al., 2001). As a result, family-level taxonomic resolution is used in many countries for routine bioassessment of surface waters (Alba-Tercedor and Sanchez-Ortega, 1988; Barbour et al., 1999; Smith et al., 1999; Environment Agency, 2008). However, for many indices use of the higher levels of taxonomic identication can result in misclassication of the ecological status of investigated sites, with variable deviations from the species-level results, and is therefore not suitable (e.g. AQEM Assessment Software, SchmidtKloiber and Nijboer, 2004). Results of the present study have shown that the SPEAR(fm)pesticides index based on the family level of taxonomic resolution can be effectively used for detection of pesticide contamination. As shown by the ANCOVAs, the explanatory power of this index is only slightly and non-signicantly lower than that of the SPEAR(sp) pesticides index. However, there were occasional differences in the absolute values of SPEAR(sp)pesticides and SPEAR(fm)pesticides due to discrepancies in SPEAR denitions for some species and their respective families. Fortunately the number of such discrepancies was very small for both the basic SPEAR database (Liess et al., 2008b) and the eld-collected data set analysed in the present study. The contribution of the species differing in SPEAR denitions from the respective families (mainly abundant species A. nervosa and B. vernus, Table 2) resulted in the observed signicant differences between actual values of SPEAR(sp)pesticides and
Table 2 Analysis of the eld-collected data sets: species having SPEAR denitions different from their family-level denitions (i.e. species classied as not at risk, but from families classied as at risk) due to differences between the species- and familyspecic trait characteristics. Occurrence in samples (percentage of samples containing the species) and mean abundance (individuals per sample, standard deviations in parentheses) are given. Species Occurrence in samples, % 32.3 25.0 1.0 4.2 6.2 Mean abundance, individuals per sample (standard deviation) 46.3 15.9 22 4.0 2.8 (119.1) (24.6) (0) (2.8) (2.4)

SPEAR(fm)pesticides. Nevertheless, across the levels of impact, most of the sites were characterised by very similar values of SPEAR(sp)pesticides and SPEAR(fm)pesticides that exhibited no signicant differences concerning the slopes and intercepts in the regression models (Fig. 2). There are two possible reasons of the observed uniformity between the species- and family-level SPEARpesticides indices. The rst is that within-family variability of the biological traits used in the SPEAR system may be much lower than the between-family variability. Several studies have shown that variability in sensitivity to toxicants and biological traits within families was signicantly lower than the variability between families (or other high taxonomic levels) (Hoekstra et al., 1994; Bonada et al., 2004; Von der Ohe and Liess, 2004). The second reason is that due to a paucity of information on the traits, data on traits for some species in the SPEAR database were extrapolated from higher taxonomic levels to the species level. Therefore, the high resemblance between species and family levels could partly be an artefact resulting from the extrapolation of data. However, this hypothesis cannot be tested since for many taxa, species-level ecological information is not likely to become available in the near future. The possibility to effectively apply SPEARpesticides indicator at the family level of taxonomic resolution suggests that this bioindicator can be easily included in routing biomonitoring programmes in various countries, as family level of taxonomic identications is usually the highest taxonomic level used, and often the suggested level (Alba-Tercedor and Sanchez-Ortega, 1988; Barbour et al., 1999; Smith et al., 1999; Environment Agency, 2008). Besides, usage of the family-level data in comparison to the species-level data requires much less resources (Marshall et al., 2006; Chessman et al.,

80 a

Finland France Germany b b

SPEAR(fm)pesticides

60

40

20 n= 12 0 < -4 Reference -4 to -2 Slightly > -2 Highly 5 5 0 9 7 0 2 8

Toxic Units(Daphnia magna) Contamination levels

Fig. 3. Comparison of the family-level SPEAR(fm)pesticides indices for the study sites in Finland, France, and Germany characterised as uncontaminated, slightly, and highly contaminated (TU < 4, 4 < TU < 2, and TU > 2 respectively). Sites with and without recovery areas are analysed together. Different letters indicate signicant differences between the site groups (p < 0.05, two-way ANOVA, Games-Howell posthoc test).

Anabolia nervosa Baetis vernus Cloeon dipterum Micropterna lateralis Oligostomis reticulata

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Species level 66 Familiy level

SPEARpesticides

55 High 44 Good 33 Moderate 22 Poor 11 Bad 0 0 10 20 30 40 50

Sample sites (ranked)

Fig. 4. Values of the SPEAR(sp)pesticides and SPEAR(fm)pesticides indices ranked in increasing order (considered independently) and boundaries for the ecological status classes according to EU Water Framework Directive.

Applicability of the SPEAR(fm)pesticides index for detecting and quantifying of pesticide effects in owing waters indicates that this indicator is a promising tool for pesticide post-authorisation monitoring programmes according to EU-directive Concerning the placing of plant protection products on the market (European Commission, 1991). Particularly, the SPEAR(fm)pesticides index can be used to link observed exposure patterns with effects on biological communities and to quantify ecological risks of the pesticide applications. Successful application of the SPEAR approach to detect and quantify contamination with organic toxicants in watercourses of Western Siberia (Beketov and Liess, 2008a) indicates that the index SPEAR(fm)pesticides can be applicable in different continents beyond Europe. Adaptation of this index to specic regions will require region-specic information on the life-cycle traits, which can be easily used in automatised computation of SPEAR indices (e.g. recent adaptation of SPEARpesticides for UK, Beketov et al., 2008a). 5. Conclusions

2007). All this suggests that SPEAR(fm)pesticides index is cost-effective and widely applicable bioassessment tool. Comparison of the family-level SPEAR(fm)pesticides values for the site groups in Finland, France, and Germany has shown that this index is potentially applicable over these different biogeographical regions (Fig. 4). This nding is in accordance with the results fer previously found for the species-level SPEAR bioindicator (Scha et al., 2007), and shows that this index can be used as a Europeanwide metric of pesticide contamination of owing waters. This also suggests that SPEAR(fm)pesticides is a promising candidate to be included into the biomonitoring programmes according to the EU Water Framework Directive, as indicators applicable across national borders are more desirable than local indicators (European Commission, 2000). Besides, as mentioned in the introduction, the capability of the bioassessment methods currently applied in Europe to detect effects of pesticides is very low (Liess et al., 2008a). This lays special emphasis on inclusion of the SPEARpesticides into the European bioassessment programmes, as achievement of good ecological status stipulated by European Commission (2000) is problematic without consideration of pesticide effects. To further facilitate the inclusion of the SPEAR indices into EU bioassessment programmes we have established classes of ecological status, which are consistent with those required under the EU Water Framework Directive (European Commission, 2000). The approach we used in the present study includes initial dening of the border between good and moderate classes as a lowest observed index value derived for uncontaminated sites, and subsequent division of the range into equidistant classes. The class high however is bigger than other classes, as uncontaminated sites are characterised by high variability of the SPEARpesticides values. Simple division of the entire observed range into equidistant classes without setting the border between contaminated and uncontaminated sites (i.e. setting the boundaries as 20, 40, 60, and 80% of the entire range, e.g. Hering et al., 2006) is not suitable for the present case, as it would result in the unrealistically high (42) border between the moderate and good classes and would lead to false classication of reference sites as polluted (20% of sites for our data). Moreover, the borders chosen according to our approach seem to be ecologically reasonable: the boundary of moderate class (33) corresponds to TU of 3.5 in terms of the doseresponse relationship obtained in the present study (Fig. 2). This threshold value, below which effects might occur, is in agreement with the threshold that can be derived fer from the previous eld studies (Liess and Von der Ohe, 2005; Scha et al., 2007), but is nevertheless higher than those observed in mesocosms (Van Wijngaarden et al., 2005; Beketov et al., 2008b).

This study gives an example of a stressor-specic bioassessment index based on biological traits that can be used at the family level of taxonomic resolution and over large geographical territories. This example shows that SPEAR(fm)pesticides bioindicator is an effective bioassessment tool that can be included in routing biomonitoring in different biogeographical regions of Europe and in monitoring programmes according to EU Water Framework Directive as a European-wide index. Potentially it can be also applied in other continents to detect and quantify pesticide effects in surface waters where family-level monitoring data are collected. Use of SPEAR(fm)pesticides bioindicator is now facilitated by webbased and freely available software SPEAR Calculator (http://www. systemecology.eu/SPEAR/Start.html). Acknowledgements The study was supported by the Environment Agency of England and Wales (project SC030189/SR4 Freshwater biological indicators of pesticide contamination An adaptation of the SPEAR approach for the UK) and by the European Union (project INTERACT, Marie Curie IIF contract No. MIF1-CT-2006-021860). References
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