Environmental Pollution 156 (2008) 980987

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

An indicator for effects of organic toxicants on lotic invertebrate communities: Independence of confounding environmental factors over an extensive river continuum
Mikhail A. Beketov*, Matthias Liess
UFZ Helmholtz Centre for Environmental Research, Department of System Ecotoxicology, Permoserstrasse 15, D-04318 Leipzig, Germany

Indicator for organic toxicants at community level can be independent of natural environmental factors.

a r t i c l e i n f o
Article history: Received 22 November 2007 Received in revised form 20 March 2008 Accepted 3 May 2008 Keywords: Bioassessment Macroinvertebrates Organic toxicants River continuum Sensitivity SPEAR

a b s t r a c t
Distinguishing between effects of natural and anthropogenic environmental factors on ecosystems is a fundamental problem in environmental science. In river systems the longitudinal gradient of environmental factors is one of the most relevant sources of dissimilarity between communities that could be confounded with anthropogenic disturbances. To test the hypothesis that in macroinvertebrate communities the distribution of species sensitivity to organic toxicants is independent of natural longitudinal factors, but depends on contamination with organic toxicants, we analysed the relationship between community sensitivity SPEARorganic (average community sensitivity to organic toxicants) and natural and anthropogenic environmental factors in a large-scale river system, from alpine streams to a lowland river. The results show that SPEARorganic is largely independent of natural longitudinal factors, but strongly dependent on contamination with organic toxicants (petrochemicals and synthetic surfactants). Usage of SPEARorganic as a stressor-specic longitude-independent measure will facilitate detection of community disturbance by organic toxicants. 2008 Elsevier Ltd. All rights reserved.

1. Introduction Within a river system, from headwaters to the mouth, the ecological conditions vary along a gradient of physicochemical parameters. Understanding of the structure and function of biological communities along such longitude/altitude gradients is one of the main research topics in freshwater ecology (Allan, 1995). Indices and community characteristics used in bioassessment of owing waters are inuenced by longitudinal/altitudinal environmental gradients, since in general the structure and function of communities depend on longitudinal physicochemical factors (e.g. Illies, 1961; Minshall et al., 1985; Paller et al., 2006; Vannote et al., 1980). To take this situation into account, the main method of bioassessment employed at present is to compare a given site with an undisturbed reference site (e.g. Bailey et al., 2004; Hering et al., 2004; Nijboer et al., 2004; Wright et al., 2000). The use of reference sites is stipulated by legislative instruments (e.g. European Commission, 2000). Comparison with a reference condition is also used in efciency assessment of restoration practices (e.g. Harrison et al., 2004). In river systems, the longitudinal variation in the macroinvertebrate

* Corresponding author: Tel.: 49 341 235 1498; fax: 49 341 235 2401. E-mail address: mikhail.beketov@ufz.de (M.A. Beketov). 0269-7491/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.05.005

communities is so great that a large range of reference sites must be established, to cover all the diversity of lotic habitats along a river continuum (e.g. Hering et al., 2004; Nijboer et al., 2004). But in practice establishing such a system is not only costly but also often impossible, because of the absence of undisturbed watercourses from which to derive reference conditions (mainly relatively large streams or rivers), and the great variability of stream biotic and abiotic parameters in time and space (e.g. Ehlert et al., 2002; Bailey et al., 2004; Nijboer et al., 2004). Currently two main approaches are used to isolate effects of anthropogenic disturbances from effects of natural environmental factors. One of them is application of analytical techniques in which natural variability is taken into account by mathematical methods. These are multivariate statistical methods (Leps and Smilauer, 2003) and mathematical modelling methods such as articial neural networks (Cereghino et al., 2003; Park et al., 2004). Another, more direct, approach is to nd a community metric independent of natural environmental factors and therefore stable along gradients of natural factors (e.g. river continuum), yet sensitive to anthropogenic disturbance. In order to nd such a community metric, it seems more promising to employ biological traits (e.g. generation time, body size) rather than taxonomic indices, since it has been shown that many of these traits are stable at a large spatial scale across ecoregions (Bonada et al., 2006). It has also been shown that

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a bioassessment system such as pesticide-specic SPEAR (i.e. Species At Risk), based on sensitivity to organic toxicants and life-cycle traits responsible for recovery, is relatively independent of abiotic environmental factors other than pesticides (Liess and von der Ohe, 2005) and applicable across different biogeographical regions in fer et al., 2007; Von der Ohe et al., 2007). Nevertheless, Europe (Scha to our knowledge, none of the previously analysed biological traits or trait-based indicator systems has been proved to be stable along a rivers longitudinal gradient. We hypothesised that a trait such as physiological sensitivity to synthetic organic toxicants (e.g. pesticides, surfactants, petrochemicals) can be distributed among organisms independently on the longitude/altitude environmental gradient. To our knowledge, no studies have yet been published regarding the distribution of species sensitivity in communities of uncontaminated lotic ecosystems, and nothing is known about longitudinal changes in the sensitivity distribution. The aim of this study was to investigate whether the intercommunity distribution of sensitivity to organic toxicants is independent of natural longitudinal factors, but instead depends on the degree of contamination with such toxicants. For this purpose we analysed the relationship between average community sensitivity to organic toxicants (hereafter SPEARorganic) and natural and anthropogenic environmental factors along a large-scale river continuum (the title SPEARorganic indicates afnity to the SPEAR bioindicator system designed by combining traits responsible for effect of stressors and associated recovery, Liess and von der Ohe, 2005; Liess et al., 2008). Values of the community sensitivity SPEARorganic were calculated as the average of sensitivities assigned to the species comprising the communities (Sorganic according to Von der Ohe and Liess, 2004). We analysed SPEARorganic along with several taxonomic richness and diversity metrics, which are known to be dependent on both anthropogenic stress and longitudinal gradient. These are taxa richness (TR), taxa richness of Ephemeroptera, Plecoptera, and Trichoptera (EPT), and Shannons diversity index (H0 ). We aimed to understand how SPEARorganic varies against the background of well-known community parameters such as TR, EPT, and H0 characterising longitudinal changes (e.g. Minshall et al., 1985; Paller et al., 2006). To achieve this objective we investigated a river system that includes a diverse set of undisturbed watercourses (from snow-fed alpine stream to medium-size lowland river) as well as a range of contaminated watercourses. The study was conducted in southwestern Siberia (Central Asia, Russia) because in this region it is possible to assess different types of pristine streams having no anthropogenic impact, including medium-size lowland rivers (e.g. 25 m width); this is impossible in densely populated areas (Ehlert et al., 2002). Furthermore, fauna and ecology of macroinvertebrates in this region are known (Beketov, 2006a, in press).
2. Materials and methods 2.1. Study area, environmental parameters, sampling and measurement methods The samples were collected during the ice-free periods of 2005 and 2004 in two regions in southwestern Siberia: Novosibirsk Province and Altai Republic (Fig. 1). Novosibirsk Province (Fig. 1a) is situated at the southeastern corner of the WestSiberian Plain (Fig. 1) and characterised by a at relief. In the southeastern region of this province there is an elevated area, the Salairskii Kryazh (up to 502 m above sea level (a.s.l.)). The climate of the region is moist continental mid-latitude with cold winters. Mean January temperature is 18  C, extreme winter temperatures are about 40  C; mean July temperature is 20  C. Precipitation is 500800 mm per year. During the wintertime (from November to April) most of the water bodies are covered with ice. Most of the territories studied here are in the forest-steppe vegetation zone of the province. Watercourses are supplied by a mixture of underground drainage, rain and snow. In contrast to Novosibirsk Province, the Altai Republic (Fig. 1b) is predominantly characterised by mountainous relief. The majority of the mountains are below 2000 m a.s.l. Mean January temperature is 18  C, mean June temperature is 16  C.

Precipitation is 8001600 mm per year. Taiga is the predominant vegetation type, but is replaced by alpine meadow and tundra at elevations higher than 2000 m a.s.l. Watercourses are fed predominantly by a mixture of rain and melted snow patches. The Novosibirsk Province and Altai Republic are linked by the basin of the Ob River, which originates as the Katun River in the Altai Mountains and ows into the Arctic Ocean. All the streams investigated are in this Ob River basin. Environmental parameters of the sample sites are summarised in Table 1. In total 497 samples were taken from 19 sites, at 11 of which there was no anthropogenic impact (Fig. 1). Samples were collected from June to August 2004 and 2005 (Table 2). The sample points were chosen to represent watercourses of different types along the wide range of absolute altitude and stream size (Table 1). The uncontaminated sites were selected by the absence of any human activity upstream of the sampling point with the sole exception of site 11, where there is no human activity (agriculture, industry, and urban activity) for about 30 km upstream of the sample point and further upstream no obvious sources of pollution. Presence/absence of human activity dened in this way was used as one of the environmental factors in the analyses (Table 1). Sites from 12 to 19 were the sample points used for water quality biomonitoring from the years 2002 to 2005 (Beketov, 2006b). A range of chemical parameters for these sites was assessed every 2 months by the Western Siberian Centre for Environmental Monitoring by standard methods. In uncontaminated sites a smaller range of chemical parameters (nitrate, nitrite, ammonia, and phosphate) was assessed in the eld using portable test kits (Merck KGaA, Darmstadt, Germany). Here we use average values for all the sites calculated from all the values measured during the ice-free periods only (Table 1). According to the land use in the study region (lack of intensive agriculture, mechanical-engineering industry) and available chemical measurement data (Table 1), the major toxicants present in the contaminated watercourses, which are thought to have a biological effect, are petrochemicals and synthetic surfactants (explained in Section 4). Chemical measurements of these contaminants were performed according to Russian state standard methods: petrochemicals were measured as the sum of hydrocarbons, resins, asphaltens, and their derivatives by thin-layer chromatography with infrared photometry and luminescence (standard method RD 52.24.454-95); surfactants were measured as the sum of anionic synthetic surfactants (salts of organic sulphonic acids and alcohol ether sulphates) by the extraction-photometry method (standard method RD 52.24.368-95). Altitude was measured by a GPS (e-Trex, Garmin, Olathe, USA) and using maps. Water temperature was measured by a hydrargyric thermometer. Stream width was measured by tape-measures, and for large watercourses using the GPS. Substrates were assigned to seven different size classes according to Hering et al. (2003), and mean particle size of the dominant substrate type was used for the analyses (as semicontinuous variable). Surface current velocity was assessed by timing a oating device (n 3) as it moved over 5 m. Presence/absence of macrophytes was recorded as binary qualitative parameter; periphyton cover was ranked as percentage. Invertebrates were collected using D-frame net (500 mm mesh) on all the stream habitat types present at each sampling site (including stones and pebbles, submerged macrophytes, debris, and ne sediments) to the water depth of 5060 cm. Each sample was taken from a 25 50-cm area of the stream bottom. Animals were preserved in 95% ethanol and identied to the lowest possible taxonomic level in the laboratory. Most of the Ephemeroptera, Odonata, Plecoptera, Trichoptera, Heteroptera, and Coleoptera were identied to the species level, while in all other taxonomic groups the identication level varied from genus to family. The full list of the taxa (121 taxa) is given in Supplementary materials, Table 1. 2.2. Biological indices 2.2.1. Community sensitivity index SPEARorganic Average community sensitivity (SPEARorganic) was calculated as the arithmetic mean of species sensitivities weighted by the log(x 1) transformed abundance of the respective species. This was applied to reduce the weight of high abundances. Values of species sensitivities were derived from Table 1 in the paper by Von der Ohe and Liess (2004) (taxon-specic Sorganic). The taxon-specic Sorganic sensitivity values were calculated by Von der Ohe and Liess (2004) in extensive analysis of the organic compounds toxicity data accumulated in Aquatic Toxicity Information Retrieval (AQUIRE) database by U.S. Environmental Protection Agency (2002). These values reect taxon-specic sensitivity to organic toxicants in general, but not to particular toxicants. They have been calculated as taxa sensitivities relative to sensitivity of Daphnia magna according to the formula:   Si log LC50Daphnia magna =LC50i where Si is the sensitivity of a taxon i (developed by Wogram and Liess, 2001, revised by Von der Ohe and Liess, 2004). The sensitivity values derived in this way for different organic toxicants (for which data was available, 164 organic compounds assessed in 2269 tests) then were averaged to dene general sensitivity for a given taxon (for details see: Von der Ohe and Liess, 2004). D. magna was used as a reference species because toxicity of a great number of toxicants have been evaluated for this standard test species. Usage of this relative rather than absolute sensitivity allows: (i) comparison of physiological sensitivities of many taxa on a uniform scale, and (ii) derivation of a general sensitivity measure for organic toxicants. The taxonspecic Sorganic sensitivity values were calculated for different taxonomic levels with

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Fig. 1. Study areas and sampling sites (A Novosibirsk Region, B Altai Republic; uncontaminated sites are marked as empty triangles).

lowest possible levels (18, 24, 16, and 24 for order- and suborder-, family-, genus-, and species-level Sorganic values, respectively). To our knowledge, this sensitivity ranking system is the only existing system that allows comparison of sensitivities to organic toxicants taken as a whole (this system exists also for metals: Von der Ohe and Liess, 2004). In this study taxon-specic Sorganic values of different taxonomic levels (Table 1, Von der Ohe and Liess, 2004) were applied to assign sensitivity of the macroinvertebrate taxa collected. In particular, for the 121 taxa recorded we used 81, 11, 26, and 2 values of order- and suborder-, family-, genus-, and species-level Sorganic values, respectively (67, 9, 22, and 2%, respectively). When data for a particular taxon was absent, sensitivity values were taken from the higher taxonomic levels. The taxon-specic Sorganic values assigned are listed in the openly available SPEAR database (Liess et al., 2008). 2.2.2. Taxonomic indices TR, EPT, and H0 Along with the SPEARorganic we used several conventional ecological indices to describe taxonomic richness and diversity of the streams investigated. These are taxa richness (TR), EPT taxa richness (EPT), and Shannons diversity index (H0 ). The index TR is the total number of taxa collected in a particular site. EPT is the number of species of Ephemeroptera, Plecoptera, and Trichoptera. Shannons diversity index H0 was calculated as follows: H0 X pi ln pi

variability in response variables (indices), including the variability not related to the explanatory variables (environmental parameters). This method provides a better understanding of relations between the SPEARorganic and the taxonomic indices characterising biological longitudinal changes than do constrained ordination techniques. Ordination axes were admitted as interpretable using the Broken Stick criterion (Jackson, 1993). Prior to the analyses, the indices values were standardised, as they do not share the same unit of measurement. Standardisation to zero population mean, with a standard deviation equal to one, was achieved by subtracting the population mean from an individual value and then dividing the difference by the standard deviation. Analyses of the whole data set were done in two steps: rst only the uncontaminated streams were analysed (to exclude effects of contamination), and then all the streams including uncontaminated and contaminated. These analyses were made using the program CANOCO 4.5 for Windows (Wageningen, the Netherlands) according to Ter Braak and Smilauer (2002) and Leps and Smilauer (2003). In addition to multivariate analyses we correlated SPEARorganic with the detected toxicants (petrochemicals, synthetic surfactants, nitrite, and ammonia), using the linear regression method to report actual values of the index in relation to actual values of the toxicants. This analysis was performed using the program Prism 4.0c for Macintosh (GraphPad Software, San Diego, USA).

3. Results 3.1. Restricted data set: uncontaminated streams The PCA performed for uncontaminated streams showed that the rst and second ordination axes are interpretable; the investigated environmental variables could explain up to 96% of the total variability of the taxonomic biological indices (Table 3, Fig. 2). The rst principal component is correlated mainly with all the factors forming the longitudinal gradient: altitude, temperature, stream width, nutrition status, and velocity. The correlation matrix (inter-set productmoment correlations) of the environmental parameters and ordination axes is shown in Table 4. Metrics TR, EPT, and H0 are closely correlated with the rst principal component; therefore they depend on the longitudinal environmental parameters (Fig. 2). Almost all variabilities of TR, EPT, and H0 are explained by the rst axis (ts for the rst axis are 0.96, 0.94, and 0.82 of the indices variances, respectively). In contrast, SPEARorganic is mainly independent of the longitudinal factors, as it is not well correlated with the rst ordination

where pi is the fraction of individuals belonging to the species i. The indices characterising the macroinvertebrate communities of the watercourses investigated are summarised in Table 2.

2.3. Statistical analyses The number of samples collected at different observation sites varied from 8 to 49 (Table 2). To avoid an inuence of sample quantity on the biological indices, the initial data set was randomly re-sampled to obtain eight samples for each site (except site 1, from which only eight samples were initially collected, Table 2). Only the re-sampled data sets were used in the statistical analyses. Analyses of variability and relations to environmental factors of indices H0 , TR, EPT, and SPEARorganic were performed by linear unconstrained multivariate ordination techniques such as Principle Components Analysis (PCA). The ordination was chosen because it is particularly appropriate for describing continuous changes in complex systems characterised by many parameters. Biological indices and environmental parameters were used as response and explanatory variables, respectively. The linear methods were chosen because of the relatively short length of gradients found by preliminary Detrended Correspondence Analysis. The unconstrained type of ordination (PCA of the biological indices with passive projection of environmental variables) was chosen because it takes into account actual

M.A. Beketov, M. Liess / Environmental Pollution 156 (2008) 980987 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 0.817 (1.057) 0.818 (1.103) 0.807 (1.112) 1.480 (1.985) 0.624 (0.950) 0.901 (1.205) 0.995 (1.340) 0.681 (1.045)

983

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.018 (0.033) 0.089 (0.125) 0.098 (0.135) 0.041 (0.071) 0.067 (0.080) 0.108 (0.125) 0.060 (0.084) 0.011 (0.032)

Table 2 Numbers of samples collected in 2004 and 2005, number of randomly re-sampled samples, mean number of individuals per sample (data for the re-sampled samples, standard errors in parentheses), and community parameters of the sample sites (SPEARorganic average community sensitivity to organic toxicants, TR taxa richness, EPT taxa richness of Ephemeroptera, Plecoptera, and Trichoptera, and H0 Shannons diversity index) Site Initial number of samples 2004 2005 8 4 4 8 6 10 16 18 12 14 24 22 22 22 20 18 15 14 14 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 35.63 77.38 13.63 66.25 20.25 41.13 27.38 93.88 25.00 38.50 69.13 20.50 52.75 14.38 39.13 20.38 11.13 13.50 34.63 (7.58) (9.45) (3.90) (10.66) (1.93) (11.36) (5.23) (20.17) (7.70) (5.70) (16.18) (2.90) (11.14) (2.99) (8.17) (5.01) (1.85) (4.07) (6.36) Number of re-sampled samples Number of individuals per sample Community indices

NO2, mg L1 (max)

NH4, mg L1 (max)

SPEARorganic 0.229 0.329 0.245 0.213 0.103 0.307 0.322 0.321 0.254 0.258 0.338 0.361 0.413 0.464 0.533 0.515 0.589 0.475 0.435

TR 5 7 10 15 6 19 12 24 12 17 45 35 35 17 9 20 16 19 27

EPT 2 4 7 11 5 13 8 14 8 10 29 24 18 8 2 10 7 9 14

H0 1.341 1.367 1.910 1.940 1.551 2.224 1.669 2.612 1.965 2.020 2.802 3.141 2.698 1.814 1.505 2.208 2.387 2.140 2.382

Synthetic surfactants, mg L1 (max)

P.A. P.A. P.A. P.A. P.A. P.A. P.A. P.A. P.A. P.A. P.A. 0.014 (0.040) 0.014 (0.040) 0.017 (0.095) 0.017 (0.105) 0.021 (0.065) 0.025 (0.150) 0.015 (0.055) 0.015 (0.042)

Table 1 Environmental parameters of the sample sites, means (in parentheses standard errors SE (when applicable), and maximum concentrations of contaminants max)

Substrate particles mean size, cm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

5 5 5 6 24 24 27 25 24 20 18 15 14 14

Petrochemical, mg L1 (max)

Presence of human activity Habitat heterogeneity, number of habitat typesa

60 60 30 30 13 60 4 13 4 13 4 4 4 4 30 4 4 4 4

P.A.b P.A. P.A. P.A. P.A. P.A. P.A. P.A. P.A. P.A. P.A. 0.057 (0.136) 0.113 (0.276) 0.200 (0.841) 0.526 (2.767) 0.389 (1.129) 0.305 (1.950) 0.227 (1.331) 0.170 (0.866)

Four habitat types: stones and pebbles, submerged macrophytes, organic debris, and ne sediments. P.A. presumably absent anthropogenic chemicals in the pristine streams not exposed to human activity.

axis (Fig. 2). The rst and second ordination axes cumulatively explain 29% and 99% of variability in the SPEARorganic index (ts for the rst and second axes are 0.29 and 0.99 of the indexs variance, respectively). The PCA ordination diagram (Fig. 2) shows that among the nine environmental factors forming the river longitudinal gradient the parameters habitat heterogeneity and macrophytes have a greater inuence on the index SPEARorganic than other parameters, although they are correlated mainly with the rst ordination axis. 3.2. Whole data set: uncontaminated and contaminated streams The PCA performed for the whole data set, including contaminated streams, has shown that environmental variables could explain up to 84% of the total variability of the taxonomic indices; rst and second axes are interpretable (Table 5, Fig. 3). Environmental parameters elevation, temperature, velocity, substrate particle size, presence of macrophytes, periphyton cover, habitat heterogeneity, and nitrate concentration are mainly correlated with the rst ordination axis. The second axis is mainly correlated with the contaminants (petrochemicals, surfactants, ammonium and nitrites), presence of anthropogenic pressure, and stream width (see inter-set correlation matrix in Table 6).

Presence of macrophytes

Stream width, m

Temperature, C (SE)

1.7 2.6 (0.2) 5.0 (1.0) 5.0 (1.5) 4.8 (0.1) 10.65 (0.15) 20.5 21.5 21.5 15.5 (0.7) 24.6 (0.45) 24.0 (0.6) 23.0 (0.4) 23.1 (0.3) 22.0 (1.5) 22.0 (0.3) 22.0 (0.3) 22.1 (0.4) 22.0 (1.5)

1.50 2.50 2.60 3.00 1.00 1.60 3.00 10.00 0.50 1.40 25.00 30.00 30.00 35.00 3.00 1149.00 785.00 869.00 754.00

0.01 0.01 0.01 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.50 (0.14) 0.64 (0.17) 0.48 (0.13) 0.74 (0.09) 0.65 (0.02) 0.46 (0.12) 0.31 (0.02) 0.44 (0.14) 0.29 (0.06)

NO3, mg L1 (SE)

15.00 50.00 57.50 (7.50) 30.00 16.67 (7.27) 8.33 (2.12) 1.25 (0.25) 11.67 (2.19) 5.00 4.29 (0.72) 1.43 (0.03) 1.43 (0.28) 1.00 1.00 25.00 (12.36) 1.00 1.00 1.00 1.00

Periphyton cover, % (SE)

1 1 1 1 1 2 3 4 3 4 3 3 3 4 4 2 2 2 3

Table 3 Summary of the PCA for the uncontaminated streams Ordination axes 1 Eigenvalues Speciesenvironment correlations 0.893 0.980 2 0.067 0.988 3 0.038 0.958 4 Total variance 0.002 1.000 0.990

Velocity, m s1 (SE) Altitude, m a.s.l. (SE)

1977 1612 1376 1278 1323 922 (3.00) 204 196 178 135 121 116 115 110 115 115 110 105 101

2.70 2.80 2.08 2.00 1.00 1.1 0.45 0.75 0.50 0.55 0.40 0.35 0.50 0.50 0.60 0.40 0.40 0.40 0.40

(0.03) (0.04) (0.05) (0.09) (0.03) (0.2) (0.03) (0.09) (0.03) (0.03) (0.02) (0.02) (0.03) (0.06) (0.06) (0.02) (0.02) (0.03) (0.03)

Site

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Cumulative percentage variance Of species data 89.3 Of speciesenvironment relation 89.3 Sum of all eigenvalues Sum of all canonical eigenvalues

96.0 96.2

99.8 99.8

100.0 100.0 1.000 0.961

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M.A. Beketov, M. Liess / Environmental Pollution 156 (2008) 980987 Table 5 Summary of the PCA for the whole set of streams Ordination axes 1 Eigenvalues Speciesenvironment correlations 0.723 0.898 2 0.244 0.982 3 0.029 0.796 4 Total variance 0.003 1.000 0.874

Cumulative percentage variance Of species data 72.3 Of speciesenvironment relation 69.5 Sum of all eigenvalues Sum of all canonical eigenvalues

96.7 97.5

99.7 99.7

100.0 100.0 1.000 0.839

Fig. 2. Ordination plot for Principle Components Analysis of the biological indices in the uncontaminated streams. Environmental variables are passively projected onto the ordination model.

Biological metrics TR, EPT, and H0 are mainly correlated with the rst ordination axis (Fig. 3). In contrast, SPEARorganic is correlated better with the second ordination axis (Fig. 3). Hence SPEARorganic is mainly dependent on the contaminants, while TR, EPT, and H0 are dependent on the longitudinal environmental factors. When linear regressions were performed for SPEARorganic and the contaminants at contaminated sites, the only signicant correlations were found for petrochemicals and synthetic surfactants (p < 0.05, Fig. 4A and B); the correlations with nitrite and ammonia were not statistically signicant (p > 0.05). Signicant correlations of SPEARorganic with these latter contaminants were obtained only when reference sites were included in the regression model (p < 0.05). Correlations of SPEARorganic with petrochemicals and surfactants calculated for the entire data set, including the reference sites, were also signicant (p < 0.05). Remarkably, two sites with the highest levels of surfactants and petrochemicals (marked by a and b in Fig. 4) were characterised by the lowest values of SPEARorganic, and lied slightly out of the regression models for the opposite contaminant (point a in Fig. 4A and point b in Fig. 4B). This conrms the results of the PCA (Fig. 3) and suggests that, although petrochemicals and surfactants were not fully correlated (Fig. 3), the index SPEARorganic correlates with both of these two contaminants (Figs. 3 and 4). 4. Discussion 4.1. Restricted data set: uncontaminated streams The present results show that in the macroinvertebrate communities of uncontaminated watercourses the distribution of sensitivity to organic toxicants is poorly dependent on the natural

longitudinal factors, as only a small part (29%) of the variability of the SPEARorganic index can be explained by these environmental factors. As was stated in Section 1, to our knowledge distribution and variability of species sensitivity to organic toxicants in natural, undisturbed communities have not previously been investigated. The richness (TR and EPT) and diversity (H0 ) indices, assessed over the large gradient from an alpine stream to a mid-size lowland river, were much greater at low altitudes (Fig. 2). For lotic systems this trend is typical, as has been shown previously by many empirical studies (for temperate-zone streams see, e.g. Minshall et al., 1985). Also theoretical considerations such as the River Continuum Concept predict that the taxonomic richness of benthic communities changes with stream size, reaching a maximum in mid-order streams where environmental heterogeneity is greater than in headwaters or large rivers (Vannote et al., 1980; Minshall et al., 1985). Previous observations within the territory studied here revealed the same trend in longitudinal variability of Ephemeroptera and Trichoptera taxa richness. Species richness of these two orders is highest in medium-size and small lowland rivers (width from 5 to 50 m), but is lowest in small rivulets (<5 m) and in the huge lowland river Ob (width about 1000 m) (Beketov, 2004a, 2006a, in press). All this suggests that longitudinal changes in structure of invertebrate communities in southwestern Siberia are similar to those observed in various temperate-zone streams (discussed in Beketov, in press). Therefore, the relative independence between the SPEARorganic index and the richness and diversity indices observed in this study is expected to be valid for different temperate regions.

Table 4 Inter-set correlation matrix between environmental variables and ordination axes in the PCA performed for the uncontaminated streams (productmoment correlations) Axis 1 Altitude Velocity Temperature Width NO3 Macrophytes Periphyton cover Dominant substrate size Habitat heterogeneity 0.661 0.577 0.718 0.877 0.801 0.636 0.408 0.484 0.570 Axis 2 0.071 0.207 0.148 0.038 0.300 0.282 0.090 0.350 0.184 Axis 3 0.301 0.221 0.206 0.329 0.132 0.028 0.096 0.183 0.368 Axis 4 0.055 0.014 0.092 0.148 0.111 0.130 0.228 0.037 0.369

Fig. 3. Ordination plot for Principle Components Analysis of the biological indices in the whole data set, including both uncontaminated and contaminated streams. Environmental variables are passively projected onto the ordination model.

M.A. Beketov, M. Liess / Environmental Pollution 156 (2008) 980987 Table 6 Inter-set correlation matrix between environmental variables and ordination axes in the PCA performed for the whole set of streams (productmoment correlations) Axis 1 Altitude Velocity Temperature Width NO3 Macrophytes Periphyton cover Dominant substrate size Habitat heterogeneity Presence of human activity Petrochemicals Surfactants NO2 NH4 0.612 0.578 0.664 0.164 0.519 0.533 0.511 0.568 0.409 0.344 0.034 0.218 0.190 0.109 Axis 2 0.476 0.342 0.499 0.628 0.430 0.152 0.220 0.196 0.274 0.776 0.878 0.872 0.770 0.773 Axis 3 0.085 0.137 0.008 0.174 0.181 0.320 0.028 0.197 0.075 0.055 0.042 0.155 0.068 0.026 Axis 4 0.077 0.077 0.058 0.071 0.059 0.101 0.170 0.135 0.184 0.154 0.027 0.033 0.219 0.074

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4.2. Whole data set: uncontaminated and contaminated streams Richness and diversity indices assessed in the full set of watercourses (including uncontaminated and contaminated) repeated the trends found in the uncontaminated streams: they were negatively correlated with altitude (Fig. 3). Reaction of these indices to the presence of contaminants was not detected because of the pronounced longitudinal changes. In contrast, SPEARorganic was correlated with concentrations of organic contaminants such as petrochemicals and surfactants

A
SPEAR organic

-0.2 -0.3 -0.4 -0.5

r2=0.68, p < 0.05

b -0.6
-0.7

Reference sites

0.1

0.2

0.3

0.4

0.5

Petrochemicals, g/L

B
SPEAR organic

-0.2 -0.3 -0.4 -0.5 -0.6 -0.7 Reference sites 0.013 0.016 b

r2=0.73, p < 0.05

0.019

0.022

0.025

Surfactants, g/L

Fig. 4. Relations between SPEARorganic and the toxicants: A petrochemicals and B synthetic surfactants. Linear regressions are based on the contaminated sites (lled circles) with reference sites (empty circles) excluded from the analyses. Letters a and b near the data points indicate the sites with maximum concentrations of surfactants and petrochemicals, respectively.

(Fig. 4). In the present data it is difcult to distinguish the effects of organic toxicants from those of such inorganic contaminants as nitrite and ammonium, as they are inter-correlated (Fig. 3). However, it is unlikely that nitrite and ammonium had a signicant biological effect on the communities, because the observed maximum concentrations of these two contaminants (Table 1) were too low to have a toxic action on the macroinvertebrates (Berenzen et al., 2001; Beketov, 2002, 2004b). Besides, as explained in the results, the correlations between SPEARorganic and both nitrite and ammonia were not signicant when only the contaminated sites were included in the regression model. This also suggests that it is unlikely that nitrite and ammonia had an effect on the observed communities, and the negative correlations between them and SPEARorganic shown by PCA (Fig. 3) can be pseudo-correlations, as they are only driven by the differences between the reference and contaminated sites. Therefore we assume that the observed correlations of SPEARorganic were mainly determined by the petrochemicals and surfactants, because maximum recorded concentrations of these two contaminants (Table 1) were higher or within the range of acute and chronic effective concentrations of toxic compounds of these types (see for petrochemicals Dorn et al., 1991; Crossland et al., 1992; for sulphonic acid salts Lal et al., 1983, 1984; MacDonald et al., 2004; for alcohol ether sulphates Dyer et al., 2000). Furthermore, according to the land use in the study region (lack of intensive agriculture and mechanical-engineering industry) other toxicants presenting signicant biological effects (e.g. pesticides) were not expected. As mentioned in Section 1, previous studies have shown that the bioindicator system pesticide-specic SPEAR, based on sensitivity to organic toxicants (Sorganic used here for calculation of SPEARorganic) and life-cycle traits responsible for recovery (generation time and migration ability), is relatively independent of abiotic environmental factors other than pesticides (Liess and von der Ohe, 2005), and is applicable across different biogeographical regions in fer et al., 2007; Von der Ohe et al., 2007). However, the Europe (Scha stability of this bioindicator system and of the separate traits comprising this system across natural longitudinal gradients has never previously been investigated. In this study the traits responsible for post-contamination recovery used in the pesticide-specic SPEAR were not considered. The reason is that in contrast to pesticides, which are characterised by pulse input to surface waters, petrochemicals and synthetic surfactants are mainly characterised by continuous inputs (Liess and von der Ohe, 2005; Beketov, 2006b). Hence, to detect community effects of these types of contaminants, we used here the average community sensitivity SPEARorganic based on species sensitivities Sorganic (Von der Ohe and Liess, 2004), but not the traits responsible for post-contamination recovery. The correlation between SPEARorganic and toxicants found in this study, as well as the independence of this community parameter from the natural longitudinal factors, suggests that this index can be used in bioassessment to detect community effects of organic toxicants across various lotic habitats. In particular, this stressorspecic index can be employed to distinguish the effects of organic toxicants from those of natural environmental factors whenever they are so closely correlated that their effects are hardly separable by partial statistical techniques with removed effects of covariables (e.g. multiple regression or constrained ordination methods with the natural factors used as covariables). Further studies in different geographical regions with various anthropogenic impacts would be helpful to validate both the indication sensitivity and the independence of longitudinal and other natural factors. Future application of SPEARorganic in bioassessment should take into account possible uncertainties associated with computation of this index, which include: (i) extrapolations in deriving of taxonspecic Sorganic sensitivities (discussed in Von der Ohe and Liess,

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M.A. Beketov, M. Liess / Environmental Pollution 156 (2008) 980987 Beketov, M.A., 2004b. Different sensitivity of mayies (Insecta, Ephemeroptera) to ammonia, nitrite and nitrate: linkage between experimental and observational data. Hydrobiologia 528, 209216. Beketov, M.A., 2006a. Caddisies (Trichoptera) of south-western Siberia: new zoogeographical records, aquatic habitat preferences and ight periods. Braueria 33, 1316. Beketov, M.A., 2006b. Characteristic of water quality by benthic macroinvertebrates. In: Sevostyanov, P.F., Zinenko, V.I., Chirkov, V.A., Karpova, T.A. (Eds.), Annual Report of Water Quality by Hydrobiological Parameters on the Territory of West Siberian Territorial Administration of Hydrometeorology and Environmental Monitoring for 2005 Year, pp. 1732 (in Russian). Beketov, M.A. Community structure of Ephemeroptera in Siberian streams. Entomological Science 11, in press.. Beketov, M.A., Liess, M., 2008. Acute and delayed effects of the neonicotinoid insecticide thiacloprid on seven freshwater arthropods. Environmental Toxicology and Chemistry 27, 461470. Beketov, M.A., Liess, M. Potential of 11 pesticides to initiate downstream drift of stream macroinvertebrates. Archives of Environmental Contamination and Toxicology 55, in press. Berenzen, N., Schulz, R., Liess, M., 2001. Effect of chronic ammonium and nitrite contamination on the macroinvertebrate community in running water microcosms. Water Research 35, 34783482. Bonada, N.B., Prat, N., Resh, V.H., Statzner, B., 2006. Development in aquatic insect biomonitoring: a comparative analysis of recent approaches. Annual Review of Entomology 51, 495523. Cereghino, R., Park, Y., Compin, A., Lek, S., 2003. Predicting the species richness of aquatic insects in streams using a limited number of environmental variables. Journal of the North American Benthological Society 22, 442456. Crossland, N.O., Mitchell, G.C., Dorn, P.B., 1992. 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2004), and (ii) effects of possible confounding factors on SPEARorganic values. The expected confounding factors are: (a) non-continuous exposure proles with post-contamination recovery periods (the pesticide-specic SPEAR system is suggested, Liess and von der Ohe, 2005; Schriever et al., 2007), (b) effects of landscape factors fa fer cilitating recolonisation (e.g. upstream undisturbed area, Scha et al., 2007), (c) effects of toxicants with specic receptor-mediated modes of action (e.g. neonicotinoids, Beketov and Liess, 2008), and (d) specic sublethal effects of contaminants (e.g. drift-initiating action of neurotoxic insecticides, Beketov and Liess, in press). These factors can result in underestimation of the effects of organic toxicants, or hamper comparison of contaminated sites. However, all these factors are expected to inuence not only SPEARorganic, but also any of the currently applied bioassessment indices. This study gives an example of using a biological trait (sensitivity) responsible for effect (toxicity) of a particular stressor (organic toxicants) in order to detect the effect of this stressor at the community level. This example demonstrates the applicability of the SPEAR approach (stressor-specic bioindicator designed by combining relevant traits responsible for the stressors effect and associated recovery) not only for pesticides (Liess and von der Ohe, 2005), but also for petrochemicals and synthetic surfactants. 5. Conclusion We conclude that in lotic systems the distribution of species sensitivity to organic toxicants is poorly dependent on natural longitudinal environmental factors, but is highly dependent on contamination with organic toxicants. Inclusion of the SPEARorganic in current freshwater bioassessment programs as a stressor-specic and longitude-independent community measure will be helpful to detect community disturbance by organic toxicants. This study indicates the possibility to create further bioindicator tools for different stressors using the SPEAR approach (e.g. for heavy metals: sensitivity to metals Smetal as presented by Von der Ohe and Liess, 2004, similar to the Sorganic used here), and can then be used alone or in combination with other traits to derive an effective community index. Acknowledgements The authors are grateful to S.B. Ivanov, V.D. Ivanov, E.N. Kosykh fer, and C.A. Schriever for kind help at difV.Yu. Kryukov, R.B. Scha ferent stages of the study, to the anonymous reviewers for valuable suggestions regarding the manuscript, and to the West-Siberian Centre for Environmental Monitoring for providing chemical data. The research was supported by the European Union (projects HAIR, SSPE-CT-2003-501997 and INTERACT, Marie Curie IIF contract No.MIF1-CT-2006-021860). Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.envpol.2008.05.005. References
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