AUTHOR QUERY FORM Journal: STOTEN Please e-mail or fax your responses and any corrections to: E-mail: corrections.esil@elsevier.spitech.com Fax: +1 619 699 6721 Article Number: 12420 Dear Author, Any queries or remarks that have arisen during the processing of your manuscript are listed below and highlighted by flags in the proof. Please check your proof carefully and mark all corrections at the appropriate place in the proof (e.g., by using on- screen annotation in the PDF file) or compile them in a separate list. For correction or revision of any artwork, please consult http://www.elsevier.com/artworkinstructions. Any queries or remarks that have arisen during the processing of your manuscript are listed below and highlighted by flags in the proof. Click on the ‘Q’ link to go to the location in the proof. 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Please supply full details for this reference. Thank you for your assistance. Our reference: STOTEN 12420 P-authorquery-v8 Page 1 of 1 mailto:corrections.esil@elsevier.spitech.com http://www.elsevier.com/artworkinstructions http://www.elsevier.com/artworkinstructions http://www.elsevier.com/wps/find/journaldescription.cws_home/503360/authorinstructions 1Q1 2 3 4 5 6 7 8 9 10 111213 14 15 16 17 18 19 20 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 Science of the Total Environment xxx (2011) xxx–xxx STOTEN-12420; No of Pages 7 Contents lists available at ScienceDirect Science of the Total Environment j ourna l homepage: www.e lsev ie r.com/ locate /sc i totenv Taxonomic and nontaxonomic responses to ecological changes in an urban lowland stream through the use of Chironomidae (Diptera) larvae A. Cortelezzi ⁎, A.C. Paggi, M. Rodríguez, A. Rodrigues Capítulo ⁎ Corresponding author. ILPLA (CONICET La Plata/FCN 23, 5-CC 1888, Argentina. Tel./fax: +54 11 4275 7799/8 E-mail address: agus@ilpla.edu.ar (A. Cortelezzi). 0048-9697/$ – see front matter © 2011 Published by E doi:10.1016/j.scitotenv.2011.01.002 Please cite this article as: Cortelezzi A, et through the use of Chironomidae (Diptera a b s t r a c t a r t i c l e i n f o 21 22 23 24 25 26 27 28 29 30 31 32 Article history: Received 7 May 2010 Received in revised form 29 December 2010 Accepted 5 January 2011 Available online xxxx Keywords: Chironomidae diversity Chironomus calligraphus Mentum deformities Sublethal effects Environmental degradation Surveillance tool 33 34 35 36 37 38 39 40 41 42 43 Biotic descriptors — both taxonomic (diversity indices, species richness, and indicator species) and nontaxonomic (biomass, oxygen consumption/production, and anatomical deformities) — are useful tools for measuring a stream's ecological condition. Nontaxonomic parameters detect critical effects not reflected taxonomically. We analyzed changes in Chironomidae populations as taxonomic parameters and mentum deformities as a nontaxonomic parameter for evaluating a South-American-plains stream (Argentina). We performed samplings seasonally (March, June, September, and December; 2005) and physical and chemical measurements at three sampling sites of the stream (DC1 at river source, through DC3 downstream). The specimens collected in sediment and vegetation were analyzed to investigate mouth deformities in Chironomidae larvae. We identified a total of 9 taxa from Chironomidae and Orthocladiinae subfamilies. Shannon's diversity index for Chironomidae decreased from 1.6 bits ind−1 (DC1) to 0.3 bits ind−1 (DC3). The total density of the Chironomidae exhibited a great increase in abundance at site DC3, especially that of Chironomus calligraphus. Chironomidae taxonomic composition also changed among the three sites despite their spatial proximity: C. calligraphus, Goeldichironomus holoprasinus, Parachironomus longistilus, and Polypedilum were present at all three; Corynoneura and Paratanytarsu at DC1 only; Cricotopus at DC1 and DC3; Apedilum elachistus notably at DC2 and DC3; and Parametriocnemus only at DC2. C. calligraphus individuals from DC1 showed no mentum deformities; only 2 from DC2 exhibited mouth-structure alterations; while specimens from DC3 presented the most abnormalities, especially during autumn and late winter. Type-II deformities (supernumerary teeth and gaps) were the most common. Anatomical deformities are sublethal effects representing an early alert to chemically caused environmental degradation. Mentum deformities in benthic-Chironomidae larvae constitute an effective biological-surveillance tool for detecting adverse conditions in sediments and evaluating sediment-quality-criteria compliance. Taxonomic (commu- nity composition) and nontaxonomic (condition of larval mouth parts) descriptors, used together, can indicate a stream's ecological state. 44 YM-UNLP), Av. Calchaqui Km. 564. lsevier B.V. al, Taxonomic and nontaxonomic responses ) larvae, Sci Total Environ (2011), doi:10.101 © 2011 Published by Elsevier B.V. 4546 62 63 64 65 66 67 68 69 70 71 72 73 74 75 1. Introduction A combination of biotic descriptors can serve as a useful tool for measuring the ecological condition of streams. Hill et al. (2001) recognized two kinds of parameters for the biological evaluation of waters: taxonomic (diversity indices, species richness, and indicator species) and nontaxonomic (biomass, oxygen consumption and production, and anatomical deformities). Both parameters can provide information on the ecological status of a given stream site under study. The usefulness of nontaxonomic parameters resides in their ability to detect effects that are not reflected in the taxonomic analysis. For instance, some toxic pollutants cause sublethal effects that are not immediately detected by the taxonomic descriptors, but become evidenced by changes in the oxygen production, growth and 76 77 78 79 80 reproduction rate, biomass, behavioral modifications, and anatomical deformities. Recent studies based on nontaxonomic parameters have proven their usefulness in evaluating the environmental quality of aquatic systems (Pascoe et al., 2000; Bartsch et al., 2000; Fellows et al., 2003; De Lange et al., 2004). Responses to the effects of sediment contamination have been studied in macroinvertebrate communities as well as in individual species both in situ (Pinel-Alloul et al., 1996) and at the laboratory (Sibley et al., 1999). The nontaxonomic responses of organisms within the Pampean plains have been investigated only a little, with the information being limited to functional responsive studies in the laboratory on either an individual or community level (Rodrigues Capítulo, 1984a,b; de la Torre et al., 1999, 2000, 2002; Olguín et al., 2000, 2004; Demichelis et al., 2001) and with few surveys being conducted in the field (Tangorra et al., 1998; Graça et al., 2002; Sierra and Gómez, 2007; Gómez et al., 2008). Certain members of the Chironomidae family are well known as indicators of ecological conditions (Paggi, 1999, 2003), with some to ecological changes in an urban lowland stream 6/j.scitotenv.2011.01.002 http://dx.doi.org/10.1016/j.scitotenv.2011.01.002 mailto:agus@ilpla.edu.ar Unlabelled image http://dx.doi.org/10.1016/j.scitotenv.2011.01.002 Unlabelled image http://www.sciencedirect.com/science/journal/00489697 http://dx.doi.org/10.1016/j.scitotenv.2011.01.002 Original text: Inserted Text "." Original text: Inserted Text "." Original text: Inserted Text " investigated" 81 82 83 84 85 86 87 88 89 90Q2 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111Q3 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 2 A. Cortelezzi et al. / Science of the Total Environment xxx (2011) xxx–xxx larvae being especially useful for detecting polluted environments through cephalic-capsule structural alterations (Wiederholm, 1984; Warwick, 1985). Chironomus deformities, in particular, have a great potential for reflecting the sublethal effects of polluted sediments, in addition to indicating the pollution-sequence history (Janssens de Bisthoven et al., 1992, 1998; Lindegaard, 1995; Diggins and Stewart, 1998; Reynolds and Ferrington, 2001) as well as the presence of complex mixtures of contaminating substances that may cause synergistic or opposite effects (Vermeulen, 1995). Chironomus calligraphus, (Goeldi, 1905) is themost commonly found species in the neotropics (Spies et al., 2002; Nazarova et al., 2004), it havingbeen recordedmainly at the limnotopes of the Pampeanplains in Argentina (Paggi, 1979). This species is widely distributed because of its ability to colonize and develop under a range of ecological circum- stances and its relatively broad tolerance to adverse environmental conditions (Spies et al., 2002). Thus,C. calligraphus canmaintain sizeable populations in sites with strong anthropic pressure and thereby constitute a nuisance to humans (Spies, 2000). The objectives of the present study were to analyze changes in Chironomidae populations and to utilize mentum deformities in their larvae as taxonomic and nontaxonomic parameters, respectively, for evaluating the ecological well-being of an urban lowland stream within the Argentinian Pampean plains that is subjected to differing degrees of pollution. 149 150 151 152 153 Q4 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 2. Materials and methods The Don Carlos Stream (34°55′–34°50′S, 58°00′–58°03′W) is located in the northeast of the Buenos Aires Province, Argentina. Don Carlos is a small stream, of length 9 km, which flows through the Pampean plain and into the Río de la Plata estuary (Fig. 1). The composition of the riverbed is mainly clay, silt, and sand, with a lesser proportion of gravel (Cortelezzi, 2010). The presence of organic matter varies depending both on the natural contributions from nearby pastures and on the surrounding anthropogenic activity. The scant slope in the rivers of this basin, however, together with an irregular discharge of their water owing to the uneven seasonal rainfall, causes a standstill in the water flow during the summer dry season. This stagnation of water results in an increased transparency and enhanced light penetration that favor the development of many submerged and floating macrophytes within the pooled areas (Rodrigues Capítulo et al., 2003). For the present study 3 sampling sites were selected: site 1 (DC1) near the source of the river and exposed to agricultural activity, site 2 (DC2) 800 m downstream just beyond the input of a major textile- industry discharge within an urban stretch; and site 3 (DC3) 1100 m 172 173 Fig. 1. Map of the study area showing the Don Carlos Stream and sampling locations. Please cite this article as: Cortelezzi A, et al, Taxonomic and nontaxon through the use of Chironomidae (Diptera) larvae, Sci Total Environ (2 farther downstream from DC2. This last site is exposed to sewage effluents and to outflows from textile and metallurgical factories, it has been canalized and its bed and lateral banks cemented. We performed samplings seasonally (March, June, September, and December; 2005) and collected sediment and vegetation samples in duplicate at the three sampling sites of the stream (DC1, DC2, and DC3). We collected the sediment with an Ekmann dredge (100 cm2), removed vegetation within the area subsumed by a 1300-cm2 plexiglas square, and trapped the phytophilous individuals on 250- μm-mesh sieves. The following physical and chemical measurements were made: the stream speed (Cole-Parmer CZ-32922-10 Flow meter), width, and depth; temperature and pH (Hanna HI 8633), conductivity (Lutron CD-4303), turbidity (Turbidity meter 800-ESD), and dissolved oxygen (Ysi 52 dissolved oxygenmeter).Water samples for the analysis of dissolved inorganic nutrients were filtered immediately through glass fibre filters (Whatman G/FC) and, together with the samples for BOD5 and COD, these were stored at 4 °C until arrival at the laboratory. Soluble reactive phosphorus (P–PO4 −3), ammonium (N–NH4 +), nitrate (N–NO3 −), nitrite (N–NO2 −), biological oxygen demand (BOD5) and chemical oxygen demand (COD) were determined according to Mackereth et al. (1978) and American Public Health Association (APHA) (1998). Organic matter (OM) percentage in sediments was calculated by weight loss after ignition at 500 °C during 4 h from a subsample (20 g fresh weight). Triplicate samples of water and sediment of each sample site were collected for determination of Cd, Zn, Cu, Pb, Ni, and Cr and analyzed by atomic absorption spectrophotometry following acid digestion of samples (VARIAN SpectrAA 55 Atomic Absorption Spectrometer, Environmental Protection Agency, EPA, 1986). The water hardness was determined according to American Public Health Association (APHA) (1998); (M 2340 C— EDTA Titrimetric Method). Themethods used for the analysis of each heavy metal can be seen in Table 1. At the laboratory, the Chironomidae larvae collected were cleared with 10% (w/v) KOH, dehydrated stepwise with 80%, 96%, and 100% (v/v) aqueous ethanol, and finally mounted on a slide in Canadian balsam for their observation under the light microscope. The chirono- mids were expressed in number of individuals m−2 for each sampling site using the average of the five replicates. Chironomidae diversity was estimated using the Shannon Weiner Index (H′) (Shannon and Wiener, 1949). The Chironomidae genera were identified by means of the keys cited in Wiederholm (1983); Paggi (2001), and Epler (2001). The incidence and degree of deformity were quantified under a binocular Olympus BH-2 microscope at 400×. In spite of the careful analysis to determine the Chironomidae taxa and their deformities, C. calligra- phus was the only species that was present in large numbers at the sampling sites. Therefore, individuals of this species at the fourth instar were selected for analysis and quantification of deformities. In order to evaluate C. calligraphus mentum abnormalities, only those Table 1 t1:1 Physicochemical variables, average and standard deviations (SD), measured at the three sampling stations in the Don Carlos Stream (n=4). t1:2 t1:3DCI DC2 DC3 t1:4T° (°C) 15.0 (4.7) 20.4 (2.8) 18.8 (3.7) t1:5Conductivity (μS cm−1) 685 (269) 955 (184) 991 (164) t1:6pH 7.8 (0.3) 7.8 (0.2) 7.8 (0.4) t1:7OD (mg r−1) 4.0 (1.3) 2.4 (1.1) 3.8 (1.3) t1:8N02−(mg l−1) 0.1 (0.1) 0.2 (0.3) 0.2 (0.3) t1:9N03−(mg l−1) 1.5 (1.5) 1.3 (1.1) 0.6 (0.7) t1:10NH4 + (mg l−1) 0.1 (0.1) 0.6 (0.3) 0.8 (0.7) t1:11P04−3 (mg l−1) 1.0 (0.2) 0.2 (0.2) 0.3 (0.1) t1:12BOD5 (mgO2 l−1) 8.2 (3.1) 21.7 (16.3) 19.0 (15.8) t1:13COD (mgO2 l−1) 18.2 (6.8) 29.7 (22.0) 28.7 (23.7) t1:14Turbidity (NTU) 22.4 (6.1) 23.6 (34.0) 5.1 (2.3) t1:15Organic matter (%) 5.4 (1.5) 4.9 (4.3) 12.3 (2.5) omic responses to ecological changes in an urban lowland stream 011), doi:10.1016/j.scitotenv.2011.01.002 image of Fig.�1 http://dx.doi.org/10.1016/j.scitotenv.2011.01.002 Original text: Inserted Text "º" Original text: Inserted Text "º" Original text: Inserted Text "º" Original text: Inserted Text "º" Original text: Inserted Text "'" Original text: Inserted Text "'" Original text: Inserted Text "'" Original text: Inserted Text "'" Original text: Inserted Text "that " Original text: Inserted Text "X" 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 t2:1 t2:2 t2:3 t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 Table 3 t3:1 Concentrations of heavy metals and hardness (mean±SD) in the water of the Don Carlos Stream at the three sampling stations and maximum permissible according to Argentine Dangerous Wastes Law, N° 24051 (1993) for protection of freshwater life. t3:2 t3:3DC1 Water DC2 DC3 Maximum permissible amount for protection of freshwater life t3:4Pb (μg l−1) 8 (10) b2 60 (50) 2 4 t3:5Zn (μg l−1) 110 (130) 20 (10) 40 (10) 30 30 t3:6Cd(μg l−1) b0.6 b0.6 b0.6 2 3 t3:7Cu (μg l−1) 31 (4) 3 (6) 6 (1) 0.8 1.3 t3:8Cr(μg l−1) b5 b5 b5 2 2 t3:9Ni (μg l−1) 6 (5) b6 50 (20) 65 110 t3:10Hardness (CaCO3 mg l−1) 97.0 (1.1) 120.7 (1.9) 132.7 (5.3) 60–120 120–180 3A. Cortelezzi et al. / Science of the Total Environment xxx (2011) xxx–xxx characteristics different from the normal structure were considered: those alterations resulting strictly from mechanical wear though use were excluded. Mouth deformities were investigated in each specimen and the type of deformity established according to the classification by Lenat (1993) and Reynolds and Ferrington (2001): Type I mild deformity, but distinguishable from normal toothwear. Type II conspicuous deformity — supernumeraries present or teeth missing. Type III extreme deformity — combination of type-II deformities. To evaluate if the differences in density of species between the sampling sites were significant, the ANOVA on Ranks and the Tukey Test were employed because the data were not normally distributed and/or had unequal variances. 209 210 211 212 Q5 213 Q6 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 3. Results 3.1. Analysis of physicochemical parameters The main physical and chemical characteristics of the sampling stations are shown in Table 2. Phosphate and nitrate increased upstream with agricultural activity, while ammonium, COD and BOD5 increased downstream with urban and industrial activities (textile and metallurgical factories). The organic matter had the highest values in DC3, thus reflecting the impoverishment of water quality downstream. Upon analysis of the heavy metals in the water, only Cu and Zn were detected at the sampling sites exceeding the limits specified as the maximum permissible established by the Argentine Dangerous Wastes Law, N° 24051 (1993) for protection of freshwater life. Moreover, the Pb concentration was much higher than the above mentioned limits in DC3 (Table 3). All the metals analyzed, except for Cd, were detected in the sediment — i. e., Cr, Cu, Zn, Pb, and Ni. The Zn was higher upstream while Cu and Cr were elevated downstream. Especially notable were the high concentrations of Pb and Ni at DC3: there, those two elements widely exceeded the mean values for the lithosphere according Frink (1996) (Table 4). 229 230 231 232 233 234 235 236 Table 2 Methodology for the determination of heavy metals in water and sediment in the Don Carlos stream. Water Unit Detection limit Method Pb μg I−1 0.002 EPA SW 846 M 3OIOAM 7420 EAA Zn μg I−1 0.007 EPA SW 846 M 3010 AM 7950 EAA Cd μg I−1 0.0006 EPASW846M7I3OEAA Cu μg I1 0.005 EPASW 846 M 3OIOAM 7210 EAA Cr μg I−1 0.005 EPA SW 846 M 7196 A UV spectrophotometry Ni μg I−1 0.006 EPA SW 846 M 3010 AM 7520 EAA Hardness CaCQ3 mg l−1 1 Standard Method M 2340 C — EDTATitrimetric Sediment Unit Detection limit Method Pb mg kg1 0.5 EPASW846M742OEAA Zn mg kg−1 0.75 EPA SW 846 M 7950 EAA Cd mg kg−1 0.125 EPA SW 846 M 3050 A M 7130 FAA Cu mg kg−1 1 EPASW846M72IOEAA Cr mg kg−1 0.5 EPA SW 846 M 3050 A M 7130 EAA Ni mg kg−1 1.125 EPA SW 846 M 7520 FAA Please cite this article as: Cortelezzi A, et al, Taxonomic and nontaxon through the use of Chironomidae (Diptera) larvae, Sci Total Environ (2 3.2. Chironomidae throughout a polluted gradient In the present study the following 9 taxa, representing both the Chironominae and the Orthocladiinae subfamilies, were recorded: Chironominae: C. calligraphus Goeldi, 1905, Goeldichironomus holoprasinus (Goeldi) Fittkau 1965, Parachironomus longistilus Paggi,1977, Polypedilum Kieffer, 1912, Apedilum elachistus Townes, 1945, and Paratanytarsus Thienemann and Bause, 1913. Orthocladiinae: Corynoneura Winnertz, 1846, Cricotopus van der Wulp, 1874, and Parametriocnemus Goetghebuer, 1932. The total density of the Chironomidae family exhibited a great increase in abundance downstream reaching mean values of 46,550 ind/m2 at site DC3. Shannon's diversity index (1963) when applied to the Chironomidae varied inversely with the density of the taxa, from 1.6 bits ind1 at DC1 to 0.3 bits ind1 at DC3 (Fig. 2). The relative density of the Chironomidae also changed particularly at sites DC2 and DC3 when compared to site DC1; but the most notable difference was the pronounced increase in the number and proportion of C. calligraphus: While this species accounted for only 6% of the total individuals at site DC1 (8 ind/m2), C. calligraphus represented an average of 91% at DC2 (471 ind/m2) and 94% at DC3 (8786 ind/m2; Fig. 3). The taxonomic composition of the Chironomidae furthermore varied among the three sites despite their close physical proximity: C. calligraphus, G. holoprasinus, P. longistilus, and Polypedilum were present at all three sites; while Corynoneura and Paratanytarsus were recorded at only DC1, Cricotopus at both DC1 and DC3, A. elachistus notably at DC2 as well as DC3, and Parametriocnemus at site DC2 only. Table 4 t4:1 Amounts of heavy metals in streambeds sediments (mean±SD) of the Don Carlos Stream at the three sampling stations and average natural amount by sedimentary rocks and soils according to Frink (1996). t4:2 t4:3DCI Sediment DC2 DC3 Average natural amount by sedimentary rocks and soils t4:4Pb (mg kg−1) 5.2 (2.4) 29.6 (48.5) 679.5 (629.6) 19.6 t4:5Zn(mg kg−1) 80.3 (40) 36.3 (13.6) 4.5 (3.6) 40 t4:6Cd (mg kg−1) b0.125 b0.125 b0.125 0.34 t4:7Cu (mg kg−1) 25 (7.8) 56 (72.8) 50 (21.2) 29.2 t4:8Cr(mg kg−1) 5.9 (3.5) 10.2 (11.6) 7.9 (6.4) 129 t4:9Ni (mg k−1) 13.0 (1.3) 68.2 (112.4) 330.7 (260.9) 11 omic responses to ecological changes in an urban lowland stream 011), doi:10.1016/j.scitotenv.2011.01.002 http://dx.doi.org/10.1016/j.scitotenv.2011.01.002 Original text: Inserted Text "º" Original text: Inserted Text "1977 " Original text: Inserted Text "." Original text: Inserted Text "." Original text: Inserted Text "º" Original text: Inserted Text "Iimit" Original text: Inserted Text "Iimit" 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 Fig. 2.Density of Chironomidae and the Shannon diversity values at the 3 sampling sites of the Don Carlos Stream. Table 5 t5:1 Summary of the number of larvae sampled and the number and percentage of mentum deformities registered at the DC3 sampling site on the Don Carlos Stream. t5:2 t5:3DC3 N° larvae N° deformities % Deformities t5:4Autumn 1584 42 2.65 t5:5Winter 223 4 1.79 t5:6Spring 192 18 9.38 t5:7Summer 55 0 0.00 t5:8Deformities average in DC3 3.13% t5:9Total N° larvae 2054 4 A. Cortelezzi et al. / Science of the Total Environment xxx (2011) xxx–xxx 3.3. C. calligraphus mentum deformities More than 2000 C. calligraphus larvae from the Don Carlos Stream were counted and analyzed. As to mentum deformities in this species, individuals from DC1 (n=12) had no alterations in their mouth structures. At site DC2, of the 51 individuals collected, only 2 exhibited mentum deformities. Site DC3, however, harbored the greatest number of C. calligraphus mentum abnormalities, especially during the autumn (n=1584) and late winter (n=223) samplings. The abundance of Chironomidae was very low during summer (n=55) and no deformities were found during that season (Table 5). The most common deformities registered were of type II (supernumerary teeth and dental gaps, Fig. 4). Mild asymmetries were, however, well represented (Table 6). 4. Discussion 4.1. Taxonomic responses Over 90% of the individuals collected in this study belonged to the Chironomini tribe of the Chironominae subfamily, and as such represented a typical fauna pattern in the lowland areas of South America (Fittkau, 1971, 1986; Fittkau and Reiss, 1979). The Chir- onomini tribe occupies a large habitat range and can be found in both oligotrophic and eutrophic environments — in rivers and streams of different sizes and with different levels of pollution (Martinez et al., Fig. 3. Relative abundance of Chironomidae colle Please cite this article as: Cortelezzi A, et al, Taxonomic and nontaxon through the use of Chironomidae (Diptera) larvae, Sci Total Environ (2 2002). The Orthocladiinae were generally scarce mainly because of their ecological requirements for rivers and streams with high slopes, high levels of oxygen, and the predominance of a hard substrate (Pinder, 1986). Nevertheless, the detection of Parametriocnemus in most of the affected sites in this study was quite unusual since some species of this genus have been reported to be sensitive to organic contamination (Epler, 2001). Despite the predominance of Chironomidae is common in many freshwater systems (Klein and Trivinho-Strixino, 2005; Bass, 1986; Cohen, 1986), the high density of larvae may indicate environmental disturbances (Coimbra et al., 1996; Marques et al., 1999). Especially, Chironomus larvae have been observed in great amount in eutrophic environments (Frank, 1963; Learner and Edwards, 1966; Dévai, 1988; Tate and Heiny, 1995; Botts, 1997; Janssens de Bisthoven and Gerhardt, 2002). These studies agree with our investigation, the increase in total density of Chironomidae at the sites with the highest level of contamination (highest values of conductivity, COD and BOD5) and impacted physical habitat (dredging, canalized) was a result of the significant rise in C. calligraphus. While in the least stressed site (DC1) this species represented less than 10% of the individuals, at the two sites with a much higher contamination (DC2 y DC3), this species exceeded 90% of total Chironomidae. This finding indicates the necessity of avoiding generalizations in defining the entire Chironomidae family as an indicator of polluted environments: indeed, the data here would argue that only the Chironomus genus constitutes a reliable indicator (Marques et al., 1999). In relation to richness, within Argentina, Marchese and Paggi (2004) reported the presence of 20 Chironominae genera and 7 Orthocladiinae genera in the catchment area of the Río Paraná and Río de la Plata rivers. The low density of those taxa registered in our study (6 Chironominae and 3 Orthocladiinae) is coincident with the values cited by Marques et al. (1999), who recorded only 5 taxa in the most polluted sites in southeast Brazil. The low richness registered in this study would stem from the strong anthropic impact on the plains urban-stream systems in this area. The paucity of ecological studies on the relationship between the distribution of Chironomidae and the gradient of environmental cted at each site on the Don Carlos Stream. omic responses to ecological changes in an urban lowland stream 011), doi:10.1016/j.scitotenv.2011.01.002 image of Fig.�2 image of Fig.�3 http://dx.doi.org/10.1016/j.scitotenv.2011.01.002 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 Fig. 4. Types of deformity registered in C. calligraphus larvae collected in the Don Carlos Stream (Buenos Aires, Argentina). t6:1 t6:2 t6:3 t6:4 t6:5 t6:6 t6:7 t6:8 t6:9 5A. Cortelezzi et al. / Science of the Total Environment xxx (2011) xxx–xxx pollution for this region makes a comparison of these results with those from other studies impossible. We can, however, cite the presence of Corynoneura and Paratanytarsus at the least impacted site and of A. elachistus at the sites of greater organic and industrial contamination. In order to make a correct assessment of the environmental status of lowland lotic systems based on the taxonomic responses of Chironomidae, we need further bionomic studies on species within this region in order to generate a larger amount of relevant empirical data. 4.2. Nontaxonomic responses Several studies have reported the presence of deformities in Chironomus larvae at sites with a strong anthropic impact (Warwick and Tisdale, 1988; van Urk et al., 1992; Callisto et al., 2000; Heylen and De Pauw, 2002; Nazarova et al., 2004). According to Kuhlmann et al. 364 365 366 367 368 369 370 371 372 373 374 375 376 Table 6 Percentage of each type of mentum deformity registered at sampling site DC3 of the Don Carlos Stream. DC3 Types of deformities Class I Class II Class III Autumn 14 26 2 Winter 0 4 0 Spring 5 12 1 Summer 0 0 0 29% 66% 5% Please cite this article as: Cortelezzi A, et al, Taxonomic and nontaxon through the use of Chironomidae (Diptera) larvae, Sci Total Environ (2 (2000), Chironomus is particularly useful for the study of deformities because this genus can be found in any type of aquatic system, is thus widely distributed, and can furthermore tolerate adverse environ- mental conditions (such as low oxygen concentrations). Moreover, Chironomus is prone to morphological deformities. This proclivity is associated with the type of food that the genus consumes — e. g., the sediment, whose ingest exposes the specimens to organic contami- nants in the form of fine particles (detritus) as well as to inorganic particles (clay). By contrast, populations of other genera that not exhibit great deformities because exposure to metal contaminants would likely be lethal for a largenumber of individuals, thusdiminishing the surviving population down to only the strongest members (Martinez et al., 2002). Unfortunately, only a few studies have been found in the literature in relation to the heavy metal distribution in water and sediment in streams from the Pampean plain of Argentina. Previous research was focused on the analysis of heavy metals in the bottom river sediment (Manassero et al., 1998; Ronco et al., 2001; Camilión et al., 2003; Ronco et al., 2008). Most of the rivers and streams in urban areas of Buenos Aires Province contain at present a high load of urban and industrial wastes (Herkovits et al., 1996; Castañé et al., 1998). The impact produced by the human activities on these watercourses results in the alteration of the natural balance of the systems (Rendina et al., 2001). In coincidence with our study, Camilión et al. (2003) registered an increased Zn and Cu concentration at the sites where intensive agricultural practices have been performed. Moreover, in sites DC2 y DC3, Pb and Ni exceeded the average natural amount by sedimentary rocks and soils, and Pb surpassed widely the maximum permissible amount for protection of freshwater life. Especially in DC3, the Pb exceeded 34 times the values of sedimentary rocks and soils, and in the water, the Pb exceeded 15 times the permissible values for aquatic life protection. According to Krantzberg and Stokes (1989) the chironomid populations may have the ability to regulate the uptake of heavy metals. These authors, in laboratory experiments, have suggested the chironomids may regulate Cu, Ni, Mn and Zn, but not Pb and Cd. This would explain the great number of deformities registered in site DC3 where the values of Pb are very high. Studies on rivers and lakes in the Northern hemisphere have estimated that a frequency of deformities above 8% of the total number of larvae can indicate unfavorable environmental conditions (Warwick, 1988); this percentage would furthermore be the threshold indicating excessive concentrations of contaminants (Janssens de Bisthoven et al., 1992; Warwick, 1990, 1991, 1992; van Urk et al., 1992). Studies along these lines carried out in South America have shown that the percentages of deformities in response to environmental adversity are lower than those registered at other latitudes. In this study, the percentage of deformities coincides with the data of Callisto et al. (2000) from southeast Brazil (3%). In both researches, heavy-metal values were similar to or higher than the maximum concentrations considered to be safe by international environmental legislation. The frequency of Chironomus mentum deformities in the Tieté river, São Paulo, Brazil (Kuhlmann et al., 2000) showed similar values to those registered in our study in both their dry (3.7%) andwet (8.3%) seasons. During the summer, the number of C. calligraphus larvae recorded here diminished, and no deformities were recorded; which finding would be directly related to the temperature-dependent life cycle of this species, with continuously overlapping generations of short life cycles occurring in the spring and summer followed by one or two generations of longer life cycles during the winter (Zilli et al., 2008). Studies performed by Nazarova et al. (2004) in Colombia reported higher values for Chironomus deformities (12%) than those registered in this study. According to this author, the low heavy-metal values registered would point to the existence of other influences acting synergistically to increase the percentage of deformities. However, we consider that a number of unquantified stressors could have resulted in such toxicological responses. omic responses to ecological changes in an urban lowland stream 011), doi:10.1016/j.scitotenv.2011.01.002 image of Fig.�4 http://dx.doi.org/10.1016/j.scitotenv.2011.01.002 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 6 A. Cortelezzi et al. / Science of the Total Environment xxx (2011) xxx–xxx With respect to the nature of the deformities found, Hamilton and Saether (1971) suggested that the various types of abnormalities could be related to different kinds of contaminants— e. g., gaps would be induced by heavy metals (Köhn and Frank, 1980; Janssens de Bisthoven et al., 1995). This type of deformity was, in fact, the most abundant in our study, in accordance with the high concentrations of Pb and Ni registered. Nevertheless, a wide variety of chemical products act at the same time on the biota of polluted ecosystems (Statham and Lech, 1975; Hellawell, 1986; De March, 1988; Calamari and Vighi, 1992; Kraak, 1992). We assume that the exposure of Chironomidae larvae to a combination of contaminants has resulted in a unique situation causing especially high levels of deformities in this stream. Deformities are sublethal effects and may, in general, be considered an early alert to the environmental degradation caused by chemical contaminants (Warwick, 1990). Mentum deformities in benthic Chironomidae larvae, in particular, appear to be an effective biological surveillance tool for the detection of adverse conditions in sediments as well as for the evaluation of the degree of compliance with the criteria for acceptable sediment quality. We conclude that a combination of descriptors – both taxonomic (community composition) and nontaxonomic (the condition of larval mouth parts) – are useful indicators for characterizing the ecological state of a given study area. Acknowledgements This research has been financed by a grant from CONICET and PICT N° 33939 (FONCYT). The authors would like to thank Jorge Donadelli, from the Laboratory of Chemistry of the ILPLA, for the nutrient- and oxygen-demand analyses of the water samples. 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Interciencia 2008;33:767–70. omic responses to ecological changes in an urban lowland stream 011), doi:10.1016/j.scitotenv.2011.01.002 http://dx.doi.org/10.1016/j.scitotenv.2011.01.002 Original text: Inserted Text ". J" Original text: Inserted Text "C" Original text: Inserted Text "PS" Original text: Inserted Text "PS" Original text: Inserted Text "LC" Original text: Inserted Text "M" Original text: Inserted Text "C" Original text: Inserted Text "A" Original text: Inserted Text "M" Original text: Inserted Text "AC" Original text: Inserted Text "F" Original text: Inserted Text "S.K." Original text: Inserted Text "LC" Original text: Inserted Text "A" Original text: Inserted Text "A" Original text: Inserted Text "A" Original text: Inserted Text "CS" Original text: Inserted Text "M" Original text: Inserted Text "M" Original text: Inserted Text "L" Original text: Inserted Text "A" Original text: Inserted Text "N" Original text: Inserted Text "WF" Original text: Inserted Text "MS" Taxonomic and nontaxonomic responses to ecological changes in an urban lowland stream through the use of Chironomidae (Diptera) larvae Introduction Materials and methods Results Analysis of physicochemical parameters Chironomidae throughout a polluted gradient C. calligraphus mentum deformities Discussion Taxonomic responses Nontaxonomic responses Acknowledgements References