~ Pergamon PII: S0043-1354(96)00109-1 Wat. Res. Vol. 30, No. 10, pp. 2265-2272, 1996 Copyright © 1996ElsevierScienceLtd Printed in Great Britain.All rights reserved 0043-1354/96$15.00+ 0.00 METAL CONCENTRATIONS IN H Y D R O P S Y C H E P E L L UCIDULA LARVAE (TRICHOPTERA, HYDROPSYCHIDAE) IN RELATION TO THE ANAL PAPILLAE ABNORMALITIES AND AGE OF EXOCUTICLE KARI-MATTI VUORI* and JUSSI K U K K O N E N Department of Biology, University of Joensuu, P.O. Box 111, FIN-80101 Joensuu, Finland (First received May 1995, accepted in revised form April 1996) Abstract--Metal concentrations in hydropsychid larvae inhabiting an acid, metal-polluted river were studied in relation to the incidence of morphological abnormalities and variation in their stage of development The significantlyhigher AI concentrations in the abnormal larvae as compared to the levels measured in the normal larvae suggest that the aluminium accumulated in the Hydropsyche pellucidula larvae significantly contributes to the occurrence of morphological abnormalities. The comparison of metal concerLtrationsbetween unmoulted and newly moulted larvae indicates that the main proportion of the iron is adsorbed on the body surface, whereas much of the aluminium, cadmium, copper, lead and zinc is also absorbed in the tissues. The average concentrations of Cd and Cu were significantlyhigher in the newly moulted than in the unmoulted larvae. It is suggested that either moulting enhances accumulation of Cd and Cu or these metals enhance moulting of the larvae. The results indicate that in bioaccumulation and monitoring studies utilizing aquatic insect larvae, more attention should be paid to the condition of the individuals. The incidence of morphological abnormalities in hydropsychid larvae appears to be a useful tool for impact assessment and the biomonitoring of metal-polluted streams. Copyright El 1996 Elsevier Science Ltd Key words---trace metals, metal partitioning, morphological abnormalities, Hydropsyche, moulting, bioaccumulation, river biomonitoring Vuori, 1995). The hydropsychid abnormalities also offer several practical advantages over the chironoThe occurrence of morphological abnormalities in mid head capsule surveys. Due to their robust body, aquatic insect larvae has been increasingly utilized in hydropsychid larvae are easily handled and checked both biomonitoring and toxicity test studies assessing for morphological abnormalities. Whilst the specific the environmental impact of pollutants (Johnson reasons for the chironomid head capsule deformities et al., 1993). Amongst these abnormalities, the head remain unclear (Warwick, 1988; Johnson et al., capsule deformities of the Chironomidae are the most 1993), the abnormalities in the hydropsychid tracheal commonly utilized indicators of contaminant stress gills and the ion-regulatory organs, anal papillae, can (e.g. Warwick, 1988). However, recent findings on the be attributed to a disruption of the respiratory and incidence of abnorraalities in the tracheal gills and the ion regulation functions of the individual (Camargo, anal papillae of hydropsychid caddis larvae offer 1991; Vuori, 1994). Further, the relatively large size another promising tool for pollution impact assess- facilitates sampling and analysis of the concenment and the biomonitoring of stream ecosystems trations of chemicals in the larvae. The burrowing (Camargo, 1991; Vuori, 1994). Laboratory and field Chironomidae are likely to be more susceptible to exposures of hy&ropsychids to copper (Petersen, sediment contamination, whereas the hydropsychid 1986), cadmium (Vaori, 1994) and aluminium (Vuori, larvae as facultative filter feeders (e.g. Wallace, 1975; 1995, 1996) have induced increasing incidence of Xiang et al., 1984; Petersen, 1987) are more exposed darkened and reduced anal papillae. to pollutants in seston, flowing water and the organic Hydropsychid czddis larvae are widely distributed matter accumulated in riffle microhabitats. and abundant in many kinds of running waters. They In general, the environmental effects of pollutants respond readily to variations in the water quality and can be more accurately predicted from the pollutant their autecology is 'well enough known for the impact concentrations in the organisms than from the of pollutants to be distinguished (Petersen, 1986; concentrations in the water or sediment (Moriarty, *Author to whom all correspondence should be 1990; Forbes and Forbes, 1994). Indeed, the use of addressed [Fax: (358) 73 151 3590; E-mail: aquatic invertebrates as sentinel organisms for measuring the environmental concentration levels, KVUORI@JOYL.JOENSUU.FI]. ]INTRODUCTION 2265 2266 K.-M. Vuori and J. Kukkonen bioavailability and the potential effects of pollutants, has become increasingly popular (e.g. Cain et al., 1992; Hare, 1992; Johnson et al., 1993; Clements and Kiffney, 1994; M o u n t et al., 1994). The ultimate aim of the contemporary environmental risk assessment is to establish those critical body residues of chemicals which cause harmful effects in organisms (McCarty and Mackay, 1993). As lotic insect larvae occupy almost all trophic levels of consumers, having a variety of feeding habits, a rapid metabolism and variable life cycles, they have the potential to reflect both the spatial and temporal aspects of the fate and effects of chemicals with great accuracy (Cain et al., 1992; Hare, 1992; Johnson et al., 1993). Also the increase in the number of morphological abnormalities displayed by the benthic invertebrates inhabiting polluted waters is presumed to imply a correspondingly elevated concentration of pollutants in animal tissues (e.g. Warwick, 1988; van Urk et al., 1992; Lenat, 1993; Vuori, 1995). However, only a few studies have verified this hypothesis by comparing the concentrations of contaminants in the individuals displaying abnormalities with those of normal structure (Janssens de Bisthoven et al., 1992; Johnson et al., 1993). The results of both field and laboratory exposure of hydropsychid larvae to high metal concentrations suggest that the increased metal concentrations in the tissues may be the main reason for anal papillae and tracheal gill damage (Vuori, 1994, 1995, 1996). Metal concentrations measured in aquatic insect larvae includes both absorbed (within tissues) and adsorbed (on the body cuticle) metals. The adsorbed metals are considered to be physiologically inactive and should therefore theoretically contribute similarly to the metal concentrations of both the morphologically abnormal and normal larvae (Hare, 1992; Janssens de Bisthoven et al., 1992). Hence, evaluation of the relationship between morphological abnormalities and metal bioaccumulation should also include an estimation of the partitioning of metals between these two fractions. The contribution of the surface-bound metals to the overall larval metal concentrations have been earlier evaluated by (1) rinsing the larvae in acid, (2) comparing the metal quantities in exuviae with those of whole larvae or adults, and by correlating the amount of metals with the extent of iron oxide deposits on the body surface (reviewed by Hare, 1992). As the growth of the hydropsychid larvae occurs by moulting the exocuticle of the previous instar (Siltala, 1907), it is plausible to presume that in the newly moulted larvae the amount of surface adsorbed metals is lower than in the larvae with older exocuticles. Currently, there is no comparative information on the metal concentrations of freshly moulted larvae compared with those with a fully developed and chitinized exocuticle. The objective of the present study is to answer the following question: (1) Do the morphological abnormalities in the metal exposed hydropsychid larvae also imply an elevation in larval metal concentrations? (2) Are there differences in the partitioning of metals between the surface and interior of hydropsychid larvae, as reflected by newly moulted vs. unmoulted larvae? MATERIAL AND METHODS The materials used in this study consist of the 4th instar larvae of the Hydropsyche pellucidula, collected by kick net from a downstream riffle area of the River Kyrrnjoki, western Finland (62 ° 45' N, 22 ° 48' E). The larvae were collected on two separate occasions: May 1991 and May 1992. The lower reach of the River Kyr/Snjoki is heavily loaded by runoff from acid sulphate soils originating from sedimentation deposited during the Littorina stage of the Baltic Sea (5000 to 1000 BP). Extensive draining of the arable land and restructuring of the river has improved soil oxidation and increased the leaching of metals and protons into the river (Hartikainen and Yli-Halla, 1986; Vuori, 1995). A more comprehensive description of the study area is provided in Vuori (1995). Comparison of the metal concentrations between moulted and unmoulted Hydropsyche pellucidula larvae was based on specimens collected in May 1991. The larvae were kept overnight at room temperature in well-aerated water drawn from the River Kyr6njoki. Wanning of the water up to + 17°C induced moulting of the larvae. The moulting phase varied from the full shedding of the old exoskeleton to only small grooves in the apotome and pronotum. The newly moulted larvae were easily distinguished by their light yellow colour, in contrast to the dark brown colour of the yet unmoulted larvae. Three replicate samples of five fully moulted and five unmoulted larvae were separated under dissecting microscope. Most of the larvae had visually distinguishable abnormalities in the tracheal gills and anal papillae. The gut contents of the larvae were carefully dissected with a glass knife. The larvae were dried at 70°C for 6 h, cooled in a desiccator and weighed to the nearest 0.1 mg. The weighed samples were transferred to 25 ml acid-washed quartz tubes, and concentrated HNO3 added such that the sample:acid ratio was 1: 10 (w/v). The tubes were heated at 50°C for 2 h and then at l l0°C for a further 6h. The samples were made up to 20ml with distilled water according to the Finnish standard SFS 5075 (Suomen Standardisoimisliitto, 1990a). The concentrations of A1, Fe, Cu, Cd, Zn and Pb were then determined with an atomic absorption spectrophotometer (Perkin-Elmer 5100 PC) in the Oulu District Office of Waters and Environment. An air-acetylene flame was used to determine Zn levels and a HGA 500 graphite furnace for the other metals according to the Finnish standards SFS 3044, 3047 and 5502 (Suomen Standardoimisliitto, 1980a, b, 1990b). Specimens for the comparison of metal concentrations between normal and abnormal larvae were collected in May 1992. The larvae with normal, transparent and abnormal, totally darkened anal papillae were distinguished under the dissecting microscope according to Vuori (1994). Most of the larvae with abnormal anal papillae also had signs of damage in the tracheal gills. Five replicates of five normal and five abnormal larvae were then dissected, freeze-dried with labconco Lymph-Lock lyophilizer, weighed and digested, as described above. The larval AI, Cu and Zn concentrations were determined using the AAS (PerkinElmer 5000 Zeeman) in the Kokkola District Office of Waters and Environment laboratory as described above. In AAS analysis, the analytical accuracy was assessed using the Standard Reference Material (SRM 1566a, Oyster Metals and abnormalities in hydropsychid larvae Tissue) of the National Institute of Standards and Technology, U.S.A. Recoverieswere within 10% of certified values. Water samples taken during collection of the larvae were analyzed for alkalinity, chemical oxygen demand (CODMn), colour, conductivity, pH, suspended solids and nutrient and metal concentration,;. Water was collected from a swift current in polypropylene bottles. The analytical methods were those in standard use at the National Board of Waters and Environment, Finland. All analyses, except for the total metal concentrations measurement, were conducted at the Vaasa District Office of Waters and Environment laboratory. Water samples for metal analysis were taken in 125 ml acid-washed polypropylene bottles and acidifiedwith 0.5 ml concentrated, suprapurified HNO3 (Merck) in 100ml samples. Metal concentrations were determined by AAS at the Oulu District Office of Waters and Environment according to the FinrLishstandards SFS 3044, 3047 and 5502 (Suomen Standardoimisliitto, 1980a, b, 1990b). RESULTS During both sampling occasions, the levels of A1, Fe, Cu, Cd, Zn and Pb in the River Kyrrnjoki were elevated. The temperature was 10 and 11.2°C and the pH level 5.3 and :5.5 in 1991 and 1992, respectively (Table 1). During the earlier weeks of both sampling occasions, much higher metal concentrations and a pH level as low as 5.1 had been recorded. For example, total A1 concentrations reached levels of 6500 and 6000pg 1-~ in May 1991 and 1992, respectively (Vuoti, 1995; Vaasa District Office of Waters and Environment, unpublished). Considerable variation in the metal concentrations among the newly moulted larvae in particular was observed (Figs la--f). However, there were also clear differences in the average metal concentrations between freshly moulted and unmoulted Hydropsyche pellucidula larvae. The average Fe concentration was significantly higher in the unmoulted larvae (t-test, T = 3.16, df = 2, p = 0.03), whereas the average Cd and Cu concentrations were significantly higher in the freshly moulted larvae (t-test, T < - 4 . 2 1 , d f = 2, p < 0.05). The ave:rage iron concentrations were more than twice as high and the Cd and Cu concentrations lower in the unmoulted larvae as compared to the newly moulted larvae. There was a clear, although statistically insignificant difference in the average A1 2267 concentration between the unmoulted larvae and freshly moulted larva (Fig. lb, t-test, T = 2 . 0 2 , df = 2, p = 0.18). The average concentrations of Pb and Zn were quite similar in both groups (Figs le and f). The average aluminium concentrations of the abnormal and normal Hydropsyche pellucidula larvae were 1982#g A l g -~ DW and 292/~g A l g - 1 DW, respectively (Fig. 2a). The difference was statistically significant (t-test, T = 2.80, df = 4, p = 0.034). The average Zn concentrations in abnormal and normal larvae were 18.8 and 16.0/tg g-~, and Cu concentrations 9.4 and 10.8/~g g-~, respectively (Figs 2b and c). These differences were not statistically significant (t-test, T = 1.01, df = 4, p > 0.1). Unfortunately, concentrations of other metals were not measured in the abnormal and normal larvae. DISCUSSION The present results suggest that accumulation of aluminium significantly contributes to the morphological abnormalities in hydropsychid larvae in the River Kyrrnjoki. This is manifested in the significantly higher AI concentrations recorded in the abnormal larvae than found in the normal larvae. Both laboratory and field exposures of hydropsychids to increasing A1 concentrations in acid conditions have induced correspondingly increasing incidence of darkened and reduced anal papillae (Vuori, 1995, 1996). Staining techniques have also demonstrated darkening of the anal papillae of the Chaoborus larvae (Havas, 1986) and the ion-regulatory organs of some cladoceran species (Havens, 1990) due to the accumulation of aluminium. Petersen (1986) reported increased incidence of darkened anal papillae in hydropsychids exposed to increasing copper concentrations. Similarly, Vuori (1994) observed increasing frequency of anal papillae damage in hydropsychid larvae with increasing Cd concentrations. However, both of these metals are likely to be of less importance in the induction of anal papillae abnormalities than aluminium. Concentrations of Cu and Cd in the River Kyr6njoki are at relatively low levels, whereas aluminium levels are quite high compared to many other rivers contaminated by metals (Table 1, Moore Table 1. Water qualityof the RiverKyrrnjoki,RiffleKolkinkoski and Ramamoorthy, 1984; Vuori, 1995). Copper during samplingof the hydropsychidlarvaeon may 1991 and May 1992. Rangeof metalconcentrationsduringthe periodApril-Julyis concentrations in the river Kyrrnjoki usually range given in the parentheses between 2 and 8/~g 1-~ and Cd concentrations remain Variable 1991 1992 below 0.2/~g I-~, whereas A1 reach maximum Alkalinity(mmol1-~) 0.02 0.12 concentrations above 5 mg 1-~ (Vuori, 1995). Further, COD Mn (02) (mgl-'} 22 21 the larval copper concentrations were identical in Colour Pt (mgl t) 150 150 abnormal and normal larvae (Fig. 2). Nevertheless, Conductivity(mS m-~) 18 13 pH 5.3 5.7 while comparison for other metals, such as Fe, Zn, Pb Suspended solids(mg 1-~) 14 8 and Mn is lacking, their contribution to the Temperature (~'C) 10.0 11.2 morphological abnormalities cannot yet be ruled out. Total P (#g I-~) 49 55 Total AI (/~g1-~) 1860 (1300-6500) 1450(759-5900) The present results imply that iron adsorbed on the Total Fe (pg 1-~) 2154 (I 100-2700) 1200(900-2500) surface of the larval exocuticle has a significantly Total Cu (pg 1-~) 4 (3-5) 2 (1.7-7.7) TotalCd(pgl -~) 0.1 (<0.1q).l) <0.1 (<0.1-0.1) greater contribution to the overall iron concentration Total Zn (#g 1-0 23 (23-55) 20 (17-51) of Hydropsyche pellucidula larvae than the iron K.-M. Vuori and J. Kukkonen 2268 (a) 15000 \ (b) SO00 \ 10000 2000 o g m m o o "o z 1000 5000 FRESH FRESH OLD (c) 1.5 OLD EXOCUTICLE EXOCUTICLE (d) 175 O) 140 \ D) \ 1.0 105 $ g Z O t) o. "D 15 =: 0.5 Z 'O 70 @ 35 u 0.0 FRESH FRESH OLD OLD EXOCUTICLE EXOCUTICLE 160 1.5 (f) (e) A D \ ot 120 $ 1.0 e u o .Ig u ~ 2 2 •," 0.5 z _e .Q L 0.0 FRESH OLD EXOCUTICLE 5o 40 FRESH OLD EXOCUTICLE Fig. 1. The average ( +_ SE, n = 3 replicates of five pooled individuals) concentrations of Fe (a), AI (b), Cd (c), Cu (d), Pb (e) and Zn (f) in the unmoulted (OLD exocuticle) and newly moulted (FRESH exocuticle) larvae of Hydropsychepellucidula. Metals and abnormalities in hydropsychid larvae accumulated within the tissues. Iron has a strong tendency to form surface complexes, especially in acid conditions (Schindler and Stumm, 1985). Heavy iron oxide coatings and iron-humic precipitates have earlier been suggested to increase both the wholebody concentrations of Fe and other metals and the mortality rate of aquatic insect larvae (Gerhardt, 1992; Hare, 1992; Vuori, 1995). These kinds of coating and precipitates on plant and animal surfaces are commonly found in the River Kyr6njoki and other rivers receiving runoff from acid sulphate soils (Vuori, 1993, 1995). Also aluminium hydroxide precipitations on body surfaces have been observed in many studies (e.g. Herrmann, 1987; Dietrich and Schlatter, 1989). However, the present results imply that the accumulation of aluminiura, and not surface adsorption alone, also significantly contributes to the larval A1 concentrations as well as 1:othe morphological abnormalities in acid conditions. However, the clear tendency for 2269 higher AI concentrations in the unmoulted larvae compared to the moulted larvae give also some indication of the surface adsorption of Al. The similarity of the average Cd, Cu, Pb and Zn concentrations in the unmoulted and moulted larvae imply that these metals are readily absorbed in the tissues. The significantly higher Cd and Cu concentrations in the moulted rather than in the unmoulted larvae suggest that either moulting enhances Cd and Cu uptake in hydropsychids or that increased tissue concentrations of these metals enhance moulting of the larvae. The acid conditions of humic waters may particularly enhance the lipid solubility of the humic ligands and the associated cadmium (Petersen et al., 1987). In humic waters, the cadmium uptake rate has been observed to be much higher than in humic-free waters (Penttinen et al., 1995). The permeability of the soft, incompletely chitinized, thin exocuticle of the larvae immediately after moulting may be much higher than that of the chitinized exocuticle. Dressing et al. 4000 (a) -~ \ 3000 m e 1l& 2000 2 "D z G - 10oo DARK NORMAL ANAL PAPILLAE 25 15 (b) (c) 2O X \ f 10 0 J= u Q. 2 2 ~o "D •r £ 5 Z £ -R c N o 0 w _ _ 5 O DARK NORMAL ANAL PAPILLAE DARK NORMAL ANAL PAPILLAE Fig. 2. The average ( ± SE, n = 5 replicates of five pooled individuals) concentrations of AI (a), Cu (b) and Zn (c) in the Hydropsyche pellucidula larvae with darkened, abnormal (dark) and transparent, normal (normal) anal papillae. 2270 K.-M. Vuori and J. Kukkonen (1982) found that most of the equilibrium Cd content was actively accumulated by the hydropsychid larvae within 24 h. As the development of the fully chitinized exocuticle in hydropsychid larvae takes several days (Vuori, personal observations), this period may be the most critical for the accumulation of metals. Of course, in addition to or instead of the increased exocuticle permeability, factors such as the metabolic condition of the larvae during moulting may also contribute to the increase or decrease in metal concentrations. On the other hand, metal uptake may also enhance moulting of invertebrates (Bengtsson et al., 1983). In general, reduction in growth induced by exposure to sublethal concentrations of metals may be alleviated by spending less energy on metabolic defence and more on growth (Sibly and Calow, 1989). Pascoe et al. (1989) reported impaired development of the Chironomus riparius larvae exposed to high cadmium levels (0.15mg Cd 1-'), whereas Timmermans et al. (1992) observed faster larval development upon exposure to lower Cd levels (0.01 and 0.025 mg Cd 1-~) as compared to the control larvae. As there were no differences in the concentrations of copper and zinc between abnormal and normal hydropsychid larvae, it seem plausible to presume that the hydropsychids in the River Kyr6njoki are able to regulate these essential metals in a similar manner as chironomid larvae (Krantzberg and Stokes, 1989). In general, the average concentrations of AI, Cu and Zn were higher in the Hydropsyche larvae collected in 1991 than in those collected in 1992. The lower pH and higher metal levels in 1991 may contribute to this interyear variation. This could also be attributed to the different drying method used: in 1991 the larvae were dried in the oven with much higher probability of sample contamination than in the freeze-drying method used in 1992. In the sediment-dwelling Chironomidae, there is possibility that different microhabitat conditions, such as the redox microenvironments maintained by microbes (Davison and DeVitre, 1992), increases both the surface-bound metal concentrations and deformations, without strict causal relations (see Hare, 1992; Janssens de Bisthoven et al., 1992). In the shallow, fast-flowing and well-oxygenated microhabitats of the hydropsychid larvae, such small-scale variations are less likely to occur. However, microhabitat factors cannot be ignored in the case of hydropsychids, either. For example, different microhabitats supply the larvae with different kinds and amounts of food (e.g. Krawany, 1930; Schuhmacher, 1970; Rhame and Stewart, 1975; Petersen, 1987). This may cause wide discrepancies between individual physiological conditions and larval development, which may in turn affect both metal uptake and the incidence of morphological abnormalities. Further, such quantitative and qualitative differences in the food supply may also expose the individuals to different amounts of dietary metals. The distinct differences in the average metal concentrations between abnormal and normal larvae, as well as between moulted and unmoulted larvae, have important implications for future research and biomonitoring applications. Such differences in metal uptake between different kinds of individuals, as the present results demonstrate, suggest that care should be taken to base any bioaccumulation analysis on morphologically similar individuals with similar stages of larval development. Both the physiological condition and genetic properties of aquatic animals are known to strongly influence the metal uptake and its toxicity in individuals (Moore and Ramamoorthy, 1984; Klerks and Levinton, 1989). Much attention has been paid to the physiological adjustment of the impacts of metals on fishes (e.g. Sj6beck et al., 1984), whereas in aquatic invertebrates, this area is as yet largely unexplored. In the case of aquatic invertebrates, it has been emphasized that the pooling of species at higher taxonomic levels may cause great variation in the bioaccumulation data, due to the wider interspecies differences in behaviour and physiology (Cain et al., 1992; Hare, 1992). The present results suggest that more attention should be paid to these kinds of differences among individuals. CONCLUSIONS Metal concentrations in the Hydropsyche pellucidula larvae inhabiting the acid, metal-polluted River Kyr6njoki are strongly influenced by the stage of development, and abnormalities in the ion-regulatory organs, anal papillae. Iron is mainly adsorbed on the surface of the larval exocuticle, whereas anal papillae abnormalities indicate a strong absorbtion of aluminium in the tissues. The significantly higher cadmium and copper concentrations in the newly moulted larvae as compared to the unmoulted larvae implies that either moulting enhances the accumulation of these metals or these metals enhance moulting of the larvae. The incidence of morphological abnormalities in hydropsychid larvae appears to be a useful tool for impact assessment and the biomonitoring of metal-polluted rivers. However, more research is needed on the relations between metals and their effects on the larvae. Acknowledgements--This study was supported by the Vaasa and Kokkola District Offices of Waters and Environment and by the Academy of Finland. We would like to thank Olle Sir6n and Jukka Palko for the metal analysis, Esa Koskenniemi and Toivo Ollila for their help in the field and Tiina Ristola for comments on the manuscript. REFERENCES Bengtsson G., Gunnarsson T. and Rundgren S. (1983) Growth changes caused by metal uptake in a population of Onychiurus armatus (Collembola) feeding on metal polluted fungi. OIKOS 40, 216-225. Metals and abnormalities in hydropsychid larvae Cain D. J., Luoma S. N., Carter J. L. and Fend S. V. (1992) Aquatic insects as bioindicators of trace element contamination in cobble-bottom rivers and streams. Can. J. Fish. Aquat. Sc,!. 49, 2141-2154. Camargo J. A. (1991) Toxic effects of residual chlorine on larvae of Hydropsyche pellucidula (Trichoptera, Hydropsychidae): a proposal of biological indicator. Bull. Environ. Cont. Toxicol. 47, 261-265. Clements W. H. and Kiffney P. M. (1994) Integrated laboratory and field approach for assessing impacts of heavy metals at th~ Arkansas River, Colorado. Environ. Toxicol. Chem. 13, 397-404. Davison W. and DeVitre R. (1992) Iron particles in freshwater. In Environmental particles Vol. I, Environmental Analytical and Physical Chemistry Series (Edited by Buiite J. and van Leeuwen H. P.), pp. 315-355. Lewis Publishers, Boca Raton. Dietrich D. and Schlatter C. (1989) Aluminium toxicity to rainbow trout at low pH. Aquatic Toxicol. 15, 197-212. Dressing S. A., Maa:~ R. P. and Weiss C. M. (1982) Effect of chemical specia'Iion on the accumulation of cadmium by the caddisfly, Hydropsyche sp. Bull. Environ. Contam. Toxicol. 28, 172-180. Forbes V. E. and Forbes T. L. (1994) Ecotoxicology in Theory and Practice. Chapman and Hall, London. Gerhardt A. (1992) Subacute effects of iron (Fe) on Leptophlebia marginata (L.) (Insecta: Ephemeroptera). Freshwater Biol. 27, 79-84. Hare L. (1992) Aquatic insects and trace metals: bioavailability, bic,accumulation and toxicity. Crit. Rev. Toxicol. 22, 327-369. Hartikainen H. and Yli-Halla M. (1986) Oxidation-induced leaching of sulphate and cations from acid sulphate soils. Water Air Soil Pollut. 27, 1-13. Havas M. (1986) A hematoxylin staining technique to locate sites of aluminium binding in aquatic plants and animals. Water Air Soil Pollut. 30, 735-741. Havens K. E. (1990) Aluminium binding to ion exchange sites in acid-sensitive versus acid-tolerant cladocerans. Environ. Pollut. 6,1, 133-141. Herrmann J. (1987) Aluminium impact of freshwater invertebrates at low pH: a review. In Speciation of Metals in Water, Sediment and Soil Systems. Lecture Notes in Earth Sciences II (Edited by Landner L.), pp. 157-175. Springer-Verlag, Berlin. Janssens de Bisthoven L. G., Timmermans K. R. and Ollevier F. (1992) The concentration of cadmium, lead, copper and zinc in Chironomus gr. thummi larvae (Diptera, Chironomidae) with deformed versus normal menta. Hydrobiologia 239, 141-149. Johnson R. K., Wiederholm T. and Rosenberg D. M. (1993) Freshwater biomonitoring using individual organisms, populations and species assemblages of benthic macroinvertebrates. In Freshwater Biomonitoring and Benthic Macroinvertebrates (Edited by Rosenberg D. M. and Resh V. H.), pp. 40-125. Chapman & Hall, New York. Klerks P. L. and Levinton J. S. (1989) Effects of heavy metals in a polluted aquatic ecosystem. In Ecotoxicology: Problems and Approaches (Edited by Levin S. A., Harwell M. A., Kelly J. R. and Kimball K. D.), pp. 41-67. Springer-Verlag, New York. Krantzberg G. and :Stokes P. M. (1989) Metal regulation, tolerance and body burdens in the larvae of the genus Chironomus. Can. J. Fish. Aquat. Sci. 46, 389-398. Krawany H. (1930) Trichopterenstudien im Gebiete der Lunzer See. Int. Rev. Ges. Hydrob. Hydrogr. 23, 417-427. Lenat D. R. (1993) Using mentum deformities of Chironomus larvae to evaluate the effects of toxicity and organic loading in streams. J. N. Am. Benthol. Soc. 12, 265-269. McCarty L. S. and Mackay D. (1993) Enhancing ecotoxicological modeling and assessment. Environ. Sci. Technol. 27, 1719--1726. 2271 Moore J. W. and Ramamoorthy S. (1984) Heavy Metals in Natural Waters. Applied Monitoring and Impact Assessment. Springer-Verlag, New York. Moriarty F. (1990) Ecotoxicology: The Stu@ of PoUutants in Ecosystems. Academic Press, London. Mount D. R., Barth A. K., Garrison T. D., Barten K. A. and Hockett J. R. (1994) Dietary and waterborne exposure of rainbow trout (Onchorhynchus mykiss) to copper, cadmium, lead and zinc using a live diet. Environ. Toxicol. Chem. 13, 2031-2041. Pascoe D., Williams K. A. and Green D. W. J. (1989) Chronic toxicity of cadmium to Chironomus riparius Meigen-effects upon larval development and adult emergence. Hydrobiologia 175, 109-115. Penttinen S., Kukkonen J. and Oikari A. (1995) The kinetics of cadmium in Daphnia magna as affected by humic substances and water hardness. Ecotox. Environ. Safety 30, 72-76. Petersen L.-B. M. (1987) Field and laboratory studies on the biology of hydropsychids (Trichoptera, Hydropsyckidae). Dissertation, Dept. Ecology/Limnology, Univ. Lund, Sweden. Petersen R. C. (1986) Population and guild analysis for interpretation of heavy metal pollution in streams. In Community Testing (Edited by Cairns J. Jr.), pp. 180-198. ASTM STP 920, American Society for Testing and Materials, Philadelphia, PA. Petersen R. C., Hargeby A. and Kullberg A. (1987) The biological importance of humic material in acidified waters. A summary of the chemistry, biology and ecotoxicology of aquatic humus in acidified surface waters. National Swedish Environ. Prot. Board, Report 338, Solna, Sweden. Rhame R. E. and Stewart K. (1975) Life cycles and food habits of three Hydropsychidae (Trichoptera) species in the Brazos river, Texas. Trans. Am. Ent. Soc. 102, 65-99. Schindler P. W. and Stumm W. (1985) The surface chemistry of oxides, hydroxides and oxide minerals. In Aquatic Surface Chemistry. Chemical Processes at the Particle-Water Interface (Edited by Stumm W.), pp. 83110. John Wiley & Sons, New York. Schuhmacher H. (1970) Untersuchungen zur Taxonomie, Biologie und Okologie einiger K6cherfiiegenarten der Gattung Hydropsyche Pict. (Insecta, Trichoptera). Int. Rev. ges. Hydrobiol. 55, 511-557. Sibly R. M. and Calow P. (1989) A life-cycle theory of responses to stress. Biol. J. Linnean Soc. 37, 101-116. Siltala A. J. (1907) Trichopterologische Untersuchungen 2.: Obcr die post-embryonale Entwicklung der Trichopteren larven. Zool. Jb. Suppl. 9, 413-427. Sj6beck M.-L., Haux C., Larsson A. and Lithner (3. (1984) Biochemical and hematological studies on perch, Perca fluviatilis, from the cadmium contaminated river Em~n. Ecotoxicol. Environ. Safety 8, 303-312. Suomen Standardisoimisliitto (1980a) Metal content of water, sludge and sediment determined by flame atomic absorption spectrometry. Principles and practical instructions, Standard SFS 3044. Suomen Standardisoimisliitto, 1980, Helsinki. Suomen Standardisoimisliitto (1980b) Metal content of water, sludge and sediment determined by flame atomic absorption spectrometry. Special guidelines for cadmium, cobalt, copper, lead, nickel, iron and zinc, Standard SFS 3047. Suomen Standardisoimisliitto, 1980, Helsinki. Suomen Standardisoimisliitto (1990a) Water analysis. Metal content of biological material determined by atomic absorption spectrometry. Digestion. Standard SFS 5075. Suomen Standardisoimisliitto, 1990, Helsinki. Suomen Standardisoimisliitto (1990b) Metal content of water, sludge and sediment determined by flameless atomic absorption spectrometry. Atomization in a graphite furnace. Special guidelines for aluminium, 2272 K.-M. Vuori and J. Kukkonen cadmium, cobalt, chromium, copper, lead, manganese, nickel and iron, Standard SFS 5502. Suomen Standardisoimisliitto, 1990, Helsinki. Timmermans K. R., Peeters W. and Tonkes M. (1992) Cadmium, zinc, lead and copper in Chironomus riparius (Meigen) larvae (Diptera, Chironomidae): uptake and effects. Hydrobiologia 241, 119-134. van Urk G., Kerkum F. C. M. and Smit H. (1992) Life cycle patterns, density and frequency of deformities in Chironomus larvae (Diptera: Chironomidae) over a contaminated sediment gradient. Can. J. Fish. Aquat. Sci. 49, 2291-2299. Vuori K.-M. (1993) Influence of water quality and feeding habits on the whole-body metal concentrations in lotic trichopteran larvae. Limnologica 23, 301-308. Vuori K.-M. (1994) Rapid behavioural and morphological responses of hydropsychid larvae (Trichoptera, Hydropsychidae) to sublethal cadmium exposure. Environ. Pollut. 84, 291-299. Vuori K.-M. (1995) Species- and population-specific responses of translocated hydropsychid larvae (Tri- choptera, Hydropsychidae) to runoff from acid sulphate soils in the River Kyr6njoki, western Finland. Freshwater Biol. 33, 305-318. Vuori K.-M. (1996) Acid-induced acute toxicity of aluminium to three species of filter feeding caddis larvae (Trichoptera, Arctopsychidae and Hydropsychidae). Freshwater Biol. 35, 179-188. Wallace J. B. (1975) The larval retreat and food of Arctopsyche; with phylogenetic notes on feeding adaptations in Hydropsychidae larvae (Trichoptera). Ann. Ent. Soc. Am. 68, 167-173. Warwick W. F. (1988) Morphological deformities in Chironomidae (Diptera) larvae as biological indicators of toxic stress. In Toxic Contaminants and Ecosystem Health; a Great Lakes Focus (Edited by Evans M. S.), pp. 281-320. Wiley and sons, New York. Xiang V. J., Schr6der P. and Schwoerbel J. (1984) Phfinologie und Nahrung der Larven von Hydropsyche angustipennis und H. siltalai (Trichoptera, Hydropsychidae) in einem SeebfluB. Arch. Hydrobiol. (Suppl.) 66, 255-292.