Journal of Environmental Science and Health Part A (2007) 42, 1–9 Copyright C Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934520601015255 Effects of cadmium and tributyltin on development and reproduction of the non-biting midge Chironomus riparius (Diptera)—baseline experiments for future multi-generation studies CHRISTIAN VOGT, DANIELA BELZ, SIMONE GALLUBA, CARSTEN NOWAK, MATTHIAS OETKEN and JORG¨ OEHLMANN Johann Wolfgang Goethe-University Frankfurt am Main, Department of Aquatic Ecotoxicology, Frankfurt am Main, Germany In this study the effects of tributyltin (TBT) and cadmium (Cd) were tested on the freshwater arthropod Chironomus riparius (Diptera) in life-cycle experiments. To this end, the OECD guideline 218 was extended with reproduction relevant parameters (e.g. number of fertile egg masses per female). Based on these reproduction data the number of larvae for next generation and the population growth rate were calculated. Experiments were performed using environmentally relevant concentrations of the test substances in the sediment (nominal ranges: 50–200 µg Sn/kg and 0.2–2.1 mg Cd/kg, on a dry weight basis). Quartz sand was used as sediment in order to develop a test system for a following research project, focusing on the effects of TBT and Cd as model stressors on C. riparius populations in multi-generation studies. Both model stressors caused significant effects on development and reproduction of C. riparius. Larval mortality proved to be a sensitive parameter and a clear concentration-response relationship was observed with a significant increase (P < 0.001) at the highest TBT concentration and at a Cd concentration of 1.17 mg Cd/kg dw (P < 0.05). The two highest TBT concentrations emergence was significantly (P < 0.05) delayed compared to the solvent control. For Cd a clear prolonged average main emergence interval was observed, but not for TBT. TBT and Cd inhibited also oviposition. These effects resulted in a concentration-depended reduction of the population growth rate. Keywords: Chironomus riparius,Tributyltin, Cadmium, Life-cycle experiments, Multi-generation studies. Introduction native field sediments with larvae being exposed to a mix- ture of native and man-made substances.[1−3] Furthermore, Various studies with C. riparius have shown that this or- two guidelines were developed for the routine testing and ganism is suitable for the testing of sediment toxicity in assessment of chemicals, wherein the larvae were exposed the laboratory. The main advantages of chironomids as test to substances via water[4] or via sediment.[5] These guide- species are that they (I) are easy and cheap to culture in lines are well established in ecotoxicological research and the laboratory; (II) spend a large part of their life-cycle standard testing, because various life-cycle parameters can as a larva in the sediment; (III) are model organisms for be observed to detect possible impacts of substances on the a widespread and species-rich taxonomic order in aquatic development and reproduction of chironomids.[6−8] ecosystems; (IV) are sensitive to pollutants; (V) have a short Therefore, we used C. riparius as a model organism for life-cycle with completing 10 or more generations per year aresearch project, in which the relation between pollution under standard conditions and (VI) have a sexual mode of and effects on the genetic diversity of exposed populations reproduction. Thus, C. riparius is becoming more popular is investigated. In this project, effects of model substances as a standard organism for bioassays. Several monitoring like tributyltin (TBT) and cadmium (Cd) on ecotoxicologi- studies were performed for the assessment of the toxicity of cal and genetic endpoints are analysed by performing multi- generation experiments according to the OECD guideline [5] Address correspondence to Christian Vogt, Johann Wolfgang 218. The guideline was extended with reproduction rele- Goethe-University Frankfurt am Main, Department of Aquatic vant parameters such as the number of fertile egg masses per Ecotoxicology, Siesmayerstrasse 70, D-60323 Frankfurt am female, the clutch size and the hatchability of egg masses. Main, Germany. E-mail: [email protected] Furthermore, the sediment described in the OECD guide- Received May 26, 2006. line was modified and a sediment free of organic matter 2 Vogt et al. containing only pure quartz sand was used. This modified into 24-microwell plates for hatching (2 mL reconstituted sediment was selected to avoid fungi or bacterial infections water per cavity). Newly hatched larvae from different egg and to reduce the risk of a possible extinction of the chi- ropes were combined to randomly select 20 and 50 first ronomid population during the multi-generation studies. instar larvae for the TBT and Cd experiments respectively. The aim of the baseline experiments reported here was The larvae were transferred to each test vessel using a to determinate suitable test concentrations for TBT and Pasteur glass pipette and a stereo microscope (Olympus SZ Cd for the following multi-generation studies. In this paper 40, Hamburg, Germany). The aeration was stopped for 24 the concentration-response curve for TBT and Cd in an hinorder to let the larvae dig into the sediment. The larvae extended life-cycle test according to the modified OECD were fed with a ground TetraMin suspension (Tetra GmbH, guideline 218 is shown. Because endpoints determined on Melle, Germany) according to OECD guideline 218. As the individual level have an insufficient power to extrapolate from the 12th experimental day on, the number of dead for population effects of pollutants,[9−11] an integrated ap- larvae and pupae, as well as the number of emerged imag- proach of single parameters was applied, which facilitated ines were counted once a day. Successfully emerged midges the calculation of the population growth rate. were collected with an exhauster and transferred into a breeding container, which consisted of a glass aquarium (30 × 20 × 20 cm) closed with a stainless steel gaze (mesh Materials and methods size 0.5 mm). Each breeding container contained a plastic dish (11.5 × 11.5 × 5.5 cm), filled with 400 mL of recon- Test organisms stituted water for oviposition. The egg ropes were removed daily out of the breeding container. The number of eggs per All Chironomus riparius larvae used in the experiments were egg mass was counted and each egg mass was placed in a obtained from our in-house mass culture, which was es- tube of a 24-microwell plate. After 3 days the hatchability tablished in 2004 with egg ropes of 11 different labora- was determined. Finally, the number of dead larvae and tory stocks from 7 countries (Bulgaria, Germany, France, dead pupae in the sediment was counted. At the end of the Finland, Great Britain, the Netherlands and the United TBT experiment (at day 32), dead imagines were collected States of America). The culture was maintained in a cli- ◦ ◦ and dried at 36 Cfor 3 days. The dry body weight of each mate room with a temperature of 20 ± 1 C, 70% relative animal was determined with a sensitive electronic balance humidity and a light:dark rhythm of 16:8 hours. The test (Sartorius 4401, Gottingen,¨ Germany) to the nearest µg. conditions were equal to the culturing conditions. The number of eggs per clutch were recorded, based on the method of Benoit et al.[13] for Chironomus tentans with the modification that the number of spirals per egg mass Experimental procedures wasmultiplied with the number of eggs from the middle In this study, static life-cycle experiments were conducted spiral (ring counting method). Prior to the experiment the according to the OECD Guideline 218.[5] Midges were ex- estimation was evaluated using a calibration curve. For this posed to the test substances via sediment starting with the purpose the number of eggs from 76 clutches was first calcu- first larval stage until emergence. For establishing a suitable lated according to the described method and then the same test design for the following multi-generation studies two egg masses were dissolved by using 40% sulphuric acid and different glass dishes with different numbers of larvae per all eggs from each clutch were counted separately. Very few beaker and sediment:water ratio were used. First, 600 mL egg masses did not show a regular spiral-shape so that their glass beakers (Simax, Czech Republic) containing 100 g egg number could not be estimated with the ring counting quartz sand (grain size 0.1–0.4 mm, Quick Mix Group, Os- method. nabruck,¨ Germany), 400 mL reconstituted water (pH-value 7.9–8.4; conductivity 540 µS/cm)[12] and 20 animals were Chemicals and residual analyses used for the TBT experiment. Secondly, 2 L crystallising dishes (Schott, Mainz, Germany, ∅ 19 cm) filled with 100 g Prior to the insertion of the larvae, the sediment was spiked quartz sand and 1 L reconstituted water were conducted with tributyltin (TBT-Cl, CAS-number 1461-22-9, Merck, for the cadmium experiment. In this experiment 50 animals Darmstadt Germany, > 97% purity, nominal concentra- per crystallising dish were exposed. Each exposure group tions: 50, 80, 100 and 200 µg Sn/kg dw) or cadmium ∗ consisted of five (TBT) or two (Cd) replicates. Prior to the (CdSO4 8H2O, CAS-number 7790-84-3, Merck, Darm- experiment the vessels were prepared with sediment and stadt Germany, > 99% purity, nominal concentrations: 0.2, water and finally covered with gaze (mesh size 0.75 mm). 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, 2.1 mg Cd/kg dw). Because Moreover, test vessels were gently aerated through Pasteur of the low water solubility of tributyltin, ethanol (Merck glass pipettes, which were attached to an air compressor (40 Schuchardt Chemicals, Darmstadt, Germany, > 99.5% pu- A, Die Pumpe, Holm, Germany) via PVC tubes.