Available online at www.sciencedirect.com Chemosphere 71 (2008) 1928–1936 www.elsevier.com/locate/chemosphere Effects of environmental contaminants on hemoglobin of larvae of aquatic midge, Chironomus riparius (Diptera: Chironomidae): A potential biomarker for ecotoxicity monitoring Mi-Hee Ha, Jinhee Choi * Faculty of Environmental Engineering, College of Urban Science, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743, Republic of Korea Received 27 September 2007; received in revised form 29 December 2007; accepted 3 January 2008 Available online 6 March 2008 Abstract The effects of environmental contaminants (i.e., nonylphenol, bisphenol A diglycidyl ether, benzo[a]pyrene, chlorpyriphos, paraquat dichloride, and lead nitrate) on Chironomus hemoglobin were investigated in the 4th instar larvae of Chironomus riparius (Diptera: Chi- ronomidae), with respect to the total hemoglobin contents, individual globin gene expression, individual globin protein expression and hemoglobin oxidation. In our studies, 7 and 6 globin isoforms were preliminarily characterized by molecular weight and isoelectric point, respectively, in the 4th instar larvae of C. riparius. Most chemicals were unable to modify the total hemoglobin contents, however, the expression patterns of the globin transcript and proteins suggest that C. riparius globin exists in both inducible and consecutively expressed forms, with multiplicity that may allow this animal to better adapt toward stressful environmental conditions, including pollution stress. The oxyhemoglobin was observed to be downregulated in C. riparius on exposure to bisphenol A and chlorpyriphos, probably reflecting its increased autoxidation to methemoglobin. The overall results would suggest that globin can be a target molecule of environmental contaminants, and of the tested parameters, the alteration of individual globin levels (i.e., mRNA or protein levels) may have potential for the development of a biomarker for ecotoxicity monitoring. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Chironomus riparius; Globin; Hemoglobin multiplicity; Ecotoxicity; Biomarker 1. Introduction 2001; Bettinetti et al., 2002; Crane et al., 2002; Lee and Choi, 2006; Lee et al., 2006). The aquatic larvae of non-biting midges (Chironomidae, One of the main particularities of Chironomus, however, Diptera) are distributed globally, and are the most abun- is they possess hemoglobin (Hb) during their larval stage. dant group of insects found in freshwater ecosystems. They Species of the genus Chironomus exhibit stage-specific hold an important position in the aquatic food chain, being and tissue-specific single-chain globin syntheses throughout a major food source for fish and other vertebrates and the four larval stages. The Hb is synthesized in the larval invertebrates (Cranston, 1995). They are sensitive to many fat body, and then secreted into the hemolymph (Bergtrom pollutants, easy to culture and have a short lifecycle; thus, et al., 1976; Saffarini et al., 1991). Although Hb is widely are used extensively to assess the acute and sublethal distributed throughout the animal kingdom, its occurrence toxicities of contaminated sediments and water (Matthew in invertebrates is restricted to only a few representatives of and David, 1998; Choi et al., 2000, 2002; Matthew et al., the large taxonomic groups, known as Insecta or Crusta- ceans. The Chironomid Hb members of insect respiratory proteins have been extensively studied, and found to be very attractive research materials as they offer the simplest * Corresponding author. Tel.: +82 2 2210 5622; fax: +82 2 2244 2245. model for more complex Hb; analysis of their structure E-mail address: [email protected] (J. Choi). may provide key information in understanding the action 0045-6535/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.01.018 M.-H. Ha, J. Choi / Chemosphere 71 (2008) 1928–1936 1929 mechanism of haem proteins, but their high heterogeneity aquaria. All larvae used in the experiment originated from provides many evolutionary problems for molecular biolo- the same egg mass, and were collected at the same time gists (Osmulski and Leyko, 1986). From an ecotoxicologi- after egg hatching to obtain an age-synchronized popula- cal point of view, Hb possessing organisms are often tion. From our previous experiments, 4th instar develop- considered as candidate sentinel species for biomonitoring ment of C. riparius larvae persisted for 22–38 days after (Osmulski and Leyko, 1986). Studies on the biomarkers of the eggs had hatched, and the measurements of the cellu- xenobiotic pollution using Hb-related parameters, have lar and extracellular proteins were conducted on the lar- recently been expanded into include invertebrate systems vae during the middle of the 4th instar developmental (Choi and Roche, 2004; Lamkemeyer et al., 2005; Rider stage (i.e., 32 days after the eggs had hatched). For the et al., 2005; Lee et al., 2006; Rider and LeBlanc, 2006). chemical treatment, 10 of the 4th instar larvae of C. ripa- Considering the potential of Chironomus larvae as bio- rius were transferred into 200 ml beakers, containing monitoring species, and the physiological particularities 100 ml of dechlorinated tap water, and treated with chem- of Chironomus Hb, this invertebrate respiratory pigment icals to assess the sublethal exposure. For each experi- has considerable potential as a sensitive biomarker for ment, 0.1 ml of the test solution was added to the environmental monitoring. In this study, the changes of experimental beakers prior to larvae introduction. Ace- the Chironomus Hb in 4th instar larvae of Chironomus ripa- tone was used as the solvent for NP, BPA, B[a]P and rius Mg. (Diptera: Chironomidae), due to exposure to 6 CP, and water was used for PQ, and Pb. Our preliminary environmental chemicals, with different modes of action; tests indicated that acetone provoked no significant effects namely, nonylphenol (NP), bisphenol A diglycidyl ether during the experiments (data not shown). Exposure was (BPA), benzo[a]pyrene (B[a]P) chlorpyriphos (CP), para- carried at constant temperature (20 ± 1 °C), with a 16:8- quat dichloride (PQ), and lead(II)nitrate (Pb) were evalu- h (light:dark) photoperiod for all experiments. For the ated. Nonylphenol is used in the polymer industry analysis of each Hb parameter, 10 larvae from the control (European Union, 2002), BPA is an intermediate in the and experimental tanks were pooled, with hemolymphs production of polycarbonate and epoxy resins (European withdrawn by opening the body wall, and the body fluids Union, 2003), BaP is a ubiquitously distributed polycyclic transferred into eppendorf cups containing ice-chilled aromatic hydrocarbons (PAH) (Juhasz and Naidu, 2000), physiological solution (NaCl 10.3 mM). Three replicates CP is an organophosphorous insecticide, PQ is an oxygen were conducted for each experimental compound for all radical generating herbicide, and Pb is commonly found experiments. heavy metal. The effects of these environmental contami- nants on Hb parameters were investigated in 4th instar lar- 2.3. Acute toxicity test vae of C. riparius with respect to; firstly, the total Hb contents, using a cyanometHb procedure, secondly, indi- Three groups of 10 larvae were exposed to four concen- vidual globin gene expression, with reverse transcription- trations of each chemical, whereas other groups were kept polymerase chain reaction (RT-PCR), thirdly, individual as a control. Acute toxicity was determined after 24 h of globin protein expression using polyacrylamide gel electro- exposure, using death of individuals as an endpoint. Log- phoresis (PAGE) and electrophoresis of isoelectric focusing Probit transformation of the data was used in order to esti- (IEF), and finally, Hb oxidation employing multi-wave- mate 10%, median and 90% 24 h lethal concentration length rapid-scanning spectrophotometry. (LC10, LC50 and LC90) values and the corresponding 95% confidence intervals. 2. Materials and methods 2.4. Body dry weight measurement 2.1. Organisms For the measurement of body dry weights, 10 larvae col- Using an original strain of C. riparius provided by the lected after 24 h of exposure to the compounds. The fresh Toxicology Research Center of the Korea Research Insti- weights were immediately measured. The dry body weight tute of Chemical Technology (Daejeon, Korea), larvae of the larvae was measured after they were exposed to a were obtained from adults reared in our laboratory. The temperature of 105 °C for 24 h. The weights were rounded larvae, fed fish flake food (Tetramin, Tetrawerke, Melle, off to the nearest 0.1 mg. Germany), were reared in a 2 l glass chamber, containing dechlorinated tap water and acid-washed sand, with aera- 2.5. Protein and hemoglobin contents measurement tion under a 16–8 h light-dark photoperiod at room tem- perature (20 ± 1 °C). The total Hb contents of the hemolymphs were esti- mated via the cyanometHb procedure (Tentori and Sal- 2.2. Exposure conditions vati, 1981), using a plasma Hb kit (Sigma–Aldrich Chemical, St. Louis, MO, USA). The protein content Hemoglobin related parameters were assessed using was measured using the Bradford method (Bradford, groups of 4th instar larvae collected from the rearing 1976). 1930 M.-H. Ha, J. Choi / Chemosphere 71 (2008) 1928–1936 2.6. Globin gene expression analysis and Bryan (1977). Briefly, the proteins were subjected to electrophoresis on a set of gels, containing various poly- The 10 larvae pooled from the