Article pubs.acs.org/est
Impact of Tributyltin on Immune Response and Life History Traits of Chironomus riparius: Single and Multigeneration Effects and Recovery from Pollution Thomas M. Lilley,†,* Lasse Ruokolainen,‡ Ari Pikkarainen,† Veronika N. Laine,† Janne Kilpimaa, Markus J. Rantala,† and Mikko Nikinmaa† †
Department of Biology, University of Turku, FI-20014 Turku, Finland Department of Biological and Environmental Sciences, University of Helsinki, Viikinkaari 1, P.O. Box 65, FI-00014 Finland
‡
ABSTRACT: Chironomids play an important role in the detritus cycle and as a component in brackish- and freshwater benthic and terrestrial food webs. If TBT is present in their environment, then they may accumulate tributyltin (TBT) during their juvenile period, which negatively affects many of their life history characteristics. The aim of this experiment is to test the effects of three TBT sediment concentrations (nominal 30, 90, and 180 μg/kg) on life history traits (development time, survival, fecundity, and weight) and immune response (number of hemocytes and phenoloxidase activity) of the nonbiting midge, Chironomus riparius. These responses were recorded immediately after one generation of TBT exposure, and in the long run during five consecutive generations. We also assessed recovery from pollution after four generations of TBT exposure. In a single generation, TBT affected all measured parameters, except phenoloxidase activity, when compared to the control. Long-term-effects of TBT lead to extinction of all treatments after the fifth generation. Again, all measured variables significantly differ from the control, although TBT had varying effects on the measured variables. Most of the effects of TBT on population viability were not evident during recovery, once TBT was removed from the sediment. The effect of previous TBT contamination was observed only in delayed larval development, suggesting that TBT has only limited maternal/epigenetic effects on individual condition. However, altered schedules in the life-cycle can have unexpected ecological impacts. TBT decreases the viability of Chironomus riparius and the effect will become stronger if exposure to TBT continues for many generations. Yet, the harmful effect of TBT disappears quickly as the TBT is removed from the environment. juvenile period.8 TBT can exert an affect on many life history traits,9−11 which can have important ecological consequences.12 Furthermore, chironomids are an important food source for fish, birds, and bats, and TBT uptake via food may be an important accumulation route. Due to rapid biotransformation, chironomid species with fast TBT metabolism relieve their predators from high TBT uptake.8 However, in northern latitudes, metabolism is slow and chironomids may take several months to develop during the winter.13 Under these conditions, TBT uptake can be expected to be stronger, leading to accumulation in predatory animals. Very high concentrations have been linked with decreased fecundity, body weight, and larval survival in Chironomus riparius.10 Insects, such as chironomids, are excellent model organisms for studying effects of pollution on immune function, as their immune defense system is far less complex than the vertebrate
1. INTRODUCTION Human-introduced organic tin compounds, such as tributyltin (TBT) and triphenyltin (TPT) in marine and fresh-water ecosystems, have long been of interest in environmental toxicology due to their detrimental effects on nontarget organisms.1,2 Organic tin compounds have been utilized extensively in industry for the last 60 years as heat and light stabilizers for polyvinyl chloride (PVC) products, pesticides, fungicides, and marine antifouling paints.3 Although organic tin compounds are rapidly decomposed in aerated water columns, they appear to be quite stable, present for up to tens of years in anaerobic sediments.4 As a consequence, concentrations in sediments from harbors can be high even at present, although organic tin compounds have been completely banned in IMO member states since 2008.5 Chironomid species, inhabiting aquatic sediments during their larval period, are a very important group ecologically,6 playing an important role in the detritus cycle and as a component in benthic and terrestrial food webs. Due to their high tolerance of anoxia,7 chironomids are able to live in TBTcontaminated sediments and bioconcentrate TBT during their © 2012 American Chemical Society
Received: Revised: Accepted: Published: 7382
February 14, 2012 May 31, 2012 June 4, 2012 June 4, 2012 dx.doi.org/10.1021/es300536t | Environ. Sci. Technol. 2012, 46, 7382−7389
Environmental Science & Technology
Article
is reared in a clean environment, due to the possible maternal and epigenetic effects of TBT.
immune system, even though many components are homologous.14 In insects, the major humoral immune effector system, responsible for resistance to parasites and pathogens, is the phenoloxidase (PO) cascade.15 PO is expressed and regulated in response to the presence of foreign materials or pathogens in the hemocoel and the activation of this enzyme results in the melanization and death of the pathogen.16 In the cellular response, hemocytes attach to the intruder, and in the process, the foreign object may become completely encapsulated in layers of hemocytes, which die and subsequently become melanized, thereby isolating the intruder from host tissue.15 Although the strength of different components of the insect immune system has been found to be heritable, many studies have shown that the strength of the insect immune defense is heavily influenced by environmental conditions.17 For example, recent studies on insects have demonstrated the influence of heavy metal pollution on the immune system.18−20 However, studies testing whether organic tin compounds impact insect immune system are lacking. Even moderate amounts of organic tin compounds can be lethal to organisms.21 The effect of TBT in marine invertebrates is expressed as disorders in growth, development, and reproduction.21,2,22 These findings eventually led to the banning of these compounds. Organisms in their early developmental stages are less tolerant to the effects of organic tin compounds.21 However, the effects of organic tin compounds on the larvae of benthic insects have been little studied10 and experimental studies, where individuals have been exposed to pollutants for multiple generations, are even more scarce.11 Due to possible maternal and epigenetic effects, which have not been studied in detail with TBT, the detrimental effect of pollutants could accumulate in subsequent generations.23,24 Environmental pollution can also act as a strong selective agent, leading to species adaptation and increased viability in polluted environments (for adaptation of insects to pollution see, e.g., ref 25). Furthermore, studies testing how populations recover from exposure to pollution are lacking. If pollutants have maternal or epigenetic effects, then one could expect the harmful effect of pollutants to remain long after contamination has been removed from the environment. The aim of this experiment was to test the effects of TBT on the chironomid, Chironomus riparius, in three stages: (1) To be able to compare our results with previous experiments,10 we tested whether a single generation exposure to three, relatively low concentrations of TBT has an effect on adult size, survival, fertility, larval development time, and immune function (phenoloxidase activity and number of hemocytes). (2) We continued to test the effects of TBT contamination for five consecutive generations. (3) Finally, we tested whether the measured effects of TBT are lost when a population exposed to TBT for multiple (four) generations is reared in a clean environment; i.e., to test for recovery potential after contamination removal. The hypothesis was that high concentrations of TBT in the sediment result in delayed emergence of imogens and reduce adult weight and egg production by females. Moreover, high sediment TBT concentrations should disrupt the immune function leading to a decrease in phenoloxidase activity and the number of hemocytes.26 We expected that the effect would be stronger if exposure continues for consecutive generations. In addition, we expected that the effect of pollution on performance would not cease if a population exposed to TBT for multiple generations
2. MATERIALS AND METHODS The Model Organism. We used the nonbiting midge, Chironomus riparius (Diptera: Chironomidae) as the model organism to study the effect of TBT. Chironomids are an ecologically important group of benthic macro-invertebrates playing an important role in detritus processing and recycling organic matter and are also important prey species for fish, bats, and aquatic birds.7,27 The larvae of Chironomus riparius occupy a wide variety of aquatic and semiaquatic environments including fresh- and brackish water, such as in the Archipelago Sea (5 psu) in S−W Finland. It is a collector-gatherer feeding on sediment-deposited detritus with a life-cycle of approximately 22 days.28 We chose C. riparius as our study species, because it has been used extensively in the past in sediment contamination tests29 30 31 and standard methods have been described for conducting toxicity tests with the C. riparius and the related species Chironomus dilutus.32−34 All C. riparius larvae used in the experiment were obtained from our in-house mass culture, which was established in 2009 with egg ropes from the University of Eastern Finland and University of Frankfurt, to avoid effects of inbreeding. The culture was maintained in a climate room with a temperature of 18 °C and a relative humidity of approximately 70%. The light:dark rhythm was at 16:8 h. The water used was run through ion exchange and active carbon filters (pH-value 7.9− 8.4; conductivity 540 μS/cm). The test conditions were equal to the culturing conditions. First Generation Effects. To test the single generation effect of sediment TBT on other life history traits and immune defense, we used three exposure groups [low (30 μg/kg), intermediate (90 μg/kg) and high (180 μg/kg TBT in sediment dry weight)], a control and a solvent control with four replicates of each. The selected TBT treatments were based on data collected from a random sampling of the Archipelago Sea, in South-Western Finland, where the mean measured sediment TBT concentration is 42 μg/kg with 95% of observation fitting within the range from 0.7 μg/kg to 353.9 μg/kg.35 To reduce the number of variables and simplify the interpretation of the results, we opted to only spike the sediment and not food in addition. Tributyltin chloride was measured (μg/kg sediment) and mixed into the sediment in 500 mL EtoH, which was also added to the solvent control to test for the effects of EtoH. The EtoH was allowed to evaporate for three days in a laminar hood. The sediment used in the experiment was a mixture of Finnish caolin clay (Kerapro, Helsinki) and fine sand (grain size 0.1−0.4 mm, Biltema) in a ratio of 1:1 (Loss on ignition 4%, median grain size 138.1 μm, measured according to Lilley et al. 201212). New batches of sediment were produced for every generation using the same stock TBT-solutions. We used custom-built aquaria (30 × 20 × 20) for the experiment covered with a wire-mesh frame (30 × 20 × 20) and aerated through Pasteur glass pipettes, which were attached to an air pump (Sera) via PVC tubes. The aquaria contained 1250 g of the sediment and 3000 mL of water. Water and sediment was allowed to settle for 48 h before addition of larvae. Three days before the start of experiment, freshly laid (≤24 h) egg ropes were taken from the mass culture and transferred to Petri dishes. We randomly took five individual freshly 7383
dx.doi.org/10.1021/es300536t | Environ. Sci. Technol. 2012, 46, 7382−7389
Environmental Science & Technology
Article
+1 L distilled water) for hemocyte counting. The anticoagulantbound hemolymph was immediately placed in a Bürker chamber and the number of hemocytes per milliliter was estimated without staining. To measure PO activity, a hemolymph sample of 2 μL was collected into a plastic micropipet. The obtained hemolymph was immediately mixed with 50 μL of phosphate-buffered saline solution (pH 7.5) with 1 mM PMSF (phenylmethylsulfonylfluoride, Sigma-Aldrich), vortexed, and stored at −80 °C prior to enzymatic assay. PO was measured in duplicates of 10 μL using 200 μL of 10 mM L-DOPA as substrate and following the changes in absorbance at 495 nm for 30 min with a multilabel counter (EnVision 2103 Multilabel Reader, Wallac, Turku, Finland) at 20 °C at 1-min intervals. PO activity was expressed as the total change in optical density. PO activity is often expressed as units per milligram of hemolymph protein,37 and for this, the total protein concentration was quantified. Total protein concentration was measured using the BioRad protein assay kit based on the Bradford method.38 In the statistical analysis, we used both the absolute PO activity (dAbs min−1 min−1) and the specific PO activity (U/mg protein). Sediment TBT Analyses. The LC/MS−MS-based analysis methods for sediment samples, analyzed as they were after preparation for the fifth generation, are described in Lilley et al.35 The results of the sediment TBT concentrations indicated the following concentrations for our treatments 24 μg/kg (30), 117 μg/kg (90), and 231 μg/kg (180). We opted for a single measurement of TBT concentrations in sediment due to its resistance to degradation39 and use of the same TBT stock to prepare the sediments in each generation. Statistical Analyses. In the case of the single-generation exposure and recovery scenarios, deviation of individual TBT treatment levels from control was tested using general linear models (glm). The multigeneration data were tested for an interaction between generation time and TBT treatment using MANOVA. All p-values
