Environ. Sci. Technol. 2007, 41, 6137-6141
Concentration Changes of Organochlorine Compounds and Polybromodiphenyl Ethers during Metamorphosis of Aquatic Insects M I R E I A B A R T R O N S , * ,†,‡ JOAN O. GRIMALT,‡ AND JORDI CATALAN† Limnology Unit (CSIC-UB), Centre for Advanced Studies of Blanes (CEAB-CSIC), Acce´s Cala St. Francesc, 14, 17300 Blanes, Catalonia, Spain, and Department of Environmental Chemistry (IIQAB-CSIC), Jordi Girona, 18, 08034 - Barcelona, Catalonia, Spain
The role of insect larvae and pupae as sources of organochlorine compounds (OCs) and polybromodiphenyl ethers (PBDEs) in freshwater food webs for high predators such as fish is evaluated. Trichoptera and diptera have been taken as organisms of choice for such comparison because they are common in benthic aquatic habitats and accumulate substantial amounts of these compounds. Hexachlorobenzene, hexachlorocyclohexanes, 4,4′-DDE, 4,4′DDT, polychlorobiphenyls, and PBDEs have been measured. The results show a nonselective enrichment of OCs and PBDEs from larvae to pupae. These concentration increases may result from the weight loss of pupae during metamorphosis as a consequence of mainly protein carbon respiration and lack of feeding. Despite the lack of change in total amount, the concentration increases from larvae to pupae are very relevant for the pollutant ingestion of the higher predators. The intakes of OCs and PBDEs by trout are between 2- and 5-fold higher per calorie gained when predating on pupae than on larvae. Since pollutant concentration, energy reward, predation susceptibility, and duration of life stage are very different between these two insect stages, and none of them is irrelevant for the incorporation of OCs or PBDEs to higher levels, bioaccumulation food-web models should distinguish between the two sources.
Introduction Organochlorine compounds (OCs), namely hexachlorobenzene (HCB), hexachlorocyclohexanes (HCHs), 4,4′-DDE, 4,4′DDT, polychlorobiphenyls (PCBs), and polybromodiphenyl ethers (PBDEs) are toxic, semivolatile molecules of widespread occurrence in the environment (1-3). As a result of their persistence and hydrophobicity, OCs and PBDEs accumulate in biological tissues and biomagnify in food webs to levels sometimes approaching or in excess of human consumption guidelines (4-7). Some of these compounds can be partially biotransformed, e.g., 4,4′-DDT into 4,4′-DDE (8) or PBDEs debromination (5), but in general their high * Corresponding author phone: 34 972 33 61 01; fax: 34 972 33 78 06; e-mail:
[email protected]. † Limnology Unit (CSIC-UB), Centre for Advanced Studies of Blanes (CEAB-CSIC). ‡ Department of Environmental Chemistry (IIQAB-CSIC). 10.1021/es0703271 CCC: $37.00 Published on Web 08/01/2007
2007 American Chemical Society
chemical stability ensures that most of them remain unchanged in the aquatic ecosystems being exchanged between different environmental compartments and organisms without significant loss (9). Insects play a significant role in freshwater benthic food webs because of their biomass, and large number of taxonomic groups and species. With a few exceptions, most of the groups are holometabolous, meaning they undergo a complete change, and immature stages are quite different in shape from adults. They generally constitute aquatic forms in the early metamorphic stages (larva, pupa) and aerial forms in the adult life. The growth of these insects occurs during the larval stage throughout several moults. During the last instar (pupa), a histolysis of tissues permits the formation of the adult structures. Larvae and pupae show contrasting ways of living. Larvae show active feeding and mobility, whereas pupae generally do not feed, have low or only passive mobility, and dark pigmentation. Thus, pupae have an increased probability of fish predation. Although the duration of pupa stage is shorter than larva stage (10), pupae are usually present at periods of high fish activity, being an attractive food item for them and an important component of freshwater fish diet (9, 11-13). This paper is aimed to assess the effects of pupa stage compared to larvae in the distribution and accumulation of OCs and PBDEs in insects from freshwater lakes and the relevance of these changes for high predators, particularly fish, and their eventual implications for bioaccumulation. The study is focused on trichoptera and diptera, which are common organisms in benthic aquatic habitats and have been shown to accumulate substantial amounts of persistent organic pollutants (POPs) (14-16).
Materials and Methods Study Site and Sampling. Samples were collected in two high mountain lakes from the Catalan Pyrenees: Llebreta (42.55° N 0.89° E; 1620 m asl) and Xic de Colomina (42.52° N 0.99° E; 2425 m asl). Both lakes are softwater, oligotrophic, and have long ice cover periods (from ca. 4 to 7 months). Water temperatures are cold during the ice-free periods. These conditions involve relatively simple food webs, scarce biomass, and slow insect development. Sampling was performed in July 2004. Extensive kick sampling (17) was carried out throughout the perimeter of both lakes. All sorts of habitats were considered. Samples were kept frozen (-20 °C) until examination. A total of 22 initial samples was considered. Sampled larvae and pupae of trichoptera belonged to the polycentropodidae and limnephilidae families. Sampled diptera belonged to the chironomidae and ceratopogonidae families. Family was the highest common taxonomic resolution that could be achieved. However, it is worth mentioning that polycentropodidae only included specimens of Polycentropus flavomaculatus, whereas limnephilidae included individuals of Annitella sp., Potamophylax sp., and a third species of the tribe limnephilini. All chironomidae analyzed corresponded to the subfamily tanypodinae. Stable Isotope Analysis. Nitrogen signatures were used to identify the larvae mean trophic level occupied by these species in the lake food webs (18). Larvae carbon signatures were currently indicating the primary source of carbon (e.g., benthic, pelagic, or allochthonous) in each species diet (19). Nitrogen and carbon signature differences between larvae and pupae were used to investigate exchanges with the medium during metamorphism. VOL. 41, NO. 17, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Samples were freeze-dried overnight in an oil-free device. Stable isotopes were determined in a Delta C Finnigan MAT mass spectrometer (Bremen, Germany) coupled online with a Carlo Erba CHNS (Milan, Italy) elemental analyzer, via a Finnigan conflo 2 interface. Specific standards from the International Agency of Atomic Energy (IAEA) were used for calibration of the isotopic signal. Sucrose (IAEA CH6), polyethilene (IAEA CH7), and graphite (IAEA-USGS 24) were used for carbon. Ammonium sulfate (IAEA-USGS 25, IAEAN1, and IAEA-N2) and potassium nitrate (IAEA-NO3) were used for nitrogen (20). A complete batch of standards was run at the beginning and at the end of each analytical session. In addition, IAEA CH6, IAEA CH7, IAEA-N1, and IAEA-NO3 were run every 12 samples for linearity control. Special care was taken in weighting the samples and the standards so that both had similar amplitudes. Results are reported using atmospheric nitrogen and Pee Dee Belemnite carbonate as references. Reproducibility was better than 0.1 and 0.2‰ for δ13C and δ15N, respectively. Chemicals. Residue analysis, n-hexane, dichloromethane, isooctane, concentrated sulfuric acid, and acetone were from Merck (Darmstadt, Germany). PCB Mix 3, PCB#30, PCB#142, PCB#194, PCB#200, PCB#206, PCB#209, Pesticide Mix 11, and Pesticide Mix 164 were from Dr. Ehrenstofer (Augsburg, Germany). Standards of thirteen polybromodiphenyl ethers (BDE#17, BDE#28, BDE#47, BDE#66, BDE#71, BDE#85, BDE#99, BDE#100, BDE#138, BDE#153, BDE#154, BDE#183, and BDE#190) and BDE#209 in isooctane were purchased from Cambridge Isotope Laboratories (Andover, MA). OCs and PBDEs Analysis. The extraction and clean up procedure is described in detail elsewhere (21). Briefly, samples were spiked with PCB#30 and PCB#209 standards and extracted by sonication with hexane/dichloromethane (4:1) in four successive steps of 15 min. Cleanup was performed by sulfuric acid oxidation (4 times). The solutions were concentrated to near dryness under a gentle flow of nitrogen and redissolved in 50 µL of isooctane. Before chromatographic analysis, internal standards of PCB#142 and PCB#200 were added. Samples were analyzed for OCs by gas chromatography coupled to electron capture detection (GC-ECD, HewlettPackard 5890 series II) with a 60 m × 0.25 mm i.d. DB-5 capillary column (J&W Scientific, Folsom, CA) coated with 5% phenyl/95% methylpolysiloxane (film thickness 0.25 µm). The GC operated in splitless mode. The oven temperature program started at 90 °C (held for 2 min), ramped to 150 °C at 15 °C‚min-1 and then to 290 °C at 4 °C‚min-1 (holding time 20 min). Injector and detector temperatures were 280 and 310 °C, respectively. Helium and nitrogen were used as carrier (1.5 mL‚min-1) and makeup (60 mL‚min-1) gases, respectively. PBDEs were analyzed by GC coupled to negative ion chemical ionization mass spectrometry (GC-MS-NICI) in a TRACE GC ULTRA (Thermo Electron, Milan, Italy) coupled to a MS DSQ (Thermo Electron, Austin, Texas). The system was equipped with a DB-5MS capillary column (15 m × 0.25 mm × 0.1 µm film thickness) coated with phenyl arylene polymer that is virtually equivalent to 5% phenyl/95% methylpolysiloxane stationary phase. The oven temperature program was from 140 °C (held for 1 min) to 325 °C at 10 °C‚min-1 (held for 10 min). Helium was used as a carrier gas (1 mL‚min-1) and ammonia was used as ionization gas (2.4 × 10-4 Pa). Transfer line temperature was 300 °C. Quantification was performed from the intensities of the m/z 79 ion [Br]-. Confirmation ions were m/z 81 [Br]-, 161 [HBr2]-, and 327, 405, 483, 563, and 643, corresponding to either [M]or [M - HBr2]-. BDE-209 was measured from the intensities of the m/z 487 ion [C6Br5O]- and the confirmation ion was m/z 489 [C6Br5O]-. 6138
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FIGURE 1. Carbon and nitrogen stable isotopic composition of larvae and pupae of polycentropodidae, chironomidae, limnephilidae, and ceratopogonidae from Colomina and Llebreta lakes. Bars indicate range.
Sample dry weight was estimated by drying in a vacuumsealed drier at 20 °C until constant weight. Lipid content was estimated as previously described (9). Quality Assurance. Quantification was performed by reference to external standards injected at different concentrations. PCB Mix 3, PCB#194, and PCB#206 were used for the PCB congeners, Pesticide Mix 11 was used for HCHs and HCB, and Pesticide Mix 164 was used for 4,4′-DDE and 4,4′-DDT. PBDEs were quantified using the above-mentioned mixture of 13 congeners and BDE#209. Procedural blanks were analyzed for every set of six samples, which corresponds to periods of 12 h of sample handling. The recoveries of the surrogate standard, PCB#30 and PCB#209, were calculated for each sample, being 61 ( 11% and 84 ( 13% (average ( standard deviation), respectively. The recoveries of these surrogates were used for correction of the concentrations of the PCB and PBDE congeners in each sample. Relative responses to PCB#142 (for OCs) and PCB#200 (for PBDEs) were used to correct for instrumental injection variability. All samples were blank corrected.
Results and Discussion Stable Isotope Composition. The differences in isotopic composition between larvae and pupae can be used to illustrate changes in insect metabolism during metamorphism. The observed δ13C values in the aquatic insects considered for study decrease from larvae to pupae exhibiting shifts of about 3.59‰, 0.82‰, and 3.06‰ in polycentropodidae, limnephilidae, and ceratopogonidae, respectively (Figure 1), which are statistically significant for polycentropodidae and ceratopogonidae (p < 0.05). Chironomidae pupae show a large variability, and the mean difference values between the two metamorphic stages are not statistically significant. Lipid content is similar between larvae and pupae, only in limnephilidae the content is apparently lower in pupae than larvae, but variability in larvae is high (Table 1). These observations are in agreement with what is known on metabolism during metamorphism in insects. Biosynthesis is intensive in pupae because of metamorphism requirements. However, there is no dietary intake. Lipid stores are scarcely used as they will support energy demands imposed by reproduction and flight during the early adult phase (22). Instead, the major source of amino acids and energy for metabolism is provided by the own insect proteins (23). These proteins are strongly enriched in 13C relative to whole-body value (24, 25). The δ13C decrease observed in pupae with respect to the larvae indicates that the CO2 enriched in heavy carbon produced from protein use diffuses out of the bodies with respiration. This effect overruns the 13C enrichment
TABLE 1. Comparison of the Concentrations of Organochlorine Compounds and Polybromodiphenyl Ethers between Larvae and Pupae lipidsa (%)
taxon polycentropodidae larvae pupae limnephilidae larvae pupae chironomidae larvae pupae ceratopogonidae larvae pupae a
( standard deviation.
b
31 ( 7 33 ( 5 55 ( 15 22 ( 6 27 ( 11 26 ( 8 23 ( 4 35 ( 7
ΣOCsb ΣPBDEsb (ng g-1 dw) (ng g-1 dw) 19* 26* 2.5** 12** 15* 80* 26* 41*
13** 27** 0.65** 9.3** 0.00* 3.9* 1.7 5.2
*, p < 0.05; **, p < 0.001.
produced by the carbon isotopic fractionation from acetyl respiration (26). Protein synthesis within pupae is derived from catabolism. In principle, since this source has already been enriched in 15 N relative to diet, additional enrichment in the metabolic nitrogen pool could be expected during metamorphism (27). However, when larvae tissues are destructed to construct the adult body, excretion of the expected nitrogen from the ureotelic pathway does not occur until the first day of imaginal live (28). Thus, the whole process results in lack of nitrogen differentiation between the larvae and pupae. Accordingly, the δ15N differences between polycentropodidae, chironomidae, and ceratopogonidae were smaller than 0.5‰ (Figure 1), being not significant (p . 0.05). The larger differences for limnephilidae were not significant (p . 0.05) due to the high dispersion values of larvae. Consistently with ureotelic excretion, an increment in δ15N should be observed in the adults after emerging from pupal case. This was effectively the case for the only adult that was captured during sampling, δ15N in pupae of the limnephilid Potamophylax cingulatus was 0.61‰ and in the adult stage it was 1.28‰. OCs and PBDEs Concentrations. OCs and PBDEs concentrations were measured in larvae and pupae of the four taxonomic groups considered for study (Figure 2). Principal component analyses of the OCs and PBDEs were performed
to summarize the main trends of variability (see Supporting Information). The first principal component explains 90% and 89% of the variance in OCs and PBDEs composition, respectively, and all compounds show positive loadings on it indicating that the differences are due to concentration changes rather than to relative composition. The rest of the axes explain low amounts of variance ( 0.05) because of the high variability in the PBDEs pupa/larva ratios, probably due to the small amounts occasionally found in larvae, which increases quantification uncertainties. For those compounds with low variability (e.g., BDE#71 and BDE#153) the observed ratios are similar to those found for OCs (Figure 3). To the best of our knowledge, no previous studies considering the larval and pupal composition of OCs or PBDEs in the same taxonomic groups or species are available for comparison of the results of the present study. Enhanced concentrations of trans-chlordane by the midge Chironomus decorus in the adult stage were observed when comparing to larvae, although in this case pupae were not analyzed (29). In any case, these results are also consistent with the larval-pupal differences found in Figure 2. Pupae Role in Food Web Bioaccumulation. The concentration differences of organohalogen compounds between larvae and pupae have implications for bioaccumulation through food web. Thus, the amounts of OCs and PBDEs acquired by trout present in these lakes will be significantly different whether predating on pupae or larvae. According to the results of Figure 2 and the characteristic energy content per individual (Table 2), the OCs and PBDEs concentration acquired by trout is between 2- and 5-fold higher per calorie gained when predating on pupae than on larvae. In addition, even though pupae have shorter duration of life stage (i.e., degree-days required for development) and less energy content per individual (a difference that is more remarkable in trichoptera), they usually have a larger contribution to diet than larvae (Table 2) because of the lower predation effort required for their capture. 6140
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This work has been supported by the EU Project Euro-limpacs (GOCE-CT-2003-505540) and Spanish MEC project Trazas (CGL2004-02989). Technical assistance in instrumental analysis by R. Chaler, D. Fanjul, and P. Alabart, field help by L. Camarero, M. Bacardit, and G. Mendoza, and taxonomic help by G. Mendoza is acknowledged. M.B. is thankful for a FPU grant of Ministerio de Educacio´n y Ciencia.
Supporting Information Available Table SI1 includes concentration data for each compound. Tables SI2 and SI3 include principal component analyses (PCA) for OCs and PBDEs, respectively. Figure SI1 and SI2 include plots of the first principal component scores for larvae and pupae of each taxonomic group for OCs and PBDEs PCAs, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.
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Received for review February 8, 2007. Revised manuscript received June 13, 2007. Accepted June 22, 2007. ES0703271
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