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Are Accumulated Sulfide-Bound Metals Metabolically Available in the Benthic Oligochaete Tubifex tubifex? Maarten De Jonge,* Marleen Eyckmans, Ronny Blust, and Lieven Bervoets Department of Biology, Ecophysiology, Biochemistry and Toxicology Group, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
bS Supporting Information ABSTRACT: The present study evaluates the relationship between metalbinding sediment characteristics like acid volatile sulfides (AVS), metal accumulation, and internal metal distribution in the benthic oligochaete Tubifex tubifex and relates this accumulation to the induction of metallothionein-like proteins (MTLPs). In total, 15 Flemish lowland rivers were sampled. Cd, Cu, Zn, Pb, Ni, As, Cr, Co, and Ag concentrations were measured in environmental fractions (water and sediment) and worm tissue (both total and subcellular fractions). Furthermore, total cytosolic MTLP concentrations were measured in the worm tissue. Our results showed that Cd, Pb, Ni, and Cr were mainly stored as biological detoxified metal (BDM) while Cu, Zn, As, and Ag were mostly available in the metal sensitive fraction (MSF). A remarkable difference in the subcellular distribution of accumulated Cd, Ni, and Co between anoxic (SEMMe - AVS < 0; mostly stored as BDM) and oxic (SEMMe - AVS > 0; mostly stored in the MSF) sediments was noticed. Moreover, a rapid increase in MTLP induction was found when SEMTot - AVS > 0. Our results indicate that the accumulated sulfide-bound metals were detoxified and little available to the metabolism of T. tubifex under anoxic conditions.
1. INTRODUCTION One of the most important factors controlling metal availability in anoxic sediments is the amount of acid volatile sulfides (AVS). AVS is operationally defined as the concentration of sulfides volatilized by the addition of 1 N HCl and mainly consists of iron and manganese sulfides.1 The metals which are associated with AVS are called simultaneously extracted metals (SEM). SEM is generally determined as the metal fraction that describes the sum of molar concentrations of toxicologically important, cationic metals (Cu, Pb, Cd, Zn, Ni, and also Cr and Ag) which are extracted together with AVS. From this, Di Toro et al.1 formulated the SEM-AVS model for estimating metal toxicity from contaminated sediments. This model predicts that when AVS concentrations in sediments, on a molar basis, exceed SEM concentrations (SEMMe - AVS < 0), all metals will be bound to sulfides and the sediment pore water is considered to be nontoxic. In contrast, when the sediment contains an excess of SEM (SEMMe - AVS > 0), metals will be released into the pore water and become potentially toxic to the aquatic life. Although the SEM-AVS model has been validated for both acute and chronic toxicity,1-3 significant evidence has been gained that benthic invertebrates can accumulate metals in large amounts, even when SEMMe - AVS < 0.2,4-9 This enhanced bioaccumulation can be explained by the fact that benthic invertebrates, which live most of the time in the sediment and ingest sediment particles as their main food source, regardless of AVS.4-9 However, it remains unclear to which extent these r 2011 American Chemical Society
accumulated sulfide-bound metals can be actually harmful to the affected organism. This because the amount of accumulated metal concentrations is not necessarily related to the development of toxic effects.10,11 In general, trace metal accumulation in aquatic invertebrates can be interpreted in terms of two categories. First of all, metals can be present in a metabolically available form, which can inappropriately interact with physiologically sensitive target molecules, like small peptides, enzymes, or organelles (e.g., mitochondria and nuclei).12,13 Second, metals can be bound to metal-rich granules (MRG) or metal-binding proteins like metallothioneins (MT),14-16 which have been detoxified and are no longer available to play any role in the organisms’ metabolism, essential or deleterious.10 Furthermore, metallothionein-like proteins (MTLP) have been suggested as a reliable biomarker for the exposure and toxicity of trace metals in aquatic invertebrates, including Tubifex tubifex.16,17 Rainbow10 suggested that the onset of toxic effects depends only on the concentration of accumulated metals in metabolically available form. Therefore, subcellular partitioning of metal accumulation has been receiving increasing attention during recent years,18,19 resulting in the attendance of the subcellular partitioning model (SPM).11,13 However, the links between Received: November 10, 2010 Accepted: February 11, 2011 Revised: February 11, 2011 Published: March 04, 2011 3131
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Environmental Science & Technology environmental conditions, metal accumulation, and subsequent subcellular distribution need to be investigated. Studying the internal metal distribution in relation with SEM-AVS will give more insight whether accumulated metals in aquatic invertebrates will be metabolically available or not. This information is crucial in evaluating the SEM-AVS concept with respect to metal accumulation. The main objective of the present study was to evaluate the relationship between SEM-AVS, metal accumulation, and internal metal distribution in the benthic oligochaete T. tubifex under natural field conditions and relate this accumulation to the induction of metallothionein-like proteins (MTLP).
2. MATERIALS AND METHODS 2.1. Study Area and Sampling Design. In total, 15 sites of Flemish lowland rivers were sampled in the spring of 2008 (Supporting Information, Table S1). At every sampling site, pH, temperature, dissolved oxygen, and electrical conductivity (EC) of the surface water were measured in situ using a Hach HQ30d multiparameter field kit; each time 3 water samples were collected for the analysis of trace metals, major cations, and dissolved organic carbon (DOC). Also, 3 field blanks consisting of ultrapure water (Milli-Q) were transported to the field in order to detect possible contamination while taking and transporting the samples. Sediment samples (the top 2 cm) were collected in triplicate using nitrogen-purged polypropylene vials. At the lab, the samples were immediately frozen at -20 °C, which is the recommended storing procedure to maintain AVS concentrations within the sampled sediment.20 Samples of T. tubifex were collected using a stainless steel Petit Ponar grab sampler (Wildco cat. no. 1728; 235 cm2). In the lab, the sediments were sieved over a 1 mm sieve, and a subsample of the collected Tubificids (25%) was identified to species level following the work of Brinkhurst.21 Each time 15 individual worms were selected for equal size and were depurated by placing them for 24 h in artificial OECD (Organization of Economic Cooperation and Development) water (2 mM CaCl2 3 2H2O, 500 μM MgSO4 3 7H2O, 771 μM NaHCO3, and 77.1 μM KCl). According to Gillis et al.22 a gut-clearance time of 24 h is recommended for the benthic oligochaete T. tubifex. 2.2. Water and Sediment Characterization. DOC in the water samples was measured using a total organic carbon analyzer (TOC-5000/5050, Shimadzu Corporation, Kyoto, Japan). In order to get an indication of the water hardness, Ca and Mg concentrations were measured together with the trace metals (see section 2.4). AVS were extracted from wet sediment using the modified diffusion method of Leonard et al.;23 the extracted amount of sulfides was measured with an ORION 96-16 ionselective sulfur electrode (Ionplus, Beverly, MA). Afterward, the wet/dry ratio of the sediment sample was determined to convert the measured sulfide concentrations into dry weight. The organic matter content of the sediment was determined through loss on ignition (LOI). For this purpose, dry sediment was incinerated at 550 °C for 4 h. In order to quantify the sediment’s clay content, its particle size distribution was analyzed via laser diffraction (Malvern Mastersizer S., Worcestershire, U.K.).24 The total metal content was measured by drying the sediments at 60 °C for 48 h and adding a mixture of HNO3 (69%) and HCl (37%)(1:3, v/v); subsequently, samples were transferred to Teflon bombs and digested in a microwave oven (ETHOS 900 Microwave Labstation, Milestone, Italy).25 After digestion, samples were filtered using a 0.20 μm cellulose acetate filter (Schleicher and Schuell
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MicroScience GmbH, Dassel, Germany) and diluted with ultrapure water (Milli-Q) up to 50 mL. 2.3. Subcellular Fractionation and Metallothionein Quantification. The worm tissue was fractionated according to the protocol of Wallace et al.12 with minor modifications.26 Tissues were homogenized in six volumes of homogenization buffer [20 mM Tris-HCl, 5 mM β-mercaptoethanol (β-ME), 0.1 mM phenylmethanesulphonylfluoride (PMSF), pH 7.4]. Homogenate (1 mL) was centrifuged at 1450g (Eppendorf Centrifuge 5804 R, Hamburg, Germany) for 15 min. The supernatant was carefully removed for further fractionation. The pellet was suspended in 500 μL ultrapure water and heated at 100 °C for 2 min. NaOH (1 M, 500 μL) was added and the samples were incubated for 60 min at 65 °C. After incubation, the samples were centrifuged at 10 000g (Sorval Discovery 90 Ultra speed centrifuge, Newton, CT) for 30 min, and the supernatant was transferred to another tube. The pellet was washed with 0.5 M NaOH and centrifuged again at 10 000g for 30 min. The supernatant was added to the previous supernatant fraction and contained the cellular debris. The pellet contained the metalrich granules (MRG). The supernatant obtained by the first centrifugation step was ultra centrifuged at 100 000g (Sorval Discovery 90 Ultra speed centrifuge, Newton, CT) for 90 min. The resulting pellet contained organelles (nuclear, mitochondrial, and microsomal fractions).12 The supernatant containing the cytosolic fraction was heated at 80 °C for 10 min and then cooled on ice for 60 min. This fraction was centrifuged at 30 000g for 15 min, resulting in a supernatant containing heat stable proteins (HSP) and a pellet containing heat denaturated proteins (HDP). In summary, 5 fractions were retrieved, i.e., MRG, debris, organelles, HDP, and HSP. Subsequently, the samples were dried for 48 h at 60 °C. Afterward, the biological material was digested with HNO3 following the procedure of Blust et al.27 and stored until analysis. Concentrations of metal accumulation in worm tissue are expressed as μmol/g dry weight. Total cytosolic MTLP concentrations in whole worm tissue were measured using the cadmium thiomolybdate saturation assay from Klein et al.28 Cytosol (50 μL) was mixed with 10 μL of 300 mM ZnSO4 3 7H2O and incubated with 10 μL of 140 mM βME. After incubation with 70 μL of acetonitrile for 3 min, 500 μL of 10 mM Tris-HCl, 85 mM NaCl at pH 7.4 (buffer A), and 100 μL of CM-Sephadex [66% (v/v) suspension in buffer A] were added. The mixture was incubated with 50 μL bovine serum albumin (30 mg/L, freshly prepared) and with 20 μL of ammonium tetrathiomolybdate (500 μM, freshly prepared in buffer A). After shaking with 100 μL of DEAE-Sephacel [66% (v/v) suspension in buffer A], the precipitate was removed by centrifugation at 8000g for 5 min. Subsequently, 600 μL of the supernatant was saturated with 10 μL of 109Cd-labeled CdCl2 (1 mM, 740 kBq/ml, specific activity). The excessive 109Cd(II) was complexed and removed by Chelex-100. Following centrifugation at 8000g for 5 min, 500 μL of the supernatant was incubated with 500 μL acetonitrile for 3 min. The precipitate was removed by centrifugation, and the 109Cd(II) bound to MTLP was measured with a Wizard 3 1480 automatic gamma counter (Perkin-Elmer, Zaventem, Belgium). The MTLP concentrations were calculated on the basis of a molar ratio of Cd/MT of 7.29 2.4. Metal Analysis. Trace metal concentrations (Cd, Cu, Zn, Pb, Ni, As, Cr, Co, and Ag) and other important cations (Al, Ca, Na, K, Fe, Mn, and Mg) were measured in total surface water and in samples filtered through a 0.20 μm cellulose acetate filter. 3132
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Figure 1. Distribution of metal accumulation among the different subcellular fractions. Average concentrations (n = 3) and standard deviations are presented. Each time the percentage of metal accumulation in a certain fraction is given for the whole range of SEM - AVS (n = 15), samples where SEMMe - AVS < 0 (n = 6) and samples where SEMMe - AVS > 0 (n = 9). Regarding Ag, all sample points had an excess of AVS. Different letters represent significant differences between fractions and conditions.
Furthermore, trace metals were measured in sediment (total as well as SEM) and worm tissue (total as well as subcellular fractions). Metal analysis in the environmental fractions were performed using an inductively coupled plasma mass spectrometer (ICP-MS, Varian UlatraMass 700, Victoria, Australia), with germanium (Ge) as internal standard. Total and subcellular metal concentrations in the worm tissue were analyzed using high resolution ICP-MS (Thermo Scientific, Finnigan element 2, Waltham, MA). Analytical accuracy was verified by the use of blanks and certified reference material (CRM) of the Community Bureau of Reference (European Union, Brussels, Belgium) during all the above-mentioned destruction protocols. The latter includes standards for trace elements in river sediment (CRM 320) and mussel tissue (CRM 278). Recoveries were within 10% of the certified values. 2.5. Statistical Analysis. Prior to analysis, all data were tested for normality with the Shapiro-Wilkinson test. Analysis of variance (ANOVA) with posthoc Tukey’s range test was used to compare averages between different groups. To determine the relation between metal accumulation in whole tissue, subcellular fractions and environmental variables, the Pearson correlation-coefficient was used. The significance level is
represented as * p < 0.05; ** p < 0.01; *** p < 0.001. All statistical analyses were performed using the software package SAS (SAS 9.2, SAS Institute Inc., Cary, NC).
3. RESULTS 3.1. Metal Accumulation and Subcellular Compartmentalization. Figure S1 presents the metal accumulation measured in
whole tissue and subcellular fractions of T. tubifex. The amount of metals in the metal sensitive fractions (MSF; organelles þ HDP) and the biological detoxified fractions (BDM; HSP þ MRG) of the worms were calculated for all the sample sites separately. The relative distribution of metal accumulation among the different subcellular fractions is presented in Figure 1. In each instance the percentage metal accumulation in a certain fraction is given for the whole range of SEM - AVS, samples where SEMMe - AVS < 0 and samples where SEMMe - AVS > 0. This figure shows that Cd, Pb, Ni, and Cr accumulation are mainly stored as biological detoxified metal (MRG þ HSP), while Cu, Zn, As, and Ag are mostly available in the metal sensitive fraction (organelles þ HDP); e.g., 91.8 ( 6.01% of the total Cd accumulation was stored as BDM under anoxic conditions while this was only 3133
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Figure 2. MTLP induction as a function of whole tissue and subcellular metal accumulation in T. tubifex. r2 values and level of the significant relations are presented. * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001. Only for Cr was a significant correlation between metal accumulation in the HSP fraction and MTLP induction found.
37.1 ( 3.05% when SEM exceeded AVS. In contrast to the latter, Cd accumulation in the metal-sensitive fraction increased from 7.89 ( 1.12% when SEMCd - AVS < 0 to 61.9 ( 4.32% when SEMCd - AVS > 0. Similar results were observed for Ni and Co. Regarding Cu, accumulation in the MSF increased when SEM exceeded AVS (from 62.3 ( 4.36% to 83.8 ( 7.66%), while Cu storage as BDM decreased (from 37.3 ( 2.74% to 15.5 ( 1.29%). Zn accumulation in the MSF decreased from 62.1 ( 4.68% when SEMZn - AVS < 0 to 51.4 ( 4.50% when SEMZn AVS > 0; meanwhile, Zn accumulation as BDM slightly increased from 37.4 ( 2.12% to 47.9 ( 3.56%. Metal accumulation in the rest fractions (cellular debris) was generally very low, except for the metalloid As, and showed no significant changes under the different conditions for all the metals measured. Figure S2 presents the metal distribution in the different subcellular fractions as a function of whole tissue metal accumulation. From this graph it seems that, with increasing Cd concentrations in the whole tissue of T. tubifex, Cd accumulation as BDM simultaneously increases until saturation is reached whereupon Cd starts to accumulate in the MSF as well. An increase in metal storage as BDM with whole tissue concentration is also found with Pb, Ni, and Cr. Regarding Cr, availability in the metal sensitive fractions did not increase with whole tissue concentration. Concerning the metals Cu and Zn an increase in both the MSF as the BDM was observed with increasing total tissue accumulation. For Co, As, and Ag, only accumulation in the metal sensitive fraction increased with whole tissue concentration.
Pearson’s correlation coefficients among metal concentrations in T. tubifex and environmental fractions are presented in Table S4. Metal accumulation in both whole tissue and subcellular fractions was generally best correlated with total metal concentrations in the sediment normalized for clay particles and organic matter content. Non-normalized metal concentrations in the sediment correlated only with total tissue accumulation for the metals Cd, Zn, and Pb and the metalloid As. Cd, Zn, Pb, Cr, and Co accumulation in the metal sensitive fraction significantly correlated with SEMMe-AVS or SEMMe-AVS/OM in the sediment. Total and dissolved concentrations in the surface water significantly correlated with Cd, Cu (only with dissolved), Pb, and As accumulation in T. tubifex. No significant correlations were found between metal storage as BDM and concentrations in environmental fractions. 3.2. Metallothionein Induction. Figure S3 presents the induction of metallothionein-like proteins (MTLP) measured in the HSP fraction of T. tubifex. MTLP induction was generally below 20 nmol/g. In comparison to the other sites examined, very high concentrations were found at sites 5 (75.3 ( 5.07 nmol/g), 11 (102 ( 9.66 nmol/g), and 12 (50.1 ( 4.50 nmol/g). Regarding sample points 2 and 14, not enough worm tissue was collected to determine MTLP concentrations. MTLP induction as a function of whole tissue and subcellular metal accumulation is presented in Figure 2. Significant correlations were found between Cd, Cu, Ni, and Cr accumulation in the metal sensitive fraction and MTLP induction in the HSP 3134
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Figure 3. MTLP induction as a function of SEMTot-AVS concentration in the sediment. SEMTot-AVS was calculated as the molar sum of Cd, Cu, Zn, Pb, Ni, Cr, and Ag/2.
fraction. No significant correlations were found between MTLP concentrations and whole tissue accumulation; a significant correlation between accumulation as BDM and MTLP induction was only found for Cr. Figure 3 presents the MTLP induction as a function of SEMTot-AVS concentration in the sediment. SEMTot-AVS was calculated as the molar sum of Cd, Cu, Zn, Pb, Ni, Cr, and Ag/2. This graph clearly shows that MTLP induction rapidly increases when SEM exceeds AVS. At the sampling sites where SEMTotAVS < 0, little MTLP induction was noticed.
4. DISCUSSION The present study measured high metal concentrations in the whole tissue of the aquatic oligochaete T. tubifex. Cd, Cu, Zn, Pb, and Ni accumulation was very high compared to the levels measured in collected oligochaetes from clean and metal contaminated field sediments30 and unexposed laboratory-cultured T. tubifex.17 Metal accumulation in whole tissue of T. tubifex was generally comparable with levels measured in Tubificid worms from analogous metal-polluted Flemish lowland rivers in the study of De Jonge et al.8 Metal distribution among the subcellular fractions clearly differed between metals. Cd, Pb, Ni, and Cr were mainly stored as BDM while Cu, Zn, As, and Ag were mostly available in the MSF. These results are in accordance with the study of Voets et al.,31 who found that Cd accumulation in Dreissena polymorpha was mainly detoxified (mostly bound to MTLP) whereas Cu and Zn were more equally distributed between the subcellular fractions. Furthermore, other authors found that Zn accumulation in marine bivalves and freshwater insect larvae was primarily associated with the MSF while Cd was mainly stored as BDM.12,32 The enhanced binding of Zn and Cu to the MSF can be ascribed to the fact that these elements play an essential role in certain metabolic processes (e.g., Zn in the enzyme carbonic anhydrase and Cu in hemocyanin), which makes binding to organelles and enzymes necessary.12,33 As all nonessential elements, Cd and Pb do not play a role in the metabolism of aquatic organisms and are therefore expected to be stored as BDM.10 Sequestration in insoluble MRG is in fact a very common detoxification strategy for the storage of Cd and Pb in aquatic invertebrates.10,34,35 Regarding the element Ni, our results are in accordance with the findings of Dumas and Hare,36
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who found that 47 ( 2% of the total accumulated Ni in T. tubifex was stored as BDM. Our findings that Ag is mostly available in the MSF are in contrast with a lot of other literature; in a study with green mussels, accumulated Ag was mostly associated with the insoluble, metal detoxified fraction.37 Furthermore, Berthet et al.38 found that up to 80% of Ag in marine bivalves was bound to MRG. Our results indicate that detoxification mechanisms in T. Tubifex got saturated, most likely due to an elevated Ag uptake at certain sample sites. Following the metal distribution in the subcellular fractions as a function of whole tissue accumulation, this study clearly showed that Cd storage as BDM reached saturation when Cd levels in the whole tissue increased. After saturation was reached, Cd accumulation in the MSF started to increase. More or less the same pattern was found for Pb, Ni, and Cr; however, for these metals no exact saturation point was observed. Steen Redeker et al.26 also noticed saturation of detoxification mechanisms and increasing Cd availability in the MSF with increasing whole tissue accumulation in T. tubifex. A gradual increase of metal storage in both the MSF and BDM with increasing whole tissue concentration was found for Cu and Zn. A similar increase in Cu and Zn accumulation in the MSF and the BDM was found in zebra mussels.31 In their field study, Cain et al.32 also observed an increase in Zn accumulation in both metal-binding proteins and non-metal-binding proteins in the mayflies Baetis sp. and Epeorus albertae. However, cytosolic Zn concentrations in caddisflies did not increase with whole-body concentration, which might be evidence of Zn regulation.32 In the present study differences in internal distribution between metals accumulated from anoxic (SEMMe - AVS < 0) and oxic (SEMMe - AVS > 0) sediments were observed. Under anoxic conditions, Cd in T. tubifex was mainly stored as BDM while it became more available in the MSF when SEM exceeded AVS. A similar pattern was found for Co and Ni. Co distribution changed from an even distribution under anoxic conditions to an increased availability in the metal sensitive fraction when SEMCo - AVS > 0; Ni distribution changed from a major storage as BDM (SEMNi - AVS < 0) to an equal distribution when SEM exceeded AVS. Pb and Cr were mainly detoxified under anoxic conditions, and metal distribution did not change when SEMMe - AVS > 0. Furthermore, significant correlations were found between Cd, Pb, Cr, and Co accumulation in the metal sensitive fraction and SEMMe-AVS concentrations in the sediment. These results indicate that, although elevated metal concentrations were measured in the whole tissue, Cd, Pb, Ni, Cr, and Co are mainly stored as BDM in T. tubifex when these metals are accumulated from sulfide-rich sediments. The metals Cu, Zn, As, and Ag were available in high amounts in the MSF when SEMMe - AVS < 0. Regarding the essential metals Cu and Zn, no real shift in metal storage was found between anoxic and oxic conditions. However, a limited increase in Cu availability in the MSF was noticed when SEMCu - AVS > 0 while Zn accumulation in the MSF decreased. The metalloid As generally did not change between anoxic and oxic conditions in the sediment. MTLP levels measured in the present study were high compared to levels measured in oligochaetes from natural metalpolluted sediments30 and Cd exposed T. tubifex in a laboratory experiment.17 Significant correlations were found between Cd, Cu, Ni, and Cr concentrations in the metal sensitive fraction and MTLP levels in worm tissue, suggesting that the induction of MTLP is a response to enhanced metal exposure.15,32 It has been 3135
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Environmental Science & Technology extensively demonstrated that exposure to both Cd and Cu can induce MTLP concentrations in T. tubifex.17,22,39 Barka et al.40 found a significant increase in MTLP after exposure to Ni in the copepod Tigriopus brevicornis. It has been proved that Ni has in fact a very high affinity for cysteine.16,41 To our knowledge, no evidence exists concerning Cr activated MTLP induction in aquatic invertebrates. However, Solis-Heredia et al.42 demonstrated a pancreatic MT increase in rats after injection with Cr. In this study no significant correlations between Zn or Ag accumulation and MTLP induction in T. tubifex was found. Nevertheless, a body of literature has proved the interactions with these metals and MTLP induction in a broad spectrum of aquatic invertebrates (ref 16 and references therein). An increase in MTLP induction was found when SEMTot AVS > 0. In contrast at sites where AVS exceeded SEM little MTLP induction was found. However, MTLP induction measured at these sites was still high compared to levels of unexposed laboratory-cultured T. tubifex in the study of Gillis et al.17 Gillis et al.30 also found elevated MTLP concentrations in oligochaetes at relatively clean sites but suggested that this induction is likely the result of the combined exposure to low levels of multiple metals. Our findings that MTLP induction increases when SEMTot - AVS > 0 suggest that in this study the accumulated sulfide-bound Cd, Pb, Ni, Cr, and Co are little available to the metabolism of T. tubifex. Similar results were found in the study of Pesch et al.,3 which showed that no significant toxicity occurred when Cd and Ni were accumulated from sulfide-rich sediments in the polychaete Neanthes arenaceodentata. Hare et al.2,7 also noticed significant Cd accumulation in benthic invertebrates when SEMCd - AVS < 0; however, no toxicity occurred. Furthermore, Sundelin and Eriksson43 described the lack of toxicity in the deposit feeding amphipod Monoporeia affinis when sulfide-bound Pb was accumulated. Our results (little MTLP induction under anoxic conditions) also imply that Cu and Zn, which are mainly associated with the MSF, are not inducing harmful effects when AVS exceeds SEM. In general, we can conclude that accumulated sulfide-bound metals were detoxified and were little available to the metabolism of T. tubifex. When SEM exceeded AVS, detoxification mechanisms became saturated and accumulation in the MSF started to occur, resulting in an enhanced MTLP induction. These results indicate that metal uptake through the ingestion of sulfide-bound particles is mainly sequestered as BDM. This applies to nonessential metals like Cd and Pb in particular. However, this also concerns elements like Ni, Cr, and Co, which are needed in little amounts for the metabolism of aquatic invertebrates. On the other hand, Cu and Zn, which are necessary in larger amounts, are metabolically available when accumulated from anoxic sediments but are not in such excess that they are able to induce MTLP in T. tubifex. It has already been stated that the internal distribution of assimilated metals is not just controlled by the quantity of the concentrations but also by the physicochemical form.10,13 Although this theory has been widely demonstrated with respect to food chain transfer (preypredator relations) in aquatic invertebrates,18,19 our results prove that it can be applicable to sediment-deposit feeder interactions as well. By assessing internal metal distribution rather than looking at total metal accumulation, this study revealed more insights in the gap between results from acute/chronic toxicity testing and accumulation studies with respect to the SEM-AVS concept.
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’ ASSOCIATED CONTENT
bS
Supporting Information. More information regarding the location of the different sampling sites together with results regarding general water and sediment characteristics, metal concentrations in environmental fractions, metal accumulation and distribution in subcellular fractions of T. tubifex, MTLP induction, and Pearson correlations. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: þ32 3 265 3533; fax: þ32 3 265 3497; e-mail: maarten.
[email protected].
’ ACKNOWLEDGMENT We would like to express special thanks to Steven Joosen and Katrien Wijnrocx for their assistance in the laboratory. Furthermore, we thank Dr. Valentine Mubiana for the execution of the metal analyses and Lien Van Gool for the English revision. Maarten De Jonge is supported by the Agency for Innovation by Science and Technology (IWT). ’ REFERENCES (1) Di Toro, D. M.; Mahony, J. D.; Hansen, D. J.; Scott, K. J.; Hicks, M. B.; Mays, S. M.; Redmond, M. S. Toxicity of cadmium in sediments: the role of acid volatile sulfides. Environ. Toxicol. Chem. 1990, 9, 1487–1502. (2) Hare, L.; Carignan, R.; Huerta-Diaz, M. A field study of metal toxicity and accumulation by benthic invertebrates—implications for the acid-volatile sulphide (AVS) model. Limnol. Oceanogr. 1994, 39, 1653–1668. (3) Pesch, C. E.; Hansen, D. J.; Boothman, W. S.; Berry, W. J.; Mahony, J. D. The role of acid-volatile sulfide and interstitial water metal concentrations in determining bioavailability of cadmium and nickel from contaminated sediments to the marine polychaete Neanthes arenaceodentata. Environ. Toxicol. Chem. 1995, 14, 129–41. (4) Lee, B.-G.; Griscom, S. B.; Lee, J.-S.; Choi, H.-J.; Koh, C.-H.; Luoma, S. N.; Fisher, N. S. Influences of dietary uptake and reactive sulfides on metal bioavailability from aquatic sediments. Science 2000, 287, 282–284. (5) Lee, B.-G.; Lee, J.-S.; Luoma, S. N.; Choi, H.-J.; Koh, C.-H. Influence of acid volatile sulfide and metal concentrations on metal bioavailability to marine invertebrates in contaminated sediments. Environ. Sci. Technol. 2000b, 34, 4511–4516. (6) Lee, J.-S.; Lee, B.-G.; Yoo, H.; Koh, C.-H.; Luoma, S. N. Influence of reactive sulfide (AVS) and supplementary food on Ag, Cd and Zn bioaccumulation in the marine polychaete Neanthes arenaceodentata. Mar. Ecol.: Prog. Ser. 2001, 216, 129–140. (7) Hare, L.; Tessier, A.; Warren, L. Cadmium accumulation by invertebrates living at the sediment-water interface. Environ. Toxicol. Chem. 2001, 20, 880–889. (8) De Jonge, M.; Dreesen, F.; De Paepe, J.; Blust, R.; Bervoets, L. Do acid volatile sulfides (AVS) influence the accumulation of sedimentbound metals to benthic invertebrates under natural field conditions? Environ. Sci. Technol. 2009, 43, 4510–4516. (9) De Jonge, M.; Blust, R.; Bervoets, L. The relation between acid volatile sulfides (AVS) and metal accumulation in aquatic invertebrates: Implications of feeding behavior and ecology. Environ. Pollut. 2010, 158, 1381–1391. (10) Rainbow, P. S. Trace metal concentrations in aquatic invertebrates: Why and so what? Environ. Pollut. 2002, 120, 497–507. 3136
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