Article pubs.acs.org/est
Importance of Subcellular Metal Partitioning and Kinetics to Predicting Sublethal Effects of Copper in Two Deposit-Feeding Organisms Olivia Campana,*,†,‡ Anne M. Taylor,§ Julián Blasco,† William A. Maher,§ and Stuart L. Simpson‡ †
Instituto de Ciencias Marinas de Andalucía (CSIC), Campus Universitario Rio San Pedro, s/n 11519 Puerto Real, Cádiz, Spain Centre for Environmental Contaminants Research, CSIRO Land and Water, Locked Bag 2007, Kirrawee, New South Wales 2232, Australia § Ecochemistry, Institute for Applied Ecology, University of Canberra, Canberra 2601, Australia ‡
S Supporting Information *
ABSTRACT: The role of subcellular partitioning of copper on the sublethal effects to two deposit-feeding organisms (41-day growth in the bivalve Tellina deltoidalis and 11-day reproduction in the amphipod Melita plumulosa) was assessed for copper-spiked sediments with different geochemical properties. Large differences in bioaccumulation and detoxification strategies were observed. The bivalve accumulated copper faster than the amphipod, and can be considered a relatively strong net bioaccumulator. The bivalve, however, appears to regulate the metabolically available fraction (MAF) of the total metal pool by increasing the net accumulation rate of copper in the biologically detoxified metal pool (BDM), where most of the copper is stored. In the amphipod, BDM concentration remained constant with increasing copper exposures and it can be considered a very weak net bioaccumulator of copper. This regulation of copper, with relatively little stored in detoxified forms, appears to best describe the strategy applied by the amphipod to minimize the potential toxic effects of copper. When the EC50 values for growth and reproduction are expressed based on the MAF of copper, the sensitivity of the two species appears similar, however when expressed based on the net accumulation rate of copper in the metabolically available fraction (MAFrate), the bivalve appears more sensitive to copper. These results indicate that describing the causality of metal effects in terms of kinetics of uptake, detoxification, and excretion rather than threshold metal body concentrations is more effective in predicting the toxic effects of copper. Although the expression of metal toxicity in terms of the rate at which the metal is bioaccumulated into metabolically available forms may not be feasible for routine assessments, a deeper understanding of uptake rates from all exposure routes may improve our ability to assess the risk posed by metal-contaminated sediments.
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INTRODUCTION The prediction of metal toxicity to benthic organisms is difficult because of the various factors influencing the bioavailability of metals from sediments1,2 and the complex metal handling and sequestration strategies of different aquatic organisms.3,4 The bioavailability is influenced by partitioning among the dissolved phase, major metal-binding sediment phases (e.g., acid-volatile sulfide (AVS), particulate organic carbon (OC), and iron and manganese oxyhydroxides5−7), and the exposure pathways specific to organisms (e.g., pore water, burrow water, or overlying water, and sediment particles or food via diet8−10). Metals bind to a range of biological ligands at sites that have different functions and potentially cause different forms of toxicity within an organism.8,11−14 Information on internal metal partitioning within organisms can be gathered by measuring five major subcellular fractions of metals within organisms: metal-rich granules, nuclei and cellular debris and organelles (which include mitochondria, microsomes, and © 2015 American Chemical Society
lysosomes), heat-denaturable proteins (also referred to as heat-sensitive proteins), and metallothionein-like proteins (also referred to as heat-stable proteins).15,16 Wallace et al.11 proposed that a metal-sensitive fraction (MSF), which is the combination of the ORG and HDP fractions, is the metabolically active metal fraction of the total metal pool in cells, and that a biologically detoxified metal pool, which combines MRG and MTLP, is considered to alleviate toxicity. Understanding both the rate of metal uptake (from all exposure routes) and the metabolically active concentration of metals within an organism may considerably improve the ability to predict the toxicity of metals.12,15,17,18 In invertebrates capable of detoxification of metals through storage in Received: Revised: Accepted: Published: 1806
June 18, 2014 January 12, 2015 January 15, 2015 January 15, 2015 DOI: 10.1021/es505005y Environ. Sci. Technol. 2015, 49, 1806−1814
Article
Environmental Science & Technology
in the 0.05) (Figure 1f). Further evidence of the difference in the subcellular distribution of BDM (SI Figure S3) is demonstrated by the copper detoxification capability of these two species, calculated as the 1810
DOI: 10.1021/es505005y Environ. Sci. Technol. 2015, 49, 1806−1814
Article
Environmental Science & Technology
Figure 3. Logistic sigmoidal regressions between sublethal end points and copper in MAF and MAFrate for T. deltoidalis (a and c), and M. plumulosa (b and d). Mean ± SE, n = 4 for T. deltoidalis, n = 2 for M. plumulosa.
the copper bioaccumulation gradient and no significant correlation with pCu was observed (p > 0.05 for both organisms). These results indicate that the bioaccumulation strategies of these two organisms for copper are quite different. The values for Cu-biorate show that the bivalve accumulates copper faster than the amphipod, as copper concentration in the particulate phase increased, however, they have similar MAFs. The bivalve can be considered a relatively strong net bioaccumulator of copper. The observation that the bivalve maintains a constant MAFrate along the pCu gradient may indicate the ability of bivalve to regulate this fraction, while not regulating the total accumulated copper. The bivalve appears to achieve this by increasing the BDMrate over this range (Figure 1e). The fractionation indicates that the majority of the copper initially accumulated by the bivalve occurs in an inert form as granules, i.e. type B granules originating from the lysosomal system that is known to accumulate copper and other metals, e.g. zinc.12,15 As further copper is accumulated, the mechanism may change to involve a greater role of the metallothioneins, a metalbinding cytoplasmatic process (SI Figure S4). Barnacles have a similar bioaccumulation strategy, where the vast majority of accumulated copper is detoxified in form of type B Cu-rich granules, probably resulting from lysosomal breakdown of metallothionein-bound copper.3,15,38 The amphipod has a slower copper uptake rate and faster efflux rate than the bivalve,6,13,26,31,37 and this results in the net accumulation rate being much lower for the amphipods than the bivalve (Figure 2). This indicates that the amphipod would be better classified as a regulator than an accumulator, or a very weak net accumulator. Limiting copper net uptake through an efficient excretion process, rather than BDM storage, appears to best describe the predominant strategy applied by the amphipod to minimize potential toxic effects. For the amphipod, along the pCu gradient the MAFrate increases, while BDMrate remains constant, indicating that excretion
slope of the linear regression of biologically detoxified copper against total bioaccumulated copper, which was 55 ± 4% (mean ± SE) for the bivalve (r2 = 0.96, p < 0.001) and 36 ± 16% (mean ± SE) for the amphipod (r2 = 0.40, p ≤ 0.05). These results highlight the greater ability of the bivalve to detoxify accumulated copper compared to the amphipod. The relative contributions of each subcellular copper fraction to the MAF and BDM pools were also investigated (SI Figure S4). In the bivalve, at the lowest bioaccumulated copper concentration (∼190 μg g−1), 84 ± 4% (mean ± SE) of bioaccumulated copper was detoxified by the formation of granules (MRG fraction) and 15 ± 4% (mean ± SE) by the MTLP fraction. As the copper concentration in the bivalves increased, the subcellular partitioning of copper to the MTLP fraction progressively increased (r2 = 0.47, p ≤ 0.01), and to the MRG fraction decreased. Approximately 50% of the copper is detoxified at the highest copper concentration bioaccumulated by the bivalve (∼900 μg g−1). It is unclear whether the apparent shift in partitioning within the BDM pool (from MRG to MTLP) indicates there is a limit to the amount of copper that can be stored in the MRG fraction, or what the implications are for the observed toxicity to bivalve growth. The detoxification strategy of the amphipod appeared quite different from that of the bivalve. As total copper bioaccumulation increased, the relative contribution of MTLPs and MRG fractions did not change significantly (p > 0.05), and averaged values were 78 ± 3% and 22 ± 3% (means ± SE), respectively. In both species, copper partitioning in the MAF pool, between the ORG and HDP, followed the same pattern. In the bivalve, the relative contributions were 83 ± 1% and 17 ± 1% (means ± SE) for ORG and HDP, respectively, despite the very different ranges in total bioaccumlated copper (190−900 μg g−1 for bivalve; 70−120 μg g−1 for amphipod) in the same sediments. A slight difference in averaged values was observed for the amphipod, with 70 ± 2% and 30 ± 2% (means ± SE) for ORG and HDP, respectively. In both species these values were constant along 1811
DOI: 10.1021/es505005y Environ. Sci. Technol. 2015, 49, 1806−1814
Article
Environmental Science & Technology
on pCu, expressed as OC-normalized copper concentrations in the
