Bioconcentration and Intracellular Storage of Hexachlorobenzene in

We investigated the bioaccumulation of hexachlorobenzene (HCB, LogKOW = 5.5) ... of intracellular accumulation of hexachlorobenzene (HCB) in Chara rud...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/est

Bioconcentration and Intracellular Storage of Hexachlorobenzene in Charophytes and Their Potential Role in Monitoring and Remediation Actions Susanne C. Schneider† and Luca Nizzetto*,†,‡ †

Norwegian Institute for Water Research, Gaustadalléen 21, NO-0349, Oslo, Norway Research Centre for Toxic Compounds in the Environment (RECETOX), Kamenice 126/3, CZ-62500 Brno, Czech Republic

‡

S Supporting Information *

ABSTRACT: It has been hypothesized that highly hydrophobic substances (LogKOW > 5) including many persistent organic pollutants cannot overtake protective tissues and diffuse inside the body of plants due to steric hindrance or very slow diffusion. We investigated the bioaccumulation of hexachlorobenzene (HCB, LogKOW = 5.5) in a benthic charophycean macro-alga: Chara rudis. Chara species are a group of common freshwater algae with a complex body structure encompassing a protective layer of cortex cells surrounding large internode cells. The charophyte cell wall has many features in common with that of higher plants; therefore, they are useful models to investigate bioaccumulation mechanisms in general. We found that HCB diffused through the cortex and reached the cytoplam of internode cells. More than 90% of the HCB mass found in the organism was in the cortex and 10% in the internode cell cytoplasm. The cortex reached a pseudoequilibrium partitioning with water, and the bioconcentration factor was in the same range as that of lower aquatic organisms such as phytoplankton. Charophytes are therefore very efficient accumulators of hydrophobic compounds. Based on the structural and ecological features of charophytes, we speculated on their possible use as biomonitors and bioremediation tools.

■

microscopy techniques.5,7,8 Phenanthrene from an artificially contaminated atmosphere was shown to diffuse inside epidermis and mesophyll cells in terrestrial plant leaves. In aquatic organisms, intracellular transport of hydrophobic substances (namely PCBs) has been shown to occur only for a unicellular phytoplankton species (namely, Chlamydomonas reinhardtii). This relatively simple organism lacks a protective surface tissue. For Chlamydomonas reinhardtii, bioconcentration dynamics may be controlled by diffusion of chemicals through the cell membrane followed by a slow accumulation in subcellular structures such as the thylakoids.9−11 In contrast to unicellular phytoplankton species, higher aquatic plants and some macroscopic algae have a more complex multicellular structure with protective surface tissues. Charophytes are benthic algae and represent an intermediate evolutionary step between unicellular algae and higher plants of which they are close ancestors.12 They are therefore a good study model to investigate chemical uptake mechanisms in organized multicellular autotrophs. Within the charophytes,

INTRODUCTION Persistent organic pollutants (POPs) are a class of ubiquitous bioaccumulative chemical contaminants representing a global concern for human and environmental health. POPs are generally characterized by LogKOW values > 5, 1 conferring them affinity for the biotic phase.2 In addition, binding to the organic matrix (particularly in primary producers in water) influences their environmental behavior by controlling multimedia distribution3 and therefore their overall fate. It has been hypothesized that steric hindrance or kinetic limitation may inhibit permeation and diffusion of highly hydrophobic substances through surface structures of autotrophs (such as leaf cuticles, cell walls, or membranes). This may be the reason why root intake and translocation of compounds with logKOW > 5 were not observed in terrestrial vascular plants4,5 (though some exceptions exist).4,6 Such a mechanism would imply that the internal tissues (and their intracellular environment) of a multicellular autotroph may not be directly exposed to hydrophobic toxicants, therefore preventing the insurgence of possible acute toxicity responses mediated by intracellular receptors. The steric hindrance hypothesis was recently challenged. Cellular intake of phenanthrene (a compound with LogKOW of about 4.5)1 was qualitatively documented using fluorescence © 2012 American Chemical Society

Received: Revised: Accepted: Published: 12427

June 4, 2012 September 27, 2012 October 12, 2012 October 12, 2012 dx.doi.org/10.1021/es302185g | Environ. Sci. Technol. 2012, 46, 12427−12434

Environmental Science & Technology

Article

Chara rudis is a perennial, benthic species with a protective layer of cortex cells surrounding the internode cells (Figure 1).13 Chara species are important components in lakes and

cytoplasm of the internode cells; ii) provide a measure of the overall capacity of Chara to accumulate HCB; and iii) quantitatively assess the distribution of HCB in different plant parts.

■

Figure 1. Schematic representation of the transversal section of a Chara internode. The cortex cells create a tight layer surrounding a single large internode cell which can be up to several cm long. Most of the inner volume of the internode cell is occupied by the vacuole, which is surrounded by the tonoplast. In this representation proportions are approximately correct for the cortex and internode cells. The size of the subcellular organelles (with the exclusion of the tonoplast) were intentionally exaggerated for illustrative purposes.

BIOLOGICAL MODEL DESCRIPTION (CHARA) Charophytes (Charales, Charophyceae) are submerged green algae with a macroscopic thallus and an Equisetum-like growth form (Figure S1). They are important in lake, stream, and pond ecosystems throughout the world13,15 because they provide food and habitat for various animals and influence the chemical and physical properties of water and sediments.16 Chara has a complex morphology with apical growth and differentiation of the thallus into nodes and internodes. In Chara rudis, the internode consists of a single large (i.e., up to several centimeters long) internode cell corticated by a layer of elongated and much smaller cells called cortex cells (Figure 1). Inside the internode cell there is a single large vacuole which is surrounded by a thin peripheral layer of cytoplasm.17 Despite being an alga, the charophyte cell wall has many similarities in chemical composition and nanoscale arrangement of polymers to that of higher plants, including the presence and arrangement of cellulose and various polysaccharides.18,19 For this reason Chara species can be a useful model to investigate the mechanisms of chemical uptake by other aquatic macrophytes.

rivers around the world. They can form dense beds on lake sediments with a biomass up to 1 kg (dry weight) m−2.14 In these conditions they play a crucial role in lakes by controlling nutrient cycles and biogeochemistry while serving as food for a number of organisms. Wherever charophytes dominate primary production in a lake they will probably also play an important role in controlling overall fate and distribution of hydrophobic pollutants since it can be expected that, by taking up chemicals from water, they can reduce concentrations in the dissolved phase, as already shown to occur during phytoplankton blooms.3 Traditionally, chemical exposure assessment in fresh water ecosystems has mainly focused on phytoplanktondominated systems and neglected the importance of benthic autotrophs. No information is available on bioaccumulation factors and uptake processes for charophytes. Charophytes can be used to test the steric hindrance hypothesis. Quantitatively measuring the intracellular fraction of contaminants in organisms is challenging due to the need for heavy manipulation of the samples.9 This may easily result in cross contamination from the extracellular material. The peculiar structure of Chara rudis, and in particular the large dimension of the internode cells, makes it possible to handle the cytoplasm of even single cells and allows separate analysis of intracellular material from the internode cell and surface material (i.e., the cortex cells plus the internode cell wall) with relatively limited sample manipulation. In this study we investigated the occurrence of intracellular accumulation of hexachlorobenzene (HCB) in Chara rudis. HCB is a widely distributed POP with LogKOW of about 5.5,1 providing an example of a hydrophobic substance for which kinetic limitation or steric hindrance is expected to occur during the transfer across protective tissues. The study aimed to i) evaluate whether a high level of structural organization (compared to phytoplankton) and the presence of protective cortex cells can prevent radial diffusion of HCB into the

EXPERIMENTAL SECTION Algae Collection and Handling. Chara rudis was collected in September 2010 in Lake Vassjøtjern, a small, calcium-rich lake approximately 50 km north of Oslo, Norway. Samples were stored in a refrigerator for a few days before the beginning of the experiment. For the exposure test only the topmost 4−5 internodes were used, corresponding to approximately 10 cm length. The selected subsamples were acclimatized for one week at similar temperature (15 °C) and light conditions. A modified, diluted (1:4) Z8 medium20 was used for the experiments, providing essential inorganic nutrients for growth of algae. Microcosms and Exposure Method. The exposure was conducted using the principles of the Multi-Media Test Chamber (MMTC),21 an approach which allows solving of the multimedia distribution of the chemical in the exposure microcosm. Briefly, microcosms consisted of 0.5 L glass bottles with polytetrafluoroethylene (PTFE) screw caps (Figure S2). A small metal alligator clamp fixed to the caps held a 25-mL glass vial in the microcosms head space, above the exposure medium. This vial, hereafter called the ‘probe’, was filled with 5 mL MilliQ water. The exposure study was performed using 14C12 radiolabeled HCB (Institute of Isotopes Co., Ltd., Budapest, Hungary). The chemical and radiochemical purity were >99.5%. The specific activity was 2.58 KBq/μg. HCB was chosen as a model compound since its high persistence prevented the influence of possible confounding factors associated with degradation in the algal tissue. In addition, HCB is sufficiently volatile to be used in measurements involving the MMTC approach. About 1666 Bq of 14C12 HCB (corresponding to about 640 ng) were diluted in 1 mL of dichloromethane and added to the glass surface at the base of the microcosm. The solvent was evaporated gently to dryness by swirling the bottle before introducing 200 mL of the medium.The nominal final

■

12428

dx.doi.org/10.1021/es302185g | Environ. Sci. Technol. 2012, 46, 12427−12434

Environmental Science & Technology

Article

to visually ensure structural integrity of the internode cell. In addition, charophytes react to damages by quickly producing a wound plug22 which prevents the capillary from entering completely into the cell. Only undamaged internode cells were sampled, in which the capillary could be inserted to the full cell length. Each cytoplasm containing capillary was thoroughly externally rinsed with Milli-Q water and transferred to a scintillation vial containing 5 mL of dichloromethane. The capillary was finely broken into pieces by means of a glass mortar and 25 mL of SL was added. The length of each sampled internode was measured from the extruded empty “tube” of cortex cells (including the internode cell wall). These samples were then transferred into scintillation vials and mixed with 5 mL of dichloromethane. They were broken down using the mortar and 20 mL of SL was added. The HCB radioactivity in cytoplasm and cortex cell samples was then measured by β decay counting. The dry weight of cortex cells was determined using a set of samples of empty cortex tubes of different lengths from other specimen of Chara rudis sampled from the same lake at the same time as the ones used in the exposure test and covering the length range of the cortex tube samples. From these samples, a linear regression curve (N = 7, P < 0.01, R2 = 0.96) was obtained between the tube lengths and their dry weight, and the associated regression function was applied to the analyzed samples to derive their dry weight from their respective lengths. The volume of the analyzed internode cell content was determined by multiplying the length of the liquid column in the capillary with its squared inner radius (0.16 mm) and π. The total length of the specimens used in the test was measured from pictures taken directly after sampling. β Decay Counting. After transferring the samples into the scintillation vials, they were kept overnight in the dark before proceeding with radioactive decay counting. This was performed over 10 min using a Canberra Packard Tri Carb 2300 TR, UK and standard calibration and quench correction techniques, as described earlier.21 Quality Assurance and Control. All media and reagents were measured for background β decay emissions. These values were used for blank correction. Method detection limits (3.18 bq) were calculated as three times the standard deviation of the β decay emissions of blanks (n = 10). A set of three positive controls, (i.e., tests carried out without the addition of the Chara samples and in which Milli-Q water was used both in the probe and as medium in the microcosm) were run simultaneously with the tests to ensure the occurrence of equilibrium between the probe content and the medium during the exposure time. After the 10 days incubation no significant differences were observed in the positive controls between CP and the concentration in the Milli-Q, indicating occurrence of equilibrium partitioning among the liquid phases and the head space. The mass of the chemical adsorbed on the probe vial glass was below detection limits, and therefore it did not affect measurements of CP. The occurrence of artifacts associated with the use of the glass pipet during sampling of the bulk water phase in the microcosms were checked by rinsing (immediately after sampling) the pipet with acetone directly into a scintillation vial in order to extract the chemical adsorbed on the inner glass surface. No measurable levels of radioactivity were detected.

concentration in the exposure medium (before the introduction of the sample, assuming no adsorption of chemical on the glass surfaces) was therefore 3.2 μg L−1, close to the reported water solubility value at 25 °C.2 This ensures that after the addition of the Chara specimen and the subsequent uptake of HCB, dissolved phase concentrations were below the solubility. Microcosms were maintained at 15 °C under slow stirring conditions for 24 h on an orbital shaker to facilitate equilibration of the chemical between the glass surface and the medium, before the Chara specimen was introduced. Incubation lasted 10 days during which the microcosm was kept sealed, under simulated sunlight conditions and at a constant temperature of 15 °C. During the incubation, the chemical underwent multimedia partitioning between the glass surface, the medium, and the Chara parts. It also degassed through the water/headspace interface and from the gas phase into the Milli-Q water in the probe. This process continued until a steady state was reached. Assuming no degradation of the chemical (HCB has water degradation half-lives in the order of magnitude of years)1 and no substantial growth of the algae, this state corresponds to multimedia single equilibrium partitioning among the accessible phases readily engaging in diffusive chemical exchange (namely the water phase, dissolved organic phase, the organism external structures, gas phase, and probe content). In particular, once equilibrium partitioning was reached between the gas phase and the probe content, the concentration of HCB in the probe (CP, μg L−1) corresponded to the concentration in the truly dissolved phase (CTDP) of the medium, as follows: CP = CTDP

(1)

The information on CTDP is key in chemical exposure assessment, given that the fraction bound to the organic matrix dissolved in the medium is virtually not available for bioconcentration. A previous study21 demonstrated that in the MMTC the water and gas phases reached partitioning equilibrium in a relatively short time (e.g., < 72 h), even in the presence of an organic carbon rich medium. Sampling from Microcosms. At the end of the exposure, the MMTCs were opened and probe vials were collected. Fifteen mL of scintillation liquid (SL) (Ultima Gold XR) was added directly to the probe and mixed with the Milli-Q to determine CTDP. Five mL of the medium was also collected using a glass pipet and transferred into a 25 mL vial together with SL for the decay counting, in order to determine the concentration of the bulk water phase (CBulk, μg L−1). Chara samples were collected and rinsed with Milli-Q water to remove the nonbound fraction of chemicals in the residual water adhering to the external surface. Sampling of the internode cell content (intracellular fraction) was achieved by cutting the internode in proximity to a node and carefully inserting a glass capillary into the internode. The outer diameter of the glass capillary (0.4 mm) corresponded to or was slightly smaller than the inner diameter of the internode cell. When the capillary was inserted to the full length of the internode, the internode cell was cut at the other end. The cell content was contained in the capillary, while the cortex cells and the internode cell wall remained as a tube which was carefully extruded. This procedure allowed obtaining both samples of single internode cell cytoplasm (and associated subcellular structures) and of the cortex cell tissue separately. This method ensured collecting cytoplasm from nondamaged internode cells. Chara rudis, in fact, is large enough 12429

dx.doi.org/10.1021/es302185g | Environ. Sci. Technol. 2012, 46, 12427−12434

Environmental Science & Technology

Article

Table 1. Summary of Results for HCB Concentration and Mass Distribution in the Water Phase, Cortex, and Internode Cell Content sample ID

mass of HCB in the cytoplasm, ng

mass of HCB in the cortex, ng

conc. HCB in cytoplasm, ng/ μL

conc. HCB in cortex, ng/g dw

conc. HCB in cortex (volumetric), ng/μL

water truly dissolved conc. CTDP, ng/L

water bulk conc. CBulk, ng/L

HCB found on microcosm glass walls, ng

3N1 6N3 8S2 8N2 5S1 7BN1 5N1

0.83 nd nd 0.22 nd 0.24 0.21

8.32 2.12 0.85 7.73 1.10 5.08 5.91

0.223 nd nd 0.053 nd 0.072 0.043

881 1489 882 1287 331 873 521

0.451 0.460 0.223 0.617 0.140 0.416 0.272

809 819 867 867 453 837 453

780 872 932 932 498 872 498

49 46 42 42 64 51 64

liquid phase (medium + probe) was about 25% of the amount originally added to the microcosms, about 65% of the total amount of chemical added to the microcosm was bioaccumulated by the algae tissues. Occurrence of Intracellular Transport. HCB was detected at measurable levels in four out of seven internode cell samples, demonstrating that HCB can overtake surface structure resistances and reach subcellular structures in the innermost part of the Chara organism. To our knowledge this is the first time transport through surface protective tissues and structures and subsequent intracellular accumulation in the internode tissue has been shown to occur for compounds with LogKOW> 5. Notably, all the cases in which HCB was found in the internode cell content below detection limits coincided with small internode cells (e.g., internode cells shorter than 12 mm); these had a sampled cytoplasm volume 3.3 μL cytoplasm. In these samples HCB activity was only a maximum factor of 2 higher than detection limits. The lack of detection in the small samples is therefore due to the instrument sensitivity limits. Concentration of HCB in the internode cell cytoplasm (CCyt) ranged 43 and 72 to 223 pg μL−1 (Table 1). One outlier was found having CCyt = 223 pg μL−1 (sample 3N1, Table 1). This sample was from a small Chara specimen whose overall length was a factor of 2 shorter than the average. Differences in cortex thickness and internode structure and volume among specimens of different age and dimensions may be one driver of the observed high concentration of HCB. CCyt were a factor 60 to 280 higher than in the medium surrounding the algae, suggesting that cell internal structures serve as bioaccumulators. However, no significant correlation was found between CCyt and CTDP. Although this may be simply a result of poor statistical power in our test (due to the low number of positive samples), it may also suggest that the time frame of the experiment was not sufficient to reach the equilibrium between internode cell content and the outer water environment. No significant relationship was found between CCyt and the concentration in the cortex (Ccor, ng g−1dw). This led to the hypothesis that, in contrast to observations in unicellular phytoplankton, after 10 days of incubation the cytoplasm of the Chara internode cells did not reach equilibrium partitioning with the external medium. This hypothesis is supported by the fact that there was no significant relationship between the measured mass of HCB in the cytoplasm and the volume of the cytoplasm itself. The different kinetics of chemical intracellular transport observed for unicellular phytoplankton and Chara is not surprising, given that, assuming no mechanisms of active transport are in place for HCB, Chara surfaces offer a much

The amount of chemical adsorbed to the microcosm jar surface was monitored at the end of the exposure tests by rinsing the microcosms with methanol and collecting the solvent in scintillation vials.

■

RESULTS AND DISCUSSION Exposure Test Quality and Chemical Mass Balance. Three out of eight specimens used in the exposure test died during incubation. These were excluded. In the remaining five specimens, occurrence of apical growth was observed suggesting the good state of the specimens. Some internode cells, however, were damaged or had too small a diameter for the capillary to be inserted and consequently were not sampled. Overall, seven samples of internode cytoplasm and the respective cortex were successfully collected from five different specimens. For two specimens, cortex and internode samples were collected twice, i.e. from two different internodes of the same plant (namely, the samples identified by the number 5 and 8 in the ID code, Table 1). These were the specimens with overall larger dimensions. CTDP was between 809 and 867 ng L−1 in four out of five tests (Table 1), while in the remaining test (that with specimen identified by the number 8 in the ID code, Table 1) CTDP was a factor of 2 lower. This outlier is explained by the fact that this test was conducted with a Chara specimen roughly twice as long as the others (the total plant length including side branches was 16 cm, while the four other samples were between 7 and 9 cm long) (Table S1). The higher amount of organic material in these tests clearly led to more depleted water concentrations. The relative difference between CBulk and CTDP ranged between 3% and 9%. Considering the expected variance of real replicate samples of bulk water measured through the MMTC method,21 these differences are not statistically significant. This suggests that i) the two phases were at a common equilibrium state with the gas phase, ii) adsorption of HCB on the dissolved organic matter present in the medium or exudated by the algae during the incubation was negligible, and iii) HCB did not interact or react with the medium components. Measured water concentrations were a factor of 5 to 7 lower than the HCB solubility reported in the literature.1 Tests were therefore carried out considerably below the limit of saturation, a key prerequisite for good quality bioconcentration experiments.2 Adsorption of HCB on the microcosm glass walls accounted for less than 10% of the amount found in the water phase (Table 1). Assuming no degradation or other losses of HCB, assuming a negligible fraction was present as gas in the head space, and considering that the bulk of the chemical in the 12430

dx.doi.org/10.1021/es302185g | Environ. Sci. Technol. 2012, 46, 12427−12434

Environmental Science & Technology

Article

Figure 2. Regression plots of Ccor vs CTDP (A); HCB mass in cortex vs cortex sample dry weight (B); Ccor vs cortex sample dry weight (C).

cortex and the dry weight of the cortex samples (P < 0.05; Figure 2B), while Ccor was independent of the sample mass (Figure 2C). Together, these data support the hypothesis that after a 10-day exposure, the cortex was approaching (or reached) partitioning equilibrium with the water phase. Based on the value of the cortex/water (truly dissolved phase) equilibrium partition coefficient KA (L kg1−) was determined by calculating the ratio between Ccor and CTDP. Given that HCB in the cortex cells represented about 90 to 97% of the total HCB found in the plant, and given that the dry mass of the internode cell cytoplasm was negligible compared to that of the cortex, the cortex-water equilibrium partitioning coefficient likely approximates the equilibrium partitioning coefficient for the whole plant. KA calculated by dry weight for 7 independent observations ranged from 0.73 × 103 to 1.8 × 103 L kg1− (mean = 1.19 × 103 L kg1−) with a normal distribution (P < 0.05) (Table S2). The box and whisker plot in Figure 3 summarizes these results. To our knowledge this is the first report of KA for HCB in charophytes. The magnitude of KA was consistent with previously reported data for phytoplankton,25 suggesting similarities between planktonic and benthic multicellular taxa

higher resistance to diffusive transport due to the more complex structure and the much longer diffusion path (in the scale of several hundreds of micrometers). Two possible pathways can be hypothesized by which HCB can enter the internode cell. These are (based on the transversal section in Figure 1) as follows: i) diffusion in and out the cortex cell through cell walls and membrane, followed by intake by diffusion through the internode cell wall and membrane, and/or ii) diffusion through the conjunction between two cortex cell walls followed by diffusion through the internode cell wall and membrane. Using a two photon excitation microscopy (TPEM) technique, Wild et al. observed that radial movement of phenanthrene did not extend beyond the epidermal cells to reach the vascular tissues of terrestrial plants.7,8 This may have occurred in our study, as cortex cell conjunctions are usually tight in Chara rudis. Cell walls in charophytes are different from vascular plants in that they lack xyloglucan, a hemicellulosic polysaccharide which binds to the surface of cellulose microfibrils.23 This could explain the observed differences between our results and the results of Wild et al.7 Our results agree, however, with Keyte et al.,24 who showed that phenanthrene can migrate across the cell membrane into adjacent cells in a moss species, and Wild et al.,8 who demonstrated that phenanthrene can enter the internal mesophyll of maize and spinach leaves. Radial diffusion was not observed by Wild et al. in root tissues,7 perhaps because roots have a tight endodermis whose function is to prevent diffusion of “unwanted” substances. In contrast, the cortex cells of Chara have a simpler structure and may only function as mechanical protection of the internode.13 In the upper Chara parts used in our experiments, the cortex cells usually close tightly and do not leave any interstitial space.13 It is unknown whether the cortex cells in Chara provide any chemical protection similar to the endodermis, but given their simple structure, they probably have a less sophisticated function than then endodermis of higher plants. Distribution of HCB in Chara and Relationship to the Equilibrium. The mass of chemical associated with an internode section of the Chara cortex was typically 10 to 35 times higher than that found in cytoplasm of the underlying internode cell (Table 1). During the time frame of this study, the cortex appeared therefore to constitute the principal storage compartment for HCB in Chara. The concentration in the cortex (Ccor, ng g−1, dw) ranged from 300 to 1500 ng g−1 (Table 1). Unlike Ccyt, the variability of Ccor was correlated (P < 0.05) with CTDP (Figure 2A). A statistically significant correlation was also found between the mass of HCB in the

Figure 3. Box plot of results for the equilibrium algae-water partitioning coefficient, KA (N = 7). 12431

dx.doi.org/10.1021/es302185g | Environ. Sci. Technol. 2012, 46, 12427−12434

Environmental Science & Technology

Article

penetrate deeply into the substrate. This suggests that uptake and redistribution of chemical pollutants from sediments is limited, and chemicals found in the algae body are waterborne. Potentially, charophytes may also be useful for remediation and pollution control. In some water bodies, in fact, charophytes constitute the largest component of biomass.34 Especially in oligotrophic or mesotrophic ecosystems they grow in very dense meadows up to two meters tall13 and can cover a large fraction of the substrate. Under these conditions, charophytes exert a fundamental control on ecosystem functions such as nutrient cycles, OC storage, and energy flows,35,36 and therefore, expectedly, also on chemical fate and distribution. As with phytoplankton, uptake by charophytes may control truly dissolved phase concentrations of hydrophobic contaminants in water, 3 reducing direct exposure to other organisms and moving chemicals to storage in sediments through litter deposition. Charophytes serve as food for a number of organisms (such as herbivorous fish,37,38 snails,39 waterfowl,40,41 and crayfish42), including some consumed by humans. Although uptake of POPs by charophytes can move chemicals to biomagnification in trophic webs, unlike phytoplankton, charophytes serves as food for herbivorous vertebrates or benthic invertebrates, rather than zooplankton. Therefore they are at the base of shorter trophic chains. From a holistic perspective this may result in a reduced exposure in predators. Charophytes grow from the top and die from the base, transferring organic matter and the associated contaminants to the substrate. Their meadows are able to stabilize the sediment bed43 inhibiting mechanical erosion and therefore also chemical pollutant resuspension. Recent studies have shown a low decomposition rate of charophyte litter, in comparison to the relatively high growth rate,14,44 suggesting that charophyte dominated systems can serve as a net sink for organic matter. This drives the hypothesis that they can also promote efficient transfer and stabilization of contaminants in the sediments. It is worth investigating the potential usefulness of these organisms for noninvasive bioremediation or restoration actions. This research must, of course, take into consideration all ecological aspects, including those concerning interaction with other elements of the ecosystem and in particular chemical trophic transfer and exposure of benthic organisms. On the other hand, harvesting of charophytes after accumulation of POPs in a remediation site is totally feasible and can be done with a minimum disturbance of the sediment. A carefully planned harvest would lead to a net removal of chemicals from contaminated sites, preventing trophic transfer and transfer to sediments. The contaminated biomass could be thereafter processed by appropriate disposal procedures. A recent study performed in controlled mesocosms demonstrated that benthic macrophytes can strongly affect the fate and distribution of chemicals in water, thus abating levels in the bulk water phase and in predators.45 Charophytes may represent a good, inexpensive, and noninvasive solution to monitoring and remediation problems for a variety of freshwater and brackish water ecosystems of high naturalistic and economic value system. They can provide ecosystem services combining the removal or sequestration of toxic chemicals in stabilized sediments with the abatement of bioavailable nutrients and the benefits of overall ecosystem quality restoration.

with respect to the structures serving as reservoir for hydrophobic chemicals. On a volumetric basis the concentration of HCB inside the internode cell was estimated to be only a factor 2 to 12 (mean = 7) lower than in the cortex (Table S2, Figure S3). This result is remarkable since cell cytoplasm is expected to be a waterdominated compartment with relatively low organic phase, in contrast to the cortex system. The hydrophobic matrix of algae cells mainly consists of the phospholipids of cell membrane and membranes of internal organelles. Jabusch et al.9 indicates that chloroplasts and tylacoids are the main storage compartment for hydrophobic chemicals in unicellular phytoplankton. In Chara internode cells, the hydrophobic parts consist of different kinds of membranes, including cell membrane, tonoplast and membranes of internal organelles, as well as of “lipid droplets”26 (Figure 1). “Lipid droplets” exist in virtually all eukaryotic cells and are assumed to be storage sites for energy.27 A large variety of different hydrophobic secondary metabolites and lipids have been identified in Chara.28 The relatively high concentrations in the cytoplasm may therefore either be related to storage of chemicals on internal membranes and/or in the lipid droplets. In contrast, the organic compartment of the much smaller cortex cells likely consists mainly of long-chained polysaccharides which are typical components of cell walls in autotrophic cells. These differences in organic matter quality may account for the higher capacity of internode cells in storing hydrophobic chemicals. Potential Role of Charophytes in Fresh Water Ecosystem Monitoring and Remediation. Charophytes are key components in freshwater ecosystems. They occur globally, growing completely submerged in the water of wetlands, rivers, streams, lakes, estuaries, and swamps.13 Charophytes compete with phytoplankton for nutrients. Indeed, the occurrence of negative relations between macrophyte and phytoplankton biomass has been documented extensively,29,30 serving as backbone of the “alternative stable state” theory.31,32 The results of this study suggest that charophytes are effective and rapid bioaccumulators of hydrophobic chemicals. These characteristics could be potentially exploited by using these organisms as natural in situ equilibrating passive samplers of truly dissolved water concentration. The relatively short equilibration time observed for HCB (in comparison to the equilibration time of many synthetic passive samplers, such as LDPE, SPMD, or silicon rubber)33 may be the result of their characteristic habitus which promote surface water exchange by expressing a very high surface to volume ratio. Charophytes may represent a useful addition or alternative to existing artificial passive samplers, especially to monitor hydrophobic chemical concentrations (in a noninvasive way) in particularly sensitive environments such as natural drinking water reservoirs, areas of high naturalistic value, etc. Potential benefits include the following: no need of phase preparation in the lab before deployment, no need of complex mooring operations, easy collection, and a nearly global distribution in fresh water ecosystems. In contrast to most other submerged macrophytes, many charophytes stay winter-green, especially in oligotrophic environments13 possibly guaranteeing monitoring services throughout the year. Finally, while benthic macrophytes can take up chemicals directly from sediments through the roots, charophytes lack true roots. They only possess small rhizoids which do not 12432

dx.doi.org/10.1021/es302185g | Environ. Sci. Technol. 2012, 46, 12427−12434

Environmental Science & Technology

■

Article

(14) Rodrigo, M. A.; Rojo, C.; Alvarez-Cobelas, M.; Cirujano, S. Chara hispida beds as a sink of nitrogen: Evidence from growth, nitrogen uptake and decomposition. Aquat. Bot. 2007, 87 (1), 7−14. (15) Wood, R. D. Monograph of the Characeae; J. Cramer: Weinheim, 1965. (16) Van den Berg, M. S.; Scheffer, M.; Van Nes, E.; Coops, H. Dynamics and stability of Chara sp and Potamogeton pectinatus in a shallow lake changing in eutrophication level. Hydrobiologia 1999, 408, 335−342. (17) Martin, G.; Torn, K.; Blindow, I.; Schubert, H.; Munsterhjelm, R.; Henricson, C. Introduction to charophytes. In Charophytes of the Baltic Sea; Schubert, H., Blindow, I., Eds.; Gantner Verlag: Ruggell, 2003. (18) Domozych, D. S.; Sorensen, I.; Pettolino, F. A.; Bacic, A.; Willats, W. G. T. The cell wall polymers of the Charophycean green alga chara corallina: Immunobinding and biochemical screening. Int. J. Plant Sci. 2010, 171 (4), 345−361. (19) Garvey, C. J.; Keckes, J.; Parker, I.; Beilby, M.; Lee, G. S. H. Polymer nanoscale morphology in Chara australis Brown cell walls studied by advanced solid state techniques. Cryptogam.: Algol. 2006, 27 (4), 391−401. (20) Staub, R. Ernährungsphysiologisch - autökologische Untersuchungen an der planktischen Blaualge Oscillatoria rubescens DC. Schweiz. Z. Hydrol. 1961, 23 (1), 82−198. (21) Nizzetto, L.; Gioia, R.; Galea, C. L.; Dachs, J.; Jones, K. C. A novel system for the controlled investigation of the environmental partitioning of hydrophobic compounds in water. Environ. Sci. Technol. 2011, 45 (18), 7834−7840. (22) Foissner, I. The relationship of echinate inclusions and coated vesicles on wound-healing in Nitella-Flexilis (Characeae). Protoplasma 1988, 142 (2−3), 164−175. (23) Popper, Z. A.; Fry, S. C. Primary cell wall composition of bryophytes and charophytes. Ann. Bot. (London, U. K.) 2003, 91 (1), 1−12. (24) Keyte, I.; Wild, E.; Dent, J.; Jones, K. C. Investigating the foliar uptake and within-leaf migration of phenanthrene by moss (Hypnum Cupressiforme) using two-photon excitation microscopy with autofluorescence. Environ. Sci. Technol. 2009, 43 (15), 5755−5761. (25) Barber, J. L.; Sweetman, A. J.; van Wijk, D.; Jones, K. C. Hexachlorobenzene in the global environment: Emissions, levels, distribution, trends and processes. Sci. Total Environ. 2005, 349 (1−3), 1−44. (26) Foissner, I. Fluorescent phosphocholine-a specific marker for the endoplasmic reticulum and for lipid droplets in Chara internodal cells. Protoplasma 2009, 238 (1−4), 47−58. (27) Goodman, J. M. The gregarious lipid droplet. J. Biol. Chem. 2008, 283 (42), 28005−28009. (28) Bankova, V.; Stefanov, K.; Dimitrova-Konaklieva, S.; Keremedchieva, G.; Frette, X.; Nikolova, C.; Kujumgiev, A.; Popov, S. Secondary metabolites and lipids in Chara globularis Thuill. Hydrobiologia 2001, 457, 199−203. (29) Muylaert, K.; Perez-Martinez, C.; Sanchez-Castillo, P.; Lauridsen, T. L.; Vanderstukken, M.; Declerck, S. A. J.; Van der Gucht, K.; Conde-Porcuna, J. M.; Jeppesen, E.; De Meester, L.; Vyverman, W. Influence of nutrients, submerged macrophytes and zooplankton grazing on phytoplankton biomass and diversity along a latitudinal gradient in Europe. Hydrobiologia 2010, 653 (1), 79−90. (30) Zhang, S. Y.; Liu, A. F.; Ma, J. M.; Zhou, Q. H.; Xu, D.; Cheng, S. P.; Zhao, Q. A.; Wu, Z. B. Changes in physicochemical and biological factors during regime shifts in a restoration demonstration of macrophytes in a small hypereutrophic Chinese lake. Ecol. Eng. 2010, 36 (12), 1611−1619. (31) Kosten, S.; Lacerot, G.; Jeppesen, E.; Marques, D. D.; van Nes, E. H.; Mazzeo, N.; Scheffer, M. Effects of submerged vegetation on water clarity across climates. Ecosystems 2009, 12 (7), 1117−1129. (32) van Nes, E. H.; Scheffer, M.; van den Berg, M. S.; Coops, H. Dominance of charophytes in eutrophic shallow lakes - when should we expect it to be an alternative stable state? Aquat. Bot. 2002, 72 (3− 4), 275−296.

ASSOCIATED CONTENT

S Supporting Information *

Summary tables with concentration data and information on HCB mass distribution in cortex and internode samples together with additional data on sample characteristics and an illustrative representation of Chararudis structures and habitus. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +47 98215393. Fax: +47 22185200. E-mail: Luca. [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Camilla Hagman, NIVA, is gratefully acknowledged for providing the growth medium and logistics for plant exposure experiment.

■

REFERENCES

(1) Mackay, D.; Shiu, W. Y.; Ma, K. C. Illustrated handbook of physical-chemical properties and environmental fate for organic chemicals; Lewis Publisher: Boca Raton, 1991. (2) Arnot, J. A.; Gobas, F. A. P. C. A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms. Environ. Rev. 2006, 14 (4), 257−297. (3) Nizzetto, L.; Gioia, R.; Li, J.; Borga, K.; Pomati, F.; Bettinetti, R.; Dachs, J.; Jones, K. C. Biological pump control of the fate and distribution of hydrophobic organic pollutants in water and plankton. Environ. Sci. Technol. 2012, 46 (6), 3204−3211. (4) White, J. C. Plant-facilitated mobilization and translocation of weathered 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (p,p ’-DDE) from an agricultural soil. Environ. Toxicol. Chem. 2001, 20 (9), 2047− 2052. (5) Wild, E.; Dent, J.; Barber, J. L.; Thomas, G. O.; Jones, K. C. A novel analytical approach for visualizing and tracking organic chemicals in plants. Environ. Sci. Technol. 2004, 38 (15), 4195−4199. (6) Hulster, A.; Muller, J. F.; Marschner, H. Soil-plant transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans to vegetables of the cucumber family (Cucurbitaceae). Environ. Sci. Technol. 1994, 28 (6), 1110−1115. (7) Wild, E.; Dent, J.; Thomas, G. O.; Jones, K. C. Direct observation of organic contaminant uptake, storage, and metabolism within plant roots. Environ. Sci. Technol. 2005, 39 (10), 3695−3702. (8) Wild, E.; Dent, J.; Thomas, G. O.; Jones, K. C. Visualizing the airto-leaf transfer and within-leaf movement and distribution of phenanthrene: Further studies utilizing two-photon excitation microscopy. Environ. Sci. Technol. 2006, 40 (3), 907−916. (9) Jabusch, T. W.; Swackhamer, D. L. Subcellular accumulation of polychlorinated biphenyls in the green alga Chlamydomonas reinhardii. Environ. Toxicol. Chem. 2004, 23 (12), 2823−2830. (10) Skoglund, R. S.; Stange, K.; Swackhamer, D. L. A kinetics model for predicting the accumulation of PCBs in phytoplankton. Environ. Sci. Technol. 1996, 30 (7), 2113−2120. (11) Skoglund, R. S.; Swackhamer, D. L. Fate of hydrophobic organic contaminants - processes affecting uptake by phytoplankton. Adv. Chem. Ser. 1994, 237, 559−573. (12) McCourt, R. M.; Delwiche, C. F.; Karol, K. G. Charophyte algae and land plant origins. Trends Ecol. Evol. 2004, 19 (12), 661−666. (13) Krause, W. Charales (Charophyceae); Gustav Fischer Verlag: Jena, 1997. 12433

dx.doi.org/10.1021/es302185g | Environ. Sci. Technol. 2012, 46, 12427−12434

Environmental Science & Technology

Article

(33) Mayer, P.; Tolls, J.; Hermens, L.; Mackay, D. Equilibrium sampling devices. Environ. Sci. Technol. 2003, 37 (9), 184a−191a. (34) Kvet, J.; Pokorny, J.; Cizkova, H. Carbon accumulation by macrophytes of aquatic and wetland habitats with standing water. Proc. Natl. Acad. Sci., India, Sect. B 2008, 78, 91−98. (35) Boros, G.; Sondergaard, M.; Takacs, P.; Vari, A.; Tatrai, I. Influence of submerged macrophytes, temperature, and nutrient loading on the development of redox potential around the sedimentwater interface in lakes. Hydrobiologia 2011, 665 (1), 117−127. (36) Hargeby, A.; Andersson, G.; Blindow, I.; Johansson, S. Trophic web structure in a shallow eutrophic lake during a dominance shift from phytoplankton to submerged macrophytes. Hydrobiologia 1994, 280, 83−90. (37) Lake, M. D.; Hicks, B. J.; Wells, R. D. S.; Dugdale, T. M. Consumption of submerged aquatic macrophytes by rudd (Scardinius erythrophthalmus L.) in New Zealand. Hydrobiologia 2002, 470 (1− 3), 13−22. (38) Miller, S. A.; Provenza, F. D. Mechanisms of resistance of freshwater macrophytes to herbivory by invasive juvenile common carp. Freshwater Biol. 2007, 52 (1), 39−49. (39) Baker, P.; Zimmanck, F.; Baker, S. M. Feeding rates of an introduced freshwater gastropod Pomacea insularum on native and nonindigenous aquatic plants in Florida. J. Mollus. Stud. 2010, 76, 138−143. (40) Noordhuis, R.; van der Molen, D. T.; van den Berg, M. S. Response of herbivorous water-birds to the return of Chara in Lake Veluwemeer, the Netherlands. Aquat. Bot. 2002, 72 (3−4), 349−367. (41) Schmieder, K.; Werner, S.; Bauer, H. G. Submersed macrophytes as a food source for wintering waterbirds at Lake Constance. Aquat. Bot. 2006, 84 (3), 245−250. (42) Cirujano, S.; Camargo, J. A.; Gomez-Cordoves, C. Feeding preference of the red swamp crayfish Procambarus clarkii (Girard) on living macrophytes in a Spanish wetland. J. Freshwater Ecol. 2004, 19 (2), 219−226. (43) Vermaat, J. E.; Santamaria, L.; Roos, P. J. Water flow across and sediment trapping in submerged macrophyte beds of contrasting growth form. Arch. Hydrobiol. 2000, 148 (4), 549−562. (44) Siong, K.; Asaeda, T. Calcite encrustation in macro-algae Chara and its implication to the formation of carbonate-bound cadmium. J. Hazard. Mater. 2009, 167 (1−3), 1237−1241. (45) Roessink, I.; Koelmans, A. A.; Brock, T. C. M. Interactions between nutrients and organic micro-pollutants in shallow freshwater model ecosystems. Sci. Total Environ. 2008, 406 (3), 436−442.

12434

dx.doi.org/10.1021/es302185g | Environ. Sci. Technol. 2012, 46, 12427−12434