Uptake of Lipophilic Organic Cu, Cd, and Pb Complexes in the Coastal Diatom Thalassiosira weissf/ogii Jonathan T. Phlnneyt and Kenneth W. Bruland'p*
Department of Biology and Institute of Marine Science, University of California, Santa Cruz, California 95064 Short-term uptake experiments using the coastal diatom Thalassiosira weissflogii demonstrated that low molecular weight, lipophilic, organic Cu, Cd, and P b complexes enter the cell by diffusion across the plasma membrane. This passive uptake mechanism acts in parallel to facilitated transport, which involves an equilibrium between free metal ions in solution and surface transport sites. Neither the observed permeability coefficents nor the calculated theoretical permeability coefficents for these lipophilic organic complexes account for the increased cellular Cu concentration observed over 5 h. Furthermore, permeability measurements cannot explain the discrepancy in cellular concentrations between Cu, Cd, and P b in the lipophilic organic metal treatments. Rather, we hypothesize that the rate-limiting step for the uptake of lipophilic organic metal complexes by unicellular algae is the ability of the metal to dissociate from the transport ligand and chelate intracellular ligands and not their flux across the membrane.
Introduction Low molecular weight, lipophilic organic compounds are known to be taken up by microorganisms from the environment by passive diffusion across the plasma membrane of the cell (1-3). The lipid bilayer acts as a barrier for aqueous hydrophilic molecules, but will allow small lipophilic molecules to readily diffuse through the membrane. It is also known that low molecular weight synthetic organic ligands such as 8-hydroxyquinoline (Ox-) and diethyldithiocarbamate (DDC-) can bind metal cations such as Cu2+,Cd2+,and Pb2+forming neutral, lipophilic metal-ligand complexes, e.g., M(Ox)z0 and M(DDC)zO. Therefore, similar to lipophilic compounds, metals existing as lipophilic organic metal complexes can likely diffuse through the plasmamembrane. However, unlike lipophilic compounds, metals that are chelated as lipophilic organic metal complexes can potentially become biologically available if they dissociate from the transport ligand and complex with intracellular binding sites. Diffusion across a membrane by lipophilic organic metal complexes is an uptake mechanism that needs to be
* Author to whom all correspondenceshould be addressed.E-mail address:
[email protected]. 7 Department of Biology. Institute of Marine Science. 0013-936X/94/0928-1781$04.50/0
0 1994 Amerlcan Chemical Society
considered in addition to the facilitated uptake by the free metal ion (4, 5). According to the free ion model, equilibrium is attained between the inorganic metal species in solution and cell surface transport sites. Subsequently, metal complexed to the surface sites is transported across the cell membrane and into the cytosol. In contrast to the inorganic species, metals chelated to organic ligands are generally considered to be not directly utilized by the cell. Instead, the organic complex acts as a reservoir or buffer for the free metal in solution. The early laboratory studies that addressed the free ion mechanism used low molecular weight synthetic organic ligands such as ethylenediaminetetracetate (EDTAQ or nitrilotriacetate (NTAS) that form anionic hydrophilic complexes such as CuEDTA2-(Figure 1)and CuNTA-. In contrast to CuEDTA2- or CuNTA-, several studies have demonstrated that lipophilic organicmetal complexes [e.g., Cu(Ox)2O, Cu(DDC)2O (Figure 11, and Cd(DDC)2OI can be directly assimilated by microorganisms. C u ( 0 x ) ~ ~ has been shown to be toxic to bacteria (6),phytoplankton (7, 8),and amphipods (9). Cu(DDC)2O and Cd(DDC)2O have not been as well studied as Cu(Ox)2O in microorganisms, but have been found to be toxic as well (10, 11). Using the pivotal work by Florence and co-workers as a foundation, we set out to clarify the mechanism of metal assimilation when the metal is chelated as a lipophilic organic metal complex. Elucidation of this uptake mechanism is critical to a more general understanding of the bioavailability of trace metals to microorganisms. Presently, there is only passing reference to such complexes in reviews of the free ion model. Sunda (12)and Morel et al. (13) point out that exceptions to the free ion model exist; for example, specific transport ligands such as siderophores or lipophilic metal complexes. In addition, HgClzO and CH3HgC1° have been implicated in entering the cell by diffusion (12,14). Only recently, a review by Campbell (15) summarized the literature (all laboratory studies) on lipophilic organic metal complexes. The lack of mechanistic studies and the dearth of field data demonstrating the importance of these complexes in the environment may have limited the inclusion of passive uptake into a more general model of metal-microorganism interactions. However, the assimilation of lipophilic organic metal complexes may not be just a laboratory phenomenon. We have found one paper demonstrating Envlron. Scl. Technol., Vol. 28, No. 11, 1994 1781
complex) between the outside and the inside of the cell (mol ~ m );-A ~is the cell surface area (cm2cell-l); Az is the membrane thickness (cm). However, the distribution of lipids and proteins within membranes can vary throughout an individual cell as well as among species of microorganisms. In addition, the thickness of the outer membrane is not uniform around the cell. Therefore, D,, and Az can vary from place to place within the membrane. Because of these uncertainties, the permeability coefficient, P m , has been used to describe the flux of a compound across a membrane (2):
0
CUEDTA~'
2-
0
I
// S
CH3CH\ CH,CH,
/ N --c
'\
/'
S\ '
\s:
S
I
/CH2CH3
// C - N
.cu2+
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Because P m encompasses the partition coefficient (KlW), it correlates with the partition of a compound between an organic solvent such as octanol and water. For organic compounds, log-log plots of the permeability coefficent versus KO,(octanol-water partitioning coefficent) are well established (2);and the solvent/water partition coefficient has been used as an analog to predict the permeability coefficent of a compound into cells (18). In order for a compound to permeate a cell, it must first diffuse across the unstirred layer associated with a membrane as well as diffuse across the membrane itself. If there is only one primary chemical species (for instance, if the metal is entirely complexed by the organic ligand), then the total permeability, Pt, of the compound across a cell can be described as (19)
\C H 2CH
Cu(DDC); (Diethyldithiocarbamate)
Flgure 1. Structures of the four metal ligand complexes used in this study. Cu is used as an example because It complexes with all the ligands.
that these types of ligands may be present in natural fresh waters (16). In addition, the extensive use of dithiocarbamates (which have a similar structure to DDC) as a fungicide on agricultural fields (17)suggests that this type of ligand could potentially enter natural waters adjacent to these fields.
(3)
where Pulis the unstirred layer permeability coefficient and P, is the membrane permeability. For a small unicellular algal cell shaped as a sphere, Pul = Dw/r (20), where D, is the diffusivity of the compound in the solution (cm2 s-1) (3) and r is the radius of the spherical cell. Overall Uptake Kinetics. Passive uptake of lipophilic compounds by microorganisms can also be represented by a single biological removal rate constant, kbio. This removal rate can be described as (3) d[MLl -- -kbio dt
Background
Passive Uptake of Lipophilic Organic Compounds by Microorganisms. The flux of lipophilic compounds through the plasma membrane of a cell involves two steps: (1)the partitioning of substrate molecules from the aqueous solution into the lipid membrane and (2) the diffusion of molecules from a higher to a lower concentration across the lipid bilayer (2) . The flux of a compound across a uniform membrane has been defined as (3)
[Cells][MLI
(4)
where -kbio is the observed second-order uptake rate constant (L cell-l h-1) for a population of microorganisms; [cells] and [ML] are the concentration of cells and metalligand complex in the culture. The constant kbio encompasses the surface area of a cell as well as the permeability. When the rate-limiting step in uptake is the transport to or through the membrane, kbio is proportional to pt (kbio =PtA), If the rate-limiting step is intracellular rather than the transport into a cell, for instance, the complexation of a metal to intracellular ligands, then kbio includes the transport as well as the intracellular chelation. Materials and Methods
where KI, is the partition coefficient of the solute from the aqueous solution into the lipid portion of the memis the diffusivity coefficient within the brane; D,, membrane (cm2d ) ; A[ML] is the difference in permeant concentration (in this case the lipophilic metal-ligand 1782 Environ. Sci. Technol., Vol. 28, No. 11, 1994
Culture Procedures. Cultures of the centric, coastal diatom Thalassiosira weissflogii (Clone Actin) were obtained from the Provasoli-Guillard Culture Collection (CCMP) at Bigelow Laboratory, Boothbay, ME. Batch cultures were grown at a temperature of 22 "C under
continuous, cool fluorescent light (100 pEinstein m-2 s-l). The cultures were not axenic. Major nutrient and vitamin concentrations were the same as f/2 (21) and Aquil (22): 300 pM nitrate, 10 pM phosphate, 100 pM silicate, 5 X 10-7 g L-1 biotin, 5.5 X g L-l vitamin B12, and 1X lo4 g L-1 thiamin. Relative to these standard culture media, the cells were grown in a lower concentration of EDTA or citrate-buffered medium as well as lower trace metal concentrations. Final ligand and trace metal concentrations for the growth medium were as follows: for experiment A, 90 nM EDTA, 90 nM Fe, 20 nM Mn, 34 nM Zn, and 0.5 nM Cu; for experiment B, 300 nM citrate, 20 nM Fe, 20 nM Mn, 34 nM Zn, and 0.5 nM Cu. Co was added only as vitamin BIZ. Metal stock was added from separate solutions. These solutions were made from atomic absorption standards (J. T. Baker) and stored in Milli-& water at a pH of 2 using trace metal-grade HC1. In all cases, Fe was added as a FeEDTA or Fe-citrate complex. We used low concentrations of metals and organic ligand in the growth medium in order to minimize potential competition between the background Cu-organic ligand in which the culture was grown and the experimental Cuorganic ligand complex. In all experiments, the cultures were grown using trace metal clean protocol. Cell cultures were grown in 1.5 L of medium in 2.5-L polycarbonate bottles. The bottles have gone through a rigorous washing procedure: rinsing in Micro solution (Baxter Scientific), rinsing in HPLCgrade methanol, and soaking for a t least 1week in 3 N HC1 followed by rinsing in Milli-Q water. The seawater used for the experiments was collected from Monterey Bay, and organics and cationic trace metals were removed (23). This seawater solution is referred to as UVSW. Before the nutrients and trace metals are added, the UVSW is sterilized in a microwave oven (24). Metal Speciation Calculations. The organic ligand concentrations required to complex the metals in each treatment were calculated using the equilibrium program TITRATOR (25). These calculations took into account the background metals and organic ligand in the culture medium as well as the added metal and experimental ligand. In all cases, enough experimental ligand was added to ensure that at least 90% (and in most cases greater than 99%) of the total Cu, Cd, and P b were chelated as the organic metal complex. The results of equilibrium speciation calculations for the experimental media are found in Table 1. The medium for experimental incubations was designed so that (1)the free metal ion concentration in the presence of the lipophilic ligands was lower than when complexed to inorganic ligands or EDTA, (2) there was minimal organic complexation in the free metal/inorganically complexed treatments (M’) (this was achieved by growing the cells in a citrate-buffered medium because citrate does not appreciably bind to Cu, Cd, or P b at the low citrate concentrations used), and (3)there was sufficient organic ligand added to the experimental medium to fully complex the metals of interest with minimal concentrations of intermediate species such as Cu(Ox)+,Cu(DDC)+,or Cu(Sox)O. From the equilibrium calculations, Cd2+and Pb2+ in seawater were found not to bind appreciably with Oxor 8-hydroxyquinoline sulfonate (Sox2-) at micromolar concentrations of the ligand. The stability constants for inorganic complexes used in the equilibrium calculations were taken from refs 26 and
Table 1. Experimental Medium Speciation Calculations. metal ligand experiment A cu treatment (1)citrate (2)EDTA (3) sulfoxine EDTA* (4) oxine EDTA* (5) DDC EDTA*
total concn
cu speciation
% total
log [Cu2+1
Cu’ CuEDTA” Cu(Sox)z”
94 >99 >99
-9.8 -13.2 -14.7
CU(OX)Z~
>99
-14.4
Cu(DDC)20
>99
-21
5 nM
40nM 5.4pM 5 pM 25 nM 5pM 25 nM 5pM 25 nM
metal/ligand
total concn
experiment B cu Cd Pb treatment (1)citrate
15 nM 9 nM 5 nM 40nM
(2) EDTA citrate*
100 pM 40 nM
(3) sulfoxine citrate* (4) oxine citrate* (5) DDC citrate*
20 pM 40 nM 20 pM 40 nM 100 pM 40 nM
Cu, Cd, and Pb speciation
Cu’ Cd’ Pb’ CuEDTA” CdEDTA2PbEDTA” Cu(Sox)~” Cu(Ox)2O
% total
log [Mn+l
96 99 99 >99 >91 >99 >99
CU-9.3 Cd-10.8 Pb-9.6 CU-13.9 Cd-11.9 Pb-13.7 CU-15.5
99
CU-14.9
Cu(DDC)2’ 99 CU-23 Cd(DDC)Z0 99 Cd-16 Pb(DDC)sO 99 Pb-18 a Final concentrations of trace metals and the calculated metal speciation in the experimental medium for experiment A, (Cut = 5 nM) and experiment B, (Cut = 15, Cdt = 9, and Pbt = 5 nM). The various organic ligands added to each treatment are grouped separately beneath the trace metal concentrations. An asterisk (*) refers to the final concentration of the background ligand used in the growth medium that was transferred to the experimental medium along with the cells. Where only citrate is listed refers to the metal treatment where no additional ligand was added to the seawater and the various metals were either complexed to the inorganic anions in solution or were as the free metal species (M’). We calculate a low concentration of CuOHCit-2 (0.3 nM) in the citrate treatment, which accounts for the Cu‘ species not being 100% of the total Cu. The final concentration of the other trace metals transferred from the growth medium into the experimental medium are as follows: for experiment A, Fe 25, Mn 5, Zn 4.5, and Cu 0.07 nM; for experiment B, Fe 2.6, Mn 2.6, Zn 4.5, and Cu 0.07 nM.
27. The stability constants for complexes with EDTA, Ox, and Soxwere taken from ref 28. The stability constants for complexes with DDC are not well known. However, estimates of these constants were taken from refs 29 and 30. All stability constants were corrected for the ionic strength of seawater (31). We added the same concentration of DDC as Ox for the Cu alone experiment (5 pM). For the Cu, Cd, and P b experiment, we added 100 pM of DDC. This concentration is adequate for the complete extraction of these three metals from seawater into an organic phase such as chloroform (32). Therefore, we are confident that greater than 99% of the Cu, Cd, and Pb added was as the M(DDC)2O complex. Experimental Manipulations. &Hydroxyquinoline, 8-hydroxyquinoline sulfonate, and citrate were purchased from Sigma Chemicals; diethyldithiocarbamate was from Fluka Chemicals; and EDTA was from GFS Chemicals. The lipophilic ligand stocks, Ox and DDC, were prepared in HPLC-grade methanol and added to the experimental Envlron. Scl. Technol., Vol. 28, No. 11, 1994
1783
medium with a 300-fold dilution of methanol to seawater. The Sox stock was added to the experimental medium in a Milli-Q water solution. EDTA was added in a 0.05 M NaC03 stock solution (22). All cell manipulations were carried out under a class 100 laminar-flow bench. At least seven cell divisions occurred in the low trace metal growth medium before the uptake experiments started. At that time, 200 mL of exponentially growing cells were transferred to 1300 mL of UVSW, which contained the desired experimental organic metal complex or the metal only. The experimental treatments were as follows (Table 1): experiment A, 5 nM Cu; experiment B, 15 nM Cu, 9 nM Cd, and 5 nM P b added to separate bottles with the various organic ligands, or UVSW only (M’), At each time point, replicate subsamples from replicate bottles were filtered. The filtration apparatus was made of Teflon and polycarbonate components that had been acid-cleaned similarly to the polycarbonate bottles. The apparatus consisted of a Teflon head unit that screwed onto the 2.5-L polycarbonate bottle. Teflon tubing and Teflon fittings connected the head unit to an in-line 47mm polycarbonate filter holder. Cells were filtered through an acid-clean (1N trace metal-grade HCl), 3-pm polycarbonate filter (Poretics). The cells were pushed through the tubing and filter with filtered nitrogen gas pressurized to approximately 30 kPa. We used the minimal filtration pressure needed to get replicate samples over a short period of time. Nonetheless, there was approximately 3-5 % of the total chlorophyll in the filtrate, demonstrating that some cells may have been lysed in this procedure. Negligible amounts of the total Cu (less than 1%)as Cu(Ox)2O or as Cu(DDC)2O were retained on polycarbonate filters with only UVSW and no cells. This result indicates that the lipophilic organic Cu complexes did not adhere to the polycarbonate filters. The filter and cells were transferred to acid-cleaned 25mL polyethylene bottles and digested in 1 mL of 7.5 N trace metal-grade nitric acid for at least 4 days. The filter samples were diluted with Milli-Q water to a final nitric acid concentration of 1.5 N before analysis. Total particulate metal concentrations were determined with a Perkin Elmer 5000 graphite furnace atomic absorption spectrophotometer using standard addition procedures. Control samples were found to have concentrations below the detection limit for P b (2 pM), confirming that these procedures were adequate in maintaining a trace metal clean environment. In addition, cells were collected for cell counts, preserved in 2 % glutaraldehyde, and refrigerated. Cell numbers were determined using an Ezone particle counter. Results
Rationale for Choosing the Organic Ligands. The structures of the four organic Cu complexes that were investigated in this study are given in Figure 1. All of these organic metal complexes are relatively small (less than 500 molecular weight) and were purposely selected for several reasons. EDTA was chosen as a model ligand because it forms well-characterized, anionic, hydrophilic complexes with the metals of interest and because of its extensive use as a metal ion buffer in biological studies. We chose Ox- because it forms neutrally charged, lipophilic complexes with Cu2+and has well established stability 1784
Envlron. Scl. Technol., Vol. 28, No. 11, 1994
constants. As a contrast to Ox, Sox was chosen to test whether the addition of a charged sulfonate side group to an Ox ligand would radically change the uptake behavior of the resultant complexes by cells. With the addition of this side group, the character of the organic Cu complex changes from a neutral, lipophilic chelate to an anionic, hydrophilic 0ne--Cu(Ox)2~ to C U ( S O X )(see ~ ~ -Figure 1). Finally, even though the stability constants for DDC- are not well-established,we selected it because it forms neutral lipophilic complexes with Cu2+, Cd2+, and Pb2+ and because of its extensive agricultural use in the class of fungicides known as dithiocarbamates (17). The results from a compilation of three laboratory experiments are reported in Figures 2-5. Figure 2 presents the results for experiment A with 5 nM Cu added to the experimental medium. Figures 3-5 give the results from experiment B measuring the uptake of 15 nM Cu, 9 nM Cd, and 5 nM Pb over a 5-h period. We present the data as percent total metal associated with T. weissflogii cells (metal retained on a 3-pm size filter) vs time and as metal per cell vs time. Both parameters are important in calculating permeability coefficients and removal rates for the different lipophilic organic metal complexes as well as for assessing the differences between treatments. The percent total metal associated with the cells indicates the degree to which the metal concentration in solution is depleted. If the solution concentration is greatly diminished, then the uptake rate by the cell will be limited by the decreasing solution concentration. Normalizing the results on a per cell basis allowed direct comparisons of the metal uptake between the different organic metal treatments and demonstrated that intracellular exchange of the metal to binding sites was occurring over 5 h. Uptake of Cu by Cells. The results with Cu demonstrate most vividly the difference in uptake rates by T. weissflogii between the lipophilic organic Cu complexes, Cu(Ox)2O and Cu(DDC)2O; the hydrophilic organic Cu complexes, CuEDTA2- and C U ( S O X )and ~ ~ the ; inorganically complexed ion, primarily CuC03O (Figures 2 and 3). (Weuse the term Cu’ to denote the inorganically complexed Cu as well as the free Cu species, Cu2+.) Table 2 gives a summary of the results. Within the first hour of the experiment, over 50 times as much Cu was taken up by the cells in the Cu(Ox)2Oand Cu(DDC)2Otreatments than in the &EDTA2- treatment (experiment A, Table 2; Figure 2). Furthermore, over 10times as much Cu was assimilated by the cells when Cu existed as the Cu(Ox)zoand Cu(DDC)20species than as CU(SOX)~~-. Finally, during the same time period, the cells in the Cu(Ox)2Oand Cu(DDC)zO solutions took up almost 3 times as much Cu as in the Cu’ treatment. By 5 h, the difference between treatments with the lipophilic organic Cu complexes and the hydrophilic organic Cu complexes increased dramatically (experiments A and B, Table 2; Figures 2 and 3). Observed permeabilities for Cu(Ox)20 and Cu(DDC)20over the first 0.5 h are very similar at approximately 6 x lo4 cm s-l (Table 3). By 24 h, the C u ( 0 x ) results ~~ for the 5 nM treatment exhibited a decrease in the cellular Cu concentration (Figure 2b). This can be explained by the fact that the reservoir of Cu(Ox)20in the solution was depleted in 5 h Figure 2a). Cell by the cells (over 60% of the CU(OX)Z~, growth, however, continued over the 24 h of this experiment, thereby diluting the Cu/cell. No 24-h point was taken in the second Cu experiment. [In another experi-
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Time (hours) 250-
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600 -
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Flgure 2. (a) Percent total Cu on cells vs time (h)and (b) Cu cell-' (amol (e)Cu(DDC)zO,(V) cell-') vs time (h) for Cut = 5 nM: (+) CU(OX)~~,
Cu', (m) CU(SOX)~-~, (0)CUEDTA-~.The data for the organic Cu treatments are from the same experiment. The Cu' data are from a separate experiment. Mean cell numbers were 13 X loE cells L-I at 5 h and 22 X 1Oe cells L-I at 24 h for the Cu-organic ligandexperiments. For the Cu' experiment, cell numbers were 13.5 X loE cells L-I by 5 h and 19 X lo6 cell L-I by 24 h. All cell numbers were f15%.
ment with 15 nM total Cu, a similar decrease in Cu/cell for the Cu(Ox)&'by 24 h was observed. These data are not presented.] Based on the percent of Cu associated with the T. weissflogii cells, there was a dramatic difference between the two experiments with lipophilic and hydrophilic organic Cu complexes. In Figure 2a with 5 nM total Cu added, approximately 90-100% of the Cu in solution as Cu(DDC)zOand over 80% of the Cu as Cu(Ox)20was taken up by the cells over a 24-h period. In contrast, only 20% of the Cu as Cu' ,6 % as CU(SOX)~~-, and 2 % as CuEDTA2became associated with T.weissflogii over the same period. In the second Cu experiment, a tripling of the total Cu
2or
I /
_-
/--+ -*
_ _ - I
0
1
I
I
I
2 3 Time (hours)
1
I
I
4
1
5
Figure 3. (a) Percent total Cu on cells vs time (h)and (b) Cu cell-' (amol cell-') vs time (h) for Cu, = 15 nM. The symbols are the same as In Figure 2. Slmllar to Figure 2, the data are from two experiments: (1) organic Cu complexes and (2) Cu'. Cell numbers were 7 X lo3 cells L-I for the experiments with the organlc Cu complexes and 13.5 X loE cells L-I for the M' experiment. Environ. Sci. Technol., Vol. 28, No. 11, 1994
1785
1.2
1
1 0.6
0.4
l i m e (hours)
1
Time (hours)
70 -
;.f
60 -
I
50 -
; -
,
_
e
-
-
40-
0
-:-+ Time (hours) Flgure 4. (a) Percent total Cd on cells vs time (h)and (b) Cd cell-' (amol cell-l)vs time (h): (e)Cd(DDC)20,(V)Cd', (0)CdEDTA-2.Cell numbers are the same as in Figure 3.
concentration to 15nM and halving the cell concentration lowered the percent uptake of solution Cu to values of 50, 11,6, 2, and 2 for Cu(Ox)2O, Cu(DDC)2O, Cu', Cu(Soxh2-, and CuEDTA2-, respectively (Figure 3a). The relative trends, however, between the five treatments remained 1786 Envlron. Scl. Technol., Vol. 28, No. 11, 1994
Time (hours) Flgure 5. (a) Percent total Pb on cells vs time (h) and (b) Pb/cell (amol/cell) vs time (h): (e)Pb(DDC)*O, (V)Pb', (0)PbEDTA-*. Cell numbers are the same as in Figure 3.
the same between the two experiments. Uptake of Cd and Pb by Cells. The same general trends as observed for Cu occurred for Cd and P b (Figures
Table 2. Cellular Metal Concentrationsa
treatment
0.5 h
8o
metal cell-’ (amol cell-9 5h lh
Experiment A 1 f 0.5 0.6 f 0.5 4 f 0.5 4 f 0.5 19 f 4 19f2 45 f 2 53 f 2 37 f 7 59 f 2 Experiment B 22 f 2 20 f 3 CuEDTA” 21 f 3 22 f 3 Cu(S0x)z” 18f4 38 f 7 CU’ 143 f 12 293 f 25 cu(ox)zo 155 13 Cu(DDC)zO 128 f 2 2f 1 1.5 f 1.2 CdEDTA” 0.5 f 0.2 0.3 f 0.1 Cd‘ 31 f 5 Cd(DDC)zO 26 f 5 0 1 f 0.3 PbEDTA22 f 0.5 2 f 0.3 Pb’ 6f1 7f1 Pb(DDC)zO
CuEDTA2cu(sox)2~CU’ cu(ox)zo Cu(DDC)zO
*
24 h 4f 1 13 f 1 52 f 4 155 f 4 218 f 9
1.5f 0.1 10f1 36 k 3 236 f 4 145 f 7
1
T
--t
1 I
34 f 7 39 f 10 63 f 12 978 f 25 226 f 13 0.8 f 0.5 2.4 f 0.7 64 f 5 2fl 4 f 0.3 9f2
a Metal cell-’ (fSD) resulta for the lipophilic metal-ligand complex for experiment A,Cut = 5 nM over 24 h; experiment B,Cut = 15,Cdt = 9,and Pbt = 5 nM over 5 h. Cu/cell for the control treatments in all experiments ranged from 4 to 17 amol/cell.
f
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - -
Table 3. Calculated Observed Permeability, Poba, Theoretical Total Permeability, Pt,Octanol/Water Coefficient, KO,,and Removal Rate, lq,ioa
treatment cu(ox)zo Cu(DDC)+ Cd(DDC)+ Pb(DDC)Z0
Pob pt 1% (x1o-Icm 8-1) (x1o-lcm 8-1) KO, 6.4 f 2.3 5.9f 1.2 >2.7 >1.1
85 90 93 99
I
1 kbio
L cell-’ h-’)
2.6 2.8 3.0
18 f 5 16+1 >5
4.0
>33
Pobs for the first 0.5 h was calculated from eq 3 with all the data normalized to 5 nM. The mean of the two experiments is listed for Cu(0x)zOand Cu(DDC)zO. Theoretical Pt was calculated from eq 4. Estimates of P, for Ptcalculations were taken from CH3HgClOuptake in T. weissflogii (14),and D,, was corrected for the differences in molar volumes (D,,, = Dw). Molar volumes for the metals were from ref 36 and for the organic ligands were calculated from ref 37. For Cu(Ox)zO, the KO, value was taken from ref 38. KO,values for Cu-, Cd-, and Pb(DDC)ZO were transformed from chloroform/water constants in ref 39 using the solvent regression equation (40). The kbio for the first 0.5 h was calculated from eq 4 with all the data normalized to 5 nM. The mean for Cu(0x)zO and Cu(DDC)# from both experiments is listed.
4 and 5). Cells in thelipophilic Cd(DDC)flandPb(DDC)ZO treatments had substantially more metal associated with them than the M’ or the MEDTA2- treatments (Figures 3 and 4). In the first 0.5 h, there was a factor of 17 and 6 greater cellular Cd and Pb, respectively, on a per cell basis when the metal was complexedto DDC than to EDTA (Table 3). Furthermore, during this same period, there was over 25 and 3 times as much Cd and P b associated with the cells as the lipophilic Cd(DDC)zOand Pb(DDC)zO complex than as inorganically complexed metal or as the free metal, Cd’ and Pb’. A comparison of the Cu(DDC)zO,Cd(DDC)Z0,and Pb(DDC)zOfrom the same experiment shows a notably different trend for the three metals. After normalizing the data to 5 nM for Cu and Cd, there is a large difference in the cellular metal concentrations between the three lipophilic complexes (Figure 6). In the first 30 min, 3 times as much Cu(DDC)zOpermeates the cells than Cd(DDC)zOand over 7 times as much as Pb(DDC)zO.
I
I
I
2 3 Time (hours)
I
I
4
I
I
5
Figure 6. Summary of the three metal-(DDC)20 complexes (metallcell vs tlme) from Figures 3-5. Cu and Cd values are normalized to 5 nM: (0)Cu(DDC)*O, (m) Cd(DDC)20,(V)Pb(DDC)20.Cell numbers are the same as in Figure 3.
Permeability Coefficients for Lipophilic Organic Metal Complexes. We calculate observed permeability coefficients, Pobs, from our initial uptake rates (eq 2). The values are summarized in Table 3. The greatest observed permeability coefficientswere measured for the Cu(DDC)zO at 5.0 X 10-4 cm s-l followed by Cd(DDC)zOat approximately half the rate, 2.7 X lo4 cm s-l, and Pb(DDC)zO at 1.1X lo4 cm s-l. Because Cd(DDC)2Oand Pb(DDC)Z0 are at equilibrium by our first time point, the Pobs for these complexes is a lower limit than the true value. kbio Results for Lipophilic Metal Organic Complexes. We calculate an initial removal rate, kbio from eq 4, for each of the lipophilic metal-ligand complexes in the first 0.5 h (Table 3). After normalizing the data to 5 nM, the results for Cu(0x)zoand Cu(DDC)zObetween the two experiments are reasonably close at between 16 and 18 X le9L cell-’ h-I. The values for Cd(DDC)zOand Pb(DDC)+ L cell-’ h-1, are significantly lower: 5 and 3 X respectively.
Discussion Free Ion Concentrations and Metal Uptake. Our results demonstrate that two separate uptake mechanisms must exist. Cu in the Cu’, CuEDTA”, and CU(SOX)~~treatments behaves as would be predicted with the free ion model of metal assimilation. According to the model, the amount of Cu transported intracellularly depends on the free Cu2+ concentration and the number of uptake sites on the cell surface. Agreater free Cu2+concentration results in a greater amount of Cu binding to the cell transport sites. This is confirmed by noting that the Environ. Scl. Technol.. Vol. 28, No. 11, 1994 1787
highest concentration of Cu/cell in these three treatments is found in the Cu' treatment, where the Cu2+concentration is the greatest (Table 1; Figures 2b and 3b). Similar to the results for Cu, the free ion concentration regulates the amount of metal that the cell can take up between the Cd' and Pb' treatments and the hydrophilic CdEDTA2- and PbEDTA" ones (Table 2). The greater the free ion concentration in the M' treatments correlates with more metal associated with the cell. There appears to be a discrepancy in the above scenario that cannot be explained simply based on Cu2+concentration. In both experiments, the Cu/cell for the Cu(S0x)2~-treatment is greater than for the CuEDTA2- treatment despite the fact that the calculated free Cu2+ concentration is lower for the Cu(Sox)~~treatment (Figures 1 and 2). Assuming that the stability constants are correct, we hypothesize that the small fraction of the neutral Cu(Sox)Ospecies that we calculated to be present in the medium (0.01 nM) may be transported across the plasmalemma of the cell. Even though the Cu(Sox)Omay be a very minor species, it could be a significant mode of Cu transport in a medium where the Cu2+concentration is very low. Diffusion of Lipophilic Organic Metal Complexes. The cellular metal in the treatments from the lipophilic organic metal complexes is substantially greater than in the treatments from the hydrophilic organic metal complexes, even though the M2+ concentration is lower. Clearly, the M/cell does not correlate with the free ion concentration. Instead, Cu, Cd, and P b in the form of these lipophilic organic metal complexes must enter the cell via a different route than the surface transport sites. We suggest that this alternate route is via direct passive diffusion of the lipophilic organic metal complexes into the cell. Within the cell there are two scenarios that could account for the lipophilic organic metal data. First, the lipophilic organic metal complex could partition into the plasmalemma, diffuse across the lipid bilayer into the cytoplasm, and become distributed among the cellular membranes and compartments as a complex. This scenario is general for any lipophilic compound. Unlike lipophilic organic compounds, lipophilic organic metal complexes can have an important second scenario: once the complex diffuses into cytoplasm, the metal can dissociate from the chelate and potentially bind to intracellular binding sites. In Table 4,we calculate the concentrations of various Cu species in different cellular compartments when at equilibrium with the medium. We chose Cu(0x)zOas an example because the equilibrium stability constants are well known. If, as in the first scenario, the Cu(0x)zo partitioned into the lipid portions of the cell without the Cu dissociating from the ligand, then on the basis of the KO, and estimates of the volume of cellular lipid, we calculate the Cu(0x)zo (lipid) concentration equal to approximately 7 X 10-2 amol/cell. If the Cu remained as a Cu(Ox)20 complex within the cytoplasm, then an equilibrium would be established by C U ( O X )between ~~ the intracellular and extracellular environments. A t equilibrium, the Cu(Ox)20(cytosol) concentration would equal 5 nM (in experiment A) or approximately 7 X 10-3 amol/ cell. What we measured was 45 amol/cell by 30 min (over 600 and 6000 times as much as the calculated values) and an equilibrium concentration of approximately 200 amol/ cell by 5 h (nearly 3000 and 30000 times as much as calculated) (Figure 2b; Table 2). We suggest that this 1788 Environ. Sci. Technoi., Vol. 28, No. 11, 1994
~
~-
Table 4. Conceptual Model for Cu(0x)zO Assimilation by T.weissflogii.
cellular Cu species
estimated cellular metal distributions (mol L-1 cell-') (amol cell-1)b
cu(ox)zo (cytosol) 5 X 1V9C 7x103 cu2+ (cytosol) 8 X 10-13d 1 x 104 CU' (cytosol) 9 x 10-"e 1 x 10"' Cu(Ox)2O (lipid) 5X10-8 7 x 10-2f Cu-L (bound intracellularly) 3 x 104 458 Calculated cellular concentrations of Cu species are for experiment A, Cu(Ox)20 = 5 nM. See the Discussion for complete details. Assumptions: (1) [CU(OX)~~I cytosol = [Cu(Ox)20] extracellular solution; (2) [H(Ox)O] cytosol = [H(Ox)O] extracellular solution; (3) cell volume = 1.44 X 10-12L cell-' (assuming a sphere with a radius of 7 pm); (4) pH (intracellular) = pH (solution) = 8; (5) KlW(membrane lipid-water partition coefficient)= K,; (6)organicC for T.weissflogii = 12 pmol of C cell-' (13);(7) 10%(total drywt) organic matter (OM) of diatom = lipid (41);(8) density of lipid = density of octanol = 827 g L-]; (9) approximately 2 g OM/g of C for diatoms (42); (IO) cells were at equilibrium with Cu(Ox)zoat 0.5 h. amol cell-' calculated from mol L-' x cell volume except for f andg. [CU(OX)~~I (cytosol) = [Cu(0x)zo] (solution) at equilibrium = 5 nM for experiment A. c U 2 + (cytosol) + 2(ox)-'(Cyt0~01) = CLI(OX)~~ (Cytosol); 8'2 [Cu(Ox)201/[Cu2+l[Ox-12; f?'z = conditional stability constant for C U ( O Xcomplex )~~ = (calculated from constant in ref 28 that was corrected for ionic strength of seawater (31);[Ox-] = 7.8 X 1 6 1 0 IfromTITRATOR (25) calculation of H(0x)Oat pH = 81. e Cu'/Cu2+ = 10.7 (26).f Klw = [Cu(Ox)zl (membrane)/[Cu(Ox)~l(aqueous)= 102.6(Table 3). For equal concentrations of lipid and aqueous solutions, Cu(Ox)$(lipid) = KlwX Cu(Ox)~o(aqueous).However, the lipid concentration in a cell is 10% of the total organic material (see assumption 5). Therefore, for Cu(0x)zo (lipid): (1)12 X 10-12 mol of C cell-' X 12 g of C mol-' X 2 g of OM g1of C = 2.9 X 10-10 g of OM cell-'; (2) 2.9 X 10-l0 g of OM cell-' X 10-l0 = 2.9 X 10-" g of lipid L of lipid cell-'; (3) 2.9 X lo-" g of lipid cell-l/827 g L-' = 3.5 X cell-'; (4) 3.5 X 10-14 L of lipid cell-' X 102.6X 5 X 10-9 mol L-1 = 0.07 amol cell-'. g Cu(Ox)20 (Figure 2b at 0.5 h). (I
large discrepancy between the measured and the calculated cellular Cu concentrations is due to the presence of intracellular binding sites that can outcompete Ox- for Cu2+. Thus, the high observed cellular Cu concentration fits only the second scenario. Based on these results, we present a conceptual model of the fate of C u ( 0 x ) ~ when ~ added to a T. weissflogii culture (Figure 7). Accordingly, Cu in the form of these small, lipophilic organic complexes bypasses the surface transport sites, X,by partitioning into the cell membrane and diffusing into the cytoplasm. Intracellularly, there are binding sites for Cu (L)throughout the cell that have a stronger affinity for Cu than Ox (e.g., sulfhydryl functional groups on proteins). The Cu dissociates from Cu(Ox)2Oand complexes with these intracellular ligands. Meanwhile, since Ox- can become protonated (HOx), it can also equilibrate with the cellular cytoplasm and lipids and the external solution. The same conceptual model can be made for Cu-, Cd-, and Pb(DDC)zO. Florence and Stauber (33) have argued that such a conceptual model may exist in microorganisms. In addition, we suggest that phytochelatins (thiol-containing metal-chelating proteins) may be one source for these intracellular ligands. The culture medium for the T. weissflogii cells had low concentrations of organic ligand and could have been stressed by the high concentration of free Cu2+ (relative to free Mn2+). The [Cu2+1/[Mn2+l in the cultures was approximately 10-l.6, which has been reported to partially limit the growth rate of marine diatoms (34). Phytochelatin concentrations of approxi-
Cu(Ox)2O + 2H'
e2HOx + CT~'
Flgure 7. Model of Cu(Oxkouptake mechanism across the plasmalemma of a phytoplankton cell. The cell wall that surrounds the plasma
membrane is omnted fromthe diagram for brevlty. Cu can enter a cell by two paths: (1) via the complexation of me Cu2+ ion to surface uptake sites, surface X. and subsequently baing transported across the plasmalemma or (2) vIa dmusion of a lipophilic Cteorganicligand complex directly into the cytosol. Cu transported by the sewnd mechanism diffuses into the cytosol, dissociates from the Ox ligand. complexes with cellular ligands (L). and remains within the cell. The O r ligand can become protonated and form a lipophilic HOx complex. This complex can diffuse back across the plasmalemma and into the external solution where it can can come to equilibrium with the Intracellular species. mately 200 amol/cell have been measured in T. weissjlogii when the cultures have been stressed by Cu2+(35).These concentrations are similar to the Culcell values in our experiments and could explain why cellular Cu concentration increases dramatically over 5 h. Disparity between Pob. and K , . The dissimilarity between trends of the observed permeability, Po&,and the corresponding K , of the complexes is consistent with the presence of these intracellular ligands (Table 3). In theory, the permeability coefficients should correlate with the K,. However, our P,,b do not. For instance, CuOX)^^ has the highest Pob but the lowest Kow. Furthermore, lipophilic partitioning alone cannot account for the differences in cellular concentrations over 5 h in the Cu-, Cd-, and Pb(DDC)zO treatments. One explanation for these differences is that the amount of cellular metal depends on the affinity of the three metals for intracellular ligands rather than their ability to diffuse into the cell. That is to say, Cu binds more readily to cellular binding sites than Cd, which in turn binds more strongly than Pb. If this scenario was not true and the three metals were complexed by cellular ligands to the same degree, then we would expect that the Cd/cell and Pb/cell concentrations would continue to increase over 5 h rather than come to an equilibrium within 0.5 h (Figure 6). Finally, the Pobfor the lipophilic organic Cu complexes are over 10times lower than the theoreticalpermeabilities, Pt (Table 3). Using T. weissjloggii, Mason and Morel (14) measuredPmforCH3HgC1equalto4.3X lVcms-'. While this value is very similar to our Pobfor Cu(0x)zoand Cu(DDC)zO(approximately6 X l V c m 8-9, theKmfor C H r HgCl is 1.7, which is over 2 orders of magnitude lower than the K , values (102.6and102.8,respectively) for these lipophilic organic metal complexes. This observation is consistent with our suggestion that the rate-limiting step for these lipophilic organic metal complexes is not their diffusionacross the membrane,buttheir ability tocomplex intracellular binding sites.
Differences i n Behavior Between Two Lipophilic Organic Cu Complexes. A comparison of experimental results from Cu(DDC)zOwith those from CU(OX)~O demonstrates that different lipophilic complexes exhibit slightly different trends. Over the initial 0.5-1 h, the Cu/ cell for the Cu(DDC)zOtreatment mirrors that of Cu(0x)zO. However, by 5 h there is a marked divergence between the two treatments. This divergence can be seen in Figure 2b, but is most dramatic in Figure 3b where there is a plateau of the Cu/cell by 5 h. The K , values for these twocomplexes are very similar (Table 3). Therefore, they should permeate the cells at about the same rate. We attribute this difference in behavior at 5 h to competition for Cu between the lipophilic organic Cu complex in the cytosol and intracellular complexation sites. The intracellular ligands may bind Cu more strongly than Ox- and, thereby, outcompete the synthetic ligand for Cu. In contrast, the cellular ligands may not be as effective in outcompetingDDC; which forms stronger complexes with Cu2+. Estimates of stability constants (corrected for the ionicstrengthofseawater)arelogpz,for Cu(DDC)zOequal to approximately 25 (29) compared to a log 8'2 for Cu(0x)zO approximately equal to 22 (28). Usingkbi.RatherThanP, for Uptakeof Lipophilic Organic Metal Complexes. Clearly, predictions of uptake rates based only upon permeability coefficients do not accurately reflect the assimilationof lipophilic organic metal complexes by microorganisms. Permeability coefficients describe only the flux of a compound across a membrane. However, our observations suggest that the rate-limiting step for cellular uptake of these lipophilic organic metal complexes appears to be the binding of the metal to intracellular binding sites and not their flux across the membrane. We believe that the removal rate, kbio (eq 4), rather than permeability, P,, is a better measure of the fate of these complexes in microorganisms. The removal rate incorporates the surface area of cells and the permeability as well as provides a measure of the fate of these lipophilic organic metal complexes in the environment.If we assume that there is no efflux of Cu from the cell, this removal rate suggests that a bloom of T.weissflogii in the environment equal to 1 X lo6 cells L-' could potentially remove approximately 40% of a 5 nM concentration of Cu(0x)zoor Cu(DDC)zOin 1day. Summary and Conclusion Similar to lipophilic compounds, lipophilicorganicmetal complexes can diffuse across cellular plasma membranes and into the cytosol. In addition, the metals in these complexes can become biologically available to microorganisms by dissociating from the transport ligand and potentially binding to intracellular binding sites. Our results suggest that the rate-limiting step for assimilation of synthetic lipophilic organic Cu, Cd, and Pb complexes is the chelation of the metal to intracellular ligands and not the flux of the complex across the plasma membrane. In our experiments, these intracellular sites hound the metalsin the followingorder: Cu> Cd> Pb. Theobserved permeabilities for the lipophilic organic metal complexes These donot correlate with the theirrespectiveKowvalues. differences appear to be due to differences in the ability oftheintracellularligandstocomplexthedifferentmetals. Because of the disparity between the calculated and theoretical permeabilities, we suggest that another constant, kbi,,, be used rather than permeability to describe Envlmn. Sd.Technol.. Vd. 28. No. il,1994 1709
the uptake of lipophilic organic metal complexes by microorganisms. Our laboratory studies demonstrate that metals existing as a lipophilic organic metal complex can diffuse into and be assimilated by cells at extremely fast rates even at low metal concentrations. Therefore, even if these complexes comprise only a small fraction of the total metal pool in the environment, they can have a disproportionately large impact on the cellular uptake of metals such as Cu, Cd, and Pb.
Acknowledgments The authors greatly appreciate discussions with R. J. M. Hudson and R. P. Mason. Thanks are also due to G. Smith and R. Franks, who provided technical assistance, and to B. Ward and M. Wells, who reviewed the manuscript. This manuscript was greatly improved by the comments of R. J. M. Hudson, W. G. Sunda, and two anonymous reviewers.
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Received for review November 15, 1993. Revised manuscript received April 20, 1994. Accepted June 9, 1994.' ~
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Abstract published in Advance ACS Abstracts, July 15, 1994.
