Environ. Sci. Technol. 1991, 25, 287-294
Biosorption of Inorganic Tin and Methyltin Compounds by Estuarine Macroa Iga e Philip J. Wrightt~~** and James
H. Weber*$+
Department of Chemistry and Jackson Estuarine Laboratory, University of New Hampshire, Durham, New Hampshire 03824
w Biosorption kinetics of total recoverable inorganic tin (TRISn), MeSn3+,Me2Sn2+,and Me,Sn+ compounds onto tissue from the macroalga Fucus vesiculosus and onto a mixed community of Enteromorpha spp. (a filamentous genus) showed that after 48-h accumulation of tin compounds followed the trend TRISn = MeSn3+ > Me2Sn2+ > Me3Sn+. Uptake of tin compounds onto F. vesiculosus consisted of three phases: rapid phase I, intermediate phase I1 (modeled by first-order kinetics), and slow phase I11 (modeled by zero-order kinetics). Uptake of tin compounds by Enteromorpha spp. occurred in rapid phase I and intermediate, saturatin phase 11, which was complete in 3 h for TRISn and MeSng+ and in 18 h for Me2Sn2+and Me3Sn+. After 24 h, [TRISn] and [MeSn3+]in dark-incubated F. vesiculosus were significantly less than in light-incubated plants, suggesting active uptake processes during phase 111. Enteromorpha spp. incubated in the dark for 1 h contained si nificantly less TRISn and MeSn3+,but not less Me2Sn and Me&+, than light-incubated plants. Interactions with different compartments of the algal thallus account for differences in amounts of biosorbed tin compounds. Initial phase I biosorption by both algae was probably adsorption onto the thallus surface. Phase I1 biosorption of TRISn and MeSn3+ by Enteromorpha spp. involved an active process, which was probably into the cell protoplasm. In contrast, accumulation of tin compounds by F. vesiculosus during phase I1 was probably by cell wall matrix polysaccharides. Phase I11 accumulation of TRISn and MeSn3+ by F. vesiculosus was also by an active process.
f+
Introduction Widespread use of organotin compounds as stabilizers, biocides, bactericides, and antifouling agents in marine plants ( I ) over the last decade has caused considerable concern over their fate in the environment. As a result, scientists developed sensitive analytical procedures for speciation of inorganic tin and organotin compounds (2-4). Consequently, several groups have determined anthropogenic n-butyltin compounds and naturally occurring methyltin compounds in the aquatic environment (5-9). Concentrations of methyltin compounds measured in bulk water from the Great Bay Estuary, NH (3),have been higher than those from other estuaries (2, 10-12). For example, in October the maximum concentration of monomethyltin was 414 ng of Sn dm-, and that of trimethyltin was 508 ng of Sn drn-,. Donard et al. (13) reported concentrations of organotin compounds in several different types of algae. Monomethyltin concentrations ranged from 30 to 47 ng of Sn 8-l fresh weight in Enteromorpha spp., Fucus spp., and Ascophyllum nodosum. Chock and Mathieson (14) estimated the mean total biomass of intertidal algae at Cedar Point (Great Bay Estuary) during a 15month period as 85 g m-2. This large biomass could play a crucial role in the distribution and cycling of inorganic 'Department of Chemistry. Jackson Estuarine Laboratory. *Current address: School of Biological Sciences, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, England. 0013-936X/91/0925-0287$02.50/0
tin and methyltin compounds in the Great Bay Estuary. Macroalgae may influence the cycling of organotin compounds in estuaries by acting as a sink for tin compounds. This process could affect cycling by (i) increasing the concentrations of methyltin compounds available to herbivores and (ii) releasing large quantities of inorganic tin and methyltin compounds into the bay as part of the detrital pool. The latter effect could result from episodic events such as shedding of the receptacles of A. nodosum, which occurs over a limited period each spring (15), or through ice-rafting during winter (16). Donard et al. (13), in a model study of the green alga Enteromorpha spp., observed cycling of tin among inorganic and methylated forms. Potential methylating agents of inorganic tin, such as methyl iodide (17, 18) and 2-(dimethylsulfonio)propionate (19,20),occur in macroalgae. Two groups have shown (21, 22) that methyl iodide methylates Sn(I1) to MeSn3+ in model studies. Previous investigators have studied biosorption of tin compounds on macroalgae (23),marine microalgae (241, and freshwater microalgae (25, 26). Ishii (23) reported accumulation of tin in marine macroalgae, but gave no quantitative analysis of data. Chiles et al. (24) investigated accumulation of tri-n-butyltin by microalgae over a 2-h incubation period, but did not investigate rates of adsorption. Consequently, no data are available on rates of biosorption of tin compounds or on whether accumulation is by thallus surface adsorption or accumulation within the cell. Such information is essential for an understanding of the role of macroalgae in the biogeochemical cycling of tin compounds in estuarine environments. This work compares bioaccumulation of inorganic tin, MeSn3+,Me2Sn2+,and Me3Sn+by a mixed community of the intertidal marine alga Enteromorpha spp. and by tissue from Fucus uesiculosus. Enteromorpha spp. and F. vesiculosus have very different structures and cell wall biochemistry. Biosorption processes of tin compounds occurred in three rate phases onto F. vesiculosus and in two phases onto Enteromorpha spp. Biosorption behavior differed for each tin compound. The multiplicity of phases mirrors different sites on the macroalgae, and significant accumulation occurred inside the cell with some macroalgae-tin compound combinations. In some cases different biosorptive behavior of light- and dark-incubated plants demonstrated involvement of metabolic processes during biosorption. Experimental Section Plant Material and Media. Enteromorpha spp. (collected as a mixed community of E. prolifera, E. intestinalis, and E. compressa) and F. vesiculosus were collected from Adams Point in the Great Bay Estuary, NH. Enteromorpha is a genus of predominantly filamentous algae, whose structure is multiseriate, tubular, and monostromatic, while F. vesiculosus is a relatively complex macroalga with high cellular heterogeneity. Plants were transported to the Jackson Estuarine Laboratory, where they were washed and cleaned of major epiphytes. Healthy plants were then selected from Enteromorpha spp. and 0.1-0.2 g of tissue was excised from between the first and
0 1991 American Chemical Society
Environ. Sci. Technol., Vol. 25, No. 2, 1991
287
second dichotomies of F. vesiculosus. The basic incubation medium used was a modified estuarine water made from the piped supply of estuarine water in the laboratory. This water was filtered, adjusted to 20 g kg-' salinity, buffered to pH 7.8 with 1 g dm-3 H E P E S [N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid], and modified by addition of nutrients (27). Adjustments in salinity were by dilution of estuarine water with distilled, deionized water. The HEPES buffer and nutrients were added a t the same concentrations to all media. Prior to use, plant material was well-aerated and maintained at a constant temperature of 20 "C, with a photon flux density of approximately 40 kmol m-2 s-l for 2 days. A mixed inorganic tin and methyltin stock solution was prepared as 500 yg of Sn cm-3 in 1.2 mol dm-3 HC1. Tin(IV), monomethyltin (MeSn3+),dimethyltin (Me2Sn2+), and trimethyltin (Me3Sn+)were added as chloride salts. Aliquots of tin compounds were added to experimental treatments as necessary. An equal volume of 1.2 mol dm-3 NaOH was added to neutralize HC1 contained in the stock solution prior to addition of plants. Biosorption Studies. Plants were blotted in a standardized manner between two pieces of Kaydry Wipers (Kimberly-Clark) by rolling a container weighing approximately 100 g over them. Between 0.1 and 0.2 g fresh weight (accurately weighed) of plant tissue was placed into 10 cm3 of 20 g kg-' modified estuarine water in a 20-cm3 borosilicate glass scintillation vial (VWR Scientific Co.). The solution was spiked with inorganic tin and methyltin compounds each with a concentration of 5 yg ~ m - ~ All. concentrations are as tin. Experiments were carried out at 20 "C with plants illuminated with a photo flux density of 40 ymol m-2 s-l (except for dark-incubated plants). All glassware used was previously cleaned, acid-washed overnight in 10% aqueous (v/v) concentrated HN03 (i.e., 1.6 mol dm-3), and thoroughly rinsed in distilled, deionized water. Plant tissue was removed from the vials after 0 (i.e., control samples a t the start of biosorption studies) 5, 15, and 30 min and 1, 3, 6, 12, 18, 24, and 48 h. These were then blotted to remove surface water and frozen until analyzed. Media samples were also frozen to allow measurement of total recoveries of inorganic tin and methyltin compound spikes from plants and media. Triplicate samples were taken a t each time for kinetics studies. Comparisons were made of adsorption onto plant material incubated in (i) light and (ii) dark and (iii) plant material treated to remove protoplasm, leaving only the cell wall. Cell wall preparations were prepared by placing tissue alternately in 0.5% Triton XlOO in modified estuarine water and 100% methanol for 12-h periods over 3 days (28) after Ritchie and Larkum (29). For the "dark" treatment, plants were preincubated in the dark for 24 h prior to and during the experiment. Biosorption of methyltin compounds onto light- and dark-incubated plants and cell wall preparations was examined by the same experimental conditions as above, but samples were only t,aken following intervals of 0, 1, and 24 h. In control experiments, plants were incubated in the absence of tin spikes to confirm the absence of contamination during the experiment. Determination of Inorganic Tin and Methyltin Compounds, Tin compounds in samples were determined by atomic absorption spectrophotometry using an electrothermal quartz furnace following hydride generation and cryogenic trapping (30),adapted from Donard et al. ( 3 ) . To improve separation of inorganic tin from monomethyltin, the cryogenic trap was changed to a 45 cm X 288
Environ. Sci. Technol., Vol. 25, No. 2, 1991
0.6 cm (i.d.1 glass Pyrex trap filled with 10% SP2100 on 80/100 Supelcoport. The column was silanized as described by Francois and Weber (30). Diethyltin chloride ( 5 ng) was always added to the hydride generation flask as an internal standard. All quantitation was based on calibration curves of analytes and the internal standard. Limits of detection were determined as the mean baseline noise f3a; they varied with the biomass of algae extracted, but were 1 ng of Sn g-l fresh weight or less. Extraction of Plant Material. Extraction of tin compounds from plant samples was carried out by grinding tissues in liquid nitrogen and then rinsing them into 30-cm3 polycarbonate centrifuge tubes with 10 cm3 of 1mol dm-3 isothermally distilled HC1 (31). Samples were sonicated for 1 h a t 60 Hz and 50 "C and centrifuged for 15 min at 3350 rpm (ICE Clinical centrifuge), and the supernatant solution was removed for determinations. Samples of media were acidified with 8 mol dm-3 isothermally distilled HC1 to give a final HC1 concentration of 1 mol dm-3. All concentrations of inorganic tin and methyltin compounds in plant material were reported as kg of tin per fresh weight of plant. Recoveries of inorganic tin and methyltin compounds from F. vesiculosus were tested in two ways. First, extraction of inorganic tin and methyltin compounds from plants was assessed by using 0.03,0.1, 1,and 6 mol dm-3 aqueous HC1 as the extraction solvent. After extraction with 6 mol dmW3HCl, acidity was decreased by NaOH to 1 mol dm-3 [H30+],which is appropriate for synthesis of tin hydrides. Second, recovery of spikes from plants was tested by adding tin compounds ( 5 ng per g of algae) to algae immediately before grinding them. Calculations of Kinetics Parameters. In most experiments inorganic tin and methyltin compounds biosorbed on plants in three phases. Phase I reaction was complete in less than 5 min, phase I1 usually occurred between 15 min and 3 h, and phase 111 occurred between 3 and 48 h. In all cases, we determined concentrations of biosorbed inorganic tin and methyltin compounds ([Sn]). A zero-order plot of [Sn] vs time for phase I11 between 3 and 48 h gives the reaction rate and intercept, which is the final [Sn] in phase I1 ( A , ) . A first-order plot of phase I1 follows eq 1,where A , and A, are [Sn] at time t and time 0 for phase 11. A , of phase I1 is also the final [Sn] of phase In ( A , - A , ) = -ht + In ( A , - A,) (1) I, which was too fast for kinetics measurements in our experiments. Phases I, 11, and 111were mathematically separated by the following method. All data was plotted as [Sn] vs time (Figures 1 and 2). For each plot the region of Phase I11 was selected by adding data points starting from the longer times of the data and continuing with linear regression until the sum of the squares of the y residuals was minimized. The remaining data points gave first-order kinetics plots for Phase I1 (Figures 3 and 4). Because y intercepts were greater than concentrations measured a t zero hours ( t test), a faster Phase I occurred.
Results Recovery of Tin Compounds. Extractions of inorganic tin and methyltin spikes from plant materials with 1 mol dm-3 HC1 resulted in maximum recoveries from 91 to 120% (f9-13%). Replicate extractions of unspiked plant material using 6 mol dm-3 HC1 yielded significantly less MeSn3+than extractions with 1mol dmv3. Use of less than 1 mol dm-3 HC1 significantly reduced extracted concentrations of both inorganic tin and methyltin compounds. All subsequent extractions therefore used 1 mol dm-3 HCl.
40
40
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0
10
20
40
30
8
Monomethyltin
0
50
1
Dimethyltin 4
10
20
30
40
50
Trimethyltin
8
0
10
20
30
40
50
Time (h) Figure 1. Rates of biosorption of inorganic tin (TRISn) and methyltin compounds by F . vesiculosus. Regression lines are for phase I11 biosorption. (See text for explanation.) Note the different scales on y axes. Concentrations are expressed as micrograms of Sn per gram fresh weight of macroalgae.
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Flgure 2. Rates of biosorption of inorganic tin (TRISn) and methyltin compounds by fnteromorpha spp. Regression lines are for phase I11 biosorption. (See text for explanation.) Note the different scales on yaxes. Concentrations are expressed as micrograms of Sn per gram fresh weight of macroalgae.
Total recoveries of spikes from media and plants 24 h after initiation of biosorption experiments varied from 93-1137'0 (&8-13%) for methyltin compounds to 79-82% (&6-97'0) for inorganic tin. Partitioning between medium and plant phases varied with the tin compound; generally a smaller proportion of the tin compound was recovered from plant material as the number of alkyl groups increased. Biosorption of Tin Compounds. Kinetics data for bioaccumulation of tin compounds of F. uesiculosus
(Figure 1)and Enteromorpha spp. (Figure 2) show three phases. Rapid phase I was complete in -5 min and intermediate phase I1 required ca. 15 min to 3 h. Slow phase I11 occurred in most experiments from ca. 3 to 48 h (last measured time), but from 18 to 48 h for Me2Sn2+and Me3Sn+ with Enteromorpha spp. Solid lines represent fitted regressions for phase I11 in both figures. Rates of biosorption of tin compounds in slow phase I11 (Table I) are the slopes of concentration vs time plots, Le., zero-order or pseudo-zero-order kinetics. The rates of Environ. Sci. Technol., Vol. 25, No. 2, 1991 289
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biosorption (kg 8-l h-l) by F. vesiculosus had the sequence MeSn3+ (0.50) > TRISn (0.35) >> Mez&?+ (0.046) > Me3Sn+ (0.015). All rates (slopes) were greater than zero ( F test, P = 0.95) except for Me3Sn+. No slope for rate of biosorption of tin compounds on Enteromorpha spp. was statistically greater than zero. Intercepts (Table I) of phase 111 plots represent the equilibrium concentration (A,) of tin compound in the plants from phase I1 biosorption. For both F. vesiculosus and Enteromorpha spp. A , values generally decreased as the number of methyl groups on tin increased. The only 290
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Environ. Sci. Technol.. Vol. 25, No. 2, 1991
exception was the A, value for MeSn3+ biosorbed on F. uesiculosus. Data for the first-order plots for phase I1 biosorption of tin compounds on F. vesiculosus (Figure 3) and Enteromorpha spp. (Figure 4)are in Table I. All rate constants (slopes) are significantly different from zero ( P = 0.05), despite great variation in biosorption of Me3Sn+ by Enteromorpha spp. (Figure 4). For both plants the rate constants have the sequence MeSn3+ > Me2Sn2+ > Me3%+. Rate constants for total recoverable Sn (TRISn) biosorption by both species were less than those for
Table I. Rate Constants (k)O for Phase 111 (Slow) a n d P h a s e I1 (Intermediate1 Biosorption of Inorganic a n d Methyltin Compounds by Fucus vrsiculosus a n d Entrromorpha spp! species
tin cumpd
F. r w c u l w \ u r
Enrermwrpha
k.
g-'
TRlSn MeSn?'
0.35
.Me,sn2* .MqSn'
m f i
THIS"
h-'
phase 111 intercept. eg g-' 6.26' 2.97 3..Il
n.ufi
1.72
1.93
1.11
i.in
0.92
UOOI
2.33 1.42
0.9h 0 65 162 0.0~6 0.027
0.96
u U(I4 u 111:1
3.33 4 U46 XYH
n.sn t).015
.Mesin"
u Ul12
Me2Sn2' hle,Sn'
phase I1 intercept. In [A. - A,]
k. h-'
A,. fig g-'
0.68' 0 0.90 0.72 2.776 1.71 0.87 0.73
0.24
0.82 0.38 -0.31
' k was calculated as the linear regreanionr m Figures 2 and 3 tphane 1111 and Figurer 3 and 5 (phaae 111. "All grams of fresh weight. 'Hold numerak rQprQSen1when b was significantly greater than zero ( P = 0.051 inorganic tin.
data expressed in terms of rorrertrd for O-h levels of
Table 11. Significant Differeacrs' of Inorganic a n d Mrthyltin Compounds Accumulated hy Fucus vesiculosus e n d Enreromorpha s p p . between Light. v e r s u ~Dark-lneuhated Plants a n d 1.ight.Incubated versus Cell Wall Extracts (c/w ext) species
comparison
F. Vesieulosus
1 h light vs 1 h dark 24 h light vs 24 h dark 1 h light vs 1 h c/w ext 24 h light vs 24 h c/w ext 1 h light vs 1 h dark 24 h light vs 24 h dark 1 h light vs 1 h c/w ext 24 h light vs 24 h e/w ext
Enteromorpha
'Comparisons made by Student's
t
tin compound Me,Sn2+
TRlSn
t e s t significant differences indicated hy
MeSn". The range of rate constant values for F. uesiculosus was 0.86 (TRISn) to 1.93 h-I (MeSn3+). Enteromorpha spp. biosorption rate constants, in contrast, were an order of magnitude lower for Me2Sn2+(0.086 h-l) and Me3Snc (0.027 h-9 than for TRISn (0.65 h-l) and Me3Sn+ (1.62 h-'). Values of A, (Table I) calculated from the intercepts of Figures 4 and 5 represent concentrations of tin compounds on the algae a t the end of very rapid (
