NISTIR 88-3852

Accumulation and

Fate of Tri butyltin

Species in Microbial

Biofilms

W. R. Blair, G. J. Olson, T. K. Trout, K. L. Jewett and F. E. Brinckman

U.S. DEPARTMENT OF COMMERCE

National Institute of Standards and Technology

(Formerly National Bureau of Standards

Institute for Materials Science and Engineering

Polymers Division

Chemical Performance and Standards Group

Gaithersburg, MD 20899

September 1988

Prepared for

David Taylor Research Center

Annapolis, MD 21402

7S Y««r» AnmncM • fVogf

NISTIR 88-3852

Accumulation and

Fate of Tri butyltin

Species in Microbial

Biofilms

W. R. Blair, G. J. Olson, T. K. Trout, K. L. Jewett and F. E. Brinckman

U.S. DEPARTMENT OF COMMERCE

National Institute of Standards and Technology

(Formerly National Bureau of Standards

Institute for Materials Science and Engineering

Polymers Division

Chemical Performance and Standards Group

Gaithersburg, MD 20899

September 1988

National Bureau of Standards became the

National Institute of Standards and Technology

on August 23, 1988, when the Omnibus Trade and

Competitiveness Act was signed. NIST retains

all NBS functions. Its new programs will encourage

improved use of technology by U.S. industry.

Prepared for

David Taylor Research Center

Annapolis, MD 21402

U.S. DEPARTMENT OF COMMERCE

C. William Verity, Secretary

NATIONAL INSTITUTE OF STANDARDS

AND TECHNOLOGY

Ernest Ambler, Director

Accumulation and Fate of Tributyltin Species in Microbial Biofilms

W. R. Blair, G. J. Olson, T.K. Trout, K. L. Jewett and F. E. Brinckman

National Institute of Standards and Technology

Chemical Performance and Standards Group

Gaithersburg, MD 20899

Abstract

Microbial biofilms, composed of microorganisms originally obtained from

estuarine harbor-exposed commercial organotin-painted panels, accumulated

tributyltin (TBT) spiked into estuarine water. Both algal or

bacterial-dominated biofilms were grown by varying nutrient conditions and were

found to accumulate TBT in excess of 300 ng/mg biomass (dry weight basis) from

estuarine water solutions containing 50 ^g/L TBT, corresponding to

bioconcentration factors of over 7000. No degradation of TBT to dibutyltin

(DBT) species was detected, either in the biofilm material or in the

surrounding solution. Thus, the microbial biofilms concentrated but did not

degrade TBT. These results suggest that microbial films on TBT-painted

structures in marine environments concentrate TBT at materials surfaces,

thereby acting as "capacitors". Such effects might make practical the use of

p paints with extremely low TBT release rates delivering antifouling service with

reduced environmental hazard.

Keywords: antifouling coatings, bioaccumulation, biodegradation, biofilms,

biofouling, organotins, tributyltin
1.0 Introduction

Organotin-based antifouling coatings are in world-wide use to prevent

attachment of marine life to ship hulls, saving time, fuel, and maintenance

costs [1]. The U.S. Navy has an interest in painting its fleet with advanced

controlled release (CR) polymer coatings that provide antifouling protection

for six years or longer [2]. However, tributyltin (TBT) , the most widely used

organotin compound for CR antifouling activity, is extremely toxic to

non-target marine life [3,4]. A number of countries including France and the

United Kingdom, and more recently individual states and the Environmental

Protection Agency in the United States, have imposed or are considering

restrictions on the use of these materials on recreational vessels [5,6],

The actual mechanism by which TBT repels fouling organisms remains to be

elucidated. Microbial biofilms rapidly develop on TBT-painted hull surfaces,

but higher forms of life (i.e. barnacles) are effectively repelled [7]. The

role of the microbial biofilm in concentrating or affecting the release rate

and form of the TBT toxicant has received very little attention. Loeb and

colleagues (unpublished data) found that the presence of microbial biofilms on

TBT-painted panels affected the release rate of the TBT into surrounding

solutions [2], Laughlin and co-workers [7] found that microbial biofilms

growing on TBT painted panels accumulated TBT. This suggests the microbial

biofilm may contribute to paint performance by reducing organotin loss, in

effect acting as a secondary controlled release medium, as well as having

protective functions toward non- target marine life [2,7]. The extent of

bioaccumulation of TBT in microbial biofilms and its possible transformations

remain uncertain, because it is difficult to measure and speciate organotins

within the biofilm matrix. The problem is further complicated by the

2
difficulty of removing biofilm from painted surfaces without also removing some

of the paint coating below.

Our earlier studies showed that estuarine heterotrophic bacteria can

rapidly accumulate TBT from solution without TBT molecular transformation [8]

.

The purpose of the current research was to determine the extent of TBT

accumulation and transformation in microbial biofilms. Methodology developed

for speciation of TBT in waters and sediments [9,10] was applied to the

analysis of butyltins in the biofilm matrix. We grew biofilms on glass slides

and exposed them to TBT in solution. While this does not precisely mimic the

leaching of TBT from a painted surface, it does offer a reliable system in

which we could be certain that none of the TBT observed in the biofilm had been

physically removed from paint. We assumed that films capable of accumulating

TBT leached from paint would also be able to accumulate TBT from surrounding

free solutions, if grown on a surface not painted with TBT. In addition, we

investigated using organotin- fluorescent ligand combinations and

epifluorescence microscopy to directly detect organotins on biofilms [11,12],

We did not find a fluorescent probe for TBT but epifluorescence microscopy of

monobutyltin in a microbial biofilm was demonstrated.

2.0 Experimental

2.1 Biofilm Growth

All biofilm experiments employed a mixed culture of estuarine

microorganisms originally obtained from the surface of a steel panel painted

with a commercial ablative copper and TBT -containing antifouling paint

(International Hysol BFA-254 copolymer). The panel had been immersed in the

Severn River at Annapolis, MD for 6.5 months at an average salinity of

3
approximately 8-10 parts per thousand (ppt) ( J . Mihm, David Taylor Research

Center, Annapolis, personal communication). The panel displayed some green

growth on the surface, suggesting the presence of algae. Upon removal from th.

river, the panel was gently rinsed with 0.5 L of Chesapeake Bay water (salinit

10 ppt), made sterile by autoclaving at 121°C for 15 min. The panel was then

gently scraped with a sterile teflon spatula over a 10 x 10 cm area, and the

scraped material was washed into a sterile polycarbonate bottle with 100 mL of

sterile Chesapeake Bay water. The wash water was immediately transported to

the laboratory, where 30 mL of the suspension was inoculated into a conical

flask (250 mL) containing 170 mL of sterilized Bay water having 10 mM ammonium

chloride and 2 mM dibasic potassium phosphate as nutrients. This flask was

placed under fluorescent lamps (intensity 350-400 foot candles) at 22 °C.

Another 30 mL of wash water was inoculated into a flask containing 170 mL of

water containing 0.03% yeast extract and 0.03% polypeptone (Difco) . These

samples were incubated at 28 °C on a gyratory shaker at 200 r.p.m. (New

Brunswick model G 76) . After one week of incubation, the contents of both

flasks were mixed and centrifuged (6000 x g, 20 min). The supernatant was

poured off and the pellet containing cells was resuspended in 70 mL of sterile

Bay water. Sterile glycerol (7.7 ml) was added, the cell suspension was

continuously stirred to mix the contents, and 1.0 mL aliquots were removed and

added to each of 60 sterile 1 . 5 mL plastic screw-capped vials designed for

cryogenic sample preservation. The vials were transported to the American Typ

Culture Collection (ATCC, Rockville, MD) , where they were frozen and stored

under liquid nitrogen. The cell suspensions contained 2.0 x 10 8 cells/mL

(Petroff -Hauser counting chamber) and 4.3 x 10 7 colony forming units/mL when

4
spread on to peptone-yeast extract (0.03% concentration of each) agar and

incubated at 28 °C for one week.

Microbial biofiims were grown on glass microscope slides (2.5 x 7.5 cm)

immersed vertically in a small glass tank (Figure 1) containing 500 mL of

sterile Chesapeake Bay water amended with peptone and yeast extract (0.05% of

each), or ammonium chloride and dibasic potassium phosphate (10 and 2 mM,

respectively) . The Bay water had been collected in a 50 L polyethylene carboy

from 1.0 m depth at the mouth of the Severn River. The sample was passed

through a 35 um filter during collection. It was stored in the dark at room

temperature in the laboratory, and aliquots were removed and autoclaved as

needed for biofilm experiments. The inoculum was prepared from frozen cells

in storage at ATCC. The cells were quickly thawed in warm water (30 °C) and

added to the biofilm growth tank. A sterile teflon stir bar was added to the

tank and the tank was placed on a magnetic stirrer in the dark or under

fluorescent lamps (intensity 350-400 foot candles), depending on the

experiment

.

2.2 Tributyltin Uptake on Biofilms

After several weeks, glass slides containing biofilms were removed from

the growth tank and immersed in smaller growth cylinders of pyrex glass

containing sterile Bay water amended with 50 ^g/L, TBT (as the cation)

.

Additionally, two groups of control slides were included, which consisted of

clean glass slides placed into TBT solutions and biofilm slides placed into

sterile Bay water minus the TBT spike. Small teflon stir bars were added to

the cylinders and the solutions were held in the dark or under fluorescent

lamps at room temperature. After incubation, the slides were dipped twice into

Bay water free of TBT and scraped with a clean razor blade. A small volume of

5
water was used to rinse the scraped material into 15 -mL glass centrifuge tube'

Scrapings from the duplicate or triplicate biofilm slides incubated with TBT

were pooled in one centrifuge tube. Aliquots were removed to separate tubes

for spiking with butyltin compounds (method of additions quantitation) and for

dry weight determinations.

2.3 Butyltin Extraction and Analysis

The procedure is a modification of a method for extraction of TBT from

sediment [10]. All concentrations of organotins are listed on a weight basis

in terms of the cations. Approximately 200 /iL of the pooled biofilm suspensic

was added to a 150 -mL round bottom flask along with an internal standard

consisting of 50 juL of tripropyltin chloride (1.5 Mg/ML in methanol).

Concentrated HC1 (0.5 mL) followed by methanol (25 mL) was added and the

mixture was refluxed for 30 min at 80-85° C. The sample was cooled to room

temperature and was extracted twice with 5-mL aliquots of cyclohexane in a 121

mL separatory funnel with shaking (10 min) on a wrist action shaker (Burrell),

The combined cyclohexane layers were evaporated to about 2 mL under a gentle

stream of air and then shaken for 45 min with 1.0 mL of 4% (w/v) aqueous NaBH<

A small aliquot of the cyclohexane layer was injected into a gas chromatograpl

equipped with a flame photometric detector operated in a tin-selective mode a:

described previously for detection of DBT and TBT [9,10].

2.4 Epifluorescence Imaging

Spectrophotometric detection of monobutyltin adsorbed to the biofilm was

successfully demonstrated by epifluorescence microscopy [11]. Monobutyltin,

which has a more intense fluorescence emission than tributyltin when complexei

with flavonol [11], was chosen for this experiment to demonstrate that the

concept of visual imaging of adsorbed organotins on the biofilm was practical

6
Glass slides covered with a well developed biofilm growth were exposed to a

solution of monobutyltin in ethanol (4.5 x 10" 4 M concentration) for

approximately 1 hour. The slides were then rinsed with ethanol and exposed to

an ethanol solution of flavonol (1.4 x 10" 4 M concentration) for 15 min.

Control slides, either exposed to monobutyltin but not to flavonol, or exposed

to flavonol but not to monobutyltin, were also prepared.

Following the above treatment, the slides were examined by epif luorescence

microscopy. The UV excitation frequency was set at 366 ran with a 28 run

bandpass. A barrier filter was used to eliminate any radiation at wavelengths

over 400 run

.

The following compounds were tested for their abilities to chelate to

butyltin species and generate luminescent emission: 3-hydroxyflavone , morin,

apigenin, galangin, emodin, flavone , chrysin, 4 '

, 5 , 7 - trihydroxyf lavone . All of

these compounds were purchased from a commercial source (Aldrich Chemical Co.,

Milwaukee, WI)

.

3.0 Results and Discussion

Preliminary experiments showed that microbial biofilms accumulated 331-

784 ng TBT/mg (dry weight) on exposure of films to Chesapeake Bay water

containing 0.344 mg TBT/L for 28 hr. Subsequent experiments were performed at

lower (50 jug/L) TBT concentrations and comparably substantial quantities of TBT

were also bound to biofilms in these experiments. The first experiment

involved biofilms grown in the dark, then immersed in the spiked Bay water and

held in the dark. After four days incubation the pooled biofilm sample

contained 353 ng TBT/mg (dry weight; Table 1, Figure 2). This represents a

bioconcentration factor of 7060. Each glass slide contained an average of

7
0.43 mg of biofilm material (dry weight) over an area of 25 cm2

. The

concentration of TBT in the film was determined by standard additions of diand tributyltin chlorides to aliquots of the pooled biofilm sample. These were

then carried through the entire extraction procedure. Samples of the solution

of Chesapeake Bay water were analyzed periodically and showed no biodegradation

of TBT to DBT, nor was any DBT detected in the biofilm material. Control slides

(clean slides exposed to TBT, or biofilm slides minus TBT) gave no butyltin

peaks in the chromatograms

Another experiment was run using biofilms that had been grown under

fluorescent lamps. These biofilms contained a large proportion of algae, as

evidenced by their green color and large numbers of red autofluorescing cells

visible under epifluorescence microscopy using 365 run excitation (owing to

chlorophyll in the cells) . These films were incubated under fluorescent lamps

for 7 days and analyzed for TBT and DBT as above. This biofilm material

contained 373 ng TBT/mg (dry weight; Table 1). This corresponds to a

bioconcentration factor of 7460. Again, there was no evidence of light- or

dark- induced degradation of TBT to DBT either in the biofilm material or in

solution during the course of the experiment. Tetrabutyltin was detected in

biofilms run in the first (dark incubated) experiment (Figure 2) . This

compound was detected as a contaminant in reagent TBT used as a spike and was

evidently bioaccumulated. This peak was not seen in the light incubated

experiments where a chromatographically purified source of TBT was employed

which did not contain tetrabutyltin. Such bioconcentration of tetrabutyltin

may be an important factor in its esturine mobility because we have also

detected this compound in harbor waters [9].

8
The concentration of TBT used in these bioaccumulation studies (50 pg/L)

greatly exceeds concentrations of TBT reported in bulk water in aquatic

environments. However, bioaccumulation of TBT in more dilute samples is also

likely, especially since biofilms accumulated nearly as much TBT from solution

at 50 pg/L as at 0.344 mg/L (see above). In addition, it is likely that the

concentration of TBT in the interstitial water of a microbial biofilm on an

organotin-painted ship hull is considerably higher than in surrounding open

waters. We therefore do not believe 50 pg TBT/L to be an unrealistic

concentration to illustrate potential microbial biofilm effects.

The TBT bioaccumulation results are similar to our previous findings with

isolated strains of estuarine bacteria (8)

.

In that study we found that the

organisms accumulated TBT to 3.7 to 7.7 pg/mg (dry weight; bioconcentration

factors in the hundreds). There was no measurable degradation of TBT to DBT.

Laughlin et al. [7] found that a microbial biofilm on a TBT painted panel

contained 20-60 pg/mg (wet weight), substantially greater than the

concentration we observed in our biofilms. This is not surprising, given the

quite different experimental systems between that study and the present

investigation. Laughlin et al. employed TBT painted panels immersed for four

months in San Francisco Bay and removed biofilm by lightly scraping the film

from the painted surface. However, both studies show that microbial biofilms

bioaccumulate TBT to a significant extent.

Previous studies have shown that TBT is degraded to DBT in the

environment, with half-lives on the order of 1-2 weeks [13,14,15]. In some

cases, microalgae have been implicated in the biodegradation process [14].

However, the current experiments show that biodegradation of TBT did not occur

in the microbial biofilm composed of organisms originally obtained from a panel

9
painted with TBT- containing paint. We believe that our past and current

results indicate biodegradation is not a common mechanism of TBT resistance in

estuarine bacteria.

Table 2 summarizes results employing EMI to detect monobutyltin in

microbial biofilms. The biofilm slide exposed to both monobutyltin and

flavonol had significantly greater fluorescence intensity than any of the

control slides. Visual examination of this slide under the epifluorescence

microscope revealed a dark blue -black background with localized areas of bright

blue fluorescence, characteristic of the monobutyltin- flavonol complex. These

images suggest that the monobutyltin moiety is not homogeneously retained in

the biofilm, but additional studies will be required to determine whether the

organotin is adsorbed chiefly by viable cells or by exocellular adhesive

polymers

Table 3 gives relative emission intensity measurements for the noncomplexed compounds, as well as the Bux Sn(4 “ x)+ complexes of the compounds.

All emission intensities were measured at the estimated wavelength of the

emission maxima of the different compounds/complexes . An excess of metal

species was used in each case to ensure complexation of all of the ligand and

guarantee that the observed emission was due to a formed complex, not excess

non- coordinated ligand. Low emission intensities of samples and multiplication

by conversion factors limited the number of significant figures for certain

species. Species which essentially did not emit gave "pseudo-emissions" at 405

ran , which actually are reflections from the 405 ran line of the mercury

excitation source lamp.

Table 3 shows that only 3 -hydroxyflavone , morin and galangin ligands form

complexes which emit sufficiently for consideration as "fluorogenic ligands"

10
for tin species. In each of these cases, it is seen that Bu3 Sn+ has a

fluorescence of only about 1/100 of that of the other tin species, perhaps due

to the UV degradation of TBT to more fluorescent species [12]. It therefore

seems very unlikely that any of these ligands could be a sensitive, selective

reagent for the detection of tributyltin species in mixtures of organotins

.

However, it seems reasonable to employ the EMI technique to survey biofilm

communities for the presence of TBT degradation compounds. In conjunction with

electron microscopy with element specific energy dispersive microanalysis,

questions could be answered concerning the location of organotin species in the

biofilm (cell mass or exocellular polymer)

.

It is interesting to note that reproducible results indicate that the

relative intensity of emissions of either morin or 3 -hydroxyflavone complexes

of butyltin species in EtOH follow the trend:

BuSn3+ > Bu2 Sn2+ > Sn4+ > Bu3 Sn+

This trend differs from the relative intensities reported for butyltin

compounds in aqueous solution [11]. The EtOH/H2 0 ratio (v/v) greatly affects

the relative emission intensities of these samples. There may be a solubility

or miscibility problem in the case of organotin- flavanoid complexes in a highly

aqueous environment. This may hinder analyses of "in-vivo" samples which

require an aqueous environment. The best use of the EMI may be to detect tin

species on a solid surface, after chromatography has been performed (i.e. paper

chromatography, TLC)

.

4.0 Summary and Conclusions

Biofilms containing microorganisms obtained from organotin painted panels

accumulated relatively large amounts of TBT from solution. There was no

11
detectable biodegradation of TBT to DBT in the films or in the surrounding

solutions. The films may thus influence the controlled-release action of

organotin coatings. Microbial biofilms may concentrate TBT on the surface of

painted ship hulls. In this manner it is conceivable that the TBT charged

microbial biofilms act as the primary repellent to settling larvae of hard

fouling organisms. If so, then organotin paints with extremely low release

rates may still provide antifouling protection by virtue of a naturallyoccurring TBT-enriched surface biofilm, and may release very little TBT to

surrounding waters. Such paints would minimize environmental impacts of the

use of TBT- containing antifouling paints. Additional experiments involving

exposure of TBT-enriched biofilms to macrofouling organisms thus are highly

warranted. Epifluorescence microscope imaging of monobutyltin in microbial

biofilms was demonstrated. The technique may be useful for the non- invasive

detection of such TBT degradation products. Direct detection of TBT in

solution or in biomass will require the development of a ligand which

specifically chelates to TBT, yielding a complex which fluoresces with

reasonable quantum efficiencies. Selected commercially available materials so

far were found to be unsatisfactory for these requirements.

5 . 0 Acknowledgments

We thank Paul Schatzberg, David Taylor Research Center (DTRC) , Annapolis,

MD , for his suggestions, ideas and support in undertaking this project. We are

grateful to George Loeb and James Mihm of DTRC for helpful discussions and for

providing the initial biofilm samples. We especially thank Lieselotte Schebek

and M.O. Andreae of the Max Planck Institute, Mainz, FRG , for assistance in

conducting the algal biofilm experiments. Portions of this work will be

12
included in the doctoral dissertation of TKT in partial fulfillment of the

requirement of the Graduate School of the University of Maryland.

13
6.0 References

[1] Ludgate , J.W. Jr., in Proc . Internat. Organotin Symp . , Oceans 87', Sept.

29 to October 1, 1987, Halifax, Nova Scotia, IEEE, Piscataway, NJ , pp . 1309-

1313 (1987).

[2] Schatzberg, P., in Proc. Internat. Organotin Symp., Oceans 87', Sept. 29

to October 1, 1987, Halifax, Nova Scotia, IEEE, Piscataway, NJ , pp . 1324-1333

(1987)

[3] Champ, M.A. and Lowenstein, F.L. Oceanus , 30, 69-77 (1987).

[4] Maguire, R.J. Appl. Organometallic Chem. 1, 475-498 (1987).

[5] Champ, M.A. and Pugh, W.L. in Proc. Internat. Organotin Symp., Oceans 87'

Sept. 29 to October 1, 1987, Halifax, Nova Scotia, IEEE, Piscataway, NJ , pp.

1296-1308 (1987).

[6] Champ, M.A. and Bell, D.F. Research Needs Concerning Organotin Compounds

Used in Antifouling Paints in Coastal Environments, Report to NOAA/National

Marine Pollution Office, Rockville, MD, March 1988, 133 pp.

[7] Laughlin, R.B. Jr., Cobet, A.B. and Guard, H.E. in Proc. Internat. Conf.

Marine Biodeterioration, in press

[8] Blair, W.R. , Olson, G.J., Brinckman, F.E. and Iverson, W.P. Microbial

Ecol . 8, 241-251 (1982).

[9] Matthias, C.L. Bellama, J.M., Olson G.J. and Brinckman, F.E. Environ.

Sci. Techno 1 . 20, 609-615 (1986).

[10] Matthias, C.L., Olson, G.J., Brinckman, F.E. and Bellama, J.M. Internat.

J. Environ. Anal. Chem., submitted.

[11] Blair, W.R.

,

Parks, E.J., Olson, G.J. and Brinckman, F.E. J. Chromatogr.

410, 383-394 (1987) .

14
[12] Trout, T.K.

,

Olson, G.J., Brinckman, F.E., Bellama, J.M. and Faltynek,

R.M.

,

submitted for publication.

[13] Seligman, P.F., Valkirs , A.O. and Lee, R.F. Environ. Sci. Technol. 20,

1229-1235 (1986).

[14] Olson, G.J. and Brinckman, F.E. in Proc . Organotin Symp., Oceans 86',

September 23-25, Washington, DC, vol. 4, IEEE, Piscataway, NJ , pp . 1196-1201

(1986)

.

[15] Clark, E.A.

,

Sterritt, R.M. and Lester, J.N. Environ. Sci. Technol. 22,

600-604 (1988).

15
TABLE 1

Bioaccumulation of TBT by Microbial Biofilms

Incubation TBT Solution TBT Accumulated Bioconc . Factor

Conditions Cone (dry wt)

Dark, 97 hr 50 jug/L 353 /xg/g 7060

Light, 168 hr 50 /ig/L 373 ng/g 7460

TABLE 2

Detection by EMI of Monobutyltin Bioaccumulation

SLIDE EMISSION MAXIMUM RELATIVE INTENSITY

No cellsno tin

no ligand

435 8 „ 58 +/- 0-25

Biofilm>

no tin

no ligand

435 8 . 54 +/- 0-27

Biofilmwith BuSn3+

no ligand

436 9.03 +/- 0.28

Biofilmwith ligand

no BuSn3 +

458 7.85 +/- 0.19

Biofilmwith BuSn3+

with ligand

452 30.39 +/- 0.87

16
TABLE 3

Relative Emission Intensities Measured at Indicated Wavelengths (solvent

was 95% EtOH)

Species CM] : [L] Relative Intensity Aem

3 -hydroxyflavone 0 55 .

5

533

SnA +: 3 -hydroxyflavone 3 413 446

BuSn3+: 3 -hydroxyf lavone 3 7669 452

Bu2 Sn2+: 3 -hydroxyflavone 3 1584 450

Bu3 Sn+

: 3 -hydroxyflavone 3 34 .

5

454

morin 0 5.17 559

SnA +: morin 3 310 497

BuSn3+: morin 3 2007 496

Bu2 Sn2+

: morin 3 603.8 496

Bu3 Sn+

: morin 3 56.3 524

apigenin 0 41.6 465

SnA +: apigenin 3 3.8 531

BuSn3+

: apigenin 3 8 538

Bu2 Sn2 +

: apigenin 3 12 461

Bu3 Sn+

: apigenin 3 72.2 476

galangin 0 1.66 405

SnA +

: galangin 3 206 475

BuSn3+

: galangin 3 417 473

Bu2 Sn2 +

: galangin 3 21 472

Bu3 Sn+

: galangin 3 2 468

emodin 0 36.8 532

SnA +

: emodin 3 11.1 532

BuSn3 +

: emodin 3 22 540

Bu2 Sn2+

: emodin 3 9.8 535

Bu3 Sn+

: emodin 3 25.3 535

17
TABLE 3, continued

Species [M] :[L] Relative Intensity Aem

f lavone 0 2.07 405

SnA +

: f lavone 3 0.7 406

BuSn3 +: flavone 3 as 00 405

Bu2 Sn2 +: f lavone 3 1 405

Bu3 Sn+: f lavone 3 0.37 405

chrysin 0 1.37 405

chrysin: Sn4 + 3 0.9 534

chrysin: BuSn3

3 2 545

chrys in : Bu2 Sn2 + 3 0 546

chrys in : Bu3 Sn+ 3 0.09 436

4 ' ,5,7- trihydroxyflavone 0 0.49 435

SnA +: 4' ,5,7- trihydroxyf lavone 3 0.9 497

BuSn3 +

: 4

'

, 5 ,

- trihydroxyflavone 3 9 495

Bu2 Sn2 +:

'

, 5 ,

- trihydroxyflavone 3 3 483

Bu3 Sn+: 4

' ,5,7- trihydroxyf lavone 3 0.63 472

18
BIOFILM GROWTH TANK

^-Plexiglass Cover

Figure 1. Apparatus used to grow microbial biofilms on glass slides.
Pr 3 Sn

Bu 3 Sn

0 5 10 mir

-

Figure 2. Chromatograms showing the bioaccumulation of TBT in microbial

biofilms after 97 hours incubation (top). Spikes of tributyltin

and dibutyltin species into biofilm matrix are shown at bottom.
^BS»1 T4A (REv. 2»8C) U.S. DEPT. OF COMM.

BIBLIOGRAPHIC DATA

SHEET (See instructions)

1. PUBLICATION OR

REPORT NO.

NISTIR 88-3852

2. Performing Organ. Report No. 3. Publication Date

SEPTEMBER 1988

4. TITLE AND SUBTITLE

Accumulation and Fate of Tributyltin Species in Microbial Biofilms

5. AUTHOR(S)

W.R. Blair, G.J. Olson, T.K. Trout, K.L. Jewett and F.E. Brinckman

6. PERFORMING ORGANIZATION (If joint or other than NBS, see instructions

)

NATIONAL BUREAU OF STANDARDS

U.S. DEPARTMENT OF COMMERCE

GAITHERSBURG, MD 20899

7. Contract/Grant No.

S. Type of Report & Period Covered

9. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street. City, State, ZIP)

David Taylor Research Center-—U.S. Navy

Annapolis, MD 21402

10. SUPPLEMENTARY NOTES

J Document describes a computer program; SF-185, FlPS Software Summary, is attached.

,1. ABSTRACT (A 200-word or less factual summary of most si gn ifi cant information. If document includes a significant

bi bl iography or literature survey, mention it here)

Microbial biofilms composed of microorganisms originally obtained from harbor

exposed organotin painted panels accumulated tributyltin (TBT ) spiked into

estuarine water. Algal and bacterial dominated biofilms were grown by varying

nutrient conditions. Both types of microbial communities accumulated TBT in

excess of 300 ng/mg biomass dry weight from solutions containing 50 ug/L TBT,

corresponding to bioconcentration factors of over 7000. No degradation of TBT

to dibutyltin (DBT) species, either in the biofilm material of in the surrounding

solution was detected in any of the experiments. Thus, the microbial biofilms

concentrated but did not degrade TBT, suggesting that microbial films on TBTpainted structures in marine environments may act to concentrate TBT at materials

surfaces, thereby acting as capacitors delivering extended antifouling service.

2. KEY WORDS (Six to twelve entries; a Iph a beti cal order; capi tal i ze only proper names; and separate key

antifouling coatings, biofilms , biofouling, organotins, tributyltin

words by semicolon s)

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