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The role of microbes in the formation of the tubeworm fouling community in Fish Harbor, Los Angeles Harbor, California

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The role of microbes in the formation of the tubeworm fouling community in Fish Harbor, Los Angeles Harbor, California

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Content TH E ROLE OF MICROBES IN TH E FORM ATION
O F TH E TUBEW ORM FOULING COMMUNITY IN FISH HARBOR,
LOS ANGELES HARBOR, CALIFORNIA
by
Minturn Tatum Wright, IV
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY O F SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Biology)
December 1991
Copyright 1991 Minturn Tatum Wright, IV
UMI Number; DP23823
All rights reserved
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UMJ
D isso r la tio n P u b lisN n q
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This dissertation, written by
Minturn Tatum Wright, IV
under the direction o f his Dissertation Committee,
and approved by all its members, has been p re
sented to and accepted by The Graduate School,
in partial fulfillment o f requirements o f the degree
of
DOCTOR OF PHILOSOPHY
pH. ID ^
' 9 1
vv 9 3
Dean of Graduate Studies
_ November 22, 1991
Date............................. ................
DISSERTATION COMMITTEE
• • •
Gerajd-J. Bakus ^ 1/ Chairman
Russel L. Zimmer X / p
.............................
Rodolfo Iturmga *
...............................................
Harrison M. Kurtz '
........
Richard Leahy
ACKNOWLEDGEMENTS
So many people have helped me in one way or another during my graduate
school years that I despair of mentioning them all and giving them proper credit.
I apologize in advance for any unintentional omission I may commit and I very
much hope that no offense is taken where none is intended.
Jon and Gil Brodie and the staff of the Institute of N atural Resources,
University of the South Pacific, provided trem endous m aterial and intellectual
support while I was in Fiji. Julie A. Silber, arriving in the nick of time, helped
salvage what could have been a nearly totally lost expedition.
William Characklis, Andreas Escher and Keith and Barbara Cooksey, all of
M ontana State University, and Douglas Caldwell, of the University of Saskatche
wan, demonstrated their image-analysis systems and gave advice on the develop
m ent of ours. Daniel Rittschof, of the Duke University M arine Laboratory, also
made helpful suggestions.
Richard Goodman and Alex Andrasi provided bacterial cultures and much
advice. Adam I. Harris suggested the idea of concurrent attraction and repulsion.
Yvon LeFebvre, of W yeth-Ayerst Laboratories, generously provided the anhy
drous ampicillin; R obert Nickol, of Hercules, provided the two special release
paint mixtures. John Shisko patiently explained culturing of and larval experimen
tation with tubew orm s. M ary Pat Eism an, through the good offices of D ale
Lorenzana, offered to perform the Microtox test.
Denise Barnes and Mohammad Yazdandoust helped in many ways with the
antibiosis experiment. Mohammad also helped in the preparation of invertebrate
ii
extracts and lent me much effort and expertise. 1 am also indebted to the Center
for E lectron Microscopy and Microanalysis and its staff, especially R obert Bils
and Alicia Thompson, who patiently endured my numerous mistakes. Many of the
staff at U.S.C.'s Fish H arbor Laboratory were vital to the success of this work,
especially Bob Bostick, who provided supplies and space, and M ikihiko Oguri,
who allowed me to use his balance.
Bernard Ormsby gave great assistance with the work, particularly with the
image processing; more importantly, his ready humor kept me sane and working
through what could have been a very difficult summer. Various graduate students,
too num erous to m ention specifically, allowed me to bend their ears and shared
many beers with me. I apologize for any cases of boredom or cirrhosis that I may
have caused.
The staff and faculty of the Department of Biological Sciences were always
there for my last-m inute equipm ent and office crises. The m em bers of my guid
ance com m ittee suffered many ignorant questions. In particular. Prof. G erald
Bakus put me up and put up with me for far longer than he probably should have.
It is largely through his judicious patience that I have reached this point.
Behind the scenes, my family and friends gave enormous amounts of love
and support, not always appreciated or even recognized at the tim e, which ena
bled me to keep working when it looked as if I would never succeed. I cannot
thank them enough.
Ill
TABLE OF CONTENTS
Page
Acknowledgem ents.............................................................................................................ii
List of T ables......................................................................................................................vii
List of Figures...................................................................................................................viii
A bstract.............................................................................................................................. xiv
I. Introduction....................................................................................................................... 1
A. History and Fouling...................................................................................................1
B. Natural Fouling Processes....................................................................................... 4
1. Steps in the Formation of a Fouling Community............................................5
a. Microbial Fouling............................................................................................ 5
b. Macrobiotal Fouling....................................................................................... 8
2. Larval Site Selection...........................................................................................12
C. Models and Mechanisms of Succession.............................................................. 18
D. Patchiness and Gregariousness............................................................................ 23
E. Research Objectives................................................................................................25
1. Antibiosis..............................................................................................................28
2. Am picillin.............................................................................................................31
3. Settlement of Larval Tubew orm s.................................................................... 32
4. Separation of Physical and Chemical Factors................................................32
5. Image Analysis.....................................................................................................33
II. M aterials and M ethods............................................................................................... 34
A. Antibiosis..................................................................................................................34
B. W inter Ampicillin....................................................................................................39
C. Spring Ampicillin.....................................................................................................41
iv
Page
D. Separation of Physical and Chemical Factors...................................................42
E. Image Analysis.........................................................................................................46
III. Results........................................................................................................................... 49
A. Antibiosis..................................................................................................................49
B. W inter Ampicillin....................................................................................................60
C. Spring Ampicillin.....................................................................................................75
D. Separation of Physical and Chemical Factors................................................. 106
1. Thickness of Coating.........................................................................................106
2. W et and Dry Mass Increases...........................................................................109
3. The Coated/U ncoated E xperim ent.............................................................. 114
4. The Double-blank Experim ent.......................................................................136
E. Image Analysis....................................................................................................... 142
IV. D iscussion.................................................................................................................. 147
A. Antibiosis................................................................................................................ 147
B. W inter Ampicillin..................................................................................................149
1. Comparisons Between Glass Slides............................................................... 150
2. Comparisons Between Teflon Slides............................................................. 151
3. Comparisons Between Glass and Teflon Slides.......................................... 152
C. Spring Ampicillin...................................................................................................153
1. Comparisons Between Glass Slides............................................................... 154
2. Comparisons Between Teflon Slides............................................................. 155
3. Comparisons Between Glass and Teflon Slides.......................................... 156
D. Separation of Physical and Chemical Factors................................................. 157
1. Coating................................................................................................................ 157
2. The Relationship Between W et and Dry Mass Increases.........................158
V
Page
3. The Coated/U ncoated Experim ent.............................................................. 159
4. The Double-blank Experim ent.......................................................................165
E. Image Analysis..................................................................................................... 165
V. Summary and Conclusions......................................................................................169
A. Sum m ary................................................................................................................. 169
B. Conclusions............................................................................................................. 171
Bibliography..................................................................................................................... 173
VI
LIST OF TABLES
Table Page
1. Organisms used in the antibiosis experiments............................................... 35
2. Statistically significant differences in inhibition zones................................ 58
3. W et and dry mass increases of the first set of fouled slides...................... 110
4. Dry mass gains of the redeployed slides as calculated from the
(unadjusted) first set of wet-dry slides............................................................ 112
5. Dry mass gains of the redeployed slides as calculated from the
adjusted first set of wet-dry slides.....................................................................113
6. Dry mass gains of the redeployed slides as calculated from the
second set of wet-dry slides...............................................................................116
7. Dry-mass gains of the redeployed slides as calculated by sub
tracting each of the two accepted starting dry-m ass figures
from the final dry-mass gain..............................................................................118
8. M anual vs. computer counts on different panel m aterials........................ 143
9. Time expended in counting worm tu b es........................................................144
10. M anual vs. com puter figures of percent cover on M asonite
panels (brown sides only)...................................................................................145
11. C om parison of worm tube areal coverage of sm all areas:
computer measurements versus visual estim ation........................................ 145
VII
LIST OF FIGURES
Figure Page
1. The location of Fish Harbor, site of most experim ents................................. 26
2. Scanning electron micrograph of the calcareous tube of a ser-
pulid tubeworm attached to a microscope slide..............................................29
3. Photograph of a typical antibiosis plate, showing the develop
ment of the bacterial lawn (cloudy white) except where inhib
ited by the contents of certain test disks........................................................... 50
4. Photograph of an antibiosis test plate, showing the great inhibi
tion of growth of the bacterium B. subtilis by a sponge (Fasci-
ospongia) extract (MTW-87-20)......................................................................... 51
5. Mean diameters of zones of inhibition of Bacillus subtilis............................52
6. Results of dose-response testing of Fasciospongia sp. extract on
Bacillus subtilis, with ampicillin (2 /^g) included for com parison................ 53
7. Mean diameters of zones of inhibition of Pseudomonas fluore-
scens..........................................................................................................................54
8. M ean diam eters of zones of inhibition of the culture swabbed
from Port Huenem e P ie r.....................................................................................55
9. Mean diameters of zones of inhibition of Pseudomonas atlanti-
ca...............................................................................................................................56
10. Mean diameters of zones of inhibition of Vibrio nereis................................. 57
11. Mass changes of the slides used in the winter ampicillin exper
iment after dipping in paint (before deploym ent).......................................... 61
12. Mass changes of all slides used in the winter ampicillin experi
ment after two days of exposure.........................................................................62
13. Mass changes of all slides used in the winter ampicillin experi
ment after four days of exposure........................................................................63
14. Mass changes of all slides used in the winter ampicillin experi
ment after eight days of exposure.......................................................................64
viii
Figure Page
15. Mass changes of all slides used in the winter ampicillin experi
ment after eighteen days of exposure................................................................66
16. Mass changes of all slides used in the winter ampicillin experi
ment after forty-five days of exposure...............................................................67
17. Mass changes of all slides used in the winter ampicillin experi
ment after seventy-four days of exposure..........................................................68
18. Mass changes of all slides used in the winter ampicillin experi
ment after 106 days of exposure.........................................................................69
19. Mass changes over tim e of all plain glass slides used in the
winter ampicillin experiment.............................................................................. 70
20. Mass changes over time of all glass slides dipped in plain fast-
release paint in the winter ampicillin experim ent..........................................71
21. Mass changes over time of all glass slides dipped in plain slow-
release paint in the winter ampicillin experim ent..........................................72
22. Mass changes over time of all glass slides dipped in fast-release
paint containing ampicillin in the winter ampicillin experim ent................73
23. Mass changes over time of all glass slides dipped in slow-re-
lease paint containing am picillin in the w inter am picillin
experiment.............................................................................................................. 74
24. Mass changes over time of all plain Teflon slides used in the
winter ampicillin experiment.............................................................................. 76
25. Mass changes over tim e of all Teflon slides dipped in plain
fast-release paint in the winter ampicillin experim ent..................................77
26. Mass changes over tim e of all Teflon slides dipped in plain
slow-release paint in the winter ampicillin experim ent................................78
27. M ass changes over tim e of all T eflon slides dipped in fast-
release paint containing ampicillin in the winter am picillin
experiment.............................................................................................................. 79
28. Mass changes over time of all Teflon slides dipped in slow-
release paint containing ampicillin in the winter am picillin
experiment.............................................................................................................. 80
ix
Figure Page
29. M ean mass changes of the painted slides used in the w inter
ampicillin study...................................................................................................... 81
30. Scanning electron m icrograph of a slide used in the w inter
ampicillin experiment, showing the dearth of attached b a c te ria................. 82
31. Scanning electron m icrograph of a slide used in the w inter
ampicillin experiment, showing some of the diatoms attached
thereto..................................................................................................................... 83
32. Scanning electron micrograph of the surface of a 74-day winter
ampicillin slide, showing the great extent of microbial fouling...................84
33. Mass changes of the slides used in the spring ampicillin exper
iment after dipping in paint (before deploym ent)..........................................85
34. Mass changes of all slides used in the spring ampicillin experi
ment after one day of exposure.......................................................................... 87
35. Mass changes of all slides used in the spring ampicillin experi
ment after two days of exposure........................................................................ 88
36. Mass changes of all slides used in the spring ampicillin experi
ment after four days of exposure....................................................................... 89
37. Mass changes of all slides used in the spring ampicillin experi
ment after eight days of exposure...................................................................... 90
38. Mass changes of all slides used in the spring ampicillin experi
ment after fifteen days of exposure................................................................... 91
39. Mass changes of all slides used in the spring ampicillin experi
ment after thirty days of exposure..................................................................... 92
40. Mass changes of all slides used in the spring ampicillin experi
m ent after fifty-two days of exposure................................................................ 93
41. Mass changes over time of all plain glass slides in the spring
ampicillin experim ent...........................................................................................94
42. Mass changes over time of all glass slides dipped in plain fast-
release paint in the spring ampicillin experiment........................................... 95
Figure Page
43. Mass changes over time of all glass slides dipped in plain slow-
release paint in the spring ampicillin experiment...........................................96
44. Mass changes over time of all glass slides dipped in fast-release
paint containing ampicillin in the spring ampicillin experim ent................. 97
45. Mass changes over time of all glass slides dipped in slow-re
lease paint containing ampicillin in the spring ampicillin exper
im ent.........................................................................................................................99
46. Mass changes over time of all plain Teflon slides in the spring
ampicillin experiment......................................................................................... 100
47. M ass changes over tim e of all T eflon slides dipped in plain
fast-release paint in the spring ampicillin experim ent................................ 101
48. M ass changes over tim e of all T eflon slides dipped in plain
slow-release paint in the spring ampicillin experim ent...............................102
49. M ass changes over tim e of all T eflon slides dipped in fast-
release paint containing am picillin in the spring am picillin
experim ent.............................................................................................................103
50. Mass changes over time of all Teflon slides dipped in slow-
release paint containing am picillin in the spring am picillin
experiment.............................................................................................................104
51. M ean mass changes of the painted slides used in the spring
ampicillin study.................................................................................................... 105
52. N um ber of adherent organisms counted within one dissecting
microscope field on each of the laboratory test slide prepara
tions........................................................................................................................107
53. Scanning electron micrographs taken of the surface of a "killed-
community" slide before they were coated with gold-palladium
alloy for redeploym ent....................................................................................... 108
54. W et and dry mass changes of the first set of slides used in the
baseline comparison for the separation-of-factors experim ent................ I l l
55. W et and dry mass changes of the second set of slides used in
the baseline comparison for the separation-of-factors experi
m ent........................................................................................................................115
xi
Figure Page
56. W et and dry mass changes of all retained slides in both sets
used in the baseline comparison......................................................................117
57. Mass changes of separation-of-factors slides after one day of
exposure................................................................................................................120
58. Mass changes of separation-of-factors slides after two days of
exposure................................................................................................................121
59. Mass changes of separation-of-factors slides after four days of
exposure................................................................................................................122
60. Mass changes of separation-of-factors slides after eight days of
exposure................................................................................................................123
61. Mass changes of separation-of-factors slides after sixteen days
of exposure...........................................................................................................124
62. Mass changes of separation-of-factors slides after thirty-one
days of exposure................................................................................................ 125
63. M ass changes of separation-of-factors slides after sixty-one
days of exposure................................................................................................ 127
64. P hotograph of several sixty-one-day separation-of-factors
slides, showing the great extent to which many of them were
overgrown with fouling organisms, epecially tubeworm s.......................... 128
65. Mass changes of the wrapped and unwrapped glass slides over
the duration of the separation-of-factors experim ent............................... 129
66. Mass changes of the wrapped and unw rapped Teflon slides
over the duration of the separation-of-factors experim ent....................... 130
67. Mass changes of live-community slides over the duration of the
separation-of-factors experiment....................................................................131
68. Mass changes of the uncoated and coated killed-community
slides over the duration of the separation-of-factors experi
m ent...................................................................................................................... 133
69. B acterial population densities upon wrapped and unw rapped
glass slides...........................................................................................................134
xii
Figure Page
70. Scanning electron m icrograph of a sixteen-day separation-of-
factors slide, showing the extent of crowding by many varieties
of fouling organisms...........................................................................................135
71. Scanning electron m icrograph of a two-day unw rapped glass
slide, showing the clumping of bacteria......................................................... 137
72. Scanning electron m icrograph of the surface of a glass slide,
showing lines of adhered bacteria, apparently along slight
irregularities in the surface of the glass......................................................... 138
73. Scanning electron micrograph of a clump of adhered bacteria,
showing the coating that covers them ............................................................ 139
74. Scanning electron micrograph of a single bacterium adhered to
a glass slide.......................................................................................................... 140
75. M ass changes of the plain and coated glass slides over the
duration of the double-blank experim ent......................................................141
Xlll
ABSTRACT
Solvent extracts of several dozen benthic marine invertebrates from the Fiji
archipelago were assayed for antibacterial activity, using four pure cultures and
one natural population. Most extracts showed little or no antibacterial activity,
but two showed significant activity. One of these, an extract from the sponge
Fasciospongia sp., was highly effective against all bacteria against which it was
tested.
To determine the relationship between antibiosis (especially against bacte
ria) and antifouling and to determ ine if bacteria were essential to the develop
m ent of an invertebrate fouling community, am picillin-containing paint was ap
plied to slides that were placed in the sea for varying periods of tim e. Bacterial
growth was reduced on the ampicillin slides, but abundance of diatoms and inver
tebrates was not greatly affected. Laboratory tests indicate that am picillin has
minimal, if any, direct effect on larval tubeworms.
To determ ine which group of characteristics (physical or chem ical) of a
m icrobial fouling com m unity m akes the surface on which th a t com m unity is
growing m ore attractive to larval invertebrates, slides bearing m icrobial fouling
communities were dried so as to preserve their microscopic ultrastructure. Some
slides were coated with a thin layer of inert metal; others were left uncoated as a
control. T hese slides w ere retu rn ed to the w ater with clean slides and slides
bearing living fouling communities. The slides bearing coated fouling communi
ties were fouled at a much greater rate than any of the other treatm ents, which
were fouled at approximately the same rate as one another. It would appear that
the physical effects of the m icrobial community are m ore im portant for the set
tlem ent of larval invertebrates than is generally thought.
xiv
To simplify the censusing of fouling organisms on fouling panels, a com
puter-driven autom atic counting and m easuring system was developed. T he re
sults obtained are not generally as accurate as human counting, in part because of
the lim ited resolution and discrim ination ability of the im aging system , but
com puter counting is much faster. Correction constants may be used to approxi
m ate m anual results. C om puter-derived figures for areal coverage com pare
favorably with manual estimation.
XV
I. Introduction
A. History and Fouling
For nearly as long as there have been vessels and structures in the sea,
humans have observed that such objects, over time, tend to become encrusted and
overgrown with the abundant and sundry forms of marine life known today by the
general term "fouling." For nearly as long as they have been making these obser
vations, hum ans have labored mightily against the growth of fouling organisms
upon their marine artifices; the fouling organisms make the objects on which they
grow heavier and more prone to drag. Additionally, some fouling organisms bore
holes in their substrates, further weakening them. The ancient Phoenicians nailed
strips of copper to the hulls of their ships to overcom e foulers (C ham p and
Lowenstein, 1987). A ristotle lam ented the quick aggregation of "fish" on ships
and the ships' subsequent slower speeds (Fischer et a l, 1984). A thenaeus {ca.
228) described in detail the construction of the ship built by H ieron of Syracuse,
which w ork was perform ed under the supervision of no less a scientist than
A rchim edes. The hull, once waterproofed, was coated with a plating of lead,
secured with bronze rivets. The extra weight was clearly seen as being worth the
freedom from fouling and the consequently reduced drag. A similar construction
was used for "Trajan's ship," which was sheathed with lead, using copper nails
(Alberti, 1470). A R om an ship recovered from the Lake of Nemi also had lead
sheets nailed to its hull (Young, 1867).
Although m ethods changed, the them e of antifouling continued through
the ages. During the reign of Henry VIII of England, ships were coated with
pitch, over which loose anim al hair was spread; strips of sheathing wood overall
served to keep the hair in place. In a return to the past, a number of the ships of
1
Charles II (reigned 1660-1685) were sheathed in lead, using copper nails (Vissch-
er, 1927). The Age of Exploration, which saw the spread of fouling organisms to
new habitats on ships (e.g., Arakawa, 1990; see also Elton, 1958), was also an age
of discovery of new fouling and boring organisms in the newly-discovered lands
and seas, which heightened the need for effective m easures against these organ
isms. The rise of great m erchant m arine trading systems led to num erous philo
sophical and experim ental forays against fouling organisms, but success was elu
sive and never com plete: enorm ous num bers of widely varied item s and proce
dures were tried in the nineteenth century (Visscher, 1927).
The historical antifouling methods cannot have been very effective, as they
were eventually abandoned (Visscher, 1927). Despite their precautions, mariners
and shipowners were faced with the question of what to do about the organisms
that grew upon their ships' hulls. It was once a common practice to clean fouled
vessels by beaching them carefully and allowing the surf, sand, and shingle to
scour the hulls. O ther vessels were periodically sailed into fresh w ater, which
would kill attached m arine organisms, some of which would fall off as a result
(Visscher, 1927). D espite these "cures," it has been the goal from the start to
prevent the attachm ent of fouling organisms in the first place. The practice of
painting ships has endured so long that its value seems quite clear. Although ships
have likely been painted almost as long as they have been built, the composition
of the paint has varied over time. In the days of wooden ships, paints were used to
prevent rot and repel borers. As iron and steel replaced wood as the dom inant
shipbuilding material, the purpose of paint changed to prevention of corrosion as
well as fouling, and its m akeup changed similarly (iron hulls m ake lead sheeting
im practical, largely because of galvanic action in seaw ater). The trend in the
T w entieth Century has been toward paints containing such toxins as copper and
2
mercury com pounds (Visscher, 1927). In recent decades, organotin com pounds
have generally been the toxin of choice (Champ and Lowenstein, 1987; Batley et
a l, 1989a).
Despite all of the research-and-development effort concentrated on fouling
and its prevention, the atten tio n paid prior to this century was aim ed at the
"climax community," the few relatively large organisms that come to dom inate a
fouling community over long periods of time: barnacles, algae, and the like {e.g.,
Visscher, 1927). Little, if any, attention was paid to the process of development of
the fouling community, let alone to the organisms that play sundry pioneer and
interm ediate roles in the developm ent process (ZoB ell and Allen, 1935). Even
studies conducted over time tended to concentrate on the larger species, paying
little attention to the smaller organisms. Despite m odern advances, this tendency
continues to this day (Dean and Connell, 1987a).
The Tw entieth Century saw great leaps in the understanding of fouling.
Although bacteria were isolated from the sea over 150 years ago (ZoBell, 1946),
little other than descriptive work could be perform ed on m arine bacteria until
they could be cultured in the laboratory (Dr. E.G . Ruby, personal com m unica
tion). The first studies of aquatic bacteria were perform ed in the late Nineteenth
Century; bacteria have been recognized in the plankton for about as long (H ob
ble, 1988). The tendency of aquatic bacteria to foul hard surfaces and form slimes
thereon has been observed since the 1920's (Angst, 1923; Hilen, 1923), although
ZoBell and Allen (1933, 1935) are generally credited with bringing this phenom e
non to the attention of the general scientific community.
Antifouling has also seen great advances in recent decades. Currently, the
m ost p o p u lar antifouling preparations contain copper or, m ore prevalently,
compounds of organotins, especially tributyltin (TBT). While these preparations
3
have m et with considerable success, they are fraught with side-effects: chemical
antifoulants tend to be excessively lethal and over-broad in their effect. TBT has
been linked with physiological and behavioral abnorm alities in a wide range of
organisms (e.g.. Champ and Lowenstein, 1987; Johansen and M0hlenberg, 1987;
Bruno and Ellis, 1988; Bryan et a l, 1988; Emmett, 1988a; Gibbs et a l, 1988, 1990;
Batley et a l, 1989b; M atthiessen and Thain, 1989; Laurence et a l, 1989; Saavedra
Alvarez and Ellis, 1990; Spence et a l, 1990; Curtis and Barse, 1990; Ellis and
Pattisina, 1990; M eyers-Schulte and Dooley, 1990; Stickle et a l, 1990), and has
been reported to undergo biom agnification as well (Johansen and M 0hlenberg,
1987; Tsuda et a l, 1988; Batley et a l, 1989b). Numerous governments have begun
to restrict the use of TBT (Champ and Lowenstein, 1987; Emmett, 1988b; Bruno
and Ellis, 1988; U.S. Congress, 1988; Leffler, 1988; Dailey, 1989; Anonymous,
1989b, 1990a). There is also evidence that TBT can increase the growth of micro
bial foulers (Loeb et a l, 1984). A practical substitute for TBT which combines
antifouling effectiveness with negligible environmental and health hazards is the
object of much ongoing research (e.g., Costlow and Tipper, 1984, especially Fisher
et a l, 1984).
B. N atural fouling processes
The development of a marine fouling community under natural conditions
proceeds in a m anner much akin to the process of terrestrial succession (e.g.,
Farrell, 1989). Over one thousand species have been reported to play some role
in m arine fouling comm unities (W oods Hole O ceanographic Institution, 1952;
Lindner, 1984). Working only on ships' hulls, Visscher (1927) reported sixty-one
species of larger animals and sixteen types of macroscopic algae, while Bagaveeva
(1988) found forty-four species of polychaetes alone. V enugopalan and Wagh
4
(1990) recorded seventy species of invertebrates and algae and thirty-one species
of diatom s from an oil platform 170 km from shore. W hatever the exact figures
are, it is certain that a huge number of species are involved (e.g., Lindner, 1984),
and the num ber of possible species interactions is consequently enormous.
1. Steps in the Formation of a Fouling Community
a. Microbial Fouling
W hen a fresh, inert surface is placed in the sea (or a similar aquatic system,
e.g, Paerl, 1980; McGuire, 1989), it immediately begins to collect dissolved organ
ic m aterial, which seem s to be adsorbed to the surface for energetic reasons
(W ahl, 1989). The adsorbable m aterial includes proteins (M cG uire, 1989) and
D N A (Cooksey, 1987); the "conditioning film" (e.g., Stotzky, 1985) that results
from adsorption of these m olecules can greatly alter the physical and chemical
characteristics of the underlying surface (Schakenraad et a l, 1989) and conse
quently influence later successional steps (Henschel and Cook, 1990; M arshall,
1985). Adsorbed material is so ubiquitous that it has been observed that there is
probably no such thing as a truly clean surface (C osterton, 1980). The living
organisms arrive in short order; bacteria are usually the first (Henschel and Cook,
1990), generally appearing within an hour of im m ersion (ZoBell, 1939a). The
attachm ent of bacteria has been described as occurring in three steps (Marshall,
1985): first, the bacteria approach the surface, either by drifting or by locomotion.
T here appears to be a practical limit to how close the bacteria can approach: a
bacterium , with its generally negative charge and acidic pH , would probably not
actually come in contact with a surface (Wicken, 1985), but would instead hover
slightly above it at a distance determ ined by the net effect of the attractive and
repulsive forces (Daniels, 1980) to which it is subjected by the surface. Reversible
5
sorption or tem porary adhesion, the second step, seems to follow this close appo
sition (M arshall and Bitton, 1980), although the actual mechanism of adsorption
has had many explanations (Daniels, 1980). An adsorbed bacterium may detach
itself ("desorb") and move away or it may proceed to the third step, that of perm a
nent ("irreversible") attachment, or adhesion (Marshall, 1985).
Perm anent attachm ent of a bacterium , which occurs shortly after adsorp
tion (Costerton et a l, 1985), appears for the most part to be linked to the produc
tion of polysaccharides (Corpe, 1980), which form a mass around the cell wall and
probably serve as a sort of cem ent betw een the bacterium and the surface (Z o
Bell, 1943; Marshall, 1985), but some bacteria develop a definite holdfast (ZoBell
and Allen, 1933). Irreversible attachm ent of bacteria is a tim e-dependent process,
and has been reported to take two to four hours (ZoBell and Allen, 1933, 1935);
perhaps time is needed to allow synthesis and secretion of the polymeric adhesives
(Corpe, 1980). Adhesion may be nonspecific or specific; som e bacteria need
particular surfaces on which to adhere, while others can adhere to many types of
surfaces (M arshall and Bitton, 1980). There are even degrees of the perm anence
of adhesion: some bacteria cement themselves in place with capsules, slime layers,
microfibrils, and fimbriae, while others, the "gliding bacteria", can move across the
surface (M arshall and Bitton, 1980). The polymers may allow the bacterium to
"duck" under the critical contact angle of the surface (which is related to its sur
face energy), and thus attach to surfaces that could not otherwise be colonized for
energetic reasons (Rogers, 1979). The adhesion process and rate of primary bio
film form ation are dependent on the physiological state of the bacteria (Marshall,
1985) and, to a lesser extent, on the chemical nature of the surface (Costerton et
a l, 1985). Even killed bacteria have been reported to attach, but this may be an
artifact of the killing process (Marshall, 1985).
6
As the attached bacteria grow and proliferate, they produce more extracel
lular polysaccharides and other products. Because they tend to divide within the
polysaccharide matrix, most bacteria remain on the surface, forming a microcolo
ny. The m icrocolonies spread over the surface, eventually joining one another
(and any recently-arrived cells) to form the prim ary biofilm (C osterton et a l,
1985). The bacterial pioneers are joined, over time, by other bacteria (including
nonmotile forms), microalgae, and microfungi, which may come to dom inate the
microbial community (Paerl, 1980). All of these microbes comprise and produce
a microbial slime, which constitutes the advanced biofilm. The slime can protect
the microbes and trap and retain nutrients (Corpe, 1980). In time, there seems to
be little difference between biofilms formed on different varieties of hard surfaces
(Costerton et a l, 1985). Succession is clear in many biofilm communities, but the
films rem ain over long periods of time (Costerton et a l, 1985). If nutrient concen
trations fall to critical levels, attached bacteria may be able to free them selves
from the surface biofilm (M arvalin and Am blard, 1989), although in other cases
detachm ent after irreversible attachm ent may result in chemical or physical rup
ture of the attached cells (Daniels, 1980).
Although biofilms have attracted much attention because of their relation
ship to fouling by macroscopic organisms, they have also been subjected to scruti
ny in their own right. Microbial fouling can seriously ham per the effectiveness of
such equipm ent as heat exchangers (Turakhia et a l, 1984; R oe et a l, 1985; La-
lande et a l, 1989), and it has been implicated in connection with such deleterious
effects as em brittlem ent and corrosion of the substrate (Characklis and Cooksey,
1983). Although living organisms may prevent microbial fouling by decoy secre
tion of glycoproteins that competitively inhibit bacterial adsorption by mimicking
the surfaces coated by the glycoproteins (Corpe, 1980), nonliving systems do not
7
have this ability. Many surface treatm ents have been tested in an effort to dis
courage bioattachm ent to many different m aterials (Loeb, 1985), but until and
unless clearly fouling-resistant surfaces are developed for widespread use, removal
of attached bacteria, associated other microbes, and their products must rem ain
an im p o rtan t consideration (B aier, 1980). Sim ple death of the living biofilm
constituents is generally not enough to remove a biofilm from the surface (Coster
ton et a l, 1985), although some m ore active killing agents are effective biofilm
removers. Oxidizing biocides such as chlorine destroy biopolymers and thus effect
cleaning. H eating in the presence of acid can also degrade biofilms, but the re
sulting residue may be no improvement over the biofilm it replaces (Costerton et
a l, 1985). T here are a num ber of m echanical m ethods for biofilm rem oval as
well. Scrubbing and abrasion have been used for years; freeze-and-thaw cycles
can also b reak up biofilm s, although som e scrubbing may still be n eed ed to
remove the residue (Costerton et a l, 1985). One of the problems with mechanical
cleaning is the tendency of polymeric "footprints" to remain after the bacteria that
produced them have been sheared off the surfaces to which they were attached
(Corpe, 1980). W hatever the method employed, a cleaning agent or m ethod must
be able to reduce the surface tensions to approximately 20 to 30 dynes per centi
m eter (0.02 to 0.03 N /m ) to clean bacterially contaminated solids. In the failure
of this condition, rem oval of bacterial residues with solvents or abrasion will be
difficult or im practical. Clearly, developm ent of effective cleaning systems will
require understanding of surface adhesion properties (Baier, 1980).
b. Macrobiotal Fouling
In a period of tim e generally ranging from days to weeks afte r initial
exposure, sedentary macroorganisms appear on the surface. They arrive as larvae
8
(in the case of invertebrates) or spores (in the case of macroalgae). These propa-
gules attach to the surface, or m ore usually the biofilm th ereo n , in a m anner
comparable to that by which the pioneer bacteria attach: they arrive, and usually
rem ain on the site for a short period of tim e w ithout attaching. The larvae of
many invertebrates have been observed to move about on the surface, presumably
testing its suitability for later life (Visscher, 1928). These larvae may move on, or
they may stay and attach them selves to the surface. If they attach, they also
undergo m etam orphosis, eventually becom ing adults. Because many of these
organisms are unable to move once they attach, the importance of proper habitat
selection is extrem e. The ability of the propagules to effect some choice about
their prospective habitats is thus a great advantage; many have been shown to
possess this ability (e.g., the polychaete Lygdamis muratus— Wilson, 1977; the
mussel Mytilus galloprovincialis—Igic, 1988). Although barnacles will attach to all
varieties of objects in the sea (Visscher, 1928, citing Darwin, 1853), given the right
conditions, (with very few exceptions, Visscher, 1927), they still exhibit habitat
selection: individual species of barnacles exhibit far m ore dem anding site selec
tion than does the Class Cirripedia as a whole (Visscher, 1928). Nearly every hard
surface is acceptable to some species of barnacle, but not every species will settle
on any given surface. D espite the ability of invertebrate fouling organisms to
make habitat choices as larvae, some of them make poor choices, sometimes dying
as a result (Holm, 1990). Clearly, the ability to make a choice is not a guarantee
of success.
The order in which macrobiotal propagules arrive and develop into adults
varies widely among places and times; larval settlem ent factors can affect and
even determ ine subsequent community structure and species densities and distri
butions (Wilson, 1990). As a result, species composition of fouling communities
9
shows marked spatial and temporal variation. In the central M editerranean, for
example, the pioneer invertebrates are hydroids, serpulids, and barnacles (Ardiz-
zone et a l, 1989). Rock oysters and tubeworms are the m ost-frequent early re
cruits to PVC plates off Oahu, Hawaii, although bryozoans and algae dom inate
after a year (Bailey-Brock, 1989). Naval mines in Queensland were dominated by
mollusks, although corals, serpulids, bryozoa, and algae were also present (Allen,
1950). B arnacles, m ollusks, and ascidians are the m ajor foulers in T uticorin
H arbor, India (M arutham uthu et a l, 1990). Mussels and oysters tend to dominate
estuarine systems (H obbie, 1988). The horse mussel M odiolus modiolus is the
most im portant sessile organism in the subtidal reaches of the G ulf of M aine
(O jeda and D earborn, 1989), and mussels are the dom inant secondary (later-
arriving) invertebrates in the central M editerranean (A rdizzone et a l, 1989).
M ore locally, the Los A ngeles H arb o r hard-substrate fouling com m unity is
m arked early on by serpulid tubeworms, whose abundance peaks in a few months,
and barnacles, which experience a less drastic decline over tim e (Bergen, 1985).
Mussels, barnacles, and tunicates eventually dominate the fouling community on
floating docks (Crippen and Reish, 1969). Later arrivals must compete for space
and other resources with those organisms already present; many latecom ers settle
directly upon the e arlie r arrivals, while others attem p t to force them aside.
Numerous fouling organisms foul oysters, for example (Arakawa, 1990), and some
species of barnacles are able to undercut or crush other species (Connell, 1961a;
Carefoot, 1977). The competition in a fouling community has the potential to be
very intense; different species of the same functional group may help one another
in com petition with species from other functional groups (W oodin and Jackson,
1979).
10
Once they find their preferred habitat, the strengths with which inverte
brates adhere to their substrates can be quite great. Limpets can adhere to slate
with a force of 1-3 x 10^ newtons per square meter, but they are not truly sessile;
this strength is largely muscular, not adhesive. The tenacity of the sea anem one
Actina equina (which can exert muscular control of attachm ent and detachm ent as
well as chem ical adhesion) has been m easured at 4.6 x 10^ N/m ^. The byssus
pads of mussels can adhere to slate with a force of 5-8.6 x 10^ N/m ^ (Yule and
W alker, 1984a). Semibalanus balanoides temporarily attaches to slate, glass, and
plastics with a force betw een 0.6 and 3.0 x 10^ N/m ^ (Dougherty, 1990). Young
Balanus balanoides can withstand forces of 1.7 x lO^N/m^, while older individu
als endure up to 9.3 x 10^ N/m^ (Yule and W alker, 1984a). O n slate, the same
species has a reported maximum adhesion of 2-3 x 10^ N/m ^ (Yule and Crisp,
1983). Dougherty (1990) reported that Chthamalus fragilis, once detached, can
reattach to polystyrene with a force averaging 1,05 x 10^ N/m ^ and w ithstand
centrifugal forces of up to 1,800 G before detaching. By way of com parison, a
standard atmosphere is 1.01 x lO^N/m^, and commercial adhesives have tensile
strengths on the order of 10^N/m^(Yule and Walker, 1984a).
The inverse relationship between force of adhesion and ease of removal of
fouling organisms is obvious. Interestingly, the force with which an organism
attach es to a surface can depend on a num ber of factors (Y ule and W alker,
1984b). For example, Balanus balanoides adheres more firmly to dark panels than
to light ones. Some proteins, when coating the substrate, can result in greater
adhesive forces, and at least som e p art of the force w ith which a cyprid larva
adheres to a surface is controlled by the cyprid's "willingness" to detach (Yule and
W alker, 1984b). M ore interesting in antifouling, the strength of the adhesive
bond is partly dependent on the nature (com position and roughness) of the sur
11
face to which the organism is attached. Some plastics can depress adhesive
strengths, but even these strengths can be increased by roughening the surface of
the substrate or treating it with a barnacle extract (Yule and W alker, 1984b).
2. Larval Site Selection
As the planktonic larvae of many fouling organisms have the ability to
choose surfaces on which to settle (e.g., Cole and Knight Jones, 1939, 1949; Pawlik
and H adfield, 1990), it becom es a subject of inquiry just w hat factors they can
detect and respond to in making that choice, or what factors influence the amount
and patterns of settlem ent of these larvae. On a broad level, these factors could
be physical or chemical. Physical factors include the roughness of the surface, the
velocity of the adjoining fluid, and the local light intensity (e.g., Bakus, 1988);
chem ical factors would be any molecules that would attract or repel larvae. On
the scale at which quantum mechanics becomes a consideration, the physical and
chemical realms are essentially merged, but at larger scales, the two are distinct.
Clearly, an organism cannot react to a stimulus which it cannot detect, but it may
still be affected, however indirectly, by factors it is unable to detect, such as large-
scale currents.
N um erous physical factors have been reported or theorized to affect the
distribution of fouling invertebrates (e.g., Bakus, 1988, and cites therein). Vissch
er (1927) observed that if barnacles and bryozoans were found on the hulls of
ships, they were found in the seams where the hull plates were joined. This could
be because of a larval preference for these locations or because of b etter post
settlem ent survivorship. Chthamalus anisopoma cyprids tend to settle in small pits
on a hard surface (Raim ondi, 1990), suggesting that there is som e sort of larval
preference for depressions. D epressions may be favored because of the protec
12
tion from shear forces they afford: Mytilus galloprovincialis prefers substrates
protected from stronger currents (Igic, 1988). Substrate texture may also be impor
tant: for example, larvae of Protodrilus rubropharyngeus prefer sand betw een 0.5
and 1.0 mm in size (Gray, 1967b).
Early work indicated that light intensity and color were also im portant in
fouling: light intensity seem s to play a role in the vertical zo n atio n of fouling
organisms on ships, particularly on the most-heavily-fouled ships. The undersides
of ships and darker-colored substrates are m ore heavily fouled with anim als
(Visscher, 1927); barnacle cyprids exhibit negative phototaxis. In the lighted
areas, the topm ost portions of subm erged hulls were m ore heavily fouled, espe
cially by plants (Visscher, 1927), presumably because of the need for light among
algae. W ithin Los Angeles H arbor, the orientation of fixed fouling panels is an
im portant factor in fouling growth (Bergen, 1985). A nother factor is am bient
tem perature, which has been reported to be positvely correlated with diversity and
num ber of species (Brankevich et al., 1988).
The physical aspects of algal mat habitats may be more im portant than the
biological factors; the physical complexity of the community provides many varied
niches as well as protection from wave stress and sim ilar physical factors (D ean
and Connell, 1987b,c). Larval gastropods settle more in seagrass beds than on
nearby open sand, apparently because the currents are w eaker there (W ilson,
1990). The settlem ent of Sargassum near stands of the same species seems simi
larly m ediated: waves wash away those individuals unlucky enough to settle in
wave-swept areas (Toda et al, 1989b).
N um erous chemical compounds have been reported to affect the settle
m ent and m etam orphosis of fouling invertebrate larvae {e.g., K irchm an et a l,
1982b; Bonar et a l, 1985; W einer et a l, 1989). Some of these factors encourage
13
and even induce settlem ent, while others retard or prevent it. As exam ples, ex
tracts of the red algae that will later serve as a food source induce settlem ent of
Haliotis larvae, as does the neurotransm itter gamma-amino-butyric acid (GABA)
and some of its congeners (Morse et a l, 1979). The natural inducer of Crassostrea
gigas settlem ent may be L-Dopa or a mimetic molecule (Fitt et a l, 1989). On the
negative side, dead organic m atter on sand can repel Ophelia bicornis larvae
(W ilson, 1955), ^nd Mytilus galloprovincialis settlem ent is reduced by toxic coat
ings, especially those for wood (Igic, 1988). Mild carbon steel exerts a negative
effect on the development of a fouling community: foulers settle on it m ore slowly
and cover less area than they do on black Perspex (Schmidt and W arner, 1989).
S ettlem en t rates of barnacles and bryozoans are inversely co rre lated
(R ittschof and Costlow, 1989); their surface-energy preferences are opposite
(R oberts et a l, m anuscript). Settling cyprids avoid substrates which a predator
occupied earlier; the repulsion seems to be chemical. Prior occupation of a sur
face could change it by removal of substances, alteration of structures, or addition
of m aterials (Johnson and Strathmann, 1989). It is im portant to bear in mind that
the processes of settlem ent on a substrate and metamorphosis from larva to adult
are different, and may well be induced by different factors (W einer et a l, 1989).
These discussions of the relative importance of the different chemical and
physical factors in the settlem ent of fouling larvae are germ ane to the m icrobial
fouling community. The relationship betw een the developm ent (and even exist
ence) of the microbial fouling community and the subsequent development of the
invertebrate fouling community has been researched for over fifty years (Angst,
1923; ZoB ell and Allen, 1935), but many of the issues and questions raised have
not been resolved. It has long been known that the m icrobial comm unity aug
ments the attractiveness of a surface to such algae as Enteromorpha (Dillon et a l ,
14
1989) and such larval invertebrates as Bugula (M iller et a l, 1948), oysters
(H enschel and Cook, 1990; Ostrea edulis— Cole and Knight Jones, 1939, 1949;
Crassostrea gigas and C. virginica— W einer et a l, 1989), polychaetes {Ophelia
bicornis— W ilson, 1955; Lygdamis m uratus— W ilson, 1977; Janua brasiliensis
— K irchm an et al., 1982a; Spirorbis borealis— M eadow s and W illiam s, 1963),
barnacles (Knight-Jones and Crisp, 1953), and in general (ZoBell, 1939a; Wood,
1950; D aniel, 1955; Crisp and Ryland, 1960; Corpe, 1970b; M itchell and Young,
1972; Crisp, 1974), although the degree of the increase in attractiveness differs
among invertebrates (H enschel and Cook, 1990). W hat is not known is w hat
about the microbial community is so attractive to the larvae. Films of some bacte
ria are m ore attractive than others (generally— M eadows and W illiams, 1963;
oysters— W einer et a l, 1989; annelids—Gray, 1966, 1967a), which indicates that
the attraction may have an element of specificity.
The attractiveness of bacterial films is m anifested in increased rates of
attachm ent of many fouling organisms. D irect observation of still-planktonic
larvae has shown that biofilms can influence settlem ent behavior of many inverte
brates, such as bryozoans (M iller et a l, 1948; M aki et a l, 1989), serpulid tube
worms (Knight-Jones, 1951), and barnacles (Angst, 1923; Bergen, 1985). Most of
the fouling larvae tested show a m arked preference for surfaces covered with a
healthy m icrobial community. Bugula larvae have a strong preference for film-
covered painted surfaces, regardless of the com position of the underlying paint;
sim ilar results occur with ground glass (M iller et a l, 1948). Larvae of Spirorbis
borealis show a g reat preference for film ed glass, and this p referen ce has an
im p o rtan t co rrelatio n with m etam orphosis (K night-Jones, 1951). T he m ost
im portant factor in the settlement of Ophelia bicornis larvae on sand grains is the
presence of living organisms (Wilson, 1955), and the interstitial annelid Lygdamis
15
muratus prefers "non-sterile" surfaces (Wilson, 1977). Interestingly, the presence
of too much m icrobial growth can be as repellent as not enough (W ilson, 1955;
Bergen, 1985).
The question remains: is the microbial community's attractiveness to larval
invertebrates the result of chemical or physical factors? T he pioneer organisms
may chemically alter the new surface and thus increase its attractiveness to later-
stage organisms. On the other hand, the pioneers may merely roughen or other
wise alter the surface's microscopic topology and m ake it m ore attractive in a
purely physical sense. Moreover, both forms of attraction may exist at once.
E vidence for the influence of chem ical factors is both th eo retical and
em pirical {e.g., W ilson, 1970; M orse et al, 1979; Bonar et a l, 1985; Bakus et a l,
1986; W einer et a l, 1989; Pawlik and Hadfield, 1990; Morse, 1990; Henschel and
Cook, 1990; R oberts et a l, m anuscript). The settlem ent o i Janua brasiliensis on
bacterially-film ed surfaces appears to be m ediated by lectins (K irchm an et a l,
1982b). The settlem ent of oysters is not only induced by compounds produced by
bacteria, but the production of these com pounds varies over tim e (F itt et a l,
1989). T heoretical considerations include concentration or production of nutri
ents by the m icrobes and short-circuiting of the invertebrates' neurochem ical
pathways, with settlem ent the result.
Em pirical evidence for the im portance of physical characteristics of the
m icrobial slime upon the settlem ent of invertebrates is less abundant. Knight-
Jones (1951) deem ed it likely that Spirorbis larvae settle on film ed surfaces be
cause they can crawl upon these surfaces because of the surfaces' stickiness—the
larvae could only leave with some difficulty. The correlation betw een invertebrate
abundance and physical depressions is high, especially if the overall population
density is not so great that the substrate is completely covered {e.g. Visscher,
16
1927). This "preference" for physical depressions may simply be a result of eddy
ing in a current: the larvae collect passively in hollows (Dr. David W ethey, pers.
comm.).
Despite all of the evidence for larval preference for microfouled surfaces,
there is little evidence directly pertaining to w hether or not the presence of the
microbial community is actually required for invertebrate settlem ent. ZoBell and
A llen (1935) pointed out that it had not been proved that fouling invertebrates
will not attach without a microbial film and this remains true today (Little, 1984).
A lthough doubts have been raised about some organisms {e.g., Bugula, M iller et
a l, 1948, and barnacles, Little, 1984) and these doubts have been extrapolated to
the fouling community as a whole (M iller et a l, 1948; Little, 1984), the need or
lack thereof for microbial fouling as a general prerequisite to invertebrate fouling
still has not been resolved (Bakus et a l, 1986). To the contrary, the general
assum ption, frequently u nstated, seem s to be that a m icrobial com m unity is
generally needed for norm al fouling developm ent (see, e.g.. Crisp and Ryland,
1960). This assum ption appears to arise from the observation, dating back to
ZoBell and Allen (1935), that the microbial community almost invariably appears
first in nature. Some later work supported this assumption: Knight-Jones (1951)
reported that larvae encountering a clean glass surface would generally swim off
immediately and would settle only after a significant delay, which might be neces
sary and sufficient for a microfouling community to develop. Furtherm ore, a large
proportion of larvae kept in clean beakers developed abnormally, although little
supporting follow-up work appears in the literature.
17
c. Models and Mechanisms of Succession
Succession, the replacement of species in a community over time, has been
studied for decades on land (Krebs, 1985), but only recently has m uch attention
been paid to the idea that fouling communities may also display succession. As a
result, the established successional m odels may exhibit a bias tow ard terrestrial
communities and factors. For example, terrestrial communities are dom inated by
plants (which are the large sessile organisms), and the successional m odels de
rived from observations of and experim entation with these com m unities tend to
emphasize factors affecting plants. Benthic marine communities, while sometimes
hospitable to significant or even luxuriant plant growth, are often dom inated by
sessile animals, which play many of the roles of plants (such as being the physical
ly dom inant organisms, Connell and Slayter, 1977), but are affected by a different
set of factors. Applications of terrestrially-derived successional m odels to the
m arine benthos without adjustment may yield confusing or even contrary results.
The earliest workable successional model grew out of the work of W arming
(1896) and Cowles (1899, 1901), and was summed up in modified form by Clem
ents (1916, 1936). Clem ents' interpretation was so satisfying to ecologists (e.g.,
Odum, 1969) that it has rem ained the dom inant m odel (Connell and Slayter,
1977). The m odel postulates that later-stage species are unable to colonize an
area in an earlier stage of succession because the factors or conditions necessary
for the later-stage species are not present. The earlier-stage species alter the
factors present in the earlier stages, thus allowing the later-stage species to colo
nize and eventually exclude the earlier-stage species. For this reason, this model
has been term ed the "facilitation" model (Connell and Slayter, 1977). A hallmark
of the facilitation model is that it is impossible for a later-stage species to colonize
an area too early in the successional process; such species m ust w ait until the
18
earlier-stage organisms sufficiently alter the habitat. There is therefore an order
ly, even hierarchical system of change (Krebs, 1985). Additionally, Clem ents
predicted that a community would, in the absence of disturbance, invariably tend
toward a single climax community (the "monoclimax hypothesis"; see also Krebs,
1985).
The second major succession theory was propounded by Egler (1954), who
postulated that succession was a scramble; the first species to colonize an area
would hold it against all comers for the life of the pioneer organisms, but any
species can colonize at any time. A bare area would thus tend to be dom inated at
first by organisms adapted for quick dispersal and reproduction, but over time the
longevous organisms would, simply by virtue of their longevity, eventually come to
dom inate the area. The basic tenet of this m odel is that once established, an
organism cannot be dislodged during its lifetime; its simple presence is sufficient
to thw art other colonists. This "inhibition" model (C onnell and Slayter, 1977)
holds simply that the race is to the swift (Krebs, 1985) and that "possession is
eleven parts of the law" (Connell and Slayter, 1977, citing C ibber, 1777). The
organism s typical to the climax community may appear in the earliest stages,
although th eir slow er grow th rates, less-frequent rep ro d u ctio n , and perh ap s
poorer dispersive abilities will make more than an occasional pioneer appearance
quite unlikely. Species replacem ent is not entirely orderly.
Interm ediate betw een these two models (Krebs, 1985) is the third m ajor
successional model, that of Connell and Slayter (1977). This model holds not only
that any species can colonize at any time, but that some species can actively dis
place others. The com petition is thus not for speed, but for ability to displace
earlier arrivals and resist displacem ent by later arrivals; the battle is thus to the
strong. Connell and Slayter applied the term "tolerance" to their model. O rgan
19
isms typical of later successional stages can be the first to arrive; if they arrive
later, they must displace those organisms already present. T he new arrivals will
survive until and unless they are displaced by organisms that are better competi
tors (and thus typical to a later stage). T here is thus no set order to the species
and communities, and some species may not appear at all in a given area (Krebs,
1985).
Research in marine fouling communities has not shown a clear pattern of
succession. Indeed, even the patterns of replacem ent are in dispute. N um erous
studies have shown that replacement of species is orderly; num erous other studies
have shown that it is disorderly (Bakus et a l, 1986). The idea of a single climax
community has likewise come under fire in recent years (Osman, 1977; Sutherland
and Karlson, 1977). Even the definition of succession has been subject to revi
sionary pressures. O dum (1969) argues that succession is (1) an orderly, even
d irectional, process of com m unity developm ent through p red ictab le, orderly
change (2) resulting from modification of the physical environm ent and (3) culmi
nating in a stable ecosystem with high biomass. Bergen (1985), on the other hand,
uses the term to m ean a change in species over time, with no assum ption made as
to the orderliness of the change or the mechanisms that brought it about. Huston
and Sm ith (1987) define succession as a sequential change in the relative abun
dance or abundances of the dominant species in a community; they further define
dom inant species in terms of biomass.
T here is a large body of evidence supporting one or another of these suc
cessional models (Bakus et a l, 1986), but detailed consideration of it all is beyond
the scope of this discussion. Connell and Slayter (1977) argue that if invertebrate
larvae prefer fouling plates already covered by a film of m icro-organism s, this
could be evidence of classical succession. Mytilus is rarely a pioneer; it may be an
2 0
example of a player in a classical succession scheme. The cause-and-effect expla
nation for the sequence of fouling community developm ent has been advanced
many times (Wahl, 1989); the facilitation model is sometimes taken to be the only
m odel {e.g.. Little, 1984), but sometimes the older models are overlooked (Huston
and Smith, 1987). The inhibition model, on the other hand, is likely supported by
the findings of Sousa (1979a), who reported that some algal colonists tend to
inhibit their replacements, which succeed despite the pioneers; grazing m ade the
difference because the grazers preferentially fed upon the pioneers, leaving the
later-stage algae alone. Sutherland and Karlson (1977) also m ake specific argu
m ents for inhibition. Krebs (1985) believes that the rocky intertidal as a whole is
a good example of inhibition-model succession. The tolerance model has had less
em pirical support, but this may be because of its com parative newness. Bergen
(1985) reports no apparent links between different species of fouling organisms in
Los Angeles Harbor, which would tend to support the tolerance model. She also
points out that inhibition seems to be a more prevalent mechanism of succession
than facilitation.
For all of the parallels, the development of the marine fouling community
(under natural conditions) does not strictly conform to Odum 's (1969) narrow
definition of terrestrial succession (Little, 1984). The third requirem ent (a final,
stable ecosystem ) is generally met, but the orderliness and predictability of the
first requirem ent (species changes) vary, calling into question the nature of the
processes driving the com m unity changes and thus the validity of the second
requirem ent (modification of the environment by the earlier arrivals) as well.
In light of all of the evidence so far discovered, it seem s th a t all three
m echanism s may operate in m arine com m unities (Bergen 1985). Indeed, m ore
than one mechanism of succession may be present in the same fouling community
21
(Bergen, 1985; Bakus et a l, 1986); Turner ( 1983a,b) even reported facilitation and
inhibition in the sam e sequence. Furtherm ore, some patterns of m arine succes
sion do not appear to follow any theoretical m odels (D ean and Connell, 1987a);
m ore m odeling may be necessary. The m ajor m odels do not account for the ef
fects of predation or grazing, perhaps because so little is known about the effects
of predators on succession (Connell and Slayter, 1977); it is clear that these effects
can greatly affect succession, at least in the fouling comm unity (Sousa, 1979a;
Bergen 1985). There is still no general theory encompassing the processes found
in succession (Huston and Smith, 1987).
Clearly, m arine succession may differ fundam entally from terrestrial suc
cession. N ot only are the dom inant organism s often not plants, as discussed
previously, but it is possible that the higher rates of m atter exchange and absence
of hum us buildup on wave-swept rocks are differences of too fu n d am en tal a
nature to allow meaningful comparison (Deans and Connell, 1987a). Sutherland
and K arlson (1977) m aintain that fouling invertebrates do not alter their sub
strate, have no way to store propagules (seeds, in terrestrial succession), and have
adults th at are usually too short-lived to allow classical succession. L ittle (1984)
entirely rejects terrestrial succession as a m odel for m icrobial fouling, but only
considers the facilitation m odel in so doing. O f perhaps greater concern is the
concept of a single, stable climax community. Some studies have found no stable
endpoint to succession (Bergen, 1985), although the stability of a fouling commu
nity certainly depends on the time scale used to judge it and is not a simple yes-no
proposition, but instead a relative idea (Sutherland, 1981). Patch size may be of
greater im portance than commonly assum ed, as well (Farrell, 1989). Bergen
(1985) was unable to determ ine the stability (or lack th ereo f) of th e fouling
community of Los Angeles Harbor.
2 2
D. Patchiness and Gregariousness
The distribution of fouling organisms is quite patchy, and community struc
ture is extrem ely variable (R oberts et a l, m anuscript, citing Sutherland, 1978).
The am ount of fouling is rarely even over a ship's entire hull; even replicate foul
ing test panels show differences in species composition (Schoener, 1984; Bergen,
1985). Patchy distrib u tio n could result from selective h a b itat selection by or
patchy distribution of the planktonic larvae, which are largely at the mercy of
currents and internal waves, which can deposit the larvae along a shoreline in
differing am ounts (Shanks and W right, 1987). A lternately, patchy distribution
may arise from irregular survivorship among settled organism s {e.g., Connell,
1961a). Biotic or abiotic factors could enhance survival and growth at one site
and kill organisms settling at another; uneven distribution would result. (See also
Young, 1990.)
A num ber of fouling organism s exhibit gregarious settlem en t p a ttern s
(Gotelli, 1990), including barnacles, tubeworms, and oysters (Knight-Jones, 1951).
Some of these organisms are attracted to others of the same species, while others
are simply attracted to the same factors that seem to have attracted their p red e
cessors; in either case, the result is clumped or patchy distribution. Larvae of the
polychaete Sabellaria spinulosa settle on newly-settled conspecifics, although
adults are almost always found alone (Wilson, 1970); Sargassum propagules settle
near stands of Sargassum (Toda et a l, 1989b); and the scyphozoan aurita
also shows gregarious settlem ent behavior (G rondahl, 1989). Spirorbis borealis
larvae not only tend to settle near S. borealis adults, but they prefer surfaces bear
ing m ore adults to those bearing less, as long as the surface is not too crowded
(Knight-Jones, 1951); Bugula stolonifera shows a distinct bimodal density effect in
23
choosing a settlem ent site, preferring both regions of high and low density of
extant colonies (Patzkowsky, 1988). Larvae of the abalone Haliotis rufescens
p refe r to settle on crustose red algae, which are the p re fe rre d food of post-
m etam orphic forms. (Morse et a l, 1979). Gregarious settlem ent in the interstitial
annelid Protodrilus rubropharyngeus seems to be related to both a bacterial film
and an ad u lt chem ical product (G ray, 1967b). T he reef-building tubew orm
Phragmatopoma lapidosa californica responds to chem ical cues in a m anner af
fected by physical factors (Pawlik et a l, 1991).
The theoretical advantages of gregariousness are basic: clumped distribu
tion assures the presence of potential mates, and the presence of adults in a cer
tain area is a good indication that conditions prevalent in that area are suitable
for that species. Both of these considerations take on added im portance to sessile
organisms, which cannot actively search for m ates or b etter living conditions.
Some empirical evidence supports this idea: early survival of benthic invertebrates
is enhanced at g reater densities of settlers (M cG uinness and D avis, 1989).
N um erous chem ical com pounds and mixtures have been discovered to induce
settlem ent in invertebrate larvae (see above); the specificity of chem ical-receptor
induction of settling on key species indicates long-term adaptive co-evolution
(M orse et a l, 1979), which would be expected if an advantage is gained by such
activity.
D espite the presum ed advantages of gregarious settlem ent, som e fouling
larvae do not show any preference for adults of their own species. Larval Bugula
neritina do not seem to respond to the presence of juveniles or adults, although
colonies are still strongly clum ped, possibly because of poor dispersal or better
post-settlem ent survivorship (Keough, 1989). Bugula turrita also shows no prefer
ence for high colony density in settlem ent (Patzkowsky, 1988). Failure of larvae
24
to choose settlem ent sites near other m em bers of their species does not rule out
the possibility that adult distribution will be clum ped; patchy distribution may
arise from individuals' independent selection (Schoener, 1984) of advantageous
sites or increased survival in certain areas as well as poor dispersal.
E. Research Objectives
The purposes of this research project span the spectrum of fouling and
antifouling. A num ber of tests were contem plated. A central objective was to
determ ine whether or not the pioneer bacteria are truly necessary for the norm al
developm ent of the rest of the fouling community, in concentration of bacteria
and other microbes, and also in amount of macrofoulers eventually present. The
reverse of this objective is to answer the questions, does antibiosis result in anti
fouling, and might natural antibiotics also be natural antifoulants? A related and
subsidiary goal was to determ ine which successional m odel or m odels, if any,
apply to the early invertebrate fouling community. An additional goal was to
determ ine just what it is about microbes in general and bacteria in particular that
m akes them attractive to fouling invertebrates. A final objective was to develop
practical methods for rapid counting and quantifying of fouling organisms, using
computer-driven image analysis techniques.
My w ork co ncentrated on the polychaete tubew orm s of Fish H a rb o r
(Latitude 33° 44' North, Longitude 118° 16' West), a small branch of Los Angeles
H arbor extending into Terminal Island (Figure 1), and the site of a University of
S outhern C alifornia m arine laboratory. A lthough Los A ngeles-L ong B each
H arbor is one of the busiest in the world. Fish H arbor is relatively calm.
25
Figure 1. The location of Fish Harbor, site of most experiments.
Each of the inset m aps shows the location of the next-larger-scale map.
U pper left: California; upper right: the Los A ngeles area; bottom : Los
Angeles and Long Beach Harbors; Fish H arbor is marked. N orth is at the
top of all three maps.
26
LONG
BEACH
WILMINGTON
Fish Harbor
SAN PCDRO
This study was largely concerned with annelid worms of the family Serpuli-
dae, which build and inhabit calcareous tubes (Figure 2) on solid surfaces in the
subtidal region. Numerous species of serpulids inhabit Los Angeles-Long Beach
H arbor; some of the most im portant are Janua brasiliensis^ Pileolaria pseudomilita-
ris, P. marginata, and Protolaeospira ?eximia {Janua— Shisko, 1975; P. pseudomili-
taris— Beckwitt, 1979; P. marginata and P. ?eximia—Leslie Harris, pers. comm.).
In this harbor, the early invertebrate fouling community is marked, indeed dom i
nated by serpulid tubeworm s (Bergen, 1985), which eventually give way to m us
sels, barnacles, and tunicates as the community m atures (C rippen and Reish,
1969). The Serpulidae have been subject to considerable taxonom ic revision,
including the proposal of a new, separate family, the Spirorbidae (Pillai, 1970, in
Shisko, 1975), and much taxonomic confusion rem ains {e.g., Shisko, 1975; Beck
witt, 1979). The taxonomy of serpulid tubeworm s is beyond the range of this
work, and thus no position will be taken on any aspect thereof: all of these species
will be known as tubeworm s, and assum ed for the sake of simplicity to be in the
family Serpulidae.
1. Antibiosis
The im petus for much of this work was the idea that an antibiotic, m ost
likely a n a tu ra l com pound, could be used as an antifouling p re p a ra tio n , th at
d isru p tio n or prev en tio n of the form ation of the m icrobial com m unity could
prevent macro-fouling as a secondary result. The first step in the execution of this
idea was to find a natural antibiotic by collecting likely m arine organism s and
testing their extracts for antibiotic activity. The second step would be to test any
antibiotic so discovered for antifouling properties. For practical reasons, I decid-
28
F igure 2. Scanning electro n m icrograph of the calcareous tu b e of a serpulid
tubeworm attached to a microscope slide.
Note the abundance of smaller organisms also attached to the substrate.
29
ed to test a commercial antibiotic first, progressing to a natural antibiotic once the
antifouling techniques were perfected.
A ntibiotics and antibiotic effects have been reported from such diverse
sources as leaf extracts (Tewari et a l, 1988; Pandey et a l, 1989; Pathak et a l,
1989); tea and coffee (Toda et a l, 1989a); herbs such as sum m er savory, Satureja
hortensis (D eans and Svoboda, 1989), and the carpetw eed Mollugo pentaphylla
(H am burger et a l, 1989); pond algae (Pratt et a l, 1944; Levina, 1961; Cannell et
a l, 1988) and num erous higher plants (M itscher et a l, 1972); bacteria collected
from elm xylem (Schreiber, 1988) and chicken crests (K ellner et a l, 1988); olive
mill wastewater (Rodriguez et a l, 1988); trout (Austin and McIntosh, 1988); and
even some artificial sponges (Llabres and Rose, 1989).
M arine organisms (and even, to a lim ited extent, seaw ater itself, ZoB ell
and Feltham , 1934; ZoBell, 1936; Sieburth and Pratt, 1962) have also attracted
attention as sources of antibiotics {e.g, Okami, 1986), which have been discovered
in algae (Burkholder et a l, 1960; Sieburth, 1964), both free-living (Cannell et a l,
1988; Kellam et a l, 1988; Kellam and Walker, 1989) and endosymbiotic (Cieresz-
ko, 1962); phytoplankton (Steem ann Nielsen, 1955a,b; Sieburth, 1959; Sieburth
and Pratt, 1962); seaweeds (Pratt et a l, 1951; Chesters and Stott, 1956); seagrasses
(Bernard and Pesando, 1989); sponges (Jakowska and Nigrelli, 1960; Burkholder
and Ruetzler, 1969; Bergquist and Bedford, 1978; Am ade et a l, 1982); gorgonian
corals (B urkholder and Burkholder, 1958; Ciereszko et a l, 1960); abalone (Li,
1960a,b); oysters (Li et a l, 1962); sea hares (Kamiya et a l, 1988); and sea cucum
bers (N igrelli and Jakowska, 1960), am ong other organism s (see also Nigrelli,
1958, 1962). Antibiotic substances discovered in the course of other research may
prove of interest to pharm aceutical researchers as well (see R inehart et a l, 1981).
30
T he existence and use of n atu ral an tib acterial substances can provide
ecological insights. C om petitive advantages gained from the elim in atio n of
com petitors or confounding their metabolic processes can completely change the
m akeup of a community and effect or prevent successional changes (see, e.g.,
Nigrelli, 1962). Specifically, the possible relationship between natural antibiosis
and natural antifouling may be elucidated as a by-product of the discovery of
natural antibiotics produced by m arine organisms, or vice versa. The first step in
this work was to collect m arine invertebrates that might contain antibiotic and
antifouling compounds (Wright, 1991; Wright, in prep.).
2. Ampicillin
I also undertook to test the hypothesis that an obligatory relationship exists
betw een micro-foulers and macro-foulers, and that prevention of the form ation of
a microbial fouling community, or of one key stage therein—perhaps even disrup
tion of the pioneer bacterial comm unity— can result in the prevention of subse
quent fouling by macro-organisms. The plan was to use any antibiotics discovered
in m arine invertebrates to kill (or perhaps repel) any bacteria that may be present
and growing on (and thus presumptively attached to) a new surface in the marine
environm ent (see Henschel and Cook, 1990). The formation, if any, of the subse
quent fouling community could be m onitored in one of a num ber of different ways
(e.g., biomass increase, thickness) and the resulting community could be watched
for signs of abnorm al developm ent (e.g., unusual dom inant species or species
order of appearance or dom inance). Because so little of the antibiotic extracts
rem ained after antibiosis testing, the commercial antibiotic ampicillin was used in
these experiments instead. The first series of ampicillin experiments was under
taken in the w inter of 1989-90. The slides in the w inter series took so long to
31
develop fouling com m unities that the results might have been skewed by such
factors as loss of paint from the slide surfaces. A second set of experim ents was
perform ed in the spring of 1990, when fouling rates were higher.
3. Settlement of Larval Tubeworms
The field experiments did not consider the direct effect, if any, of ampicil
lin on the Fish H arbor tubeworms. The possibility rem ained that the field results
m ight be m ore reflective of the result of the larval worm s' reactio n s to the
presence of am picillin (and perhaps the release paint) than to the absence of
bacteria. As a check on the field experiments, an additional experiment involved
m aintaining adult tubeworms in the laboratory and exposing their larval offspring
to these substances under close observation. The additional factors present in the
field, which might confuse the results of field experim ents, were thus avoided as
much as possible, leaving as experimental factors the susceptibility or lack thereof
of the Fish H arbor tubeworms to ampicillin and the release paint.
4. Separation of Physical and Chemical Factors
T his w ork included an attem p t to sep arate the physical and chem ical
changes wrought upon a surface by the microbial fouling organisms and to deter
mine which, if either, of the two sets of factors is more im portant in the attraction
of other fouling organisms, especially the tubeworms of Fish H arbor. The answer
could have significance in the developm ent of surface treatm ents to discourage
and retard fouling, such as antifouling paints. Additionally, it could provide in
sights into the mechanisms of site selection for planktonic larvae of fouling organ
isms. F urtherm ore, it might also yield clues into the nature of the processes of
community change that occur in a fouling community and identify which, if any, of
32
the popular m odels of succession is most applicable to the Fish H arbor fouling
community.
5. Image Analysis
O ne of the g reatest obstacles to antifouling research is the enorm ous
am ount of time required to quantify fouling by counting the fouling organisms or
m easuring the area covered by them: manually censusing the organisms on a 20 x
20 cm fouling panel can take six to eight hours per panel side (Bakus et a l, 1990).
W hen a full-scale research program envisions using large num bers of experimen
tal treatm ents, sim ple m ultiplication is sufficient to show th at a huge am ount of
tim e and effort will be required to provide the num bers needed for scientific
confidence; the result is a significant research bottleneck. O ne solution to the
problem is to use computer-based image processing and image analysis to perform
the quantification automatically (Hàder, 1988). An autom ated image-processing
(C roft and Jafek, 1988; Ravich, 1987) and -analysis system, should one prove
practical, w ould possess several advantages over m anual q u an tificatio n {e.g.,
V erran et a l, 1980). First and foremost, such a system would be fast (Shakespeare
and V erran, 1988), particularly if it is equipped with parallel processors. F u r
therm ore, com puter counting may be m ore consistently accurate than m anual
counting, as errors arising from fatigue, eyestrain, and boredom would be elim i
nated {e.g., Sieracki et a l, 1989). Crucial to this project was the developm ent of
such a counting and measuring system as part of a m icrocomputer.
The problems raised here comprise some of the questions at the center of
fouling and antifouling research. The work presented herein addresses som e of
these questions and issues.
33
II. Materials and Methods
A. Antibiosis
Benthic invertebrates (sponges, gorgonians, and soft corals) were collected
from coral reef habitats in the Fiji Archipelago. M ost of the collections w ere
m ade in B eqa (M bengga)* L agoon (L atitu d e 18° 20' South, L ongitude 178°
O ' E ast) and L aucala (L authala) Bay (18° 10' S, 178° 25' E); the rest w ere m ade
near these sites (Table 1). Preferred specimens were free of obvious large exter
nal fouling growth; clean-surfaced m arine sponges exhibit g reater antim icrobial
activity than do those with fouled surfaces (W alker et a l, 1985), and fouling-free
organism s are logically m ore likely to have greater antifouling capability. The
specimens were preserved in m ethanol (occasionally after freezing) and returned
to the laboratory for extraction and identification. Extracts w ere p rep ared by
soaking the collected organisms in fresh m ethanol for two or m ore days. The
m ethanol was poured off and retained and the specim en soaked in additional
fresh methanol. On occasion, the specimen was later soaked in dichlorom ethane
(C H 2CI2 ), a non-polar solvent, to ensure the extraction of all soluble organic
compounds (Dr. P. Crews, personal communication).
As the extracts accumulated, their antibiotic capabilities and characteristics
were tested in a standard m anner (a modified form of that in U.S. Food and Drug
Adm inistration, 1979): petri dish disk assays, which are generally reliable indica
tors of antim icrobial activity (R inehart, 1988). In light of the potentially enor
mous num ber of disks required for broad-scale dose-response testing, I decided to
* The Fijian language, while using the Rom an alphabet, assigns different sounds
to some of the letters. In this dissertation, where a difference exists betw een the
Fijian and phonetic English spellings of a place name, the Fijian spelling (used on
m ost land maps) appears first, with the English spelling (used on British Adm iral
ty charts, the best available for Fijian waters) appearing after it in parentheses.
34
Table 1. Organisms used in the antibiosis experiments.
R elevant collection data are included. The Fijian spellings of place names
appear first, followed by the phonetic English spellings, where different, in paren
theses.
35
Species Name
and Collection Code
Organism Collection
Type D ate Collection Location
[unidentified] sponge
NlKM-87-00
[unidentified] sponge
MKM-87-02
Dendronephthya sp. soft coral
MKM-87-07
[unidentified] sponge
MKM-87-09
Dendronephthya cf. minima soft coral
MKM-87-10
Subergorgia mollis gorgonian
MKM-87-12
[unidentified] sponge
MKM-87-14
[unidentified] sponge
MKM-87-15
Astrogorgia sp. gorgonian
MTW-87-01
Anthogorgia sp. gorgonian
MTW-87-02
[unidentified] sponge
MTW-87-04
[unidentified] sponge
MTW-87-06
[unidentified] sponge
MTW-87-12
Siphonogorgia sp. gorgonian
MTW-87-17
Fasciospongia sp. sponge
MTW-87-20
[unidentified] sponge
MTW-87-21
[unidentified] sponge
MTW-87-25
[unidentified] sponge
MTW-87-26
[unidentified] sponge
MTW-87-30
[unidentified] sponge
MTW-87-32
[unidentified] sponge
MTW-87-37
[unidentified] sponge
MTW-87-40
8 July 1987
8 July 1987
9 July 1987
9 July 1987
9 July 1987
11 July 1987
11 July 1987
11 July 1987
25 June 1987
25 June 1987
28 June 1987
28 June 1987
10 July 1987
10 July 1987
5 August 1987
5 August 1987
7 August 1987
7 August 1987
7 August 1987
7 August 1987
7 August 1987
7 August 1987
Yanuca (Y anutha) I., Beqa
(Mbengga) Lagoon
Y anuca (Y anutha) I., Beqa
(M bengga) Lagoon
Y anuca (Y anutha) I., Beqa
(M bengga) Lagoon
Yanuca (Y anutha) I., Beqa
(M bengga) Lagoon
Yanuca (Y anutha) I., Beqa
(M bengga) Lagoon
Beqa (M bengga) Lagoon
"Side Streets"
Beqa (M bengga) Lagoon
"Side Streets"
Beqa (M bengga) Lagoon
"Side Streets"
Suva H arbor
Suva H arbor
Makuluva I., Laucala
(Lauthala) Bay
M akuluva I., Laucala
(Lauthala) Bay
Yanuca (Y anutha) I., Beqa
(M bengga) Lagoon
Yanuca (Y anutha) I., Beqa
(M bengga) Lagoon
M akuluva I., iJaucala
(Lauthala) Bay
M akuluva I., Laucala
(Lauthala) Bay
M akuluva I., la u c a la
(Lauthala) Bay
M akuluva I., Laucala
(Lauthala) Bay
M akuluva I., Laucala
(Lauthala) Bay
M akuluva I., Laucala
(Lauthala) Bay
M akuluva L, la u c a la
(Lauthala) Bay
M akuluva I., la u c a la
(Lauthala) Bay
36
concentrate on high (strong) doses, and later to concentrate on sm aller doses of
promising extracts.
The first-run doses of the extracts were prepared in the following manner:
concentrated methanol solutions of the extracts were applied to 7-mm (V^") test
ing disks (BBL Laboratories, Cockeysville, Maryland). In most cases, ten drops of
the concentrated solution were applied over a length of tim e sufficient to allow
the disks to dry m oderately to com pletely betw een applications. Some extracts
w ere so thick that they clearly saturated the disks, frequently m ounding up over
their surfaces, after fewer than ten drops were applied. In such cases, the disks
were deem ed to be saturated with less than ten drops. One or two extracts were
so pasty that the extract paste was applied to the top of the disks, and m ethanol
was dripped over them to impregnate the disks. The disks were allowed to dry for
some time, but even after several days, some remained moist and sticky.
B atches of D ifco 2216 m arine agar (Z oB ell, 1941) w ere p re p a re d and
poured into petri dishes, 20 ml per dish. These were wrapped, pasteurized (three
one-hour exposures to 60° C at twenty-four-hour intervals), and stored under
refrigeration until used. Flasks of Difco 2216 marine broth were inoculated with
the c u ltu re s to be used and in c u b a te d w ith m o d e ra te a g ita tio n (125-150
cycles/sec) overnight. Bacterial culture samples, 0.1 ml each, were applied to the
agar plates and spread over their surfaces with a glass spreader, which was steri
lized after each use. Control and test disks were applied to the plates; each plate
bore one experim ental disk, one am picillin (2 //g) disk, one tetracycline (5 pg)
disk (b o th BBL Sensi-D iscs, B ecton D ickinson and C om pany, C ockeysville,
Maryland), one solvent control disk (ten drops of methanol, then allowed to dry),
and one plain (untreated) control disk. Duplicates were m ade of each experimen
tal treatm ent. The plates were incubated overnight (24 hours). The zones free of
37
visible bacterial growth (the zones of inhibition) were m e^sujed and the plates
were photographed. To increase replicability, the later test runs were perform ed
using cultures that had been diluted to a concentration of 10 -10 cells/m l.
Several cultures of bacteria were used; four were pure strains and one was
a mixture obtained from nature. Bacillus subtilis Ehrenberg, 1835, and Pseudomo
nas fluorescens M igula, 1895, w ere obtained from the U niversity of S o u th ern
California's bacterial culture collection. A sample was taken from the low interti
dal zone of the shorew ard side of a piling beneath Port H uenem e P ier in Port
H uenem e, California, with a sterile cotton swab and cultured in the laboratory.
Pseudomonas atlantica Humm, 1946 and Vibrio nereis (Baumann et a l, 1971) were
purchased from the American Type Culture Collection (Rockville, M aryland) {P.
atlantica, culture No. 19262, V. nereis, culture No. 25917). Bacillus subtilis is a very
common gram-positive bacterium, and P. fluorescens is a common gram-negative
species (Buchanan and Gibbons, 1974). Pseudomonas atlantica and V. nereis are
m arine fouling bacteria (Corpe, 1980; V enugopalan et a l, 1988), and a culture
swabbed from a pier piling is presumably largely composed of fouling bacteria as
well. The U.S.C. cultures were incubated at 26° C, as were the pier culture and V.
nereis', P. atlantica was cultured in a 14-15° C cold room on a magnetic stirrer (the
w arm th of the stirrer raised the tem perature of the culture slightly above th at of
the cold room ) and incubated at ambient tem perature, which averaged 22-23° C.
The diam eters and free radii of the zones of inhibition w ere tabulated,
although only diam eters are presented in the tables and figures. T he separate
readings were averaged and the standard deviations of the results were calculated.
The Mann-W hitney U-test (Campbell, 1989) was employed to determ ine the sta
tistical significance of differences in means and m easurem ent sets.
38
B. Winter Ampicillin
T o test the effects of the antibiotic am picillin on the fo rm atio n of the
fouling community, ordinary 1" x 3" (25 x 76 mm) glass microscope slides (VW R
Scientific, Inc., San Francisco, California) were used as micro-panels. Additional
m icro-panels of the sam e size were cut from a sheet of Teflon approxim ately 1
mm (1/32 inch) thick; these will hereinafter be known as Teflon slides. (The sheet
of "Teflon" used is actually a "CR-5 product" m ade from reprocessed resins of
Teflon, Halon, and Fluon [Norton Performance Plastics, Wayne, New Jersey]; the
mixed m aterial is herein referred to as Teflon for simplicity.) The slides w ere
inscribed for identification, scrubbed clean with Alconox, rinsed thoroughly, dried,
and w eighed. The weights were recorded and the slides w ere sterilized in an
autoclave. Slides w ere grouped in sets of seven for handling and deploym ent.
The num ber seven was originally chosen because it allowed doublings of deploy
m ent times (one day, two days, four days, etc.) over the expected duration of the
form ation of the early fouling community. The num ber becam e the standard
because the bags used to wrap some slides for deployment could comfortably hold
seven slides. As a result, all deployments were of multiples of seven slides.
C o n tro lled -release pain t was obtained from H ercules In co rp o ra te d
(W ilm ington, D elaw are). Two paint form ulations w ere used: form ulation X-
28339-53 was designed for fast release of the incorporated m aterials; form ulation
X-28339-54 was designed to afford a slow er release (R o b e rt G. N ickol, pers.
comm .). A pproxim ately 0.1 g of anhydrous am picillin (W yeth-Ayerst L ab o rato
ries, Princeton, New Jersey) was mixed with 60 ml of paint. Sterilized slides were
dipped into the paint-ampicillin mixtures; approximately 80-85% of each slide was
covered. Tow ard the end of each batch of am picillin-containing paint, the paint
level in the dipping container was sufficiently low that dipping was im practical.
39
T he remaining slides were coated by smearing paint on their surfaces and allow
ing the excess to drip off. T he coated slides were suspended by their uncovered
ends under a dust cover, indirectly exposed to the open air, until the paint dried.
Paint controls were m ade by the same procedure using paint (both fast- and slow-
release) with no ampicillin mixed in it; plain control slides were not dipped at all.
Slide suspension devices w ere constructed of w ooden snap clothespins. Silicone
rubber was applied to the tips of the jaws of the clothespins to improve their grip,
and polypropylene twine was tied through the coil of the springs.
W hen the dipped slides had dried, they w ere weighed again. The slides
w ere all w rapped in sterile polypropylene bags to keep them sterile and prevent
contact with the bacteria of the neuston. Surfaces exposed to the neuston can
hold up to three orders of m agnitude m ore attached b acteria than non-exposed
surfaces (DiSalvo, 1973). The slides were deployed in Fish H arb o r in January,
1990. B efore placem ent, the bags were opened enough to allow the suspension
devices to be attached to the slides; the bags were re-closed for placem ent. Once
the slides were in the w ater (well below the surface), the bags w ere re-opened,
and w ater allowed to flow in. W hen the w ater had equilibrated, the bags were
rem oved and the slides were allowed to hang free. The slides were suspended one
m eter below the surface of the water, hanging from the north side of the large
mooring float at Fish H arbor Laboratory. Duplicates of each preparation (plain
glass slides, glass slides with plain fast-release paint, glass slides with plain slow-
release paint, glass slides with am picillin in fast-release paint, glass slides with
ampicillin in slow-release paint, plain Teflon slides. Teflon slides with plain fast-
release paint. Teflon slides with plain slow-release paint. Teflon slides with ampi
cillin in fast-release paint, and Teflon slides with ampicillin in slow-release paint),
twenty slides in all, were removed from the water at intervals of 2, 4, 8, 18, 45, 74,
40
and 106 days of exposure. Duplicates were used in preference to larger num bers
of replicates in most of these experiments because the law of diminishing returns
applies to fouling: many replicates are not needed because m ost organism s will
appear on the first few (Schoener, 1984).
R ecovered slides were rinsed in fresh w ater, to rem ove excess salt and to
rinse off any organism s that might be present on the slides but not attached to
them (Z oB ell and Allen, 1933), but not fixed in alcohol, lest the alcohol extract
any com pounds from the rem aining paint. T he rinsed slides w ere d ried in a
w arm ing oven for at least tw enty-four hours (d ried to constant w eight) and
w eighed once again. T heir dry-weight gains (or, in some instances, losses) w ere
calculated and recorded. D ried slides w ere subjected to exam ination under a
dissecting microscope to determine the composition of their fouling communities
and in a scanning electron microscope to evaluate the composition of the m icrobi
al community, if any, on their surfaces (see Fletcher and Floodgate, 1976).
C. Spring Ampicillin
G lass and Teflon slides were prepared plain and also dipped in fast- and
slow -release paint with and w ithout am picillin in the sam e m anner as before.
W hen the dipped slides had dried, they w ere weighed again. The slides w ere all
w rapped in sterile polypropylene bags and placed in Fish H arbor in May of 1990.
The slides w ere suspended one m eter below the surface of the w ater, hanging
from the north side of the large m ooring float at Fish H arbor L aboratory. The
bags w ere not rem oved until the slides w ere below the surface of th e w ater.
D uplicates of each of the ten preparations, twenty slides in all, w ere rem oved
from the w ater at intervals of 1, 2, 4, 8, 15, 30, and 52 days of exposure.
41
R ecovered slides w ere rinsed in fresh w ater and dried in a w arm ing oven
for at least twenty-four hours (to constant weight) and weighed once again. Their
dry-weight gains (or, in some instances, losses) were calculated and recorded.
Selected slides were subjected to examination in a scanning electron microscope
to determ ine the extent and nature of m icrofouling that had occurred on their
surfaces.
As a check on the direct effects of am picillin on the settlem ent of tube-
worm larvae, plain glass, control fast-release paint, and fast-release paint with
am picillin slides were prepared as for the am picillin field experim ents. A dult
tubeworm s {Janua brasiliensis) were collected from Los Angeles H arbor and Port
H uenem e. L ate-term gravid individuals were rem oved from their tubes and the
larvae liberated from the brood chambers (see Shisko, 1975). One of each variety
of the p repared slides was placed in the bottom of a petri dish or finger bowl,
which was partially filled with seawater. Liberated tubeworm larvae were added
to the w ater and observed. Settled organisms were counted after several hours
and after one day, with care taken to note where they had settled and usually how
completely they had metamorphosed. The separate results were com pared.
D. Separation of Physial and Chemical Factors
Twenty-eight m icroscope slides w ere num bered, cleaned, weighed, and
then autoclaved as before. They were placed in glass slide-holders from histologi
cal staining dishes and suspended one m eter below the surface of Fish H arbor for
15 days. They were allowed full contact with the neuston during the deployment
procedure. At the end of the test period, the slides were recovered and returned
to the laboratory in seaw ater. They w ere fixed in a 2.5% solution of glutaralde-
hyde in seawater (as an osmotic buffer), rinsed in additional seawater, and dehy
42
drated with increasingly pure (to absolute) ethanol. They were then subjected to
critical-point drying using carbon dioxide as the transitional medium (Anderson,
1951, 1956). T he slides were again weighed, and several w ere exam ined with a
scanning electron microscope (Cambridge Stereoscan).
To determ ine the amount of coating required to effect a watertight seal, a
suspension of potassium iodide (KI) crystals in a saturated solution of KI was
prepared. Several drops of this suspension were applied to the surface of each of
twenty-six fresh, clean glass microscope slides, which were placed under a cover to
protect them from settling dust and allowed to dry. The slides were subjected to
varying am ounts of coating with a gold-palladium (A u/Pd, 60% gold, 40% palla
dium ) alloy in a sputter coater or a vacuum evaporator with varying angles of
coverage and amounts of rotation. A suspension and solution of corn starch was
prepared and applied to the coated surfaces of the slides with an eyedropper.
Some applications were to the centers of the m ounds, some to the edges of the
mounds, and some to areas away from the mounds. The slides were examined for
signs of penetration of the coating by the starch suspension. Such signs included
blistering of the coating and especially the appearance of the purple color typical
of the com plex form ed by iodine and starch in w ater. Two or th ree replicate
slides, each with several mounds, were used for each treatm ent.
Once the requisite coating thickness was determined, half of the slides that
had been suspended in Fish H arbor were coated with m etal of this thickness; the
other half were left bare. These slides, along with a similar num ber (each of glass
and T eflon) of cleaned, autoclaved slides, were w rapped in polyethylene bags.
A dditional groups of the same num bers of unw rapped glass and T eflon slides
w ere deployed alongside the w rapped slides at the sam e tim e. A d d itio n al
(cleaned, weighed, and sterilized) slides had been suspended in Fish H arb o r for
43
fifteen days, and then retrieved and weighed wet. H alf w ere fixed in m ethanol,
dried, and weighed again. The other half were returned to the w ater alongside
the coated and uncoated slides, which had just been deployed. The wet and dry
mass increases of the first group were correlated to provide a basis from which to
calculate the masses of the organisms already on the slides returned to the water.
The slides were removed from the harbor for the last time, in groups of two
(per treatm ent), at 1, 2, 4, 8, 16, 31, and 61 days after they were redeployed (in the
initial mass deployment). Each batch of slides was returned to the laboratory in
seawater; the slides were fixed in methanol overnight and then dried and weighed.
The increases in mass were tabulated, and observations m ade about the slides
(surface condition, size and m akeup of the com m unity growing thereon) were
recorded.
To provide m ore data points from which the relationship betw een the wet
and dry mass increases of fouled slides might be determ ined, an additional forty-
nine glass slides w ere num bered, cleaned, weighed, and sterilized. These were
suspended in Fish H arbor for fifteen days, and then retrieved. They w ere re
turned to the laboratory, shaken or allowed to drip until they were merely moist,
and weighed. They were then rinsed in fresh water, fixed in methanol, and dried
in the warm ing oven. W hen dry, they were weighed again and the dry weights
correlated with the wet weights.
To observe and m easure the presence of bacteria on their surfaces, m any
slides w ere subjected to scanning electron microscopy (SEM ) after exposure to
the sea. The presence or absence of bacteria, as well as their nature, shape, and
distribution, w ere recorded, keeping in m ind the possibility of shrinkage during
preparation for electron microscopy (e.g., Fuhrman, 1981). Estim ates of bacterial
44
p o p u latio n density w ere m ade in the follow ing m anner. T he SEM stage was
aligned visually so that the microscope was directed, as closely as possible, at the
cen ter of the slide; no scanning was perform ed during visual alignm ent, thus
avoiding possible bias. An area of twenty-five scanning fields (arranged in a 5 x 5
pattern) was viewed. W ithin each field, which measured approximately 8.5 x 5.7
jbim, b acteria were counted and recorded. The counts for the entire scan area
were totaled and the m ean and standard deviation count per scanning field were
calculated. Arbitrarily-chosen other field areas were also examined, to see if they
w ere significantly different. If great differences in substrate m akeup w ere ob
served, 5 x 5 counts w ere perform ed on each substrate type. Som e longer-ex-
posed slides were too covered with invertebrates to allow viewing of their surfaces
at the chosen point. These slides were scanned until a sufficiently large open area
was discovered, and a census was perform ed on that area instead. O ver forty
m icrographs were taken.
To test (and ensure) the reported inertness of the gold-palladium alloy,
twenty-eight new glass microscope slides were num bered, cleaned, weighed, and
sterilized. H alf of these were coated with the sam e gold-palladium alloy as be
fore, and half were not. These slides were suspended in Fish H arbor in a m anner
that allowed only one of their large faces to be exposed to the water. A fter peri
ods of 1, 2, 8, 16, and 36 days, they were retrieved and returned to the laboratory.
They w ere rinsed in fresh w ater, fixed in m ethanol, and dried in the warm ing
oven. W hen dry, they were weighed and their weights were compared.
45
E. Image Analysis
F ouling panels w ere p rep ared in a num ber of d ifferen t m an n ers from
num erous m aterials. Several d ifferen t m ethods of applying substances to be
tested for antifouling activity were used as well. Combinations of panel m aterials
and application m ethods used in the first experim ent, which chiefly com pared
substrates, were as follows.
1. An ordinary (not specially treated) piece of high-quality Ys"
(9.5 m m ) plywood, cut to a size of SV»" (22.5 cm) on a side and
routed with two grooves on each side (see Bakus et a l, 1990).
2. Thin ( 1/ 3 2", 0.8 mm) sheets of Teflon, cut to size and affixed
to the faces of plywood panels with nylon bolts.
3. Strips of polyester transparency sh eet (TF-30, N ashua
Corporation, Nashua, New Hampshire), cut to size and affixed ("top"
side out) to the faces of plywood panels with nylon bolts. W ashers
separated the sheeting from the nuts to minimize torsional wrinkling
and tearing.
4. Two pieces of window glass, connected back-to-back by
nylon bolts through holes bored near diagonally-opposite corners.
5. Two pieces of Lucite-L (E.I. D uPont de N em ours & Co.,
Inc., Wilmington, Delaware), connected back-to-back by nylon bolts
through holes bored near diagonally-opposite corners.
6. A plywood panel similar to No. 1 above, but with two round
alum inum weighing dishes (5 cm in diam eter x 1 cm deep) attached
with brass screws at diagonally-opposite corners of the area of inter
est. To prevent electrolytic corrosion in seaw ater, the screws w ere
separated from the dishes with nylon washers.
46
In the cases of panels with extra layers (Numbers 2, 3, 4, and 5), a bead of silicone
sealant was run around the edges of the working surface to prevent intrusion by
w ater and fouling organisms beneath the surface.
Fifteen millilitres of thawed freshwater extract of Callyspongia dijfusa were
poured on the experimental surface of Panels 2-5 and the panels were dried using
mild heating. This procedure was repeated once when the first application dried
for a total of 30 ml of extract per panel. The control sides of these panels were
left untreated. Fifteen millilitres of extract, mixed with ten grams of abietic acid
(C 2 0H 3 0O 2 ) in rosin (MCB code AX-5; see, e.g., Bakus et a l, 1990), were poured in
each groove or pan on the experim ental faces of Panels 1 and 6 for a total of 30
ml of extract per panel. The control sides were treated with a mixture of abietic
acid (also in rosin) and water. W hen dry, the panels were frozen at 0° C until they
w ere used. W hen all panels were ready, they were thaw ed, set into a Plexiglas
fram e, and suspended vertically one m eter below the surface in Fish H arb o r on
May 8 , 1989. They were checked after one week and retrieved on May 23, after a
total of fifteen days in the water.
A second set of experim ents, chiefly to com pare binders, was conducted
using M asonite showerboard panels (cut in 16.5 cm squares) only. Abietic acid (in
rosin), rosin alone, agar, and Quetol 651 (a water-soluble polymer. Polysciences,
Inc., W arrington, Pennsylvania) were used as experimental binders. Sponge GJB-
89-04 was used in the experimental binder mixtures; the binders were used alone
as controls, and som e panels were left plain as a further control. The binders
were applied in mounds around the edges of the panels (some on the brown side
of the show erboard, some on the white side) and suspended in Fish H arbor on
47
July 12, 1989. They were checked after one week and retrieved on July 24, after a
total of twelve days in the water.
A com plete im age-processing and image analysis system was assem bled.
T he heart of the system was a PC Vision fram egrabber board (Imaging Technolo
gy, Inc., W oburn, Massachusetts; Imaging Technology, 1985a), installed in an IBM
A T microcom puter, which was equipped with an Intel 80287 m athem atics coproc
essor. A ttach ed to the fram eg rab b er board was an R C A TC1005 television
cam era for photography and a Sony PV M -1271Q color m onitor for view ing
im ages. The fram egrabber board was driven by Im ageA ction im age processing
software (Imaging Technology, 1985b) and Im ageM easure image analysis software
(Microscience, Inc., Federal Way, Washington), specifically m odule 5100 (count
ing and m easuring). P hotographic filters could be attac h ed to the cam era in
various combinations.
To p rev en t edge effects, a w orking area of 12.5 x 8 cm (100 cm^) was
designated on each panel surface, away from the sides. The working areas w ere
p h o to g rap h ed electronically, using a polarizing filter, and the im ages stored.
Image processing techniques were used to enhance the images, and the objects on
the panels w ere counted by com puter. N um erous image processing algorithm s
were devised and tested on a trial-and-error basis. The calcareous worm tubes in
the w orking areas were also counted m anually or the percentage coverage was
estim ated m anually using a counting grid, as a baseline and com parison; the fig
ures were com pared to establish a correlation. The time needed for counting was
noted as well. Sm aller areas (2x2 cm) were chosen from panels with typically
high or low w orm tube densities and counted similarly. M anual counts of worm
tubes in these areas were made using two different counting screens, one with a 1
cm X 1 cm grid, the other with a 2 mm x 2 mm grid. The counts were compared.
48
III. Results
A. Antibiosis
The m ean diam eters of the zones of inhibition around the disks (Figures 3,
4) and the standard deviations of the measurements are presented in Figures 5-10.
The statistical significances of these results are presented in Table 2. The diam e
ters are taken to be proportional to the degree of inhibition of the bacterium in
question (see Figures 3, 4). Against Bacillus subtilis (Figure 5), m ost extracts
perform ed less well than two micrograms of ampicillin; only one, that of Fascios
pongia sp. (M 20 in the figures), was stronger (Figure 4) and one m ore, that of
specimen MTW-87-06 (M 6 in the figures), was roughly as effective as ampicillin.
In dose-response testing of Fasciospongia against B. subtilis (Figure 6 ), effective
ness fell slowly with decreasing concentration until one drop of concentrated
extract, at w hich p oint effectiveness fell m ore rapidly. E ven at th a t strength,
however, the extract compares favorably with 2 pg of ampicillin.
A gainst Pseudomonas fluorescens (Figure 7), none of the extracts tested
was as effective as ampicillin, although the differences were not significant in four
cases. T he Fasciospongia extract was unavailable for testing. A gainst the pier
culture (Figure 8 ), most extracts were essentially ineffective, as were the com m er
cial antibiotics; extract MTW-87-12 (M12 in the figures) and Fasciospongia were
substantially m ore effective than the others. Many of the extract test disks showed
thinner bacterial growth around them, however. These zones of presum ed partial
inhibition w ere circular, just as were the zones of inhibition on the other test
plates; the difference was the growth of some bacteria within this zone. The pier
culture test series was the only instance in which another extract was m ore effec
tive than that of Fasciospongia.
49
atJantJca
TW
Figure 3. Photograph of a typical antibiosis plate, showing the developm ent of the
bacterial lawn (cloudy white) except w here inhibited by the contents of
certain test disks.
The tetracycline (5 /(g) disk is to the upper left, the solvent control disk is
to the upper right, the plain control disk is in the center, the experim ental
ex tract disk is to the low er left, and th e am picillin ( 2 /(g) disk is to the
lower right.
50
[
V , / / > /
1 O d r o p s
M T W -87
Figure 4. Photograph of an antibiosis test plate, showing the great inhibition of
grow th of the bacterium B. subtilis by a sponge {Fasciospongia) extract
(MTW-87-20).
T he am picillin (2 pg) disk is to the upper left, the experim ental sponge
extract disk is to the upper right, the plain control disk is in the center, the
solvent control disk is to the lower left, and the tetracycline (5 /(g) disk is to
the lower right.
51
4 0
D i a m e t e r o f i n h i b i t i o n z o n e ( m m )
35 -
30 -
25
20 4
15
10 4
5
f
0
KO K2 KIO K12 K13 K15 M4 M6 M12 M17 M20 M21 A T
E x tr a c t (code)
Figure 5. M ean diam eters of zones of inhibition of Bacillus subtilis.
E rro r bars indicate standard deviation; the horizontal line indicates the
diam eter of the test disks. In this and subsequent graphs in this section, the
extract codes are abbreviated as follows: KO = MKM-87-00, etc.; M4 =
MTW-87-04, etc. A = ampicillin (2 /(g); T = tetracycline (5 /(g).
52
D i a m e t e r o f i n h i b i t i o n z o n e ( m m )
5 0
40
30
20
10
10 3 7 8 9 2 5 6 0 1 4
D rops of e x t r a c t (MTW-87 —20)
E xtract M T W -87-20 • A m p icillin (2 g)
Figure 6 . Results of dose-response testing of Fasciospongia sp. extract on Bacillus
subtilis, with ampicillin ( 2 //g) included for comparison.
53
45
40
35 4
30
25
20
15 H
10
5
0
D i a m e t e r o f i n h i b i t i o n z o n e ( m m )
KO K2 KIO K12 K15 M6 M17
E x tra c t (code)
Figure 7. M ean diam eters of zones of inhibition of Pseudomonas fluorescens.
E rro r bars indicate standard deviation; the horizontal line indicates the
diam eter of the test disks.
KO - MKM-87-00, etc.; M 6 = MTW-87-06, etc. A = ampicillin (2 //g); T
= tetracycline (5 //g).
54
2 0
15
D i a m e t e r o f i n h i b i t i o n z o n e ( m m )
10
o -
KIO K13 M4 M12 M20 M21
E xtract (code)
Figure 8 . M ean diam eters of zones of inhibition of the culture swabbed from Port
H uenem e Pier.
E rro r bars indicate standard deviation; the horizontal line indicates the
diam eter of the test disks.
KIO = MKM-87-10, etc.; M4 = MTW-87-04, etc. A = ampicillin (2/^g).
55
3 0
D i a m e t e r o f i n h i b i t i o n z o n e ( m m )
25 -
20 -
15
10 -
5 -
*
*
rh
r f i
* f f
0 7 9 10 12 13 14 1 ^ 1 4 6 12 17 20 21 25 26 30 32 34 37 40 A T
E xtract (code)
Figure 9. M ean diam eters of zones of inhibition of Pseudomonas atlantica.
E rro r bars indicate standard deviation; the horizontal line indicates the
diam eter of the test disks.
KO = MKM-87-00, etc.; M l = MTW-87-01, etc. A = ampicillin (2 /^g); T
= tetracycline (5 /^g).
56
3 0
D i a m e t e r o f i n h i b i t i o n z o n e ( m m )
25 -
20 -
15 -
10 -
5 -
rîn
0 7 9 10 12 13 14 15 1 4 12 17 20 25 26 30 32 34 37 40 A T
K: M :
E x tra c t (code)
Figure 10. M ean diam eters of zones of inhibition of Vibrio nereis.
E rro r bars indicate standard deviation; the horizontal line indicates the
diam eter of the test disks.
KG = MKM-87-00, etc.; M l = MTW-87-01, etc. A = ampicillin (2 //g); T
= tetracycline (5 ^g).
57
Table 2. Statistically significant differences in inhibition zones.
Codes in colum ns under the nam es of bacteria indicate the direction and
significance (M ann-W hitney U -test, 95 % confidence level) of differences of
diam eters of inhibition zones of extracts compared with ampicillin (A) and tetra
cycline (T). An A-, for example, indicates that the extract inhibited the indicated
bacterium significantly less than ampicillin; a T + indicates that the extract showed
significantly m ore inhibition than tetracycline. A zero indicates that the differ
ence, if any, was not statistically significant. The code "N.T." indicates that the
extract was not tested against the bacterium. An asterisk indicates that only one
successful observation was m ade for the indicated com bination of extract and
bacterium ; the U -test is thus insignificant.
58
Species Name
and Collection Code
Bacillus
subtilis
Pseudomonas
fluorescens
Dock
culture
Pseudomonas
atlantica
Vibrio
nereis
[unidentified]
MKM-87-00 A-X + A-T- N.T. A-TO A-TO
[unidentified]
NlKNI-87-02 A-T+ AOTO N.T. A + T + A-TO
Dendronephthya sp.
MKM-87-07 N T . N.T. N.T. AOT+ A-T+
[unidentified]
MKM-87-09 N.T. N.T. N.T. A + T +
*
Dendronephthya cf. minima
MKM-87-10 A-T+
*
A+T+ A-TO
*
Subergorgia mollis
MKM-87-12 A-T+ AOTO N.T. AOTO A-T+
[unidentified]
MKM-87-14 A-TO N.T. A+T+ AOT+ A -T+
[unidentified]
MKM-87-15 AOT+ AOTO N.T. AOT+ AOT+
Astrogorgia sp.
MTW-87-01 N.T. N.T. N.T. A-TO A-TO
Anthogorgia sp.
MTW-87-02 A-T+ N.T. N.T. A + T + N.T.
[unidentified]
MTW-87-04 A -T+ N.T. AOTO A-TO A-T-
[unidentified]
MTW-87-06 A-TO AOTO N.T. A-TO N.T.
[unidentified]
MTW-87-12 A-T+ N.T. A+T+ A + T + A-T-
Siphonogorgia sp.
MTW-87-17 A-TO A-T- N.T. AOTO A-TO
Fasciospongia sp.
MTW-87-20 A +T+ N.T. A+T+ A + T + A + T+
[unidentified]
MTW-87-21 A-TO N.T. AOTO A-TO N.T.
[unidentified]
MTW-87-25 N.T. N.T. N.T. A +T+ AOT+
[unidentified]
MTW-87-26 N.T. N.T. N.T. A-TO A-TO
[unidentified]
MTW-87-30 N.T. N.T. N.T. A +T+ A-T+
[unidentified]
MTW-87-32 N.T. N.T. N.T. AOT + A-TO
[unidentified]
MTW-87-37 N.T. N.T. N.T. A + T + AOT+
[unidentified]
MTW-87-40 N.T. N.T. N.T. AOTO A-TO
59
A gainst Pseudomonas atlantica (Figure 9), num erous extracts equaled or
exceeded ampicillin in effectiveness. Eight were significantly m ore effective than
am picillin, four of these substantially so. Two, extract MTW -87-12 and Fascios
pongia, w ere far m ore effective. Against Vibrio nereis (Figure 10), three extracts
w ere approxim ately as effective (insignificant differences) as am picillin. Only
Fasciospongia was significantly more effective. The other extracts were less effec
tive than ampicillin. Tetracycline showed little or no inhibition in any of the tests.
B. Winter Ampicillin
T he mass gains (m eans and standard deviations) attributed to dipping in
paint are shown in Figure 11. The slow-release paint, which was noticeably thick
er than the fast-release formulation, added between two and three times as much
mass to the slides. The addition of am picillin to the paint had negligible effects
on the m asses of the coats form ed on the slides. T he slightly higher standard
deviations in the mass increases of the slides coated with am picillin-containing
paint probably arise from those slides' being manually coated ("painted") with the
paint instead of being dipped in it, as it was in too-short supply to form a pool
deep enough for dipping.
A num ber of the painted slides lost some or all of their paint coatings after
some tim e in the water; these were om itted from consideration (although they are
represented in the graphs). The mass changes (m eans and ranges) of all slides
after two days of exposure are shown in Figure 12. Only the sm allest sm udges
w ere observed on the surfaces of the slides. T he mass changes of all slides after
four days of exposure are shown in Figure 13. Small specks or clumps of m icroal
gae were present on the slides. The mass changes of all slides after eight days of
exposure are shown in Figure 14. A fine, although not even, layer of micro algae
60
M a s s i n c r e a s e ( g r a m s )
0.8 -
0.4 -
0.2
GFC TFC GFA TFA GSC TSC GSA TSA
T re a tm e n t
Figure 11. M ass changes of the slides used in the w inter am picillin experim ent
after dipping in paint (before deployment).
Bars show m ean changes, error bars show one standard deviation. In this
and subsequent graphs in this section, the slide classes (base m aterials and
treatm ents) are abbreviated as follows:
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
61
3 0
M a s s i n c r e a s e ( m i l l i g r a m s )
20 -
10
-1 0 -
-20
-30
-40
-æ -
-Î-
GPC TPC GFC TFC GSC TSC GFA
T re a tm e n t
TFA GSA TSA
Figure 12. M ass changes of all slides used in the w inter am picillin experim ent
after two days of exposure.
Bars show mean; error bars show range.
G PC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
62
10
M a s s i n c r e a s e ( m i l l i g r a m s )
-1 0
-2 0 -
-30 -
-40
T
GPC TPC GFC TFC GSC TSC GFA TFA GSA TSA
T re a tm e n t
Figure 13. M ass changes of all slides used in the w inter am picillin experim ent
after four days of exposure.
Bars show mean; error bars show range.
G PC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
T FA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
63
M a s s i n c r e a s e ( m i l l i g r a m s )
-5 -
-1 0 -
-15 -
-2 0 -
-2 5
GPC TPC GFC TFC GSC TSC GFA TFA GSA TSA
T re a tm e n t
Figure 14. Mass changes of all slides used in the w inter am picillin experim ent
after eight days of exposure.
Bars show m ean; error bars show range.
G PC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
T FA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
64
could be seen with the naked eye; a few worm tubes were barely discernible. The
mass changes of all slides after eighteen days of exposure are shown in Figure 15.
The m icroalgal layer was thicker, and a few tiny ascidians and tubew orm s were
visible. The mass changes of all slides after forty-five days of exposure are shown
in Figure 16. The slides had considerable microalgal cover, some worm tubes, and
a few bryozoans, mostly Bugula sp. T he mass changes of all slides after seventy-
four days of exposure are shown in Figure 17. There Were num erous worm tubes,
mostly small, amid the microalgae; ascidians and bryozoans were also present in
significant, although lesser num bers, and a small num ber of tiny M ytilus shells
w ere also observed. The mass changes of all slides after 106 days of exposure are
shown in Figure 18. M ost of the slides in this batch bore m uch bryozoan over
growth, which dom inated the slides. W orm tubes w ere m uch less obvious than
bryozoans on the whole, and tended to be small and clum ped together. A few
Mytilus and barnacles were also observed.
The mass changes present a different picture when viewed over time. The
mass changes (over time, means and ranges) of the plain glass slides are shown in
Figure 19. The mass changes of glass slides dipped in plain fast-release paint are
shown in Figure 20. O ne of the 18-day slides, one of the 45-day slides, and all
su b seq u en t slides in this series lost significant am ounts of p ain t. T he m ass
changes of glass slides dipped in plain slow-release paint are shown in Figure 21.
O ne of the 106-day slides lost a significant am ount of paint. The mass changes of
glass slides dipped in am picillin-im pregnated fast-release p a in t are show n in
Figure 22. B oth of the 74-day slides and both of the 106-day slides lost m ost of
their paint. T he mass changes of glass slides dipped in am picillin-laden slow-
release paint are shown in Figure 23. Both 74-day slides lost a small, but probably
significant, am ount of paint; one 106-day slide lost a similarly sm all am ount of
65
M a s s i n c r e a s e ( m i l l i g r a m s )
3 0
20 -
10 -
-1 0 -
-20
GPC TPC GFC TFC GSC TSC GFA TFA GSA TSA
T re a tm e n t
Figure 15. M ass changes of all slides used in the w inter am picillin experim ent
after eighteen days of exposure.
Bars show m ean; error bars show range.
G PC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
6 6
1 5 0
100
50
M a s s i n c r e a s e ( m i l l i g r a m s )
-Î-
-50 -
- 1 0 0
GPC TPC GFC TFC GSC TSC GFA
T re a tm e n t
TFA GSA TSA
Figure 16. Mass changes of all slides used in the w inter am picillin experim ent
after forty-five days of exposure.
Bars show mean; error bars show range.
G PC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
67
M a s s i n c r e a s e ( m i l l i g r a m s )
2 0 0
100 1
-1 0 0 -
-200 -
-300 -
-400 -
-500
GPC TPC GFC** GSC TFC TSC GFA** GSA TFA TSA
T re a tm e n t
Figure 17. Mass changes of all slides used in the w inter am picillin experim ent
after seventy-four days of exposure.
Bars show m ean; error bars show range. Asterisks indicate slides that have
lost paint; each asterisk represents one slide.
G PC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
6 8
M a s s i n c r e a s e ( m i l l i g r a m s )
1 0 0 0
500 -
-500 -
-1000
TSA TSC GFA** GSA* TFA GPC TPC GFC** GSC* TFC
T re a tm e n t
Figure 18. Mass changes of all slides used in the w inter am picillin experim ent
after 106 days of exposure.
Bars show mean; error bars show range. Asterisks indicate slides that have
lost paint; each asterisk represents one slide.
GPC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
69
8 0 0
M a s s i n c r e a s e ( m i l l i g r a m s )
600 -
400
200 -
4 8 18 45 74
D uration of exposure (days)
106
Figure 19. Mass changes over tim e of all plain glass slides used in the w inter
ampicillin experiment.
Bars show mean; error bars show range.
70
100
M a s s i n c r e a s e ( m i l l i g r a m s )
5 0 -
-5 0
-100 -
-150 -
-200 -
-250
] [
-e-
18* 45* 74** 106**
D uration of exposure (days)
Figure 20. Mass changes over time of all glass slides dipped in plain fast-release
paint in the winter ampicillin experiment.
Bars show m ean; error bars show range. Asterisks indicate slides that have
lost paint; each asterisk represents one slide.
71
M a s s i n c r e a s e ( m i l l i g r a m s )
D uration of exposure (days)
200
100 -
-100 -
-200 -
-3 0 0 -
- 4 0 0
2 4 8 18 45 74 106*
Figure 21. Mass changes over time of all glass slides dipped in plain slow-release
paint in the winter ampicillin experiment.
Bars show mean; error bars show range. Asterisks indicate slides that have
lost paint; each asterisk represents one slide.
72
4 0 0
M a s s i n c r e a s e ( m i l l i g r a m s )
200 -
— 200 -
- 4 0 0 -
-6 0 0
4 8 18 45* 74**
D uration of exposure (days)
106'
Figure 22. Mass changes over time of all glass slides dipped in fast-release paint
containing ampicillin in the winter ampicillin experiment.
Bars show m ean; error bars show range. Asterisks indicate slides that have
lost paint; each asterisk represents one slide.
73
200
M a s s i n c r e a s e ( m i l l i g r a m s )
-200 -
-4 0 0
-6 0 0 -
-8 0 0
4 8 18 45 74
D uration of exposure (days)
106'
Figure 23. Mass changes over time of all glass slides dipped in slow-release paint
containing ampicillin in the winter ampicillin experiment.
Bars show m ean; error bars show range. Asterisks indicate slides that have
lost paint; each asterisk represents one slide.
74
paint; the other lost nearly all. The mass changes of the plain T eflon slides are
show n in F igure 24. T he m ass changes of T eflon slides d ip p ed in p lain fast-
release paint are shown in Figure 25. The mass changes of Teflon slides dipped in
plain slow -release paint are shown in Figure 26. The m ass changes of Teflon
slides dipped in ampicillin-impregnated fast-release paint are shown in Figure 27.
O ne of the 74-day slides was lost entirely, and the other lost a sm all am ount of
paint. The mass changes of Teflon slides dipped in ampicillin-laden slow-release
paint are shown in Figure 28. As an overview, the m ean mass changes of all of the
painted slides used in the winter experiment are depicted in Figure 29.
Surprisingly little bacterial growth was observed in exam ination of the
short-duration (eight days and less) slides with a scanning electron m icroscope
(Figure 30). M ost scan fields showed no bacteria at all. T here w ere, however,
num erous diatom s of sundry species (Figure 31). These ap p eared to be rath er
firmly attached to the substrate. Longer-duration slides had little or no bare slide
surface to examine (Figure 32).
C. Spring Ampicillin
T he mass gains (m eans and standard deviations) attrib u ted to dipping in
paint are shown in Figure 33. The addition of ampicillin to the paint continued to
have little effect on the sizes of the coats left on the slides, but the difficulties
caused by the dwindling supplies of paint did leave a few slides with slightly less
paint than norm al, especially the Teflon slides coated in am picillin-containing
paint, which were the last to be coated and thus the most subject to short-weight
ing.
A num ber of the painted slides lost some or all of their paint coatings after
some time in the water; these were om itted from consideration although they are,
75
1 0 0 0
M a s s i n c r e a s e ( m i l l i g r a m s )
800 -
600
4 0 0 -
200 -
-Î-
4 8 18 45 74
D uration of exposure (days)
106
Figure 24. Mass changes over tim e of all plain T eflon slides used in the w inter
ampicillin experiment.
Bars show mean; error bars show range.
76
600
500 -
400 -
300 -
200 -
100
0
-100
M a s s i n c r e a s e ( m i l l i g r a m s )
±
4 8 18 45 74
D uration of exposure (days)
106
Figure 25. Mass changes over time of all Teflon slides dipped in plain fast-release
paint in the winter ampicillin experiment.
Bars show m ean; error bars show range.
77
6 0 0
M a s s i n c r e a s e ( m i l l i g r a m s )
500 -
400 -
300 -
200 -
100 -
±
- 1 0 0
4 8 18 45 74
D uration of exp o su re (days)
106
Figure 26. M ass changes over tim e of all Teflon slides dipped in plain slow -re
lease paint in the winter ampicillin experiment.
Bars show m ean; error bars show range.
78
7 0 0
M a s s i n c r e a s e ( m i l l i g r a m s )
600 -
500 -
400 -
300
200 -
100 -
0
-100
-Î-
4 8 18 45 74'
D uration of exposure (days)
106
Figure 27. Mass changes over tim e of all Teflon slides dipped in fast-release paint
containing ampicillin in the winter ampicillin experiment.
Bars show mean; error bars show range. Asterisks indicate slides that have
lost paint; each asterisk represents one slide.
79
1000
M a s s i n c r e a s e ( m i l l i g r a m s )
800 -
600 -
400 -
200 -
-200
4 8 18 45 74
D uration of exposure (days)
106
Figure 28. Mass changes over tim e of all Teflon slides dipped in slow -release
paint containing ampicillin in the winter ampicillin experiment.
Bars show m ean; error bars show range.
80
M a s s i n c r e a s e s ( m i l l i g r a m s )
8 0 0
600 -
400 -
200 -
-2 0 0 -
- 4 0 0
106 2 4 8 18 45 74
D uration of exposure (days)
G F C
T F C
G S C
T S C
G F A
T F A
G S A
T S A
Figure 29. M ean mass changes of the painted slides used in the winter ampicillin
study.
Slides that lost paint are included.
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
81
3
Figure 30. Scanning electron micrograph of a slide used in the winter ampicillin
experiment, showing the dearth of attached bacteria.
Salt crystals may be identified by their straight edges and flat faces.
82
m
%
Figure 31. Scanning electron micrograph of a slide used in the w inter ampicillin
experiment, showing some of the diatoms attached thereto.
83
f
À
Figure 32. Scanning electron micrograph of the surface of a 74-day winter ampi-
cillin slide, showing the great extent of m icrobial fouling.
84
1 .8
M a s s i n c r e a s e ( g r a m s )
1.6
1.4
1.2 —
1
0.8 —
0.6 —
0.4
0.2 +
0
- = F -
GFC TFC GFA TFA G SC
T re a tm e n t
TSC GSA TSA
Figure 33. M ass changes of the slides used in the spring am picillin experim ent
after dipping in paint (before deployment).
Bars show m ean changes, error bars show one standard deviation.
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
85
where possible, represented in the graphs. The m ean and range of mass changes
(gains and losses) of all slides after 1 day of exposure are shown in Figure 34. A
few tiny reflective flecks, later identified as being the dried remains of gelatinous
ascidians, were present on the slides. The mass changes of all slides after 2 days
of exposure are shown in Figure 35. M ore ascidians w ere present, along with
som e tiny calcareous worm tubes. T he mass changes of all slides after 4 days of
exposure are shown in Figure 36. M ore ascidians and tubes were present, but they
could not be term ed num erous. The mass changes of all slides after 8 days of
exposure are shown in Figure 37. Algae were visible, forming a thin covering over
most slides; ascidians and tubes were also present. The mass changes of all slides
after 15 days of exposure are shown in Figure 38. Algae, ascidians, and worm
tubes w ere all increasing in num ber, size, and density. The mass changes of all
slides after 30 days of exposure are shown in Figure 39. A few bryozoans (both
encrusting and stolonate) and the occasional barnacle were now in the comm uni
ty, w hich was com ing to be dom inated by algae and tubew orm s. T he m ass
changes of all slides after 52 days of exposure are shown in Figure 40. Tubeworms
and algae were prevalent on most slides; some slides were already populated with
num erous barnacles (Balanus and Tetraclita).
The mass changes (over time, means and ranges) of the slides used in the
spring experim ent are shown in Figures 41-50. The mass changes of plain glass
slides are shown in Figure 41. The mass changes of glass slides dipped in plain
fast-release paint are shown in Figure 42. Only one 15-day slide was recovered;
the 30- and 52-day slides all lost at least som e paint. The mass changes of glass
slides d ip p ed in plain slow -release p ain t are show n in F igure 43. T he m ass
changes of glass slides dipped in am picillin-im pregnated fast-release pain t are
shown in Figure 44. One 52-day slide lost a significant am ount of paint; both 30-
86
M a s s i n c r e a s e ( m i l l i g r a m s )
- 5 -
-1 0
-1 5 -
-2 0 -
-2 5 -
- 3 0
TPC GFC GPC TFC GSC TSC TFA GFA GSA TSA
T re a tm e n t
Figure 34. Mass changes of all slides used in the spring am picillin experim ent
after one day of exposure.
Bars show m ean; error bars show range.
G PC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
T FA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
87
M a s s i n c r e a s e ( m i l l i g r a m s )
0
- 5 -
-1 0 -
-1 5
-2 0 4
-2 5
- 3 0
- 3 5
-æ -
GPC TPC GFC TFC GSC TSC GFA
T re a tm e n t
TFA GSA TSA
Figure 35. Mass changes of all slides used in the spring am picillin experim ent
after two days of exposure.
Bars show mean; error bars show range.
G PC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
88
M a s s i n c r e a s e ( m i l l i g r a m s )
- 5 -
- 1 0 -
- 1 5 -
-2 0 -
- 2 5 -
- 3 0 -
- 3 5
TPC GFC GPC TFC GSC TSC GFA TFA GSA TSA
T re a tm e n t
Figure 36. Mass changes of all slides used in the spring am picillin experim ent
after four days of exposure.
Bars show m ean; error bars show range.
G PC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
89
M a s s i n c r e a s e ( m i l l i g r a m s )
10
-1 0 -
- 2 0 -
- 3 0 -
-4 0
TSA TFA GSA GFA GSC TSC TPC GFC TFC GPC
T re a tm e n t
Figure 37. Mass changes of all slides used in the spring am picillin experim ent
after eight days of exposure.
Bars show mean; error bars show range.
G PC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
T FA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
90
M a s s i n c r e a s e ( m i l l i g r a m s )
3 0
20 -
10 -
-1 0
-20
TPC GFC TFC TSC GFA TFA GSA TSA GPC GSC
T re a tm e n t
Figure 38. Mass changes of all slides used in the spring am picillin experim ent
after fifteen days of exposure.
Bars show mean; error bars show range.
G PC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
91
5 0
M a s s i n c r e a s e ( m i l l i g r a m s )
i
-5 0 -
- 1 0 0 -
-1 5 0 -
-200
-2 5 0
GPC TPC GFC* TFC GSC TSC GFA TFA GSA TSA
T re a tm e n t
Figure 39. Mass changes of all slides used in the spring am picillin experim ent
after thirty days of exposure.
Bars show mean; error bars show range. Asterisks indicate slides that have
lost paint; each asterisk represents one slide.
GPC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
92
2 0 0 0
M a s s i n c r e a s e ( m i l l i g r a m s )
1500 -
1000 -
500
GPC TPC GFC* TFC GSC TSC GFA* TFA GSA TSA
T re a tm e n t
Figure 40. Mass changes of all slides used in the spring am picillin experim ent
after fifty-two days of exposure.
Bars show m ean; error bars show range. Asterisks indicate slides that have
lost paint; each asterisk represents one slide.
GPC = glass plain control
G FC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TPC = Teflon plain control
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
93
1600
1400 H
1200
1000 -
800 -
6 0 0 -
400
200 H
M a s s i n c r e a s e ( m i l l i g r a m s )
2 4 8 15 30
D uration of exposure (days)
52
Figure 41. Mass changes over time of all plain glass slides in the spring ampicillin
experiment.
Bars show m ean; error bars show range.
94
1000
M a s s i n c r e a s e ( m i l l i g r a m s )
800 -
600 -
400 -
200
0
-200 4
-4 0 0
2 4 8 15 30**
D uration of exposure (days)
52'
Figure 42. Mass changes over time of all glass slides dipped in plain fast-release
paint in the spring ampicillin experiment.
B ars show m ean; error bars show range. Only one fifteen-day slide was
recovered. A sterisks indicate slides th a t have lost p ain t; each asterisk
represents one slide.
95
7 0 0
M a s s i n c r e a s e ( m i l l i g r a m s )
600 -
500 -
4 00 -
300 -
200 -
100 -
0
-100
] [
2 4 8 15 30
D uration of exposure (days)
52
Figure 43. Mass changes over time of all glass slides dipped in plain slow-release
paint in the spring ampicillin experiment.
Bars show m ean; error bars show range.
96
1000
M a s s i n c r e a s e ( m i l l i g r a m s )
800 -
600 -
4 0 0 -
-200
200 -
2 4 8 15 30**
D uration of exposure (days)
Figure 44. Mass changes over time of all glass slides dipped in fast-release paint
containing ampicillin in the spring ampicillin experiment.
Bars show m ean; error bars show range. Asterisks indicate slides that have
lost paint; each asterisk represents one slide.
97
day slides may also have lost some. The mass changes of glass slides dipped in
ampicillin-laden slow-release paint are shown in Figure 45. The mass changes of
the plain Teflon slides are shown in Figure 46. The mass changes of Teflon slides
dipped in plain fast-release paint are shown in Figure 47. O ne 30-day slide may
have lost som e paint. The mass changes of Teflon slides dipped in plain slow-
release paint are shown in Figure 48. The mass changes of Teflon slides dipped in
am p icillin -im p reg n ated fast-release p ain t are show n in F igure 49. T he m ass
changes of Teflon slides dipped in ampicillin-laden slow-release paint are shown
in Figure 50. As an overview, the m ean mass changes of all of the painted slides
used in the spring experiment are depicted in Figure 51.
Scanning electron microscopy revealed very few bacteria on the slides it
was possible to scan (see Figure 30). The great m ajority (> 7 5 % ) of scan fields
showed no bacteria at all.
A n interesting, if unexpected, observation m ade in the course of these
experiments concerns the fouling of the field apparatus by tunicates. Given suffi
cient tim e (a m onth or m ore in winter, several weeks in spring), virtually every
apparatus was quite heavily fouled with these organisms. At the end of the exper
iment, many clothespins were unrecognizable, so thick was their cover. W hat was
interesting about this fouling was that the tunicates w ere attached and growing
entirely on the wood of the clothespins, or on that portion of the slide imm ediately
adjacent to the wood, close enough for the tunicates to have contact with it. None
of these organism s was observed on any other portion of the entire apparatus.
This preference for wood over glass. Teflon, paint, and am picillin is interesting
and invites further investigation, which is, unfortunately, beyond the scope of this
study.
98
M a s s i n c r e a s e ( m i l l i g r a m s )
1 4 0 0
1200 -
1000 -
800 -
6 0 0 -
400
200 -
-200
2 1 4 8 15 52 30
D uration of exposure (days)
Figure 45. Mass changes over time of all glass slides dipped in slow-release paint
containing ampicillin in the spring ampicillin experiment.
Bars show mean; error bars show range.
99
2000
M a s s i n c r e a s e ( m i l l i g r a m s )
1500 -
1000
500 -
2 4 8 15 30
Duration of exposure (days)
Figure 46. Mass changes over time of all plain Teflon slides in the spring ampicil
lin experiment.
Bars show mean; error bars show range.
1 0 0
1600
M a s s i n c r e a s e ( m i l l i g r a m s )
1400 -
1200 -
1000 -
800 -
600 -
400 -
200 -
0 —
- 2 0 0 —
2 4 8 15 30*
D uration of exposure (days)
52
Figure 47. Mass changes over time of all Teflon slides dipped in plain fast-release
paint in the spring ampicillin experiment.
Bars show mean; error bars show range. Asterisks indicate slides that have
lost paint; each asterisk represents one slide.
101
2000
M a s s i n c r e a s e ( m i l l i g r a m s )
1500 -
1000
500 -
-5 0 0
2 4 8 15 30
D uration of exposure (days)
Figure 48. Mass changes over time of all Teflon slides dipped in plain slow-re
lease paint in the spring ampicillin experiment.
Bars show mean; error bars show range.
1 0 2
6 0 0
M a s s i n c r e a s e ( m i l l i g r a m s )
-1 0 0
500 -
400 -
300 -
200 -
100 -
2 4 8 15 30
D uration of exposure (days)
Figure 49. Mass changes over time of all Teflon slides dipped in fast-release paint
containing ampicillin in the spring ampicillin experiment.
Bars show mean; error bars show range.
103
1600
M a s s i n c r e a s e ( m i l l i g r a m s )
1400
1200 -
1000 -
800 -
600 -
400 -
200 -
0
-200
2 4 8 15 30
D uration of exposure (days)
52
Figure 50. Mass changes over time of all Teflon slides dipped in slow-release
paint containing ampicillin in the spring ampicillin experiment.
Bars show mean; error bars show range.
104
M e a n m a s s i n c r e a s e ( m i l l i g r a m s )
1400
1200 -
1000
800 -
600 -
400 -
200 -
-200
2 1 4 8 15 30 52
D uration of exposure (days)
G F C
T F C
T r e a t m e n t
G S C G F A
T S C T F A
G S A
T S A
Figure 51. Mean mass changes of the painted slides used in the spring ampicillin
study.
Slides that lost paint are included.
GFC = glass, fast-release control
GSC = glass, slow-release control
G FA = glass, fast-release ampicillin
GSA = glass, slow-release ampicillin
TFC = Teflon, fast-release control
TSC = Teflon, slow-release control
TFA = Teflon, fast-release ampicillin
TSA = Teflon, slow-release ampicillin
105
T he num ber of organism s (i.e., tubeworm s, with an occasional ascidian)
counted within a dissecting microscope field on each of the laboratory test slides is
depicted in Figure 52. Only small differences were noticed. Num erous problem s
with worm larvae prevented using large num bers of larvae in an experim ent or
perform ing m ultiple repetitions of these experiments. O n several occasions, the
larvae all died before settling. The w ater in the petri dishes used in one such
failed test was subjected to Microtox toxicity testing (Bulich, 1979; Qureshi et al,
1982; R eteuna et al, 1986; Walker, 1986); each sample proved to be not only non
toxic, but actually stimulatory to the activity of the assay bacteria.
D. Separation of Physical and Chemical Factors
1. Thickness of Coating
A fter their original exposure to the sea, the preexposed slides (those for
the killed-community study) were observed to bear a fairly diverse community of
bacteria and diatom s, as yet largely unidentified (Figure 53). Som e algae and a
few sm all worm tubes w ere also present. The m axim um elevation of the organ
isms above the surface of the glass was less than 0.5 mm. At roughnesses approx
im ating that of the dried 15-day fouling community on the slides, a A u/P d coating
300 nm in depth seem ed adequate to seal the substrate away from the environ
m ent; coatings 200 nm or less in thickness were penetrated by the starch solution.
Angled coating and coating of rotating slides did not appear to improve the results
or achieve im perm eability at lesser thicknesses. T he application of too m uch
m etal led to flaking and wrinkling of the coating, thus compromising the integrity
of the seal. F or these reasons, a A u /P d coating 300 nm in thickness was chosen
for covering the fouled slides.
106
N u m b e r o b s e r v e d i n o n e f i e l d
glass paint control
T re a tm e n t
paint + a m p icillin
Figure 52. N um ber of adherent organism s counted w ithin one dissecting m icro
scope field on each of the laboratory test slide preparations.
T he organism s w ere tubew orm s, occasionally w ith a few ascidians; the
m agnification was 15 x . Bars indicate standard error.
107
.S --
■ • N
% ' • V
Figure 53. Scanning electron m icrograph of the surface of a "killed-com munity"
slide before coating with gold-palladium alloy for redeploym ent.
The microbial fouling community is dom inated by bacteria and diatoms.
108
2. Wet and Dry Mass Increases
Several of the slides used to calculate a baseline for the others bearing a
live fouling community were lost during the exposure period; although sixty-three
slides w ere deployed (fo u rteen in the first deploym ent and forty-nine in the
second), forty-six w ere recovered (fourteen from the first group, thirty-tw o from
the second). Two of the slides in the first group appeared to have been damaged
(T able 3). W hen plotted on a C artesian coordinate system, the points from the
first group form the arrangem ent depicted in Figure 54; the suspect nature of the
values for the questionable slides becom es readily apparent. N earest-neighbor
analysis served to confirm these suspicions.
Because the data points of both sets of slides (those removed and dried and
those returned to the water) are so scattered, it is unrealistic to use the m ean dry
mass increase of the dried slides as the calculated dry mass increase of the slides
returned to the w ater. I decided instead to correlate the wet and dry m asses of
each of the baseline slides to derive a m athem atical relation betw een the two
mass increases. The relation could then be applied to the w et m ass increases of
the returned slides to calculate their likely dry-mass increases. Substitution of the
wet m ass increases of the redeployed slides into the regression form ula derived
from the dried slides yields the presum ed dry mass increases listed in T able 4.
The average increase is negative, and thus an average decrease. This result is
clearly inconsistent with observed reality: organisms grew on the slides. If the two
suspect data points are om itted, however, the situation im proves dram atically
(Table 5). The positive average increase indicates a better correlation with reali
ty, and the sm aller num ber of negative individual values, com bined with their
small absolute values, strengthens the conclusion. T he two suspect points were
not used in subsequent calculations and are not considered further.
109
Table 3. W et and dry mass increases of the first set of fouled slides.
Slide num ber wet mass increase (g) dry mass increase (g)
43 0.1853 0.0131
44* 0.2096 0.0228
45 0.2615 0.0120
46* 0.1393 0.0219
47 0.4596 0.0228
48 0.4003 0.0208
49 0.3692 0.0178
50 0.4138 0.0159
51 (T3881 0.0124
52 0.4334 0.0246
53 0.3871 0.0216
54 0.3023 0.0121
55 0.3784 0.0116
56 (13885 0.0127
m ean (n = 14) 0.3369 0.0173
m ean (n = 12) 0.3640 0.0165
* The values for these slides are to be viewed with suspicion: the slides appeared
to have lost a portion of the adherent com m unity (see text). M ean values are
calculated for the group as a whole (n = 14) and for the group without these slides
(n = 12).
1 1 0
W e t m a s s i n c r e a s e ( g r a m s )
0 . 5
0.4
SB
0.3
0.2
0.015 0.011 0.019 0.023 0.027 0.035 0.031 0.039
Dry m a ss in c rease (g ram s)
accep ted data — se t 1 all d a ta---- s e t 1
Figure 54. W et and dry mass changes of the first set of slides used in the baseline
comparison for the separation-of-factors experiment.
The regression lines are shown for all points and for accepted points (see
Tables 4 and 5).
I l l
Table 4. Dry mass gains of the redeployed slides as calculated from the (unad
justed) first set of wet-dry slides.
num ber wet mass increase (g) dry mass increase (g)
29 0.3468 0.0225
30 0.2971 -0.0035
31 0.3692 0.0342
32 0.3609 0.0299
33 0.3501 0.0242
34 0.2651 -0.0203
35 0.2348 -0.0362
36 0.2896 -0.0075
37 0.3299 0.0260
38 0.1781 -0.0659
39 0.1963 -0.0563
40 0.1818 -0.0639
41 0.1419 -0.0848
42 0.2228 -0.0424
(n = 14) 0.2689 -0.0174
The equation used,
(dry mass increase) =
(wet mass increase) - 0.304
1.91
grams,
was derived from the regression line for all points shown in Figure 54.
1 1 2
Table 5. Dry mass gains of the redeployed slides as calculated from the adjusted
first set of wet-dry slides.
num ber wet mass increase (g) dry mass increase (g)
29 0.3468 0.0147
30 0.2971 0.0095
31 0.3692 0.0170
32 0.3609 0.0161
33 0.3501 0.0150
34 0.2651 0.0062
35 0.2348 0.0030
36 0.2896 0.0088
37 0.3299 0.0129
38 0.1781 -0.0028
39 0.1963 -0.0009
40 0.1818 -0.0024
41 0.1419 -0.0065
42 0.2228 0.0018
(n = 14) 0.2689 0.0066
T he equation used,
(dry mass increase)
(wet mass increase) - 0.205
9.65
grams,
was derived from the regression line for accepted points shown in Figure 54.
113
The data points from the second group of slides form a m ore cohesive mass
(Figure 55) than the first group, although the slides they represent have, on the
whole, a greater dry weight than the slides in the first group. Substituting the wet
mass increases of the redeployed slides into the equation derived from the second
group of slides yields the calculated dry mass increases listed in Table 6. The line
so d escrib ed is not congruent to th at from the first group of points, b ut has a
slightly lower slope (Figure 56). Consequently, there is no single line defining the
calculated dry mass increases of the slides that w ere returned to the w ater. In
stead, these two lines were used to define the range of the base mass increases for
calculations of subsequent mass increases (T able 7). Because of the distance
betw een these lines over most of the range at which they were used, the calculated
ranges of mass differences used were far larger than the mass ranges observed in
the other treatm ents, which w ere all derived from actual weighings. W here it is
necessary to exclude the live-community figures from discussion of mass increases,
the term "m easured" is used to indicate such exclusion; "calculated" is used to
stress that the masses so labeled were not m easured directly.
3. The Coated/U ncoated Experiment
The first-exposed slides, those that would becom e the coated and uncoated
killed com m unity slides, bore a complex m ixture of m icro-organism s on their
surfaces (see Figure 53). Bacteria and diatoms were common; a small num ber of
young tubew orm s w ere also observed. T he slides bearing "fresh" (live) fouling
communities showed more growth than those bearing preserved fouling comm uni
ties, at least at first. The gold-palladium alloy coating began to flake off the sur
faces of the coated slides in some places. By the end of one m onth in the w ater,
the slides showed enorm ous differences in the extent to which they w ere fouled.
114
0 . 5
W e t m a s s i n c r e a s e ( g r a m s )
0.4 -
0.3 -
0.2 -
0.1
X
X
X
X
X
X
X
X X X
X
X
X
X
X X
X
X
X
........
X X
-X T
X
X
0.011 0.015 0.019 0.023 0.027 0.031 0.035 0.039
Dry m a ss in crease (g ram s)
Figure 55. W et and dry mass changes of the second set of slides used in the base
line comparison for the separation-of-factors experiment.
The regression line is also shown (see Table 6).
115
Table 6. Dry mass gains of the redeployed slides as calculated from the second set
of wet-dry slides.
num ber wet mass increase (g) dry mass increase (g)
29 0.3468 0.0411
30 0.2971 0.0251
31 0.3692 0.0483
32 0.3609 0.0456
33 0.3501 0.0421
34 0.2651 0.0148
35 0.2348 0.0050
36 0.2896 0.0227
37 0.3299 0.0356
38 0.1781 -0.0132
39 0.1963 -0.0073
40 0.1818 -0.0120
41 0.1419 -0.0248
42 0.2228 0.0012
(n = 14) 0.2689 0.0160
The equation used,
(wet mass increase) - 0.219
(dry mass increase) = --------------------------------------------grams,
3.11
was derived from the regression line shown in Figure 55.
116
W e t m a s s i n c r e a s e ( g r a m s )
0 . 5
0.4
0.3
X X
0.2
0.015 0.011 0.019 0.023 0.027 0.031 0.035 0.039
Dry m a ss increase (gram s)
a ccep ted d a ta — set 1 -x - d a ta — se t 2
Figure 56. W et and dry mass changes of all retained slides in both sets used in the
baseline comparison.
The regression lines (shown; see Table 7) for the two groups were used to
define the range of calculated baseline values. The lines intersect at 0.0021
g dry, 0.226 g wet.
117
Table 7. Dry-mass gains of the redeployed slides as calculated by subtracting each
of the two accepted starting dry-mass figures from the final dry-mass gain.
num ber final dry mass (g) less first (g) less second (g)
29 0.0116 -0.0163 -0.0295
30 0.0166 0.0071 -0.0085
31 0.0136 -0.0034 -0.0350
32 0.0277 0.0116 -0.0179
33 0.7449 0.7299 0.7028
34 1.1072 1.1010 1.0924
35 5.7874 5.7844 5.7824
36 0.0157 0.0069 -0.0070
37 0.0224 0.0095 -0.0132
38 0.0329 0.0357 0.0461
39 0.1614 0.1693 0.1687
40 0.2865 0.2889 0.2985
41 2.3690 2.3755 2.3938
42 7.1617 7.1599 7.1605
118
Some slides were as heavily encrusted as the clothespins that held them, perhaps
m ore, while others, generally Teflon slides, w ere hardly covered. This disparity
continued through the second month (see below).
T he m ean mass changes and their ranges for this experim ental series at
each retrieval tim e are depicted in Figures 57-63. The mass gains (and losses)
after one day's exposure are shown in Figure 57. The coated slides lost more mass
than did the uncoated slides; the mass losses were probably the result of dissolu
tion of organic m atter in the case of the uncoated community and a small am ount
of flaking away of the gold-palladium alloy in the case of the coated slides. The
results after two days' exposure appear in Figure 58. The glass slides show a slight
increase in mass and the Teflon slides straddle the line betw een increase and
decrease; in these cases, the mass differences are approxim ately equal to the
reproducibility of the balance used.
T he situation after four days in the w ater is depicted in Figure 59. The
plain slides have gained a small amount, but the killed-community slides still show
a net mass loss. The situation changed by the eighth day, with all slides in all
treatm ents registering mass gains, with the possible exception of the live commu
nity (Figure 60). The next testing interval saw many comparisons change: by Day
16 (F igure 61), m any slide treatm en ts th at had gained m ore m ass th an th eir
counterparts earlier in the experim ent no longer had greater mass gains. The
m ost rem arkable result was that the coated com m unity registered the greatest
m easured mass increase, more than double the next-greatest m easured increase.
By 31 days, the results showed som e stability (Figure 62). The live slides,
th eir range finally becom ing com parable with those of the o th er trea tm e n ts,
showed a m ass gain sim ilar to those of several other treatm ents. T he uncoated
killed com m unity com pared with the glass slides, and the coated com m unity
119
2 0
M a s s i n c r e a s e ( m i l l i g r a m s )
10 -
- 1 0 -
-2 0
-3 0
- T -
U— glass W— glass U — Teflon W— Teflon Live
T r e a t m e n t
U n coated Coated
Figure 57. Mass changes of separation-of-factors slides after one day of exposure.
Bars indicate m ean changes, error bars indicate range. The live-communi
ty figures are from a calculated beginning point. T he le tte r U signifies
unwrapped slides; the letter W signifies wrapped slides.
1 2 0
10
M a s s i n c r e a s e ( m i l l i g r a m s )
-5 -
-1 0 -
-1 5
- Î -
U— glass W— glass U— Teflon W— Teflon Live U ncoated Coated
T re a tm e n t
Figure 58. M ass changes of separation-of-factors slides after two days of expo
sure.
Bars indicate m ean changes, error bars indicate range. The live-communi
ty figures are from a calculated beginning point. T he letter U signifies
unw rapped slides; the letter W signifies wrapped slides.
1 2 1
6 0
40
20 -
M a s s i n c r e a s e ( m i l l i g r a m s )
-20 -
-4 0
U -g la ss W -glass U -T eflon W -Teflon Live
T re a tm e n t
U ncoated Coated
Figure 59. Mass changes of separation-of-factors slides after four days of expo
sure.
Bars indicate m ean changes, error bars indicate range. The live-communi
ty figures are from a calculated beginning point. T he letter U signifies
unwrapped slides; the letter W signifies w rapped slides.
1 2 2
2 0 0
150
100
50
M a s s i n c r e a s e ( m i l l i g r a m s )
-5 0
U -g la s s W -glass U— Teflon W -Teflon Live U ncoated Coated
T re a tm e n t
Figure 60. M ass changes of separation-of-factors slides after eight days of expo
sure.
Bars indicate m ean changes, error bars indicate range. The live-communi
ty figures are from a calculated beginning point. T he le tte r U signifies
unwrapped slides; the letter W signifies wrapped slides.
123
M a s s i n c r e a s e ( g r a m s )
0 . 8
0.6 -
0.4 -
0.2 -
U -g la s s W -glass U -T eflon W -Teflon U ncoated Coated Live
T re a tm e n t
F igure 61. M ass changes of sep aratio n -o f-facto rs slides a fte r sixteen days of
exposure.
Bars indicate m ean changes, error bars indicate range. The live-communi
ty figures are from a calculated beginning point. T he letter U signifies
unw rapped slides; the letter W signifies w rapped slides.
124
M a s s i n c r e a s e ( g r a m s )
8
7
6
5
4
3
2
1
0
U -g la ss W— glass U -T eflon W-Teflon Live U n coated Coated
T re a tm e n t
Figure 62. M ass changes of separation-of-factors slides after thirty-one days of
exposure.
Bars indicate m ean changes, error bars indicate range. The live-communi
ty figures are from a calculated beginning point. T he letter U signifies
unwrapped slides; the letter W signifies wrapped slides.
125
posted over three tim es the m ean mass increase of any other treatm ent. By the
end of the experiment, sixty-one days later, the differences betw een the extremes
w ere a bit m odulated (Figure 63). T he growth of tubew orm s was so great on
som e of the slides that the slides w ere unrecognizable as such (Figure 64). The
two pairs of plain-slide treatm ents were nearly identical, with unw rapped gaining
a small am ount m ore than wrapped in each case. The Teflon slides continued to
show about half the mass increase as the glass slides. The live community, contin
uing to settle into the m iddle of the experim ent, show ed a (ca lcu late d ) m ass
increase virtually identical to that of the uncoated com m unity and only slightly
m ore than th at of unw rapped glass. T he coated killed com m unity continued to
enjoy a com m anding lead over all other treatm ents, but its m ass gain was only
twice th at of the uncoated killed com m unity at the experim ent’s end, so th at the
gap betw een them was closed somewhat.
Parallels can be seen when the separate treatm ents are view ed over tim e
(Figures 65-66). Most treatm ents displayed the phases of slower growth and then
faster growth typical of many populations, although an im portant p a rt of this
growth is size increase of individuals. The w rapped and unw rapped glass slides
(Figure 65) show this well, although it is unclear if the region of environm ental
resistance has been reached by the end of the experimental period. The extent of
the similarity of the two sets of mass gains can be seen in the figure: the curves are
alm ost identical. W rapped and unw rapped Teflon (Figure 66) show m uch the
sam e p attern as glass, but the m agnitudes of the mass increases w ere far less,
enough so that the minuscule errors arising from the balance used (mass losses on
several of the slides) can be seen quite plainly. Overall, the Teflon slides were far
less heavily fouled than the glass slides.
126
M a s s i n c r e a s e ( g r a m s )
1 6
14 -
U -g la s s W— glass U -T eflon W-Teflon U ncoated Coated Live
T re a tm e n t
Figure 63. M ass changes of separation-of-factors slides after sixty-one days of
exposure.
Bars indicate m ean changes, error bars indicate range. The live-communi
ty figures are from a calculated beginning point. T he letter U signifies
unwrapped slides; the letter W signifies wrapped slides.
127
F igure 64. P h o to g rap h of several sixty-one-day sep aratio n -o f-facto rs slides,
show ing th e g reat exten t to w hich m any of th em w ere overgrow n w ith
fouling organisms, especially tubeworms.
128
M a s s i n c r e a s e ( g r a m s )
7
6
5
4
3
2
1
70 20 50 60 40 10 30
D u ra tio n of e x p o s u re (days)
w rapped --e i-- un w rap p ed
Figure 65. M ass changes of the w rapped and unw rapped glass slides over the
duration of the separation-of-factors experiment.
129
M a s s i n c r e a s e ( g r a m s )
2.5
0 .5 -
10 20 30 40 50 60 70 0
D u ra tio n of e x p o su re (days)
w rapped u n w rap p ed
Figure 66. Mass changes of the w rapped and unw rapped T eflon slides over the
duration of the separation-of-factors experiment.
130
M a s s i n c r e a s e ( g r a m s )
4 -
2 -
2 4 8 16 31
D u ra tio n of e x p o s u re (days)
61
Figure 67. Mass changes of live-community slides over the duration of the separa
tion-of-factors experiment.
B ars indicate m ean changes, erro r bars indicate range. In this treatm en t
only, the starting mass of each slide was calculated and not m easured.
131
The live slides (Figure 67), were beset with (calculated) mass losses at first
and large ranges in m ass changes over m ost of the course of the experim ent,
although the relative magnitude of the ranges decreased as the masses increased.
D espite these problem s, these slides still show ex p o n en tial m ass increases.
U ncoated slides bearing a killed comm unity (Figure 68) lost a slight am ount of
mass in the first days, rebounding to pull slightly ahead of the plain glass slides,
the usual m ass-increase leaders. T he cham pion reb o u n d ers w ere the co ated
slides, which posted the greatest (m easured) mass loss (Day 4) and the greatest
mass gain (Day 61). The greatest difference between pairs of similar treatm ents
was displayed by the uncoated and coated slides. The coated slides were so thick
ly overgrown with tubeworms that it is questionable if the uncoated slides would
ever catch up to them, although the slowing in the growth rate of the coated slides
toward the end of the experiment may be an early indication of such a possibility.
Not all slides' surfaces could be examined in the scanning electron m icro
scope: the longer-duration slides were too heavily fouled to show any original
surface at all. Teflon slides were extremely difficult to image, probably because of
their extremely poor electrical conductivity. W hat Teflon slides could be imaged
showed noticeably greater surface irregularities than the plain glass slides, but the
collection of microscopic fouling organisms and debris did not appear to be great
ly affected. T he counts of bacteria on the glass slides appear in Figure 69. The
m ean population density on wrapped slides increases with time. M ean bacterial
population densities on unwrapped glass increase at first, and then decrease. This
may be related to the difficulty in finding a sufficiently open area to count; this
difficulty increased over time. The unwrapped slides becam e so overgrown with
larger fouling organism s (principally diatom s, and later sm all invertebrates as
well) and debris that it was not possible to find an area of sufficient openness and
size to allow a census of bacteria on the 16-day slide (Figure 70). T he bacteria
132
M a s s i n c r e a s e ( g r a m s )
15
10 -
50 60 30 40 20 10 0
D u ra tio n of e x p o s u re (days)
u n c o a te d m e a n c o a te d m e a n
Figure 68. Mass changes of the uncoated and coated killed-community slides over
the duration of the separation-of-factors experiment.
133
B a c t e r i a p e r s c a n f i e l d
2 4
D u ra tio n of e x p o s u re (days)
w rapped slid e s ^ un w rap p ed s lid e s
Figure 69. B acterial population densities upon w rapped and unw rapped glass
slides.
E ach symbol represents twenty-five scan fields (n = 25). Points indicate
means; vertical bars indicate one standard deviation.
134
Mi
W : ,
Figure 70. Scanning electron m icrograph of a sixteen-day separation-of-factors
slide, showing the extent of crowding by m any varieties of fouling organ
isms.
135
present on the unw rapped slides showed considerable clum ping as well (Figure
71), to a much greater extent than those on the wrapped slides.
Few er bacteria (by a factor of two to six) w ere counted on slides that had
been w rapped before deployment than on those that were deployed unwrapped,
with full contact with the neuston. No com parison was possible after the eighth
day. T he differences w ere not as great as some other researchers {e.g., DiSalvo,
1973; Little, 1984) have reported. On some slides, the bacteria showed a m arked
tendency to gather along small cracks, grooves, or fissures in the surface of the
glass (Figure 72). Clum ping of bacteria was common, even on sm ooth areas of
glass, and was especially noticeable on the slides with one or two days' exposure
(see Figure 71). This clumping, particularly com m on on the unw rapped slides,
was responsible for the large standard deviations in the population counts of those
slides. T he clum ped bacteria were som etim es covered with a thin, blanket-like
coating, presum ably comprised of bacterial exudates and probably serving to hold
the bacteria together, if not to the surface of the slide as well (Figure 73). Single
bacteria did not seem to have such a coating (Figure 74).
4. The Double-blank Experiment
Some flakes of the A u /P d coating were observed peeling off the coated
plain slides, especially at the start of the experiment, but the overall coverage did
not seem to be im paired. This flaking would appear to have resulted from too-
heavy coating in the vacuum evaporator. The plain glass slides (Figure 75) gained
mass slowly at first, and then with increasing rapidity. The coated glass slides lost
mass at first (probably from flaking away of the coating), but gained mass after
th at (starting on Day 2). The gains were slightly slower than those of the plain
glass slides at first, but they increased to the point that the two treatm ents w ere
136
e
'X f .'.
" ' T
T>fyS
■:?i
Figure 71. Scanning electron m icrograph of a two-day unw rapped glass slide,
showing the clumping of bacteria.
137
y
«
Figure 72. Scanning electron m icrograph of the surface of a glass slide, showing
lines of adhered bacteria, apparently along slight irregularities in the sur
face of the glass.
138
Figure 73. Scanning electron micrograph of a clump of adhered bacteria, showing
the coating that covers them.
139
?ir
Figure 74. Scanning electron micrograph of a single bacterium adhered to a glass
slide.
140
M a s s i n c r e a s e ( m i l l i g r a m s )
100
80 -
60 -
40 -
20 -
-20
10 0 20 30 40
D u ra tio n of e x p o s u re (days)
c o a te d u n c o a te d
Figure 75. Mass changes of the plain and coated glass slides over the duration of
the double-blank experiment.
141
approximately even by Day 16; by Day 36, the coated slides had a small lead, but
the ranges still overlapped slightly. The similarities betw een these two treatm ents'
mass-increase curves can be seen in the figure.
E. Image Analysis
The results of manual and computer-based counts of tubeworms attached
to the different fouling panel types used in the first comparison are listed in Table
8. In every case, the com puter counts were less than the m anual counts; in most
cases, the com puter total was 30-50% of the m anual total. The Teflon sheets were
too white for the worm tubes to stand out against them ; m anual counting was thus
very difficult and tim e-consum ing, and com puter counting was im possible. The
polyester sheet discolored somewhat, but this did not seem to affect the results
greatly; the difficulty in counting the worms arose at least as much from problem s
in handling the very flexible sheet as from other difficulties. T he other panel
types were counted without much trouble.
T he tim e n eed ed to count the worm s on each p an el type (co n tro l and
ex p erim en tal) by hand and by com puter is show n in T able 9. T he resu lts of
m anual and com puter-based estimates of areal coverage by tubeworms attached
to the different fouling panel faces (all M asonite) used in the second comparison
are listed in Table 10. W orm tubes on the white sides of the showerboard could
not be counted by computer, as they did not have enough contrast with the back
ground. The results of manual and computer-based estim ates of areal coverage of
small areas by worm tubes are shown in Table 11. G rid mesh size m ade an im por
tant difference in results; the com puter-derived figures com pare favorably with
the m anual figures. These and other results and their implications appear and are
discussed in W right et a l (1991).
142
Table 8. M anual vs. computer counts on different panel materials.
Panel # & Type of Num ber of worm tubes: Percent Correction
com position treatm ent manual com puter difference decrease factor*
2. Teflon control 116 0 116 100.0 N. A.
experiment 342 0 342 100.0 N. A.
3. Polyester control 590 245 345 582) 2.41
experiment 888 343 545 61.4 2.59
5. Lucite control 159 92 67 42.1 1.73
experiment 306 183 123 40.2 1.67
4. Glass control 160 59 101 63.1 2.71
experiment 269 199 70 26.0 1.35
1. W ood w. control 386 141 245 63.5 2.73
grooves experiment 386 120 266 68.9 3.21
6. W ood w. control 552 173 379 68.7 3.19
cups experiment 543 197 346 63.7 2.75
* The "C orrection factor" is the num ber by which the com puter count m ust be
m ultiplied to yield the manual count number. "N. A." = not available.
143
Table 9. Time expended in counting worm tubes.
Panel Difficulty Time expended Tim e expended
Pairs of on manual on com puter
counting counts (minutes) counts (minutes)
Teflon high 155
*
polyester m oderate 150 10
Lucite easy 125 10
glass easy 120 10
wood w. grooves easy 70 10
wood w. cups easy 90 10
Total tim e for m anual counts: 710 minutes (11 hours, 50 minutes)
not including Teflon: 555 minutes (9 hours, 15 minutes)
Total time for com puter counts: 50 minutes (not including Teflon)
Percent saving: 93% (assuming development time am ortized already)
not including Teflon: 91% (same assumption)
* The Teflon sheets could not be censused autom atically because they w ere so
white that the worm tubes did not contrast enough to be recognized. See text.
144
T able 10. M anual vs. com puter figures of p ercen t cover on M asonite panels
(brown sides only).
Panel Type of
description treatm ent
Coverage by worm tubes:
manual com puter difference
Abietic acid experimental 8.2 7.7 0.5
Rosin experimental 8.4 6.4 2.0
Rosin control 46.0 35.9 10.1
Agar experimental 27.6 18.9 8.7
T able 11. C om parison of worm tube areal coverage of sm all areas: com puter
m easurem ents versus visual estimation.
Relative
density of tubes
on panels
Percentage cover:
by visual estimation by com puter
(1 cm X 1 cm grid) (2 mm x 2 mm grid)
high
low
55
14
44
7
57
11
145
IV. Discussion
A. Antibiosis
M ost of the extracts show ed little effect on the grow th of the b a c te ria
against which they w ere tested. Two extracts seem ed to be effective generally;
one of these, that of the sponge Fasciospongia sp., was significantly m ore effective
than eith er am picillin or tetracycline in the strengths used in all of the tests. In
sm aller doses, it is alm ost as effective as in larger doses, although the potency
appears to drop off near the lower lim it of strengths tested (Figure 6). This ex
tract bears further investigation. The antibacterially active com ponent of this
extract would appear not to be a protein, as it survived exposure to fairly high
tem peratures (approximately 60° C) and retained its effectiveness after prolonged
exposure to air. O ther recently-discovered m arine antibacterial com pounds have
been rep o rted to have carbon-chain backbones {e.g.^ Okam i, 1986; V an Alstyne
and Paul, in press); it may be that this is another.
The effectiveness of the dose-response testing of the Fasciospongia extract
(F igure 6) is lim ited by the unknow n identity, chem ical n a tu re , an d thus size
(m olecular mass) and concentration of the active compound or com pounds in the
extract. Not knowing which compounds are effective, I have no way of determ in
ing the concentrations and am ounts of those com pounds in the extract or in the
testing disks. As a result, there is no way to compare the strength of the antibiotic
(or antibiotics) in the extract with that of ampicillin. Consequently, dose-response
testing is necessarily of limited usefulness.
Tetracycline appeared only m arginally effective, if at all, in all of these
experim ents. It is quite possible that the bacteria used were resistant to tetracy
cline, as tetracycline resistance is common, especially among Pseudomonas species
146
(Dr. H arrison M. Kurtz, pers. comm.). A nother possibility is that either the batch
used was poor or it lost its effectiveness in transit or storage.
The possible ecological and evolutionary significance of production of
antibacterial com pounds by m arine invertebrates seem s clear. T hese organism s
may well derive a benefit, direct or indirect, from killing or retarding the growth
of b acteria and other microbes. They m ight elim inate com petition for food or
even for space— a sort of m arine allelopathy (Tanaka and Asakawa, 1988). A nti
biotic-producing organisms might prevent bacterial fouling (and clogging) of their
surfaces {e.g.. Vaqué et a l, 1990), through which num erous im portant physiologi
cal tran sactio n s are conducted. P revention of b a cterial fouling m ay possibly
retard fouling by later successional stages of biofilm -dependent or biofilm-loving
fouling organism s as w ell, and it will certainly p rev en t b a cterial exudation of
slimes (C haracklis and Cooksey, 1983) over the surface of the organism in ques
tion. The production of antibiotics by microbes could prove particularly useful in
competitive circumstances (Steem ann Nielsen, 1955a,b; Proctor, 1957), allowing
an organism that grows slowly to hold its own against or even out-com pete organ
isms capable of rapid growth, such as bacteria {e.g., Sieburth and Pratt, 1962), and
could as a result have profound effects on the species m akeup of a m icrobial
community (Nigrelli, 1962).
O ther, undiscovered antibacterial com pounds may exist in the organism s
used in this series of antibiotic tests. M ore sophisticated preservation {e.g., flash-
freezing of living organism s in liquid nitrogen), extraction {e.g., freeze-drying,
Wright, in prep.), and preparation methods might have yielded a greater num ber
of positive results, but this is conjectural.
In light of these m any possible uses for an tim icro b ial substances, and
further in light of the ecological interactions occurring in the sea, a new question
147
is raised. Many studies (e.g., ZoBell and Allen, 1935) have drawn a link between
the settlem ent and growth of m arine bacteria on surfaces on the one hand and
subsequent growth of other microbes and later growth of fouling invertebrates on
the same surfaces. Might the production of antimicrobial compounds result in an
indirect benefit to the producing organism in preventing the settlem ent of larval
invertebrates and the consequent developm ent of a fouling com m unity on the
surface of the organism in question? This intriguing possibility proved to be the
driving force for much of this research.
T he extracts, all of m arine invertebrates, showed a range of effects on a
range of bacteria. The two m ost effective extracts in these experim ents were
extracts of sponges. These were both effective against the fouling bacteria against
which they w ere tested, although the extract of specim en M TW -87-12 showed
indifferent activity against V. nereis. It stands to reason that these sponges may
produce their antibacterial compounds to protect themselves from bacterial foul
ing of their surfaces. Fouling of these surfaces by bacteria could greatly im pede
the m etabolic processes of the host sponge, especially its filter-feeding and respi
ration. Keeping these surfaces clean of fouling overgrowth is thus of great im por
tance to the sponge; a chem ical m eans of accom plishing this end is probably
superior to achieving cleanliness by continuous or interm ittent sloughing of exter
nal tissue (as in Halichondria panicea—Barthel and W olfrath, 1989). Because of
their activity against fouling bacteria, these extracts may prove useful in fouling
and antifouling studies.
Because of the extrem ely short supply of Fasciospongia extract rem aining
after this experiment, I decided to save the extract for crucial work; not only were
m ore antibiosis tests contem plated, but the chem ical stru c tu re of the active
com pound or com pounds should also be determ ined. O ne or m ore com m ercial
148
antibiotics would be used instead for testing the relationship betw een antibiosis
and antifouling in the short term. Once the techniques were perfected, and if the
results w ere prom ising, the Fasciospongia extract could be substituted for the
com m ercial antibiotic for a last test of antibiosis and antifouling. T he results of
the antibiosis testing indicated that am picillin was effective against a range of
m arine fouling bacteria and might be a good choice for the prelim inary antibiosis-
antifouling experiments.
B, W inter Ampicillin
The variation in masses of dried paint is not great within single treatm ent
classes, but differs m arkedly betw een som e treatm ents (see Figure 11). Surpris
ingly, there is very little difference betw een glass and Teflon slides given the same
treatm ent, despite the great differences in the hydrophilic n atu re of these two
surfaces. O n the other hand, the differences between fast- and slow-release paint
are large indeed; the slow-release paint added betw een two and three tim es as
m uch mass as did the fast-release paint. This indicates that the base m aterial of
the slide will be of little consequence to fouling once paint is applied, but the
paint form ulation may prove to be of great importance. T here was thus likely to
be m ore ampicillin in the experimental treatm ents of slow-release paint as well.
The plain slides, glass and Teflon, may be taken as baselines for com pari
son with the painted slides as a whole. Both sets showed m ass increases, both
m arginal and overall, at every step of the way. Mass increases were small at first,
as the pioneer fouling organisms settled and attached; later increases were more
rapid, as the first organism s grew and later arrivals settled on the slides. This
indicates and attests to the presence and settlem ent ability of num erous fouling
organism s, including serpulid tubew orm s, during the period of this experim ent
149
(January through M arch). Bugula was particularly reproductively active during
this period.
All of the paint-dipped slides, control and experimental, lost mass at first.
The rate of loss was initially rapid; losses then slowed and eventually reversed
them selves. This is alm ost undoubtedly the result of the confluence of two fac
tors: dissolution and settlem ent. The dissolved solutes would be expected to
dissolve away most quickly at first, when their concentration in the paint is the
highest and their concentration in the surrounding w ater is lowest. M eanw hile,
the mass gains resulting from fouling of the surfaces of the slides and subsequent
growth of the fouling organisms would be expected to begin slowly and then speed
up, eventually tapering off as the carrying capacity is reached (see Figures 19 and
24, see also part IV.D.3.). The sum of these two processes would be a quick initial
loss of mass, gradually but with increasing m om entum replaced by a mass gain.
This p attern may be seen in each of the treatm ents that did not lose paint too
quickly.
1. Com parisons Between Glass Slides
The glass-plain-control slides (Figure 19) show consistently g reater mass
increases than do slides from either of the glass paint control sets (Figures 20 and
21); this difference becam e especially m arked after the painted slides began to
lose their paint. The plain glass slides also show much greater mass increases than
glass-fast-am picillin (Figure 22) and glass-slow -am picillin (F igure 23), w hich
seems to lag a m onth or more behind the plain set.
The two sets of control paint on glass showed sim ilar results over tim e.
F a st-rele ase p ain t (Figure 20) lost m ore m ass in two days, but slow -release
(Figure 21) caught up with it by four days. Both showed sm all tim e-based mass
150
increases after that. Fast-release appears to have had a slight lead at the tim e
paint loss began. The glass-fast-control slides (Figure 20) lost less mass than the
glass-fast-ampicillin slides (Figure 22) for as long as there was paint. O ne distinct
sim ilarity betw een the two glass-fast sets was the speed with which they lost the
paint applied to them . The different treatm ents of slow -release paint (control,
Figure 21, and ampicillin. Figure 23) were quite similar, with a small "advantage"
to the control paint. A comparison was difficult to make at 106 days, as only one
slide rem ain ed from each set; th ere was, perhaps, a sm all "advantage" to the
ampicillin slide. In any case, there was virtually no difference.
B ecause of paint losses, the two varieties of am picillin-containing paint
could only be com pared during the first eighteen days. T he fast-release paint
slides (Figure 22) lost less mass than did the slow-release paint (Figure 25), except
at Day 8, w hen large ranges in the fast-am picillin-treated slides' mass changes
brought the two sets into overlap. T here was also overlap at 45 days, but paint
losses had set in by then, rendering conclusions conjectural.
2. Comparisons Between Teflon Slides
The T eflon-plain-control slides (Figure 24) gained m ore mass faster than
any of the painted Teflon slides. Slow-release am picillin caught up to a rough
parity in the later days of the experiment, after Day 45.
Com parison of the two control paints shows an exchange of position. The
slow-release paint slides (Figure 26) initially lost m ore mass and then gained more
than fast-release paint (Figure 25). The two fast-release paint treatm ents (Figures
25 and 27) had sim ilar results. T heir mass change ranges overlap at each point
except at Day 4 and Day 74, with am picillin-containing paint tending to show
slightly more mass toward the end of the experiment. W hen the two slow-release
151
paints (Figures 26 and 28) are compared, ampicillin loses m ore at first, and then
shows greater mass increases after Day 45, although the ranges overlap. The two
ampicillin-containing treatm ents (Figures 27 and 28) were not very different from
one another either.
3. Comparisons Between Glass and Teflon Slides
T he two plain control slide sets (Figures 19 and 24) w ere quite sim ilar to
one another at each stage in this experiment; the increase ranges overlap and are
nearly coterm inous. The two different substrates of control fast-release paint
(glass. Figure 20, and Teflon, Figure 25) showed alm ost no difference betw een
them , as long as there was paint on their surfaces. The control slow-release paint
on both substrates (Figures 21 and 26) also showed very little difference until the
106th day, when paint loss prevented a proper comparison.
In the same theme, there was almost no difference betw een the fast-ampi
cillin paints (Figures 22 and 27) applied to the two substrates. The two sets of
slow-am picillin slides (Figures 23 and 28) followed the sam e general trend of
sim ilarity. The principal difference betw een the two slide types was the success
with which they retained the fast-release paint applied to their surfaces; as m en
tioned previously, glass lost paint much more readily than did Teflon.
The dearth of bacteria on the surfaces of all varieties of slides, control and
am picillin, suggests that the practice of wrapping the slides before deploym ent,
only unwrapping them when they were below the surface of the water, prevented
contam ination of the slides with bacteria from the neuston. O ther m arine m icro
organism s, such as diatom s, would appear to have had no trouble colonizing the
slide surfaces. Active w aterborne m arine bacteria are concentrated at the sea
152
surface (Tsyban, 1971); bacterioneuston far outnum ber bacterioplankton (Sie
burth, 1965, in DiSalvo, 1973). By deploying the slides in a way that avoided any
contact with the sea surface and neuston, I may have nearly elim inated bacterial
se ttlem e n t and grow th on them (see D iSalvo, 1973), although the possibility
rem ains th at bacteriovorous grazers are responsible for the observed d earth of
bacteria.
C. Spring Ampicillin
As in the winter experiment, the variation in masses of dried paint in spring
is not great within single treatm ent classes, but differs m arkedly betw een som e
treatm ents (Figure 33). The plain slides, both glass and Teflon, may again be
taken as baselines for com parison with the painted slides as a whole. M ass in
creases were small at first, as the pioneer fouling organisms settled and attached;
later increases w ere m ore rapid, as the first organism s grew and later arrivals
settled on the slides. The rate (or magnitude) of the mass increases indicates and
attests to the presence of num erous fouling organism s during the period of this
experim ent. The em erging dom inance of the serpulid tubew orm s in this experi
m ent confirms their importance as fouling organisms in Fish H arbor (C rippen and
Reish, 1969; Bergen, 1985).
All of the paint-dipped slides, control and experimental, lost mass at first.
At least half of the greatest loss occurred in the first day of exposure; m ost slide
groups had positive average mass changes by Day 15. The loss-and-gain p attern
resem bles that displayed by the winter slides, and probably arises from the same
causes. T he mass changes for the first thirty days are all dw arfed by the mass
increases of the fifty-two day slides, which are at least an order of m agnitude
greater (and frequently far m ore) than any other mass increases. T he control
153
paint slides lost slightly less mass than did the am picillin paint slides, with their
one additional solute. The slow-release paint slides lost far m ore m ass than did
their fast-release counterparts, even losing slightly more mass in proportion to the
mass they gained by being painted. The Teflon slides tended to lose slightly more
m ass th an did the glass slides, b u t the m ass-loss ranges g en erally o v erlapped
betw een treatm ents that were the same except for substrate m aterial.
T he distinct differences betw een treatm ents at the start (Figures 34-36)
becom e less so by eight days (Figure 37) and are lost by fifteen days (Figure 38).
The ranges also increase as the means increase, making determ inations of relative
m agnitude very difficult. Some patterns rem ain after thirty days (Figure 39), but
the fifty-two day slide graph (Figure 40) resem bles a disorganized m élée, with
little, if any, pattern to it. The presence of barnacles on the last slides contributes
greatly to this problem . A barnacle of the size encountered on the 52-day slides
can weigh over 100 m illigram s dry, far m ore than the other fouling organism s
observed in this series of experiments; even one of these individuals can seriously
affect a slide's mass, and they rarely appeared alone.
Fewer spring slides lost paint than did their winter counterparts, probably
because of the shorter duration of the experiment and the warmer, m ilder w eath
er. W hat losses did occur, how ever, did so in a p a tte rn m uch like th a t of the
w inter slides: glass slides lost far more paint than Teflon slides. The fast-release
paint was m ore easily lost than was the slow-release paint.
1. Com parisons Between Glass Slides
The glass-plain-control slides show greater mass increases than do the glass
paint control slides until the accum ulations of organisms by Day 52 blur the dis
154
tinctions. The plain slides also have far greater mass increases than do the glass-
paint-ampicillin slides.
The two sets of glass slides with control paint showed similar mass-change
patterns, although the magnitudes were different by a factor of two to three times.
Paint losses prevent meaningful comparisons after Day 15. The two sets of glass
slides coated with fast-release paint (control and ampicillin) show similar parallel
tracks; these two treatm ents were the most prone to paint loss. The two sets of
glass in slow -release paint behaved in sim ilar m anner. T he two glass-am picillin
treatm ents are also rem arkably parallel; they start to change mass quickly and
reach the greatest loss at Day 8.
2. Comparisons Between Teflon slides
The plain Teflon slides gained distinctly m ore mass than did the painted
slides until Day 30, when Teflon-slow-ampicillin drew close, thanks in large part to
its wide mass range. T hree barnacle-laden painted T eflon treatm ents (fast-con-
trol, slow-control, and slow-ampicillin) had mass means and ranges com parable to
that of plain Teflon on Day 52. The coated Teflon slides showed uniform mass
losses until Day 15, w hen most showed slight mass gains or at least no net loss;
mass gains were the order after that.
The two Teflon paint control treatm ents have similar mass change curves,
but the fast-control paint reaches its greatest mass loss later than the slow-control.
T he two T eflo n -fast-release trea tm e n ts are even m ore closely p arallel, both
achieving maximum mass loss on Day 8, and approximately half of that on Day 1.
The slow -release paints reach their greatest mass loss on Day 2, and slowly gain
mass from then on. The two am picillin-containing T eflon treatm ents behaved
m uch m ore in keeping w ith th eir p ain t types th an w ith th e ir so lu te m akeup.
155
A lthough Teflon-slow -am picillin lagged behind at 15 days, it posted the greatest
gain of any painted slide treatm ent on Day 30 and still led Teflon-fast-ampicillin
by Day 52.
3. Comparisons Between Glass and Teflon Slides
Substrate composition made almost no difference betw een any of the slide-
m aterial group pairs. The fast-release-paint slides were quite alike one another,
as w ere the slow-release slides. The affinities of the treatm ents for one another
w ere m ost pronounced w hen the solutes are considered separately for the fast-
release paint: the two fast-control and the two slow-control treatm ents' plots are
nearly coterm inous. The general resem blance of the m ass gain patterns of the
different paint compositions, particularly in the early days of the experiment, can
be seen clearly in Figure 50. In the later days of the experiment, paint losses and
eventually barnacle attachm ent and growth tend to disrupt, or at least confuse, the
clear patterns of the early days.
In hindsight, the worm-settlement experiment may not have been absolute
ly necessary. The results of the two sets of am picillin experim ents indicate that
neither ampicillin nor either form ulation of release paint has a negative effect on
the norm al settlem ent and subsequent growth of local serpulid tubeworms. This is
in keeping with the findings of Kirchman et a l (1982a,b). The communities on the
slides grew and developed seemingly without regard to the presence or absence of
ampicillin or even paint, after the initial few days.
Fouling bacteria were very scarce on these slides, but this absence appears
not to have disrupted the developm ent of the invertebrate fouling comm unity.
C onsequently, it w ould a p p ea r th at there is little o r no rela tio n sh ip betw een
156
presum ptive antibiosis and antifouling, at least in Fish H arb o r (see also W right,
1991). As a result, it seems that not using the last of the Fasciospongia extract was
a good idea— the extract would have been depleted, and the experim ent would
alm ost certainly have failed through lack of antifouling activity. T he rem aining
extract can still be subjected to chemical and other analysis.
D. Separation of Physical and Chemical Factors
1. Coating
G old and palladium are two quite in ert m etals, and do n o t rea ct w ith
seaw ater (D r. R obert F. Bils, pers. comm.). This minim izes the chance th at the
coating used will exert a chem ical effect on the system and processes exam ined.
A lthough m olecular carbon is known for its lack of grain in coating by vacuum
evaporation {e.g., Bils, 1974), the possibility that carbon might possibly be exploit
ed as a nutrient or exert some other chemical effect on the experim ent led to the
m etal alloy being chosen instead.
T he coating thickness required to seal crystals of potassium iodide, up to
approxim ately 1 mm in size, in a watertight fashion is 300 run. Slides covered with
thinner coatings had purple blotches w hen tested with starch solution, and the
coatings were frequently observed to have blistered as the solution penetrated the
coating and spread underneath it. Clearly, local topology plays a part in determ in
ing the requisite thickness. Lower and sm oother rises will be m ore easily sealed
by a thinner coating than greater and m ore precipitous rises.
The KI mounds were considerably larger than almost any of the organisms
that fouled the initial slides, and at least as rough. Consequently, it would appear
that a gold-palladium coating of 300 nm was sufficient to seal the pioneer fouling
com m unity from the seaw ater into which it was later lowered. As 300 nm (0.3
157
//m ) is sm aller than all but the sm allest bacteria (bacteria range from approxi
mately 0.1 yw m to over 5.0 yw m in diameter, Brock and Madigan, 1988; m ore to the
point. Vibrio species range from 1.5-3.0 yw m , Colwell, 1977, and Pseudom onas
species range from 0.5 fum to 1.0 jum across by 1.5 jum to 4.0 p m long, Palleroni,
1977), a coating of this thickness would not be likely to obscure the overall surface
topology of a pioneer fouling community.
2. The Relationship Between Wet and Dry Mass Increases
The w et-and-dry slide correlations are som ew hat m ore com plicated than
the results of any other slide treatm ent. If the two troublesom e data points are
included, the regression line for the first group has a negative slope, indicating an
inverse relationship betw een wet and dry weights. The absurdity of this result
(especially in combination with the observed damage) m ade it quite easy to elim i
nate the two points from consideration. Additionally, the second group of slides,
used because of the small num ber in the first group, does not simply reiterate the
results of the first, but shows considerably m ore dry-mass increase for w et-m ass
increases of sim ilar m agnitude. No confounding factor that would explain this
discrepancy was observed, although there are possible differences in weighing and
balances; the difference was probably a result of a slightly d ifferen t fouling
com m unity com position, which was in tu rn the resu lt of the la te r tim e of the
second group's deployment. Two regression lines, one for each set of data points,
were used to delineate the range of possible relationships. The results (the calcu
lated dry masses of the slides returned to the water. Tables 5 and 6) seem em piri
cally reasonable.
158
3. The Coated/Uncoated Experiment
One clear trend in all of the treatm ents in the separation-of-factors exper
im ent was the instantaneous rate of increase of biom ass as the separate fouling
communities grew and developed. Masses increased rapidly at first, approaching
exponential growth for a time; the "lag" and "log" phases are visible in some of the
graphs {e.g.. Figure 68). Later in the experiment, the rates of increase slowed and
the masses increased less rapidly, the growth curve departing from the exponential
p a tte rn of theory. T he m ass increases slowed over tim e, and th e plots of the
increases began to resem ble the typical sigm oid shape of th e logistic curve
(V erhulst, 1838; Pearl and Reed, 1920; Pearl, 1927; all in K rebs, 1985), but the
experim ent did not continue long enough for the net mass increases to level off.
The separate treatm ents w ere not all at the sam e point in the presum ed logistic
curve at the term ination of the experim ent; some com m unities had proceeded
farther along the curve than had others.
T he glass slides gained mass at all points of the ex p erim en t (w ith one
possible exception). Each of the two treatm ents showed approximately exponen
tial growth for the first eight to sixteen days of the exposure period (Figure 65).
A fter that point, however, the curves flattened to linear increases, presum ably
because environm ental resistance was causing their logistic d ep artu re from the
exponential form. W rapping the slides, and thus preventing their being contam i
nated with bacteria from the neuston, had little or no effect on the rate of mass
increase; the wrapped slides merely achieved linearity slightly earlier than did the
unwrapped slides. It would therefore appear that the presence or absence of the
film of bacteria associated with the neuston (Tsyban, 1971) and resulting from
passing through it (DiSalvo, 1973) had little effect on the subsequent developm ent
of the (macroscopic) fouling community on the glass. The m acrofoulers arrived,
159
settled, attached, and grew with little regard for w hat cam e or did not com e b e
fore. This is in keeping with other researchers' observations, especially on barna
cles (Little, 1984; Roberts et a l, manuscript).
The T eflon slides in both treatm ents appeared to lose a sm all p ortio n of
their initial m ass gain after placem ent, but this counter-intuitive result would
appear to be the product of erroneously high day-one mass increases. In any
event, the masses and differences in question are at the very limit of the ability of
the balance used to repeat itself and are vanishingly small in relation to the later-
occurring m ass changes (see Figure 66). A fter the start, th e slides displayed
nearly exponential rates of mass increase, which declined from the exponential in
tim e (the decline was m ore dramatic in the wrapped slides). Overall, there seems
to be little difference betw een Teflon slides placed in Fish H arbor through the
neuston and those so placed protected from such contact. T he presence or ab
sence of the bacteria of the neuston would appear to have little or no effect on the
settlem ent and subsequent growth of tubeworms on Teflon.
T he glass slides gained m ore mass, at a faster rate, than did the Teflon
slides. The differences between glass and Teflon were far greater than the differ
ences betw een w rapped and unw rapped slides in each case (betw een slides of
each m aterial). G lass is strongly hydrophilic, while Teflon is very hydrophobic;
their differing surface energies and consequently different w ettabilities could
effect different results in the attraction and attachm ent of planktonic foulers {e.g.,
R ittschof and Costlow, 1989). This effect might be direct, a result of the w ettabili
ty of the naked surface, or it might be indirect, an effect of the m icro-organism s
ad h eren t onto surfaces of differing w ettabilities (Schakenraad et a l, 1989). D if
ferent species' larvae show widely different preferences for wettability (Rittschof
and Costlow, 1989); the Fish H arbor foulers may be showing a similar disparity.
160
T he live-com m unity slides are the most difficult from which to draw con
clusions because of the uncertainty of their starting masses and the range of mass
gains resulting from this uncertainty. D espite this difficulty, som e clear trends
may be noticed, particularly using the m idpoints of the extra m ass-gain ranges.
Although they tend to increase as time passes, the ranges becom e relatively small
er w ith tim e, as th e m ass gains increase m ore rapidly; eventually the ranges
becom e indistinguishable from the mass ranges of the o th er treatm ents. T he
slides gain mass rapidly, although not exponentially, showing the familiar sigmoid
shape of the logistic curve. The head start that these slides enjoyed over the other
glass-slide treatm ents by having a living early fouling com m unity in place at the
beginning of the experiment made little difference in the longer run.
The uncoated slides bearing the killed early fouling community also had an
advanced start, but their com m unities' growth was then arrested by fixing, dehy
dration, and drying. N ot only did the living fouling com m unity have to resum e
from the beginning, with only the (unextracted) chemical and physical cues from
the previous community, but the killed community was subject to further dissolu
tion and extraction of what compounds were still present in the (organic) remains
of the organism s. Furtherm ore, the dead, dried organism s w ere quite brittle;
extra care was needed in handling them . It therefore comes as no surprise that
the uncoated slides lost mass im m ediately after deploym ent: losses (presum ably
from dissolution and perhaps abrasion as well) exceeded mass gains from settle
m ent and growth of new colonists. Once the initial deficit was overcome, howev
er, the uncoated slides gained mass rapidly and com pared favorably to the other
glass-slide treatm ents. W hat differences did exist w ere positive: the uncoated-
com m unity slides' new com m unities were slightly m ore massive than those that
colonized bare glass slides, while they were almost identical to those on the live-
161
com m unity slides. The presence of the killed pioneer com m unity m ade only a
small difference in the attraction and settlem ent of new colonists; the slides were
slightly attractive. The sum of the chemical and topological factors is thus a slight
attraction.
T he coated pre-fouled slides were the anomaly. T he coated slides lost
m ore mass and then gained m ore mass (with the possible exception of the live-
com m unity slides) than slides in any other treatm ent—up to three tim es m ore
than the treatm ent with the next-greatest gain. They also showed the most com
plete sigmoid curve. The initial mass losses certainly resulted from flaking away
of small portions of the A u/P d coating: a few silvery flakes were observed in the
w ater im m ediately after deploym ent of coated slides. The com pleteness of the
sigm oid curve may well be the result of the slides' having so m uch m ore growth
than the others— the com m unities they bore were simply closer to the carrying
capacity of the slides, having grown more rapidly during the course of the experi
m ent. T he growth was so great that the slides them selves w ere unrecognizable
b eneath the worm tubes late in the experim ent (see Figure 64). Fouling organ
isms, especially tubeworms, showed great affinity for the coated community. The
isolated physical (topological) factor would thus appear to be a strong attraction.
Com paring the uncoated and coated slides leads to the m ost surprising
conclusions in this experim ental set. The coating was planned to conceal the
chemistry of the previously-existing fouling community while preserving its m icro
scopic-level topology. This would separate two major possible avenues by which
the pioneer fouling community might exert its attractive effect on the later-arriv
ing organisms. If the chemical cues were m ore im portant than the physical cues in
the attraction of fouling organisms, the uncoated slides would be expected to show
greater growth than the coated slides. The degree of difference in growth results
162
would reflect the extent of the relative importance of the chemical cues: if physi
cal cues were of no importance whatever, the coated slides would experience mass
gains sim ilar to those of the plain (w rapped) glass slides. If, on the o th er hand,
the topological cues were m ore im portant, then the coated slides w ould not lag
behind the uncoated slides as much as the bare slides. If topological cues w ere
all-im portant, and chemical cues of no im portance at all, the coated and uncoated
slides, which both exhibit the topological effects of a pioneer fouling community,
would be expected to show identical patterns and am ounts of growth, each sur
passing the plain glass slides. The results of this series of experim ents do not fit
either of these scenarios. Instead, the "physical-only" test did far b e tter than did
the "both-chem ical-and-physical" and the "neither-chem ical-nor-physical" tests,
and "both" was only m arginally ahead of "neither" at the end of two m onths. It
would therefore appear that the physical effects of the pre-fouled slides are quite
strong, while the chem ical effects of these slides exerted a negative influence on
the Fish H arbor foulers, specifically the tubeworms. The sum of the physical and
chemical effects was only slightly more attraction than plain glass.
O n the pioneer and m icroscopic level, w rapping the slides m ade a differ
ence in their coverage with bacteria, but less of a difference than has som etim es
been reported (DiSalvo, 1973; Little, 1984). In no case was the observed bacterial
p o p u latio n density of the unw rapped slides as m uch as ten tim es th a t of the
w rapped slides (see Figure 69); in other areas, exposure to the surface layer has
been rep o rted to result in up to a thousand-fold increase in bacterial population
(Sieburth, 1965; DiSalvo, 1973). It is possible that wastes from the tuna canneries
at Fish H arb o r have enriched the local w ater to the point that local bacterial
populations, and especially distributions, are atypical of most of the m arine envi
163
ronm ent. A nother possibility is that grazing by bacteriovores reduced the popula
tion. As the experiment progressed, not only did the differences betw een the two
treatm ents decline, but the m ean bacterial population densities did as well. This
co u n terin tu itiv e resu lt may well be a side-effect of the success of the fouling
com m unity as a whole: as the slides becam e m ore covered, the increasingly few
areas w ith little enough grow th to allow censusing of b a c te ria directly on the
surfaces had correspondingly little settlem ent. Even the m arginal areas w ere
eventually overgrown. O f particular interest is the tendency of the bacteria on
these slides to be found in clumps (see Figures 71 and 73), particularly those on
unwrapped slides and especially in the early treatm ents. The clumps were proba
bly present in the surface film, and were transferred to the slides' surfaces w hen
the slides were deployed.
The presence or absence (and population density) of bacteria on a slide
m ade little difference in the size and makeup of its ultim ate invertebrate comm u
nity. The planktonic larvae of the macroscopic invertebrates arrived, settled, and
grew with little or no regard to the m icrobial condition of the slides. This is an
observation that has been made on other organisms in other locations (e.g.. Little,
1984). Roberts et a l (manuscript) report that invertebrate larvae can attach to a
new surface in the m arine environm ent as quickly as bacteria, and perhaps even
faster. T he b acteria would appear unnecessary, at least in som e cases. T heir
being the first organism s observed on fouling p lates and slides w ould fu rth er
appear to be an artifact of the methods used to place those objects in the water. If
an object is deployed unwrapped, it will almost of necessity pass through a layer of
bacteria in the neuston, many of which are certain to stick to the surface of the
object. Consequently, bacteria are the first organisms to appear on (unwrapped)
fouling slides.
164
4. The Double-blank Experiment
Plain, untreated glass slides gain mass through fouling alm ost as readily as
do plain glass slides coated with a layer of gold-palladium alloy. As time passes,
the co ated slides a p p ea r to take a sm all lead, but the d ifferen ce b etw een the
groups of unfouled (at the sta rt of the experim ent) slides is far less th an it is
betw een the groups of uncoated and coated previously-fouled slides. In com pari
son, the m etals used to form the coating have little or no effect on the rate of mass
increase attributable to the settlem ent and growth of fouling organisms.
This finding supports the contention of Dr. R obert F. Bils (pers. comm .)
that the alloy is non-reactive in and with seaw ater and suggests th at vacuum-
evaporation with gold and palladium results in the form ation of a coating surface
with negligible (or nearly so) chemical properties in seawater. Such a coating, and
the ability to place such a coating over a substrate while m aintaining the m icro
scopic roughness of the surface of the substrate, is of potential utility in fouling
studies, especially studies that concentrate on initial events in the form ation of a
fouling community. M ore to the point of this work, this finding should rem ove
suspicion that any m ore than a slight am ount of the additional m ass increases
enjoyed by the coated killed-community slides was an artifact of the alloy used in
the coating process. Even if such an artifact is given the full benefit of any doubt
rem aining after the double-blank experiment, the dominance of the coated fouled
slides is clear and definite.
E. Image analysis
The best image-processing algorithm depends on the nature and gray-scale
brightnesses of the substrate and fouling organisms thereto attached; which algo
rithm is the best therefore will vary with substrate and other circumstances. The
165
single best background for image analysis was brown Masonite, whose even, dark
color and ease of handling make it nearly ideal for tubeworm studies. The sheets
of M asonite used w ere too thin for practical routering of grooves, b ut several
sheets may be stacked and bolted together to give a sufficiently thick panel for
routering.
In counting calcareous structures such as worm tubes, which are generally
quite white, maximum success will be obtained by darkening the background (and
any non-target organisms) as much as possible. This will cause the worm tubes to
contrast with their background. If clear (e.g., glass) panels are used, colored paper
can be placed behind them to alter the color of the background. If the panels are
opaque, they may be darkened, in actuality or appearance, by m anipulation of the
cam era ap ertu re and lighting, by wetting the panel, and by using colored filters.
Some image-processing algorithms may also be used: the Sobel operator (Imaging
Technology, 1985b; Hader, 1988) serves to brighten object edges and darken more
hom ogeneous areas, but care must be exercised in selecting the quantizer bin size.
W hite Teflon and the white side of M asonite show erboard are unsuitable back
grounds against which to view worm tubes: the white tubes simply do not contrast
with the w hite surfaces. W hile a hum an counter may, with difficulty, be able to
discern the tubes, to a computer (or a television cam era) they simply disappear.
To avoid false readings, the background (panel face) should be as hom o
geneous as possible. For this reason, wooden panels are not ideal: the grain tends
to interfere with the edge detection process. Subtraction of an image of the grain
alone (taken from the unfouled panel) from the image of the fouled panel will
only work if the two images are identical and aligned exactly. A m isalignment by
so m uch as one pixel in any direction may not only defeat the purpose of subtrac
tion, but it may even worsen the problem . Panels m ade of m aterials of single,
166
even colors are m ore easily scanned. Glass, Lucite, and M asonite panels, with
their even com position, are thus preferable to such m aterials as wood. Panels
m ade of clear m aterials have an additional advantage in that colored paper may
be placed behind them, to manipulate the apparent background color and maxi
mize the contrast of the adherent organisms. Unfortunately, these m aterials have
a disadvantage in the ease with which they are cracked or scratched, and the slick
ness of their surfaces may be problem atic as well.
The conditions present during photography can also affect the composition
of the im age and thus the ease and accuracy with which the panels can be cen-
sused. As before, w ettable panels may be darkened by keeping them dam p, but
they may becom e shiny (reflective) if they are too wet. R eflections from such
organisms as ascidians may be avoided entirely be keeping the organisms dry, but
this runs counter to keeping the panel surface wet. Reflections (glare) from wet
ascidians can be reduced by illuminating the panel from the lowest angle possible
and using a polarizing filter on the camera. Extraneous light should be reduced as
much as possible. Averaging several images of the sam e panel can reduce elec
tronic "noise" and resulting aberrations. W ith luck (and experience), a photogra
pher may be able to make images requiring little or no image-processing m anipu
lation before they are scanned.
If the worm tubes are sufficiently dense or large that they touch each other
or overlap, it is difficult or im possible to count them individually (E rh ard t et a l,
1980, in H ader, 1988), which will reduce counts; this is one of the m ost difficult
problem s in electronic im age analysis today (Dr. R ichard Leahy, pers. comm.),
and is simply beyond the ability of Im ageM easure. A real coverage rem ains dis
cernible, however. Estimations and calculations of percent cover are therefore of
greater utility in this type of experiment. Not only are they much easier to obtain,
167
but they may be of greater intrinsic usefulness in dealing with colonial or highly
gregarious organisms. F or this reason, the general trend in the developm ent of
this autom ated system was away from counts of individuals and tow ard areal
coverage.
The discovery of the ideal panel materials and preparation m ethods, condi
tions for photography, im age-processing algorithm s, and com puter-scanning
p aram eters takes an enorm ous am ount of tim e and effort, even if the image-
analysis system works perfectly. This expenditure can be recouped through the
tim e savings realized in making scans of the panels, but the initial investm ent is so
large th at m any panel scans will be needed for recom pensation. As technology
im proves and operator experience increases, the initial investm ent may well be
lessened by a considerable amount.
168
V. Summary and Conclusions
A. Summaiy
O f the two dozen invertebrates whose extracts w ere tested for antibiotic
activity, two (both sponges) were generally inhibitory to the bacteria used; one
(Fasciospongia) was highly effective. Because the identities of the active princi
ples of these extracts are unknown, it is not possible to determ ine effective doses.
Ampicillin made little or no difference in invertebrate fouling of slides, be
they glass or T eflon or th eir p ain t fast-release or slow -release. Fish H a rb o r
tubeworm s do not appear to be affected, directly or indirectly, by the presence of
ampicillin on the surfaces available to them. In time, many of the slides lost paint,
indicating that bonding of paint to its substrate will be an im portant consideration
in antifouling work. G row th (mass increase) was roughly sigmoid, as far as it
could be traced ; d ifferen t rates of increase w ere a p p aren t, how ever. In each
comparison, plain-control slides gained more mass than painted slides, regardless
of the composition of the release paint. The difference was probably the result of
leaching from the paint. T here was little difference betw een the fouling of glass
and Teflon, except that Teflon held the paint better.
Very few bacteria were present on the slides (which w ere all deployed
w rapped), regardless of w hether or not they were treated with am picillin. The
growth of the tubew orm -dom inated fouling comm unity progressed despite the
lack of a bacterial community, indicating that a microbial community is not neces
sary for the settlem ent or m aturation of tubeworms.
T he spring results were much like the w inter results, but faster, with less
resultant paint loss. The winter slides had a problem with bryozoan accumulation,
while barnacles were a particular problem for the spring slides. These organisms
showed strong seasonality of relative fouling rates. The tubew orm s exhibited
169
seasonal fouling, but to a lesser extent. This seasonality is typical of many fouling
organisms.
Because m any invertebrate larvae possess the ability to colonize a site on
their own, it would appear that the oft-reported prim acy of bacteria and other
microbes on a fresh surface in the marine environment arises as a result of differ
ing population densities and abilities to arrive at the new surface. As most m arine
bacteria are found in the neuston, and slides deployed w ithout contact with the
neuston b e a r few er b a cteria than those deployed w ith such contact, it w ould
fu rth er a p p e a r th at the early ap p earan ce of b a c te ria on new surfaces in the
m arine environm ent is largely an artifact of the m ethods used to deploy these sur
faces. This artifact need not invalidate all fouling research, however: it is of prac
tical im portance because most objects placed in the sea are so placed through the
sea surface and its bacteria.
A gold-palladium coating of 300 nm thickness can coat m icrobes while
leaving their topology minimally masked; thus, the chemical and physical charac
teristics of the m icrobial fouling com m unity can be sep arated . T he coated-
com m unity slides gained far m ore mass than did uncoated-com m unity or plain
slides, indicating that the physical characteristics of the microbial fouling com m u
nity play a m ore im portant role than chemistry in the attractiveness of microbially-
fouled slides to larval invertebrates in Fish H arbor. This is not to say that current
attention to chemical factors is misguided; it merely overlooks another im portant
factor.
The wet and dry mass increases of the early and middle fouling community
can be correlated to one another. This allows mass increases to be determ ined
without im m ediate weighing of a wet fouling community and the attendant extra
equipm ent and procedures.
170
It is possible to su b stitu te com puter-driven im age analysis for m anual
counting of fouling panels and other fouled surfaces. There is a high initial time
cost of development, but this investment can be recovered in tim e saved in scan
ning many panels. The accuracy of com puter scanning is not yet as high as that of
m anual counting, but results are generally proportional, allowing the use of a
correction factor to yield acceptably accurate figures. With gregarious organisms
such as tubeworms, scanning for areal coverage often gives the best results.
B. Conclusions
T he "pioneer" bacteria do not seem to be necessary for the norm al devel
opm ent of the fouling community of Fish Harbor. The invertebrate m em bers of
the fouling community will appear, settle, and grow regardless of the presence or
absence of bacteria on a substrate.
Because these bacteria are not necessary, antibiosis does not effect anti
fouling goals. Even if the pioneer bacteria are killed or prevented from attaching
to a surface, invertebrate larvae can and will settle and attach. Although extracts
of tropical m arine invertebrates may have antibiotic an d /o r antifouling properties,
the two are not necessarily linked. Consequently, the classical "facilitation" model
of succession would not appear to apply to the Fish H arb o r fouling community.
The other im portant successional models may apply, but it is possible that a new
m odel is needed.
The physical (topological) characteristics of the microbial fouling commu
nity appear to exert a greater attractive effect on the Fish H arbor tubeworms than
does the net sum of the chem ical characteristics. Future investigations into the
factors attracting larval invertebrates should not ignore the physical characteristics
of surfaces.
171
Com puter-driven image analysis may be used in place of m anual counting
of fouling panels and other fouled surfaces, although a large-scale experim ental
program may be needed to make the substitution worthwhile. Scanning for areal
coverage often gives the best results for quantifying tubeworms.
172
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Creator Wright, Minturn Tatum, IV (author)

Core Title The role of microbes in the formation of the tubeworm fouling community in Fish Harbor, Los Angeles Harbor, California

School Graduate School

Degree Doctor of Philosophy

Degree Program Biology

Degree Conferral Date 1991-12

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biological sciences,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c30-250325

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Identifier DP23823.pdf (filename),usctheses-c30-250325 (legacy record id)

Legacy Identifier DP23823.pdf

Dmrecord 250325

Document Type Dissertation

Rights Wright, Minturn Tatum

Type texts

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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