'■■:■'

SCANNING ELECTRON MICROSCOPE
OBSERVATIONS OF MARINE MICRO-
ORGANISMS ON SURFACES COATED
WITH ANTIFOULING PAINTS

Patrick R. Kelly

NAVAL POSTGRADUATE SCHOOL

Monterey, California

THESIS

SCANNING ELECTRON MICROSCOPE
OF MARINE MICROORGANISMS ON
COATED WITH ANTIFOULING

OBSERVATIONS

SURFACES
PAINTS

by

Patrick R. Kelly

June 1981

Thesis

Advisor

: E

. C. Haderlie

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Scanning Electron Microscope Observations
of Marine Microorganisms on Surfaces
Coated with Antifouling Paints

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June 1981

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Patrick R. Kelly

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Naval Postgraduate School
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It. SUPPLEMENTARY NOTES

K. KEY WOROS fCanilnu* on ravaraa tlda II nacaataty and Idantlty by block nianbar)

Electron Microscopy
Marine microfouling
Marine fouling
Marine microorganisms
Primary film

Slime film
Microbial film
Antifouling paints

20. ABSTRACT (Conllnua on ravaraa alda li nacaaaawy and Idantlty oy block itumbar)

Scanning electron microscopy was used to observe microbiological
primary fouling of glass slides and slides coated with U. S. Navy
antifouling paints exposed in Monterey harbor. Four paints were
tested, three of which contained copper or tin as their toxic
ingredient and one which used a chlorinated pesticide, an organic
compound, as the anti-fouling ingredient. Samples removed at
regular intervals, of days up to several weeks, showed that

DO , :°r7, 1473
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#2 0 - ABSTRACT - CONTINUED

bacterial slimes populated the glass and heavy-metal based
paints early and in great numbers throughout the study, but
the surfaces painted with the organic compound toxicant
were free of all microfouling organisms. A succession of
periphytic microorganisms was observed on glass and the heavy-
metal based painted surfaces which began with bacteria followed
by diatoms and later by protozoans.

>D 1 J_aOj3_.14!-.. o UNCLASSIFIED

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Scanning Electron Microscope Observations
of Marine Microorganisms on Surfaces
Coated with Antifouling Paints

by

Patrick R. Kelly
Lieutenant Commander, United States Navy
B.S., United States Naval Academy, 1971

Submitted in partial fulfillment of the
requirements for the degree of

MASTER OF SCIENCE IN METEOROLOGY AND OCEANOGRAPHY

from the

NAVAL POSTGRADUATE SCHOOL
June 1981

ABSTRACT

Scanning electron microscopy was used to observe micro-
biological primary fouling of glass slides and slides coated
with U. S. Navy antifouling paints exposed in Monterey harbor.
Four paints were tested, three of which contained copper or
tin as their toxic ingredient and one which used a chlorinated
pesticide, an organic compound, as the anti-fouling ingredient.
Samples removed at regular intervals, of days up to several
weeks, showed that bacterial slimes populated the glass and
heavy-metal based paints early and in great numbers throughout
the study, but the surfaces painted with the organic compound
toxicant were free of all microfouling organisms. A succession
of periphytic microorganisms was observed on glass and the
heavy-metal based painted surfaces which began with bacteria
followed by diatoms and later by protozoans.

TABLE OF CONTENTS

I. INTRODUCTION 14

A. GENERAL DISCUSSION OF MICROFOULING 14

1. Brief History of Microfouling Research - 16

B. MOLECULAR FOULING 16

C. SORPTION OF MICROFOULING ORGANISMS 18

1. Bacterial Film Formation 18

2. Sorption of Diatoms and Other
Microfoulers 20

D. PRIMARY FILM AND SUBSEQUENT MACROFOULING 21

II. OBJECTIVES 23

III. MATERIALS AND METHODS 25

A. GENERAL 25

1. Antifouling Paints 25

B. EXPERIMENT 1 26

1. Experimental Substrates 26

2. Sample Collection 30

3. Sample Preparation 33

a. Fixation 34

b. Dehydration 34

c. Freeze Drying 35

d. Gold Coating 35

C. EXPERIMENT 2 36

1. Experimental Substrates 36

2. Sample Collection 37

3. Sample Preparation 37

D. EXAMINATION USING THE SEM 38

IV. RESULTS 40

A. EXPERIMENT 1 40

1. General 40

2. Organisms Sorbed to Glass 43

3. Organisms Sorbed to Formula 196D 48

4. Organisms Sorbed to Formula 1114 48

5. Temperatures and Salinities 49

B. EXPERIMENT 2 49

1. General 49

2. Organisms Sorbed to Glass 53

3. Organisms Sorbed to Formula 196D 55

4. Organisms Sorbed to Formula 1114 56

5. Organisms Sorbed to Formula 170 57

6. Organisms Sorbed to Formula 121 58

7. Temperatures and Salinities 59

V. DISCUSSION 61

VI. CONCLUSIONS 68

APPENDIX A: NAVY STANDARD FORMULA 121 RED VINYL

ANTIFOULING PAINT 70

APPENDIX B: NAVY STANDARD FORMULA 170 BLACK

CAMOUFLAGE VINYL ANTIFOULING PAINT "71

APPENDIX C: DTNSRDC EXPERIMENTAL ANTIFOULING

PAINT FORMULA 2844-1114 "72

APPENDIX D: ORGANOTIN EPOXY GEL FORMULA 19 6D

DTNSRDC EXPERIMENTAL ANTIFOULING PAINT 73

APPENDIX E: PHOTOGRAPHS 74

LITERATURE CITED 126

INITIAL DISTRIBUTION LIST 130

6

LIST OF FIGURES

1. Microscope slide box 28

2. Exposure array showing the arrangement of

slide boxes 29

3. Location of test site 31

4. Relationship of arrays to sea surface and

each other 32

5. Numbers of bacteria sorbed to surfaces

in Experiment 1 44

6. Temperature and salinity values recorded

during Experiment 1 50

7. Numbers of bacteria sorbed to various

surfaces in Experiment 2 54

8. Temperature and salinity values recorded

during Experiment 2 60

9. Results of bacterial attachment seen on
glass in Experiments 1 and 2 and results
of similar tests made on glass or plexi-
glass by three different research groups 62

LIST OF TABLES

1. Settlement Sequence of Dominant Fouling

Organisms observed in Experiment 1 41

2. Settlement Sequence of Dominant Fouling

Organisms observed in Experiment 2 51

LIST OF PHOTOGRAPHS

Plate

1. Glass coated with paint Formula 196D immersed

6 days, 3000X. Rod-shaped bacteria 74

2. Glass immersed 6 days, 3000X. Rod-shaped

and ring-forming bacteria 75

3. Glass immersed 8 days, 1500X. Two centric
diatoms, filamentous microorganisms, and
rod-shaped bacteria 76

4. Glass immersed 14 days, 5000X. Two

vorticellid protozoans 77

5. Glass immersed 83 days, 2000X. Close-
up of a spiral-stalked protozoan 78

6. Glass immersed 26 days, 1500X. Pennate
diatom, filamentous microorganisms,

bacteria, and debris 79

7. Glass immersed 22 days, 1500X. Unusual

mass of cylindrical objects 80

8. Glass immersed 49 days, 500X. The

colonial ciliated protozoan Zoothamnium 81

9. Glass immersed 83 days, 500X. Two shelled
protozoans called foraminifera 82

10. Glass immersed 49 days, 30X. Bryozoan

colony surrounded by protozoans 83

11. Glass immersed 49 days, 150X. Close-up
of a bryozoan colony showing four

opercula 84

12. Glass immersed 83 days, 3000X. Centric

diatom with a coccolith plate 85

13. Glass immersed 49 days, 280X. A
Zoothamnium colony and some singular

protozoans 86

14. Glass immersed 49 days, 700X. The protozoan
Acineta tuberosa 87

15. Glass immersed 49 days, 940X. Protozoan

possibly of the genus Acineta 88

Plate

16. Glass immersed 49 days, 110X. Two hydroids

of the genus Obelia surrounded by protozoans 89

17. Glass immersed 49 days, 250X. Close-up

of the hydroid Obelia 90

18. Glass immersed 49 days, 130X. Large unknown
organism surrounded by protozoans 91

19. Glass immersed 83 days, 1600X. The

planktonic diatom Nitzszhia closterium 92

20. Glass immersed 83 days, 2000X. The skeleton
of a single-celled flagellated plant known

as a silicof lagellate 93

21. Glass immersed 83 days, 300X. Several
peritrichous ciliates within protective
loricas or houses and two unknown

ciliated bodies 94

22. Glass immersed 83 days, 2700X. Close-up of

an unknown ciliated body 95

23. Glass coated with paint Formula 19 6D immersed
30 days, 1500X. Rod-shaped bacteria, debris,

and a filamentous microorganism 96

24. Glass coated with paint Formula 196D immersed
10 days, 1500X. Two planktonic diatoms,
bacteria, and debris 97

25. Glass coated with paint Formula 196D immersed
83 days, 800X. Several centric diatoms, a

broken coccolithophorid, and debris 98

26. Glass coated with paint Formula 196D immersed
83 days, 1500X. Unknown bulbous-like

organism 99

27. Glass coated with paint Formula 19 6D immersed
83 days, 1000X. Sponge spicule possibly from

the genus Leucosolenia 100

28. Glass coated with paint Formula 1114 immersed
4 days, 1500X. Surface composed of paint
artifacts, salt crystals, and some debris 101

29. Glass coated with paint Formula 1114 immersed
83 days, 1000X. The planktonic diatom,
Biddulphia longicruris 102

10

Plate

30. Glass coated with paint Formula 1114 immersed
83 days, 4000X. Close-up of the diatom

Biddulphia longicruris 103

31. Glass coated with paint Formula 1114 immersed
83 days, 1500X. Several broken diatoms of

the genus Thalassiosira 104

32. Glass immersed 14 days, 500X. Linked

rectangular diatoms of the genus Thalassionema 105

33. Glass immersed 14 days, 8 00X. Centric diatoms,
Skeletonema costatum on the surface of a worm

tube casing 106

34. Glass immersed 14 days, 1500X. Unknown object
possibly a transparent sack containing eggs 107

35. Glass immersed 14 days, 700X. Unknown

attached organism 108

36. Glass immersed 30 days, 300X. One colonial
protozoan surrounded by several solitary

protozoans 109

37. Glass immersed 30 days, 1500X. Protozoans

known as choanof lagellates 110

38. Glass immersed 30 days, 38 OX. Unknown

ciliated organism 111

39. Glass coated with paint Formula 196D immersed
24 hours, 400X. Salt and other crystals

forming an unusual geometric pattern 112

40. Glass coated with paint Formula 196D immersed
30 days, 1500X. A foraminifera surrounded

by broken diatoms 113

41. Glass coated with paint Formula 196D immersed

30 days, 100X. Worm tubes 114

42. Glass coated with paint Formula 170 immersed

4 days, 1500X. Bacteria 115

43. Glass coated with paint Formula 170 immersed
8 days, 600X. Centric diatom, Skeletonema

costatum 116

44. Glass coated with paint Formula 17 0 immersed
30 days, 1500X. Coccolithophoroid with a few

plates missing 117

11

Plate

45. Glass coated with paint Formula 121 immersed
6 days, 5000X. Pennate diatom possibly

Amphora 118

46. Glass coated with paint Formula 121 immersed
6 days, 8000X. Close-up of Skeletonema

costatum 119

47. Glass coated with paint Formula 121 immersed
6 days, 4000X. The planktonic diatom,

Chaetoceros radicans 120

48. Glass coated with paint Formula 121 immersed
14 days, 600X. Diatoms, protozoans, and

some debris 121

49. Glass coated with paint Formula 121 immersed

30 days, 4000X. Stalked, ciliated protozoan 122

50. Glass coated with paint Formula 121 immersed

30 days, 3000X. Coccolithophorid 123

51. Glass coated with paint Formula 121 immersed

30 days, 12,000X. Coccolithophoroid 124

52. Glass coated with paint Formula 121 immersed

30 days, 10,000X. Coccolithophoroid 125

12

ACKNOWLEDGMENTS

I express my indebtedness to Mr. Chris Patton (Hopkins
Marine Station) for his patience and guidance in teaching me
the operation of the scanning electron microscope and provid-
ing valuable assistance and advice; to Miss Bonnie Hunter
(Naval Postgraduate School, Monterey) who worked with me
throughout the study providing the necessary laboratory and
technical help; to Professors Isabelle A. Abbott (Hopkins
Marine Station) and Thomas D. O'Neill (Ventura College) for
their assistance in identifying many of the organisms I
photographed; to Mrs. Jean Montemarano and Mr. Steve Rogers
(David W. Taylor Naval Ship Research and Development Center)
for providing detailed information on the paints tested; to
Mr. Vincent Costelli (David W. Taylor Naval Ship Research and
Development Center) for providing the requirements for this
project and supplying the paints for testing; to Professor
Eugene C. Haderlie (Naval Postgraduate School, Monterey) for
his guidance and many valuable suggestions; and finally to
my wife for her understanding and help during the course of
this study.

13

I. INTRODUCTION

A. GENERAL DISCUSSION OF MICROFOULING

Marine microfouling is a process which involves the inter-
action of living and non-living materials with solid surfaces
submerged in seawater resulting in the establishment of a
complex film (Corpe, 1977) . This film, composed mainly of
bacteria and diatoms plus secreted extracellular materials
and accumulated debris, has been referred to as the "primary
film", "bacterial fouling film", or "slime layer" (Horbund
and Freiberger, 1970) . The latter name has been applied because
the film ultimately becomes thick enough to feel slippery or
slimy (Haderlie, 1977) . The composition of this film may
also include yeasts, fungi, and protozoans. The settlement
sequence of these constituents and the specific make-up of
the film depends on several factors including location, season
and year, depth, and proximity to previously fouled surfaces
(O'Neill and Wilcox, 1971) and other physico-chemical
parameters .

Early investigators noted that the presence of a slime
film seemed to facilitate the attachment of larger fouling
organisms such as bryozoans and barnacles (Woods Hole Oceano-
graphic Institution, 1952; Horbund and Freiberger, 1970;
Zobell, 1939) . If the nature of the film was understood and
if its characteristics controlled, fouling by macroscopic
organisms might be substantially reduced. Aside from the

14

obvious benefits that control of macrof ouling would have to
shipping and other areas of marine engineering, control of
the growth of primary slime film itself has become an area
of concern with regard to Ocean Thermal Energy Conversion
(OTEC) Systems and long term oceanographic instrumentation
(Dexter, 1977) .

Corpe (1977) stated that fouling by microbial films have
two broad functions, (a) to provide a surface favoring the
settlement and adhesion of animal larvae and algal cells, and
(b) to provide a rich source of both particulate and soluble
food material that could sustain and/or enhance the development
of fouling populations. Further, Zobell (1939) wrote that
bacteria might promote the fouling of submerged surfaces by:

1. Affording the larval forms of larger fouling organisms
a foothold or otherwise mechanically facilitating
their attachment.

2. Serving as food.

3. Discoloring bright or glazed surfaces.

4. Increasing the alkalinity of the film-surface interface
thereby favoring the deposition of calcareous cements.

5. Influencing the e.m.f. potential of the surface.

6. Increasing the concentration of plant nutrients at the
expense of the organic matter which the bacteria
decompose.

The study of microf ouling organisms, therefore, is an impor-
tant contribution to the complete understanding of marine
fouling.

15

1. Brief History of Microfouling Research

The study of marine microfouling has its roots in the
1930' s and 1940 's with research primarily conducted by Zobell
and his colleagues. Zobell' s research focused primarily on
the early stages of settlement and growth of microorganisms,
particularly bacteria on solid substrates immersed in the sea.
From that time until the early 19 60"s only sporadic studies
were conducted because there were very few investigators
interested in the bacteriology of the ocean. Since the mid-
1960' s and especially since 1970 research has increased tre-
mendously in marine bacteriology and the broad problems of
bioadhesion in general and specifically in the initial stages
of microfouling on solid substances (Haderlie, 1977).

Notable contributions to our fundamental understanding
of marine periphytic bacteria and primary bacterial films
have been made by Corpe and his colleagues at Columbia Uni-
versity, and Mitchell and his co-workers at Harvard. In addi-
tion, Baier of Cornell and Neihof and Loeb at the Naval Re-
search Laboratory have helped explain the nature of the
"molecular fouling" layer that precedes the attachment of
bacteria to solid surfaces in the sea. The work, of these
investigators and many others will be drawn upon in discussing
the results of the experiments conducted in the present study.

B. MOLECULAR FOULING

Molecular fouling or "surface conditioning" is the sorption
of organic matter dissolved or suspended in sea water to solid

16

surfaces. This was first demonstrated by Zobell (1943) and
since has been confirmed by many other investigators including
Loeb and Neihof (1975, 1977). The sorption of dissolved
material creates changes in the surface of the substrate (con-
ditioning) which are favorable for subsequent biological
settlement.

Organic materials dissolved in sea water originate as the
end-products of bacterial decay, execretory products, dissolu-
tion from seaweeds, etc., and consist principally of sugars,
amino acids, urea, and fatty acids (Taylor, 1977). Much of
this non-living organic matter in the sea occurs in the form
of small aggregates which are formed by the sorption of dis-
solved organic matter upon bubbles and other naturally-
occurring surfaces (Riley, 1963) .

Loef and Neihof (1975, 1977) have determined that molecular
films may form within minutes after the substrate enters the
water and continue to grow in thickness, leveling off after
about 20 hours. They confirmed that molecular films were
organic, electronegative, and composed of humic materials.

Baier (1973) determined that the initial events in biologi-
cal adhesion are influenced by the texture, chemistry, and
charge of the substrate surface. He reported that the adhesion
of organisms to surfaces submerged in sea water depended in
large measure on the "critical surface tension" or wettability
of the substrate. When any clean solid object, whether it is
glass, metal, wood, stone, or plastic, is immersed in natural

17

seawater, a layer of non-living organic matter immediately
sorbes to the surface (Loeb and Neihof , 1975, 1977) . This
initial non-living film is a monolayer of glycoprotein which

o

in a few hours may develop to a thickness of 200 A (Baier,
1973) . As molecules are absorbed they are changed from a
3-dimensional to a 2-dimensional form thus modifying their
reactivity. The "critical surface tension" of the coated sur-
face may be modified so that strong bonding is possible with
the mucopolysaccharides exuded by the film forming bacteria
(Baier, 1973) .

C. SORPTION OF MICROFOULING ORGANISMS
1 . Bacterial Film Formation

Bacteria have been found securely attached to sub-
strates immersed in seawater after just a few hours (Corpe,
19 72; Dempsey, 19 81; Dexter, 1977; Gerchakov et a., 197 6;
O'Neill and Wilcox, 1971; Zobell and Allen, 1935). Two stages
have been identified in the development of bacterial fouling
communities, initial colonization by rod-shaped bacteria,
followed by stalked forms within 24 hours (Marshall et al . ,
1971b) to 48 or 72 hours (Corpe, 1973; Dempsey, 1981) .

The initial colonizers have been identified as psuedo-
monads, principally species of Psuedomonas , Flavobacterium,
and Achromobacter (Corpe, 1973; Corpe and Winters, 1972;
O'Neill and Wilcox, 1971). Gram-negative species dominated
the populations with only 10 to 15 percent being gram-positive
(Dempsey, 1981) . The secondary colonizers, in general, were

18

stalked, budding, or filamentous types identified as species
of Caulobacter , Hypomicrobium and Saprospira (Dempsey, 1981;
Marshall et al., 1971b). Zobell and Allen (1935) reported
that between 40 and 50 species of marine bacteria could be
isolated from the surface of glass slides immersed in seawater
for a few days .

Marshall et al., (1971a) has defined two stages of
sorption of bacteria to solid surfaces. Reversible sorption
is an essentially instantaneous attraction of bacteria to a
surface. Such bacteria are held weakly near the surface; they
still exhibit Brownian motion and are readily removed by
washing the surface. Irreversible sorption involves the firm
adhesion of bacteria to the surface; they no longer exhibit
Brownian motion and are not removed by washing.

The first stage, reversible sorption, involves physi-
cal forces which attract the cell to the surface. These forces
include Van Der Waals forces of mass attraction and electro-
static forces due to the interaction between ionic groups on,
or surrounding, the approaching cell and substrate surfaces
(Dempsey, 1981) . There is an apparent equilibrium condition
between the electrostatic repulsion forces and the attractive
influences of the Van Der Waals forces.

In irreversible sorption the bacteria, especially rods,
produce extracellular bridging material called acid mucopoly-
saccharides (Corpe, 1970b) . These high molecular weight poly-
mers are believed to be important in the firm adhesion of the

19

organism to solid surfaces (Corpe, 1975) . Adhesive extra-
cellular polysaccharides have been observed with the aid of
the transmission electron microscope (TEM) (Marshall et al.,
1971a) and the scanning electron microscope (SEM) (DiSalvo
and Daniels, 1975; Gerchakov et al. , 1977) . These polymers
are quite resistant to dislodgement and appear to be sticky
in nature as evidenced by the accumulation of algae and bits
of debris on their surfaces (Corpe, 1970b). The tackiness of
this secretion may encourage the settlement of other fouling
species by providing a base to which organisms may readily
attach and obtain nourishment.

Although investigations of the role of surface and
extracellular polymers in bacterial aggregation has generally
implicated polysaccharides, other polymeric material, notably
nucleic acids and proteins typically excreted or introduced
to the medium by cellular lysis, frequently have been shown
to play a significant role in bacterial aggregation (Harris
and Mitchell, 1973) .

After attachment the bacteria reproduce by binary
fission; each half grows to an average size of 1 to 2 urn and
divides again. Bacterial counts on developing colonies indi-
cate that at 20 degrees centigrade a population can double
every 4 hours (Woods Hole Oceanographic Institution, 1952).
2 . Sorption of Diatoms and Other Microfoulers

Following the establishment of the initial film of
bacteria and their secreted extracellular polymers on a solid

20

substrate, additional bacteria and other microorganisms may
attach. Debris, organic material, and other particular matter
may also adhere to the surface creating an environment of
intense biochemical activity. The other microfoulers which
may now settle include benthic diatoms, filamentous micro-
organisms, and protozoans. These organisms, especially the
diatoms, may contribute to the so-called primary film and
eventually become a distinctive part of the microfouling
community (Corpe, 1972; Woods Hole Oceanographic Institution,
1952). These diatoms, which can also live in suspension in
the sea, are unicellular plants encased in siliceous shells.
Although the presence of a bacterial film may facilitate the
attachment of diatoms it is not essential. Like bacteria,
reproduction and colonization by diatoms is a rapid process
(Woods Hole Oceanographic Institution, 1952). The colonial
diatom Lichmophora and the colonial ciliate protozoan
Zoothamnium are often conspicuous as microfoulers (Haderlie,
1977) .

D. PRIMARY FILM AND SUBSEQUENT MACROFOULING

Many studies have reported on the existence of some kind
of ecological succession of fouling communities beginning with
film forming bacteria, diatoms, and protozoans, and ending
with barnacles, tunicates, mussels, and seaweeds (Haderlie,
1974; Horbund and Freiberger, 1970; O'Neill, 1975; Woods Hole
Oceanographic Institution, 1952). There seems to be a general
agreement among investigators in this area that one group of

21

organisms in some way changes or conditions the surface so
that a second community can develop, and so on to the climax
fouling community (Haderlie, 1977). However, it has not yet
been proven that microfouling is a necessary prerequisite of
heavy, destructive fouling, since barnacles and other such
organisms have not been cultivated in the complete absence
of microorganisms (Corpe, 1977).

22

II. OBJECTIVES

The primary objective of this study was to determine how
well four U. S. Navy antifouling paints function in preventing
or limiting the attachment of microfouling organisms in
Monterey Bay. Three of the four paints tested were experi-
mental and as such an evaluation of their ability to prohibit
the formation of "slime film" has not yet been made. The
project was proposed by the David W. Taylor Naval Ship Research
and Development Center, Annapolis, Maryland to fulfill a Navy
requirement to evaluate these paints.

One of the four paints, Formula 121, also known as Copper
Oxide, was not an experimental paint but has been in general
use by the Navy for many years. This paint's effectiveness
against micro and macrofoulers has been determined by other
researchers and was tested here again for comparison with the
other experimental paints.

Microscope slides were coated with the paints and exposed
to the harbor waters in Monterey for varying time intervals.
A scanning electron microscope (SEM) was used to view the
samples. The slides were examined to identify the attached
organisms to genus and species where possible and to get a
rough quantitative estimation of density of settlement of

V. J. Costelli, phone interview, June 1980. Mr. Costelli
is the senior task scientist in charge of controlled release
polymer formulations for antif ouing paints .

23

some of them. From the types and numbers of organisms found
on the test surfaces, a determination was made as to how
effective each paint was in preventing microfouling .

Secondary objectives included verifying the sequence of
microfouling organisms on uncoated glass slides in Monterey
Harbor, developing field test procedures for exposing anti-
fouling paints, and evaluating techniques for the employment
of the SEM in observing microorganisms on such surfaces .

24

III. MATERIALS AND METHODS

A. GENERAL

Two experiments were carried out. Experiment 1 tested
two antifouling paints and ran from 18 November 1980 to 17
February 1981. Experiment 2 tested four antifouling paints
and was conducted from 18 February to 2 0 March 1981. The
experiments consisted of suspending glass microscope slides
coated with antifouling paints in the waters of Monterey Bay
for periods ranging from 24 hours to several weeks. After
recovery from the water the slides were chemically fixed and
then prepared for viewing under the Scanning Electron Micro-
scope (SEM) . Observations and photographs of the slide sur-
faces made with the SEM were used in determining general num-
bers and kinds of organisms present for each immersion period

Prior to Experiment 1, tests were conducted to determine
the best laboratory and field test methods to be followed.
As expected, some difficulties developed resulting in the
elimination of two antifouling paints in the first experiment
These problems, however, were resolved and all four paints
were tested in the second experiment.

1 . Antifouling Paints

The four antifouling paints evaluated in this study
included:

1. Navy Standard Formula 121 Red Vinyl Antifouling Paint.
This paint incorporates cuprous oxide as the antifouling

25

ingredient and is intended for use on shipbottom exterior
surfaces (Appendix A) .

2. Navy Standard Formula 170 Black Camouflage Vinyl Anti-
fouling Paint. The antifouling ingredients of this paint
include tributyltin oxide and tributyltin fluoride (Appendix
B).

3. DTNSRDC Experimental Antifouling Paint Formula 2844-
1114. This is a two component, lead peroxide cured poly-
sulfide antifouling paint. It is intended for use primarily
on rubber, incorporating Nopcocide N-96 (2 , 4 , 5 , 6, -tetrachloro-
isophthalonitrile) as the antifouling ingredient (Appendix C)

4. Organotin Epoxy Gel Formula 196D DTNSRDC Experimental
Antifouling Paint. This is a two component paint intended
for use on glass reinforced plastic (GRP), incorporating
Tributyltin as the antifouling ingredient (Appendix D) .

B. EXPERIMENT 1

Experiment 1 tested two experimental antifouling paints,
Formula 1114 and Formula 196D.

1. Experimental Substrates

Standard glass microscope slides one inch by three
inches were used as the surfaces upon which the paints were
applied and exposed to seawater. Glass slides were used be-
cause they were easy to manipulate throughout all phases of
testing and because results, especially from the uncoated

David Taylor Naval Ship Research and Development
Center.

26

glass slides, could be compared with similar work performed
by other investigators including Corpe (e.g., 1970a, 1970b,
1972, 1975, 1977), Dempsey (1981), Dexter (1976), DiSalvo and
Daniels (1975), Gerchakov et al., (1976), Tosteson and Corpe
(1975), Winters and Corpe (1972), and Zobel (1939). Corpe
(1970a) noted that glass slides have been used by many inves-
tigators as the traditional method for study of aquatic bac-
teria because they can be examined by microscope and cultures
can be isolated from the surfaces.

Common nylon paint brushes 2.5 centimeters wide were
used to apply the paints to the slide surfaces. Care was
taken to ensure that no oil or grease contaminated the slides
and interfered with paint adhesion. Two coats of paint were
applied directly over the smooth glass surfaces. The slides
were air-dried at a temperature of about 18.0 degrees centi-
grade for a minimum of five days before exposure in the water.

Microscope slide boxes of 100 slide capacity were used
to hold the slides during field exposure. To ensure a free
flow of water around the slides two windows 18.0 centimeters
by 4 . 0 centimeters were cut out of the front and back of the
boxes (Figure 1) . Two slide boxes were assigned to hold the
coated slides for each paint. A maximum of six slides were
placed in each box. Limiting the capacity of each slide box
allowed sufficient horizontal separation of approximately 8.0
centimeters between samples to avoid concentration of anti-
fouling toxicant between adjacent slides.

27

£=k

win i inn 1 1) Vi i ii i p j \ \^\

wnmnm gnmnnia

Figure 1. Microscope slide box showing
positions of painted slides

Two boxes assigned to each paint were attached to
a polyvinylchloride (PVC) pipe by nylon cord so that the boxes
were fixed about 31.0 centimeters apart. The entire assembly
was suspended by polyethylene rope which had a 14.0 kg weight
on the lower end (Figure 2) .

All of the uncoated control glass slides were held in
one slide box, each slide separated from one another by about
2.5 centimeters. The control slide box was attached directly
to the polyethylene rope and anchored with a 14.0 kg weight.
The arrangement of the slide box(s) with the rope, weight,

28

Figure 2 . Exposure array showing the
arrangement of slide boxes

29

with or without PVC pipe, will hereafter be referred to as
an array .

The boxes with slides were exposed with the slides
oriented vertically. A horizontal arrangement of slides was
not considered necessary because other investigators, especially
O'Neill (1975), have shown that the number of microfoulers on
horizontally placed slide surfaces did not differ from ver-
tically arranged slides having the same immersion periods.

The arrays were suspended in Monterey harbor beneath
the tide station of Muncipal Wharf No. 2 (Figure 3) . This
location was in a shaded site which seldom received direct
sunlight. The arrays were anchored in the water so that the
slide boxes were 3.0 meters from the bottom and approximately
3.5 meters below mean lower low water. The control array and
the arrays containing painted slides were separated by a mini-
mum horizontal distance of 1.5 meters to limit the possibility
of contamination between adjacent sets of arrays (Figure 4) .
2 . Sample Collection

One uncoated control slide and one sample from each of
the two paints under study were removed after immersion periods
of: (1) 2 4 hours, (2) 4 8 hours, (3) 4 days, (4) 6 days,
(5) 8 days, (6) 10 days, (7) 14 days, (8) 18 days, (9) 22 days,
(10) 26 days, (11) 30 days, (12) 49 days, and (13) 83 days.
Because initially the total number of slides exposed for each
paint was limited to twelve, the long-range test of 8 3 days
did not begin on 18 November 1980. Slides for this test were

30

CHART OF
MONTEREY HARBOR

Figure 3. Location of test site (denoted by arrow)

31

0 --

DEPTHS

IN
METERS

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MEAN LOWER-LOW WATER

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FORMULA 19 6D FORMULA 1114 CONTROL

Figure 4. Relationship of arrays to sea
surface and each other

32

put into the exposure boxes on 26 November 1980. Since four
sample recovery periods had passed, the addition of these
slides still allowed for more than sufficient horizontal sep-
aration between samples to avoid the concentration of toxicant

These exposure periods were chosen because the early
periods matched exposure times of other investigators. Immer-
sion periods from 24 hours up to and including the 14th day
of this experiment match the exposure times of O'Neill (1975)
in his research into primary film formation. This permitted
comparisons to be made with O'Neill's work as well as that of
other investigators including Corpe (1972), Dempsey (1981),
Gerchakov et al., (1976), O'Neill (1971), O'Neill and Wilcox
(1971), and Marshall et al., (1971).

To avoid artificially increasing the bacterial popu-
lations of the exposed slides, the sample boxes were recovered
and returned to the water sealed within plastic bags , avoid-
ing contact with the neuston at the air-sea interface.

Water samples for salinity determination and surface
bucket temperatures were taken at the exposure site every two
days up to and including exposure period 11 (day 30) of Experi-
ment 1. The seawater samples were tested for salinity using
a Plessey Salinometer Model 6230N. Temperatures were deter-
mined using a standard bucket thermometer.
3 . Sample Preparation

After the slide samples were removed from the water
they were chemically preserved and prepared for study under
the SEM. The steps followed in sample preparation included:

33

a. Fixation

b. Dehydration

c. Freeze-drying

d. Gold plating

a. Fixation

Immediately after recovery from the water the
slides were put into a coplin jar containing two percent
gluteraldehyde and fixed for two hours. The gluteraldehyde
was diluted with filtered seawater from the exposure site.
A 0.2 ym milipore filter was used to filter the water. Dilu-
tion with this seawater was done to ensure an isotonic environ-
ment for fixation.

b. Dehydration

The dehydration process was employed in order to
preserve the shape of the microorganisms through the freeze-
drying process. Following fixation the samples were washed
in distilled water several times and then immersed in a graded
series of aqueous acetone (dimethyl ketone) solutions, for
five minutes in each concentration. The acetone was diluted
with distilled water. The dehydration sequence followed was
consistent with experimental procedures of other researches
Hayat (1978) and Taylor (1977) . The dilutions used were as
follows:

1. 10 percent acetone

2. 30 percent acetone

3. 50 percent acetone

34

4. 70 percent acetone

5. 90 percent acetone

6. 100 percent acetone (2 changes)

Slides coated with Formula 1114 and Formula 196D
showed varying degrees of blistering and cracking after dehy-
dration. This effect became more pronounced as the immersion
time in the water increased. This problem, however, did not
exclude any slides from being studied under the SEM. The
other two antifouling paints which were to be tested in Experi-
ment 1, Formula 170 and Formula 121, were eliminated from this
test because dehydration in either acetone or another similar
solution, methyl alcohol, severely affected the painted sur-
faces. Formula 170 was completely dissolved in both dehydra-
tion agents while the entire surface of slides painted with
Formula 121 cracked and pealed when dipped into liquid nitrogen
following dehydration.

c. Freeze Drying

Following the dehydration step, slides were plunged
into liquid nitrogen and freeze-dried at -50°C for four hours
in a model 10-141 Unicool, manufactured by the Virtis Company,
Gardiner, New York. After freeze drying the samples were
stored in a desiccator until final preparation for viewing
under the SEM.

d. Gold Coating

Nonconductive specimens, like those examined in
this study, cannot rapidly channel the excess primary electrons

35

away from the scanned area and a local charge may build up on
the specimen's surface. This increases abnormally the second-
ary electrons emitted to the collector and creates a localized
glow which destroys the imaging of the microscope (Hayat,
1978). To avoid this situation, an extremely thin conductive

o

coating of gold, approximately 100 A thick, was applied to
the slides by vaporizing a gold disc in an evacuated chamber.

The plating process was accomplished by a DSM-5
Cold Sputtering Module mounted inside a bell jar vacuum evapor-
ator known as a Denton DV-502. This equipment was manufact-
ured by the Denton Vacuum Corporation, Cherry Hill, New Jersey.
The size of the pedestal upon which the plating was done per-
mitted only one slide to be coated at a time. Due to the ex-
pense associated with the process, all slides were cut in
half in order to plate one side of two different samples at
a time.

C. EXPERIMENT 2

Experiment 2 tested all four paints of interest:

1. Formula 170

2. Formula 1114

3. Formula 19 6D

4. Formula 121

1. Experimental Substrates

Because of paint adhesion problems experienced in
Experiment 1, the surfaces of all the glass slides to be
painted were roughened prior to coating. This was accomplished

36

by passing them across a belt sander. The texture of the
painted slide surfaces, however, showed no evidence of the
slightly roughened surface below and had the same texture as
the paint applied to smooth glass. Paint on the surfaces of
the slides in Experiment 2 adhered well, showing no signs of
the cracking which occurred in Experiment 1.

The slides were painted and exposed to the water in
the same manner as in Experiment 1, with the exception that
there were now a total of five arrays involved in the test.

2 . Sample Collection

Sample collection steps followed exactly those used
in the first experiment except that instead of thirteen collec-
tion periods only eight were used: (1) 24 hours, (2) 4 8 hours,
(3) 4 days, (4) 6 days, (5) 8 days, (6) 10 days, (7) 14 days,
and (8) 30 days.

Sea-surface bucket temperatures and water samples were
taken with each slide sample recovered instead of every two
days as earlier.

3 . Sample Preparation

All preparation steps followed in the first experiment
were used in the second except that the dehydration step was
eliminated. In morphological studies the dehydration step is
important in preserving the shape of microorganisms. Deleting
this step in sample preparation may cause some of the micro-
organisms to appear flattened, but they retain their basic
shape Hayat (1978) . Since the aim of this study was to simply

37

examine the numbers and types of organisms present and not
their morphology, elimination of this step still permitted
the goals of the study to be achieved. There was no notice-
able change, however, in the shape of the organisms viewed
under the SEM between the two experiments. A similar result
regarding the effects of dehydration was recorded by Dempsey
(1981) .

A summary of the steps followed in sample preparation
in Experiment 2 included:

1. Fixation

2. Freeze drying

3. Gold plating

D. EXAMINATION USING THE SEM

The gold-coated slide samples were viewed with an Hitachi
S-4 50 SEM using an accelerating voltage of 15KV. Initially,
a quick look over most of the slide surface was made using
magnifications ranging from 63X to 5000X and various SEM scans
This was done to get a view of the types of organisms present
and whether or not they were uniformly distributed. Areas
near the edge of the slides were avoided because fouling popu-
lations may be anomalously high there.

In both experiments the only microorganisms which appeared
to be uniformly distributed were rod-shaped bacteria. This
uniform distribution agrees with studies conducted by DiSalvo
and Daniels (1975) and O'Neill (1975) who stated that bacterial

38

cells appeared to be uniformly spaced over the test surfaces
rather than grouped in aggregates.

After this general scan, two or more locations were select-
ed as being representative of the surface and photographs taken
Polaroid Type 55 positive-negative 4X5 inch land film was
used for all photographic work. This is a fine-grain panchro-
matic film, yielding a positive print and a negative of high
resolution. Photographs were also taken of any unusual or
interesting organisms.

Magnifications of 1,500X or 3,000X were employed exten-
sively in photographing bacteria and other organisms. The
number of photos taken of each slide surface were limited due
to cost and the uniform distribution of bacteria.

A quantitative estimation of the numbers of bacteria was
made for each immersion period of both experiments using SEM
photographs. The bacterial count was an average for the
photo (s) of that exposure period.

Where possible, identification of organisms to genus and
species was made. When this was not possible, identification
was limited to general groups of organisms (bacteria, fila-
mentous microorganisms, hydroids, bryozoans, diatoms, fungi,
protozoans, etc.).

39

IV. RESULTS

A. EXPERIMENT 1
1 . General

A succession of periphytic microorganisms was observ-
ed on uncoated glass slides during Experiment 1 (Table 1) .
This succession included rod-shaped bacteria followed by dia-
toms, filamentous microorganisms, and finally protozoans.
Slides coated with Formula 196D or Formula 1114 did not
exhibit a settlement sequence. Formula 196D surfaces were
colonized early and throughout the experimental period by
bacteria. No other organisms appeared on this surface until
diatoms were seen after 83 days of exposure. Glass slides
coated with Formula 1114 were free of all fouling organisms
until after 83 days of immersion when diatoms and macrofouling
organisms such as bryozoans became evident.

When bacteria were seen on the test surfaces in both
Experiments 1 and 2 they appeared to be uniformly distributed
which permitted an estimate of their populations to be made
from two or three SEM photographs. The word sorbed will be
used to refer to the adhesion or attachment of bacteria and
other microorganisms to solid surfaces, a term used by other
researchers in this area including Corpe (1977) and Marshall
et al. , (1971a) .

Throughout the presentation of results from both
experiments, attention has been directed to photographic

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42

plates to help clarify the text material. Each photographic
plate contains one or more references, indicated in parenthe-
sis, which were used to identify the organisms. The scale
on each photograph is in microns.
2 . Organisms Sorbed to Glass

The major changes on the glass surfaces with respect
to the microfouling organisms occurred during the first four
weeks of exposure, a result which agrees with similar obser-
vations by Gerchakov et al., (1976). The first organisms to
appear were rod-shaped bacteria approximately 1.0--2.0 ym long
They were evident following the first immersion period of
24 hours. Plate 1 presents a good example of the size and
the appearance of these organisms although the picture is
from a coated slide. The bacteria increased up to 48 hours
then decreased slightly, leveling off until the 14th day
(Figure 5) . No bacterial counts on any surfaces in either
Experiment 1 or 2 were made beyond 14 days. This was because
the presence of so many organisms and so much organic debris
after 14 days of immersion negated the possibility of counting
bacteria, a condition which agrees with research results of
O'Neill (1971) and O'Neill and Wilcox (1971) .

The numbers of bacteria sorbed to the glass surfaces
as illustrated in Figure 5 must be considered an approximation
of low confidence because of the few SEM pictures used for
counting in each exposure period. The graph of the results,
however, does show a characteristic accelerated growth period

43

10 —

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NUMBER OF DAYS EXPOSURE

Figure 5. Numbers of bacteria sorbed to
surfaces in Experiment 1

44

during the initial 24 to 48 hours of exposure, followed by a
leveling off with a slightly decreasing population. This kind
of growth profile agrees with research and observations by
Bott and Pinherio (1976), Dexter (1976), Marshall et al. ,
(1971a), O'Neill (1971,1975), and O'Neill and Wilcox (1971).

By the end of the first week ring-f orming bacteria,
unknown filamentous microorganisms, and solitary diatoms
became evident on the glass surface (Plate 2) . These organisms,
although present throughout the remaining exposure periods,
were not uniformly distributed and so no attempt at estimating
their numbers was made. The most conspicuous of the diatoms
present were the centric forms Thalassiosira and Coscinodiscus
(Plate 3) . Identification of bacteria to genus and species
was not attempted. Ring forming bacteria, however, had the
size and appearance of Flectobacillus (Sieburth, 1975) .

At the end of the second week of exposure a greater
number and variety of diatoms were seen. Several pennate
diatoms especially Cocconeis populated the surface along with
centric types. The surface also became littered with a large
amount of organic debris. It was around these organic deposits
that the greatest diatom density was seen. Bacteria in the
form of rods and rings as well as filamentous microorganisms
continued to sorb to the glass throughout the remaining
exposure periods of Experiment 1.

At 18 days exposure the surface was dotted with
various kinds of protozoa, most notably the ciliated protozoan,

45

Cilophoran (possibly C. carchesium or C. ephelota) (Plate 4) .
Small numbers of planktonic algae known as coccolithophoroids
were also seen. A worm tube casing was observed firmly
attached to the surface giving evidence to the possible
beginning of fouling by macroscopic organisms. Organic
debris continued to increase over the surface and provided
areas of active bacterial and diatomaceous activity.

After 22 and 2 6 days of immersion the population of
protozoa, like the vorticelled ciliate shown in plate 5,
increased, while the number of bacteria and diatoms appeared
to decrease slightly. The surface landscape was a mixture of
diatoms, protozoa, filamentous microorganisms, and debris
(Plate 6) . An unusual mass of very small cylindrical objects,
believed to be a group of protozoans was seen for the first
and only time after 22 days exposure (Plate 7) .

Following 30 days of exposure there was a dramatic
change in the numbers and types of organisms seen on glass.
The entire surface was covered with many algal filaments and
protozoans as well as several bryozoan colonies. The protozoans
included many Zoothamnium colonies (Plate 8) and a few foramin-
ifera (Plate 9). About eight bryozoan colonies were observed
attached to the slide surface (Plates 10 & 11) . The develop-
ment of the bryozoan colonies may be looked upon as the real
beginning of macrofouling . There was a noticeable decrease in
the number of bacteria and diatoms sorbing to glass. Generally,
many of the diatoms in evidence were fragmented and appeared

46

to be decaying. Only a few diatoms were complete like the
centric form seen in Plate 12.

After 49 days of immersion the number and variety of
protozoa had increased noticeably. Zoothamnium colonies and
peritrichous ciliates, some housed within loricas, continued
to be the dominant organisms of the surface population (Plate
13) , as well as the suctorian Acineta (possibly A. tuberosa)
(Plates 14 & 15) . Another organism which had not been observed
before on glass was identified as the hydroid Qbelia (Plates
16 & 17) . These organisms were seen scattered across the
glass surface in small numbers. The number of bacteria and
diatoms sorbed to the surface continued to be few in number.
One unidentified organism was also observed (Plate 18) .

After 8 3 days of exposure the glass surface was a
densely-layered environment which included both micro and
macrofouling organisms as well as large amounts of debris.
Algae filaments and protozoa were the dominant organisms
present along with some hydroids and a few bryozoan colonies.
Again the number of bacteria and diatoms seen remained few
in number.

Three organisms which were seen for the first time
included the planktonic diatom Nitzschia (possible N. closterium)
(Plate 19) , a skeleton of a flagellated plant known as a
silicof lagellate (Plate 20) , and many unknown ciliated bodies
(Plates 21 & 22) .

47

3 . Organisms Sorbed to Formula 19 6D

The first microfouling organisms to appear were the
ubiquitous rod-shaped bacteria, which were not seen until
after 48 hours of exposure (Table 1) . These organisms seemed
to thrive on the Formula 196D paint surface. Like the bacteria
sorbed on glass, these rod-shaped bacteria exhibited an initial
rapid growth period followed by a slower but steadily increas-
ing population (Figure 5). However, after four days of exposure,
there was a significant increase in the numbers of bacteria
seen on the Formula 19 6D surfaces as compared with glass immers-
ed in seawater for the same period of time. These results
indicate that the bacteria may have a preferential attraction
for or be stimulated by this paint. This condition was noted
through the second week of exposure testing until bacterial
counts were discontinued.

The bacteria remained the major colonizers of this
painted surface until the 18th day of exposure when filamen-
tous microorganisms joined them (Plate 23) . Diatoms were not
seen until the 12th week of exposure and included planktonic
types Chaetoceros (Plate 2 4) , pennate forms Cocconeis , and
centric forms Thalassiosira (Plate 25) . Several unknown
bulbous-like organisms also became visible after 83 days of
immersion (Plate 26) along with a few sponge spicules (Plate 27) .

4 . Organisms Sorbed to Formula 1114

No organisms of any kind were seen attached to the
surface of paint Formula 1114 until after 83 days of exposure.

48

For the entire experimental period this painted surface had
a very "rocky" appearance, composed of paint artifacts, salt
crystals, and some debris (Plate 28). At this time several
bryozoan colonies were observed without the aid of a micro-
scope. The SEM also revealed a great number and variety of
diatoms, including a small number of a species not seen before,
such as Biddulphia longicururis (Plates 29 & 30) , along with
many familiar centric forms, Thalassiosira (Plate 31). No
bacteria, however, were seen on this paint during Experiment 1.
5 . Temperatures and Salinities

Temperature and salinity values collected for the
first 30 days of Experiment 1 have been graphed in Figure 6.
The average surface water temperature for the period was
12.6°C and the average salinity was 33.56 %0 .

B. EXPERIMENT 2
1 . General

A succession of periphytic microorganisms was observed
on four out of the five test surfaces exposed in Experiment 2
(Table 2) . The types of organisms and the times of their
initial settlement on glass slides matched almost exactly the
initial settlement time and succession of microfoulers seen on
glass surfaces in Experiment 1. The settlement sequence on
slides painted with Formula 196D, Formula 170, and Formula 121
consisted of bacteria followed by diatoms and finally protozoa.
Slides coated with Formula 1114, on the other hand, became
colonized early in the second week and throughout Experiment 2

49

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52

exclusively by solitary diatoms. In general, a greater number
and variety of diatoms were seen in this experiment than in
Experiment 1 .

This section includes detailed results from exposure
testing of glass slides coated with Formula 170 and Formula
121, two paints which were not examined in Experiment 1.
For the test surfaces of uncoated glass, Formula 196D, and
Formula 1114, a detailed listing of microorganisms and their
settlement sequence will be made only when these results differ
from those of Experiment 1. Similar results from the two
experiments will be noted with general comments.
2 . Organisms Sorbed to Glass

As in Experiment 1, the first organisms to appear
were rod-shaped bacteria. These microfoulers were seen
throughout all exposure periods of this experiment. An
accelerated growth period, similar to the bacterial growth
observed in Experiment 1, was again seen in the initial 24 to
48 hours (Figure 7) . Unlike the results of Experiment 1, the
number of bacteria continued to increase slowly beyond the
initial growth period until counting was no longer possible.
The number of bacteria graphed in each immersion period agrees
well with the bacterial counts of corresponding periods in
Experiment 1.

Diatoms became evident on glass after 14 days of
immersion. In general, the same centric diatoms Thalassiosira
and Coscinodiscus as well as pennate forms Cocconeis , were
seen. Two additional diatoms were identified which had not

53

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O GLASS

D FORMULA 19 6D

A FORMULA 17 0

-fr FORMULA 121

A 1 f-

^ 1 h

1 2 3 4 5 6 7 8 9 10 11 12 13 14

NUMBER OF DAYS EXPOSURE

Figure 7. Numbers of bacteria sorbed to various surfaces
in Experiment 2 .

54

been observed in Experiment 1. These included several groups
of rectangular diatoms, Thalassionema (Plate 32), and centric
diatoms, Skeletonema costatum (Plate 33), seen scattered about
the glass surface. As in Experiment 1, diatom density was
concentrated around deposits of debris. Also, a variety of
ciliated protozoa, the same seen in Experiment 1, began to
populate the glass. These organisms were joined by two uniden-
tified forms not seen before. Several clusters of what appeared
to be transparent sacks containing eggs dotted the surface
(Plate 34) , along with several attached organisms (Plate 35) .

After 30 days of immersion in Monterey Bay the glass
slide surface was again populated by colonies of bryozoans
and Zoothamnium as well as a large number of vorticelled cili-
ates (Plate 36) . The surface also contained many protozoans
known as Choancf lagellates which were not seen before in either
experiment (Plate 37). Several unknown organisms, possibly
ciliates, were also seen for the first time (Plate 38). As
in Experiment 1, the number of bacteria and diatoms sorbed to
the surface appeared to have decreased noticeably at this
exposure period.

3 . Organisms Sorbed to Formula 196D

After 24 hours of exposure unusual geometric forma-
tions, not seen before, were observed scattered about the
painted surface (Plate 39) . They did not appear to be
organic but rather crystalline in nature.

Rod-shaped bacteria became visible after 48 hours,
colonizing the surface until the conclusion of the experiment.

55

The bacteria again exhibited a rapid growth period followed
by a slower but steadily increasing population (Figure 7) .
As in Experiment 1, after four days of exposure, there was
a significant increase in the number of bacteria seen on the
Formula 196D surface as compared with corresponding glass
samples .

Diatoms settled on Formula 19 6D after only 6 days
as compared to 8 3 days in Experiment 1. The diatom population,
however, was sparse, composed mainly of centric forms,
Thalassiosira. These organisms were joined after 8 days and
throughout the remaining experimental period by planktonic
diatoms, Chaetoceros .

After 14 days of exposure the centric diatom Skeletonema
costatum joined the developing population along with several
silicof lagellates .

On the 3 0th day of the experiment, the Formula 19 6D
surface was littered with a large number of shelled protozoans
identified as foraminifera (Plate 40) . Several worm tube
casings were also seen (Plate 41). Unlike uncoated glass,
the number of bacteria and diatoms continued to populate the
Formula 196D surface in large numbers.
4 . Organisms Sorbed to Formula 1114

Solitary diatoms were sparsely settled across the
Formula 1114 surface after 8 days of immersion (Table 2) . They
continued to populate the surface in increasing numbers through-
out the remaining exposure periods. Diatoms identified

56

included pennate forms Amphora and centric types Thalassiosira,
Cosinodiscus , and Skeletonema costatum. Diatoms were the
only organisms which settled on paint Formula 1114 in Experi-
ment 2.

5 . Organisms Sorbed to Formula 170

No organisms were seen until after 4 days of immersion
(Table 2) . The first microfoulers which appeared at this time
were identified as rod-shaped bacteria (Plate 42) . Like the
bacteria sorbed to glass and Formula 19 6D, the bacteria on
this surface exhibited an early rapid growth period followed
by a slight decrease in population for a few days (Figure 7) .
From day 4 through day 10 of the bacteria counted on the sur-
face of Formula 170, agreed well with the number of bacteria
counted on Formula 19 6D and glass for the same exposure times.
However, by day 14 a significantly greater number of bacteria
were counted, giving evidence of another rapid growth period.

The bacteria were joined after 8 days of immersion
by a sparse population of solitary diatoms including pennate
forms and centric types Skeletonema costatum (Plate 43) . The
diatoms which were seen throughout the remainder of this
experiment continued to be solitary and few in number.

After 14 days a few unidentified stalked organisms,
believed to be protozoa were seen.

Following 30 days of exposure the Formula 170 surface
contained a dense population of bacteria and organic debris .
Diatoms and protozoa, although few in number, were also seen
along with some coccolithophorids (Plate 44) .

57

6 . Organisms Sorbed to Formula 121

Rod-shaped bacteria became visible on this painted
surface after 4 days of exposure (Table 2) . The bacteria
increased significantly from day 4 to day 6 and then leveled
off through to day 8. Beyond the 8th day the bacteria appeared
to be grouped in layers covered by a film which made counting
them impossible.

After 6 days of exposure a large number and variety
of diatoms were also seen. The forms identified included
pennate types (possibly Amphora) (Plate 45) , centric types
Skeletonema costatum (Plate 46) , and some other planktonic
diatoms like Chaetoceros (Plate 47) . As noted in Experiment
1, diatom density was most intense around organic debris
deposits .

The number of diatoms, bacteria, and debris continued
to increase through days 8, 10, and 14. After day 14 the
surface was littered with debris, bacteria, diatoms, and some
protozoans (Plate 48) . This condition was again seen following
30 days of immersion with the exception that the debris and
organisms mentioned were more numerous and two additional
organisms were identified. Many solitary stalked microfoulers
believed to be ciliated protozoans were seen (Plate 49), along
with several coccolithophorids (Plates 50, 51, & 52). The
three coccolithophorids pictured in these plates, although
probably of different species, may be exhibiting three differ-
ent stages of decay. Plate 50 may be a healthy coccolithophorid

58

that was killed at the time the sample was fixed, while
Plates 51 and 52 show increasing stages of decay.
7 . Temperatures and Salinities

Temperatures and salities recorded for Experiment 2
have been graphed in Figure 8 . The average surface water
temperature for the test was 13.3°C and the average salinity
was 3 3.37 %Q .

59

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60

V. DISCUSSION

The glass surfaces tested in both Experiments 1 and 2
showed what may be considered typical microfouling sequences.
The succession included rod-shaped bacteria followed by fila-
mentous microorganisms, diatoms, protozoa, and finally
bryozoans. Similar sequences and times of initial settlement
of fouling organisms on glass have been reported by other
investigators including Corpe (1970a, 1972), Dempsey (1981),
Dexter (1976), Gerchakov et al., (1976), Marshall et al.,
(1971), O'Neill (1971), and O'Neill and Wilcox (1971).

It was desired to compare the number of bacteria seen on
glass in Experiments 1 and 2 with the results of similar work
by other researchers. Figure 9 displays bacterial counts
made on glass from Experiments 1 and 2 and the results of
counts of bacteria made on glass or plexiglass from research
conducted by Dexter (1976), O'Neill (1977, unpublished), and
O'Neill and Wilcox (1971) .

O'Neill's unpublished research was conducted in Monterey
harbor from December 1976 to January 19 77 using polymethylmeth-
acrylate (plexiglass) as the test substrate. Dexter conducted
his tests using a variety of substrates including glass immers-
ed in Woods Hole harbor, Massachusetts, from July through
August 1975, during the peak of the fouling season at that
location. The average sea surface temperature was 22.6°C.
O'Neill and Wilcox conducted their bacterial counts using a

61

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O GLASS - EXPERIMENT 1

n
0

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GLASS - EXPERIMENT 2

GLASS - O'Neill S Wilcox (1971)

Glass - DEXTER (1976)

PLEXIGLASS - O'Neill (1977
unpublished)

3 4 5 5 7 8 9 10 11 12 13 14
NUMBER OF DAYS EXPOSURE

Figure 9. Results of bacterial attachment seen on glass in
Experiments 1 and 2 and results of similiar tests made on
glass or plexiglass by three different research groups .

62

variety of substrates including glass immersed in the harbor
at Port Hueneme, California from July to September 1965. The
water temperature at the time of their test ranged from 13.0
to 18.0 °C.

Although there is a large difference in the number of
bacteria noted in corresponding periods, all three research
results showed an initial rapid growth period followed by a
leveling off and then a slowly increasing or decreasing popu-
lation. The growth trend exhibited by Experiments 1 and 2
agrees well with these research results. This trend is the
only common factor between all these curves.

The bacterial counts of Dexter, made in Woods Hole harbor,
and O'Neill and Wilcox made in Port Hueneme harbor agree more
with the results of Experiments 1 and 2 than that of O'Neill
which was conducted in Monterey harbor. The factors influ-
encing bacteria settlement are many. It is not possible here
to evaluate these factors in explaining the agreement or dis-
agreement of these research results with Experiments 1 and
2. The graphed data from the three researchers was provided
only to give a general view of the number of bacteria which
may sorb to glass surfaces, the variability of bacterial popu-
lations with location and time of year, and some typical growth
profiles .

Microfouling seen on all but one of the antifouling paints
followed a similar succession sequence to that observed on
glass. Formula 196D, tested in both experiments, along with
Formula 170 and Formula 121, tested in Experiment 2, exhibited

63

a fouling sequence which included bacteria, followed by
diatoms, and finally protozoa. These coatings appeared to
have altered the times for the initial settlement of these
organisms as well as their abundance. Although bacteria were
not seen until 48 hours of immersion on the Formula 19 6D sur-
face and after 4 days on the slides painted with Formula 170
and Formula 121, their numbers quickly increased to the point
where they were significantly more abundant than the bacteria
on glass slides. Paint Formula 1114, however, showed no se-
quence of organisms and was free of bacteria throughout both
experiments. The only microfoulers observed on this paint
were diatoms.

To explain the bacterial numbers and the microfouling
sequences it was necessary to discuss the toxicants used in
each paint formulation and their effects on microorganisms.
The paints tested and their toxic ingredients are summarized
below:

Formula 196D - Tributyltin

Formula 170 - Tributyltin oxide and Tributyltin fluoride

Formula 121 - Cuprous oxide

Formula 1114 - Nopcocide N-9 6

There has been some discussion by various researchers con-
cerning the effects that paints containing tributyltin and
copper have on microfouling. O'Neill (1975) tested an anti-
fouling paint containing tributyltin oxide (TBTO) against the
settlement of bacteria, diatoms, and barnacles under controlled

64

laboratory conditions. He concluded that the presence of TBTO
appears to inhibit the development of a primary film of bac-
teria and diatoms and does hinder later barnacle attachment.

A different result was reported by Dempsey (1981) who said
that tolerance to heavy metal poisons is a common phenomenon
in microorganisms. He stated that organotin antifouling paints
are not effective against gram-negative bacteria, although
they are highly toxic to gram-positive species. Furthermore,
in laboratory tests of another tin-based antifouling paint
containing triphenyl tin fluoride (TPTF) , Dempsey found that
extensive bacterial communities developed on the TPTF paint
after 4 weeks of exposure.

Corpe (1977) also reported that primary film forming bac-
teria are little affected by metallic paints or other toxic
coatings such as organotin. In tests conducted on slides sub-
merged in the sea for 2 4 and 9 6 hours, Corpe indicated that
the same kinds and numbers of bacteria were isolated from the
test surfaces whether they were cotaed with copper and mercury
paint, tributyltin, or uncoated.

The sorption of microorganisms observed on Formula 196D,
Formula 170, and Formula 121, seem to agree with the results
of Corpe and Dempsey. Not only did these paints appear to have
very little effect on these microfoulers in general, but they
appear to actually stimulate the growth or attraction of bac-
teria. An explanation of these results is provided by Corpe
(1975) who reported that copper or lead in a concentration of

65

-4
4 x 10 M actually stimulated growth of bacteria when the

nutrient concentration was high.

Approximately 85 to 95 percent of fouling bacteria are
gram-negative (Corpe, 1973) . A major characteristic of these
bacteria is the presence of a lipopolysaccharide (LPS) outer
cell layer which may act as a penetration barrier, especially
to hydrophobic compounds (Dempsey, 1981; Corpe, 1977).
Therefore, tolerance to antifouling paints containing metals
such as copper, tin, lead, or mercury probably results from
the LPS layer acting as a penetration barrier. Evaluation
of the presence of such an outer cell layer was beyond the
laboratory capabilities used in Experiments 1 and 2. However,
many of the bacteria, on glass and painted surfaces, exhibited
holdfast structures known as polymeric fibrils (Marshall et
al., 1971). The extracellular appendages were more evident
from bacteria on the heavy metal paints than from those sorbed
to glass.

No macrofouling organisms (i.e., bryozoans) were seen
on any of the antifouling paints for the first 30 days in
either experiment. However, after 83 days of exposure, Formula
1114 had many well developed bryozoan colonies scattered
across the painted surface. The toxicant used in Formula
1114 was Nopcocide N-96. This compound is a chlorinated
pesticide and is considered to be organic in nature containing
no heavy metals. The results of Experiments 1 and 2 suggest
that this substance may be effective in controlling or preventing

66

marine bacterial settlement and/or growth. However, the
establishment of the bryozoan colonies on this paint may
indicate that Nopcocide is not as effective in preventing
macrof ouling.

67

VI . CONCLUSIONS

Of the four paints tested in this study only Formula 1114
can be said to be effective in preventing and limiting the
early development of microfouling organisms. This paint,
however, may not be as effective in limiting the sorption of
macroorganisms such as bryozoans . The results of Experi-
ments 1 and 2 also suggest that the establishment of a com-
plete microfouling community which includes bacteria, diatoms,
and protozoa is not a necessary precursor to the settlement
of macrof oulers .

The other three paints with copper or tin as their toxic
ingredient only delayed the onset of microfouling by a few
days. The bacterial populations seemed to thrive on these
heavy metal paints, achieving populations greater than those
observed on glass. Much circumstantial evidence has been
gathered to support the possibility that bacterial fouling
layers with their extracellular mucilage secretions may render
antifouling paints less efficient. It has been recognized
that Formula 121, for example, is effective in preventing the
settlement of barnacles and other macrof ouling organisms.
However, the effective life of the paint may be shortened by
microf oulers . If the experimental paints, Formula 170 and
Formula 196D prove to be effective in combating macrof ouling,
their surfaces will most likely be covered with bacteria and

68

other microfouling organisms, a condition which may also
shorten their service life.

This study also established that there is a definite
sequence to the sorption of marine microfoulers on glass and
even on some antifouling paints. This sequence begins with
the settlement of bacteria and is followed by filamentous
microorganisms, diatoms, and finally protozoa.

Future research in this area should concentrate on longer
exposure periods ranging from 6 months to several years .
Test substrates should include hull steel and glass reinforced
plastic and any other surfaces for which the end application
is intended.

69

APPENDIX A

NAVY STANDARD FORMULA 121
RED VINYL ANTIFOULING PAINT

INGREDIENTS AMOUNT IN POUNDS1

Cuprous oxide 144 0

Rosin 215

2

Vinyl resin 55

Tricresyl phosphate 50

Methyl isobutyl ketone 165

Xylene 115

Antisettling Agent 5 to 9

Source: Department of the Navy Military Specification
Mil-P-15931C, Paint, Antifouling, Vinyl (Formula Numbers
121 and 129)

Notes: 1. The formula, given slightly in excess of 100
gallons to allow for manufacturing loss, may be proportioned
to the size batch desired.

2. The resin shall be a vinyl chloride-vinyl
acetate copolymer. It shall contain 85 to 88 percent
vinyl chloride and 12 to 15 percent vinyl acetate. The
resin shall have a specific gravity of 1.35 to 1.37.

70

APPENDIX B

NAVY STANDARD FORMULA 17 0
BLACK CAMOUFLAGE VINYL ANTIFOULING PAINT

INGREDIENTS AMOUNT (parts by mass)1

2
Vinyl resin -. 150

Bis (tributyltin) oxide (TBTO) 36

Tributyltin fluoride (TBTF) J 155

Carbon black 18

Titanium dioxide 6

Ethylene glycol monoethyl ether

acetate . 2 6

4

Normal propanol 9 5

Normal butyl acetate 370

856

Source: Department of the Navy Military Specification
DOD-P-24588, Paint, Antifouling Vinyl, Camouflage (Formula
numbers 170, 171, 172, and 173), 2 May 1979.

Notes: 1. Use of kilograms as mass units results in a
volume slightly in excess of 833 liters. Use of pounds as
mass units results in a volume slightly in excess of 100
gallons .

2. The resin shall be a copolymer of vinyl
acetate and another monomer which contains carboxyl groups.
It is manufactured by Air Products Chemical Company, Allen-
town, Pennsylvania under the name VINAC ASB-516.

3. Manufactured by M&T Chemicals Incorporated,
Rahway, New Jersey, or by Cincinnati Milacron Chemicals
Incorporated, 500 Jersey Avenue, New Brunswick, New Jersey.

4. This material contains a minimum of 9 7 percent
normal propanol with a minimum boiling point of 9 5 degrees
centigrade .

71

APPENDIX C

DTNSRDC EXPERIMENTAL ANTIFOULING PAINT
FORMULA 2844-1114

INGREDIENTS

POUNDS GALLON
PER 103 PER 103
GALLONS GALLONS

COMPONENT A

Liquid polysulfide polymer

(Thiokol LP-2) 1
Toxic (Nopococide N-96) (2,4,5,6-

tetrachloroisophthalonitrile) 2
Carbon black ..

Adhesion promoter (Durez 10694)
Thixotrope (Cabosil M-5) 4
Solvent (Xylene)

385

35.9

385

26.7

11.6

0.8

19.2

1.8

11.6

0.6

269.4

37.2

1081.6

102.9

COMPONENT B

Lead paste Mixture for 103
gallons of component A^

80.83

5.29

Source: David Taylor Naval Ship Research and Development
Center Purchase Description (draft) , Antifouling Paint,
DTNSRDC Experimental Formula 2844-1114, December 1980.

Notes: 1. Thiokol LP-2, manufactured by Thiokol Chemical
Company, 93 0 Lower Ferry Road, P. 0. Box 129 6, Trenton,
New Jersey.

2. Nopcocide N-9 6 is manufactured by the Diamond
Shamrock Chemical Company, process chemicals division, 350
Kemble Avenue, Morristown, New Jersey.

3. Durex 10 69 4 is manufactured by the Hooker
Chemical Corporation, durez plastics division, 14120 Walck
Road, North Tonawanda, New Jersey.

4. CAB-O-SIL M-5 is manufactured by Cabot Corpora-
tion, 125 High Street, Boston, Massachusetts.

5. Lead Paste Mixture:

Parts by Lbs. to make Gal. to make

wt. 1 Gal. 1 Gal.

Lead Peroxide 90-95% PB02

Plasticizer (Thiokol TP-680)

Stearic acid

112.5

7.64

0.10

109.5

7.44

0.87

3.0

0.20

0.03

215.1

15.28

1.00

72

APPENDIX D

ORGANOTIN EPOXY GEL FORMULA 19 6D
DTNSRDC EXPERIMENTAL ANTIFOULING PAINT

INGREDIENTS AMOUNT (parts

by weight)

COMPONENT A

EPON 8282 - 30

EPI-REZ 505 50
60% Tributyltin ester of

SMA 10004 160

Lampblack 2.2 6

Titanium dioxide 0.52

COMPONENT B

DMP-305 8

Source: David W. Taylor Naval Ship Research and Develop-
ment Center Technical Manual TM-28-80-105, Investigation of
the Sprayability of Organotin Epoxy Gel Coats, by J. A.
Montemarano, S. A. Cohen, A. M. Ross, and A. R. Parks, 27
June 1980.

Notes: 1. Use of kilograms as mass units results in a
volume of approximately 232 liters. Use of pounds as mass
units results in a volume of approximately 28 gallons of
paint.

2. EPON 828 is an epoxy resin with a density of
1.15 g/cc . It is manufactured by the Shell Chemical Company.

3. EPI-REZ 505 is an epoxy resin with a density
of 1.01 g/cc. It is manufactured by the Celanese Corporation.

4. The organotin component of this formulation is
the 60 percent tributyltin ester of SMA 1000. The SMA 1000

is a copolymer of styrene: maleic anhydride (1:1 molar ratio),
manufactured by the Atlantic Richfield Corporation.

5 . This is a curing agent known as TRIS

( dimethyl aminomethyl) Phenol, distributed by Miller-Stephenson
Company .

73

APPENDIX E

Plate 1. Glass coated with paint Formula 196D immersed 6
days, 3000X. Rod-shaped bacteria, some showing hold-fast
structures .

74

Plate 2. Glass immersed 6 days, 3C00X. Rod-shaped and

ring-forming bacteria.

Ring-forming bacteria (possibly
Flectobacillus) (Sieburth, 1975)

Diatom frustle fragment

Rod-shaped bacteria

75

Plate 3. Glass immersed 8 days, 1500X. Two centric
diatoms, filamentous microorganisms, and rod-shaped
bacteria .

Centric diatom Coscinodiscus (possibly
C. marginatus; (Cupp, 1943)

Rod-shaped bacteria

Centric diatom Thalassiosira (Cupp, 1943)
Filamentous microorganism (Sieburth, 1975)

76

Plate 4. Glass immersed 14 days, 5QQQX. Two vorti<
cellid protozoans CI. Abbott, personal communicat-
ion) .

77

Plate 5. Glass immersed 33 days, 2000X. Close-up of a
spiral-stalked protozoan. Note the cilia extended from
the top of the organism. It moves these fine hair-like
structures in a rhymthic manner creating a vortex thus
drawing food into itself CCupp, 1943).

78

Plate 6. Glass immersed 25 days, 1500X. Pennate diatom,
protozoan, filamentous microorganisms, bacteria and debris

Pennate diatom Navicula (Cupp, 194 3)

Rod-shaped bacteria
Debris

Filamentous microorganisms (Sieburth, 1975)

Ciliated protozoan Carchesium or Ephelota
(T. B. O'Neill, personal communication)

79

Plate 7. Glass immersed 22 days, 15Q0X. This unusual
mass of cylindrical objects was identified as possibly
being the exogenus buds of a ciliated protozoan,
Ephelota gemminif era (T. 3. O'Neill, personal commun-
ication) .

80

Plate 8. Glass immersed M-9 days, .5 0QX,
protozoan, Zoothamnium (Sieburth, 1975

Th.e colonial
19.79) .

81

Plate a. Glass immersed 83 days, 5QQX. Two shelled
protozoans called foraminifera CStinemeyer and
Reiter, 195 8).

82

Plate 1Q . Glass immersed 49 days, 3QX. Bryozoan colony
surrounded by many microscopic protozoans CMorris et.
al., 1930).

83

Plate 11. Glass immersed 49 days, 150X. Close-up of
a bryozoan colony showing four opercula.

84

Plate 12. Glass immersed 33 days, 3Q0QX. Centric
diatom with a coccolith plate on the surface CCupp,
194 3) .

85

Plate 13. Glass immersed 49 days, 280X. Zoothamnium
colony and some singular protozoans.

Zoothamnium colony (Sieburth, 1979)

Stalked lorica housing peritrichous ciliate
(Sieburth, 1979)

Protozoan, Acineta tuberosa (T. 3. O'Neill,
personal communication)

86

■ >v^8sca

Plate 14. Glass immersed 49 days, 70 OX. The protozoan
Acineta tuberosa CT. 3. O'Neill, personal communicat-
ion; Sieburth, 1279).

87

Plate 15. Glass immersed 43 days, 940.X. A solitary
protozoan, possibly Acineta CT. 3. O'Neill, personal
communication-, Sieburth, 1979).

88

PfP!k i •fiiMBIMlHi'TiJiS

Plate 16. Glass immersed 4 9. days, 110X. Two hydroids
of the genus Obelia surrounded by protozoans.

Two hydroids, genus Obelia (Sieburth, 1975).

Protozoan, Acineta tuberosa (T. 3. O'Neill

personal communication)

Zoothamnium colony (Sieburth, 1979)"

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89

"^-%^«l&gg3f$

! V -V .4, » ' '

H

'S:/:.;?i-i° ^

^

Plate 17. Glass immersed 49 days, 250X. Close-up of
the hydroid Obelia. Mote what appear to be spikes
which cover the surface of the tentacles. These are
the triggering mechanisms for the stinging cells or
the nematocysts (Sieburth, 1975).

90

Plate 18. Glass immersed 49 days, 13QX. Large unknown
organism surrounded by orotozoans .

Unknown organism

Zoothamnium colony (Sieburth, 1979)

Vorticellid protozoans (Sieburth, 1975)

V

91

^■:.,

Plate IS. Glass immersed 33 days, 1630X. T^ie planl<
diatom Mitzschia closterium CCupp , 1943).

:omc

92

■S&

m^* t :,*ft>f^&;

Plate 20. Glass immersed 33 days, 200CX. The skeleton
of a single-celled flagellated plant known as a
silicof lagellate Cl. Abbott, personal communication;
Sieburth, 1975) .

93

*f^'"',*afl£i

WE-W*%,

^

Mey?

'*m M

.*■ K*

Plate 21. Glass immersed 3 3 days, 300X. Several peritrichous

ciliates within protective Icricas cr houses and two
unknown ciliated bodies.

Loricas housing peritrichous ciliates
(Sieburth, 1979)

Unknown ciliated organisms

94

- .> ' • ■>

mm0&m

^'i' ■■■•' ' -'<''- '■

Plate 22. Glass immersed 33 days, 27Q0X. Close-iio of
an unknown ciliated body.

95

Plate 23. Glass coated with paint Formula 196D immersed
30 days, 15C0X. Rod-shaped bacteria, debris, and a
filamentous microorganism.

96

Plate 24. Glass coated with paint Formula 19-6 D immersed
10 days, 15 OCX. Two planktonic diatoms, bacteria, and
debris .

Rod-shaped bacteria

Planktonic diatoms Chaetoceros (Cupp, 1943)

Debris

97

Plate 25. Glass coated with paint Formula 196B immersed
8 3 days, 800X. Several centric diatoms, a broken
coccolithophorid , and debris.

Centric diatoms, Thalassiosira (Cupp, 1943)

Centric diatom dividing (E. C. Haderlie,
personal communication)
Broken coccolithophorid (Sieburth, 1979)

98

HHS^ '-ItHH

■^flliPP

4!^% v^

*:.;.'■:■:

«*~1s

i;0^»^

^^^^^^la^A^^I^.-te^-w^^^^^^^jy^^^ya^.

Plate
ed 33

26. Glass coated with paint Formula 19.6 D iminers
days, 150QX. Unknown bulbous-like organism.

99

Plate 27. Glass coated with paint Formula 196D immers
ed 33 days, 10.0 QX. Sponge spicule possibly from the
genus Leucosolenia (Light 1975).

100

Plate 23. Glass coated with paint Formula 1114 immers-
ed 4 days, 1500X. Surface composed of paint artifacts,
salt crystals, and some debris .

101

Plate 29. Glass coated with, paint Formula 1114 immerS'
ed 33 days, 10GQX. The planktonic diatom, Biddulphia
Ion gri cruris CCupp , 1943).

102

IP I ISP

s# •**■ ^"s.-

,\! '•"

*#:;

;*'<a..:^,' ^ije'i'iJilAii.^.-i-vV^:-!, -y-i '■ ■ : l^'rif- :. '.■t'M.^ik^ '>V--S5S^^r.^Y±?,^i^^.^M^K£. .'•; .^L-.-iV

Plate 30. Glass coated with paint Formula 1114 immers^
ed 3 3 days, 4QQ0X. Close-up of the diatom Biddulphia
longicruris CCupp, 1343).

103

■■ v/."-"^--^'.--";^-;"- cr

Plate 31. Glass coated with paint Formula 1114 immers-
ed 33 days, 150.0X. Several broken diatoms of the genus
Thalassios ira CCupp , 19 43).

104

Plate 32. Glass immersed 14 days, 500X. Linked rec-
tangular diatoms of the genus Th-alassionema CI. Abbott,
personal communication; Cupp, 13 4 3) .

105

Vi*

tllPlls*!

V

tw .',:& >,>»

si#P"*

111* !,W* ^*--f«**.^

K HI

y-mmasak ^-mm

Plate 33. Glass immersed 14 days, 300X. Centric diatoms,
entangled with debris on the surface of a worm casing.

Centric diatoms, Skeletonema costatum
(Cupp, 1943)

Worm tube casing

106

v '• m

>.

W^^^^^m^M

Plate 34. Glass immersed 14 days, 1500X. Unknown
object, oossiBly a transparent sack containing eo-o-s

107

,.;Wr

Wmsmisgiki; <l ..-.■ --*V:>°<- *■■■ >*■'"'■.

&aS ifefcs&is&fesffii;

Plate 35. Glass immersed 14 days, 7G0X. Unknown attach'
ed organism. Mote the hold-fast structures.

108

^Mw

WsSm 5-'*s|' «m^

•.•;>■'.'** ^-.,:.«&, .>' ,,-,,1

wmmk 3

!/i ^S3%sk%i w

MrM

'$

isJI

Plate 36. Glass immersed 30 days, 300X. One colonial
protozoan surrounded by several solitary protozoans .

Vorticellid protozoans (Sieburth, 1975)

Zoothamnium colony (Sieburth, 1979)

"V

.rf°

109

Plate 37. Glass immersed 30 days, 1500X. Several
stalked protozoans known as choanof lagellates
CSieburth, 19 79) .

110

■ ";*

Ml

■» "jiijs^

>C#

Plate 33. Glass immersed 3Q days, 330X
ciliated organism.

Unknown

111

S&SS$?5£1?^CS!.; ,

"'' t::' ■:;«:.";:'-'■-:-

wBB W 1

5%' ■■."'.-;i*

&■?

,wa£J

.1

•i .i; -V-.f

i;p

■

weT ^x*vv-i-\;. £ -

Plate 39. Glass coated with paint Formula 19.SD immers-
ed 24 hours, 400.X. Salt and other crystals forming

an unusual geometric oatter-n

112

Plate 40. , Glass coated with, paint Formula 12L6B immers-
ed 30 days, 1500X. A foraminifera surrounded By broken
diatoms CStinemeyer and Reiter, 1953).

113

Plate 41. Glass coated with paint Formula 19.6D imme.rS'
ed 30 days, 100X. Worm tubes CHaderlie, personal
communication) .

114

Plate 42. Glass coated with paint Formula 170 immers-
ed 4 days, 1500X. The irregular surface is the texture
of the oaint itself. The oaint is ccmDletely covered

£ £ £

by rod-shaped bacteria.

115

Plate 43. Glass coated with paint Formula 170 immers^
ed 8 days, S00X. A long chain of the centric diatom
Skeletonema costatum Cdupp , 134 3)

116

Plate 1+1+ . Glass coated with paint Formula 170 immersed
30 days, 1500X. A coccolithophorid with a few plates

missing

Broken coccolithophorid (Sieburth, 1979)

117

m^^^^^^mi^m^'^^^m ■■ &*&mm&iagm

Plate 45. Glass coated with paint Formula 121 immerS'
ed 6 days, 5G0OX, Pennate diatom, possibly of the
genus Amphora CI. Abbott, personal communication).

118

Plate 46. Glass coated with, oaint Formula 121 imners-

4-

ed 6 days, 30Q3.X. Close-up of the centric diatom
Skeletcnema costatum. Note the rod-shaoed Bacteria on

the surface of the diatom.

119

■-■3HHBHH6^'''':';: "'■?> v";^«"V".' ■3^E^^^S*!S5s??*jG!':??"-'"Tr'.-"''"Ji"

;'^:''. ;.*?*<;^: —.'""> :

^^^

f

■ ft

«&• :

w

Plate 47. Glass coated with paint Formula 121 immers-
ed 5 days, 400QX. The planktonic diatom Chaetoceros
radicans CCupp, ISM-SI.

120

«^^!^^^^ai^^^^^E^p^y:y^^^"';;i:?^f^ 'Oilf j^^^^^^^^^^^P^

Plate 4 3. Glass coated with paint Formula 121 immersed
1M- days, 500X. Diatoms, protozoans, and some debris.

Pennate diatoms (Cupp, 1943)

Lorica housing a protozoan (Sieburth, 1979)
Centric diatom, girdle view (Cupp, 194 3)

121

v#

Plate 49.
ed 30 days

.ass coated with, paint Formula 121 immers-
40CQX. Stalked, ciliated protozoan.

122

Plate 50. Glass coated with paint Formula 121 immers<
ed 30 days, 3Q0.Q.X. Coccolithophorid (.Sieburth, 13 79)

123

Plate 51. Glass coated with paint Formula 121 immers-
ed 30 days, 12.QQ0X. Coccolithophorid CSieburth, ia79)

124

^^^Sm^^f^mi^mismsmii^mt^smii^SmSS

mmfm

Plate 52. Glass coated with paint Formula 121 immers-
ed 30 days, 10,QQ0X. Coc'colithophorid CSieburth., 197 9)

125

LITERATURE CITED

Baier, R. E. 1973. Influence of the initial surface condition
of materials on bioadhesion. pp. 633-639. In: Acker,
R. F., et al . , (eds . ) , Proceedings of the Third Inter-
national Congress on Marine Corrosion and Fouling.
Northwestern University Press, Evanston, 111.

Bott, T. R. and Pinherio, M.M.P.S. 1976. Biological fouling-
velocity and temperature effects. Canadian Society for
Chemical Engineering, paper No. 76-CSMS/CSHE-25 .

Corpe, W. A. 1970a. Attachment of marine bacteria to solid
surfaces, pp. 73-87. In: Man ley, R. S. (ed.), Adhesions
in biological systems. Academic Press, N. Y.

1970b. An acid polysaccharide produced by a primary

film-forming marine bacterium. Developments in Industrial
Microbiology. 11:402-412.

1972. The attachment of microorganisms to glass slides

submerged in San Diego Bay, with special reference to a
colonial protozoan. Office of Naval Research Technical
Report No. 8-10-72.

1973. Microfouling: The role of primary film forming

marine bacteria, pp. 598-609. In: Acker, R. F. et a_l
(eds.) , Proceedings of the 3rd International Congress on
Marine Corrosion and Fouling, Northwestern University
Press, Evanston, 111.

1975. Metal binding properties of surface materials

from marine bacteria. Developments in Industrial Micro-
biology. 16: 249-255.

1977. Primary bacterial films and marine micro-

fouling, pp. 97-100. In: Romansky, V. (ed.), Proceedings
of the 4th International Congress of Marine Corrosion
and Fouling.

Corpe, W. A. and Winters, H. 19 72. Hydrolytic enzymes of
some periphytic marine bacteria. Canadian Journal of
Microbiology. 18:1484.

Cupp, Easter E. 1943. Marine plankton diatoms of the west
coast. University of California Press. Berkeley,
California.

David Taylor Naval Ship Research and Development Center Purchase
Description (draft). 1980. Antifouling paint DTNSRDC
experimental formula 2844-1114.

126

Dempsey, M. J. 1981. Marine bacterial fouling: A scanning

electron microscope study. Marine Biology. 61: 305-315.

Department of the Navy Military Specification DOD-P-24588.
1979. Paint, antifouling vinyl, camouflage (formula
numbers 170, 171, 172, and 173).

Department of the Navy Military Specification MIL-P-15931C .
Paint, antifouling, vinyl (formula numbers 121 and 129) .

Dexter, S. C. 1977. Influence of substrate wettability on the
formation of bacterial slime films on solid surfaces
immersed in natural sea water, pp. 131-138. In: Romanovsky,
V. (ed.), Proceedings of the 4th International Congress on
Marine Corrosion and Fouling.

DiSalvo, L. H. and Daniels, G. W. 1975. Observations on

estuarine microfouling using the scanning electron micro-
scope. Microbial Ecology. 2: 234-240.

Gerchakov. S. M. , Marszalek, D. S., Roth, F. J. and Udey, L.
R. 1977. Succession of periphytic microorganisms on
metal and glass surfaces in natural sea water. PP • 193-200.
In : Romanovsky, V. (ed.) , Proceedings of the 4th Inter-
national Congress on Marine Corrosion and Fouling.

Haderlie. E. C. 1974. Growth rates, depth preference, and
ecological succession of some sessile marine inverte-
brates in Monterey harbor. Veliger 17 (supplement): 1-35.

1977. The nature of primary organic films in the

marine environment and their significance for ocean thermal
energy conversion (OTEC) heat exchange surfaces. Naval
Postgraduate School, Monterey, California Technical
Report No. NPS-68HC77021 . 44 pp.

Harris, R. H. and Mitchell, R. 1973. The role of polymers

in microbial aggregation. Annual Review of Microbiology.
27: 27-50.

Hartman, Willard D. 1975. Phylum porifera. pp. 32-58. In:
Smith, R. I. and Carlton, J. T. (eds.), Light's Manual:
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University of California Press. 716 pp.

Horbund, H. W. and Freiberger, A. 1970. Slime films and

their role in marine fouling. Ocean Engineering 1: 631-
634.

Hyat, M. A. 1978. Introduction to biological scanning electron
microscopy. University Park Press. 323 pp.

127

Loeb , G. I. and Neihof, R. A. 1975. Marine conditioning

films. Advances in chemistry Series, No. 145: 319-335.

1977. The interaction of dissolved organic matter

in seawater with metallic surfaces, pp. 359-361. In:
Romanovsky, V. (ed.), Proceedings of the 4th International
Congress on Marine Corrosion and Fouling.

Marshall, K. C, Stout, R. and Mitchell, R. 1971a. Mechanism
of the initial events in the sorption of marine bacteria
to surfaces. Journal of General Microbiology. 68: 337-
348.

1971b. Selective sorption of bacteria from sea water

Canadian Journal of Microbiology. 17: 1413-1416.

Montemarano, J. A., Cohen, S. A., Ross, A. M. , and Parks, A.
R. 1980. Investigation of the sprayability of organotin
epoxy gel coats. David W. Taylor Naval Ship Research
and Development Center Technical Manual TM-28-80-105 .
9 pp.

O'Neill, T. B. 1971. The influence of surface roughness on

the formation of primary film. Naval Civil Engineering

Laboratory Technical Note N-Z. ROOl-01-01-111 , Port
Hueneme, California.

1975. Some experiments concerning the influence of

the primary film on subsequent macroscopic fouling.
Naval Civil Engineering Laboratory Special report 52-76-
04, Port Hueneme, California.

O'Neill, T. B. and Wilcox, G. L. 1971. The formation of a
"primary film" in materials submerged in the sea at
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Riley, G. A. 19 63. Organic aggregates in sea water and the

dynamics of their formation and utilization. Limnology and
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Sieburth, J. M. 1975. Microbial seascapes. University Park
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1979. Sea microbes. Oxford University Press.

Soule, J. D., Soule, D. F. , and Abbott, D. P. 1980. Bryozoa
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128

Stinemeyer, E. H. and Reiter, M. 1958. Ecology of the

foraminif era of Monterey Bay, Calif ornia--Report Part
I, report No. 1621. Shell Oil Company Exploration
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Taylor, J. E. 1977. Marine microfouling in Monterey harbor:
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Master's thesis. Naval Postgraduate School, Monterey,
California. 66 pp.

Tosteson, T. R. and Corpe, W. A. 1975. Enhancement of adhesion
of the marine Chlorella vulgaris to glass. Canadian
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Woods Hole Oceanographic Institution. 1952. Marine fouling
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Zobell, C. E. 1939. The role of bacteria in the fouling of
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1943. The effect of solid surfaces upon bacterial

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129

INITIAL DISTRIBUTION LIST

No. Copies

1. Defense Technical Information Center 2
Cameron Station

Alexandria, Virginia 22314

2. Library, Code 0142 2
Naval Postgraduate School

Monterey, California 93940

3. Department Chairman, Code 68Mr 1
Department of Oceanography

Naval Postgraduate School 93940

4. Professor Eugene C. Haderlie, Code 68Hc 1
Department of Oceanography

Naval Postgraduate School
Monterey, California 93940

5. LCDR Patrick R. Kelly 2
NOCD, Box 6302

APO

San Francisco, CA 96519

6. Dr. Thomas B. O'Neill, Code L52 1
Naval Civil Engineering Laboratory

Port Hueneme, California 93043

7. Mrs. Jean Montemarino, Code 2 844 2
David W. Taylor Naval Ship Research

and Development Center
Annapolis, Maryland 21402

8. Mr. Steve Rogers, Code 2841 1
David W. Taylor Naval Ship Research

and Development Center
Annapolis, Maryland 21402

9. Miss Bonnie Hunter, Code 68 1
Department of Oceanography

Naval Postgraduate School
Monterey, California 93940

10. Mr. Dana Austin, Code 68 1

Department of Oceanography
Naval Postgraduate School
Monterey, California 93940

130

11. Professor Isabella A. Abbott
Hopkins Marine Station
Pacific Grove, California 93950

12. Mr. Chris Patton
Hopkins Marine Station
Pacific Grove, California 93950

13. Mrs. Anne Harrington
Hopkins Marine Station
Pacific Grove, California 93950

14. Library

Hopkins Marine Station

Pacific Grove, California 93950

15. LCDR C. R. Dunlap, Code 68DU
Assistant Professor
Department of Oceanography
Naval Postgraduate School
Monterey, California 93940

131

/

L_

Thesis

K2863 Kelly 193240
Scanning electron
microscope observa-
tions of marine
microorganisms on
surfaces coated with
antifouling paints.

Thesis

K2863 Kelly 193240

c-l Scanning electron
microscope observa-
tions of marine
microorganisms on
surfaces coated with
antifouling paints.

JS•l«w■*w»E!iSSS,

3 2768 002 11245 0

DUDLEY KNOX LIBRARY