Indian Journal of Geo-Marine Sciences

Vol. 43(11), November 2014, pp. 2067-2074

A Short Review of Laboratory and Field Testing of Environmentally Benign

Antifouling Coatings
Chin Sing, LIM*1, Serina Siew Chen, LEE1, Wai, LEONG1, Ying Xian NG2, Serena Lay Ming, TEO1
1
Tropical Marine Science Institute, National University of Singapore, Singapore 119227
2
Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 119260*
[E-mail: tmslimc@nus.edu.sg]

Received 25 April 2014; revised 11 August 2014

Unlike traditional biocide-based antifouling coatings, emerging antifouling concepts combining

environmentally benign materials and biomimetic concepts may require new antifouling assay methods for
evaluating activity before the transition from lab to dynamic field test environment. As a result, many new
antifouling assays have been introduced in the past few years, each designed to measure specific parameters.
However, it is important to keep in mind that emerging novel antifouling systems leverage on synergism between
more than one antifouling mechanisms of action. Thus, to understand antifouling activity, it is necessary to evaluate
them in different laboratory and natural field environment conditions as polymers may behave differently in static
controlled environment and dynamic conditions of the natural marine environment. For field tests, new ways to
elucidate performance in smaller scale experiments before transition to larger panel performance tests are needed. In
this paper, we discuss the emerging challenges for antifouling testing for R&D.

[Keywords: bioassays, field testing, antifouling, coatings, dynamic tests]

Introduction ships. Candidate coatings selected for vessel tests

Traditional antifouling (AF) solutions
typically incorporate the heavy use of copper-based
biocide formulations in AF coatings1.
Commercialization of a product is usually
undertaken by a paint developer who oversees
development from coating formulation, in-situ field
demonstration to the latter stages of vessel patch
test and full vessel test. Following the discovery of
a biologically active compound in a research
laboratory, developers then undertake the onerous
task of coating formulation, which involves a
complex process to evaluate coating performance in
a laboratory or field testing environment to identify
the best performing coatings. Due to biological
variation in locales and seasonal changes, this
process can be long-drawn and may involve
repeated trials in the laboratory or field environment
to verify its AF performance. The latter stages of
vessel patch test and full vessel test are driven by
performance demonstration goals, requiring coating
developers to apply promising candidate coatings
on a small patch of a vessel hull to demonstrate its
AF efficacy in natural operating conditions.
Successful candidates may then further progress to
performance demonstration on a full vessel, with
applications ranging from yachts, barges and finally
present an expensive gamble for coating developers.
As traditional biocide coatings were largely based
on a toxic activity mechanism, the number of test
standards and criteria that have been developed for
evaluation of AF coatings were designed to assess
coatings that release bioactive substances2. Several
ASTM standards were developed for evaluation of
biocide based coatings 3, 4.
The shift towards the development of
environmentally benign AF solutions has raised
new challenges for AF testing 2. Some of these
“non-toxic” AF solutions may involve multiple
synergistic mechanisms of action leading to an AF
effect5. Emerging non-toxic AF concepts include
biomimetic or “bio-inspired” concepts 6, 7, 8, novel
bioactive molecules9, 10, foul-releasing or ‘no-foul’
surfaces5 and fouling resistant surface micro-
topographies 11, 12, 13. These novel concepts require a
new close cooperation between biologists,
engineers and polymer chemists to elucidate
mechanism of action and ensure appropriate
experiments are designed and used to validate the
merits of the technology. Due to the diversity and
interplay of multiple AF mechanisms, greater
discretion is required compared to the mass
screening approach taken with traditional biocides-
2068 INDIAN J. MAR. SCI., VOL 43, NO.11 NOVEMBER 2014
based coating. Because of the synergy between
mechanisms, it is important to study the AF activity
under both laboratory and field environment
conditions, as polymers and surfaces often behave
differently in static controlled environment as
compared to the dynamic conditions of the natural
marine environment14, 15. The assumption that direct
correlations should exist between laboratory and
field tests is a misnomer 15 as each serves a different
purpose in understanding AF behavior. Testing of
novel AF design under dynamic conditions (that is,
under hydrodynamic shear in moving water), may
reflect more closely natural physical conditions in
the environment.
The importance of appropriate selection of
experimental design, controls and benchmarks
cannot be over-emphasized. For the novice, it may
seem that the variation in the fouling communities
over time and space contributes some confusion in
interpreting field tests. This would not be the case if
the study was supported with robust laboratory
experiments that try to explain the AF mechanism.
Given the constraints for a single laboratory to
undertake field and laboratory tests, there is a need
to develop more ways to evaluate AF activity in
smaller scale experiments before the transition to
large scale AF performance testing. The latter is
often more effectively undertaken by industry as
part of a larger holistic effort towards product
development, which takes into account
manufacturing practice and business models.
During the early stages of coating formulation, the
coating chemistry may not as yet have been
optimized to endure long term weathering in natural
sea conditions. Moreover, materials used during
experimental stages for novel formulations are often
expensive and/or difficult to produce in large
amounts. Most research laboratories are also often
not able to tackle health and safety issues associated
with preparation of large quantities of coatings
needed for panel tests.
Thus, it is prudent to separate the
challenges associated with demonstrating
antifouling activity versus antifouling performance.
The former seeks to demonstrate an AF effect while
the latter usually addresses longer term AF
performance in the natural environment.
In this paper, we review the diversity of AF
testing methods available to practitioners new to the
research and discuss some of the challenges in
selecting the best methods for AF testing. Broadly,
we have divided the R&D process into three phases:
Discovery, Proof-of-concept and Product
Development. A solutions-based approach that
combines laboratory and field assessment is
proposed for evaluating the efficacy of new
environmentally benign AF surfaces.
Laboratory bioassays
Laboratory bioassays are normally
conducted in what may be described as the
Discovery Phase in the AF product R&D. At this
stage, short term tests are favored to rapidly identify
promising formulations for further work. The
coating material may also be fragile and not ready
to withstand physical deterioration in a field
environment i.e. weathering under sunlight and
seawater immersion. Hence, laboratory bioassays
are often introduced at this early development stage
where only a preliminary examination of AF
activity of the coating or surface interaction against
fouling organisms is desired. It is useful at this
stage to also begin to investigate material
compatibility, fabrication techniques and cost
issues.
Fouling organisms commonly used for
laboratory bioassays are classified into micro and
macrofoulers. The former consists mainly of
diatoms such as Amphora spp., Navicula spp. And
Nitzschia spp. and bacterial strains isolated from the
marine environment. Macrofoulers include barnacle
cyprids of Amphibalanus amphitrite, bryozoans
(Bugula neritina), mussel larvae (Mytilus spp.),
tubeworms (Hydroides elegans) and zoospores of
the green alga Ulva. Briand (2009) provides an
overview of the common methods used for
laboratory assays. Selecting an appropriate test
organism (e.g. algae, macroinvertebrate larvae) may
also be important in the design of a laboratory
bioassay. The choice of test organisms must take
into account considerations such as the availability
of broodstock, selection of hardy species that can
survive in laboratory culture and ease of culture
maintenance. Ideally, the biology of the target
species should be well studied. For example, larvae
of the tubeworm Hydroides elegans require a
biofilm to settle 16, 17, 18, 19, 20, 21; hence the design of
bioassays would have to take into account the
conditions for settlement of this species. It is also
useful, at the earliest possible time, to select a test
organism that is relevant to the target application.
Increasingly, emerging new technologies
are focused on developing environmentally benign
surfaces that confer AF properties through the
alteration of surface chemistry, such as surface
charges, surface energies, zwitterionic forces,
wettability22,23,24,25,26,27,28,29,30,31,32,33,
microtopography34,35,36,37,38,39, enzymatic
coatings40,41 or multifunctional strategies such as the
slippery liquid-infused porous surfaces42(SLIPSs)
 LIM et al.: LABORATORY AND FIELD TESTING OF ANTIFOULING COATINGS
that couples both texturing and chemistry. Often
these new technologies are prepared on planar
surfaces and initially presented for assays in small
size coupons for reasons mentioned afore.
Traditionally, anti-settlement assays were
designed on the basis of biocides dissolved in
seawater with assays ran in static condition for 24
hours, in containment. Many new bioassays have
been introduced to assess AF activity. An example
is the droplet assay. In this assay, 20 - 40 barnacle
cyprids are suspended in a droplet (volume ranges
from 400ul to 1.5ml depending on the size of the
material) and dispensed onto a test surface 43, 44, 30, 41
and incubated usually in the dark. Settlement
success is enumerated after 24 hours. A variation
of this test method is the apposing surface assay
developed by Petrone45, where two test surfaces trap
a droplet of cyprids solution in a specially designed
height-adjustable jack. This design is intended to
reduce cyprids-air interaction, which may interfere
with cyprids-surface interaction. In these tests, the
percentage of cyprids settlement on test surfaces,
mortality of cyprids and metamorphosed but
detached barnacle cyprids were investigated. A low
settlement count with low natural mortality
indicates that the surface has an effect against
cyprids settlement. In many cases, the duration of
assays was extended to 48 hours to ensure sufficient
settlement in controls, some examples being tests
involving PDMS34, 44, 36, Xerogel26, PEG43, 30and
PEMA copolymer films41. These design
considerations should be taken into account when
selecting an assay, for example if the materials
contain rapid biodegradable bioactive substances.
Foul release surfaces such as those
composed of silicone or fluoropolymers5 are
typically assessed for performance based on the
adhesion strength of settled macrofoulers such as
barnacles based on the46, 47presented a new assay
using laboratory-reared adult barnacles in a re-
attachment assay on foul-release surfaces, as a
means to evaluate the critical removal stress of
barnacles re-attached on these surfaces. The assay
assists to overcome some limitations for barnacle
testing in the field environment, such as low
settlement rate during poor weather conditions. A
computer controlled automated barnacle removal
test modified from the force gauge removal test47
further enabled down-selection of foul-release
coatings using a high-throughput screening
method48. Some new methods examining cyprids
exploration behavior on modified surfaces have
been introduced to understand larval interactions
with surfaces49, 50, 51. Experiments examining
2069
settlement, larvae metamorphosis, mortality and
exploratory behavior provide valuable clues to
understand mechanisms of action.
Dynamic forces existing in the natural
marine environment (e.g. from tidal streams and
boat wakes) or those occurring over moving vessels
present challenges for marine organisms attempting
to settle. Emerging non-toxic AF solutions that
incorporate modification of surface chemistries
and/or physical microtopography may interfere with
a marine organism’s interaction and subsequent
attachment with these new surfaces, potentially
resulting in lower attachment numbers or lower
adhesion strength if attachment occurred52,53,54,34.
External dynamic forces may therefore aid in the
removal of attached organisms from surfaces. Thus,
there is an incentive to include dynamic flow
elements into laboratory bioassays or field tests to
understand marine organism behavior and fouling
community development under flow conditions.
Many novel dynamic tests have been introduced to
complement our understanding of a flow effect on
AF activity or performance on surfaces. Some
examples of laboratory dynamic assay systems
include surface-biofilm studies in microfluidic flow
channels55, 56, measurement of microfouling (e.g.
algae) adhesion strength in turbulent flow channels
57,58
and measurement of adhesion strength of
macroinvertebrates like ascidians, barnacles and
bryozoans59.
Field Tests
Field immersion studies in natural seawater
is often necessary to further investigate the AF
activity as demonstrated in laboratory conditions
given the diverse chemical, biological and physical
environmental conditions that cannot be replicated
in the laboratory60. Even in the absence of high
current velocities in most static field tests carried
out in coastal or port waters, instantaneous
turbulence created by wind chops and boat wakes
may affect larval settlement61. Field testing
therefore challenges novel surfaces in an
environment subject to natural physical processes
and a diverse fouling community.
Short term field immersion tests are often
utilized in a Proof-of-Concept Phase in field
exposures lasting less than a month. Such
experiment would be sufficient to 1) expose the
coating to fouling by diverse consortia of fouling
organisms in the field, and 2) examine the physical
integrity of the coating under short term weathering
conditions. Small size coupons such as glass rods62
2070 INDIAN J. MAR. SCI., VOL 43, NO.11 NOVEMBER 2014
and microscopic slides63 have been employed.
These may be field tested with a large number of
replicates which present robust data for statistical
tests.
Like laboratory bioassays, short term field
testing may be employed to assist in a ‘screening’
process, with large arrays of coatings to gather
preliminary fouling settlement results. Large arrays
can be screened quickly and the settlement data
used to select for the best coating performer. Unlike
laboratory bioassays, physical weathering issues in
short term field test may be explored at this stage
and are critical feedback to the early stage
development process, providing information on
coating stability and polymer compatibility issues.
Direct field exposure trials have also been used for
the preliminary examination of the natural AF effect
of surface micro-topography in mollusk shells12 and
other macro-invertebrates like crabs (Cancer
pagurus), brittle stars (Ophiura texturata) and
bivalves (Mytilus edulis) 64.
After the initial evaluation of activity, the
next stage would involve a scale-up and application
of promising coating onto large panel sizes
consisting of thick multi-layered coatings that
conform to ASTM requirements65. Candidate
coatings entering the Product Development stage
are evaluated for field performance. Here, the
coating can be subjected to longer field immersion
duration (which may last one to two years) to
evaluate coating effectiveness and other scale-up
issues that may undermine coating durability. While
coatings can be applied on a standard 10 by 12
inches panel using ASTM standards for coating
field evaluation, smaller 4 by 8 inches panels could
also be adopted if size and cost is a constraint.
However, edge effects as a result of encroachment
of organisms from the substrata edges onto the
coating increase with decreasing panel or substrata
size. Selection of appropriate substrate dimension is
therefore an important design criterion. Ideally,
replication should be high but cost constraints often
result in smaller sample numbers.
In the design and conduct of a field test, a
few considerations may be warranted. The actual
duration of the field immersion tests may vary
according to the fouling community and prevalent
fouling rates in the field, as fouling communities
vary in composition and density according to
seasonality. Urban freshwater runoffs and local
changes in hydrology may also affect fouling rates.
These environmental conditions affect the design of
field tests and are important in interpreting the
results gathered from immersion tests. Thus, in any
experiment, it is essential to benchmark the results
against a control coating. It may also be useful to
benchmark results against an equivalent commercial
product as an additional measure of coating
performance. Good performing coatings may be
expected to remain foul-free for one to two years.
Inspection of biofouling coverage on surfaces is
often conducted following ASTM D6990 – 05.
Field immersion studies carried out over different
geographical locations can further yield valuable
field performance data on coating efficacy 66.
Commercially available foul-free or foul
release surfaces have been introduced as an
environmentally benign alternative to toxic coatings
to prevent or reduce fouling settlement on surfaces.
These surface coatings are mainly silicone or
fluorinated polymers and are believed to interfere
with the adhesion strength or attachment of
organisms in contact with the surface5, although the
actual mechanism is not clear. There are now also
new novel surfaces that combine micro-texture, bio-
adhesive resistance, low energy and
environmentally benign compounds that disrupt
adhesives of problem foulers67. The barnacle
adhesion test 47 is often used as a measure of the
fouling-release properties 52, 53, 48. At optimal flow
velocities on a ship vessel, loosely attached
organisms are dislodged from these surfaces. These
surfaces also allow organisms to be removed easily
from a surface with minimal cleaning forces.
Barnacle adhesion tests in accordance with the
ASTM standard, D5618-94 is normally done by
using a force measuring device such as a force
gauge to push against the base of a barnacle until
detachment occurs. To obtain good quality adhesion
data, firstly, a large number of suitably sized
barnacles must be available (usually 5 to 20 mm
diameter). This requires a large surface area for
barnacle settlement to occur and secondly, a
reasonable duration for barnacle growth before
adhesion tests can be carried out. Barnacle
settlement may also be limited by seasonal changes
in barnacle recruitment. Moreover, the quality of
the data is also dependent on the user to perform the
removal test consistently and without damaging the
barnacle. These factors may constraint the quality
of the adhesion tests.
While a laboratory dynamic test carried out
in early stage Discovery phase may involve a
preliminary demonstration of the antifouling
activity of a surface, field dynamic test in the Proof-
of-Concept or Product Development phase usually
take a longer time and involve more robust testing.
Hydrodynamic drag evaluation in foul release
coatings 68 using a rotary disc system may be one
way of investigating drag forces over surfaces in
 LIM et al.: LABORATORY AND FIELD TESTING OF ANTIFOULING COATINGS
flow. Dynamic Performance test methods include
the ASTM D4939 – 89, the rotary drum systems
such as the Poseidon Dynamic Test System
(Poseidon Ocean Sciences, Inc) and flow channel
tests69. There may be however a need to introduce
the development of additional dynamic test systems
that bridge the gap between laboratory conceptual
and commercialization phase.
As AF coatings are usually applied in a
wide variety of applications, ranging from inter alia
shipping, aquaculture to acoustic sensing
instruments, it is ideal that the designing of new
coatings take into account the desired application at
the earliest possible stage, so that durability,
lifespan and operating conditions of the application
is considered early in development. While a coating
intended for ship hull application should remain
effective against macroinvertebrate fouling
settlement for more than five years and suitably
durable for physical wear and tear, application
intended for other novel solutions in aquaculture
and sensing instruments may tolerate a less
demanding operating cycle. For many applications,
it is now useful to include maintenance regimes to
enhance coating lifespan such as the recent
introduction of concepts for hull grooming70.
Conclusion
With the increasing legislation governing
the use of biologically active materials in the
environment, there is a need to consider life cycle
costs associated with environmental fates and
effects. The development of novel non-toxic
antifouling solutions focusing on surface chemistry
or physical micro-topography to reduce organism
settlement or interactions results in an AF effect on
multiple levels, involving different mechanisms of
action. It is thus, now increasingly important that
coating developers consider what the hypothetical
mechanism of action is from the onset of research
and develop appropriate assays to test these
hypotheses. It may also be timely that the research
community considers how ecotoxicology studies
may be introduced earlier in coatings research.
Custom assays may be required to address for
example, effects of increased environmental
degradation time for bioactive molecules released
from surface bonded structures. Such studies will
facilitate and reduce the business risks associated
with the development of environmentally benign
coatings.
For most part, research and development in
novel AF surfaces or solutions employs a
conceptual developmental process that follows the
2071
Discovery - Proof-of-Concept - Product
Development pathway described earlier. This
process aims to identify and simplify a transitional
phase as a new AF product or solution is put into
development. Today, we consider the starting point
as identifying a clear hypothesis for mechanism for
action and using appropriate assays for testing these
AF activities. Mass screening approaches may no
longer be optimal as a starting point, and best left as
an approach for fine tuning formulations. Once an
effect has been demonstrated, appropriate field tests
can be employed that addresses issues with longer
term effect, coating durability and transferability to
a prototype platform for desired application.
Appropriate feedback channels should be in place in
these processes to get timely review. The
emergence of dynamic testing systems is indicative
of the growing interest to understand how
organisms interact with surfaces and their removal
under dynamic conditions. These approaches
provide valuable means to enhance antifouling
activity without the heavy-handed use of toxic
substances.
The scale up of novel coatings into
prototype applications may also present additional
challenges as the synergism or relationship between
different features may change at different scales. As
such, it is envisaged that the development of
biomimetic surfaces will require a much longer
incubation period between laboratory and field
demonstration. The introduction of novel small
scale tests will be useful to bridge the gap between
laboratory and field (static/dynamic) tests.
Lastly, given the extent of data collection
needed for commercialization and registration
process (e.g. European Union Directive 98/8/EC), it
is useful to consider target application early in the
research phase and work closely with industry
partners to gather data in the relevant format as
early as possible.
Acknowledgements
The authors acknowledge the research
support from A*STAR-SERC "Innovative Marine
Antifouling Solutions" Program Grant No. 102-166-
0102 and MIMO Program Grant No. 112300.
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