Coral-Sunscreen Report FD PDF

(CONFIDENTIAL REPORT)
Photos by Sabine R. Alshuth-Dugger and Durwood M. Dugger © 2013
An Initial Study of Patrick's Sunscreen Effects

On Six Reef Building Hard Coral Species
(A new experimental protocol for skin and hair protection product testing on reef species.)
October 30, 2013
Prepared for: Kelley Stark, Pres., Patrick's Sunscreen

24418 Main St., Ste. 405
Carson City, CA 90745
Tel. 310-294-5030, Cel. 951-312-1525
kelley@patrickssunscreen.com
Prepared by: Durwood M. Dugger and Sabine R. Alshuth-Dugger, Ph.D.

BCI, Inc.
947 Sandpiper Lane
Vero Beach, FL 32963
Tel. 772-332-1046, Skype - Biocepts
ddugger@biocepts.com
An Initial Study Of Patrick's Sunscreen Effects On Six Reef Building Hard Coral Species Page i
Abstract
To examine the effects of Patrick's Sunscreen mineral shield™ on six species of Pacific reef
building hard corals (20 specimens each) produced by aquaculture and purchased from a
local producer, the corals were placed in six specially designed coral life-support systems,
each system containing three tanks. The corals and system were acclimated for two weeks
while water quality was managed, measured daily (temperature, salinity, pH, ammonia
nitrogen and carbonate hardness), recorded and photos of each coral were recorded. Then
five of the six sets of corals were dosed at 5 different sunscreen dilutions of Patrick's
Sunscreen mineral shield™ from 0.025 - 10.0 ppm. One of the six systems was designated
as a control, and received the same conditions as all the other tanks, but received no
sunscreen treatment. Post-dosing the water quality and the corals continued to be
monitored and photo recorded daily for the following week. Seven days after exposure all
corals were removed from the tanks and visually described/rated according to a
predetermined coral health descriptive rating system. Coral bleaching was not observed in
any of the six treatments. A Univariate Analysis of the corals' health condition one week
after their dosing produced no statistical differences between the Patrick's Sunscreen
mineral shield™ treated corals and the untreated control corals.
Copyright: © 2013 Durwood M. Dugger and Sabine R. Alshuth-Dugger, Ph.D.
Funding: This work was funded by Patrick's Sunscreen, Inc. The funder had no role in study
design, data collection and analysis, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Correspondence: ddugger@biocepts.com
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947 Sandpiper Lane Vero Beach, FL 32963 *** 772/332-1046 *** www.biocepts.com *** ddugger@biocepts.com
An Initial Study Of Patrick's Sunscreen Effects On Six Reef Building Hard Coral Species Page ii
Acknowledgements
We would like to thank the following people and organizations for their support, assistance
and help in the preparation of this report:
• Mr. Kelly Stark, President of Patrick Sunscreen and OP Products, Inc. for funding this
study.
• Dr. Junda Lin, Director and Ms. Nancy Pham, Site Manager of Florida Institute of
Technology for making space and resources available at their Vero Beach Marine Lab
on short notice to house our testing systems.
• Mr. William Hoffman, Smithsonian Marine Ecosystem Exhibit for his suggestions in hard
coral holding system design and help in evaluating the post-experimental coral health
conditions.
• Mr. Jason Dwinell, ORA, Inc. for his help in evaluating the post-experimental
photographs of the studies' coral specimen health conditions, and particularly in defining
typical types of coral tissue necrosis, their differences and diagnostics, and possible
causes.
• Mr. Adeljean L.F.C. Ho, Ph.D. Candidate, Fish Biology/Aquaculture Laboratory, Florida
Institute of Technology, Melbourne, Florida, USA for his help in the statistical analysis of
the study results.
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An Initial Study Of Patrick's Sunscreen Effects On Six Reef Building Hard Coral Species Page iii
Contents
Acknowledgements ............................................................................................................. ii
Contents............................................................................................................................... iii
Table 2. End-Of-Study, Observable Coral Health Evaluation and Ranking (Six Species)... vii
Figures ................................................................................................................................ vii
1 Executive Summary ....................................................................................................... 1
1.1 Introduction and Background...................................................................................... 1
1.2 Methodology ............................................................................................................... 1
1.3 Key Findings ............................................................................................................... 2
1.4 Key Recommendations .............................................................................................. 3
2 Introduction..................................................................................................................... 4
2.1 Background and review of current literature ............................................................... 5
2.1.1 Danovaro et al. (2008), Sunscreens Cause Coral Bleaching by Promoting Viral
Infections .......................................................................................................................... 5
2.1.2 Antonio Tovar-Sánchez et al., (2013) Sunscreen Products as Emerging

Pollutants to Coastal Waters ............................................................................................ 9
2.2 SHPP concentration levels in context with population density and usage - and actual
coral reefs.......................................................................................................................... 11
2.3 Objectives ................................................................................................................. 16
3 Methods ......................................................................................................................... 18
3.1 Research Questions ................................................................................................. 18
3.2 Research Design ...................................................................................................... 18
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3.3 Test System Design ................................................................................................. 20
3.4 Primary Data Collection Instruments ........................................................................ 24
3.5 Test Population......................................................................................................... 25
3.6 Data Collection ......................................................................................................... 26
3.7 Data Analysis............................................................................................................ 27
3.8 Limitations ................................................................................................................ 27
4 Results and Discussion ............................................................................................... 28
4.1 Study Data and Results ............................................................................................ 28
4.2 External Study Variables .......................................................................................... 31
4.2.1 Tissue Necrosis (TN) .......................................................................................... 31
4.2.2 Experimental Coral Sources ............................................................................... 35
4.3 Additional experimental observations - incidental test species ................................ 36
4.3.1 Incidental coral species that experienced dosing and survived.......................... 36
4.3.2 Incidental shrimp species that experienced dosing and survived....................... 37
4.3.3 Incidental brittle star species that experienced dosing and survived.................. 37
4.4 Proposed explanations for Patrick's Sunscreen mineral shield's™ general lack of
toxicity ............................................................................................................................... 38
5 Recommendations........................................................................................................ 41
6 References .................................................................................................................... 42
7 Appendices ................................................................................................................... 43
7.1 Appendix - Understanding sunscreen concentrations .............................................. 43
7.2 Appendix - Water Quality.......................................................................................... 45
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An Initial Study Of Patrick's Sunscreen Effects On Six Reef Building Hard Coral Species Page v
7.2.1 Daily Tank - Temperature Record ...................................................................... 45
7.2.2 Daily Tank - pH Record ...................................................................................... 46
7.2.3 Daily Tank - Salinity Record ............................................................................... 47
7.2.4 Data Logger Control Tank Temps./30 min. ........................................................ 48
7.3 Appendix - Coral/Patrick's Sunscreen Study Statistical analysis.............................. 49
7.3.1 Page 1. ............................................................................................................... 49
7.3.2 Page 2. ............................................................................................................... 50
7.3.3 Page 3. ............................................................................................................... 51
7.3.4 Page 4. ............................................................................................................... 52
7.3.5 Page 5. ............................................................................................................... 53
7.3.6 Page 6. ............................................................................................................... 54
7.3.7 Page 7. ............................................................................................................... 55
7.3.8 Page 8. ............................................................................................................... 56
7.3.9 Page 9. ............................................................................................................... 57
7.3.10 Page 10. ........................................................................................................... 58
7.3.11 Page 11. ........................................................................................................... 59
7.3.12 Page 12. ........................................................................................................... 60
7.3.13 Page 13. ........................................................................................................... 61
7.4 Appendix - Pre-dosing and Post-dosing Photo Record ............................................ 62
7.4.1 Photo Comparison Tank 1. ................................................................................. 63
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Tables
Table 1. Study activity timeline.
Table 2. End-Of-Study, Observable Coral Health Evaluation and Ranking (Six Species).
Figures
Figure 1. Concentrations of sunscreen ingredients in waters of Palmira Beach, Mallorca.
Figure 2. High population beaches and marine environment/human SHPP interaction.
Figure 3. Population density for some of the world's most popular coral reefs.
Figure 4. Coral holding study system and component comparison collage.
Figure 5. Coral Species Placement Pattern For All Tanks.
Figure 6. Reef building hard coral species selected randomly from study's photo records.
Figure 7. The Coral Color Reference Card developed for standardizing changes in coral
Figure 8. Tissue necrosis one day after receiving corals from ORA and before dosing.
Figure 9. Various stages of coral tissue necrosis from treated and non-treated tanks.
Figure 10. Extraneous species observed incidentally participating in the experiment.
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An Initial Study Of Patrick's Sunscreen Effects On Six Reef Building Hard Coral Species Page viii
Acronyms
SHPP Skin and Hair Protective Products
ORA Ocean Reefs and Aquariums, Inc.
PS Patrick's Sunscreen mineral shield product™
VBML Vero Beach Marine Lab of the Florida Institute of Technology
TCC Treasure Coast Corals, Inc.
SML Surface Micro-Layer
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An Initial Study Of Patrick's Sunscreen Effects On Six Reef Building Hard Coral Species Page 1
1 Executive Summary
1.1 Introduction and Background

In 2008 a scientific paper was published that arguably claimed that sunscreen products and
other skin and hair protective products (SHPP) were broadly "toxic" to corals at the levels
that are normally found in proximity with coral reefs. The study arguably did show that
sunscreen compounds produced stress in hard corals directly at the excessive levels used
in the study. The study did show that when the corals' symbionts were sufficiently stressed
(with or without endemic virus infections), it caused their coral hosts to eject them. This
ejection of the corals symbionts is the basic definition of coral bleaching, but it is also the
corals' reaction to a number of forms of metabolic stress beyond toxicity.
The results of Danovaro's 2008 study caused wide spread alarm in the environmental
activist community, genuine concern amongst marine scientists and serious concern among
many SHPP manufacturers and sales distributors. Many marine scientists and coral reef
experts questioned some of Danovaro's assumptions and cautioned that further
investigation was needed before accepting its results. However, even without examination
of these 2008 study's assumptions, or any attempts to replicate it, and or further studies to
verify, confirm, clarify and or expand its results - the studies' unverified claims were recycled
ad nauseum in the environmental, popular and social medias. Consequently, potential
SHPP toxicity has become a major reef conservation and associated marketing concern to
environmentally sensitive sunscreen users and those product's producers.
The study reported herein was funded by, and because of, one such sunscreen
manufacturer's concerns. Our study was conducted to determine if Patrick's Sunscreen
mineral shield™ effects the six species of reef building hard corals selected for the study.
1.2 Methodology
Six species of Pacific reef building hard corals (20 specimens each) that were produced by
commercial aquaculture fragmentation technology (asexual propagation) were purchased
from Ocean Reefs and Aquariums, Inc. They were placed in six specially designed coral life-
support systems, each system containing three tanks. Two of these tanks formed the coral
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holding and interconnected life-support sump system. The corals were placed equidistantly
(about 3 inches) apart from each other on a plastic matrix with one-half inch openings that
could receive and or support the plastic bases of the cultured coral specimens. The third
upper tank in the vertical sets of three was a separate tank with only aeration. This tank was
used for dosing the corals. All corals were monitored and photo recorded daily through the
following week.
Seven days after exposure, all corals were removed one at a time from the tanks and
visually described/rated according to a predetermined coral health descriptive rating system
that was based on the observable coral tissue/polyp health and comparisons of coral
specimen color immediately before dosing and one week after dosing - comparing each
coral's color to the Siebeck Coral Color Reference Card.
1.3 Key Findings

• There were no mass bleachings of any coral species or individual specimens. There
were three species out of the six species used in the experiment where individual coral
specimens evinced various levels of partial to complete tissue necrosis generally
progressing from the base up. The reasons for their poorer performance appeared to be
unrelated to sunscreen exposure. We believe there were two reasons for their poor
performance in the test system:
o These corals were apparently recently fragmented, as they did not show
comparable base overgrowth establishment - as did other species in the study.
o Generally all of the poorer performing species were branched hard corals - known
to require high levels of water movement - indicating possible insufficient water
movement for them in the study's coral holding system.
• Three of the coral species had no abnormal health implications (100% healthy
specimens at the end of the study) regardless of the level of sunscreen dosing indicating
that water quality itself was not an issue. The three other coral species in the study had
various levels of tissue necrosis from minor in two species' specimens, to complete
necrosis of several individuals in one species.
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Given their spatial distribution within each tank, and that the necrosis incidence was also
recorded in the control tank, it was unsurprising that there was no significant statistical
relationship between dosage concentrations and the control which was verified by
Univariate statistical analysis.
1.4 Key Recommendations

• Additional tests need to be performed on a broader array of key reef building coral
species and other phyla critical to maintenance of a healthy coral reef complex.
• While the designed system served the initial study quite well, a pulse dose/constant flow
through system would be required to demonstrate the effects of longer term and repeat
exposures. A properly designed flow-through test system (with adequate seawater
resources available) using a precise metering of the product to be tested could emulate
the daily pulse dose of SHPP as shown in recent studies on coastal sunscreen
concentration levels, and to emulate specific and accurate concentration levels at highly
visited coral reefs - once they are known. There are currently no such studies available.
• Additional SHPP concentration studies need to be made in the field adjacent to and
verified around high visitation coral reefs, to aid in SHPP formulation guideline
development and or coral reef management guideline changes before they are made.
While some published studies show the presence of typical sunscreen ingredients in
coastal waters of high density public beach areas in the Mediterranean, there are no
known studies documenting the levels of coral sunscreen even in popular coral reef
areas, or studies showing a direct linkage between sunscreens in coral reef waters to
any negative coral health conditions.
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2 Introduction
In recent years the confluence of factors have caused concerns about sunblock chemicals
in the marine environment. Some of the primary factors are:
• A growing awareness of UV radiation hazards and the increased use of UV blocking

chemical in sunscreens, cosmetics, shampoos, conditioners and other in human health
maintenance.
• The increased use of the same and or chemically related industrial UV blocking agents
in paints, coatings, clothing, dyes and plastics - all of which find their way into coastal
runoff and are typically found in global sewage outfall systems.
• The increased air travel service (and other modes of transportation) to more remote
locations allowing more coral reef/human interactions than ever before.
While the above chemical pollutant and access factors have created greater concerns
regarding potential SHPP toxicity, the increased access for more snorkelling and SCUBA
diving on coral reefs has had a positive aspect as well. Increased travel to more remote
tropical areas with coral reefs has increased our knowledge and awareness of coral reef
ecosystems. Indeed, this increased access to, and visitation of coral reef areas around the
globe have largely been responsible for the awareness of global coral reef health changes
and potential anthropogenic activity impacts.
Unfortunately, a general lack of baseline surveying to determine and establish what actual
exposure concentration levels of SHPP are at high visitation coral reefs has led to
inaccurate and biased claims of those concentrations by environmental alarmists pursuing
their own agendas that occasionally are seen as self-serving regarding their own research
funding. Consequently, some studies have used grossly unrealistic levels of SHPP, and as
well confuse chemical metabolic toxicity with physical stress symptoms caused by their
unrealistic dosage concentration levels. This has created confusion regarding the validity
and the accuracy of their results by alarming environmental activists, environmental
managers and the general public regarding the responsible use of SHPP such as
sunscreens. The potential damage to the health of the public who has difficulty weighing
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SHPP health advantages with poorly established and unverified environmental affects of
SHPP is a very real problem. If the potential negative environmental affects of SHPP are not
accurately assessed and carefully weighed against their human health benefits, the moral
and ethical liability of increased illness and premature death rates from excessive UV
radiation exposure becomes the burden of those (scientists and media) who pre-maturely
and without confirming studies create unnecessary alarm.
2.1 Background and review of current literature
2.1.1 Danovaro et al. (2008), Sunscreens Cause Coral Bleaching by Promoting Viral
Infections
In 2008 Roberto Danovaro et al. published a paper that arguably demonstrated evidence
that sunscreen products were broadly "toxic" to corals. The study questionably showed that
sunscreen compounds were directly toxic to hard corals. The study's dosage levels were
orders of magnitude over sunscreen levels recorded in very high population density
beaches. Physical stress was created by the coral study's holding system's water quality.
The study's results are further obscured by its failure to document water quality during the
study. As well, these high dosage level induced stress factors could have also caused
"bleaching" - zooxanthellae ejection, and the rapid multiplication of endemic virus breakage
found in the corals' symbiont zooxanthellae - even without sunscreen exposure. While
Danovaro's study primarily focuses on the zooxanthellae/ viral activity, unfortunately the
general lack of detail recorded in the Danovaro report, particularly regarding to coral holding
methodology, water quality maintenance in the holding systems and the grossly excessive
sunscreen dosage levels (10-100 ppm), makes it impossible to accurately separate physical
stress factors from chemical toxicity factors and assess the validity of reported results (see
Appendix 7.1).
The Danovaro study proposed that the UV sunblock chemicals caused viral breakage and
ultimately caused the ejection of the virally infected zooxanthellae by the host coral polyps.
According to the author - the study showed that the endemic virus living in the
zooxanthellae were sensitive to some of the most common sunblock compounds found in
commercial sunscreen products and that the virus responded to exposure to these
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compounds by multiplying and making the zooxanthellae sick which caused the coral to
eject them (bleaching), ultimately leading to the death of those coral polyps.
It is possible for physical stress (induced by heat, changes in pH, high ammonia nitrogen,
low dissolved oxygen, high water column particulates, etc.) to produce the same effects in
the corals causing them to eject zooxanthellae. You can't simply drop a coral polyp colony in
a plastic bag without a water quality/life support management system - including adequate
water quality monitoring and then try to separate resulting stress affects from any toxic
affects created by the actual chemical products being tested.
Regardless of the Danovaro study's debatable scientific merit, this 2008 study produced a
general alarm among environmentalist, environmental NGOs, made for "hot copy" in the
popular environmental media and even some of the mainstream media. The venerable
National Geographic did not wait for replication and verification of Danovaro's study and
confirmation of its claims regarding SHPP toxicities. Remarkably few, if any additional
studies have been done since 2008, to replicate Danovaro's work or broaden the
understanding of sunscreens' environmental effects. Consequently, potential SHPP toxicity
has become a major reef conservation issue and associated marketing concern to
environmentally sensitive sunscreen users and those product's producers.
The Danovaro study's primary contributions were:
1. Providing further environmental concern regarding overpopulation effects and potential

SHPP chemical toxicities to coral reef species interactions.
2. Demonstrating the general confusion and scientific inadequacy regarding the differences
between public beach human population densities and typical coral reef visitation.
3. Demonstrating the necessity of understanding what real world seawater levels are for
SHPP chemicals near coral reefs - before doing a study. Understanding not only the
SHPP concentration levels, but as well the SHPP concentration levels' dilution rates by:
filtration, break down, binding, and or interaction with their proximate environments.
4. Demonstrating the need to determine real world SHPP component concentration in the
typical coral areas around the world, again before beginning testing for toxicity.
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5. The need for a reproducible coral experimental system wherein SHPP products can be
tested with adequate water quality maintenance and without handling stresses becoming
significant bias and or variables within the experiment.
"Reef Safe" certification implausibility - "Sunscreens cause the rapid and complete
bleaching of hard corals, even at extremely low concentrations. The effect of sunscreens is
due to organic ultraviolet filters, which are able to induce the lytic viral cycle in symbiotic
zooxanthellae with latent infections." Following publication of the above study and the quote
from it above, and the resulting popular and social media regurgitation, there has been a
demand for SHPP "reef safe" certification by buyer/consumers from SHPP producers to
assure them that specific sunscreen products were not generally hazardous to coral reef
environments. This demand by consumers has caused some sunscreen producers to
logically seek "reef safe - scientific certification" of their products as being non-hazardous to
the reef environment.
As a response to the "Reef Safe" demand some organizations such as MARINEPOSITIVE

(of which Roberto Danovaro is coincidently now Chief Scientist and Co-founder) are
providing "reef safe" testing of SHPP and other products on reef species.
MARINEPOSITIVE has an even broader business model and in addition to SHPP testing - it
also does reef safe live style assessments for individuals, organizations and businesses - all
for an appropriate fee of course. After paying professional expenses Marine Positive's
remaining "profits" are donated to the "Going Blue Foundation" - another "non-profit"
organization founded, owned, operated and we only assume "expensed" by some of the
same people as MARINE POSITIVE including Roberto Danovaro.
Why "Reef Safe" certification isn't credible - The concept and the purpose of "Reef Safe"
certification - is more than logically and scientifically questionable. Hard corals are members
of a large class of organisms with over 6,000 species - about 1,000 of which are reef-
building corals - that live in the coral reef ecosystem complex. This coral reef system
complex has been estimated to contain nearly a million species. While hard corals
(Scleractinians) are primary reef structure builders, many other species are required to build
a "reef" - including various calcareous algae, molluscs, crustaceans and other calcium
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carbonate secretors that all contribute to reef structure concretions. Coral reefs are also
dependent on many non-anthropogenic factors:
1. Their inherent geology (geological up thrust can push reefs above the ocean surface, or
geologic subsidence can sink reefs below critical light and temperature tolerances).
2. Their specific location regarding currents, extreme weather exposure (i.e. extreme
temperature).
3. Fluctuations in oceanic chemistry and resulting transparency from land runoff, short and
long cyclical ocean upwelling processes, and or volcanoes and other geothermal events.
4. Storm wave energy erosion and or damage.
5. Ecological shifts in coral predator and or competitor balances.
Each factor is important and potentially limiting to a coral reef's overall health and longevity.
Consequently, some coral reefs have more success and or natural degradation than others
- regardless of proximate human activities. It is important for everyone to understand that
not all coral reef health woes are human caused and that natural negative processes need
to be separated from anthropogenic causes.
Currently there are no scientifically accepted standard protocols to test for "Reef Safe"
certification because of coral reef's broad speciation and testing difficulties listed.
Additionally there have been far too few studies addressing actual SHPP concentration
levels experienced by coral reefs and other sensitive marine ecosystems to claim that
sunscreen chemicals are potential threats to corals or other organisms. Even if competent
studies were to eventually determine that UV sunblock chemicals were toxic, the source of
those chemicals in the environment would need to be first determined. Determining how to
separate and compare quantitatively the much higher global volumes of chemicals similar to
SHPP chemicals commonly found in coastal runoff and sewage ocean-out-falls (cosmetics,
shampoos, conditioners, hair styling agents, industrial UV inhibitors in paints, plastics, dyes,
coatings, etc.) would need to be accomplished in order to prioritize the threat from
sunscreen on beach bathers and water recreationists.
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2.1.2 Antonio Tovar-Sánchez et al., (2013) Sunscreen Products as Emerging

Pollutants to Coastal Waters
Perhaps the most illustrative information regarding typical sunscreen dilutions in the water
near a crowded beach is an open source report by A. Tovar-Sánchez et al. (Sunscreen
Products as Emerging Pollutants to Coastal Waters. Published June 15, 2013). Besides the
Tovar-Sánchez study results showing the primary influence of the tested SHPP were in the
Surface Micro-Layer (SML) as opposed to the water column beneath (where corals
generally exist, excepting their surface exposure during major tidal variations such as the
perihelion in combination with spring tides), of particular interest was their tracking of not
just major organic UV sunblocks - such as benzophehone 3 (BZ-3) and 4-
methylbenzylidene camphor (4-MBC), but metallic oxide sunblocks such as TiO2 and ZnO in
the coastal waters off the mildly crowded Palmira Beach (but very densely populated
compared to most coral reef areas), and down current along the Island of Mallorca
(Majorca) in the Mediterranean. The Palmira Beach area does not have coral reefs, but
does provide a specific water column and SML model for sunscreen use pulse peaks and
the following dilution, reduction and conversion (physical, chemical and biological) over the
beach day - at least under the specifics of similar beaches with similar processes and
characteristics.
Of particular note in Tovar-Sánchez study results are how the Zinc and Titanium peak with
the solar/bathing sunscreen application. As well, how the highest levels (less 3.5 ppm in the
SML) tend to be limited to the seawater's surface interface, while the subsurface levels are
barely affected (unlike the BZ-3), and how even the SML of sunscreen rapidly (about 6-7
hours) dilute to background seawater levels of less than 120 ppb by the end of the
"sunbathing day" and at which these compound toxicities are all undemonstrated (see
Figure 1).
The purpose of examining both the Danovaro and the Tovar-Sánchez studies is relevant - if
not essential to setting realistic dosing for SHPP testing. The 2008 report by Danovaro
stated sunscreen testing levels in µ-liters/Liter - or parts per million. The Danovaro study
dosage levels of 10-100 µl/L (10-100 parts per million), and the report stated that
"Sunscreens cause the rapid and complete bleaching of hard corals, even at extremely low
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concentrations."
Compared to Danovaro's study dosages, A. Tovar-Sánchez, et al. (2013) were only able to
measure organic sunscreen levels (BZ-3 and 4 MBC) in the seawater column levels ranging
from 10 ng/L (10 parts per trillion) and to the highest concentrations in the SML of under 600
ng/L.
Figure 1. Concentrations of sunscreen ingredients in waters of Palmira Beach, Mallorca.

(BZ-3 (A), 4-MBC (B), Zn (C), Ti (C) and nutrients (D) in the unfiltered (Total) and filtered (<0.22 µm; Diss)
fraction of the Surface micro-layer (SML) and Subsurface (1 cm) seawater (SW) samples from Palmira Beach.
(Adapted from Antonio Tovar-Sánchez et. al. June, 2013))
Of particular interest to this study's designers, A. Tovar-Sánchez, et al. (2013) were only
able to measure metallic sunscreen levels (ZnO and TiO2) in the seawater column ranging
from 0.02 µg/L (parts per million) and at the highest concentrations in the SML of under 3.5
µg/L (parts per million).
The point here is that when you compare the maximum real world recorded sunscreen
levels of less than of 0.0000006-3.5 ppm (depending on sunscreen formula) in the seawater
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column (where corals spend most if not all of their time depending on species) - as in the
Tovar-Sánchez study - it puts the Danovaro study design and results in very questionable
light - specifically regarding the lack of research in the experimental design of the
methodologies and appropriate dosages used in their study. Consider that Danovaro's
study dosage levels were water column levels of 10-100 ppm of sunscreen, which were at
3-30 orders of magnitude higher than dosages shown to be actually present in the water
column off a highly used Mediterranean beach resort community of Palmira Beach by the
Tovar-Sánchez study. Very probably even less comparable to the additionally lower orders
of magnitude levels of SHPP likely to be present on the more typically remote and lower
human density usage coral reef systems.
2.2 SHPP concentration levels in context with population density and

usage - and actual coral reefs
To make sunscreen evaluation studies even more difficult to perform accurately, sun and
beach bathing, water sports recreation, and sunscreen/water interactions vary in the
extreme from activity to activity and area to area. In addition, the vast majority of coral reefs
lie well offshore from dense tourist beach bathing areas and even when proximity is close -
the dilution factors between bathing areas and coral reefs are usually extremely great. Even
at dense beach bathing areas there is continual mixing by wave action and on-shore and
lateral moving currents and as well the filtration of inshore water by waves casting surface
components up on the intertidal beach and rack lines.
Reefs are primarily affected by those humans who come within close proximity of the reefs -
such as snorkelers and scuba divers. It should be noted that many water recreationists and
particularly snorkelers wear rash guard clothing to protect them against stinging cells and -
the sun and therefore are far lower potential sources of sunscreens of any kind. All human
SHPP concentration levels and their respective environmental impact factors vary by both
wind and tidal influences compounding the difficulty in estimating the dilution
concentrations. It would be highly misleading to rely on the broadly and highly differing
sunscreen usage data from high-density public beach usage and concentrations as a
baseline reference or assumptions for coral reefs in general. Each specific coral reef should
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be assessed based on current chemical analysis of sunscreen materials in its surrounding

waters.
Additionally, the physical parameter variability of coral reefs also affects SHPP
concentration. While sunscreen chemical concentrations may be perfectly safe in an area
where high mixing and dilution occurs rapidly, even a mildly toxic chemical could be
hazardous to the coral reef in a small lagoon system where much less water flow might
allow concentrations levels to build up and dilution might not occur rapidly enough. Again,
very few studies have been done which report the actual levels of human body originating
SHPP (as opposed to similar industrial chemicals used to block UV in paints, plastics, and
other products) in the proximate waters of coral reefs - or the factors that affect their dilution.
No current studies exist that adequately define sunscreen usage with and as a function of
water recreational type, respective population density and those densities' relationship to
environmentally sensitive areas. In other words they do not distinguish levels found near
metropolitan public beaches from those of dramatically lower populated and used coral
reefs - and most especially the relative concentrations of SHPP in those areas.
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The photos below are of some of the world's most crowded bathing beaches. None are
immediately proximate to coral reefs, and only one has any information regarding actual
sunscreen levels in the associated waters.
Figure 2. High population beaches and marine environment/human SHPP interaction.
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Now examine views of some of the world's most popular coral reefs - as swimming,
snorkelling, and SCUBA diving destinations:
Figure 3. Population density for some of the world's most popular coral reefs.
Above, Figures 2 and 3 are in no way intended to be a scientific analysis, to diminish, or

deny the threats to coral reefs from global over-population, or specific anthropogenic
chemical pollution. They are offered to gain a realistic perspective in the differences
between popular public beach population densities - and the much lower popular coral reef
population densities (and their typical proximity to public beaches) and with respect to their
vast potential differences in SHPP concentration levels in the surrounding waters.
Understanding the differences between public beach SHPP levels and coral reef levels are
essential to designing meaningful studies to determine SHPP affects on coral reefs.
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The photos in Figure 3 show some of the world's most popular coral reef snorkelling and
SCUBA diving sites, and are offered to contrast their visual and respective population
densities/usage with those of high population public beaches in Figure 2. The Palmira
Beach photo (lower right corner) in Figure 2 is included because it is the location of an
actual study of that area's coastal sunscreen levels and to provide population density
comparisons to the popular dive sites in Figure 3.
Of the high visitation coral reef sites included in Figure 3 the island of Bonaire in the Dutch
Caribbean - may have the best statistical information of diver usage as any place in the
world. Bonaire is known for its 42 miles of coastal dive sites, of which about 60 are
accessible for shore snorkelling and diving - unlike most snorkelling and dive sites that
require boat access. Bonaire has been one of - if not the top diving destination in the
Western Hemisphere for the past five decades. Bonaire's coral reefs continue to be rated as
some of the healthiest coral reefs on the planet. Additionally the island's coast offers very
little in the way of large public bathing beaches that might obscure diving related SHPP
impacts on local coral health. Bonaire receives about 50,000 snorkelers and divers each
year because of its stable tropical temperatures, being located out of the Hurricane belt, and
the usually crystal clear waters which are accessible for diving year round.
If we divide the Bonaire's average 50,000 divers a year by 365 days in a year we get an
average of 137 divers in the water each day generally for about 1-3 hours (one to two dives
a day) per day. Bonaire's diving takes place over approximately 42 miles of east/south-
western coastline (including Klein Bonaire Islet) and Lac Bay on its southeastern coast,
while most of Bonaire's eastern and north coast are too rough for safe diving. These 42
miles contain about 90 marked dive sites of which at least 50-60 are accessible from the
shore of Bonaire (Klein Bonaire Islet accessible by boat only). All of which means that on
the average there are only 3.4 divers per mile (1.3 divers divided over Bonaire's 90 dive
sites) per day diving in the 42 miles and 90 dive sites of Bonaire. Look again at the public
beach photos and the coral reef population density photos. Again, the point here is to
understand that there are dramatic differences between the population density usage and
potential SHPP concentration levels for typical tourism exploited coral reefs.
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If the previous review of the comparative differences between actual sunscreen

concentrations as measured in the Tovar-Sánchez study - to that of the dosages used by
the Danovaro coral toxicity study aren't convincing that Danovaro's dosage levels made his
study effectively meaningless, consider a representative dosage analogy that the average
person might be better able to visualize. Imagine a standard Olympic size swimming pool.
The Olympic standard requires it to be fifty by twenty-five meters and two meters deep
containing 2,500,000 liters (660,502 gallons) of water. Using Danovaro's own assumption
sources, we find that it would take 102,596 people wearing sunscreen at USDA
recommended levels to bring the pool's water to 100 ppm over a period of one hour. The
amount of space given to each person to stand would be 0.01 meters square (0.13 square
feet) - which is a spatial impossibility for normal sized humans. Essentially, to achieve
Danovaro's highest coral dosage levels calculated as coming from bathers' bodies in an
Olympic Pool dimensioned area - would effectively require so many bathers it would leave
no space in the pool for water or corals. Obviously, far exceeding any possible human water
recreational densities - anywhere. (For the basis of these statement's calculations, see
Appendix 7.1 Real world sunscreen concentrations.).
2.3 Objectives
The objectives of this study are:
1. To responsibly help protect global coral reefs from potential negative human SHPP
affects by increasing the available information for the accurate design and implementation
of such studies.
2. To determine if the formula of Patrick's Sunscreen mineral shield ™ product that was
supplied for the respective tests would produce any specific observable abnormal
responses in the respective hard coral species being tested.
3. To record, quantify, compare, analyse and interpret study results and report them
accurately and without bias to the client by:
a. Developing a reproducible physical test system design that as close as possible
mimics coral reef water quality conditions in velocity, salinity, pH, carbonate
hardness, ammonia nitrogen levels, temperature and light characteristics.
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b. Determining realistic levels of SHPP that might be experienced at real world coral
reefs and developing suitable SHPP testing dosages that cover those levels.
c. Designing an experimental testing system that can be constructed from readily
available equipment, materials and supplies.
d. Sourcing water chemistry protocols that use as much as possible of pre-packaged
water quality testing kits and or instruments for that purpose and are commonly used
by successful coral culturists.
e. Having zero impact on the environment by using aquacultured hard coral species
that are available through the marine aquaria hobbyist trade and which don't further
impact coral reef systems as wild collected coral testing obviously does.
f. Accomplishing this study using more realistic levels/dosages of SHPP that might be
actually experienced by coral reefs - as opposed to those experienced by high-
density public beach waters.
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3 Methods
3.1 Research Questions
The research questions to be answered by this study are:
a. Do previous studies on sunscreen toxicity represent accurate assessments of sunscreen

affects on the marine environment?
b. Have previous studies used doses of sunscreen that are appropriate and representative
of real world sunscreen exposure to coral reefs?
c. Or, have previous studies used doses of sunscreen that are actually more representative
of real world sunscreen exposure - at typical high population density public beaches
rather than coral reefs?
d. Can an effective coral holding system with the necessary life support be designed and
built cost effectively that allows separation of captivity and handling stresses from the
potential SHPP toxicity effects to be tested?
e. Considering all of the aforementioned questions does Patrick's Sunscreen mineral
shield™ product produce any statistically negative impacts on the specific reef building
hard coral species being tested?
3.2 Research Design

To answer the above research questions and previously stated objectives:
a. A literature review was made of existing literature regarding SHPP and coral reef
interactions, and SHPP concentration ranges.
b. A literature review was made for various types of SHPP concentration levels found in
coastal waters with respect to how they might compare to previous SHPP/coral
interactions and effect studies - specifically Danovaro's 2008 study.
c. By comparing the current, but still limited studies of SHPP concentration levels in near
shore coastal waters, we were able to design a more appropriate test system with study
dosage concentration levels to be more comparable to those dosage levels potentially
experienced by high visitation coral reefs - rather than those of high population density
public beaches.
d. Based on existing technology and the available resource limitations an economical coral
holding and life support system was designed and built from materials and equipment -
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available from local reef aquarium shops and off the internet. This system demonstrated
its adequacy in holding and testing corals for the duration of this initial SHPP/coral study.
e. Six species of aquaculture propagated corals were selected as being typical and
representative of Pacific reef building hard coral species, purchased, acclimated, pre-
dosing monitored, a baseline water quality, health and photographic record created.
Then the respective coral test specimens were dosed with Patrick's sunscreen mineral
shield ™ at five different dosages concentration levels (excluding the untreated control) -
and then post-dosed monitored, health condition evaluated and condition-ranked and the
results statistically analysed.
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3.3 Test System Design
Coral testing system - A coral holding/life support system was designed and built based on
adaptations of the "Standard" and "Berlin" Method of coral/reef tank culture systems. The
coral testing holding system consisted of a three shelf - ABS plastic vertical shelf rack
wherein each shelf held one 20-gallon (76 litre) black polypropylene tank (see Figure 4).
Figure 4. Coral holding study system and components.

First row photos, left to right: Lab room with coral study system with T5 programmed lighting, higher
perspective, individual system. Second row photos, left to right: Water level siphon/overflow, siphon starter,
protein skimmer, fluidized bed biofilter/aragonite calcium reactor. Third row photos, left to right: Current
direction shifting valve, coral holding matrix, matrix with corals.
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System tanks - The molded plastic tanks were slightly tapered with rounded corners and
consisted of the following internal dimensions: length 27 inches (68.58 cm), width - 18
inches (45.72 cm), and depth of 17 inches (43.18 cm). However, the operating depth was
11 inches. Due to the taper and lack of a perfect rectangular shape, the tanks were filled
with 20 gallons of water, weighed for volume determination/confirmation and the operating
level marks (11 inches [27.94 cm]) were painted on each tank with a permanent and non-
toxic paint pen.
The top third tank was not connected to and separate from the bottom two tank's
interconnected water maintenance/life support system. The top dosing tank was equipped
with only low-pressure air and two large aquarium type air diffusers and the necessary
tubing and valving.
The bottom and middle tanks were connected by a recirculating plumbing system. The
bottom and middle tank formed the coral holding and life support system and contained a
total operating capacity of 40 gallons (152 liters).
The bottom tank of the rack functioned as a water management sump designed to maintain
water quality at levels typically required by hard corals. The middle tank was for coral
holding. A third tank was placed on the top of each shelf to be used as a holding tank for
dosing (see Figure 4).
Study time line - A general timeline is given for the primary activity components of the study
(see Table 1).
Water circulation - A 750 GPH/12.5 GPM (47.3 LPM) Danner Mag-Drive submersible pump
delivered the sump water to the coral holding tank. Because corals require substantial flow
from multiple directions, the water entering the coral holding tank was diverted 180 degrees
at entry approximately every 30 seconds, by a 3iQVentures - SCWD (Switching Current
Water Director) wave maker geared valve device.
Water filtration - The water maintenance sump consisted of a circulation pump that powered
a dual purpose fluidized bed bio-filter with an aragonite sand media to provide additional
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calcium required for optimum coral health and as a pH buffer. Additionally the sump tank
contained a ASM Mini-G Protein Skimmer to further reduce system nitrogen loading through
protein foam air bubble striping.
Water Chemistry - Three water chemistry factors (salinity, pH, and carbonate hardness)
were manually controlled throughout the study by:
1. Daily adjustments of individual test system's salinity using commercial Reverse

Osmosis water purchased for this purpose to lower salinities (above 35 g/L (ppt -
parts per thousand) because of salt concentration from evaporative water losses.
2. Periodic adjustments of individual test system's potential ionic imbalances by using
facility seawater coming in at 35 ppt salinity consistently.
3. Adjusting of individual test system's pH and carbonate hardness of 10 and a pH of
8.2 by the addition of NaCO3.
Lighting - The coral holding system was lighted by a Solar Extreme Deep Blue
programmable T5 quad (156 W-39x4) lighting system containing two daylight spectrum
bulbs and two blue actinic bulbs - all specifically designed for holding and growing corals.
The lights were programmed to provide an average of 12 hours of simulated daylight per 24
hours consisting of 11 daylight bulb hours and 13 hours of actinic illumination.
Temperature recording - The lab room housing the experiment was temperature controlled
and air within it sufficiently mixed to maintain temperatures in typical coral reef optimum
ranges. A Sper Scientific Min-Max thermometer recorded room air temperatures. Individual
daily tank water temperatures were recorded manually with a Hanna combination pH,
temperature, and conductivity meter. In addition and for continual temperature confirmation,
a data logger (EasyLogger - EL-USB-1) was used to record the control tank's temperature
every 30 minutes.
Seawater Resource - The seawater provided by the VBML comes from wells horizontally
drilled under the adjacent beach surf zone at a depth of approximately 30 feet beneath the
surface of the sand/shell beach. The seawater quality is further confirmed by the VBML
broad research and commercial tenants/partners that culture and produce a variety of
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species from several different marine phyla. Incoming seawater had a constant salinity of 35
ppt, a pH of 8.0-8.1, a carbonate hardness of 7-8, and an incoming temperature of 27 C
through the entire study.
Coral specimen matrix - Each coral holding tank had one plastic matrix (an adapted egg
crate fluorescent light fixture diffuser panel) measuring 30.4x60.9x1.2 cm (12x24x0.5
inches). The matrix grid rectangular openings were on 1.27 cm (0.5 inches) centers. This
allowed the coral fragment plastic culture bases to fit in (bayonet base for coral fragments)
and or rest on the grid (disc base for coral fragments). The corals were set into a specific
pattern to as much as possible expose each of the three individual specimens of the six
coral species to the tank's various water flow characteristics (see Figure 5).
Figure 5. Coral Species Placement Pattern For All Tanks.
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Representative species photographs from the study's photo records are given below (see
Figure 6).
Figure 6. Reef building hard coral species selected randomly from study's photo records.
Of particular note and consideration is the color/health considering that these photos were
randomly taken from all the tanks on Oct. 8th (dosing was on Oct. 1) - the day before the
end of the post-dosage monitoring period. Most of the coral specimens regardless of
treatment status remained in good health throughout the study.
3.4 Primary Data Collection Instruments
• Aquatic EcoSystems, Inc. automatic temperature compensating salinity refractometer

• API KH Carbonate Hardness Test Kit
• EasyLogger - EL-USB-1, Temperature Data Logger
• Hanna combination pH, temperature, TDS, and conductivity meter.
• LaMotte Ammonia Nitrogen Test Kit (1-8 ppm)
• Siebeck's Coral Bleaching Color Shift Monitoring Chart
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• Sper Scientific - Min-Max Thermometer

• Canon PowerShot ELPH 100 HS with a Canon WP-DC39 U/W housing
3.5 Test Population
Previous Coral Toxicity Studies
Most coral toxicity studies to date have consisted of going to various coral reefs in tropical
regions around the world and collecting hard corals from the reefs by breaking or cutting off
small fragments, or "nubbins" of specific reef building coral specimens to be tested, testing
them onboard, or transporting them to land where they were tested using various limited
equipment and materials to test and monitor the SHPP in question. Generally, there has
been very inadequate care taken to eliminate uncontrolled variables in the collection,
fragmentation, handling, and water quality maintenance during the collection and the testing
of the representative coral species used.
Aquacultured Coral Test Species
This study population consisted of all aquacultured coral species. There were six species of
Pacific reef building hard corals (see Figure 6). Three specimens of each species (total 120
corals/20 corals of each species) were purchased from ORA and each specimen was
individually packed in small (approximately 500 ml) heat sealed plastic bags and labelled in
commercial shipping containers. The total time that the corals spent packed in their
respective bags and shipping containers was less than four hours - starting from the time
the corals were packed, picked up, delivered to VBML and acclimation was started. The six
experimental coral holding systems were pre-adjusted to the same seawater chemistry
parameters (temperature, pH, salinity, and carbonate hardness) that ORA reported the
corals had been living in - and were packed in.
Test Species Handling and Preparation
Acclimation consisted of floating the corals in their respective pre-adjusted test system coral
holding tanks for approximately one hour. Acclimation technique consisted of making two
small holes (0.5 mm) in each bag - one at the top for venting of the bag and one in the
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bottom to allow water to fill it. The holes allowed system seawater to enter slowly (about
twenty minutes to fill each bag), effecting a gradual introduction of the new system's
seawater and chemistry. There was some variation caused by weight/density differences
between coral specimens and species. This required lighter bags to have additional holes
for filling and venting.
Each tank matrix received three sets of the six species of corals totalling eighteen
specimens. In addition each tank received two extra specimens (18+2) that would be used
to offset any handling and shipping losses among the corals - prior to dosing. This would
allow dosing of only those specimens unaffected by shipping and handling stresses.
3.6 Data Collection
Durwood M. Dugger and Sabine R. Alshuth-Dugger, Ph.D. accomplished the data collection
for this study. Data collection consisted of the following parameters:
• Daily water quality monitoring of test area air temperature with Sper Min-Max
thermometer (see Appendix 7.2.1).
• Pre-dosing bi-daily water analysis of each individual coral holding system consisting
of current temperature, salinity, pH, carbonate hardness, and total ammonia nitrogen
and entered into the appropriate form (see Appendix 7.2.1-7.2.3).
• Post-dosing daily water analysis of each individual coral holding system consisting of
current temperature, salinity, pH, carbonate hardness, and total ammonia nitrogen
and entered into the appropriate form (see Appendix 7.2.1-7.2.3).
• Constant temperature data logging of control tank (see Appendix 7.2.4).
• Pre-dosing bi-daily photographic record of each coral specimen (two photos each)
and entered into the appropriate form (see Appendix 7.4).
• Post-dosing daily photographic record of each coral specimen (two photos each)
using a Canon PowerShot ELPH 100 HS inside of its Canon WP-DC39 U/W housing
(see Appendix 7.4).
• End-of-study manual observation, out-of-water, of each coral specimen for
abnormalities (see Table 2).
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3.7 Data Analysis

The raw data and its basic summation are listed in Table 2. The current study was designed
to be an initial test of Patrick's Sunscreen mineral shield ™ impacts on reef building corals.
"Initial" in that demonstrated toxicity would have made additional and more in depth studies
unnecessary. The initial basis of the study and its focus on limiting experimental stress
variables were also practically limited by both costs/funding and the respective availability of
laboratory facilities with the necessary spatial and seawater resources. As such, this study
could not be designed with the replications necessary for Analysis of variance (ANOVA)
often used for standard toxicity type studies. It was statistically analysed using Univariate
analysis commonly used in the first, descriptive stages of research, before being
supplemented by more advanced, inferential bivariate or multivariate analysis found in
larger studies (see Appendix 7.3).
3.8 Limitations
Available financial, physical and temporal resources and their interrelationships limited the
study - as is often the case. These limitations more specifically consisted of economic,
physical and temporal constraints regarding:
• The lack of existing local marine laboratory testing facilities (two marine labs) and their
particular physical seawater resource limitations.
• The number of species of reef building hard corals tested (six) and as well the number of
other critical reef complex phyla (fish, crustaceans, and molluscs).
• The number of tanks necessary for a larger scale study with replication, randomization
and other elements necessary for optimal statistical analysis.
• Design limitations created by a general lack of environmentally controlled local lab space
with existing filtered and temperature controlled seawater volumes necessary for a
periodic and pulsed dose flow through test system design.
• Inability to predict the future need and duration for extending similar studies and the
"initial study" and potential temporary nature of the design limitations that this placed on
all design parameters.
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4 Results and Discussion
4.1 Study Data and Results

This initial study was researched, designed and developed over about a three-month period.
Its actual implementation, testing, data analysis and reporting required about two of those
months (see Table 1).
Table 1. Study activity timeline.
At the end of the study each coral was removed from its respective tank, held and visually
inspected in all dimensions. The observations were recorded on the basis of a
predetermined health class rating system, which consisted of evaluating the corals' primary
differences in health levels. This study produced no "bleaching" conditions as noted in
Danovaro's 2008 paper. The primary normal health limitation consisted of a small
percentage of specimens from three of the coral species presenting necrotic tissue lesions
(see additional discussion on coral tissue necrosis under section 4.1 External variables).
This study's raw coral health data results are summarized in Table 2 (below):
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Table 2. End-Of-Study, Observable Coral Health Evaluation and Ranking (Six Species).
(Coral Health Ranking: Normal = 100%, Necrosis/Base = 75%, Necrosis/Base + Stem = 50%, Necrosis/Base +
Stem + Branches = 25%, No living tissue - total necrosis = 0%.)
The results of the study answered most if not all of the previous Objectives and Research
Questions. The ability of the coral testing system design to provide and maintain stable and
adequate water chemistry during the study can be seen in Appendix 7.1 and in the general
overall survival of the corals in Table 2 (above). The study's highest priority Research
Question was the potential toxicity of Patrick's Sunscreen's mineral shield.™ The
experimental results can be stated in summary as follows:
1. A Univariate Analysis was carried out (see Appendix 7.3.1-7.3.13) on the data given in
Table 2. Statistical significance was set at p = < 0.05 (see Appendix 7.3.7). There was
no statistical difference between treated (dose 0.025 - dose 10 [ppm]) and non-treated
(dose 0.000) coral health conditions (see Appendix 7.3.11).
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2. No statistically significant difference was found between the general health of the
untreated control and dosed treatments at dosage levels of 0.025, 0.5, 1.0, 1.0, and 10
ppm. We propose that this would mean that Patrick's Sunscreen mineral shield™
product was not toxic within the given parameters of this study.
3. Coral species used in the study showed no detectable overall color shifts from bleaching
as indicated by the Siebeck Coral Color Shift Reference Card, and or as reported to
have occurred in the Danovaro 2008 study on sunscreens (see Figure 7). We propose
that this would mean that no bleaching occurred when the corals were exposed within
the parameter of the experiment to Patrick's Sunscreen mineral shield™ product.
4. A significant statistical difference in health quality was demonstrated by three branched
coral species in both treated and untreated controls to non-treatment variables (see
Section 4.1 and Appendix 7.3.12). These non-treatment variables presented varying
observable rates of necrotic tissue abscesses generally starting on the coral specimen
base (see Figure 8).
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Figure 7. The Coral Color Reference Card developed for standardizing changes in coral color.
(The hues are given for the four different color categories arranged in groups around the sides of the chart.
Brightness and saturation values are given for one hue only since they are identical for the same numerical
color score for each of the other hues. (Adapted from Siebeck, U. E. et. al. 2006).
4.2 External Study Variables

4.2.1 Tissue Necrosis (TN)
There are a number of differences between "coral bleaching" and "coral tissue necrosis."
Coral bleaching is the relatively gradual and relatively uniform fading - color shifting of coral
tissues caused by ejection of their symbiont zooxanthellae.
Coral tissue necrosis is described by two syndromes: Rapid Tissue necrosis (RTN) and
Slow Tissue Necrosis (STN). Both of these conditions - and unlike bleaching - represent a
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loss of polyp tissue down into the coral skeleton. Corals may recover from bleaching. Tissue
necrosis allows no regeneration of the stricken coral polyps and actually requires new coral
polyps to overgrow the bare skeleton.
There are different reasons for coral tissue necrosis:
1. Aggression by other corals - using their long "sweeper" stinging tentacles e.g. Favites
pentagona.
2. Protozoan, bacterial and or other pathogens.
3. Physical damage from the surrounding environment - predators, storms, or shipping and
handling injuries.
The tissue necrosis lesion conditions were found in all six coral test systems, including the
untreated control. The tissue necrosis condition was evaluated and ranked when the corals
were removed at the end of the study. They were carefully taken from the water, observed,
and ranked on an appropriate data sheet on the following basis:
• Normal - zero observable health issues = 100%.

• Tissue necrosis in and around the branched coral base = 75%.
• Tissue necrosis in the base and branched coral main stem = 50%.
• Tissue necrosis in the base, stem and the coral branches, but with some significant
amounts of coral polyps = 25%.
• Tissue necrosis and dissolution of all observable tissue on the coral skeleton = 0%.
There were a few necrotic lesions observed and photographed in the purchased corals and
before the study started (see Figure 7). Given the time constraints of the study and the
supplier's lack of additional Acropora species for replacement, and the extra two specimens
of each species as part of the experimental design to replace shipping and handling
damaged corals - the study continued as planned. Any observed specimens with necrosis
were replaced with the extra specimens prior to dosing.
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Figure 8. Tissue necrosis one day after receiving corals from ORA and before dosing.
Some progressive tissue necrosis development continued in some (but not all) of the three
branched coral species (Acropora tenuis, A. microphthalma, Montipora digitata) during the
study - in both treated and untreated (control) experimental elements (see Figure 9).
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Figure 9. Various stages of coral tissue necrosis from treated and non-treated tanks.
Consequently, we hypothesize that this condition could have been due to several factors
unrelated to sunscreen dosing. We have ranked these factors by our opinion of their
probability of occurrence below - although it is not impossible that all three factors were
present and active during the study:
• Branched corals are typically found high on the reef in areas of greater water movement.
The preference of the three affected branched coral species for higher water velocity
conditions to maintain growth/good health and keep their bases free of fouling would
make them more susceptible to opportunistic infections by endemic and opportunistic
organisms such as vibrio bacteria.
• The amount of time allowed for a particular species' growth, recovery and base
regeneration after the coral supplier propagated (fragmented) the particular species'
specimens before selling them, which in this case may have been inadequate. There
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was an observed relationship between branched coral species with the least base
overgrowth and presentation of necrotic tissue lesions - when compared to those that
had the more established base overgrowth. This was particularly the case in Scripps
Green (Acropora microphthalma) specimens which had only 63% survival compared to
the overall average survival of 93% of which few specimens had any base overgrowth
(see Figure 9 and Table 2).
• An unknown and progressively infectious necrosis agent specific for branched corals
was already present in the ORA corals and continued in our system. While all of the
coral species used in this study came through ORA's aquacultured coral quarantine
system, we have photographically identified at least six branched specimens with
necrotic base tissues on the first day (September 21st) after the corals were received
from ORA and transferred into the coral holding systems (see Figure 8).
• It was suggested that aggressive interactions between some of the coral species in the
experiment (for example the sweeper tentacles of the brain coral species Favites
pentagona) might have caused the tissue necrosis lesions in question. We did
photographically document sweeper tentacles of at least about 50+ mm (two inches).
However, the corals on the matrix tended to have more than 75 mm (3 inches) of
separation. Additionally, some specimens with tissue necrosis were also documented to
be impossibly distant from any Favites pentagona specimens and their sweeper
tentacles (see Figure 4, bottom right image).
4.2.2 Experimental Coral Sources
It has been argued that the use of aquaculture produced corals in experimental testing
protocols produces an experimental bias:
• It has theorized that aquacultured corals are more tolerant to many environmental
stresses because of the inherent natural selection processes in nature that also make
them naturally hardy and suitable for the rigours of marine "reef tank" life.
• Additionally, it has been theorized that aquacultured corals have become increasingly
hardy due to selection of survivors for propagation.
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It would be difficult to argue that there are no inherent selection processes for coral species
created by the difficulties of surviving in the often less than optimum marine environments,
especially when those optimum environments are similar to the less than optimal
environments of many marine reef tanks. At the same time it would be equally hard to be
able to document specifically - how aquaculture corals acquire any increased resistance to
commercial chemical HSPP formulations or other anthropogenic pollutants - while being
reared over repeated generations in the highly filtered seawater systems of commercial
aquaculture coral propagation systems. One could argue that aquaculture corals would
actually become less resistant to SHPP and other ocean pollutants due to their lack of
regular SHPP and other pollutant challenges. Certainly less than their wild counter parts
that may be exposed to some SHPP daily if they are in high human population density
areas.
Regarding the "increasing" hardiness of aquaculture propagated corals, the previous

argument also fails basic logic since we know of few commercial coral farmers that
challenge their broodstock with SHPP. In addition given that coral sexual breeding
techniques and genetic selection are in their early development and we don't know of any
commercial coral farmers that are sexually breeding corals as part of their commercial
production program, it is unlikely that genetic enhanced resistance to toxins of any kind
might be passed between generations. The coral supplier (ORA) for this study propagates
its corals exclusively through asexual methods, so no sexual genetic selection has
occurred. Consequently we do not see theoretical SHPP resistance to SHPP and other
chemicals as a valid reason not to use aquaculture-propagated corals in experiments - such
as the one reported herein.
4.3 Additional experimental observations - incidental test species
4.3.1 Incidental coral species that experienced dosing and survived
It was noted that a soft coral species of the genus Cespitularia was incidental to the
experiment and growing on one of the brain corals Favites pentagona in Tank #1 which
received the 1 ppm dosing of Patrick's Sunscreen mineral shield™ (see Figure 10). It
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apparently experienced no toxic affects from its dosing with Patrick's Sunscreen mineral
shield™ and survived beyond the end of the study.
4.3.2 Incidental shrimp species that experienced dosing and survived
In an informal observation of two species of shrimp (used to load the biofilters of the
experimental study tank systems) - four specimens of Farfantepenaeus duorarum and 10
specimens of Latreutes fucorum (both occur in tropical seas and coral reef areas) were
placed in the two dosage tanks containing 10 ppm, and the 1.0 ppm doses respectively -
after the corals had been removed from their treatment dosing. The shrimp were left in the
dosage tanks for 48 hours with only aeration, no water filtration, or adjustments. Both
species had 100% survival when removed alive and returned to their respective water
maintenance sumps. While these observations were not part of the experimental design
they could be considered as additional indications of Patrick's Sunscreen mineral shield ™
general lack of toxicity in other coral reef complex species (see Figure 10).
4.3.3 Incidental brittle star species that experienced dosing and survived
One species of brittle star (tentatively identified as Ophiocoma pumila) was observed
beneath several of the coral matrix platforms - which were all transferred together into their
respective dosing tanks. These brittle stars were observed in Tank # 2 (0.05 ppm) and Tank
5 (0.1 ppm) and received their respective dosing of Patrick's Sunscreen mineral shield™
along with their respective test subject coral companions. There was no beginning count of
these brittle star specimens, so no quantitative value could be assigned to their survivals
even though they were present and well at the end of the study (see Figure 10).
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Figure 10. Extraneous species observed incidentally participating in the experiment.
4.4 Proposed explanations for Patrick's Sunscreen mineral shield's™

general lack of toxicity
Regarding the potential toxicity of TiO2 - The metabolic routes and potential toxicity of
nanoparticle metal oxides in the environment and respective cellular biology are just
beginning to be studied. Given the amount of conflicting data on toxicity in the literature for
these substances, they are clearly still not well understood. However, the general
conclusion is that microparticles of metal zinc and titanium oxides are far less toxic than
their nanoparticles.
According to the Scientific Committee on Consumer Safety's (SCCS) July 22, 2013 opinion
they seem to believe that TiO2 is a relative non-toxic substance as long as it stays on the
outside of cell membranes in humans:
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"From the limited relevant information provided in the submission, and the information from
open literature, the SCCS considers that TiO2 nanomaterials in a sunscreen formulation are
unlikely to lead to:
• Systemic exposure to nanoparticles through human skin to reach viable cells of

the epidermis, dermis, or other organs.
• Acute toxicity via dermal application or incidental oral ingestion. This, however,
does not apply to sprayable applications that may lead to inhalation exposure of TiO2
nanomaterials which may result in lung inflammation.
• Skin irritation, eye irritation, or skin sensitization when (repeatedly) applied on healthy
skin (except possible photoxicity of insufficiently coated nanomaterials)
• Reproductive effects when applied on healthy skin."
Given the results of our study - it would appear that the corals are capable of keeping TiO2
particles from entering the interior of their cells where it might become a potential problem.
Regarding the potential toxicity of ZnO: According to Lenntech - one of the major authorities
on marine water chemistry and its treatment: "Zinc is naturally present in water. The
average zinc concentration in seawater is 0.6-5 ppb. Rivers generally contain between 5
and 10 ppb zinc. Algae contain 20-700 ppm, sea fish and shells contain 3-25 ppm, oysters
contain 100-900 ppm and lobsters contain 7-50 ppm."
According to both Lenntech and a paper by Y. Chang in 2012, Zn in its many compound
forms are a part of seawater and the natural marine environment, ecosystems and its
organisms. While this doesn't mean some Zn compounds aren't toxic - like TIO2, there are a
number of complex key factors such as compound co-element characteristics, particle size,
surface characteristics, dissolution, product specific coatings, binders and exposure routes
that mediate many toxic effects of this element's many organic compounds.
Consequently, we theorize the use of micro-particulate Zn and Ti oxide sizes (rather than
nanoparticles) in Patrick's Sunscreen mineral shield™ and the coating action of its water
proofing ingredients act to limit the materials' toxicity to the six species of reef building hard
corals and other creatures that survived the dosing in this experiment. Broad assumptions
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have been made about the toxicity of many sunscreen products and their components.
However, hard data regarding the actual concentration levels of the many different forms of
UV sunblocks and their effects on marine creatures are far from complete. Testing the final
composition of SHPP for toxicity as was accomplished in this study, is the ultimate guideline
on these products' appropriateness for use in an around the marine environment and
ecosystems.
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5 Recommendations
Based on the results from the current study the following recommendations are made:
• Additional studies should be made to determine the effects of periodic repeat dosing
that more closely mimics the real world daily pulses of sunscreen within the
concentrations as documented in the 2013 Tovar-Sánchez study and experienced in
highly populated water recreation areas of the Mediterranean. Such studies will
necessarily require flow-through systems, preventing the water maintenance/life
support system's characteristic of closed systems from removing and or otherwise
interfering with dosage levels. Consequently these flow-through test systems will
need to be appropriately designed and built at facilities with adequate flow through
seawater volumes.
• Current information on sunscreen levels in the water around coral reefs is inadequate
to access the level of threat these products might pose and or to base additional
toxicity studies upon. More studies are needed to document actual sunscreen
dosages experienced at the coral reefs with the most human interaction. There
appear to be no studies that distinguish UV sunblock chemicals from non-sunscreen
sources. For example, studies that distinguish sunblock chemicals coming from very
similar if not the same chemicals used by the cosmetic, paint and plastic industries
that are common to ocean outfall sewage disposal systems.
• Studies are needed to determine uptake of isotopic labelled sunscreen ingredients to
understand the pathways that they might enter corals' and other creatures'
metabolism and the coral reef complex in total.
• The tropical coral reef complex contains an estimated one million species. Additional
studies should also include representatives from a broader range of critical reef
complex species and phyla.
• Additional studies should be made with sufficient resources (physical and fiscal) to
provide statistical replication and analysis.
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6 References
Chang Y, Zhang M, Xia L, Zhang J, Xing G. (August 2012) The Toxic Effects and
Mechanisms of CuO and ZnO Nanoparticles. Materials 2012, 5, 2850-2871;
doi:10.3390/ma5122850
Danovaro, Roberto, Bongiorni L, Cinzia Corinaldesi C, Giovannelli D, Damiani E, Astolfi P,

Greci L, Pusceddu A. (2008). Sunscreens Cause Coral Bleaching by Promoting Viral
Infections. Environ Health Perspect. 2008 April;116(4): 441–447. Published online 2008
January 3. doi: 10.1289/ehp.10966
Gago-Ferrero, P., Díaz-Cruz, M. S., & Barceló, D. (2012). An overview of UV-absorbing

compounds (organic UV filters) in aquatic biota. Analytical and Bioanalytical Chemistry,
404(9), 2597-2610.
Huffington Post, 12 Most Overcrowded Beaches In The World (07/29/2010). Available:

http://www.huffingtonpost.com/2010/07/29/12-most-overcrowded-
beach_n_659706.html#s118638&title=Girguam_Beach_
Lenntech, Concentrations of Zinc and Titanium in seawater (2013) Available:

http://www.lenntech.com/periodic/water/zinc/zinc-and-water.htm
Siebeck UE, Marshall NJ, Kluter A, Hoegh-Guldberg O. (2006). Monitoring coral bleaching
using a colour reference card. Coral Reefs 25(3):453–460; 10.1007/s00338-006-0123-8
Scientific Committee on Consumer Safety (SCCS). (22 July 2013) OPINION ON Titanium
Dioxide (nano form) COLIPA n° S75
Stime J (2013) Reef Filtration Methods (2013) Available:

http://www.aquarium-design.com/
Tovar-Sánchez A, Sánchez-Quiles D, Basterretxea G, Benedé JL, Chisvert A, et al. (June

2013) Sunscreen Products as Emerging Pollutants to Coastal Waters. PLoS ONE 8(6):
e65451. doi:10.1371/journal.pone.0065451
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7 Appendices
7.1 Appendix - Understanding sunscreen concentrations
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7.2 Appendix - Water Quality

7.2.1 Daily Tank - Temperature Record
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7.2.2 Daily Tank - pH Record
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7.2.3 Daily Tank - Salinity Record
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0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
19/09/2013 13:09:38
20/09/2013 09:09:38
21/09/2013 05:09:38
22/09/2013 01:09:38
22/09/2013 21:09:38
23/09/2013 17:09:38
24/09/2013 13:09:38
25/09/2013 09:09:38
26/09/2013 05:09:38
27/09/2013 01:09:38
27/09/2013 21:09:38
28/09/2013 17:09:38
29/09/2013 13:09:38
30/09/2013 09:09:38
1/10/13 5:09
2/10/13 1:09
2/10/13 21:09
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3/10/13 17:09
7.2.4 Data Logger Control Tank Temps./30 min.
4/10/13 13:09
5/10/13 9:09
6/10/13 5:09
7/10/13 1:09
Data Logger Control Tank Temps./30 min.
7/10/13 21:09
8/10/13 17:09
9/10/13 13:09
An Initial Study Of Patrick's Sunscreen Effects On Six Reef Building Hard Coral Species
Celsius(°C)
Page 48
7.3 Appendix - Coral/Patrick's Sunscreen Study Statistical analysis

7.3.1 Page 1.
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7.3.2 Page 2.
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7.3.3 Page 3.
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7.3.4 Page 4.
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7.3.5 Page 5.
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7.3.6 Page 6.
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7.3.7 Page 7.
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7.3.8 Page 8.
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7.3.9 Page 9.
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7.3.10 Page 10.
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7.3.11 Page 11.
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7.3.12 Page 12.
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7.3.13 Page 13.
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7.4 Appendix - Pre-dosing and Post-dosing Photo Record
All the photos in Appendix 7.4 were taken by hand using a Canon PowerShot ELPH 100 HS
with a Canon WP-DC39 U/W housing on its "Underwater" and "Macro" settings. Because of
the corals" individual configuration, the ability to approach each coral at the same distance
and from the same angle were limited by those individual configuration differences.
All photos in Appendix 7.4 are shown without any computer image adjustment, photo image
enhancement or color photo editing. Changes and variations seen in background color are
as seen at the time the photo was taken. Some back ground color changes (whiter areas)
are caused by aragonite filter sand being ejected from the fluidized beds and settling on the
bottom of the coral holding tanks.
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7.4.1 Photo Comparison Tank 1.

Favites pentagona
Before Treatment After Treatment
Specimen 1.
Specimen 2.
Specimen 3.
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Photo Comparison Tank 1. Acropora tenuis
Specimen 1.
Specimen 2.
Specimen 3.
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Photo Comparison Tank 1. Acropora microphthalma
Specimen 1.
Specimen 2.
Specimen 3.
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Photo Comparison Tank 1. Montipora digitata
Specimen 1.
Specimen 2.
Specimen 3.
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Photo Comparison Tank 1. Stylophora pistillata
Specimen 1.
Specimen 2.
Specimen 3.
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Photo Comparison Tank 1. Pavona frondifera
Specimen 1.
Specimen 2.
Specimen 3.
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Favites pentagona
Specimen 1.
Specimen 2.
Specimen 3.
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Photo Comparison Tank 2.

Acropora tenuis
Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Favites pentagona
Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Favites pentagona
Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Favites pentagona
Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Favites pentagona
Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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Specimen 1.
Specimen 2.
Specimen 3.
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(CONFIDENTIAL REPORT)

Photos by Sabine R. Alshuth-Dugger and Durwood M. Dugger © 2013

An Initial Study of Patrick's Sunscreen Effects

October 30, 2013

Prepared for: Kelley Stark, Pres., Patrick's Sunscreen

Prepared by: Durwood M. Dugger and Sabine R. Alshuth-Dugger, Ph.D.

Copyright: © 2013 Durwood M. Dugger and Sabine R. Alshuth-Dugger, Ph.D.

Figures ................................................................................................................................ vii

1 Executive Summary ....................................................................................................... 1

1.1 Introduction and Background...................................................................................... 1

1.2 Methodology ............................................................................................................... 1

1.3 Key Findings ............................................................................................................... 2

1.4 Key Recommendations .............................................................................................. 3

2.1 Background and review of current literature ............................................................... 5

2.1.2 Antonio Tovar-Sánchez et al., (2013) Sunscreen Products as Emerging

2.3 Objectives ................................................................................................................. 16

3.1 Research Questions ................................................................................................. 18

3.2 Research Design ...................................................................................................... 18

3.3 Test System Design ................................................................................................. 20

3.4 Primary Data Collection Instruments ........................................................................ 24

3.5 Test Population......................................................................................................... 25

3.6 Data Collection ......................................................................................................... 26

3.7 Data Analysis............................................................................................................ 27

3.8 Limitations ................................................................................................................ 27

4 Results and Discussion ............................................................................................... 28

4.1 Study Data and Results ............................................................................................ 28

4.2 External Study Variables .......................................................................................... 31

4.2.1 Tissue Necrosis (TN) .......................................................................................... 31

4.2.2 Experimental Coral Sources ............................................................................... 35

4.3 Additional experimental observations - incidental test species ................................ 36

4.3.1 Incidental coral species that experienced dosing and survived.......................... 36

4.3.2 Incidental shrimp species that experienced dosing and survived....................... 37

7.1 Appendix - Understanding sunscreen concentrations .............................................. 43

7.2 Appendix - Water Quality.......................................................................................... 45

7.2.1 Daily Tank - Temperature Record ...................................................................... 45

7.2.2 Daily Tank - pH Record ...................................................................................... 46

7.2.3 Daily Tank - Salinity Record ............................................................................... 47

7.2.4 Data Logger Control Tank Temps./30 min. ........................................................ 48

7.3 Appendix - Coral/Patrick's Sunscreen Study Statistical analysis.............................. 49

7.3.1 Page 1. ............................................................................................................... 49

7.3.2 Page 2. ............................................................................................................... 50

7.3.3 Page 3. ............................................................................................................... 51

7.3.4 Page 4. ............................................................................................................... 52

7.3.5 Page 5. ............................................................................................................... 53

7.3.6 Page 6. ............................................................................................................... 54

7.3.7 Page 7. ............................................................................................................... 55

7.3.8 Page 8. ............................................................................................................... 56

7.3.9 Page 9. ............................................................................................................... 57

7.3.10 Page 10. ........................................................................................................... 58

7.3.11 Page 11. ........................................................................................................... 59

7.3.12 Page 12. ........................................................................................................... 60

7.3.13 Page 13. ........................................................................................................... 61

7.4 Appendix - Pre-dosing and Post-dosing Photo Record ............................................ 62

7.4.1 Photo Comparison Tank 1. ................................................................................. 63

7.4.2 Photo Comparison Tank 2. ................................................................................. 69

7.4.3 Photo Comparison Tank 3. ................................................................................. 75

7.4.4 Photo Comparison Tank 4. ................................................................................. 81

7.4.5 Photo Comparison Tank 5. ................................................................................. 87

7.4.6 Photo Comparison Tank 6. ................................................................................. 93

Table 1. Study activity timeline.

Figure 1. Concentrations of sunscreen ingredients in waters of Palmira Beach, Mallorca.

Figure 2. High population beaches and marine environment/human SHPP interaction.

Figure 4. Coral holding study system and component comparison collage.