FLORIDA INTERNATIONAL UNIVERSITY
Miami, Florida
OCCURRENCE AND DISTRIBUTION OF IRGAROL 1051 AND ITS NATURAL
METABOLITES IN BIOTIC AND ABIOTIC MARINE SAMPLES
A thesis submitted in partial fulfillment of the
requirements for the degree of
MASTER OF SCIENCE
in
CHEMISTRY
by
Charles Edward Maxey IV
2006
To: Interim Dean Mark Szuchman
College of Arts and Sciences
This thesis, written by Charles Edward Maxey IV, and entitled Occurrence and
Distribution of Irgarol 1051 and its Natural Metabolites in Biotic and Abiotic Marine
Samples, having been approved in respect to style and intellectual content, is referred to
you for judgment.
We have read this thesis and recommend that it be approved.
_______________________________________
Bruce McCord
_______________________________________
Rudolf Jaffe
_______________________________________
Piero Gardinali, Major Professor
Date of Defense: November 16, 2006
The thesis of Charles E Maxey IV is approved.
_______________________________________
Interim Dean Mark Szuchman
College of Arts and Sciences
_______________________________________
Dean George Walker
University Graduate School
Florida International University, 2006
ii
Copyright 2006 by Charles Edward Maxey IV
All rights reserved.
iii
ACKNOWLEDGMENTS
I would like to thank the members of my committee for all of their comments and
insight towards my thesis and research. I would especially like to thank my major
professor, Dr. Piero Gardinali for over six years of support, dedication, kind words, and
encouragement. Also, thanks are given to any and all research group members that have
assisted in the completion of this work. Finally, thanks are owed to Ciba Specialty
Chemicals for partial funding of this research
iv
ABSTRACT OF THE THESIS
OCCURRENCE AND DISTRIBUTION OF IRGAROL 1051 AND ITS NATURAL
METABOLITES IN BIOTIC AND ABIOTIC MARINE SAMPLES
by
Charles Edward Maxey IV
Florida International University, 2006
Miami, Florida
Professor Piero Gardinali, Major Professor
Marinas and coral reefs from the Florida coast were sampled to monitor surface
water, seagrass, and sediment for the antifouling biocide Irgarol 1051 and its metabolites.
Concentrations of Irgarol in surface water and seagrass ranged from N.D. to 1239 ng/L
and 2.35 ng/g to 225 ng/g, respectively. Concentrations of Irgarol in sediments were
negligible (< 10 ng/g). BCF values for Irgarol uptake into seagrass ranged from 60 to
31588. While Irgarol was found at the majority of the marinas sampled, no Irgarol was
found at any coral reef, the specie reported to be the most sensitive to Irgarol.
Of the three known metabolites of Irgarol, M1 was the only one found in any
appreciable amount (N.D. to 429 ng/L in surface water). M2 was not found, and M3 was
negligible (<2.5 ng/L). Also, a previously undetected metabolite, CA30-0156, was found
in surface waters up to 137 ng/L.
v
TABLE OF CONTENTS
CHAPTER .................................................................................................................. PAGE
I. INTRODUCTION .......................................................................................................... 1
1.1. The Economics of Fouling ....................................................................................... 1
1.2. A Brief History of Antifouling Products ................................................................. 2
1.3. Irgarol 1051: A Review ........................................................................................... 4
1.3.1. Occurrence ........................................................................................................ 4
1.3.2. Environmental Fate ........................................................................................... 7
1.3.3. Toxicity ........................................................................................................... 16
1.4. Scope and Objectives of Study .............................................................................. 20
II. Occurrence of Irgarol 1051 and its Major Metabolite, M1, in Florida Marine
Environments .................................................................................................................... 22
2.1. Study Areas around the Coast of Florida ............................................................... 22
2.2. Experimental .......................................................................................................... 27
2.2.1. Sample Collection ........................................................................................... 27
2.2.2. Sample Extraction ........................................................................................... 29
2.2.2.1. Surface water samples.............................................................................. 29
2.2.2.2. Submerged vegetation samples ................................................................ 29
2.2.2.3. Sediment extraction ................................................................................. 31
2.2.3. Sample analysis ............................................................................................... 31
2.2.4. Method Performance and Statistical Analysis ................................................ 32
2.2.5. Chemicals ........................................................................................................ 34
2.3. Results .................................................................................................................... 35
2.3.1. Surface water samples..................................................................................... 35
2.3.1.1. Key Largo Harbor .................................................................................... 35
2.3.1.2. Biscayne Bay ........................................................................................... 38
2.3.1.3. Other Coastal Marinas ............................................................................. 42
2.3.1.4 Coral Reef Sampling ................................................................................. 60
2.3.2. Seagrass Samples ............................................................................................ 61
2.3.3. Sediment Samples ........................................................................................... 69
2.4. Conclusions ............................................................................................................ 71
III. GC/MS Analysis for Nitrogen Based Pesticides and Herbicides ............................... 78
3.1. Triazine Herbicides ................................................................................................ 78
3.2. Expansion of Method to Include Additional Analytes .......................................... 80
3.2.1. Method Conditions.......................................................................................... 80
3.2.2. Figures of Merit .............................................................................................. 83
3.3. Environmental Application .................................................................................... 84
3.4. Conclusions ............................................................................................................ 86
IV. Environmental Detection of Recently Discovered Irgarol Metabolites ..................... 87
4.1. Objectives .............................................................................................................. 87
vi
4.2. Experimental .......................................................................................................... 87
4.2.1. Materials ......................................................................................................... 87
4.2.2. Sample Extraction ........................................................................................... 88
4.2.3. Data Acquisition ............................................................................................. 88
4.3. LC/MS Method Development................................................................................ 89
4.3.1. MS Optimization............................................................................................. 89
4.3.2. Characterization of Authentic Standards ........................................................ 90
4.3.3. Chromatographic Separation .......................................................................... 95
4.3.4. Instrument Calibration .................................................................................... 96
4.3.5. Method Performance ....................................................................................... 97
4.3. Results .................................................................................................................... 98
4.4. Conclusions .......................................................................................................... 107
V. Conclusions ............................................................................................................... 109
REFERENCES ............................................................................................................... 112
Appendix A. Concentrations of Irgarol and M1 in Key Largo Harbor ......................... 119
vii
LIST OF TABLES
TABLE
PAGE
Table 1.1: Worldwide Irgarol concentrations in marine environments .............................. 8
Table 1.2: Concentration of the family of Irgarol compounds in Hong Kong ................ 16
Table 1.3: Toxicity data for Irgarol 1051.......................................................................... 18
Table 2.1: SIM mass table for Irgarol analysis ................................................................. 32
Table 2.2: Recoveries of target compounds in fortified blanks ....................................... 34
Table 2.3: Summary of results from the KLH area ......................................................... 36
Table 2.4: Concentrations of Irgarol and M1 in the Biscayne Bay area .......................... 41
Table 2.5: Concentrations of Irgarol and M1 in samples collected from the west coast of
Florida ............................................................................................................................... 43
Table 2.6: Concentrations of Irgarol and M1 in coral reefs and nearby marinas ............ 60
Table 2.7: Concentrations of Irgarol and M1 in seagrasses collected from Key Largo
Harbor and Coconut Grove*.............................................................................................. 62
Table 2.8: Irgarol bioconcentration factors in Key Largo Harbor and Coconut Grove
Marina ............................................................................................................................... 66
Table 2.9: Irgarol and M1 concentrations in sediments .................................................... 69
Table 3.1: Structures of some commonly used triazine herbicides ................................. 79
Table 3.2: Retention times and SIM ions for nitrogen based pesticides .......................... 81
Table 3.3: Recoveries and MDLs for Nitrogen Pesticides .............................................. 83
Table 4.1: MS paramaters for APCI+ mode ..................................................................... 88
Table 4.2: Molecular Ions and SRM ions for analytes .................................................... 89
Table 4.3: Average recoveries and MDLs for LC/MS analysis........................................ 97
Table 4.4: Distribution of CA30-0156 in each sampling site ........................................ 105
viii
Table A-1: Concentrations of Irgarol and M1 in Key Largo Harbor .............................. 119
ix
LIST OF FIGURES
FIGURE
PAGE
Figure 1.1: Irgarol 1051 ..................................................................................................... 4
Figure 1.2: Irgarol's major metabolite, M1 ...................................................................... 12
Figure 1.3: Photodegradation pathway of Irgarol 1051 in natural environments (Plasencia
2001) ................................................................................................................................. 14
Figure 1.4: Structure of the new Irgarol metabolite M2 .................................................. 15
Figure 1.5: Structure of the new Irgarol metabolite M3 .................................................. 16
Figure 2.1: Study areas along the coast of Florida........................................................... 24
Figure 2.2: Coconut Grove Marina overlaid with a graphical representation of seagrass
beds. Darker greens indicate more dense seagrass patches. White lines in the water are
docking piers, some of which are covered by the seagrass layer. ..................................... 25
Figure 2.3: Key Largo Harbor overlaid with a graphical representation of seagrass beds.
Darker shades of green indicate denser patches of seagrass ............................................. 26
Figure 2.4: Location of coral reef sample sites in the Florida Keys ................................. 27
Figure 2.5: Thalassia testudinum, Turtle Grass ............................................................... 28
Figure 2.6: Syringodium filftorme, Manatee Grass ......................................................... 28
Figure 2.7: Chromatogram of a calibration standard ....................................................... 33
Figure 2.8: Calibration curves for Irgarol and M1. Curves were generated by plotting the
concentration ratio of the analyte and surrogate versus the area ratio of the analyte and
surrogate. ........................................................................................................................... 33
Figure 2.9: Distribution of Irgarol in Key Largo Harbor, 2/5/2004................................. 37
Figure 2.10: Concentrations of Irgarol throughout the 24-hour sampling period (black)
overlaid with tidal patterns (blue). Average concentrations during high and low tide are
shown in red and green, respectively. ............................................................................... 37
Figure 2.11: Miami River and Biscayne Bay, March 2006 .............................................. 39
Figure 2.12: Coconut Grove surface water sampling, August 2006 ................................. 40
x
Figure 2.13: Dania Beach Inlet/Port Everglades, Fort Lauderdale, September 2006 ....... 46
Figure 2.14: Collier County marinas, Naples, February 2003 ......................................... 47
Figure 2.15: Locations of Fort Myers sampling locations relative to Gulf waters ........... 48
Figure 2.16: Fort Myers Yacht Basin, January 2005 ........................................................ 49
Figure 2.17: Liberty Yacht Club, Fort Myers, January 2005............................................ 50
Figure 2.18: Peppertree Point Marina, Fort Myers, January 2005.................................... 51
Figure 2.19: Tarpon Point Marina, Ft Myers, January 2005 ............................................ 52
Figure 2.20: Gasperilla Marina, Charlotte Harbor, January 2005 .................................... 53
Figure 2.21: Palm Island Marina (bottom) and Charlotte Harbor Marina Basin (top),
Charlotte Harbor, January 2005 ........................................................................................ 54
Figure 2.22: Marina Jack's, Sarasota Bay, January 2005.................................................. 55
Figure 2.23: Longboat Key Moorings, Sarasota Bay, January 2005 ................................ 56
Figure 2.24: Boca Ciega Yacht Club, Tampa Bay, May 2005 ......................................... 57
Figure 2.25: The Harborage Marina, Tampa Bay, May 2005 .......................................... 58
Figure 2.26: St Petersburg Municipal Marina, Tampa Bay, May 2005 ............................ 59
Figure 2.27: Percentile plot of Irgarol concentrations in surface water showing the 90th
percentile Irgarol concentration and 3 concentrations that affect small marine organisms
(a. 63ng/L; b. 100 ng/L; c. 136 ng/L) ............................................................................. 73
Figure 2.28: Percentile plot of Irgarol concentrations in surface waters showing the 90th
percentile Irgarol concentration calculated without Key Largo Harbor and 3
concentrations that affect small marine organisms. (a. 63 ng/L; b. 100 ng/L; c. 136
ng/L) .................................................................................................................................. 73
Figure 2.29: Concentrations of Irgarol in selected stations in Biscayne Bay, 1999-2006
........................................................................................................................................... 75
Figure 2.30: Box plots comparing the concentrations of the two different specie of
seagrass ............................................................................................................................. 76
Figure 2.31: 90th Percentile graph for seagrass BCF values ............................................ 77
xi
Figure 3.1. Structures of other nitrogen based pesticides ................................................ 79
Figure 3.2: Chromatogram of a mixture of nitrogen based pesticides, Irgarol, and its
metabolites ........................................................................................................................ 82
Figure 3.3: Sample location for nitrogen pesticide monitoring ........................................ 84
Figure 3.4: Concentrations of nitrogen based pesticides in the L-31 canal ...................... 85
Figure 4.1: Structures of M2 (left) and M3 (right) .......................................................... 87
Figure 4.2: Chromatograms of M1 (A) and M2 (B) from injection into the standard
GC/MS method for Irgarol analysis. ................................................................................. 92
Figure 4.3: Chromatogram of M2 injected into the GC/MS using a Siltek® deactivated
liner ................................................................................................................................... 93
Figure 4.4: MS/MS Spectrum of Irgarol 1051 ................................................................. 93
Figure 4.5: MS/MS Spectrum of M1 ................................................................................ 94
Figure 4.6: MS/MS Spectrum of M2 ............................................................................... 94
Figure 4.7: MS/MS spectrum of M3 ................................................................................ 95
Figure 4.8: Chromatogram of mixture of Irgarol and its metabolites ............................... 95
Figure 4.9: Calibration curves for analytes ....................................................................... 96
Figure 4.10: Chromatogram of a sample screened for M2 and M3 .................................. 99
Figure 4.11: M2 Stability in water in laboratory conditions ........................................... 100
Figure 4.12: Comparison of new metabolites in a calibration solution versus an archived
sample showing that the unknown peak is not M2. ........................................................ 100
Figure 4.13: Structure of CA30-0156 ............................................................................. 102
Figure 4.14: Mass spectrums showing the fragmentation patterns of the unknown peak in
the archived samples and the "M2" peak in the calibration solutions. ........................... 103
Figure 4.15: Comparison of the unknown peak in a sample with that of a standard
solution of CA30-0156 (note: retention times shifted about 0.3 minutes. Atrazine d-5
shown for reference) ....................................................................................................... 104
xii
Figure 4.16: Mass spectrum of CA30-0156 obtained from our GC/MS ........................ 106
xiii
I. INTRODUCTION
1.1. The Economics of Fouling
Fouling is defined as the settlement and growth of marine organisms on
submerged surfaces. There are an estimated 2500 different marine organisms that have a
role in the fouling of surfaces submerged in water (Anderson et al. 2003). Fouling is
recognized as a major problem by the commercial shipping and recreational boating
industries. As little as a 1 mm increase of hull thickness can cause drag on a submerged
hull to increase up to 80%. On a larger scale, tanker fuel efficiency can drop by up to
40%, causing an overall voyage cost to increase by up to 77% due to the increased
friction caused by fouling (Champ 2000). This increased fuel consumption leads to an
increase of greenhouse gas emissions, which is believed to be a cause of global warming.
In addition, regional biological communities can also be affected by fouling, due to the
transport of non-native species on fouled hulls. In San Francisco Bay, 150 non-native
species have been documented (Cohen and Carlton, 1995). Other areas show similar
findings: 100 exotic species in Pearl Harbor, Hawaii (Coles et al., 1997); 50 exotic
species in Puget Sound, Washington (Cohen et al., 1998); 100 non-native species in Port
Phillip Bay, Australia (Hewitt et al., 1999).
This is particularly adverse in marine
communities where the introduction of unchallenged exotic species leads to alteration of
biodiversity of marine species. The utility industry is also affected by fouling. Power
plants that use seawater as a cooling agent in steam condensers can experience fouling as
well.
Fouling of condenser cooling water tubes decreases the efficiency of the
condensers, which in turn decreases the overall generation of power for the public. It is
estimated that as much as 4% of temporary station shutdowns at power plants generating
1
more than 600 megawatts are due to fouling problems (Meesters et al., 2003).
Commercially, fouling can have a large impact on consumers, but environmentally
speaking, fouling has many other disadvantages.
1.2. A Brief History of Antifouling Products
Some of the initial attempts at preventing marine fouling were to use chemicals
that are now known to be highly toxic, such as arsenic, organo-mercury, DDT, and lead.
All of these compounds were voluntarily pulled from the market in the 1960’s due to
studies showing that they were harmful to valuable resources and highly persistent in the
environment after the eventual release from the surfaces they were applied to.
The next step in the prevention of fouling was the development of tributyltinbased antifouling paints. These products quickly grew in popularity and effectiveness
and their use spread across the globe in little time. By 1985, the production of TBT
antifouling agents was in the range of 8-10000 metric tons per year (Alzieu et al. 1991).
While first used to support copper-based paints, which were also shown to help in the
prevention of fouling, tin compounds quickly became the main active ingredient in
marine paints. Most formulations in the 1960’s and 1970’s were based on the “free
association form,” which are formulations in which the active ingredient is not
chemically bound to the paint binder but rather freely mixed into the solution. Paints of
this type usually have high leaching rates of the active ingredient, causing environmental
levels of the compounds to be quite high. Due to these high leaching rates, ships had to
be dry-docked more frequently for repainting and maintenance, causing reduced
efficiency and higher costs to the shipping industry.
2
It was not long after the use of TBTs became a worldwide practice that concerns
of its environmental impact arose. Sexual disorders in gastropod species exposed to
TBTs were widely described in the early 1990’s (Stewart 1996; Adelman, 1990). TBTs
were also shown to accumulate in fish and sediment (Guruge et al. 1997; Kannan and
Falandysz 1997; Tanabe 1999; el Hassani et al. 2005). Due to the detrimental impacts of
TBTs to the environment, the International Maritime Organization (IMO) passed
legislation in 2001 restricting the use of TBT antifouling products on vessels less than 25
meters in length. As of 2003, the IMO resolutions say that there is now a complete ban
on the use of TBT based paints for marine use and further efforts are being made to
safely remove TBT from contaminated waters and from applied hulls. The deadline for
complete removal of TBT from hulls is January 1st, 2008 (Champ 2003).
When TBTs were found to be a potential environmental problem, alternative
chemicals started to be developed. It was found that copper based formulations were still
the most effective in the fight against fouling. However, some organisms, microalgae in
particular, were found to be resistant to copper.
Microalgae colonization led to
subsequent growth of seaweed on hulls (Gough et al., 1994). To combat these resistant
biota, organic booster biocides were added to paint formulations. One of the most
commonly used organic boosters is Irgarol 1051 (2-methylthio-4-tert-butylamino-6isopropylamino-s-triazine).
Irgarol (Figure 1.1) belongs to the family of s-triazine
herbicides and has a structure similar to that of the common agricultural herbicide
Atrazine.
3
S
N
N
H
N
N
N
H
Figure 1.1: Irgarol 1051
Irgarol is a white powder with a relatively low water solubility of 7 mg/L (ppm).
Irgarol was first registered with the EPA in the United States in 1994, but was in use in
Europe before that time. EPA registrations for marine paints containing Irgarol were
obtained by paint manufacturers beginning in 1998. Irgarol has been shown to be more
effective than any other antifoulant at inhibiting growth of fresh or saltwater algae, even
at levels as low as 10 ppb (Ciba-Giegy 1995). In light of the gradual restrictions imposed
on TBTs in the 1980’s and 1990’s, the use of Irgarol steadily increased worldwide.
Consequently, concerns regarding the environmental impact of Irgarol quickly arose and
numerous studies pertaining to environmental occurrence, toxicity and fate have been
performed over the last 15 years (Konstantinou and Albanis 2004).
1.3. Irgarol 1051: A Review
1.3.1. Occurrence
The first environmental detection of Irgarol was reported by Readman et al. in
surface waters taken from marinas off the coast of Côte d’Azur, France at levels up to
1700 ng/L (ppt) (Readman et al. 1993). Since that initial report Irgarol has been found
throughout the world’s coastal environments, and is now considered to be a ubiquitous
substance in most coastal areas where important boating activity exists. The main source
4
of the compound to the environment is leaching from applied surfaces during the lifetime
of the paint (Thomas et al., 2002). The highest environmental concentration if Irgarol
currently found in the literature is around 4000 ppt off the coast of Singapore (Basheer et
al., 2002). Irgarol concentrations have also been correlated to both seasonal changes and
number of boats present in sampling locations. Surface water collected during summer
months, or months associated with increased boating activity, contain generally higher
concentrations of Irgarol than months with little or no boating activity (Gough et al.,
1994; Rogers et al., 1996; Tolosa et al. 1996; Scarlett et al. 1997; Hall et al. 1999). Areas
containing mostly recreational yachts also follow a similar trend showing higher Irgarol
concentrations than commercial ports, most likely due to the fact that the 2001
restrictions of TBTs only affected vessels < 50 ft (Readman et al. 1993; Gough et al.
1994; Rogers et al., 1996; Tolosa et al. 1996; Hall et al. 1999; Gardinali et al. 2002;
Gardinali et al., 2004).
Most of the Irgarol occurrence literature concentrates on coastal areas along
Europe and Asia, but recently a comprehensive survey of the United States coast has
been undertaken (Gardinali et al. 2002; Gardinali et al., 2004; Hall and Gardinali 2004).
Although there is an abundance of data surveying the aquatic concentrations of Irgarol,
there is far less data dealing with its accumulation in other marine matrices, namely fish,
submerged vegetation, and sediment.
There have been two literature publications describing the uptake of Irgarol into
submerged vegetation.
Scarlett et al. proposed that Zostera marina can have
bioconcentration factors (plant tissue concentration ÷ water concentration) reaching as
high as 25000 (Scarlett et al. 1999a). Off the coast of Queensland, Australia, two types
5
of vegetation, namely eel grass (Zostera marina) and shoal grass (Halodule), were
collected from 10 stations and analyzed for Irgarol. Concentrations ranged from nondetect (N.D.) to 118 ng/g. Irgarol was found in 9 out of the 10 seagrasses sampled
(Scarlett et al. 1999b).
Sediment work is more abundant in the literature when compared to submerged
vegetation. Studies have determined that Irgarol has a relatively high octanol-water
partitioning coefficient of log Kow = 3.95 (Bard and Peterson, 1992). This suggests that
there is a great possibility for Irgarol to partition into sediment. However, studies have
shown that this is not the case. Irgarol shows a low affinity for particulate matter when it
comes to partitioning into sediment matter. Log KD and log Koc were determined to be
3.4 and 3.0, respectively (Voulvoulis et al., 2002; Comber et al. 2002). Log Kow was
calculated to be 3.4 (Plasencia, 2001). These data suggest that most of the Irgarol in
marine environments will remain dissolved in the water column. However, Irgarol was
found to partition well into the sediments when it was still associated with paint particles.
Paint chips containing Irgarol that break off from ship hulls will remain as particulate
matter and associate themselves with sediment. Irgarol is then slowly released through
dissolution processes into the sediment (Thomas et al., 2002). Most areas from which
sediments were sampled had relatively low concentrations of Irgarol (< 100 ng/g), with a
few stations topping the 100 ng/g mark. The highest concentration of Irgarol found in
sediments so far is 1011 ng/g in a marina from Orwell Estuary in the UK (Boxall et al.
2000). This marina holds in excess of 350 boats and has low water circulation with a
water half-life of around 10 days.
6
A list of coastal environment settings and their corresponding Irgarol
concentrations are listed in Table 1.1. While this table is not a comprehensive collection
of all surveys ever undertaken, it clearly shows that the occurrence of Irgarol is
widespread.
1.3.2. Environmental Fate
Due to the fact that Irgarol has been found in many coastal areas around the entire
world and because of it’s potentially toxicity to some key species, it is important to know
the fate of Irgarol once it reaches the environment. As previously mentioned, Irgarol has
been shown not to partition well into the sediment phase, with the exception of paint
particle association, but rather stays in solution. Hence, the major modes of removal
from the environment are biodegradation and photodegradation from the water column.
Studies pertaining to the biodegradation of Irgarol suggest that it is not readily
biodegradable (Liu et al. 1997). Irgarol was found to be poorly biodegraded by bacteria.
A 5 month incubation of bacteria native to Lake Ontario showed no biodegradation or
biotransformation of Irgarol.
Biotransformation using the white rot fungus
Phanerochaete chrysosporium caused Irgarol to undergo N-dealkylation at the
cyclopropylamino group forming Irgarol’s major metabolite, M1 (2-methylthio-4-tertbutylamino-6-amino-s-triazine), shown in Figure 1.2. M1 was also found to be stable in
regards to biotransformation, with no depletion of the compound after a 6 month
exposure to P. chrysosporium. No half-life for either compound was given for this study.
7
Table 1.1: Worldwide Irgarol concentrations in marine environments
Location
Date
Water (ng L-1)
Sediment (ng g-1)
Reference
United Kingdom
Marinas
Kent, Sussex, Hampshire
Sutton Harbour
Plymouth Sound
Conwy, Wales
Southern coast
Brighton
Humber
Orwell
Hamble
August 1993
April - October 1998
July - August 1995
January - March 1999
January - October 1998
November 1999 - January 2001
April - September 1995
September 1998 - February 1999
September 1998 - February 1999
52 - 500
< 1 - 69
28 -127
7 - 543
< 1 - 1421
< 1 - 964
169 - 682
5.6 - 201.4
18.3 - 61.1
n.s.
n.s.
n.s.
n.s.
n.s.
< 1- 77
n.s.
< 10 - 1011
< 10
Gough et al. (1994)
Thomas et al. (2001)
Scarlett et al. (1997)
Sargent et al. (2000)
Thomas et al. (2001)
Bowman et al. (2003)
Zhou et al. (1996)
Boxall et al. (2000)
Boxall et al. (2000)
July – September 1993
September 1998 – February 1999
April – October 1998
April– September 1995
August 1993
October 1998 – June 1998
April – October 1998
Summer 1997– Spring 1998
April– October 1998
End boating season, 1998
Summer 2000
January–October 1998
12 – 190
7.3 – 17.9
< 1 – 141
< 1 –39
4 – 18
150– 680
< 1 –49
< 3 –10
< 1 –403
n.s.
< 1 –305
< 1 –32
12 - 132
< 10
n.s.
n.s.
n.s.
3.3– 222
n.s.
n.s.
n.s.
< 1 –40
0.3– 3.5
n.s.
Gough et al. (1994)
Boxall et al. (2000)
Thomas et al. (2001)
Zhou et al. (1996)
Gough et al. (1994)
Voulvoulis et al. (2000)
Thomas et al. (2001)
Scarlett et al. (1999a)
Thomas et al. (2001)
Thomas et al. (2000)
Thomas et al. (2002)
Thomas et al. (2001)
Estuaries
Hamble
Hamble
Hamble
Humber
Medway
Blackwater, Essex
River Crouch
Yealm and Salcombe
Southampton Water
Southern coast
8
Table 1.1 (continued)
Location
Date
Water (ng L-1)
Sediment (ng g-1)
Reference
Ports, Coastal areas
Kent, Sussex, Hampshire
August 1993
9 – 14
n.s.
Gough et al. (1994)
July–September
< 2 –11
n.s.
Gough et al. (1994)
France
Marinas
Coˆte d’ Azur
Riviera, Monaco
June 1992
May– June 1995
110– 1700
22– 640
132– 275
n.s.
n.s.
n.s.
Readman et al. (1993)
Tolosa et al. (1996)
Tolosa and Readman (1996)
Ports
Coˆte d’ Azur
Riviera, Monaco
June 1992
May– June 1995
< 5 –280
13.8– 264
n.s.
n.s.
Readman et al. (1993)
Tolosa et al. (1996)
Beaches
Coˆte d’ Azur
Riviera, Monaco
June 1992
May– June 1995
negligible
< 1.5– 1
n.s.
n.s.
Readman et al. (1993)
Tolosa et al. (1996)
1996–1997
January–August 1999
April 1996–January 1999
June 2000
February 1997– June 1998
7 – 325
< 50
15– 320
n.s.
ND-119
25– 450
< 10– 50
n.s.
n.s.
n.s.
< 0.2–88
3 – 57
n.s.
n.s.
Ferrer et al. (1997)
Martinez et al. (2000)
Ferrer and Barcelo (1999)
Martinez and Barcelo (2001)
Ferrer and Barcelo (2001)
Aguera et al. (2000)
Pocurull et al. (2000)
Spain
Marinas
Catalonia
Barcelona (Masnou)
Almeria
Tarragona-Cambrils
March– June 1999
9
Table 1.1 (continued)
Location
Southeast Spain
Greece
Marinas
Piraeus-Elefsina
Thessaloniki
Patras
Chalkida
Igoumenitsa-Aktio
Ports
Piraeus
Thessaloniki
Patras
Netherlands
Marinas
Dutch coast
Rotterdam coastal area
Estuaries
Western Scheldt
Water (ng L-1)
Sediment (ng g-1)
Reference
50– 1000
n.s.
Hernando et al. (2001)
October 1999– September 2000
ND-90
ND-68
12– 24
ND
ND-27
ND-690
75– 350
ND-37
ND-88
ND-74
Sakkas et al. (2002a)
Albanis et al. (2002)
October 1999–September 2000
10– 24
ND
ND
ND-19
ND-11
ND-11
Sakkas et al. (2002a)
Albanis et al. (2002)
April–November 2000
End boating season, 1998
8– 90
n.s.
n.s.
<1
Lamoree et al. (2002)
Thomas et al. (2001)
April 1996
April 1996–March 1997
1.6– 10
8– 37
5– 42
n.s.
n.s.
n.s.
Steen et al. (1997)
Steen et al. (2001)
Hall et al. (1999)
August 1994–April 1995
September 1999
2.5– 145
ND-135
2.5– 8
Toth et al. (1996)
Nystrom et al. (2002)
Date
Sas Gent Schaar van Ouden
Switzerland
Lake Geneva
Lake Geneva
10
Table 1.1 (continued)
Location
Date
Water (ng L-1)
Sediment (ng g-1)
Reference
Germany
Marinas
North Sea
Baltic Sea
July–September 1997
July–September 1997
11 – 170
80– 440
3 – 25
4 – 220
Biselli et al. (2000)
Biselli et al. (2000)
Portugal
River water (Ponte Aranha)
April–July 1999
10– 260
n.s.
De Almeida Azevedo et al. (2000)
Sweden
Marinas
Fiskebäckskil (West coast)
June 1993– September 1994
30– 400
n.s.
Dahl and Blanck (1996)
Karlslund, Sth Stockholm
April 1996–November 1996
4– 125
2–9
Haglund et al. (2001)
March 1999– September 2000
March 1999– September 2000
March 1999– September 2000
September - October 2001
Summer 2001
< 1 – 15.2
< 1 – 1.1
< 1 – 60.9
< 1 - 182
12.2–144.2
n.s.
n.s.
n.s.
n.s.
n.s.
Gardinali et al. (2002)
Gardinali et al. (2002)
Gardinali et al. (2002)
Gardinali et al. (2004)
Owen et al. (2002)
Summer 2003 - 2004
Summer 2003 - 2004
5 - 1816
ND - 85
n.s.
n.s.
Hall et al. (2005)
Hall et al. (2005)
ND
n.s.
Liu et al. (1999b)
USA
Biscayne Bay
Marinas
Ports
Miami River
Florida Keys Marinas
Florida
East Coast Marinas
Back Creek/Severn River
Carolinian Province
Canada
Marinas and Ports
1996– 1997
n.s.: Not Sampled; N.D.: Not Detected
11
S
N
N
N
H
N
NH2
Figure 1.2: Irgarol's major metabolite, M1
Photodegradation
studies
have
shown
that
Irgarol
readily
undergoes
photodegradation in the environment. There have been conflicting results on the actual
degradation time, however. Data from the manufacturer states that the half-life of Irgarol
in seawater and freshwater is 100 and 200 days, respectively (Ciba-Giegy, 1995).
Laboratory tests under various conditions show that the actual degradation rates for
Irgarol is faster than initially predicted. Photodegradation of Irgarol in seawater and river
water was first confirmed by Okamura et al., in 1999, but no half-lives were given for
their testing. One study was devoted to assess the photodegradation rates of Irgarol under
natural and simulated conditions. Simulated conditions involved exposing pure and
natural water containing Irgarol to 350nm UV light in a photoreactor. Half-lives for the
pure and natural waters were close to 8 days and 4 days, respectively. Natural conditions
involved submerging these same waters in a pond and leaving then exposed to natural
sunlight for a period of time. Results of the natural exposure testing were similar to those
of the simulated conditions with half-lives on the order of 6 to 10 days. (Placensia,
2001). Recent literature corroborates these results (Amine-Khodja et al. 2006).
Published data also suggests that the degradation of Irgarol can be influenced by
dissolved organic matter in natural systems. This was confirmed in two separate studies.
Humic and fulvic material were shown to increase the Irgarol degradation rate to a range
12
of 2 to 9 hours, depending on the concentration of the DOM. Photosensitizers, such as
benzophenone, acetone, and tryptophan have also been shown to influence the
degradation of Irgarol (Sakkas et al., 2002).
In all studies (both laboratory and natural conditions), it was shown that M1 was
the major metabolite produced from photodegradation. All intermediate products formed
during the reaction had M1 as their endpoint when subject to further stress.
Photodegradation rates of M1 were slower than the parent compound itself. Research
shows that the half-life of M1 is around 200 days, suggesting that M1 will persist in the
environment longer than Irgarol (Placensia, 2001; Thomas et al., 2002). Some minor
products formed from photodegradation include oxidation of the parent compound
producing a sulfone and further degradation of M1 which involves cleavage of the t-butyl
group. A proposed degradation pathway of Irgarol is shown in Figure 1.3 (Placensia,
2001).
Abiotic transformation of Irgarol is another area of importance relating to its
environmental fate. Irgarol has been shown to be very stable under hydrolysis. A
hydrolysis reaction carried out at 50 C for 7 days showed no appreciable degradation of
Irgarol. Also, autoclaving at 120 C showed no degradation. It was only after a 6 week
period of continuous hydrolysis that the Irgarol concentration decreased by 20%.
According to ASTM procedures, if 10% of a compound degrades after 7 days of
hydrolysis, its half-life is 6 months or less. It is with this caveat that Irgarol is considered
to be unaffected by direct hydrolysis (Okamura et al., 1999).
13
O
SCH3
N
SCH3
N
HN
N
NH
N
N
H2 N
NH
GS 26575
“M1”
N
N
N
N
H2 N
SCH3
NH
H2 N
IRGAROL 1051
O
S O
N
N
N
NH
OH
N
H2 N
N
N
N
H2 N
NH
GS 28620
N
N
NH
CGA 234575
Figure 1.3: Photodegradation pathway of Irgarol 1051 in natural environments (Plasencia 2001)
Under catalyzed hydrolysis, however, Irgarol behaves quite differently. Heavy
metal catalysis using CuCl2, AgNO3, CdCl2, PbCl2, and ZnCl2 showed 0% Irgarol
hydrolysis in solution, even at concentrations reaching 100 mg/L. Mercuric chloride on
the other hand, was shown to completely hydrolyze Irgarol at low concentrations (10
mg/L). This reaction was not dependant on pH or any other factors. All tests showed
100% degradation. Once again, the final end point of the parent compound was M1 (Liu
et al. 1999).
Until recently, it was thought that the environmental fate of Irgarol was
thoroughly understood. It seems that this is no longer the case. In 2004, a paper was
published characterizing a new degradation product formed during the mercuric chloride
catalyzed hydrolysis, designated M2
(3-[4-t-butylamino-6-methylthiol-s-triazine2-
14
ylamino]-propionaldehyde; Lam et al. 2004). It is hypothesized that this degradation
product was not detected before, due to the fact that in GC/MS systems, M2 has been
shown to degrade into M1. Also, current liquid chromatographic separation methods
cause the M2 peak to broaden to the point where it is unnoticeable when concentrations
are very low in the reaction mixture. The addition of the ion-pairing reagent sodium 1heptanesulphonate during HPLC-UV chromatography allows for narrow peak shapes and
good resolution of M1 and M2. Structure elucidation of M2 was carried out using
preparative chromatography of the hydrolysis mixture, followed by high resolution
tandem mass spectrometry (m/z = 270.1385) and nuclear magnetic resonance. The
proposed structure of M2 is shown in Figure 1.3 (Lam et al. 2004).
S
N
N
H
N
N
O
N
H
H
Figure 1.4: Structure of the new Irgarol metabolite M2
There has been one more Irgarol related compound that has been characterized
within the past two years. This compound was first detected during aqueous TiO2catalyzed photodegradation.
It was considered a minor intermediate during the
photodegradation process (Konstantinou et al., 2001). High resolution MS-MS, NMR
and careful preparative chromatography have characterized this compound to be a side
product of the industrial production of Irgarol. It has since been designated M3 (Figure
1.4) and has a chemical name of N,N’-di-tert-butyl-6-methylthiol-s-triazine-2,4,-diamine
(Lam et al. 2005).
15
S
N
N
H
N
N
N
H
Figure 1.5: Structure of the new Irgarol metabolite M3
Along with characterizing the new metabolite and impurity, Lam et al. also found
these compounds in surface waters around the Pearl River Estuary off the coast of Hong
Kong. A summary of concentrations of Irgarol, M1, M2, and M3 found in these samples
during the studies are shown in Table 1.2. There is not yet any data on the environmental
stability of these two compounds.
Table 1.2: Concentration of the family of Irgarol compounds in Hong Kong
Station
Concentration (ng/L)
Irgarol-1051 M1
M2
M3
January 2004
S1
600
8,160
11,090
n.a.
S2
360
7,890
20,280
n.a.
S3
1,410
24,050
15,390
n.a.
May 2004
S1
1,320
S2
520
S3
110
S4
1,620
S5
640
n.a.: Not analyzed for this sample
122,870
36,820
204,970
104,820
259,000
n.a.
n.a.
n.a.
n.a.
n.a.
280
390
60
120
30
1.3.3. Toxicity
Studies have shown Irgarol to be a strong photosynthesis II inhibitor in marine
algae. Inhibition of the PSII process causes less uptake of CO2 by algae, likely producing
death. Toxicity tests show that small aquatic plant systems seem to be the most affected
by Irgarol. Concentrations as low as 63 ng/L affect the carbon uptake of inshore corals
(Madracis mirabilis) and concentrations as low as 100 ng/L can reduce the net
16
photosynthesis of intact corals after an 8 hour exposure (Owen et al. 2002). Chronic
effects to periphyton communities can be seen with concentrations ranging from 60 ng/L
to 250 ng/L (Dahl and Blanck, 1996). All of these effects were also shown to be
reversible with the removal of the stress on the organisms. Effect levels for other
photosynthetic biota range from 136 ng/L for the diatom Naviculla pelliculosa (Hughes
and Alexander 1993) to 8100 ng/L for the seaweed Lemna minor (Okamura et al. 2000).
Toxicological results for M1 show that the metabolite is far less toxic than the parent
compound itself. For example, EC50 values for Naviculla pelliculosa and Lemna minor
are 71,000 ng/L and 190,000 ng/L, respectively. A more comprehensive list of toxicity
data of Irgarol and M1 can be found in Table 1.3.
Toxicological data seems to indicate that the effects of Irgarol decrease in higher
forms of marine life. Based on these observations, it is expected that corals could be the
species that is most likely to be affected by Irgarol. It is important to note, however, that
to date there is no published report showing concentrations at locations populated by
coral reefs that are at or above the levels that have been shown to affect said systems (≥
63 ng/L).
17
Table 1.3: Toxicity data for Irgarol 1051
Class
Test Organism
Irgarol
Irgarol
M1 EC/LC50
Toxicity Index EC50a or LC 50b NOECc or LOECd (ng/L)
Reference
Seaweed
Pophyra yezoensis
4-day EC50
600000
≤ 300c
17000
Okamura et al. (2000b)
Eisenia bicyclis
4-day EC50
2.6e6 - 7.4e6
3200c
32000
Okamura et al. (2000b)
Seagrass
Zostera marina
10-day EC50
2500000
Algae
Closterium ahrengergii
5-day EC50
2500000
Microphytes
5000 ± 900d
Okamura et al. (2000b)
Chlorococcum sp.
EC50
420
Hoberg (1998b)
Dunaliella tertiolecta
EC50
560
Hoberg (1998d)
Scenedesmus subspicatus
EC50
4030
Fernández-Alba et al. (2002)
Rufli (1988)
Elodea canadensis
EC50
1.7e7 - 5.2e7
2500 - 25300
Potamogeton pectinatus
EC50
10000000
2500d
d
Nyström et al. (2002)
49000
d
Nyström et al. (2002)
EC50
442000 - 647000 25 - 647
Navicula pelliculosa
EC50
136
190000
Hughes and Alexander (1993a)
Skeletonema costatum
EC50
420
16150
Hoberg (1998b)
Skeletonema costatum
EC50
386
Emiliania huxleyi
72-hrs EC50
250
Synecochoccus sp.
72-hrs EC50
160
Rhodomonas salina
Lemna gibba
7-day EC50
7-day EC50
-1
EC50 = effect concentration (ng L )
LC50 = lethal concentration (ng L-1)
100
c
d
Devilla et al. ( 2005)
Devilla et al. ( 2005)
c
d
Zamora et al. (2006)
c
d
Zamora et al. (2006)
c
d
Zamora et al. (2006)
441 ; 963
7-day exposure
19-day exposure
Nyström et al. (2002)
Hughes and Alexander (1993e)
12-day exposure
Scrippsiella sp.
Lemna minor
b
86000
1.08e7 ± 1.7e6
Synecochoccus sp.
a
Scarlett et al. (1999b)
Selenastrum capricorniatum 3-day EC50
Phytoplankton various species
Duckweed
10000
d
350 ; 800
640 ; 836
1.1e7 – 1.2e7
120000
Okamura et al. (2000b)
7.3e6- 8.9e6
71000
Okamura et al. (2000b)
-1
NOEC (ng L ) = no observed effect concentration
d
LOEC (ng L-1) = lowest observable effect concentration
18
Table 1.3 (continued)
Class
Bacteria
Test Organism
Vibrio fischeri
Toxicity Index
15-min EC50
Crustacean
Daphnia magna
48-hrs EC50
Daphnia magna
48-hrs EC50
Daphnia pulex
24-hrs LC50
Thamnocepharus platvurus 24-hrs LC50
Artemia salina
Corals
Madrasis mirabilis
Rainbow trout
Sea urchin
Oncorhynchus mykiss
Anthocidaris crassispina
a
EC50 = effect concentration (ng L-1)
LC50 = lethal concentration (ng L-1)
b
24-hrs LC50
7-day LC50
Pluteus formation
32-hrs
c
Irgarol
EC50a or LC 50b
50800000000 ±
7800000000
7300000000 ±
1200000000
6700000000 ±
10000000000
5100000 –
6300000
1100000 –
13000000
> 4 x 107
Irgarol
M1 EC/LC50
NOECc or LOECd (ng/L)
Reference
10000000 ± 900000d
Fernández-Alba et al. (2002)
2400000 ± 300000d
Fernández-Alba et al. (2002)
63d
Owen et al. (2002)
10000c
Okamura et al. (2002)
Kobayashi and Okamura (2002)
25000000
NOEC (ng L-1) = no observed effect concentration
LOEC (ng L-1) = lowest observable effect concentration
d
19
Only one study involving Irgarol toxicity to fish was found in the literature. This
study showed that Irgarol toxicity in fish was very low. Average 24-h EC50 values for
suspension-cultured fish cells were found to be greater than 100 ppm. Average LC50
values at 7 days and 28 days for the rainbow trout Oncorhynchus mykiss were 25 and
0.88 ppm, respectively (Okamura et al. 2002).
Since M2 and M3 were recently discovered, there is not yet any data in the
literature pertaining to toxicity towards any type of marine organisms.
1.4. Scope and Objectives of Study
Since Irgarol seems to be ubiquitous in areas where important boating activity is
present, Florida is a model area for all facets of Irgarol research. Since Irgarol has been
shown to persist in the water column for a reasonable amount of time, especially in areas
with high concentrations of recreational boats, there is a possibility for it to affect biota in
the area. The close presence of all potentially impacted marine species, such as corals
and submerged vegetation, makes assessing risk to these organisms easier. It is also
important to revisit the fate of Irgarol, since there have been recent reports of new
metabolites being detected in surface waters. The primary goals of this research are as
follows:

Develop a method to quantify Irgarol, M1, M2, and M3 in a single analysis and
determine if the newly discovered metabolites are present in the US coastal
environment.

Conduct an environmental assessment of Irgarol, M1, M2, and M3 along the
majority of the Florida Coastline.
20

Determine whether offshore coral reef systems are in possible danger due to
Irgarol contamination of nearby marinas.

Determine the concentration of Irgarol and its major metabolites in submerged
aquatic vegetation at two model areas, Key Largo Harbor and Coconut Grove
Marina.

Expand the current protocol for Irgarol analysis to include a larger group of
compounds in the nitrogen pesticide and triazine herbicide families.
21
II. Occurrence of Irgarol 1051 and its Major Metabolite, M1, in Florida Marine
Environments
2.1. Study Areas around the Coast of Florida
While there are many publications in the literature pertaining to the occurrence of
Irgarol, the majority of research is focused on the coastal areas of Europe and Asia.
Research in the United States has been primarily focused on two main areas: Port
Annapolis and the Severn River in Annapolis, Maryland, and the southern coast of
Florida, which includes Biscayne Bay, the Miami River, and Key Largo Harbor
(Gardinali et al. 2002; Gardinali et al., 2004; Hall et al. 2005; Hall et al., 2004). Since
Irgarol seems to be present in the majority of coastal environments in Europe and Asia, it
is important to expand Irgarol sampling to the entire coast of the US. It is particularly
important to know whether or not Irgarol is present in such high concentrations as to
affect sensitive marine organisms, such as coral reef systems and submerged vegetation.
South Florida is an ideal area to study marine pollution, due to the fact that the
climate is such that it facilitates year round boating activity. Since boating is a popular
activity in Florida, the state is home to thousands of areas for boat storage. These include
public and private marinas, wet and dry-docking facilities, wet and dry repair yards,
personal docks behind waterfront homes, and commercial and industrial ports.
As
previously stated, it has been shown that Irgarol concentrations are higher at areas where
recreational boating occurs and lower at commercial and industrial facilities, due to the
higher restrictions placed on TBT predecessors on small vessels. For this reason, the
study areas for monitoring Irgarol were concentrated on large marinas (> 100 wet slips)
along the Florida coastline.
The marinas sampled all have varying geometric
22
configurations, including enclosed marinas where water circulation was well restricted
and open marinas that consisted of piers extending out into open waters. Figure 2.1
shows the locations of some of the study areas through Florida. Of particular importance
are Biscayne Bay and the Florida Keys.
Biscayne Bay contains two important sampling locations: the Miami River and
Coconut Grove Marina. The Miami River is a six-mile long river that discharges into
Biscayne Bay. It is approximately 50 feet across its widest point. The river is home to
mostly large shipping operations and large commercial vessels. This is an ideal location
for assessing commercial and industrial contributions, or lack thereof, of Irgarol into
Biscayne Bay. Coconut Grove is the area with the largest marina concentration in
Biscayne Bay, and has the capacity to hold in excess of 1000 recreational vessels ranging
from small personal watercraft to large pleasure craft (110 feet or greater). There is also
a large sailboat mooring area located in the southern portion of the marina. The channels
leading out from Coconut Grove Marina are home to many seagrass beds. Figure 2.2
shows the location of the seagrass beds in relation to the docking areas. At low tide,
water flows out of the marina past the seagrass beds. This puts the seagrass at potential
risk of exposure to any Irgarol that is present in the water in the marina. While the
marina is indeed large and houses many boats, it can still be considered to be an “open”
marina, due to the fact that water circulation is quite high. Previous research has shown
that Irgarol concentrations in the area have been consistent at about 40 – 60 ng/L since
1999 (Gardinali et al., 2004).
The Florida Keys is the other area of importance in the South Florida
environment. The Florida Keys is a collection of over 800 separate islands, of which the
23
1.
2.
3.
4.
5.
6.
7.
8.
Tampa Bay
Sarasota Bay
Charlotte Harbor
Ft Myers
Collier County
Key Largo Harbor
Biscayne Bay/Miami River/Coconut Grove
Dania Beach/Port Everglades
1
2
4
3
8
5
7
6
Figure 2.1: Study areas along the coast of Florida
24
Sailboat Areas
Figure 2.2: Coconut Grove Marina overlaid with a graphical representation of seagrass beds.
Darker greens indicate more dense seagrass patches. White lines in the water are docking piers,
some of which are covered by the seagrass layer.
highest elevation point is approximately 18 feet. This area is home to the location that
has been reported to have the second highest Irgarol concentrations in the United States
(around 200 ng/L; Owens et al., 2002; Gardinali et al., 2004), Key Largo Harbor (Figure
2.3). Key Largo Harbor is a mostly residential area, comprised of approximately 200
houses with personal docking areas along the harbor. It also is home to a few marine
service facilities, hotels and restaurants. While this area does not have near the capacity
for boat storage as Coconut Grove Marina, its geometric configuration is completely
different. The area consists of one main channel that runs north/south, with multiple
minor channels branching out to the west of the main channel. It is these minor channels
that contain the residential areas with boats. The main channel turns to the west at the
extreme north of the harbor. It is in this area where the marine facilities, restaurants, and
hotels are located. The area has only one exit, which is located to the extreme south of
25
the main channel. The geometry of the area is such that water circulation is very poor,
especially in the north of the harbor. Another interesting aspect of this location is that
located just outside the exit, there are large, healthy seagrass beds with abundant
coverage. This makes the area another ideal location for determining the potential impact
of Irgarol on submerged vegetation.
Figure 2.3: Key Largo Harbor overlaid with a graphical representation of seagrass beds. Darker
shades of green indicate denser patches of seagrass
Due to the low concentrations (< 100 ng/L) reported to cause a reversible net
effect of photosynthetic processes of intact corals (Owen et al. 2002), it was thought
necessary to conduct a survey of various reef systems off the coast of the Florida Keys to
see if there was a possible risk associated with their proximity to nearby contaminated
marinas. Nine reef sites were sampled (Figure 2.4) for this particular part of the research.
Two reefs in the Middle Keys (Sombrero and E. Wasserwoman) are in close proximity
with two marinas named Boat Key Harbor and Sombrero Marina. The five reefs in the
Upper Keys (Carysfort, Elbow, Molasses, Pickles, and Conch) are offshore of Key Largo
26
Harbor. The Dry Tortugas is a large reef system in the middle of the Gulf of Mexico.
Looe Key is another large reef system that is well off the coast of the Lower Keys.
Dry Tortugas
1. Carysfort Reef
2. Elbow Reef
3. Molasses Reef
4. Pickles Reef
5. Conch Reef
6. E. Wasserman Reef
7. Sombrero Reef
8. Looe Key
1
2
3
4
5
7
6
8
Figure 2.4: Location of coral reef sample sites in the Florida Keys
2.2. Experimental
2.2.1. Sample Collection
All samples were collected from a boat with no antifouling paint. For aqueous
samples, four (4) liter subsurface water samples were collected in precleaned amber glass
bottles. Submerged vegetation samples of turtle grass (Thalassia testudinum, Figure 2.5)
and manatee grass (Syringodium filiforme, Figure 2.6) were collected by hand by a diver
or by using a grab sampler. Samples were collected in triplicate, wrapped in solvent
rinsed aluminum foil, and placed in zip-lock bags. Sediment samples were also collected
by a diver or a grab sampler and stored in 250 mL precleaned, certified I-CHEM jars. A
majority of the pore water was decanted before sealing the jar.
Environmental
descriptors such as depth, pH, salinity, dissolved oxygen, and temperature were recorded
27
Picture Courtesy of Dr.
Brigitta Tussenbroek and Dr.
Jim Fourqurean, FIU Dept of
Biological Sciences
Figure 2.5: Thalassia testudinum, Turtle Grass
Picture Courtesy of Dr.
Brigitta Tussenbroek and Dr.
Jim Fourqurean, FIU Dept of
Biological Sciences
Figure 2.6: Syringodium filftorme, Manatee Grass
28
at each sampling site. Upon return to the laboratory, aqueous samples were stored in a
dark refrigerator at temperatures between 1 C and 4 C. Submerged vegetation and
sediment samples were stored in a freezer at temperatures below -10 C.
2.2.2. Sample Extraction
2.2.2.1. Surface water samples
Aqueous samples were extracted using basic liquid-liquid extraction techniques.
Two (2) liters of each water sample were filtered through a 0.45 m membrane filter
before extraction. Samples were quantitatively transferred to a 2 liter separatory funnel.
Samples were then fortified with the appropriate amount of the surrogate standard,
Atrazine-d5 (100 L of a 1 ppm solution). Twenty grams of sodium chloride was added
to each sample to increase the ionic strength of the samples and increase extraction
yields. Samples were then extracted sequentially with three fractions of 50 mL portions
of methylene chloride. The fractions were combined, dried over anhydrous sodium
sulfate, and collected in a 250 mL round-bottom flask. The organic layers were then
concentrated to 10 mL using a water bath and a glass distillation column. Samples were
again quantitatively transferred to a Kimax 25mL concentrator. The samples were then
further evaporated to a final volume of 0.5 mL, after exchanging solvent to hexane. An
appropriate amount of the internal standard solution, tetrachloro-m-xylene (TCMX, 100
L of a 1 ppm solution) was added to all samples, followed by quantitative transfer to a 2
mL amber target vial for analysis.
2.2.2.2. Submerged vegetation samples
Seagrass samples were extracted using accelerated solvent extraction (ASE)
followed by solid phase extraction (SPE). Before sample processing, all submerged
29
vegetation samples were separated from their roots and freeze-dried to remove any water
still present in the sample. The blades of each seagrass sample were then crushed to
make a homogenous mixture. 1.5 grams of the homogenous dry powder was weighed out
and dispersed with diatomaceous earth (DE) powder. The mixture was then transferred
into a 33 mL ASE cell (Dionex Industries). The cell was filled with additional DE
powder. The samples were then fortified with the appropriate amount of the Atrazine-d5
surrogate solution (200 L of a 1 ppm solution). The cells were fitted on each end with a
0.45 m filter paper and sealed. The ASE instrument was operated at a pressure of 1500
psi. The oven was operated at 100 C. The samples were extracted with a 90:10 mixture
of methanol and distilled, deionized (DDI) water. Samples were heated for 5 minutes and
then left static for 10 minutes. The extracts were then purged from the cell for 2 minutes,
using pure nitrogen. Extracts were collected in clean 50 mL glass vials. The 45 mL
extracts were then centrifuged to remove any material that precipitated after extraction.
In order to obtain an aqueous extract for the SPE processing, samples were
evaporated using a Rotovap to remove the methanol. An Oasis HLB SPE cartridge was
conditioned with 10 mL of methanol at a rate below 2 mL/min. The cartridge was air
dried for 1 minute, then equilibrated with 5 mL of DDI water. The SPE cartridge was
fitted with a Whatman GF/B glass fiber filter to trap any remaining particulate matter left
in the samples. The now aqueous sample was loaded onto the cartridge at a rate of 1
mL/min. All the above liquids that passed through the SPE cartridge in the conditioning
and loading steps were collected as waste and discarded. The cartridge was once again
air dried to remove any traces of water left. Analytes were eluted into a 15 mL test tube
using 10 mL of methylene chloride at a rate of 1 mL/min. If there was still water present
30
in the test tube, a small amount of sodium sulfate was added to dry the extract. Samples
were then quantitatively transferred into a 25 mL Kimax concentrator tube and
evaporated to a final volume of 1.0 mL in hexane, after solvent exchange. 100 L of the
1 ppm TCMX internal standard solution was added, and samples were transferred into a
target vial for GC/MS analysis.
2.2.2.3. Sediment extraction
The sediment extraction procedure is identical to the submerged vegetation
procedure, with the exception of pre-extraction sample preparation. In this case, thirty
(30) grams of wet sediment was weighed out into a glass beaker. The sediment was dried
and dispersed using DE powder and transferred to an ASE cell. From this point forward,
the extraction protocol for the seagrass samples was followed.
2.2.3. Sample analysis
All three matrices were analyzed using the same procedure. All samples were
analyzed using a Gas Chromatography/Mass Spectrometry (GC/MS) system operating in
selected ion monitoring (SIM) mode. At least two ions were scanned for each analyte, a
quantitation ion and at least one confirmation ion. For Irgarol, the total ion current (TIC)
of the three major fragments was used for quantitation with these three fragments being
used as confirmation ions. Table 2.1 shows the ions that were monitored for each
analyte.
Two L of each sample extract were injected using splitless injection into a
Thermo Trace Ultra GC interfaced with a Finnigan Trace DSQ MS. Analyte separation
was carried out using a 30 m x 250 m I.D. x 0.25 m film thickness DB5-MS fused
silica capillary column (Agilent). Helium was used as a carrier gas at a constant flow of
31
1.2 mL/min. The GC oven temperature program was as follows: the initial temperature
of 100 C was held for one minute, ramped to a final temperature of 300 C at a rate of
15 C/min and held for 0.67 minutes. The MS transfer line and ion source temperatures
were 280 C and 250 C, respectively.
Table 2.1: SIM mass table for Irgarol analysis
Quantitation
Confirmation
Analyte
Ion
Ion 1
TCMX (IS)
244
242
Atrazine-d5
205
222
Atrazine
200
215
M1
213
198
Irgarol
TICa
182
a. Total Ion Current
Confirmation
Ion 2
246
--217
157
238
Confirmation
Ion 3
--------253
The total run time per sample is 15 minutes. A chromatogram of a 1 ppm calibration
standard is shown in Figure 2.7.
2.2.4. Method Performance and Statistical Analysis
Analytes were quantitated using a 9 point calibration curve. Calibration solutions
ranged from 2.5 pg/L to 1000 pg/L. In order to ensure quality data processing, Rsquared values for all calibration curves for each sample set analyzed must be greater
than 0.995 to pass method QA/QC parameters. Example calibration curves for Irgarol
and M1 are shown in Figure 2.8.
For aqueous samples, analytical performance was checked by running artificial
seawater blanks (DDI water with the addition of 20 grams of sodium chloride) and
fortified blanks. Fortified blanks were spiked with 50 L of a 2 ppm solution of a
mixture of all analytes. For vegetation and sediment samples, blanks were made by
packing an ASE cell with DE powder. Only fortified blanks and matrix spiked samples
were spiked with 200 L of a 2 ppm solution of a mixture of all analytes to assess the
32
performance of the extraction method. Recoveries for both the surrogate compound and
target compounds are listed in Table 2.2.
A b u n d a n c e
Atrazine
T IC :
d a ta .c d f
M3 (see chapter 4)
3 .8 e + 0 7
3 .6 e + 0 7
Atrazine-d5
3 .4 e + 0 7
3 .2 e + 0 7
3 e + 0 7
Sea-Nine 211
2 .8 e + 0 7
(not discussed)
2 .6 e + 0 7
2 .4 e + 0 7
M1
2 .2 e + 0 7
2 e + 0 7
1 .8 e + 0 7
TCMX
Irgarol
1 .6 e + 0 7
1 .4 e + 0 7
1 .2 e + 0 7
1 e + 0 7
8 0 0 0 0 0 0
6 0 0 0 0 0 0
4 0 0 0 0 0 0
2 0 0 0 0 0 0
8 .5 0
9 .0 0
9 .5 0
1 0 .0 0
1 0 .5 0
1 1 .0 0
1 1 .5 0
1 2 .0 0
1 2 .5 0
T im e - - >
Figure 2.7: Chromatogram of a calibration standard
M1 Calibration Curve; R2=0.997
40
8
30
6
Response Ratio
Response Ratio
Irgarol Calibration Curve; R2=0.999
20
10
4
2
0
0
-2
-10
0
2
4
6
8
10
0
12
2
4
6
8
10
12
Amount Ratio
Amount Ratio
Figure 2.8: Calibration curves for Irgarol and M1. Curves were generated by plotting the
concentration ratio of the analyte and surrogate versus the area ratio of the analyte and surrogate .
Recoveries for all three matrices were good. Seagrass samples showed a higher
relative standard deviation (% RSD) than the other two matrices. Irgarol % RSD in
surface water and sediment were 12 and 14, respectively, while in seagrass samples the %
RSD was 28. The results for M1 were similar; 16 and 10 for surface water and sediment,
33
respectively, and 30 for seagrass. This was expected, due to the high matrix interferences
of extractable plant tissue matter (i.e., lipids, pigments, etc). Method detection limits
(MDL) for surface water samples were set at 1 ng/L. The MDL for seagrass and
sediments was set at 1 ng/g. Target compound concentrations in artificial seawater
blanks, seagrass blanks, and sediment blanks were either non-detect (N.D.) or below the
MDL parameters.
Table 2.2: Recoveries of target compounds in fortified blanks
Compound % Recovery % R.S.D.a # of samples
Surface Water
Atrazine-d5b 104
22
428
Irgarol
106
12
30
M1
100
16
30
Seagrass
Atrazine-d5b
Irgarol
M1
88
92
90
19
28
30
154
20
20
Sediment
Atrazine-d5b 103
23
144
Irgarol
97
14
17
M1
103
10
17
a. Relative Standard Deviation (Standard Deviation/Average * 100)
b. For the surrogate, recoveries were calculated using samples, blanks, and
fortified blanks
2.2.5. Chemicals
Irgarol 1051®, and its major metabolite, M1, were obtained from Ciba Specialty
Chemicals (Tarrytown, New York, USA). Atrazine-d5 used as a surrogate standard was
purchased as a certified standard solution from Dr. Ehrenstorfer GmbH (Augsburg,
Germany). All glassware used in the experiments and sample processing was rinsed and
combusted at 450 C for 4 hours to remove all organic residues. All solvents were
34
purchased from Fisher Scientific (Fair Lawn, New Jersey, USA), were of pesticide grade,
and were used without further purification.
2.3. Results
2.3.1. Surface water samples
2.3.1.1. Key Largo Harbor
Key Largo Harbor is an area that has been routinely monitored for the presence of
Irgarol. Previous studies have shown that the concentrations of Irgarol in the area had
increased over a three year period from a maximum concentration of 71.4 ng/L in 2000 to
a maximum concentration of 182 ng/L in 2001 (Gardinali et al. 2002; Gardinali et al.,
2004). Similar levels have been reported by other research groups (Owen et al. 2002).
Results for the 2003-2006 KLH survey showed that Irgarol was present in 100%
(151/151) of the samples, with concentrations ranging from 1.82 ng/L to 1239 ng/L. The
average concentration and median concentration was 188 ng/L and 99.9 ng/L,
respectively.
M1 was found in 87% (132/151) of the samples with concentrations
ranging from N.D. to 429 ng/L. The average concentration and median concentration
was 76.5 ng/L and 29.8, respectively. Concentration ranges, average concentration, and
median concentration for each individual sampling trip are listed in Table 2.3.
A
complete list of all samples collected with their corresponding locations and Irgarol and
M1 concentrations can be found in Appendix A.
35
Table 2.3: Summary of results from the KLH area
Collection Date
Irgarol
Irgarol
Range (ng/L)
Average
and Median
(ng/L)
2/15/2003, Site 1
230-1239
484; 371
2/15/2003, Site 2
144 – 575
272; 222
8/21/2003
52.5 – 448
230; 129
2/5/2004
1.82 – 450
97.4; 77.0
4/29/2004
3.32 – 266
47; 4.80
6/14/2004
5.16 – 213
57; 22.9
8/27/2004
5.02 – 229
70; 49.4
3/23/2006
8.36 – 271
91.3; 45.6
M1 Range
(ng/L)
M1 Average
and Median
(ng/L)
Number of
Samples
34.1 – 1296
73.9 – 226
20.7 – 135
N.D. – 60.1
N.D. – 74.8
N.D. – 70.8
N.D. – 78.2
5.05 – 68.2
249; 256
127; 105
63.3; 35.8
22.8; 14.0
39.9; 0.00
23.5; 8.64
28.6; 16.3
26.8; 17.0
24
20
19
19
14
22
28
16
Distributions of the analytes were similar in each sampling trip, with the highest
concentrations appearing towards the “rear” of the area where the marine facilities are,
and decreasing towards the exit of the Harbor. Starting in 2004, samples were collected
from just outside of the harbor area, where analyte concentrations were expected to
decrease rapidly. Figure 2.9 shows the distributions of Irgarol within the Harbor on
2/5/2004.
The samples collected on 2/15/2003 correspond to a 24-hour sampling trip where
one sample was collected every hour to see if any tidal trends were present. Station 1
was towards the back of the Harbor, where the water flow is restricted. Station 2 was
next to the mouth of the Harbor area, where water exchange is high. The exact locations
of the stations can be seen on Figure 2.9. While both stations show tidal influences, the
two trends are opposite. At station 1, Irgarol concentrations are higher at low tide and
concentrations are lower at high tide. Irgarol concentrations at station 2 were reversed,
with higher concentrations at high tide and lower concentrations at low tide. This could
be due to a dilution effect at station 1 while the tide at station two could be flushing
Irgarol out of the Harbor at low tide. Concentrations of Irgarol over the 24 hour period
overlaid with tidal patterns for the same period are shown in Figure 2.10.
36
24-hour
sampling
site #1
24-hour
sampling
site #2
Figure 2.9: Distribution of Irgarol in Key Largo Harbor, 2/5/2004
Site 1
Site 2
1400
2.0
1400
1200
2.0
1200
1.0
800
600
0.5
400
1.5
1000
1.0
800
600
0.5
400
0.0
200
0
12:00:00
Tide Height (meters)
1000
Tide Height (meters)
Concentration (ng/L)
Concentration (ng/L)
1.5
0.0
200
16:00:00
20:00:00
00:00:00
04:00:00
08:00:00
-0.5
12:00:00
0
12:00:00
16:00:00
20:00:00
00:00:00
04:00:00
08:00:00
-0.5
12:00:00
Time
Time
Figure 2.10: Concentrations of Irgarol throughout the 24-hour sampling period (black) overlaid
with tidal patterns (blue). Average concentrations during high and low tide are shown in red and
green, respectively.
37
2.3.1.2. Biscayne Bay
Irgarol distribution in Biscayne Bay was similar to that of Key Largo Harbor
(100% occurrence, 24/24 samples). However, analyte concentrations are much lower
than those in Key Largo Harbor. Irgarol and M1 concentrations in Biscayne Bay ranged
from 2.63 ng/L to 101 ng/L and N.D. to 35.9 ng/L, respectively. Average concentrations
of Irgarol and M1 were 32.4 ng/L and 9.67 ng/L, respectively. Median concentrations of
Irgarol and M1 were 29.8 ng/L and 12.2 ng/L, respectively.
The samples from the Biscayne Bay area can be separated into three different
categories: The Bay area, the Miami River, and Coconut Grove. Distributions of Irgarol
in the samples collected in Biscayne Bay and the Miami River during March of 2006 is
shown in Figure 2.11. Distributions of Irgarol in Coconut Grove in August 2006 are
shown in Figure 2.12. A categorized list of all samples collected in these areas, along
with their concentrations ranked from highest to lowest is shown in Table 2.4.
38
Figure 2.11: Miami River and Biscayne Bay, March 2006
39
Figure 2.12: Coconut Grove surface water sampling, August 2006
40
Table 2.4: Concentrations of Irgarol and M1 in the Biscayne Bay area
Site
Collection Date
Latitude
Longitude
Irgarol
M1
Biscayne Bay
BBKB
BBCP
BBMBM
BBRB
BBSM
BBLB
BBSL
BBBP
BBDK
BBMH
BBSB
BBKB
BBCP
BBRB
BBMBM
BBSL
BBBS
2/12/2004
2/12/2004
2/12/2004
2/12/2004
2/12/2004
2/12/2004
2/12/2004
2/12/2004
2/12/2004
2/12/2004
2/12/2004
3/20/2006
3/20/2006
3/20/2006
3/20/2006
3/20/2006
3/20/2006
25.70030
25.72370
25.76960
25.74750
25.79140
25.77710
25.79140
25.76990
25.72615
25.67917
25.72291
25.69970
25.72462
25.74659
25.76992
25.79125
25.77942
80.16990
80.15640
80.14080
80.17740
80.18330
80.18380
80.15330
80.18940
80.23638
80.25926
80.23046
80.16930
80.15704
80.17600
80.14055
80.18419
80.18504
30.8
47.3
2.63
4.55
4.90
3.83
11.0
12.3
25.7
23.3
19.4
51.6
11.3
85.8
2.17
5.42
43.0
3.64
0.58
1.80
0.00
0.00
0.00
3.38
2.20
9.02
4.69
7.25
14.1
4.17
26.0
1.26
4.50
12.2
2/12/2004
2/12/2004
2/12/2004
2/12/2004
2/12/2004
2/12/2004
3/20/2006
3/20/2006
3/20/2006
3/20/2006
3/20/2006
3/20/2006
3/20/2006
25.78790
25.79509
25.78790
25.78630
25.77990
25.78320
25.76976
25.77111
25.77994
25.78290
25.78621
25.78439
25.79023
80.22990
80.24527
80.22990
80.22380
80.20970
80.21650
80.18963
80.19977
80.20975
80.21556
80.22378
80.22920
80.23455
11.9
3.84
12.4
14.7
26.0
16.9
21.0
58.7
77.4
101
80.3
50.7
56.5
0.00
0.00
0.00
0.00
0.00
0.00
8.63
18.2
22.1
27.0
26.0
21.8
20.8
2/12/2004
2/12/2004
10/1/2004
10/1/2004
10/1/2004
10/1/2004
10/1/2004
10/1/2004
3/20/2006
3/20/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
25.72715
25.73100
25.72770
25.72652
25.72669
25.72560
25.72425
25.72915
25.72642
25.73084
25.72399
25.72167
25.72615
25.72936
25.73155
25.73348
25.71895
25.72294
80.23170
80.23080
80.23119
80.23048
80.22900
80.22770
80.22642
80.22753
80.23355
80.23091
80.23055
80.22894
80.22285
80.22797
80.22860
80.22623
80.23143
80.23940
23.9
26.8
48.0
28.0
35.1
37.0
18.2
35.9
40.9
87.4
30.4
27.9
32.6
57.7
61.0
56.8
10.6
18.8
4.79
5.99
25.0
10.8
16.0
18.7
35.9
7.84
18.4
32.9
15.4
13.6
16.2
20.8
22.5
23.7
5.54
9.14
Miami River
MRDD
MRLP
MRHC
MRYC
MRNS
MRMS
MRBP
MR95
MRNS
MRMS
MRYC
MRSF
MRCP
Coconut Grove
BBCG
BBMM
CG01
CG02
CG03
CG04
CG05
CG06
BBCG
BBMM
CG01
CG02
CG03
CG04
CG05
CG06
CG07
CG08
41
2.3.1.3. Other Coastal Marinas
Due to the well documented results that Irgarol occurrence is more frequent, and
with higher concentrations, in large marinas, the sampling strategy for the west coast of
Florida was designed specifically to target these types of areas.
As expected,
distributions of Irgarol and M1 are similar in the west coast samples as they were in the
South Florida sampling sites with 100% occurrence (67/67) for Irgarol and 89%
occurrence (57/62) for M1. Concentrations of Irgarol range from 1.16 ng/L to 110 ng/L
with an average of 40.5 ng/L. M1 concentrations range from N.D. to 48.1 ng/L with an
average of 13.2 ng/L. A complete list of Irgarol and M1 concentrations in samples
collected from the west coast of Florida can be found in Table 2.5. Satellite photos of
marinas with their corresponding concentrations can be found in Figures 2.13 through
2.26.
One marina that had Irgarol concentrations that were relatively higher than other
marinas along the west coast was the St. Petersburg Municipal Marina, located in western
Tampa Bay. This marina is quite similar to Key Largo Harbor in that it is enclosed and
only has one exit to open waters. The marina holds in excess of 600 boats and contains a
couple of public boat ramps. A map of the area along with the corresponding Irgarol
concentrations is shown in Figure 2.26. The sample with the highest concentration was
taken adjacent to the refueling dock and public ramps, where people are most likely to
clean their boats upon returning to the marina. Concentrations are also higher closer to
docking piers, and decrease towards the exit of the marina.
This trend of having
increased amounts of Irgarol closer to mooring areas and decreasing towards open waters
is a common trend in most areas sampled.
42
Table 2.5: Concentrations of Irgarol and M1 in samples collected from the west coast of Florida
Collection
Site
Description
Date
Latitude
Naples
CC01
Collier County Site 1
2/1/2003
26.13777
CC02
Collier County Site 2
2/1/2003
26.13948
CC03
Collier County Site 3
2/1/2003
26.14015
CC04
Collier County Site 4
2/1/2003
26.14132
CC05
Collier County Site 5
2/1/2003
26.14255
CC06
Collier County Site 6
2/1/2003
26.14265
CC07
Collier County Site 7
2/1/2003
26.13310
CC08
Collier County Site 8
2/1/2003
26.13215
Fort Myers
FMYB01
Ft. Myers Yacht Basin Site 1
1/22/2005
26.64689
FMYB02
Ft. Myers Yacht Basin Site 2
1/22/2005
26.64683
FMYB03
Ft. Myers Yacht Basin Site 3
1/22/2005
26.64905
LYC01
Liberty Yacht Club Site 1
1/22/2005
26.53188
LYC02
Liberty Yacht Club Site 2
1/22/2005
26.53362
LYC03
Liberty Yacht Club Site 3
1/22/2005
26.53605
PPM01
Peppertree Point Marina Site 1
1/22/2005
26.52648
PPM02
Peppertree Point Marina Site 2
1/22/2005
26.52549
TPM01
Tarpon Point Marina Site 1
1/22/2005
26.53854
TPM02
Tarpon Point Marina Site 2
1/22/2005
25.53991
Charlotte Harbor
GM01
Gasparilla Marina Site 1
1/22/2005
26.83539
GM02
Gasparilla Marina Site 2
1/22/2005
26.83475
GM03
Gasparilla Marina Site 3
1/22/2005
26.83396
GM04
Gasparilla Marina Site 4
1/22/2005
26.83357
CHMB01 Charlotte Harbor Marina Basin Site 1
1/22/2005
26.86828
CHMB02 Charlotte Harbor Marina Basin Site 2
1/22/2005
26.89510
CHMB03 Charlotte Harbor Marina Basin Site 3
1/22/2005
26.87442
PIM01
Palm Island Marina
1/22/2005
26.86960
PIM02
Palm Island Marina
1/22/2005
26.87051
PIM03
Palm Island Marina
1/22/2005
26.87111
43
Longitude
Irgarol
(ng/L)
M1
(ng/L)
81.78977
81.78987
80.79057
81.78993
81.78980
81.78747
81.79253
81.79325
25
26.6
31.7
22.8
16.9
17.2
23.7
43.5
4.50
4.76
6.14
3.41
2.72
2.95
3.78
7.22
81.86981
81.87068
81.87190
81.93984
81.93859
81.93790
81.95407
81.95590
82.00043
81.99946
60.8
11.6
7.17
7.46
9.46
5.91
13.5
5.81
43.2
16.9
25.2
6.84
7.43
4.67
4.37
5.16
6.23
4.72
7.78
6.22
81.99946
81.25023
82.25918
82.26093
82.31286
82.31407
82.31407
82.31039
82.31049
82.31222
36.7
15.4
21.6
7.68
44.8
37.9
74.2
54.4
35.8
5.43
8.37
6.25
6.02
3.59
11.4
11.2
13.2
20.7
10.7
2.13
Table 2.5 (continued)
Site
Description
St. Petersburg
MJ01
Marina Jack Site 1
MJ02
Marina Jack Site 2
MJ03
Marina Jack Site 3
MJ04
Marina Jack Site 4
LKM01
Longboat Key Moorings Site 1
LKM02
Longboat Key Moorings Site 2
LKM03
Longboat Key Moorings Site 3
LKM04
Longboat Key Moorings Site 4
Tampa Bay
BCYC01
Boca Ciega Yacht Club/Gulfport Marina Site 1
BCYC02
Boca Ciega Yacht Club/Gulfport Marina Site 2
BCYC03
Boca Ciega Yacht Club/Gulfport Marina Site 3
BCYC04
Boca Ciega Yacht Club/Gulfport Marina Site 4
STPMM01
St. Petersburg Municipal Marina Site 1
STPMM02
St. Petersburg Municipal Marina Site 2
STPMM03
St. Petersburg Municipal Marina Site 3
STPMM04
St. Petersburg Municipal Marina Site 4
STPMM05
St. Petersburg Municipal Marina Site 5
STPMM06
St. Petersburg Municipal Marina Site 6
STPMM07
St. Petersburg Municipal Marina Site 7
TV01
Tierra Verde Marina Site 1
TV02
Tierra Verde Marina Site 2
HM01
Harborage Marina Site 1
HM02
Harborage Marina Site 2
HM03
Harborage Marina Site 3
Dania Beach/Port Everglades
DB01
Dania Beach Inlet/Port Everglades Site 1
DB02
Dania Beach Inlet/Port Everglades Site 2
DB03
Dania Beach Inlet/Port Everglades Site 3
a. Sample contaminated with M1 in the lab during analysis
Latitude
Longitude
Irgarol
(ng/L)
M1
(ng/L)
1/22/2005
1/22/2005
1/22/2005
1/22/2005
1/22/2005
1/22/2005
1/22/2005
1/22/2005
27.33381
27.33438
27.33232
27.33246
27.36878
27.37177
27.37017
27.37298
82.54597
82.54733
82.54454
82.54633
82.61864
82.61085
82.61822
82.61716
11.9
2.53
12.4
6.22
31.8
29.3
18.3
9.35
5.55
2.18
4.07
2.58
6.55
4.33
5.62
3.85
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
5/7/2005
27.73982
27.74048
27.73883
27.73832
27.75952
27.76016
27.76031
27.77315
27.77209
27.77136
27.76849
27.76900
27.76799
27.77088
27.69347
27.69480
82.69640
82.69536
82.69497
82.69628
82.63507
82.63567
82.63543
82.62880
82.62998
82.62862
82.63133
82.63011
82.62741
82.62649
82.71947
82.71872
83.7
82
110
39.2
57.8
78.6
82.7
49.5
58.2
47.4
33.7
1.59
1.16
121
175
80.3
38.4
a
40.1
23.3
39.7
26.3
48.1
20.3
29.0
a
35.9
a
a
a
a
a
9/7/2006
9/7/2006
9/7/2006
26.09212
26.09254
26.09865
80.11275
80.10174
80.11926
25
20.2
37.1
8.84
5.59
11.1
Collection Date
44
Table 2.5 (continued)
Site
DB04
DB05
DB06
DB07
DB08
DB09
DB10
DB11
DB12
DB13
Description
Dania Beach Inlet/Port Everglades Site 4
Dania Beach Inlet/Port Everglades Site 5
Dania Beach Inlet/Port Everglades Site 6
Dania Beach Inlet/Port Everglades Site 7
Dania Beach Inlet/Port Everglades Site 8
Dania Beach Inlet/Port Everglades Site 9
Dania Beach Inlet/Port Everglades Site 10
Dania Beach Inlet/Port Everglades Site 11
Dania Beach Inlet/Port Everglades Site 12
Dania Beach Inlet/Port Everglades Site 13
Collection Date
9/7/2006
9/7/2006
9/7/2006
9/7/2006
9/7/2006
9/7/2006
9/7/2006
9/7/2006
9/7/2006
9/7/2006
45
Latitude
26.10321
26.10290
26.11245
26.11080
26.11129
26.11485
26.11561
26.11733
26.11733
26.12205
Longitude
80.11847
80.12274
80.12118
80.10661
80.10872
80.10777
80.10640
80.10743
80.10743
80.10812
Irgarol
(ng/L)
45.9
96.2
62.8
62.6
57.5
53.8
72.5
57.2
48.4
54.6
M1
(ng/L)
13.4
22.0
16.6
21.3
18.5
17.4
24.5
21.8
20.8
18.4
Figure 2.13: Dania Beach Inlet/Port Everglades, Fort Lauderdale, September 2006
46
Figure 2.14: Collier County marinas, Naples, February 2003
47
Tarpon Point
Marina (2.17)
Peppertree
Point Marina
(2.16)
Fort Myers Yacht
Basin (2.14)
Liberty Yacht
Club (2.15)
Figure 2.15: Locations of Fort Myers sampling locations relative to Gulf waters
48
Figure 2.16: Fort Myers Yacht Basin, January 2005
49
Figure 2.17: Liberty Yacht Club, Fort Myers, January 2005
50
Figure 2.18: Peppertree Point Marina, Fort Myers, January 2005
51
Figure 2.19: Tarpon Point Marina, Ft Myers, January 2005
52
Figure 2.20: Gasperilla Marina, Charlotte Harbor, January 2005
53
Figure 2.21: Palm Island Marina (bottom) and Charlotte Harbor Marina Basin (top), Charlotte Harbor, January 2005
54
Figure 2.22: Marina Jack's, Sarasota Bay, January 2005
55
Figure 2.23: Longboat Key Moorings, Sarasota Bay, January 2005
56
Figure 2.24: Boca Ciega Yacht Club, Tampa Bay, May 2005
57
Figure 2.25: The Harborage Marina, Tampa Bay, May 2005
58
Figure 2.26: St Petersburg Municipal Marina, Tampa Bay, May 2005
59
2.3.1.4 Coral Reef Sampling
Irgarol was found in only 5% (5/96) of the offshore reef samples, and all five of
these were less than 2 ng/L (but still above the detection limits for surface waters). All
five positive detections were from samples taken from Looe Key during an annual
Underwater Music Festival. The festival is a weekend event in which recreational boats
tie to moorings in order to let passengers dive into the reef to listen to music from
speakers placed in the reef. Since the main introduction pathway of Irgarol is leaching
over the lifetime of the paint (Thomas et al., 2002), this could explain why small amounts
of Irgarol were found while boats were present and no Irgarol was found in samples
collected before or after the festival. No M1 was found in any of the sampling sites
(0/96). These results seem to indicate that the reef areas are not impacted by the Irgarol
from nearby marinas, even though two marinas sampled contained Irgarol (100%
occurrence, 9/9 samples). Concentrations of Irgarol and M1 in the selected reefs and
nearby marinas are shown in Table 2.6.
Table 2.6: Concentrations of Irgarol and M1 in coral reefs and nearby marinas
Sampling Location
Irgarol (ng/L)
M1 (ng/L)
Reefs
Looe Key
N.D. - 1.92
N.D.
Conch Reef
N.D.
N.D.
Molasses Reef
N.D.
N.D.
Pickles Reef
N.D.
N.D.
E. Wasserwoman Reef
N.D.
N.D.
Sombrero Reef
N.D.
N.D.
Carysfort Reef
N.D.
N.D.
Elbow Reef
N.D.
N.D.
Dry Tortugas
N.D.
N.D.
# of Samples
29
10
10
5
5
6
10
5
16
Marinas
Boat Key Harbor
Sombrero Marina
22.2-50.3
36.5-45.9
60
8.42-13.1
9.86-12.4
5
4
2.3.2. Seagrass Samples
Analyses of Irgarol in seagrass samples started in April of 2004. Since that time,
seven sets of samples were collected and analyzed.
The total number of samples
analyzed in these seven sets was 97. Irgarol was found in 100% (97/97) of the samples.
M1 was found in 9% of the samples (9/97). Concentrations of Irgarol in the seagrass
samples ranged from 2.35 ng/g to 283 ng/g. The average Irgarol concentration was 23.8
ng/L. The median concentration was 8.86 ng/L. M1 concentrations ranged from N.D. to
1.374 ng/g. A complete list of concentrations for all 97 seagrass samples is given in table
2.7.
Using this data and the data obtained from the surface water samples collected at
the same time, bioconcentration factors (BCFs) were calculated. According to the EPA,
BCFs are a measurement of the ratio of the chemical concentration in a marine organism
compared to that of the surrounding water. BCFs greater than 1000 are considered to be
accumulative, while BCFs greater than 5000 are considered highly accumulative (EPA
1999). Since BCFs are reported in L/Kg, the ratio of the tissue concentration to the water
concentration must be multiplied by 1000 to obtain the correct units.
Bioconcentration factors ranged from 30 to 31588. While these numbers cover a
wide range, only five of the BCF values calculated were above 5000. Three of these 5
samples were the three samples of manatee grass collected. The bioconcentration factors
for manatee grass were 11941, 14610, and 31588. These values are on the same order of
those calculated by Scarlett et al. in their studies (Scarlett et al., 1999). Thus, it seems
that different specie of seagrass accumulate Irgarol more effectively than others. A list of
sample sites and their BCF values are given in table 2.8.
61
Table 2.7: Concentrations of Irgarol and M1 in seagrasses collected from Key Largo Harbor and Coconut Grove *
Site
Description
Latitude Longitude Collection Date Irgarol
Key Largo Harbor
NKLH-01A
North Key Largo Harbor Site 1
25.08855 80.43059
4/29/2004
9.89
NKLH-01B
North Key Largo Harbor Site 1
25.08855 80.43059
4/29/2004
8.86
NKLH-01C
North Key Largo Harbor Site 1
25.08855 80.43059
4/29/2004
6.86
NKLH-02A
North Key Largo Harbor Site 2
25.08864 80.43047
4/29/2004
12.8
NKLH-02B
North Key Largo Harbor Site 2
25.08864 80.43047
4/29/2004
10.1
NKLH-02C
North Key Largo Harbor Site 2
25.08864 80.43047
4/29/2004
11.8
NKLH-03A
North Key Largo Harbor Site 3
a
a
4/29/2004
10.4
NKLH-03B
North Key Largo Harbor Site 3
a
a
4/29/2004
8.01
NKLH-03C
North Key Largo Harbor Site 3
a
a
4/29/2004
8.67
NKLH-04A
North Key Largo Harbor Site 4
a
a
4/29/2004
7.94
NKLH-04B
North Key Largo Harbor Site 4
a
a
4/29/2004
8.83
NKLH-04C
North Key Largo Harbor Site 4
a
a
4/29/2004
3.38
SKLH-05A
South Key Largo Harbor Site 1
25.08745 80.43060
4/29/2004
10.6
SKLH-05B
South Key Largo Harbor Site 1
25.08745 80.43060
4/29/2004
9.16
SKLH-05C
South Key Largo Harbor Site 1
25.08745 80.43060
4/29/2004
9.35
SKLH-06A
South Key Largo Harbor Site 2
25.08689 80.43011
4/29/2004
5.47
SKLH-07A
South Key Largo Harbor Site 3
25.08650 80.42970
4/29/2004
3.62
SKLH-07B
South Key Largo Harbor Site 3
25.08650 80.42970
4/29/2004
2.66
SKLH-07C
South Key Largo Harbor Site 3
25.08650 80.42970
4/29/2004
5.03
SKLH-08A
South Key Largo Harbor Site 4
25.08712 80.42983
4/29/2004
4.17
SKLH-08B
South Key Largo Harbor Site 4
25.08712 80.42983
4/29/2004
4.69
SKLH-08C
South Key Largo Harbor Site 4
25.08712 80.42983
4/29/2004
4.43
NKLH-01
North Key Largo Harbor Site 1
25.08944 80.43021
6/14/2004
14.9
NKLH-02
North Key Largo Harbor Site 2
25.08810 80.43020
6/14/2004
6.91
NKLH-03
North Key Largo Harbor Site 3
25.08722 80.40080
6/14/2004
7.95
NKLH-04
North Key Largo Harbor Site 4
25.08732 80.42937
6/14/2004
3.60
NKLH-05
North Key Largo Harbor Site 5
25.08830 80.42940
6/14/2004
6.55
NKLH-06
North Key Largo Harbor Site 6
25.08957 80.42944
6/14/2004
10.7
NKLH-07
North Key Largo Harbor Site 7
25.08960 80.42834
6/14/2004
3.28
NKLH-08
North Key Largo Harbor Site 8
25.08761 80.42837
6/14/2004
2.98
62
M1
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Table 2.7 (continued)
Site
SKLH-01
SKLH-02
SKLH-03
SKLH-04
SKLH-05
SKLH-06
SKLH-07
SKLH-08
NKLH-01
NKLH-02
NKLH-03
NKLH-04
NKLH-05
NKLH-06
NKLH-07
NKLH-08
NKLH-09
NKLH-10
SKLH-01
SKLH-02
SKLH-03
SKLH-04
SKLH-05
SKLH-06
SKLH-07
SKLH-08
SKLH-09
SKLH-10
KLH-09
KLH-10
KLH-11
Description
South Key Largo Harbor Site 1
South Key Largo Harbor Site 2
South Key Largo Harbor Site 3
South Key Largo Harbor Site 4
South Key Largo Harbor Site 5
South Key Largo Harbor Site 6
South Key Largo Harbor Site 7
South Key Largo Harbor Site 8
North Key Largo Harbor Site 1
North Key Largo Harbor Site 2
North Key Largo Harbor Site 3
North Key Largo Harbor Site 4
North Key Largo Harbor Site 5
North Key Largo Harbor Site 6
North Key Largo Harbor Site 7
North Key Largo Harbor Site 8
North Key Largo Harbor Site 9
North Key Largo Harbor Site 10
South Key Largo Harbor Site 1
South Key Largo Harbor Site 2
South Key Largo Harbor Site 3
South Key Largo Harbor Site 4
South Key Largo Harbor Site 5
South Key Largo Harbor Site 6
South Key Largo Harbor Site 7
South Key Largo Harbor Site 8
South Key Largo Harbor Site 9
South Key Largo Harbor Site 10
Key Largo Harbor Site 9
Key Largo Harbor Site 10
Key Largo Harbor Site 11
Latitude
25.08920
25.08819
25.08695
25.08690
25.08791
25.08875
25.08827
25.08785
25.08904
25.08895
25.08907
25.08957
25.08970
25.08965
25.08943
25.09008
25.09299
25.09324
25.08804
25.08778
25.08748
25.08733
25.08796
25.08867
25.08894
25.08781
25.08407
25.08207
25.08792
25.08883
25.08831
63
Longitude
80.43129
80.43166
80.43150
80.43250
80.43265
80.43255
80.43306
80.73334
80.43024
80.42995
80.42944
80.42947
80.42957
80.43005
80.43046
80.42976
80.42873
80.42649
80.43143
80.43138
80.43198
80.43219
80.43262
80.43238
80.43176
80.43324
80.43505
80.43702
80.43258
80.43222
80.43131
Collection Date
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
3/23/2006
3/23/2006
3/23/2006
Irgarol
8.56
6.71
8.47
16.8
15.8
6.12
21.2
22.7
6.92
7.13
12.7
9.36
6.52
15.3
6.97
5.99
3.45
3.42
3.01
7.66
5.27
7.20
7.31
8.29
5.13
5.74
6.25
2.35
42.2
36.7
15.3
M1
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
0.499
0.611
N.D.
N.D.
0.552
1.374
0.615
0.729
0.813
N.D.
0.535
1.010
N.D.
N.D.
N.D.
N.D.
N.D.
Table 2.7 (continued)
Site
KLH-12
KLH-13
KLH-14
KLH-15
KLH-16
Description
Key Largo Harbor Site 12
Key Largo Harbor Site 13
Key Largo Harbor Site 14
Key Largo Harbor Site 15
Key Largo Harbor Site 16
Latitude
25.08919
25.08870
25.08908
25.08967
25.08967
Longitude
80.43114
80.42994
80.42977
80.42936
80.43008
Collection Date
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
Irgarol
12.9
9.74
11.1
8.24
7.64
M1
N.D.
N.D.
N.D.
N.D.
N.D.
Coconut Grove
CG01
CG02
CG03
CG04
CG05
CG06
CG01A
CG01B
CG01C
CG01D*
CG02A
CG02B
CG02C
CG03A
CG03B
CG03C
CG04A
CG05A
CG05B
CG05C
CG06A
CG06B
CG06C
CG06D*
Coconut Grove Site 1
Coconut Grove Site 2
Coconut Grove Site 3
Coconut Grove Site 4
Coconut Grove Site 5
Coconut Grove Site 6
Coconut Grove Site 1
Coconut Grove Site 1
Coconut Grove Site 1
Coconut Grove Site 1
Coconut Grove Site 2
Coconut Grove Site 2
Coconut Grove Site 2
Coconut Grove Site 3
Coconut Grove Site 3
Coconut Grove Site 3
Coconut Grove Site 4
Coconut Grove Site 5
Coconut Grove Site 5
Coconut Grove Site 5
Coconut Grove Site 6
Coconut Grove Site 6
Coconut Grove Site 6
Coconut Grove Site 6
25.72770
25.72652
25.72669
25.72560
25.72425
25.72915
25.72399
25.72399
25.72399
25.72399
25.72167
25.72167
25.72167
25.72615
25.72615
25.72615
25.72936
25.73155
25.73155
25.73155
25.73348
25.73348
25.73348
25.73348
80.23119
80.23048
80.22900
80.22770
80.22642
80.22753
80.23055
80.23055
80.23055
80.23055
80.22894
80.22894
80.22894
80.22285
80.22285
80.22285
80.22797
80.22860
80.22860
80.22860
80.22623
80.22623
80.22623
80.22623
10/1/2004
10/1/2004
10/1/2004
10/1/2004
10/1/2004
10/1/2004
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
6.25
7.97
5.44
6.81
6.43
6.56
28.6
13.0
26.8
225
23.0
26.9
33.2
32.0
44.2
26.5
106
70.4
61.2
91.7
10.0
31.1
18.3
283
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
64
Table 2.7 (continued)
CG07A
Coconut Grove Site 7
25.71895 80.23143
8/21/2006
CG07B
Coconut Grove Site 7
25.71895 80.23143
8/21/2006
CG07C
Coconut Grove Site 7
25.71895 80.23143
8/21/2006
*
CG07D
Coconut Grove Site 7
25.71895 80.23143
8/21/2006
CG08A
Coconut Grove Site 8
25.72294 80.23940
8/21/2006
CG08B
Coconut Grove Site 8
25.72294 80.23940
8/21/2006
CG08C
Coconut Grove Site 8
25.72294 80.23940
8/21/2006
*
Samples marked with an asterisk are samples of manatee grass. All others are turtle grass samples
65
100
65.0
32.1
175
27.2
27.0
22.7
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Table 2.8: Irgarol bioconcentration factors in Key Largo Harbor and Coconut Grove Marina
Collection
Site
Description
Latitude
Longitude
Date
Key Largo Harbor
NKLH-01
North Key Largo Harbor Site 1
25.08855
80.43059
4/29/2004
NKLH-02
North Key Largo Harbor Site 2
25.08864
80.43047
4/29/2004
NKLH-03
North Key Largo Harbor Site 3
a
a
4/29/2004
NKLH-04
North Key Largo Harbor Site 4
a
a
4/29/2004
SKLH-05
South Key Largo Harbor Site 1
25.08745
80.43060
4/29/2004
SKLH-06
South Key Largo Harbor Site 2
25.08689
80.43011
4/29/2004
SKLH-07
South Key Largo Harbor Site 3
25.08650
80.42970
4/29/2004
SKLH-08
South Key Largo Harbor Site 4
25.08712
80.42983
4/29/2004
NKLH-01
North Key Largo Harbor Site 1
25.08944
80.43021
6/14/2004
NKLH-02
North Key Largo Harbor Site 2
25.08810
80.43020
6/14/2004
NKLH-03
North Key Largo Harbor Site 3
25.08722
80.40080
6/14/2004
NKLH-04
North Key Largo Harbor Site 4
25.08732
80.42937
6/14/2004
NKLH-05
North Key Largo Harbor Site 5
25.08830
80.42940
6/14/2004
NKLH-06
North Key Largo Harbor Site 6
25.08957
80.42944
6/14/2004
NKLH-07
North Key Largo Harbor Site 7
25.08960
80.42834
6/14/2004
NKLH-08
North Key Largo Harbor Site 8
25.08761
80.42837
6/14/2004
SKLH-01
South Key Largo Harbor Site 1
25.08920
80.43129
6/14/2004
SKLH-02
South Key Largo Harbor Site 2
25.08819
80.43166
6/14/2004
SKLH-03
South Key Largo Harbor Site 3
25.08695
80.43150
6/14/2004
SKLH-04
South Key Largo Harbor Site 4
25.08690
80.43250
6/14/2004
SKLH-05
South Key Largo Harbor Site 5
25.08791
80.43265
6/14/2004
SKLH-06
South Key Largo Harbor Site 6
25.08875
80.43255
6/14/2004
SKLH-07
South Key Largo Harbor Site 7
25.08827
80.43306
6/14/2004
SKLH-08
South Key Largo Harbor Site 8
25.08785
80.73334
6/14/2004
NKLH-01
North Key Largo Harbor Site 1
25.08904
80.43024
8/27/2004
NKLH-02
North Key Largo Harbor Site 2
25.08895
80.42995
8/27/2004
NKLH-03
North Key Largo Harbor Site 3
25.08907
80.42944
8/27/2004
NKLH-04
North Key Largo Harbor Site 4
25.08957
80.42947
8/27/2004
NKLH-05
North Key Largo Harbor Site 5
25.08970
80.42957
8/27/2004
66
BCFb
2136
2774
1990
1904
2515
549
1059
1223
2098
186
353
153
405
2065
627
178
451
254
314
813
948
350
963
575
73
114
383
346
264
Table 2.8 (continued)
Site
NKLH-06
NKLH-07
NKLH-08
NKLH-09
NKLH-10
SKLH-01
SKLH-02
SKLH-03
SKLH-04
SKLH-05
SKLH-06
SKLH-07
SKLH-08
SKLH-09
SKLH-10
KLH-09
KLH-10
KLH-11
KLH-12
KLH-13
KLH-14
KLH-15
KLH-16
Coconut Grove
BB-01
BB-02
BB-03
BB-04
BB-05
BB-06
CG01T
Description
North Key Largo Harbor Site 6
North Key Largo Harbor Site 7
North Key Largo Harbor Site 8
North Key Largo Harbor Site 9
North Key Largo Harbor Site 10
South Key Largo Harbor Site 1
South Key Largo Harbor Site 2
South Key Largo Harbor Site 3
South Key Largo Harbor Site 4
South Key Largo Harbor Site 5
South Key Largo Harbor Site 6
South Key Largo Harbor Site 7
South Key Largo Harbor Site 8
South Key Largo Harbor Site 9
South Key Largo Harbor Site 10
Key Largo Harbor
Key Largo Harbor
Key Largo Harbor
Key Largo Harbor
Key Largo Harbor
Key Largo Harbor
Key Largo Harbor
Key Largo Harbor
Latitude
25.08965
25.08943
25.09008
25.09299
25.09324
25.08804
25.08778
25.08748
25.08733
25.08796
25.08867
25.08894
25.08781
25.08407
25.08207
25.08792
25.08883
25.08831
25.08919
25.08870
25.08908
25.08967
25.08967
Longitude
80.43005
80.43046
80.42976
80.42873
80.42649
80.43143
80.43138
80.43198
80.43219
80.43262
80.43238
80.43176
80.43324
80.43505
80.43702
80.43258
80.43222
80.43131
80.43114
80.42994
80.42977
80.42936
80.43008
Collection Date
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
BCFb
Coconut Grove Site 1
Coconut Grove Site 2
Coconut Grove Site 3
Coconut Grove Site 4
Coconut Grove Site 5
Coconut Grove Site 6
Coconut Grove Site 1 (Thallasia)
25.72770
25.72652
25.72669
25.72560
25.72425
25.72915
25.72399
80.23119
80.23048
80.22900
80.22770
80.22642
80.22753
80.23055
10/1/2004
10/1/2004
10/1/2004
10/1/2004
10/1/2004
10/1/2004
8/21/2006
130
285
155
184
353
183
1481
67
269
60
234
145
238
62
144
104
153
206
353
165
188
787
468
1141
836
610
273
1172
1328
665
346
Table 2.8 (continued)
Site
Description
Latitude
CG01U
Coconut Grove Site 1 (Syringodium)
25.82399
CG02
Coconut Grove Site 2
25.72167
CG03
Coconut Grove Site 3
25.72615
CG04
Coconut Grove Site 4
25.72936
CG05
Coconut Grove Site 5
25.73155
CG06T
Coconut Grove Site 6 (Thallasia)
25.73348
CG06U
Coconut Grove Site 6 (Syringodium)
25.73348
CG07T
Coconut Grove Site 7 (Thallasia)
25.71895
CG07U
Coconut Grove Site 7 (Syringodium)
25.71895
CG08
Coconut Grove Site 8
25.72294
a. missing gps coordinates
b. sites with triplicate seagrass samples were averaged to calculate BCF values
68
Longitude
80.23055
80.22894
80.22285
80.22797
80.22860
80.22623
80.22623
80.23143
80.23143
80.23940
Collection Date
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
8/21/2006
BCFb
14610
2037
2113
5096
2924
835
11941
11859
31588
2805
2.3.3. Sediment Samples
A total of 73 sediment samples were analyzed for Irgarol and M1 during 2004.
Out of these 73 samples, 92% (67 samples) contained Irgarol and 5% (4 samples)
contained M1. While Irgarol was present in most of the samples, there was only one
sample that contained Irgarol greater than 5 ng/g (6.89 ng/g in KLHS03, June 2003).
Irgarol was present in the remaining 66 samples at levels ranging from 0.08 ng/g to 1.81
ng/g. These results seem to be in agreement with the literature stating that Irgarol does
not partition well into sediment. A complete list of Irgarol and M1 concentrations in the
sediment samples is given in table 2.9.
Table 2.9: Irgarol and M1 concentrations in sediments
Site
Collection Date
Latitude
Longitude
KLHS01
6/19/2003
a
a
KLHS02
6/19/2003
a
a
KLHS01
8/21/2003
a
a
KLHS03
8/21/2003
a
a
KLHS04
8/21/2003
a
a
KLHS05
8/21/2003
a
a
KLH01A
4/29/2004
25.08855
80.43059
KLH01C
4/29/2004
25.08855
80.43059
KLH02A
4/29/2004
25.08864
80.43047
KLH02B
4/29/2004
25.08864
80.43047
KLH02C
4/29/2004
25.08864
80.43047
KLH03A
4/29/2004
a
a
KLH03B
4/29/2004
a
a
KLH03C
4/29/2004
a
a
KLH04A
4/29/2004
a
a
KLH04B
4/29/2004
a
a
KLH04C
4/29/2004
a
a
KLB05A
4/29/2004
25.08745
80.43060
KLH05B
4/29/2004
25.08745
80.43060
KLH05C
4/29/2004
25.08745
80.43060
KLH06A
4/29/2004
25.08689
80.43011
KLH06B
4/29/2004
25.08689
80.43011
KLH06C
4/29/2004
25.08689
80.43011
NKLH-01
6/14/2004
25.08944
80.43021
NKLH-02
6/15/2004
25.08810
80.43020
NKLH-03
6/16/2004
25.08722
80.40080
NKLH-04
6/17/2004
25.08732
80.42937
NKLH-05
6/18/2004
25.08830
80.42940
NKLH-06
6/19/2004
25.08957
80.42944
69
Irgarol (ng/L)
0.58
1.61
1.81
6.89
0.57
1.50
0.41
0.35
0.42
0.50
1.00
0.33
0.38
0.39
0.42
0.38
0.21
0.08
0.13
0.11
N.D.
N.D.
N.D.
0.41
1.15
0.24
0.22
0.18
N.D.
M1(ng/L)
N.D.
N.D.
0.35
1.01
N.D.
1.23
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Table 2.9 (continued)
Site
Collection Date
NKLH-07
6/20/2004
KLH-08
6/21/2004
SKLH-01
6/22/2004
SKLH-02
6/23/2004
SKLH-03
6/24/2004
SKLH-04
6/25/2004
SKLH-05
6/26/2004
NKLH-01
8/27/2004
NKLH-02
8/27/2004
NKLH-03
8/27/2004
NKLH-04
8/27/2004
NKLH-05
8/27/2004
NKLH-06
8/27/2004
NKLH-07
8/27/2004
NKLH-08
8/27/2004
NKLH-09
8/27/2004
NKLH-10
8/27/2004
SKLH-01
8/27/2004
SKLH-02
8/27/2004
SKLH-03
8/27/2004
SKLH-04
8/27/2004
SKLH-05
6/26/2004
NKLH-01
8/27/2004
NKLH-02
8/27/2004
NKLH-03
8/27/2004
NKLH-04
8/27/2004
NKLH-05
8/27/2004
NKLH-06
8/27/2004
NKLH-07
8/27/2004
NKLH-08
8/27/2004
NKLH-09
8/27/2004
NKLH-10
8/27/2004
SKLH-01
8/27/2004
SKLH-02
8/27/2004
SKLH-03
8/27/2004
SKLH-04
8/27/2004
SKLH-05
8/27/2004
SKLH-06
8/27/2004
SKLH-07
8/27/2004
SKLH-08
8/27/2004
SKLH-09
8/27/2004
SKLH-10
8/27/2004
BB01A
10/1/2004
BB01B
10/1/2004
BB01C
10/1/2004
BB02A
10/1/2004
BB02B
10/1/2004
BB02C
10/1/2004
BB03A
10/1/2004
Latitude
25.08960
25.08761
25.08920
25.08819
25.08695
25.08690
25.08791
25.08904
25.08895
25.08907
25.08957
25.08970
25.08965
25.08943
25.09008
25.09299
25.09324
25.08804
25.08778
25.08748
25.08733
25.08791
25.08904
25.08895
25.08907
25.08957
25.08970
25.08965
25.08943
25.09008
25.09299
25.09324
25.08804
25.08778
25.08748
25.08733
25.08796
25.08867
25.08894
25.08781
25.08407
25.08207
25.72770
25.72770
25.72770
25.72652
25.72652
25.72652
25.72669
70
Longitude
80.42834
80.42837
80.43129
80.43166
80.43150
80.43250
80.43265
80.43024
80.42995
80.42944
80.42947
80.42957
80.43005
80.43046
80.42976
80.42873
80.42649
80.43143
80.43138
80.43198
80.43219
80.43265
80.43024
80.42995
80.42944
80.42947
80.42957
80.43005
80.43046
80.42976
80.42873
80.42649
80.43143
80.43138
80.43198
80.43219
80.43262
80.43238
80.43176
80.43324
80.43505
80.43702
80.23119
80.23119
80.23119
80.23048
80.23048
80.23048
80.22900
Irgarol (ng/L)
0.30
N.D.
0.53
0.47
0.44
0.23
0.81
0.35
0.37
0.40
0.32
0.21
0.09
0.43
0.42
0.26
0.11
0.49
0.30
0.31
0.33
0.81
0.35
0.37
0.40
0.32
0.21
0.09
0.43
0.42
0.26
0.11
0.49
0.30
0.31
0.33
0.33
0.51
0.46
0.62
0.26
0.14
0.34
0.14
0.20
0.28
0.30
0.16
0.22
M1(ng/L)
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
3.34
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
3.34
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Table 2.9 (continued)
Site
Collection Date
BB03B
10/1/2004
BB03C
10/1/2004
BB04A
10/1/2004
BB04B
10/1/2004
BB04C
10/1/2004
BB05A
10/1/2004
BB05B
10/1/2004
BB05C
10/1/2004
BB06A
10/1/2004
BB06B
10/1/2004
a. Missing GPS coordinates
Latitude
25.72669
25.72669
25.72560
25.72560
25.72560
25.72425
25.72425
25.72425
25.72915
25.72915
Longitude
80.22900
80.22900
80.22770
80.22770
80.22770
80.22642
80.22642
80.22642
80.22753
80.22753
Irgarol (ng/L)
0.97
0.18
N.D.
0.20
0.19
0.26
0.27
0.22
0.38
0.23
M1(ng/L)
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
2.4. Conclusions
The above results seem to concur with the majority of what is found in the
literature. Irgarol and M1 were found to be common contaminants in all of the marinas
sampled. Concentrations of Irgarol and M1 in surface waters ranged from N.D. to 1239
ng/L and from N.D. to 429 ng/L, respectively. Although it is a ubiquitous contaminant in
the marinas, the real question posed before this research was whether or not marine life is
at risk due to this contamination. According to the literature, it seems that coral systems
are the most affected by Irgarol, with concentrations as low as 63 ng/L affecting the
carbon uptake of inshore corals (Owen et al., 2002). However, based on available data, it
does not seem possible for Irgarol from nearby marinas to affect corals, due to the fact
that concentrations seem to decrease rapidly as you increase the distance from a point
source. Moreover, no Irgarol was found in any of the coral reef systems sampled, with
the exception of an isolated event at Looe Key, where Irgarol was found in 5 samples at
levels below 2 ng/L.
Concentrations of Irgarol in Florida coastal waters seem to be small in
comparison to concentrations reported elsewhere in the world. This can be illustrated by
calculating the 90th percentile concentration using all 368 of the samples collected from
71
all sampling locations in Florida. A plot of the data on a percentile curve is shown in
Figure 2.27. The resulting 90th percentile concentration is 246 ng/L, which is well above
the effect levels determined for corals and other small marine biota. Out of the 368
samples, 101 were above the level shown to affect the carbon uptake of inshore corals (63
ng/L), 79 were above the level shown to affect the net photosynthesis of intact corals
(100 ng/L), and 69 were above the level shown to affect the diatom Naviculla pelliculosa
(136 ng/L). These results can be misleading, however, because out of the 101 samples
above these limits, 94% (95 samples) were collected from Key Largo Harbor. While the
90th percentile concentration is higher than the concentrations shown to affect the most
sensitive of species, it is close to the 10th percentile toxicity level, 251 ng/L (Hall et al.,
1999). This is the concentration that would kill 10% of the species exposed to Irgarol,
while the remaining 90% would be protected.
If the 90th percentile concentration is recalculated without including the samples
taken from inside Key Largo Harbor, the number decreases drastically to 57.8 ng/L
(Figure 2.28).
Looking at the remaining 279 samples, only 18 samples (one from
Charlotte Harbor, 8 from Tampa bay, 3 from the Miami River, 2 from Biscayne Bay, 2
from Port Everglades, and 2 from outside Key Largo Harbor) are above the 63 ng/L level,
5 samples (three from Tampa Bay, 1 from the Miami River, and 1 from outside Key
Largo Harbor) are above the 100 ng/L level, and only one sample (from Tampa Bay) is
above the 136 ng/L level. These results seem to indicate that Key Largo Harbor is an
area of concern, but that the rest of the marinas sampled are not as heavily impacted by
Irgarol. A better description of the distribution would be that there seems to be a good
chance that small concentrations of Irgarol can be found at any given marina throughout
72
a b c
Percentile
99
90
246 ng/L
70
50
30
0.1
1
10
100
1000
10000
Concentration (log10)
Figure 2.27: Percentile plot of Irgarol concentrations in surface water showing the 90th percentile
Irgarol concentration and 3 concentrations that affect small marine organisms (a. 63ng/L; b. 100
ng/L; c. 136 ng/L)
a
b c
Percentile
57.5 ng/L
90
70
50
0.1
1
10
100
1000
Concentration (log10)
Figure 2.28: Percentile plot of Irgarol concentrations in surface waters showing the 90th percentile
Irgarol concentration calculated without Key Largo Harbor and 3 concentrations that affect small
marine organisms. (a. 63 ng/L; b. 100 ng/L; c. 136 ng/L)
73
Florida. While most of the marinas sampled will have low concentrations, there are some
“hot spots” located around Florida that contain elevated amounts of Irgarol.
Other highly polluted areas, such as Port Annapolis (Hall and Gardinali 2004;
Hall et al. 2005), seem to conform to the same geographic restrictions as Key Largo
Harbor, namely small, restricted areas with very low water circulation. Areas that are
mainly open water with high circulation show very quick dissipation of Irgarol after
transport to open waters, as illustrated by the fact that Irgarol concentrations were highly
affected by tide changes.
With regards to the long-term persistence of Irgarol, overall concentrations in
large marina surface waters seem to be increasing. This can be demonstrated by plotting
histograms of selected sites over time. A plot of some stations from Biscayne Bay is
shown in Figure 2.29. Concentrations in Coconut Grove Marina and Monty’s Marina
have increased since the last sampling trips in 1999-2001. These results warrant further
monitoring of Irgarol in the future.
One area that does show grounds for concern is the uptake of Irgarol into
submerged vegetation. While the data is limited, Irgarol does seem to bioaccumulate into
seagrass, though the extent of the accumulation seems to be varied. Samples collected
during April of 2004 showed higher accumulation (750 – 2500), than samples collected
throughout the rest of the year (< 500). The higher BCF values for the April samples
seem to be due to lower Irgarol concentrations in the surface water during this period.
Concentrations of Irgarol in seagrass tissue through out the entire year seem to be
consistent, with values ranging from about 2 to around 20 ng/g. BCFs for the seagrass
samples collected in 2006 seem to be higher than those collected in 2004, but there is not
74
100
Bayside
Brickell Point
Coconut Grove Marina
Monty's Marina
Concentration (ng/L)
80
60
40
20
0
1
1
6
0
0
0
4
00
00
00
00
00
00
00
2
2
2
2
2
2
2
t,
r,
r
y,
y,
y,
h,
h,
ul
us
ar
ar
rc
rc
be
be
J
g
u
u
a
a
r
r
M
M
em
em
Au
eb
eb
pt
ec
F
F
e
D
S
9
99
1
,
ch
ar
M
9
99
1
,
Collection Date
Figure 2.29: Concentrations of Irgarol in selected stations in Biscayne Bay, 1999-2006
enough data to speculate whether or not there is an trend of increasing concentrations.
There is also a contrast in the BCF values between the two species of seagrass. Most
BCF values for Thallasia were less than 2500, with only 5 samples, all from Coconut
Grove in 2006, containing BCF values above this mark. While there were only 3 samples
of manatee grass, the BCFs for these samples ranged from 11941 to 31588. The numbers
obtained for manatee grass are comparable with those calculated by Scarlett et al., in
1999. Box plots comparing the BCF values of the two specie of seagrass are shown in
Figure 2.30. With the samples collected throughout the 2004 and 2006 campaign, a 90th
75
percentile value for BCFs with respect to seagrass was calculated to be 2,780 (Figure
2.31). Based on this number, it seems that while Irgarol does accumulate in seagrass,
most of the samples do not reach the level that the EPA considers “highly accumulative.”
More research needs to be done in regards to long-term monitoring and acute and chronic
effects, if any, Irgarol has on submerged vegetation such as promotion or prevention of
epiphytic growth.
Concentrations of Irgarol and M1 in sediment samples never rose above 2 ng/g,
with the exception of one sample that contained 6.89 ng/g.
These results are in
agreement with the current literature, and it seems that Irgarol does not partition well into
the sediment phase and other modes of removal from the water column will take
precedence. The Irgarol introduced in the water will either accumulate into submerged
vegetation or photodegrade into its metabolites.
35000
30000
BCF Value
25000
20000
15000
10000
5000
0
Turtle Grass
Manatee Grass
Figure 2.30: Box plots comparing the concentrations of the two different specie of seagrass
76
90th Percentile
2780
Percentile
90
70
50
30
10
10
100
1000
10000
BCF (Log10)
Figure 2.31: 90th Percentile graph for seagrass BCF values
77
100000
III. GC/MS Analysis for Nitrogen Based Pesticides and Herbicides
3.1. Triazine Herbicides
As previously stated, Irgarol 1051 belongs to a class of compounds called triazine
herbicides. These compounds all have a similar core structure, namely a triazine ring.
The compounds differ from each other depending on the substituents bonded to the ring.
Most of these compounds act by disrupting electron transport during the photosystem II
phase of photosynthesis. Also, many of these compounds are used in agriculture to
protect crops from various weed problems, but some, such as cyromazine, have more
specific uses. In the case of cyromazine, it is used to prevent insect growth on crops.
While certainly not a complete list of all triazine herbicides available on the market,
Table 3.1 gives a list of some of the most commonly used compounds available. One of
these compounds, atrazine, was one of the most widely used herbicides in the agricultural
industry throughout the 1990’s. As shown in Figure 2.7, atrazine is already included in
our normal Irgarol protocol, though environmental concentrations are not reported in this
work. Other commonly used pesticides and herbicides also contain nitrogen based rings,
though these are not classified as triazine rings. A few examples of these compounds are
alachlor, butachlor, metolachlor, bromacil, metribuzin, and molinate (Figure 3.1).
Since the analytical method used for Irgarol and its metabolite seems to be a
quick, robust method for the analysis of these two triazine herbicides, it was hypothesized
that this method could be expanded to include a larger selection of nitrogen based
herbicides, namely those listed in Table 3.1 and shown in Figure 3.1.
78
Table 3.1: Structures of some commonly used triazine herbicides
R1
N
N
H
H
N
N
N
R2
Herbicide
Ametryne
Atraton
Atrazine
Prometon
Prometryne
Propazine
Secbumeton
Simazine
Simetryne
Terbuthylazine
Terbutryne
R3
R1
-SCH3
-OCH3
-Cl
-OCH3
-SCH3
-Cl
-OCH3
-Cl
-SCH3
-Cl
-SCH3
R2
-i-C3H7
-i-C3H7
-C2H5
-i-C3H7
-i-C3H7
-i-C3H7
-CH(CH3)CH(CH3)CH3
-C2H5
-C2H5
-C2H5
-C2H5
R3
-C2H5
-C2H5
-i-C3H7
-i-C3H7
-i-C3H7
-i-C3H7
-C2H5
-C2H5
-C2H5
-t-C4H9
-t-C4H9
O
O
O
Cl
Cl
N
N
O
O
N
S
Alachlor
Metolachlor
Molinate
O
O
Cl
N
O
N
Br
N
N
N
O
S
NH2
H
Butachlor
O
Bromacil
Metribuzin
Figure 3.1. Structures of other nitrogen based pesticides
79
3.2. Expansion of Method to Include Additional Analytes
3.2.1. Method Conditions
With the addition of 16 compounds to the GC program, resolution becomes an
issue. The ramp of 15 C/min would likely cause many overlapping peaks. Since most
of these compounds have similar fragment ions, there will be mass overlap and in turn,
data that is inadequate for quantitation. For this reason, the temperature program of the
oven was changed to allow for better resolution of analyte peaks. The new temperature
program started at an initial temperature of 100 C and was held for 1 minute. The oven
was then raised to a temperature of 250 C at a rate of 6 C/min. The column was then
cleaned by raising it to a temperature of 300 C at a rate of 20 C/min and holding for 2
minutes. A list of retention times and SIM ions is given in Table 3.2. A chromatogram
of the compound mixture is shown in Figure 3.2.
80
Table 3.2: Retention times and SIM ions for nitrogen based pesticides
Compound
TCMX
Atrazine-d5
Molinate
Atraton
Simazine
Prometon
Atrazine
Propazine
Terbuthylazine
Secbumeton
M1
Metribuzin
Alachlor
Simetryne
Ametryne
Prometryne
Terbutryne
Bromacil
Metolachlor
M3
Sea-Nine 211
Irgarol 1051
Butachlor
Retention Time
15.04
17.34
13.57
17.03
17.24
17.24
17.41
17.52
17.88
18.57
19.12
19.73
19.97
20.10
20.23
20.34
20.76
20.85
20.24
20.24
22.62
22.85
23.810
Quantitation Ion
242
205
126
196
201
210
200
214
214
196
198
198
188
213
227
241
226
205
162
269
246
TIC
160
Confirmation Ion 1
244
220
187
211
186
225
215
229
229
225
213
199
237
170
212
184
185
188
238
254
182
182
188
81
Confirmation Ion 2
246
222
Confirmation Ion 3
169
173
168
172
173
210
157
169
160
198
170
226
241
190
198
169
238
238
213
253
RT: 13.35 - 23.97
100
q
NL:
1.13E7
TIC F: MS
8587
95
90
85
e
80
o
d
75
n
70
g
Relative Abundance
65
f
60
55
c
50
45
a
m
l
h
s
k
t
40
i
35
30
j
25
r
20
p
b
15
10
5
0
14
15
16
a. Molinate
e. Atrazine & Atrazine-d5
i. M1
m. Ametryne
q. Metolachlor & M3
17
18
19
Time (min)
b. TCMX
f. Propazine
j. Metribuzin
n. Prometryne
r. Sea-Nine 211
c.
g.
k.
o.
s.
20
Atraton
Terbuthylazine
Alachlor
Terbutryne
Irgarol
21
22
23
d. Simazine & Prometon
h. Secbumeton
l. Simetryne
p. Bromacil
t. Butaclor
Figure 3.2: Chromatogram of a mixture of nitrogen based pesticides, Irgarol, and its metabolites
82
3.2.2. Figures of Merit
In order to assess the quality of the method, 7 replicate extractions were
performed applying the same techniques used for the analysis of surface waters for
Irgarol. Using these 7 replicates, average recoveries, standard deviations, and method
detection limits (MDLs) were calculated. The results are shown in Table 3.3.
Table 3.3: Recoveries and MDLs for Nitrogen Pesticides
Pesticides
Average % RSD MDL (ng/L)
Molinate
104
0.95
0.57
Atraton
70.4
0.87
0.92
Simazine
59.7
1.10
0.99
Prometon
100
0.51
0.77
Atrazine
80.4
0.78
0.94
Propazine
94.9
0.62
0.88
Terbuthylazine
94.8
0.61
0.87
Secbumeton
90.7
0.59
0.82
Metribuzin
72.6
0.79
0.86
Alachlor
78.6
0.72
0.84
Simetryne
64.8
0.85
0.82
Ametryne
73.2
0.37
0.41
Prometryne
78.7
0.81
0.96
Terbutryne
81.4
0.44
0.54
Bromacil
n/a
n/a
n/a
Metolachlor
81.7
0.36
0.50
Butachlor
82.9
0.65
0.87
Recoveries for all compounds, except Bromacil, were acceptable.
Standard
deviation for all compounds was very low, and thus MDLs were all below 1 ng/L for all
compounds. Bromacil turned out to be a difficult compound to analyze by GC/MS. As
shown in Figure 3.2, Bromacil was not as sensitive as the other compounds in the
mixture, making integration of concentrations even as high as 50 ppb difficult. Bromacil
was also very susceptible to peak tailing, making quantitation even more difficult.
83
3.3. Environmental Application
Using the above method, samples were collected from a canal in southeast Miami
Dade County, near Biscayne National Park and Bayfront Park and Marina. The canal has
the designation L31A, given by the South Florida Water Management District
(SFWMD).
The canal in question is routinely sprayed by the SFWMD to control
vegetation growth along the sides of the canal. It is also a run-off area for agricultural
fields from the Homestead area. An overhead view of the canal is shown in Figure 3.3.
Samples were collected at the north, middle and south of the canal. On the southern side,
samples were collected from both sides of the bridge. Samples were collected once every
two weeks, over a period of 18 weeks. Plots of concentration of the detected compounds
versus time are shown in Figure 3.4.
Site 1
L31A
Canal
Site 2
Site 3
Site 4
Figure 3.3: Sample location for nitrogen pesticide monitoring
84
40
10
10
0
0
Figure 3.4: Concentrations of nitrogen based pesticides in the L-31 canal
Collection Week
Collection Week
85
Week 9: 2/6/2006
Prometon
Atrazine
Metolachlor
Week 9: 2/6/2006
Week 8: 1/23/2006
Week 7: 1/4/2006
Week 6: 12/22/2005
Week 5: 12/7/2005
40
Week 8: 1/23/2006
50
Week 7: 1/4/2006
L31 Site 3
Week 6: 12/22/2005
Collection Week
Week 5: 12/7/2005
0
Week 4: 11/23/2005
0
Week 4: 11/23/2005
10
Week 3: 11/16/2005
10
Week 3: 11/16/2005
Prometon
Atrazine
Metolachlor
Week 2: 11/2/2005
50
Week 2: 11/2/2005
20
Week 1: 10/21/2005
20
Concentration (ng/L)
30
Week 1: 10/21/2005
30
Concentration (ng/L)
40
Week 9: 2/6/2006
Week 8: 1/23/2006
Week 7: 1/4/2006
Week 6: 12/22/2005
Week 5: 12/7/2005
Week 4: 11/23/2005
Week 3: 11/16/2005
Week 2: 11/2/2005
Week 1: 10/21/2005
Concentration (ng/L)
40
Week 9: 2/6/2006
Week 8: 1/23/2006
Week 7: 1/4/2006
Week 6: 12/22/2005
Week 5: 12/7/2005
Week 4: 11/23/2005
Week 3: 11/16/2005
Week 2: 11/2/2005
Week 1: 10/21/2005
Concentration (ng/L)
L31 Site 1
L31 Site 2
50
Prometon
Atrazine
Metolachlor
30
20
Collection Week
L31 Site 4
50
Prometon
Atrazine
Metolachlor
30
20
Over the nine sampling campaigns, only Prometon, Atrazine, and Metolachlor
were frequently detected at relatively low levels. Concentrations of Prometon were
relatively constant throughout the sampling period with sites 1 and 2 showing
concentrations around 4 ng/L. Sites 3 and 4 showed concentrations of Prometon close to
1 ng/L. Concentrations of Atrazine at site 1 and 2 decreased from around 40 ng/L to
around 19 ng/L over the study period. At sites 3 and 4, concentrations were steady
around 20 ng/L. At sites 1 and 2, Metolachlor concentrations started around 15 ng/L and
decreased to close to zero by week 8. During the last sampling episode, concentrations
increased to around 27 ng/L. This could be due to heavier usage upstream during that
period of time. Concentrations of Metolachlor in sites 3 and 4 were low and close to the
detection limit throughout the study period.
3.4. Conclusions
Based on the data obtained from this small experiment, it seems that these
analyses are well suited for a wide variety of nitrogen based pesticides and herbicides.
This method only was expanded to accommodate 17 compounds, but in theory it could be
expanded even further to quantitate more compounds. To accomplish this, the GC
temperature program would have to be slowed down even further, but that is easily
accomplished.
86
IV. Environmental Detection of Recently Discovered Irgarol Metabolites
4.1. Objectives
Recently, the environmental fate of Irgarol has been brought back into the
forefront of the antifouling research community due to the reported discovery of two new
metabolites in the environment.
It is important to determine whether or not these
compounds are actually stable in the environment and able to be formed in the
environment, especially since Lam et al. reported to have detected these new compounds
in the environment at levels reaching 200 ppb. Also, since one of these compounds has
been shown to be unstable in a GC injector port, a new method needs to be developed
that can quantitate Irgarol and all its known metabolites in a single analysis.
4.2. Experimental
4.2.1. Materials
In addition to the compounds mentioned in Chapter 2 for routine Irgarol analysis,
authentic neat standards of M2 and M3 were obtained from Ciba Specialty Chemicals. It
should be noted that these standards are the only standards of their type available in the
world. All other materials used for this method development were mentioned in Section
2.2.5. Structures of M2 and M3 are shown in Figure 4.1.
S
N
N
H
S
N
N
O
N
H
N
H
N
H
N
N
Figure 4.1: Structures of M2 (left) and M3 (right)
87
N
H
4.2.2. Sample Extraction
Sample extraction techniques can be found in section 2.2.2. The only addition to
this method was that final sample extracts were evaporated to dryness and reconstituted
to 0.5 mL of methanol for LC/MS analysis.
4.2.3. Data Acquisition
Samples were analyzed using a Thermo Finnigan LCQ Advantage Max LC/MS
system. This system consists of a Surveyor Autosampler, Surveyor LC Pump, and the
LCQ Advantage MS with the Ion Max ion source housing. The MS parameters for APCI
positive mode are listed in Table 4.1.
Table 4.1: MS parameters for APCI+ mode
Vaporizer Temperature
Sheath Gas Flow
Auxiliary Gas Flow
Discharge Voltage
Capillary Temperature
Capillary Voltage
Tube Lens Offset Voltage
Number of Microscans
Maximum ion injection time
300 C
20 (arbitrary units)
13 (arbitrary units)
4.0 kV
245 C
5V
55 V
1
200 ms
Two types of analysis were performed to determine the best analytical procedure
to deal with these compounds: selected ion monitoring (SIM) and selected reaction
monitoring (SRM). In theory, SRM analysis should allow for the limit of detection to be
decreased, as well as obtaining “cleaner” chromatograms, due to the fact that all noise
should be discarded before taking an analytical scan.
The analytes of interest were detected by monitoring for the protonated molecular
ion ([M+H]+) of each compound. A list of analytes with their corresponding molecular
and SRM ion is given in Table 4.2.
88
Table 4.2: Molecular Ions and SRM ions for analytes
Analyte
Atrazine-d5 (Internal Standard)
Atrazine
Irgarol
M1
M2
M3
Molecular Ion [M+H]+
221
216
254
214
270
270
SRM Ion
179
174
198
158
214
214
The LC pump was operated using a gradient elution, starting with 40:50:10
methanol:water:0.04% ammonium acetate and ramping to 90:10 methanol:0.04%
ammonium acetate in 15 minutes. The pump was then set back to initial conditions and
allowed to equilibrate for 3 minutes before the next injection. The autosampler was
operated using the full loop mode, with an injection volume of 25L. This was done to
ensure that there was no variation in the amount of sample injected between samples.
The chromatographic separation was carried out using a Phenomenex Luna C-18 column
with dimensions of 150 x 4.6 mm (length by diameter) and a particle size of 5 microns.
4.3. LC/MS Method Development
4.3.1. MS Optimization
The MS parameters were optimized using a 1 ppm solution of a mixture of all
compounds in methanol. The LCQ has the ability to continuously bleed a solution into
the LC flow and into the MS detector using a syringe pump. Using this constant flow,
the MS software can automatically optimize all parameters important for analysis.
Unfortunately, one parameter could not be set at the optimized value. Adding auxiliary
gas seems to decrease the signal of all compounds in the detector, but without the
presence of the gas, the high flow rate coming into the ion source causes the corona pin to
short out. This causes a complete signal loss in the MS. None of the other parameters
89
gave any problems, and were all set at their optimum values. Global parameters such as
all temperatures and gas flow rates were kept constant during the analysis, but lens
voltages can be changed at specific retention times if there is a change in signal between
compounds. In this method, all of the analytes belong to the same family of compounds,
and only differ slightly in the arrangement of the substituents on the triazine ring.
Therefore, it was not necessary to change octapole and lens voltages throughout the run.
4.3.2. Characterization of Authentic Standards
All previous work done with M2 and M3 by Lam et al. was performed using
compounds that were separated and purified from the HgCl2-catalyzed hydrolysis
reaction mixture (in the case of M2), or separated and purified from commercially
available Irgarol. For this study, authentic standards of Irgarol, M1, M2, and M3 were
obtained directly from the manufacturer.
Our goal was to determine if the compounds that Lam et al. purified in their
laboratory had the same MS spectra as the authentic standards. The first point that was
addressed was the need for LC-MS analysis to quantify M2. As previously stated, Lam
et al. (2004) reported that M2 was incompatible with GC systems. M2 was shown to
decompose to M1 due to the high temperatures of the GC injector port. They proposed
that this is a possible reason why this compound was never detected in any previous
degradation or monitoring studies. To test this theory, two separate injections were made
into our GC/MS: one solution containing only M1 (Figure 4.2a) and another solution
containing only M2 (Figure 4.2b). The injection of M1 was as expected; a single peak at
around 10.21 minutes with major fragment peaks at 157, 198, and 213 m/z. The injection
of M2 showed two peaks, one corresponding to M1 and the second showing many
90
fragment ions, including some characteristic of triazine compounds.
Indeed, M2
degrades into M1, but in contrast to the conclusion by Lam that the compound
completely degrades, our experiment showed only partial degradation. The degradation
of M2 may not be entirely due to the possible instability of the compound itself, but also
the setup of the GC instrument. A new type of liner was installed in the GC injector port,
one that was less activated and is designed to help prevent breakdown of molecules
before they reach the GC column. M2 was re-run using this liner and the compound
showed much lower degradation than the previous run (Figure 4.3). There is still some
slight degradation of M2 into M1, but this shows that there is still a possibility of
analyzing M2 via GC/MS if the right instrumental configuration is obtained. Since M1 is
an important analyte in Irgarol assessments, any method that introduces false positives
due to interferences by other compounds should not be used. For this reason, it was
decided to develop a method using LC-MS/MS, which uses a softer ionization method in
which the dynamics of the ion source prevent breakdown of the analytes during analysis.
Second, MS-MS spectra of all four authentic standards were obtained and
compared to those reported by Lam et al (2004 and 2005). These spectra are shown in
Figures 4.4 through 4.7. The MS/MS spectrum of Irgarol shows a major fragment peak
at 198 m/z (loss of t-butyl group). This is consistent with typical MS/MS scans of Irgarol
found in literature (Cai et al., 2006). Typical MS/MS scans of M1 also agree with our
results, showing a loss of the t-butyl group to form a major fragment peak of 158 m/z.
An M2 spectrum obtained in our lab show fragments of 226, 214, 170 and 158. M3
showed two main fragments at 214 and 158. Both of these results agreed with those
obtained by Lam and coworkers (Lam et al., 2004; Lam et al., 2005).
91
F:\M2 runs 11-3-06\0382
11/3/2006 11:07:26 AM
M1 full scan
RT: 8.00 - 13.00
RT: 10.21
100
NL:
8.46E8
TIC F:
MS ICIS
0382
S
90
Relative Abundance
80
A
70
60
N
N
50
40
30
N
H
20
10
N
NH2
0
8.0
8.2
8.4
8.6
8.8
9.0
9.2
9.4
9.6
9.8
10.0
10.2
10.4
10.6
Time (min)
10.8
11.0
11.2
11.4
11.6
11.8
12.0
12.2
12.4
12.6
12.8
13.0
0382 #1054 RT: 10.20 AV: 1 SB: 224 10.54-11.09 , 9.48-10.02 NL: 1.14E8
T: + c Full ms [ 50.00-325.00]
198
100
90
Relative Abundance
80
157
70
60
213
50
40
30
83
20
10
110
68
56
57
74
73 75 82
58 67
0
50
60
70
85
80
99
98
91
90
116
117 125
108
100
110
120
156 158
159
140 142
130
199
150
140
150
F:\M2 runs 11-3-06\0397
160
196
171 180 182 184
170
180
190
m/z
201
200
11/3/2006 4:29:50 PM
211
214
216
210
230 233
240
230
240
220
247
256 259
250
260
271 275 284 287 292 299
270
280
290
307 313
300
310
322
320
New M2 10 ppm hexane old liner full scan
RT: 8.00 - 13.00
8.88
100
Relative Abundance
80
NL:
5.90E7
TIC F:
MS 0397
8.44
90
B
M1
8.66 8.69
8.07 8.12
8.16 8.20
70
8.84
60
8.94 8.96
9.09
9.27
M2
RT: 10.19
9.50 9.55 9.57 9.71
10.43
9.86 9.94
50
10.84 10.88
11.03
10.72
11.15
40
RT: 12.69
12.31
11.71
10.48
11.85
11.50
11.94
12.20
12.36 12.46
12.74
12.2
12.4
12.8
30
20
10
0
Relative Abundance
8.0
8.2
8.4
8.6
8.8
9.0
9.2
9.4
9.6
9.8
10.0
10.2
10.4
10.6
Time (min)
80
55
11.4
11.6
11.8
12.0
12.6
13.0
NL: 3.03E6
0397# 1052 RT: 10.19 A V: 1SB :
640 8.58-9.94 , 10.61-12.40 T: +
c Full ms [ 50.00-325.00]
213
83
68
58
74
67
110
82 85 91
99
109
116
125 129
0
100
140
150
156 158
80
163 171
181 190 196
185
170
60
74
55 58 61
73
83
82
0
60
199
80
99 105
90 93
113
124
100
120
138
152 156 158 169 171
137 142
140
160
231 234
243
253
264
276 282 285
296
309 314
324
NL: 1.18E6
0397# 1560 RT: 12.69 A V: 1
SB : 209 9.76-10.01 , 11.63-12.41
T: + c Full ms [ 50.00-325.00]
210
184
111
212 214
198
157
68
40
20
11.2
157
40
20
11.0
198
100
60
10.8
182
241
213
186 196 199
180
200
221
220
226
228 241
240
254
242 253 255
260
269
281 285
280
295 300
300
311
322
320
m/z
Figure 4.2: Chromatograms of M1 (A) and M2 (B) from injection into the standard GC/MS method
for Irgarol analysis.
92
F:\M2 runs 11-3-06\0393
11/3/2006 3:05:45 PM
New M2 10 ppm Hexane full scan
RT: 8.00 - 13.00
RT: 12.70
100
90
NL:
3.38E7
TIC F:
MS 0393
Relative Abundance
80
70
M2
M1
60
50
40
30
RT: 10.22
20
10
8.17
8.36 8.49 8.59 8.71
8.92
9.09
9.28 9.35 9.52 9.63
9.83 9.91
10.26 10.34 10.57
10.16
12.24 12.37 12.54 12.66
11.16 11.32 11.49 11.56 11.77 11.94 12.08
10.86
12.77
0
Relative Abundance
8.0
8.2
8.4
8.6
8.8
9.0
9.2
9.4
9.6
9.8
10.0
10.2
10.4
10.6
Time (min)
10.8
11.0
11.2
11.4
11.6
11.8
12.0
12.2
12.4
12.6
80
13.0
NL: 8.30E5
0393# 1058 RT: 10.22 A V: 1
SB : 533 8.63-9.88 ,
10.99-12.36 T: + c Full ms [
50.00-325.00]
198
100
12.8
157
60
213
40
83
68
20
57
74
58
111
82 85 91
99
108
116
125
0
100
156 158
135 142
182 192 196
185
171
80
199
213 214 221
236 241 247 253 261 265
278
299
311 316
NL: 2.46E6
0393# 1562 RT: 12.70 A V: 1
SB : 392 8.61-9.50 , 11.32-12.36
T: + c Full ms [ 50.00-325.00]
198
170
157
60
210
213
68
40
20
54
58
83
74
55 57
67
111
110
113
99
82 85
93
184
116 123
138
137
152
156
158 169 171
182
160
180
186 196 199
241
226
227
214
254
242 253 255
240
269
0
60
80
100
120
140
200
220
240
272 281 287 292
260
280
305 314 317 324
300
320
m/z
Figure 4.3: Chromatogram of M2 injected into the GC/MS using a Siltek® deactivated liner
0687 #782 RT: 13.38 AV: 1 NL: 2.64E7
T: + c APCI corona Full ms2 254.00@36.00 [ 100.00-300.00]
198
100
S
90
N
80
N
Relative Abundance
70
N
H
N
N
H
60
50
-56 m/z (loss of t-butyl)
40
30
20
10
0
100
197 199
120
140
160
180
200
m/z
254
226
220
240
Figure 4.4: MS/MS Spectrum of Irgarol 1051
93
260
280
300
0687 #481 RT: 9.17 AV: 1 NL: 1.53E7
F: + c APCI corona Full ms2 214.00@34.00 [ 100.00-300.00]
158
24
S
22
N
20
N
Relative Abundance
18
N
H
16
N
NH2
-56 m/z (loss of t-butyl)
14
12
10
8
6
4
2
214
159
0
120
130
140
150
160
170
180
190
m/z
200
210
220
230
240
250
260
270
Figure 4.5: MS/MS Spectrum of M1
0687 #595 RT: 10.66 AV: 1 NL: 2.86E6
F: + c APCI corona Full ms2 270.00@36.00 [ 100.00-300.00]
214
S
19
18
17
N
N
O
16
15
14
N
H
Relative Abundance
13
N
N
H
H
12
226
11
-56 m/z (loss of t-butyl)
10
9
8
7
-100 m/z (loss of t-butyl and C2H4O)
6
5
158
4
-44 m/z (loss of C2H4O)
170
3
2
213
1
0
159
160
169
170
180
190
200
210
220
m/z
230
240
Figure 4.6: MS/MS Spectrum of M2
94
250
260
270
0687 #1117 RT: 16.25 AV: 1 NL: 3.93E7
F: + c APCI corona Full ms2 270.00@36.00 [ 100.00-300.00]
214
19
S
18
17
16
N
N
15
14
N
H
Relative Abundance
13
12
158
11
N
N
H
-112 m/z (loss of 2 t-butyl groups)
10
9
8
7
6
-56 m/z (loss of t-butyl)
5
4
3
2
159
1
215
0
160
170
180
190
200
210
220
m/z
230
240
250
260
270
Figure 4.7: MS/MS spectrum of M3
4.3.3. Chromatographic Separation
HPLC separation using the solvent gradient shown above provided good
resolution of all analytes of interest. A chromatogram of a calibration standard using
SIM is shown in Figure 4.8.
RT: 7.50 - 17.60
Atrazine-d5 (8.83)
Atrazine (8.91)
Irgarol (13.65)
Relative Abundance
100
M1 (9.37)
80
60
M3 (16.45)
40
M2 (10.48)
20
0
8
9
10
11
12
13
Time (min)
14
15
16
17
Figure 4.8: Chromatogram of mixture of Irgarol and its metabolites
95
NL:
1.05E7
m/z=
220.50221.50 F:
MS ICIS 0422
NL:
9.64E7
m/z=
215.50216.50 F:
MS ICIS 0422
NL:
2.96E8
m/z=
253.50254.50 F:
MS ICIS 0422
NL:
1.34E8
m/z=
213.50214.50 F:
MS ICIS 0422
NL:
3.21E8
m/z=
269.50270.50 F:
MS ICIS 0422
4.3.4. Instrument Calibration
Calibration curves for all compounds showed good linearity (R2≥0.999). Each
analyte had 9 calibration points, ranging from 2.5 ppb to 1000 ppb.
Since the
autosampler injects 25L per sample analysis, the actual on column injections of the
calibration standards ranged from 62.5pg to 2500pg. Calibration curves for each analyte
using SIM are shown in Figure 4.9. While they are not shown here, calibration curves for
the SRM analysis were similar to the SIM curves.
Irgarol
Y = 0.159993+0.0132542*X R^2 = 0.9991 W: Equal
Atrazine
Y = -0.0271467+0.00888137*X R^2 = 0.9994 W: Equal
14
12
10
Area Ratio
Area Ratio
8
6
4
8
6
4
2
2
0
0
0
200
400
600
pg/uL
800
0
1000
200
M1
Y = 0.00355602+0.010138*X R^2 = 0.9992 W: Equal
400
600
pg/uL
800
1000
M2
Y = 0.00435976+0.00532574*X R^2 = 0.9995 W: Equal
10
4
Area Ratio
6
4
3
2
2
1
0
0
0
200
400
600
pg/uL
800
1000
0
200
400
M3
Y = 0.0495424+0.0110543*X R^2 = 0.9993 W: Equal
12
10
Area Ratio
Area Ratio
5
8
8
6
4
2
0
0
200
400
600
pg/uL
800
1000
Figure 4.9: Calibration curves for analytes
96
600
pg/uL
800
1000
4.3.5. Method Performance
Method performance was checked by running seven replicate fortified artificial
seawater samples. Using these samples, an average recovery of the analytes was found,
along with the relative standard deviation. Method detection limits (MDL) were also
calculated for the SIM analysis using EPA statistical methods (standard deviation
multiplied by 3). The results are shown in Table 4.3.
Table 4.3: Average recoveries and MDLs for LC/MS analysis
SIM
Average
%RSD MDL
n
Average
%Recovery
(ng/L)
%Recovery
Atrazine
Irgarol
M1
M2
M3
100.53
96.47
90.98
83.10
103.54
0.18
0.33
0.66
0.83
0.32
0.55
0.97
1.80
2.08
0.99
7
7
5
7
7
103.77
102.21
83.58
86.07
108.72
SRM
%RSD
4.90
3.05
7.06
7.08
2.75
MDL
(ng/L)
n
15.2
9.35
17.7
18.3
8.97
7
7
7
7
7
Recoveries of all compounds were shown to be good by both SIM and SRM
analyses. The difference in the two analyses is the % RSD between the replicates. %
RSD values for the SRM analysis are much higher than those of the SIM analysis. This
is most likely do to the ion trap having to switch scanning frequencies in order to take an
MS-MS spectrum of the different compounds, while in the SIM analysis the trap is taking
a constant scan of a group of ions. This might be partially explained by looking at the
differences of the % RSD within the SRM experiment itself. The two compounds that
are well resolved from all other peaks have lower RSD values (≤ 3) than those peaks that
partially overlap. Irgarol and M3 are separated in their own segment of the run; during
the scanning window of Irgarol and M3, only their respective ions are scanned, so the ion
trap does not switch scanning frequencies to look for other ions. This insures a more
consistent amount of ions are scanned each time. When using SRM, the detection limits
97
of the instrument decreased, allowing for the detection of sub-ppb concentrations, but
calibration solutions below 2.5 ppb were not prepared.
4.3. Results
The method described above shows that M2 and M3 can be extracted from
environmental samples using basic liquid-liquid extraction techniques.
Since this
technique has been previously employed to analyze Irgarol in surface water samples in
our lab for the past 6 years (Gardinali et al., 2002; Gardinali et al., 2004), samples that
were previously analyzed by our normal protocol for Irgarol and M1 were screened for
M2 and M3. Since little is known about the stability of these compounds in solution,
screened samples were limited to those extracted in the year 2006. While data for these
samples has not yet been published, they were all collected from sites that have already
been proven to contain Irgarol and M1 throughout the year, or the summer months in the
case of samples collected from Chesapeake Bay, Maryland, collected by our
collaborators (Gardinali et al., 2002; Gardinali et al., 2004; Hall et al., 2004; Hall and
Gardinali, 2005). Also, since SRM gives an additional degree of analyte confirmation
and because limits of detection were lower, SRM was chosen to screen for M2 and M3 in
environmental samples. A total of 127 samples were screened using this method.
Out of the 127 samples screened for M2 and M3, none contained M2 and 78
(61%) contain M3. While an M3 peak was detected in the majority of the samples
screened, none of the peaks were large enough to reach the lower limit of quantitation set
by the method calibration solutions. This means that the environmental concentrations of
M3 in the water samples ranged from N.D. to 2.5 ng/L at most. These concentrations are
negligible compared to their corresponding Irgarol concentrations, which range up to 800
98
ng/L in samples collected from Port Annapolis, Maryland. A chromatogram of a sample
screened for M2 and M3 is shown in Figure 4.10.
RT: 0.00 - 18.99 SM: 11B
Irgarol
RT: 13.47
100
NL: 4.25E7
TIC F: + c APCI
corona SRM ms2
254.00@36.00 [
197.15-198.85] MS
0654
80
60
40
20
11.76
Relative Abundance
0
100
14.67 15.18
RT: 9.16
NL: 5.84E6
TIC F: + c APCI
corona SRM ms2
214.00@34.00 [
157.15-158.85] MS
ICIS 0654
M1
80
60
40
20
RT: 4.45
0
100
RT: 9.50
NL: 2.53E5
TIC F: + c APCI
corona SRM ms2
270.00@36.00 [
213.15-214.85] MS
ICIS 0654
80
Unknown Peak
60
M3
RT: 16.27
40
20
RT: 6.23
RT: 15.36
RT: 17.22
0
0
1
2
3
4
5
6
7
8
9
10
Time (min)
11
12
13
14
15
16
17
18
Figure 4.10: Chromatogram of a sample screened for M2 and M3
It could be argued that any M2 in the samples could have degraded during storage
time. While there are no papers yet in press pertaining to the stability of M2, a solution
of M2 in methanol left unrefrigerated and exposed to light in the lab showed no depletion
of M2 over a 14 day period (Figure 4.11). While this time frame is short, archived
extracts stored in a dark freezer would degrade much slower than that of the solution
exposed in the laboratory. It is therefore safe to assume that these archived samples
contained no M2 when collected from the field.
During the SRM screening of these samples, some contained an anomalous peak
that was isolated at an m/z of 270 and also contained a fragment ion of 214 m/z. While
these ions are the same as those of both M2 and M3, the retention time of this unknown
peak matched neither compound (Figure 4.12).
99
2.5e+8
2.0e+8
Response
1.5e+8
1.0e+8
5.0e+7
0.0
0
2
4
6
8
10
12
Time (days)
Figure 4.11: M2 Stability in water in laboratory conditions
RT: 0.00 - 18.99 SM: 11B
RT: 16.43
16.23
Relative Abundance
100
80
M3
Calibration
Solution
60
NL:
8.35E7
TIC F: + c APCI
corona SRM ms2
270.00@36.00 [
213.15-214.85]
MS 0469
M2
40
RT: 10.37
20
0.59
0
100
1.44 1.91 2.53
4.15 4.54 5.24
6.87
7.87
Sample
Chromatogram
80
60
40
Unknown
peak
15.81
15.42
20
2.75
11.59
4.91 5.14 5.44 6.98 7.68 8.06
3.29
NL:
7.38E4
TIC F: + c APCI
corona SRM ms2
270.00@36.00 [
213.15-214.85]
MS 0479
M3
RT: 9.47
1.28 1.90
17.30 18.21
15.74
RT: 16.24
17.77
18.14
0
0
1
2
3
4
5
6
7
8
9
10
Time (min)
11
12
13
14
15
16
17
18
Figure 4.12: Comparison of new metabolites in a calibration solution versus an archived sample
showing that the unknown peak is not M2.
100
In order to try to determine the identity of this compound, a sample containing the
unknown peak was run in MS/MS isolating the m/z of 270 and scanning for all
fragmentation ions. This mass spectrum is quite different than the one obtained for M2,
containing fragment ions at 252, 224, 196, and 168 m/z. M2 does not contain fragment
peaks at 252 and 196 m/z. Now confirmed to not be the M2 reported by Lam et al.,
(2004) further experiments were done to try to positively identify this compound.
Previous research done in our laboratory focused on detailed photodegradation
studies of Irgarol, including reaction rates and identifying major and minor metabolites
and various pathways to these metabolites.
Fortunately, our lab also has authentic
standards of all the photodegradation products that are known to be related to Irgarol.
One of these compounds, CA30-0156 ([7]; Figure 4.13), has a molecular weight of 269,
which is 16 mass units higher than Irgarol. This is also the molecular weight of M2. As
shown in Figure 4.12, this compound could be formed by photodegradation of Irgarol in
the environment by oxidation of the sulfur atom to a sulfoxide. It was thought that since
this is a known photodegradation product of Irgarol there is a possibility that the
unknown peak in the samples could be CA30-0156. A solution of this compound was
made in methanol and injected into the LC-MS/MS under the same parameters used to
analyze the archived samples.
A full scan MS/MS spectrum (Figure 4.14) shows
fragment peaks at 252, 214, and 196 m/z. These fragments correspond to those that show
up in the unknown peak of the archived samples. Moreover, the retention times of the
unknown peak and CA30-0156 are also identical (Figure 4.15).
101
SCH3
SCH3
(7)
O
N
N
H
N
SCH3
N
N
N
H
N
300nm
N
N
N
NH
O
S O
N
N
N
H
N
350nm
254nm
N
N
N
N
H
O
SCH3
NH2
H2N
OH
N
NH
N
N
(12)
NH2
O
S O
N
N
H2N
(10)
N
NH
N
N
N
NH
N
N
N
H
(5)
NH
(6)
N
N
N
(3)
OH
N
NH
H2N
(3)
NH
OH
N
N
N
N
H2N
H2N
(9)
N
H2N
SCH3
N
N
H
N
(11)
NH
300nm
N
NH
(1)
NH
SCH3
(8)
(2)
N
H2N
N
N
NH
(4)
H2N
Figure 4.13: Structure of CA30-0156
102
N
N
(4)
NH
C:\Xcalibur\...\0687
9/11/2006 5:45:56 PM
RT: 0.00 - 18.98
RT: 0.00 - 18.99
RT: 16.23
16.19
100
S
N
70
60
N
N
H
N
N
H
50
40
M2
S
30
N
N
O
20
10
0.69
0
0
N
H
N
2.05 2.85 3.89
2
4
N
H
5.57 6.69
6
8
60
N
14
16
40
N
H
2.12
0
80
Relative Abundance
80
40
30
20
16.34
8.00
15.99
2
4
6
8
10
Time (min)
0
100
120
140
160
200
m/z
220
18
[M+H] = 270
60
50
214
40
-56 m/z (loss of t-butyl)
30
20
199 213 215
180
16
-18 m/z (loss of
water)
10
170
14
252
196
168
158
12
70
226
10
16.71
11.52
100
90
50
7.84
7.44
0693 #495 RT: 9.35 AV: 1 SB: 67 2.40-7.72 NL: 9.13E5
F: + c APCI corona Full ms2 270.00@36.00 [ 100.00-300.00]
90
60
2.92 3.08 3.23 4.18
N
H
16.21
0
18
70
N
30
[M+H] = 270
214
100
N
50
17.29
15.69
12
O
S
10
11.53
10
Time (min)
Unknown
Peak
70
20
0687 #559 RT: 10.23 AV: 1 SB: 46 4.85-8.43 NL: 7.74E6
F: + c APCI corona Full ms2 270.00@36.00 [ 100.00-300.00]
Relative Abundance
80
10.60
8.36 9.59
Sample
Chromatogram
90
RT: 10.27
H
NL:
1.87E6
TIC F: + c APCI
corona Full ms2
270.00@36.00 [
100.00-300.00]
MS 0693
RT: 9.35
100
Relative Abundance
Relative Abundance
80
NL:
5.18E7
TIC F: + c APCI
corona Full ms2
270.00@36.00 [
100.00-300.00]
MS 0687
M3
Calibration
Solution
90
CS9 MS-MS SCANS (FULL FRAGMENTATION)
284
252
240
260
280
300
0
100
126
120
137
140
157 168 172
160
185
180
197
200
m/z
-74 m/z
(loss of water and t-butyl)
224
241 251 255
220
240
260
270
280
Figure 4.14: Mass spectrums showing the fragmentation patterns of the unknown peak in the archived samples and the "M2" peak in the
calibration solutions.
103
300
C:\Xcalibur\...\0693
9/12/2006 9:00:21 AM
RT: 0.00 - 18.98 SM: 7B
NL: 1.15E6
TIC F: + c A P CI co ro na
Full ms2 221.00@38.00 [
100.00-300.00] M S ICIS
0693
RT: 8.58
100
80
60
Atrazine-d5
40
20
0
100
80
Sample
Chromatogram
60
40
80
40
20
0
100
NL: 8.74E5
TIC F: + c A P CI co ro na
Full ms2 270.00@36.00 [
100.00-300.00] M S ICIS
0695
RT: 9.09
80
CA30-0156
Standard
60
40
20
20
0
0
0
2
4
6
8
10
Time (min)
12
14
16
18
0
0693 #499 RT: 9.40 AV: 1 NL: 7.23E5
F: + c APCI corona Full ms2 270.00@36.00 [ 100.00-300.00]
4
6
8
10
Time (min)
12
14
16
18
252
100
90
90
80
70
Relative Abundance
80
214
60
50
40
30
20
70
112
126
120
143
140
50
40
30
20
196
224
168
167
160
214
60
196
10
0
100
2
0693 #499 RT: 9.40 AV: 1 NL: 7.23E5
F: + c APCI corona Full ms2 270.00@36.00 [ 100.00-300.00]
252
100
Relative Abundance
Atrazine-d5
60
NL: 1.37E6
TIC F: + c A P CI co ro na
Full ms2 270.00@36.00 [
100.00-300.00] M S ICIS
0693
RT: 9.40
NL: 5.76E5
TIC F: + c A P CI co ro na
Full ms2 221.00@38.00 [
100.00-300.00] M S ICIS
0695
RT: 8.20
100
Relative Abundance
Relative Abundance
RT: 0.00 - 18.99 SM: 7B
PA01 MS-MS Full scan of Frags
213
180
200
m/z
215
220
225 235
240
253
260
269
10
284 293
280
300
0
100
112
126
120
143
140
224
168
167
160
213
180
200
m/z
215
220
225 235
240
253
260
269
284 293
280
300
Figure 4.15: Comparison of the unknown peak in a sample with that of a standard solution of CA30-0156 (note: retention times shifted about 0.3
minutes. Atrazine d-5 shown for reference)
104
To further differentiate this compound with M2, an injection of CA30-0156 was
made into the GC/MS. This compound is indeed stable in the GC, and shows many
fragments, including those characteristic to Irgarol: 253, 238, and 182 m/z.
The
compound is retained in the column for a slightly longer time than Irgarol (13.8 minutes),
most likely due to the fact that this compound has one extra oxygen making it slightly
more polar than Irgarol. A complete mass spectrum of the compound is shown in Figure
4.16. Since this compound is stable in the GC, unlike M2, an attempt was made to
quantitate the sulfoxide compound using the GC, but the levels were well below the
detection limits of the instrument.
Quantitation using the LC/MS-MS was achieved by preparing calibration
solutions ranging from 1.0 ppb to 100 ppb. The calibration curve obtained had a R2 value
of 0.997 (not shown). Out of the 125 samples screened for CA30-0156, 86% (107/125)
were found to contain the compound above the detection limits. The average and median
concentrations of the sulfoxide were 7.40 ng/L and 2.95 ng/L, respectively.
distribution of the sulfoxide from each sample location is shown in Table 4.4.
Table 4.4: Distribution of CA30-0156 in each sampling site
Area
Collection Date
% Sulfone
Average
Detection
Key Largo
March 2006
100
7.17
Harbor
Biscayne Bay
March 2006
75
3.28
Miami River
March 2006
100
2.47
Port Annapolis
August 2006
100
116
Severn River
August 2006
93
11.6
San Francisco
September 2006
71
2.07
Bay
San Diego Bay
September 2006
93
2.62
Los Angeles
September 2006
77
4.93
All Sample Sites
2006
86
7.40
105
Median
4.12
Number of
Samples
16
3.92
2.37
121
8.86
1.68
8
6
3
15
24
2.47
3.64
2.95
27
26
125
The
F:\0284
10/2/2006 5:24:56 PM
CA30-0156
RT: 9.50 - 14.00
RT: 13.80
100
CA30-0156
Relative Abundance
50
0
100
NL:
2.82E6
m/z=
252.50-253.50
F: M S ICIS
0085
RT: 11.72
Irgarol
50
0
100
NL:
2.90E6
m/z=
212.50-213.50
F: M S ICIS
0085
RT: 11.02
M3
RT: 10.19
M1
50
NL:
2.97E8
TIC F: M S
ICIS 0284
0
9.5
10.0
10.5
11.0
11.5
12.0
12.5
13.0
13.5
14.0
Time (min)
0284 #1784 RT: 13.80 AV: 1 SB: 416 11.60-13.65 , 13.98 NL: 2.53E7
T: + c Full ms [ 50.00-325.00]
150
100
90
Relative Abundance
80
70
166
60
50
68
40
30
83
252
238
56
124
20
10
196
182
108
57
55
63
81
69 74
84
93 98
109
99
122
126
140 148
151
198
157
167
180
183
222
206
213
223
236
239
0
60
80
100
120
140
160
180
200
220
240
m/z
Figure 4.16: Mass spectrum of CA30-0156 obtained from our GC/MS
106
269
253
254
270
256
281 285
301
260
280
300
311 315 325
320
4.4. Conclusions
This work concludes that the characterization of the two new Irgarol-related
compounds found by Lam et al. is accurate (2004 and 2005). This was confirmed by
obtaining authentic standards of Irgarol, M1, M2, and M3 and duplicating the MS
characterization studies that were performed by the Lam group. Since M2 was found not
to be compatible with GC systems, a new LC/MS method was developed to quantify
Irgarol and all its metabolites in a single analysis. Analyte recoveries were between 80
and 103%, with MDLs below 1 ng/L. Analysis of 127 samples showed that the aldehyde
compound was not present in any marine environment sampled in the United States in
2006. M3 was present in 61% of the samples, though concentrations were negligible (<
2.5 ng/L).
While it was proven that M2 is a product of the HgCl2-catalyzed hydrolysis of
Irgarol 1051, it seems highly unlikely that it will appear in the marine environment, since
it does not appear during photodegradation of the parent compound, which is the main
pathway for removal of the compound in the environment. There was, however, an
unexpected detection of a second photodegradation product of Irgarol found in
environmental samples. CA30-0156 was found in 86% (107/125) of the samples with
concentrations ranging from N.D. to 137 ng/L. The average and median concentrations
were 7.40 ng/L and 2.95 ng/L, respectively. The environmental detection of a previously
unseen metabolite of Irgarol warrants re-examination of the fate of Irgarol in the
environment. All future studies should involve monitoring for not only M1, but minor
degradation products as well. Also, the toxicity of these minor products to marine life
107
should be determined since there is a possibility of them showing up in the aquatic
environment.
108
V. Conclusions
The occurrence of Irgarol 1051 in the environment is widespread, however the
majority of locations sampled did not seem to be heavily impacted by the compound.
Concentrations of Irgarol 1051 ranged from non-detect (N.D.) to 1239 ng/L. Using the
data from all 370 water samples collected, the calculated 90th percentile concentration in
surface water was found to be 246 ng/L, with almost 50% of the samples above the
LOEC shown to affect the carbon uptake of coral systems (63 ng/L). Although this fact
could be interpreted as alarming, the 90th percentile concentration is roughly equal to the
10th percentile toxicity level, meaning that 90% of the species exposed to Irgarol at this
concentration would not be affected. A close look at the data suggests that most of the
samples above the 3 lowest affect levels were obtained from inside the Key Largo Harbor
Marina, an area with many boats and low water circulation. By excluding these samples
from the 90th percentile calculations, the number decreases drastically to 57.8 and only 20
out of 281 samples are above the 63 ng/L benchmark. This suggests that Key Largo
Harbor is a special case of contamination, and a closer look at the Irgarol sources in this
area is needed.
It was also determined that the most sensitive specie, coral reef systems, are not at
a high risk of exposure to Irgarol, due to the fact that Irgarol seems to dissipate rather
quickly upon release into open waters.
This was shown by the fact that Irgarol
concentrations at the entrance to Key Largo Harbor were highly affected by the tide, with
low tide carrying Irgarol out into the open ocean. No Irgarol was found in the 100+
samples collected at various coral reef systems, with the exception of 5 samples taken
from Looe Key during the Underwater Music Festival. While Irgarol was detected at this
109
location, all 5 samples contained negligible concentrations (< 2.0 ng/L), and disappeared
quickly once all the boats left the area. Although impact to major reefs seems to be an
unlikely event, many small coral patches and sparse coral heads could be considered as a
potential target species.
The transport and accumulation of Irgarol into sediments was negligible, which
seems to be in accordance to but in slight contrast to previous literature that calculated
the Kow partition coefficients. Submerged vegetation on the other hand was shown to
readily accumulate Irgarol. Bioconcentration factors (BCFs) ranged from 60 to 31588.
The 90th percentile BCF value was calculated to be 2780. While there are only 3 samples
of manatee grass, it seems that different species have different accumulation rates than
others. One possibility for the increased accumulation of Irgarol into manatee grass
could be that the growth rate of manatee grass is faster than that of turtle grass. The BCF
values for manatee grass seem to be in agreement with those calculated by Scarlett et al.,
which were up to 25000. The fact that Irgarol has been shown to readily accumulate into
submerged vegetation is alarming, since Irgarol is a photosystem II inhibitor. There is
not yet enough data to observe any appreciable trends; however the high BCF numbers
obtained from some samples warrant further investigation into any potential effects
Irgarol posses to submerged vegetation.
Characterization of authentic standards of the HgCl2-catalyzed hydrolysis
aldehyde metabolite M2 and the Irgarol impurity M3 proved that these compounds are
indeed related to Irgarol. M3 was found in the environment at negligible concentrations
(< 2.5 ng/L). No M2 was found in any environmental samples (n = 127). While Lam et
al. (2004) reported to have detected M2 in the environment at levels reaching 20,000
110
ng/L, it seems unlikely that this compound will naturally appear in the environment. It is
highly probable that during their synthesis and purification of M2, bench tops and/or
laboratory glassware became contaminated with the sulfoxide, thus leading to false
positives of M1 (levels reaching as high as 259,000 ng/L) and M2 in their environmental
samples. This is, however, the first report of a second photodegradation product of
Irgarol, CA30-0156, occurring in the environment. This sulfoxide compound differs
from the M2 characterized by Lam et al. (2004), but has the same molecular weight.
Concentrations of the sulfoxide ranged from N.D. to 137 ng/L (n = 125). The average
and median concentrations were 7.40 ng/L and 2.95 ng/L, respectively. The detection of
this previously undetected metabolite warrants a re-examination of its fate with respect to
photodegradation in the environment. Future work should include studies of the fate and
toxicity of the sulfoxide to determine if there is any risk to marine organisms from this
compound.
111
REFERENCES
Agüera A., Piedra, L., Hernando, M.D., Fernnádez-Alba, A.R., 2000: Multiresidue
method for the analysis of five antifouling agents in marine and coastal waters by gas
chromatography-mass spectrometry with large-volume injection.
Journal of
Chromatography A, 889, 261-269.
Albanis, T. A., D. A. Lambropoulou, V. A. Sakkas, and I. K. Konstantinou, 2002:
Antifouling paint booster biocide contamination in Greek marine sediments.
Chemosphere, 48, 475-485.
Alzieu, C., P. Michel, I. Tolosa, E. Bacci, L. D. Mee, and J. W. Readman, 1991:
Organotin compounds in the Mediterranean: A continuing cause for concern. Mar.
Environ. Res., 32, 261-270.
Amine-Khodja, A., O. Trubetskaya, O. Trubetskoj, L. Cavani, C. Ciavatta, G. Guyot, and
C. Richard, 2006: Humic-like substances extracted from composts can promote the
photodegradation of Irgarol 1051 in solar light. Chemosphere, 62, 1021-1027.
Anderson, C., M. Atlar, M. Callow, M. Candries, A. Milne, and R. L. Townsi, 2003: The
development of foul-release coatings for seagoing vessels. Journal of Marine Design
and Operations, B4, 11-23.
Bard, J., Pedersen, A., 1992: Ecotoxicological evaluation of the antifouling compound 2(tert-butylamino)-4-(cyclopropylamino)-6-(methylthio)-1,3,5-triazine, Irgarol.
Solna,
Sweden: Swedish National Chemicals Inspectorate (KEMI).
Basheer, C., K. S. Tan, and H. K. Lee, 2002: Organotin and Irgarol-1051 contamination
in Singapore coastal waters. Mar. Pollut. Bull., 44, 697-703.
Biselli, S., K. Bester, H. Huhnerfuss, and K. Fent, 2000: Concentrations of the
Antifouling Compound Irgarol 1051 and of Organotins in Water and Sediments of
German North and Baltic Sea Marinas. Mar. Pollut. Bull., 40, 233-243.
Bowman, J. C., J. W. Readman, and J. L. Zhou, 2003: Seasonal variability in the
concentrations of Irgarol 1051 in Brighton Marina, UK; including the impact of dredging.
Mar. Pollut. Bull., 46, 444-451.
Boxall, A. B. A., S. D. Comber, A. U. Conrad, J. Howcroft, and N. Zaman, 2000: Inputs,
Monitoring and Fate Modelling of Antifouling Biocides in UK Estuaries. Mar. Pollut.
Bull., 40, 898-905.
Cai, Z., Y. Fun, W. Ma, M. H. Lam, and J. Tsui, 2006: LC-MS analysis of antifouling
agent Irgarol 1051 and its decyclopropylated degradation product in seawater from
marinas in Hong Kong. Talanta, 70, 91-96.
112
Champ, M., 2000: A review of organotin regulatory strategies, pending actions, related
costs and benefits. The Science of the Total Environment, 258, 21-71.
Champ, M. A., 2003: Economic and environmental impacts on ports and harbors from
the convention to ban harmful marine anti-fouling systems. Marine Pollution Bulletin,
46, 935-940.
Ciba Geigy, 1995: Irgarol 1051 material safety data sheet 6389501 Ciba Geigy. Basel,
Switzerland: Additives Division.
Cohen, A.N., Carlton, J.T., 1995: Nonindigenous aquatic species in a United States
estuary: a case study of the biological invasions of the San Francisco Bay and Delta. A
report for the United States Fish and Wildlife Service, Washington, DC, and the National
Sea Grant College Program, Connecticut Sea Grant, NTIS Report Number PB96-166525.
Cohen, A.N., Mills, C.E., Berry, H.K., Wonham, M.J., Bingham, B., Bookheim, B.,
Carlton, J.T., Chapman, J.W., Cordell, J., Harris, L.H., Klinger, T., Kohn, A.J., Lambert,
C., Lambert, G., Li, K., Secord, D.L., Toft, J., 1998: A rapid assessment survey of nonindigenous species in the shallow waters of Puget Sound. Report for the Washington
State Department of Natural Resources, Olympia, Washington.
Coles, S.L., DeFelice, R.C., Eldredge, L.G., Carlton, J.T., 1997: Biodiversity of marine
communities in Pearl Harbor, Oahu, Hawaii with observations on introduced exotic
species. Bishop Museum Press, Honolulu, HI.
Comber, S. D., G. Franklin, M. J. Gardner, C. D. Watts, A. B. Boxall, and J. Howcroft,
2002: Partitioning of marine antifoulants in the marine environment. Sci. Total Environ.,
286, 61-71.
De Almeida Azevedo, D., Lacorte, S., Vinhas, T., Viana, P., Barceló, D., 2000:
Monitoring of priority pesticides and other organic pollutants in river water from Portugal
by gas-chromatography-mass spectrometry and liquid chromatography-atmospheric
pressure chemical ionization mass spectrometry. Journal of Chromatography A, 879, 1326.
Dahl, B., and H. Blanck, 1996: Pollution-induced community tolerance (PICT) in
periphyton communities established under tri-n-butyltin (TBT) stress in marine
microcosms. Aquatic Toxicology, 34, 305-325.
Dahl, B., and H. Blanck, 1996: Toxic effects of the antifouling agent Irgarol 1051 on
periphyton communities in coastal water microcosms. Mar. Pollut. Bull., 32, 342-350.
Devilla, R. A., M. T. Brown, M. Donkin, and J. W. Readman, 2005: The effects of a PSII
inhibitor on phytoplankton community structure as assessed by HPLC pigment analyses,
microscopy and flow cytometry. Aquat. Toxicol., 71, 25-38.
113
el Hassani, L. H., A. G. Frenich, J. L. Martinez Vidal, M. J. S. Muros, and M. H.
Benajiba, 2005: Study of the accumulation of tributyltin and triphenyltin compounds and
their main metabolites in the sea bass, Dicentrachus labrax, under laboratory conditions.
Science of The Total Environment, 348, 191-198.
EPA Bulletin, Federal Register: November 4th, 1999 (Volume 64, Number 213). 6019460204.
Fernandez-Alba, A. R., M. D. Hernando, L. Piedra, and Y. Chisti, 2002: Toxicity
evaluation of single and mixed antifouling biocides measured with acute toxicity
bioassays. Anal. Chim. Acta, 456, 303-312.
Ferrer, I., B. Ballesteros, M. Marco, and D. Barceló, 1997: Pilot Survey for
Determination of the Antifouling Agent Irgarol 1051 in Enclosed Seawater Samples by a
Direct Enzyme-Linked Immunosorbent Assay and Solid-Phase Extraction Followed by
Liquid Chromatography-Diode Array Detection. Environmental Science Technology, 31,
3530 - 3535.
Ferrer, I., Barceló, D., 1999: Simultaneous determination of antifouling herbicides in
marina water samples by on-line solid-phase extraction followed by liquid
chromatography-mass spectrometry. Journal of Chromatography A, 854, 197-206.
Ferrer, I., and D. Barcelo, 2001: Identification of a new degradation product of the
antifouling agent Irgarol 1051 in natural samples. J. Chromatogr. A, 926, 221-228.
Gardinali, P. R., M. Plasencia, S. Mack, and C. Poppell, 2002: Occurrence of IRGAROL
1051 in coastal waters from Biscayne Bay, Florida, USA. Mar. Pollut. Bull., 44, 781-788.
Gardinali, P. R., M. D. Plasencia, and C. Maxey, 2004: Occurrence and transport of
Irgarol 1051 and its major metabolite in coastal waters from South Florida. Mar. Pollut.
Bull., 49, 1072-1083.
Gough, M. A., J. Fothergill, and J. D. Hendrie, 1994: A survey of Southern England
coastal waters for the s-triazine antifouling compound Irgarol 1051. Marine Pollution
Bulletin, 28, 613-620.
Guruge, K. S., H. Iwata, H. Tanaka, and S. Tanabe, 1997: Butyltin accumulation in the
liver and kidney of seabirds. Mar. Environ. Res., 44, 191-199.
Hall Jr., L. W., J. M. Giddings, K. R. Solomon, and R. R. Balcomb, 1999: An Ecological
Risk Assessment for the Use of Irgarol 1051 as an Algaecide for Antifoulant Paints. Crit.
Rev. Toxicol., 29, 367-437.
Hall, J.,Lenwood W., and P. Gardinali, 2004: Ecological risk assessment for Irgarol 1051
and its major metabolite in United States surface waters. Hum. Ecol. Risk Assess., 10,
525-542.
114
Hall, J.,Lenwood W., W. D. Killen, R. D. Anderson, P. R. Gardinali, and R. Balcomb,
2005: Monitoring of Irgarol 1051 concentrations with concurrent phytoplankton
evaluations in East Coast areas of the United States. Mar. Pollut. Bull., 50, 668-681.
Hall, L. W.,Jr, W. D. Killen, and P. R. Gardinali, 2004: Occurrence of Irgarol 1051 and
its major metabolite in Maryland waters of Chesapeake Bay. Mar. Pollut. Bull., 48, 554562.
Haglund, K., A. Petterson, M. Petterson, H. Kylin, S. C. Lord, and P. Dollenmeier, 2001:
Seasonal distribution of the antifouling compound Irgarol 1051 outside a marina in the
Stockholm Archipelago. Bulletin of Environmental Contamination and Toxicology, 66,
50-58.
Hernando, M. D., L. Piedra, A. Belmonte, A. Aguera, and A. R. Fernandez-Alba, 2001:
Determination of traces of five antifouling agents in water by gas chromatography with
positive/negative chemical ionization and tandem mass spectrometric detection. J.
Chromatogr. A, 938, 103-111.
Hewitt, C.L., Campbell, M.L., Thresher, R.E., Martin, R.B., 1999: Marine biological
invasions of Port Phillip Bay, Victoria. Centre for Research on Introduced Marine Pests,
Technical Report No. 20, CSIRO Marine Research, Hobart.
Hughes, J., and M. M. Alexander, 1993: The toxicity of Irgarol 1051 to Skeletonema
costatum. B267-582-4,
Kannan, K., and J. Falandysz, 1997: Butyltin residues in sediment, fish, fish-eating birds,
harbour porpoise and human tissues from the Polish coast of the Baltic Sea. Mar. Pollut.
Bull., 34, 203-207.
Kobayashi, N., and H. Okamura, 2002: Effects of new antifouling compounds on the
development of sea urchin. Mar. Pollut. Bull., 44, 748-751.
Konstantinou, I. K., and T. A. Albanis, 2004: Worldwide occurrence and effects of
antifouling paint booster biocides in the aquatic environment: a review. Environ. Int., 30,
235-248.
Lam, K. H., Z. Cai, H. Y. Wai, V. W. Tsang, M. H. Lam, R. Y. Cheung, H. Yu, and P.
K. Lam, 2005: Identification of a new Irgarol-1051 related s-triazine species in coastal
waters. Environ. Pollut., 136, 221-230.
Lam, K. H., M. H. Lam, P. K. Lam, T. Qian, Z. Cai, H. Yu, and R. Y. Cheung, 2004:
Identification and characterization of a new degradation product of Irgarol-1051 in
mercuric chloride-catalyzed hydrolysis reaction and in coastal waters. Mar. Pollut. Bull.,
49, 361-367.
115
Lamoree, M. H., C. P. Swart, A. van der Horst, and B. van Hattum, 2002: Determination
of diuron and the antifouling paint biocide Irgarol 1051 in Dutch marinas and coastal
waters. J. Chromatogr. A, 970, 183-190.
Liu, D., R. J. Maguire, Y. L. Lau, G. J. Pacepavicius, H. Okamura, and I. Aoyama, 1997:
Transformation of the new antifouling compound Irgarol 1051 by Phanerochaete
chrysosporium. Water Res., 31, 2363-2369.
Liu, D., G. J. Pacepavicius, R. J. Maguire, Y. L. Lau, H. Okamura, and I. Aoyama, 1999:
Mercuric chloride-catalyzed hydrolysis of the new antifouling compound Irgarol 1051.
Water Res., 33, 155-163.
Martinez, K., and D. Barcelo, 2001: Determination of antifouling pesticides and their
degradation products in marine sediments by means of ultrasonic extraction and HPLCAPCI-MS. Fresenius J. Anal. Chem., 370, 940-945.
Martinez, K., I. Ferrer, M. D. Hernando, A. R. Fernandez-Alba, R. M. Marce, F. Borrull,
and D. Barcelo, 2001: Occurrence of antifouling biocides in the Spanish Mediterranean
marine environment. Environ. Technol., 22, 543-552.
Meesters, K. P. H., J. W. Van Groenestijn, and J. Gerritse, 2003: Biofouling reduction in
recirculating cooling systems through biofiltration of process water. Water Research, 37,
525-532.
Nystrom, B., K. Becker-Van Slooten, A. Berard, D. Grandjean, J. C. Druart, and C.
Leboulanger, 2002: Toxic effects of Irgarol 1051 on phytoplankton and macrophytes in
Lake Geneva. Water Res., 36, 2020-2028.
Okamura, H., Aoyama, I., Liu, D., Maguire, R.J., Pacepavicius, G.J., Lau, Y.L., 1999:
Photodegradation of Irgarol 1051 in water. Journal of Environmental Science and Health,
Part B. 34(2), 225-238.
Okamura, H., Aoyama, I., Liu, D., Maguire, R.J., Pacepavicius, G.J. and Lau, Y.L.
2000a, Fate and ecotoxicity of the new antifouling compound Irgarol 1051 in the aquatic
environment, Water research, vol. 34, no. 14, pp. 3523-3530.
Okamura, H., Aoyama, I., Takami, T., Maruyama, T., Suzuki, Y., Matsumoto, M.,
Katsuyama, I., Hamada, J., Beppu, T. and Tanaka, O. 2000b, Phytotoxicity of the New
Antifouling Compound Irgarol 1051 and a Major Degradation Product, Marine pollution
bulletin, vol. 40, no. 9, pp. 754-763.
Okamura, H., T. Watanabe, I. Aoyama, and M. Hasobe, 2002: Toxicity evaluation of
new antifouling compounds using suspension-cultured fish cells. Chemosphere, 46, 945951.
116
Owen, R., A. Knap, M. Toaspern, and K. Carbery, 2002: Inhibition of coral
photosynthesis by the antifouling herbicide Irgarol 1051. Mar. Pollut. Bull., 44, 623-632.
Placensia, M., 2001: MSc. Thesis, Florida International University, Miami, Florida,
December, 2001.
Pocurull, E., Brossa, L., Borull, F., Marcé, R.M., 2000: Trace determination of
antifouling compounds by on-linbe solid phase extraction-gas chromatography-mass
spectrometry. Journal of Chromatography A, 885, 361-368.
Readman, J. W., L. L. W. Kwong, D. Grondin, J. Bartocci, J. P. Villanueve, and L. D.
mee, 1993: Coastal water contamination from a triazine herbicide used in antifouling
paints. Environmental Science and Technology, 27, 1940-1942.
Rogers, H.R., Watts, C.D., Jonshon, I. 1996: Comparative predictions of Irgarol 1051
and atrazine fate and toxicity. Environmental Technology, 17, 553-556.
Rouhi, A. M., 1998: The squeeze in tributyltins. Chemical Engineering News, , 41-42.
Sakkas, V. A., D. A. Lambropoulou, and T. A. Albanis, 2002: Photochemical
degradation study of Irgarol 1051 in natural waters: influence of humic and fulvic
substances on the reaction. Journal of Photochemistry and Photobiology A: Chemistry,
147, 135-141.
Sargent, C. J., J. C. Bowman, and J. L. Zhou, 2000: Levels of antifoulant irgarol 1051 in
the Conwy Marina, North Wales. Chemosphere, 41, 1755-1760.
Scarlett, A., M. E. Donkin, T. W. Fileman, and P. Donkin, 1997: Occurrence of the
marine antifouling agent Irgarol 1051 within the Plymouth Sound locality: Implications
for the green macroalga Enteromorpha intestinalis. Mar. Pollut. Bull., 34, 645-651.
Scarlett, A., P. Donkin, T. W. Fileman, S. V. Evans, and M. E. Donkin, 1999a: Risk
posed by the antifouling agent Irgarol 1051 to the seagrass, Zostera marina. Aquatic
Toxicology, 45, 159-170.
Scarlett, A., P. Donkin, T. W. Fileman, and R. J. Morris, 1999b: Occurrence of the
Antifouling Herbicide, Irgarol 1051, within Coastal-water Seagrasses from Queensland,
Australia. Mar. Pollut. Bull., 38, 687-691.
Steen, R.J.C.A., Leonards, P.E.G., Brinkman, U.A.Th., Cofino, W.P., 1997: Ultra-tracelevel determination of the antifouling agent Irgarol 1051 by gas chromatography with
tandem mass spectrometric detection. Journal of Chromatography A, 766, 153-158.
Steen, R. J., J. van der Vaart, M. Hiep, B. Van Hattum, W. P. Cofino, and U. A.
Brinkman, 2001: Gross fluxes and estuarine behaviour of pesticides in the Scheldt
estuary (1995-1997). Environ. Pollut., 115, 65-79.
117
Stewart, C., 1996: The efficacy of legislation in controlling tributyltin in the marine
environment. In Tributyltin: Case Study of an Environmental Contaminant. 264-297 pp.
Tanabe, S., 1999: Butyltin Contamination in Marine Mammals - A Review. Mar. Pollut.
Bull., 39, 62-72.
Thomas, K.V., Blake, S.J., Waldock, M.J., 2000: Antifouling paint booster biocides
contamination in UK Marine sediments. Marine Pollution Bulletin, 40, 739-745.
Thomas, K. V., T. W. Fileman, J. W. Readman, and M. J. Waldock, 2001: Antifouling
paint booster biocides in the UK coastal environment and potential risks of biological
effects. Mar. Pollut. Bull., 42, 677-688.
Thomas, K. V., M. McHugh, and M. Waldock, 2002: Antifouling paint booster biocides
in UK coastal waters: inputs, occurrence and environmental fate. Sci. Total Environ., 293,
117-127.
Tolosa, I., and J. W. Readman, 1996: Simultaneous analysis of the antifouling agents:
tributyltin, triphenyltin and IRGAROL 1051 in marina water samples. Anal. Chim. Acta,
335, 267-274.
Tolosa, I., J. W. Readman, A. Blaevoet, S. Ghilini, J. Bartocci, and M. Horvat, 1996:
Contamination of Mediterranean (Cote d'Azur) coastal waters by organotins and irgarol
1051 used in antifouling paints. Mar. Pollut. Bull., 32, 335-341.
Toth, S., S. K. Becker-van, L. Spack, L. F. Alencastro, and J. Tarradellas, 1996: Irgarol
1051: an antifouling compound in freshwater sediment and biota of Lake Geneva.
Bulletin of Environmental Contamination and Toxicology, 57, 426-433.
Voulvoulis, N., Scrimshaw, M.D., Lester, J.N., 2000: Occurrence of four biocides
utilized in antifouling paints, as alternatives to organotin compounds, in waters and
sediments of a commercial estuary in the UK. Marine Pollution Bulletin, 40 938-946.
Voulvoulis, N., M. D. Scrimshaw, and J. N. Lester, 2002: Partitioning of selected
antifouling biocides in the aquatic environment. Mar. Environ. Res., 53, 1-16.
Zamora-Ley, I. M., P. R. Gardinali, and F. J. Jochem, 2006: Assessing the effects of
Irgarol 1051 on marine phytoplankton populations in Key Largo Harbor, Florida. Marine
Pollution Bulletin, 52, 935-941.
Zhou, J.K., Fileman, T.W., Evans, S., Donkin, P., Mantoura, R.F.C., Rowland, J.S., 1996:
Seasonal distribution of dissolved pesticides and polynuclear aromatic hydrocarbons in
the Humber estuary and Humber coastal zone. Marine Pollution Bulletin, 32, 599-608.
118
Appendix A. Concentrations of Irgarol and M1 in Key Largo Harbor
Table A-1: Concentrations of Irgarol and M1 in Key Largo Harbor
Site
Description
Collection Date
KLH01-01
Key Largo 24 Hour sampling Site 1 Hour 1
2/15/2003
KLH01-02
Key Largo 24 Hour sampling Site 1 Hour 2
2/15/2003
KLH01-03
Key Largo 24 Hour sampling Site 1 Hour 3
2/15/2003
KLH01-04
Key Largo 24 Hour sampling Site 1 Hour 4
2/15/2003
KLH01-05
Key Largo 24 Hour sampling Site 1 Hour 5
2/15/2003
KLH01-06
Key Largo 24 Hour sampling Site 1 Hour 6
2/15/2003
KLH01-07
Key Largo 24 Hour sampling Site 1 Hour 7
2/15/2003
KLH01-08
Key Largo 24 Hour sampling Site 1 Hour 8
2/15/2003
KLH01-09
Key Largo 24 Hour sampling Site 1 Hour 9
2/15/2003
KLH01-10
Key Largo 24 Hour sampling Site 1 Hour 10
2/15/2003
KLH01-11
Key Largo 24 Hour sampling Site 1 Hour 11
2/15/2003
KLH01-12
Key Largo 24 Hour sampling Site 1 Hour 12
2/15/2003
KLH01-13
Key Largo 24 Hour sampling Site 1 Hour 13
2/16/2003
KLH01-14
Key Largo 24 Hour sampling Site 1 Hour 14
2/16/2003
KLH01-15
Key Largo 24 Hour sampling Site 1 Hour 15
2/16/2003
KLH01-16
Key Largo 24 Hour sampling Site 1 Hour 16
2/16/2003
KLH01-17
Key Largo 24 Hour sampling Site 1 Hour 17
2/16/2003
KLH01-18
Key Largo 24 Hour sampling Site 1 Hour 18
2/16/2003
KLH01-19
Key Largo 24 Hour sampling Site 1 Hour 19
2/16/2003
KLH01-20
Key Largo 24 Hour sampling Site 1 Hour 20
2/16/2003
KLH01-21
Key Largo 24 Hour sampling Site 1 Hour 21
2/16/2003
KLH01-22
Key Largo 24 Hour sampling Site 1 Hour 22
2/16/2003
KLH01-23
Key Largo 24 Hour sampling Site 1 Hour 23
2/16/2003
KLH01-24
Key Largo 24 Hour sampling Site 1 Hour 24
2/16/2003
KLH02-01
Key Largo 24 Hour sampling Site 2 Hour 1
2/15/2003
KLH02-02
Key Largo 24 Hour sampling Site 2 Hour 2
2/15/2003
KLH02-03
Key Largo 24 Hour sampling Site 2 Hour 3
2/15/2003
KLH02-04
Key Largo 24 Hour sampling Site 2 Hour 4
2/15/2003
KLH02-05
Key Largo 24 Hour sampling Site 2 Hour 5
2/15/2003
119
Latitude
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0575
25.0542
25.0542
25.0542
25.0542
25.0542
Longitude
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2593
80.2588
80.2588
80.2588
80.2588
80.2588
Irgarol (ng/L)
1239
380
770
836
650
292
976
230
379
332
292
355
320
430
485
408
813
373
345
382
369
328
328
296
186
176
177
180
240
M1 (ng/L)
429
163
305
360
331
43.9
99.9
34.1
265
230
216
262
223
243
264
238
394
252
231
304
274
260
260
297
101
102
92.0
102
123
Table A-1 (continued)
Site
Description
KLH02-08
Key Largo 24 Hour sampling Site 2 Hour 8
KLH02-09
Key Largo 24 Hour sampling Site 2 Hour 9
KLH02-10
Key Largo 24 Hour sampling Site 2 Hour 10
KLH02-11
Key Largo 24 Hour sampling Site 2 Hour 11
KLH02-12
Key Largo 24 Hour sampling Site 2 Hour 12
KLH02-13
Key Largo 24 Hour sampling Site 2 Hour 13
KLH02-14
Key Largo 24 Hour sampling Site 2 Hour 14
KLH02-15
Key Largo 24 Hour sampling Site 2 Hour 15
KLH02-16
Key Largo 24 Hour sampling Site 2 Hour 16
KLH02-17
Key Largo 24 Hour sampling Site 2 Hour 17
KLH02-18
Key Largo 24 Hour sampling Site 2 Hour 18
KLH02-19
Key Largo 24 Hour sampling Site 2 Hour 19
KLH02-20
Key Largo 24 Hour sampling Site 2 Hour 20
KLH01
Key Largo Harbor Site 1
KLH02
Key Largo Harbor Site 2
KLH03
Key Largo Harbor Site 3
KLH04
Key Largo Harbor Site 4
KLH05
Key Largo Harbor Site 5
KLH06
Key Largo Harbor Site 6
KLH07
Key Largo Harbor Site 7
KLH08
Key Largo Harbor Site 8
KLH09
Key Largo Harbor Site 9
KLH10
Key Largo Harbor Site 10
KLH11
Key Largo Harbor Site 11
KLH12
Key Largo Harbor Site 12
KLH13
Key Largo Harbor Site 13
KLH14
Key Largo Harbor Site 14
KLH15
Key Largo Harbor Site 15
KLH16
Key Largo Harbor Site 16
KLH17
Key Largo Harbor Site 17
KLH18
Key Largo Harbor Site 18
Collection Date
2/15/2003
2/15/2003
2/15/2003
2/15/2003
2/15/2003
2/16/2003
2/16/2003
2/16/2003
2/16/2003
2/16/2003
2/16/2003
2/16/2003
2/16/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
8/21/2003
120
Latitude
25.0542
25.0542
25.0542
25.0542
25.0542
25.0542
25.0542
25.0542
25.0542
25.0542
25.0542
25.0542
25.0542
25.05434
25.05576
25.05704
25.05771
25.05767
25.05756
25.05714
25.05716
25.05591
25.05582
25.05443
25.05465
25.05403
25.05362
25.05366
25.05352
25.05276
25.05265
Longitude
80.2588
80.2588
80.2588
80.2588
80.2588
80.2588
80.2588
80.2588
80.2588
80.2588
80.2588
80.2588
80.2588
80.25863
80.25823
80.25805
80.25794
80.25887
80.26206
80.2584
80.26039
80.2618
80.25833
80.2592
80.2618
80.26168
80.25801
80.25836
80.25877
80.25895
80.25848
Irgarol (ng/L)
248
246
221
192
144
157
178
150
575
509
491
513
393
129
263
299
219
234
165
284
239
262
220
110
90.7
102
46.7
43.3
64.4
60.8
65.3
M1 (ng/L)
102
133
107
94.0
77.2
73.9
87.5
82.4
222
199
208
226
163
35.8
50.8
65.7
65.7
80.4
41.8
77.0
61.8
50.1
43.3
24.2
21.6
21.9
15.6
10.5
21.1
16.2
20.8
Table A-1 (continued)
Site
Description
KLH040205-01
Key Largo Harbor Site 1
KLH040205-02
Key Largo Harbor Site 2
KLH040205-03
Key Largo Harbor Site 3
KLH040205-04
Key Largo Harbor Site 4
KLH040205-05
Key Largo Harbor Site 5
KLH040205-06
Key Largo Harbor Site 6
KLH040205-07
Key Largo Harbor Site 7
KLH040205-08
Key Largo Harbor Site 8
KLH040205-09
Key Largo Harbor Site 9
KLH040205-10
Key Largo Harbor Site 10
KLH040205-11
Key Largo Harbor Site 11
KLH040205-12
Key Largo Harbor Site 12
KLH040205-13
Key Largo Harbor Site 13
KLH040205-14
Key Largo Harbor Site 14
KLH040205-15
Key Largo Harbor Site 15
KLH040205-16
Key Largo Harbor Site 16
KLH040205-17
Key Largo Harbor Site 17
KLH040205-18
Key Largo Harbor Site 18
KLH040205-19
Key Largo Harbor Site 19
NKLH-01
North Key Largo Harbor Site 1
NKLH-02
North Key Largo Harbor Site 2
NKLH-03
North Key Largo Harbor Site 3
NKLH-04
North Key Largo Harbor Site 4
SKLH-05
South Key Largo Harbor Site 1
SKLH-06
South Key Largo Harbor Site 2
SKLH-07
South Key Largo Harbor Site 3
SKLH-08
South Key Largo Harbor Site 4
KLH-01
Key Largo Harbor (inside) Site 1
KLH-02
Key Largo Harbor (inside) Site 2
KLH-03
Key Largo Harbor (inside) Site 3
KLH-04
Key Largo Harbor (inside) Site 4
Collection Date
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
2/5/2004
4/29/2004
4/29/2004
4/29/2004
4/29/2004
4/29/2004
4/29/2004
4/29/2004
4/29/2004
4/29/2004
4/29/2004
4/29/2004
4/29/2004
Latitude
25.09076
25.09333
25.09533
25.09624
25.09612
25.09563
25.09412
25.09405
25.09201
25.09224
25.09102
25.09000
25.08839
25.08845
25.08859
25.08944
25.08942
25.08914
25.09570
25.08855
25.08864
n/a
n/a
25.08745
25.08689
25.08650
25.08712
25.09603
25.09616
25.09418
25.09407
121
Longitude
80.43074
80.43034
80.43007
80.42976
80.43150
80.43667
80.43235
80.43647
80.43390
80.43630
80.43392
80.43632
80.43153
80.43084
80.43008
80.42995
80.43071
80.43121
80.40388
80.43059
80.43047
n/a
n/a
80.43060
80.43011
80.42970
80.42983
80.43650
80.42979
80.43338
80.43031
Irgarol (ng/L)
86.1
135
175
178
289
450
62.4
78.4
106
99.9
54.3
77.0
9.76
9.86
4.20
4.92
16.1
12.9
1.82
4.00
4.17
4.53
3.53
3.86
3.32
3.56
3.62
202
60.3
73.8
77.7
M1 (ng/L)
12.6
27.0
36.9
35.8
33.8
60.1
11.1
21.0
14.0
24.5
15.5
19.8
3.43
2.94
0.00
1.44
3.14
2.22
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
76.1
27.5
27.7
38.4
Table A-1 (continued)
Site
Description
KLH-05
Key Largo Harbor (inside) Site 5
KLH-06
Key Largo Harbor (inside) Site 6
NKLH-01
North Key Largo Harbor Site 1
NKLH-02
North Key Largo Harbor Site 2
NKLH-03
North Key Largo Harbor Site 3
NKLH-04
North Key Largo Harbor Site 4
NKLH-05
North Key Largo Harbor Site 5
NKLH-06
North Key Largo Harbor Site 6
NKLH-07
North Key Largo Harbor Site 7
NKLH-08
North Key Largo Harbor Site 8
SKLH-01
South Key Largo Harbor Site 1
SKLH-02
South Key Largo Harbor Site 2
SKLH-03
South Key Largo Harbor Site 3
SKLH-04
South Key Largo Harbor Site 4
SKLH-05
South Key Largo Harbor Site 5
SKLH-06
South Key Largo Harbor Site 6
SKLH-07
South Key Largo Harbor Site 7
SKLH-08
South Key Largo Harbor Site 8
KLH-01
Key Largo Harbor (inside) Site 1
KLH-02
Key Largo Harbor (inside) Site 2
KLH-03
Key Largo Harbor (inside) Site 3
KLH-04
Key Largo Harbor (inside) Site 4
KLH-05
Key Largo Harbor (inside) Site 5
KLH-06
Key Largo Harbor (inside) Site 6
NKLH-01
North Key Largo Harbor Site 1
NKLH-02
North Key Largo Harbor Site 2
NKLH-03
North Key Largo Harbor Site 3
NKLH-04
North Key Largo Harbor Site 4
NKLH-05
North Key Largo Harbor Site 5
NKLH-06
North Key Largo Harbor Site 6
NKLH-07
North Key Largo Harbor Site 7
Collection Date
4/29/2004
4/29/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
6/14/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
Latitude
25.09212
25.09053
25.08944
25.08810
25.08722
25.08732
25.08830
25.08957
25.08960
25.08761
25.08920
25.08819
25.08695
25.08690
25.08791
25.08875
25.08827
25.08785
25.09210
25.09415
25.09630
25.09618
25.09591
25.09525
25.08904
25.08895
25.08907
25.08957
25.08970
25.08965
25.08943
122
Longitude
80.43385
80.43081
80.43021
80.43020
80.40080
80.42937
80.42940
80.42944
80.42834
80.42837
80.43129
80.43166
80.43150
80.43250
80.43265
80.43255
80.43306
80.73334
80.43067
80.43026
80.04299
80.43464
80.43639
80.43381
80.43024
80.42995
80.42944
80.42947
80.42957
80.43005
80.43046
Irgarol (ng/L)
58.2
3.62
7.08
37.1
22.5
23.6
16.1
5.16
5.23
16.7
19.0
26.4
27.0
20.7
16.6
17.5
22.0
39.4
68.7
158.6
213
172
189
136
95.3
62.8
33.1
27.1
24.7
56.8
115
M1 (ng/L)
25.2
0.00
3.23
8.98
4.31
6.64
4.14
0.00
1.53
6.74
5.43
7.74
0.00
32.9
30.7
18.7
8.39
9.87
19.1
47.5
70.8
57.5
66.2
38.5
28.1
15.0
13.0
14.2
11.6
18.7
32.7
Table A-1 (continued)
Site
Description
NKLH-08
North Key Largo Harbor Site 8
NKLH-09
North Key Largo Harbor Site 9
NKLH-10
North Key Largo Harbor Site 10
SKLH-01
South Key Largo Harbor Site 1
SKLH-02
South Key Largo Harbor Site 2
SKLH-03
South Key Largo Harbor Site 3
SKLH-04
South Key Largo Harbor Site 4
SKLH-05
South Key Largo Harbor Site 5
SKLH-06
South Key Largo Harbor Site 6
SKLH-07
South Key Largo Harbor Site 7
SKLH-08
South Key Largo Harbor Site 8
SKLH-09
South Key Largo Harbor Site 9
SKLH-10
South Key Largo Harbor Site 10
KLH-01
Key Largo Harbor (inside) Site 1
KLH-02
Key Largo Harbor (inside) Site 2
KLH-03
Key Largo Harbor (inside) Site 3
KLH-04
Key Largo Harbor (inside) Site 4
KLH-05
Key Largo Harbor (inside) Site 5
KLH-06
Key Largo Harbor (inside) Site 6
KLH-07
Key Largo Harbor (inside) Site 7
KLH-08
Key Largo Harbor (inside) Site 8
KLH01
Key Largo Harbor Site 1
KLH02
Key Largo Harbor Site 2
KLH03
Key Largo Harbor Site 3
KLH04
Key Largo Harbor Site 4
KLH05
Key Largo Harbor Site 5
KLH06
Key Largo Harbor Site 6
KLH07
Key Largo Harbor Site 7
KLH08
Key Largo Harbor Site 8
KLH09
Key Largo Harbor Site 9
KLH10
Key Largo Harbor Site 10
Collection Date
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
8/27/2004
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
Latitude
25.09008
25.09299
25.09324
25.08804
25.08778
25.08748
25.08733
25.08796
25.08867
25.08894
25.08781
25.08407
25.08207
25.09612
25.09610
25.09423
25.09342
25.09210
25.09227
25.09103
25.08914
25.09589
25.09614
25.09631
25.09433
25.09217
25.08936
25.08725
25.08692
25.08792
25.08883
123
Longitude
80.42976
80.42873
80.42649
80.43143
80.43138
80.43198
80.43219
80.43262
80.43238
80.43176
80.43324
80.43505
80.43702
80.43604
80.42999
80.43536
80.43027
80.43562
80.43028
80.43081
80.43067
80.43636
80.43261
80.43022
80.43025
80.43061
80.43047
80.43611
80.44111
80.43258
80.43222
Irgarol (ng/L)
25.6
23.8
14.3
48.8
53.3
50.5
47.2
35.5
23.5
31.1
30.6
7.94
5.02
192
172
74.1
196
74.8
229
100
106
152
271
234
193
112
21.7
110
163
37.0
43.9
M1 (ng/L)
15.4
18.2
10.3
17.6
15.0
17.2
13.5
13.1
9.85
14.0
12.2
3.34
2.63
78.2
66.8
41.4
78.2
43.3
76.0
49.7
45.5
47.1
56.3
46.8
41.6
29.8
9.14
41.5
68.2
14.1
16.6
Table A-1 (continued)
Site
Description
KLH11
Key Largo Harbor Site 11
KLH12
Key Largo Harbor Site 12
KLH13
Key Largo Harbor Site 13
KLH14
Key Largo Harbor Site 14
KLH15
Key Largo Harbor Site 15
KLH16
Key Largo Harbor Site 16
Collection Date
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
3/23/2006
Latitude
25.08831
25.08919
25.08870
25.08908
25.08967
25.08967
124
Longitude
80.43131
80.43114
80.42994
80.42977
80.42936
80.43008
Irgarol (ng/L)
25.1
47.3
8.31
8.36
12.4
22.1
M1 (ng/L)
13.9
17.3
5.05
5.37
6.94
9.74