Supplementary material: Bioavailability and release rates of biocides
AFFINITY STATES OF BIOCIDES DETERMINE BIOAVAILABILITY AND
RELEASE RATES IN MARINE PAINTS
MIA DAHLSTRÖM1* MARTIN SJÖGREN3, PER R. JONSSON4, ULF GÖRANSSON3,
LISELOTT LINDH5, THOMAS ARNEBRANT6, EMILIANO PINORI1, HANS ELWING2
AND MATTIAS BERGLIN1
1
Unit for Chemistry, Materials and Surfaces, SP Technical Research Institute of Sweden, Box
857, SE-501 15 Borås, Sweden
2
Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, SE-
405 30 Göteborg, Sweden,
3
Department of Medicinal Chemistry, Biomedical Centre, Uppsala University, Box 574, SE-
751 23 Uppsala, Sweden
4
Department of Biological and Environmental Sciences, Sven Lovén Centre Tjärnö,
University of Gothenburg, SE-452 96 Strömstad, Sweden,
5
Department of Prosthetic Dentistry, Faculty of Odontology, Malmö University, SE-205 06
Malmö, Sweden,
6
Biomedical Laboratory Science, Health and Society, Malmö University, SE-205 06 Malmö,
Sweden
*
To whom correspondence should be addressed. E -mail: mia.dahlstrom@sp.se, tel: +46(0)10-
5165340, (mobile) +46(0)730-785340
SUPPLEMENTARY MATERIAL
Supplementary material: Bioavailability and release rates of biocides
MATERIALS AND METHODS
Preliminary field experiment using medetomidine in four different non-toxic paints
To evaluate the antifouling performance of medetomidine in the field, four different non-toxic
paints were chosen. The trade names of the paints, their different paint technologies (as stated
by their respective manufacturer) and their respective manufacturers are listed in Table S1.
150 mg of medetomidine (Orion Oy, Finland) was dissolved in 100 µL of 95% ethanol and
added to 15 g of each of the paints to give the concentration 1% (w/w). The paints were
thoroughly mixed and 5 g was taken from the mixtures and further diluted with paint without
the addition of medetomidine in steps 1:3 to yield the concentrations 0.33%, 0.11%, 0.037%
and 0.012%. Panels of Plexiglass (110x110 mm) were painted with the five different
concentrations of medetomidine for each paint formulation. For each panel, approximately 11.5 mL of coating was used. A set of panels was painted with the coatings without
medetomidine to serve as controls. Each treatment was replicated 3 times. The panels were
coated with 1 layer of paint and were allowed to dry for 48 h before they were fixed to 4
different aluminium racks. The location of each panel on the rack was randomised. Panels
were deployed at depths between 0.6 and 2.2 m. The racks were placed in a bay outside
Tjärnö Marine Biological Laboratory, at the end of May and were maintained until the
beginning of September (12 weeks). The panels were viewed under a stereomicroscope and
an area of 100x100 mm2 was checked for the number of recruited B. improvisus.
The effects of the paints in combination with the different concentrations of medetomidine on
the recruitment of B. improvisus were evaluated in a 2-factor analysis of variance (ANOVA)
with dose and paint as fixed factors. The minimum concentrations to produce a significant
reduction in recruitment in the respective paint were analysed with means comparison
contrast (MCC). The data were checked for homoscedasticity using Cochran’s test. Where
data displayed significant heterogeneity of variances a log-transformation was successfully
Supplementary material: Bioavailability and release rates of biocides
employed. In all tests a type-I error () of 0.05 was used. The erodible paint, SPF, and the
solid paint, VC 17 NT, (Table S1) were chosen in the further studies of release rate of
medetomidine and effects on barnacle recruitment.
Surface characterisation of pristine and 24 h water-aged antifouling coatings
The surface properties of pristine and 24 h-water-aged coated surfaces were investigated with
Fourier transform infrared spectroscopy (FT-IR) using the attenuated total reflection (ATR)
technique. The coatings were applied onto plastic stripes and allowed to dry prior to analysis.
The instrument used was a Perkin-Elmer 2000 equipped with a germanium crystal as the
reflection element. The absorbance was recorded with a nitrogen-cooled
mercury/cadmium/telluride (MCT) detector from 4000 cm-1 to 750 cm-1 and at least 100 scans
were co-added for each sample. The sample chamber was purged with dry carbon dioxide
prior to/during analysis. To be able to record a spectrum of the erodible paint we removed the
pigment by centrifugation. Thus, the erodible paint was diluted in n-hexane (1:5) and
centrifuged at 4000 rpm for 30 minutes. The supernatant were used to coat the plastic stripes.
The water aging was performed in the laboratory. The samples were placed in a glass
container filled with flowing distilled water for 24 hours.
Preparation of calibration standards for HPLC analysis
Prior to the RP-HPLC analyses of the release of medetomidine and tolazoline from the
painted glass petri dishes, the UV-absorbance maxima of medetomidine and tolazoline were
determined using a UV-spectrometer (Shimadzu Instruments, Japan). Absorbance spectra
were taken between 190-500 nm (Table S2). Calibration standards were prepared at
concentrations of 0.002, 0.02, 0.2, 2.0, 20 and 200 ng µL-1 of medetomidine and of 0.0015,
0.015, 0.15, 1.5, 15 and 150 ng µL-1 of tolazoline in filtered seawater (FSW): acetonitrile
Supplementary material: Bioavailability and release rates of biocides
(9:1). Volumes of 100 µL of these standards, corresponding to 0.2, 2, 20, 200, 2000 and
20000 ng for medetomidine and to 0.15, 1.5, 15, 150, 1500 and 15000 ng for tolazoline were
injected in replicates (n=5) to create the calibration curves (Fig. S1).
Spiked samples to control for possible interactions between medetomidine and tolazoline with
released paint components
Also, spiked samples for the identification of the active ingredients medetomidine and
tolazoline in the paint samples were prepared. The spiked samples were also prepared in order
to ensure the complete separation of medetomidine or tolazoline from other substances
originating from the paints (Fig S2). Known amounts (0.5 µg mL-1) of the two compounds
were added respectively, to samples (2 mL) collected from control glass petri dishes. The
spiked samples were shaken vigorously (3 min) and were allowed to stand for 10 min for deaeration prior to HPLC analysis. 1 mL of each spiked sample was injected onto the column.
Examples of chromatograms of spiked samples added to day 1 controls are presented in
Figure S2.
RESULTS
Preliminary field experiment using medetomidine in four different non-toxic paints
The 2-factor ANOVA did not detect any significant interaction between the factors paint and
concentration (2-factor ANOVA, F15, 48=1.7, p>0.05) indicating that the four paints reduced
recruitment of B. improvisus in a similar dose-response manner. The abilities of the different
paints to deter colonization of the barnacle B. improvisus for 12 weeks in the field is
summarised in Table S2. Also, the F- and p-values of the MCC-test are presented in Table S3.
In this field test, the erodible paint, SPF, was the most efficient to reduce barnacle
colonisation over a 12-weeks period. A three-fold increase in concentration was required for
Supplementary material: Bioavailability and release rates of biocides
the paint FabiEco representing a similar paint technology as SPF or the combined
erodible/solid paint (TF) for a significant reduction of recruitment as compared to control
panels. Furthermore, a tenfold increase in concentration as compared to the erodible paint SPF
was required for the solid paint (VC 17 NT) to display a significant reduction of recruitment.
The two paints displaying the most (SPF) and the least (VC 17 NT) efficient antifouling
effect, respectively, were subsequently chosen for the further studies on release rate and
antifouling activity.
Characterisation of paints by Infrared Spectroscopy
To demonstrate a difference in chemistry of the two coatings used in the present study, we
employed Attenuated Total Reflection-Fourier Transform/Infrared Spectroscopy (ATRFT/IR). The ATR-FT/IR analysis of the pristine coatings of the erodible paint and the solid
paint revealed the difference in chemistry between the two different coatings. Peaks
originating from fluorinated alkane groups (CF2 and CF3) at 1350-1120 cm-1 were clearly
visible in the spectra of the solid paint. No such peaks were detected in the erodible paint.
Strong absorbance of aliphatic C-H stretching vibrations originating from CH2 and CH3 was
found in both coatings. No major discrepancies were detected when the spectra of 24 h wateraged coatings were compared with the spectra of pristine coatings, indicating that organic
solvents did not interfere with the spectra of the pristine coatings. It was observed, however,
that a thin whitish layer was formed on the surface of the erodible paint after 24 h incubation
in mQ water. No such observation was made with the solid paint.
Supplementary material: Bioavailability and release rates of biocides
FIGURES
Fig S1: Standard curve of medetomidine and tolazoline.
Supplementary material: Bioavailability and release rates of biocides
Fig S2. Spiked samples demonstrating the separation of medetomidine and tolazoline peaks in
the chromatograms.
Supplementary material: Bioavailability and release rates of biocides
TABLES
Table S1. The four different non-toxic paints used in the preliminary field experiment, their
different paint technologies and respective manufacturer. In the experiment, the ability of
different concentrations of medetomidine in combination with different paint technologies to
deter recruitment of the barnacle B. improvisus was evaluated.
Trade name
Paint technology
Paint manufacturer
VC 17 New
Technology
Insoluble matrix paint (solid paint)
International Coatings (Akzo Nobel, The
Netherlands)
SPF
Soluble matrix paint (erodible paint)
Lotréc, LeFant (Sweden)
FabiEco
Soluble matrix paint (erodible paint)
International Coatings (Akzo Nobel, The
Netherlands)
TF
Combined soluble matrix (erodible) paint/
insoluble matrix paint
Lotréc, LeFant (Sweden)
Supplementary material: Bioavailability and release rates of biocides
Table S2. Calibration and precision data for the HPLC analysis of medetomidine and
tolazoline with UV-detection at 220 nm.
Detection
limit (ng)
Calibration equation
R2
Linear range (ng)
Medetomidine
2
y=3.78 107x
1
2-2000
Tolazoline
15
y=2.54 107x
1
15-15000
Analyte
Supplementary material: Bioavailability and release rates of biocides
Table S3. The effect of the four different paints to which medetomidine had been added, on
the recruitment of B. improvisus for 12 weeks field exposure. The table summarises the
minimum significant dose of the respective paint to reduce recruitment of B. improvisus and
the percentage in reduction as compared to control panels. F-values and the p-values were
obtained in a 2-factor ANOVA and in a subsequent Means Comparison Contrast (MCC) test
to detect significant reductions in recruitment between the treatments with medetomidine in
the different paints and the respective controls. Fouling data were log-transformed prior to
ANOVA.
Paint
Minimum
significant
concentration
(%) in paint to
reduce
recruitment of B.
improvisus
F-value
pvalue
Reduction in
recruitment (%)
VC17 New
Technology
1.0
F1, 12=9.11
0.004
83
SPF
0.03
F1, 12=8.5
0.0055
94
FabiEco
0.1
F1, 12=20.9
0.0001
100
TF
0.1
F1, 12=16.9
0.0002
97