SK and IK are employed by Japan NUS Co., Ltd. YS is employed by Chugoku Marine Paints, Ltd. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
‡ These authors also contributed equally to this work.
A laboratory test with a flow-through system was designed and its applicability for testing antifouling paints of varying efficacies was investigated. Six different formulations of antifouling paints were prepared to have increasing contents (0 to 40 wt.%) of Cu2O, which is the most commonly used antifouling substance, and each formulation of paint was coated on just one surface of every test plate. The test plates were aged for 45 days by rotating them at a speed of 10 knots inside a cylinder drum. A behavioral test was then conducted using five mussels (
Antifouling (AF) paints should be evaluated and selected from the viewpoint of fuel consumption efficiency and effectiveness in preventing the attachment of marine fouling organisms on ships [
AF paints have been evaluated in the past mostly through field experiments on rafts and by patch-tests on ship hulls. For instance, the Efficacy Assessment Guideline of the European Chemical Agency (ECHA) clearly states that field experiments are necessary to evaluate efficiency under natural conditions where biofouling occurs [
A wide range of test organisms has been used in AF bioassay in controlled experimental conditions utilizing dominant macrofoulers. Mussels are one of the dominant macrofoulers and are recorded as major fouling organisms of marine structures and ships’ fouling, hence they are used in settlement inhibition assays [
In this paper, the authors prepared test plates coated with different AF paints and tested their antifouling efficiencies in a newly proposed laboratory-based method under controlled conditions using mussels,
The matrix polymer of the paints tested was a self-polishing hydration type copolymer that was polymerized by vinyl chloride and isobutyl vinyl ether. Cu2O was the only antifouling agent used in the test paints, because it enabled easier analysis and evaluation of the leaching phenomenon, and also eliminated the synergy effect between Cu2O and other booster biocides [
Composition |
A-0 | A-1 | A-2 | A-3 | A-4 | A-5 |
---|---|---|---|---|---|---|
Cuprous Oxide | 0 | 5 | 10 | 20 | 30 | 40 |
Xylene | 23 | 23.6 | 24 | 25 | 26 | 27 |
Methylisobutylketone | 5 | 5 | 5 | 5 | 5 | 5 |
Base polymer | 9 | 8.7 | 8.5 | 8 | 7.5 | 7 |
Rosin | 9 | 8.7 | 8.5 | 8 | 7.5 | 7 |
Barium sulfate | 50 | 45 | 40 | 30 | 20 | 10 |
Anhydrous ferric oxide | 1 | 1 | 1 | 1 | 1 | 1 |
Oxidized polyethylene wax | 1 | 1 | 1 | 1 | 1 | 1 |
Amide wax | 2 | 2 | 2 | 2 | 2 | 2 |
a) Values in the Table indicate mass %.
Polyvinyl chloride (PVC) plates (50 mm x 50 mm x 2mm) that were not coated with the test paint were used as the control plates in laboratory experiments. PVC control plates (150 mm x 100 mm x 5mm) that were used in the field experiment were also not coated with test paints, but their surfaces were scrubbed with sandpaper (#120) prior to use. For the experimental plates, PVC plates were used in the laboratory experiments, while sandblasted steel plates (Sa 2.5) were used in field experiments. These test plates had the same size as their control counterparts, and were coated with test paints on only one side. These test plates were coated first with 100 μm thickness of epoxy resin, then with a second coating of 100 μm thickness of epoxy binder, and dried at room temperature for 20 h. The dried test plates were further twice coated with 50 μm thickness of the test paints, and finally dried for 7 days at room temperature [
The novel dynamic aging system consisted of a water tank (ca. 230 L) installed with an apparatus to hold the test plates, a thermostat that kept the temperature constant at 20±3°C, a controller and a primary storage tank (ca. 300 L, the replenishing rate: ca. 0.7 L/min.), as shown in
(a) the cylinder with test plates fixed on it, and (b) the test plates inside the cylinder.
During the aging process, the cylinder apparatus holding the test plates was rotated by a motor at a plate surface speed of 5 m/sec (ca. 10 knots) for 45 days with water flow rate of 50 L/hr. This aging process was equivalent to 20,000 km of travel distance by oceangoing vessels. During its operation, the water inside the apparatus was always renewed by the continuously flowing filtered seawater in it, thereby preventing leached Cu2O from accumulating inside the apparatus and being re-absorbed into the surface of the test plates. After the aging process, the surfaces of test plates were covered with microbial biofilm. Microbial biofilm can affect the leaching rate of antifouling agents [
The concentration of Cu2O in the test water of each experimental group was measured after each experiment. Ten ml of the test solution was first desalted by solid-phase extraction method (Inert SEP ME-2, GL Science), and its Cu2O concentration was determined using ICP-MS (Agilent 7500i) after adjusting the amount of the solution to 10 ml by adding ultrapure water (Milli-Q system, Merck Millipore).
Seven-liter glass tanks were used in laboratory experiments. These tanks were each filled with 2 L of the test seawater prior to the experiments. During experiments, 1 μm mesh filtered test seawater was continuously pumped (14ml / min.) into the tank from the test seawater storage tank in a laboratory using a peristaltic pump. A drainage system was attached to each tank to allow water in the tank to flow out through a siphon tube. The rate of test seawater exchange inside the tank was adjusted to ca. 10 exchanges / day. Nine replicate tanks were prepared for one experimental group. Experiments were conducted in an incubator with a 12 h light: 12 h dark environment at a 2000 lux light intensity during the light period, simulating a light/ dark period where actual ships’ hulls are exposed. Water temperature, pH and salinity of the test water in the tanks were measured at the start and 24 h after experiment.
No specific permits were required to collect mussels in the study sites.
During acclimation, the shell movements of living mussels were observed, and were selected for the experiments. Mussels were detached from the substrate by carefully cutting their byssus threads with scissors, making sure that tissues connected to the byssus threads were not damaged. Five mussels were fixed to the surface of each test plate (50 mm x 50 mm x 2 mm), with their body axes parallel to the surface. A cyano-acrylate adhesive (Aron Alpha A, surgical grade, Daiichi Sankyo) was used to fix the mussels to the test surface. A diagram of a test plate with mussels fixed on its surface is shown in
(a), the position of five mussels fixed on the test plate; (b), top view of an individual (enlarged) with its byssus threads and attachment plaques; and (c), side view of an individual fixed on top of a filter paper glued to the test plate.
A total of nine experiments on byssus threads production of mussels were conducted; three times each in October, November and December 2013. In each experiment, three test plates for the control and one test plate each for the six experimental groups were evaluated, with each test plate having 5 mussels fixed to it. Accordingly, a total of 405 mussels were used in the nine experiments; 135 individuals in the control groups and 270 individuals in the experimental groups. No mortality was observed throughout the experiments and less than one individual per group was observed detached in the control and experimental groups.
The inhibiting effect of Cu2O on the surface of the test plate was evaluated by comparing the number of byssus threads between the experimental and control groups. The average number of byssus threads produced by an individual mussel in the control group was calculated according to Eq 1:
The average number of byssus threads produced by an individual mussel in experimental group is calculated according to Eq 2:
The ratio of byssus threads production (R), which is the average number of byssus threads produced in the experimental group in comparison to that of the control group, was calculated according to Eq 3:
Field experiments were conducted in August (Round-1, summer), October (Round-2, autum), December (Round-3, winter) of 2013, and in May 2014 (Round-4, spring) at Miyajima (Hiroshima Prefecture, Japan: St.1, 34°15'N, 132°15'E) and Tamano (Okayama Prefecture, Japan: St.2, 34°31'N, 133°59'E) experimental sites, located in the inland sea of Japan, as shown in
The source of the map is provided for free from
Test plates were coated using the same test paints shown in
Statistical analysis including one-way analyses of variance (ANOVA), nonparametric tests, Kruskai-Wallis test (p<0.05), and calculation of correlation coefficients, were conducted using PRISM version 6.0h (GraphPad Software, 2015).
The water temperature, salinity and pH of the test waters in the nine experiments were controlled at 20.3 ± 0.6°C, 30.6 ± 0.4 ‰ and 8.2 ± 0.1, respectively. The concentrations of Cu2O in the test waters of the control groups ranged from 0.2 μg/L to 0.8 μg/L in the three experiments. On the other hand, the concentrations of Cu2O in test waters of the experimental groups were 4.5 μg/L, 9.6 μg/L, 17.9 μg/L and 21.2 μg/L in the groups with 5 wt.% Cu2O content (A-1), 10 wt.% Cu2O content (A-2), 20 wt.% Cu2O content (A-3) and 30 wt. % Cu2O content (A-4), respectively, in the December 2013 experiment. The concentrations of Cu2O in the test waters of the 40 wt. % Cu2O content (A-5) groups ranged from 22.4 μg/L to 42.2 μg/L in the three experiments. Results indicate that the amount of Cu2O elution in the test water increased with increasing Cu2O content of the paints.
The concentration in the dynamic aging tank was 26 ppb (avg., n = 3) after dynamic aging. Furthermore, at the 0 wt. % Cu2O content group, the authors confirmed that the concentration after bioassay was almost the same as with the level in natural seawater (< 1 μg/L). The leaching rate of each panel was confirmed to be at a steady state after 45 days. These results suggest that there was no absorption from the water in the dynamic aging tank, and no subsequent leaching from the paint surface. Therefore, the authors concluded that the concentration build-up of Cu2O had no effect on the subsequent leaching from the surface of the test panels.
The number of byssus threads produced by the mussels differed in each of the control groups of the nine experiments conducted in October, November and December. This result is consistent with previous reports [
The R values for the paints containing different concentrations of Cu2O are shown in
The byssus threads production ratios in relation to the concentration of Cu2O in the paints, obtained in October, November and December 2013. The error bars on the open triangles indicate the SD in the ratio of byssus threads production.
Average seawater temperatures and results of the field experiments at Miyajima (St.1) and Tamano (St.2) experimental sites are shown in Figs
Hence, fouling on the plates at both experimental sites generally exhibited a similar tendency, which was a decrease in the degree of fouling with increasing Cu2O content in the paint. This tendency of decreasing degree of fouling with increasing Cu2O content in the paint was also observed in all rounds. The average rank of fouling for each experimental group of each site was calculated from values obtained from the four rounds.
In designing a laboratory experiment to assess the efficiency of AF paints, the authors took into consideration the following: (1), the condition of the test water; (2), the test organism; and (3), preparation of the test plates in a manner that simulated leaching from actual ships’ hull. In regard to the condition of the test water in bioassays, most literature available performed bioassays in a still water condition [
Previous reports have assessed the efficiency of AF paints by quantifying byssus threads produced by mussels fixed on the surface of test plates and comparing the results between the control and experimental groups in a still water condition [
In general, newly sprayed AF paints are known to have higher leaching rates, and leaching stabilizes after a certain period of time, normally after 45 days under laboratory controlled condition [
In this study, the repellant effect of AF paint on mussels was evaluated based on the production of byssus threads in a flow-through system. According to a toxicity study on mussels that was conducted in a still water condition, CuSO4 5H2O was detected to be lethal from 250 mg/L, and mussel mortality was 68% at 500 mg/L [
The quantification of byssus threads production of mussels in a flow-through system proved to be an expedient, applicable and indicative method for evaluating the efficiencies of paints containing Cu2O of varying concentrations, in terms of the equipment and methodology used, and the duration of the experiment. Differences in results observed between experiments indicate that byssus threads production of mussels vary among individuals and with the season. To minimize individual and seasonal variability of results, byssus threads production can be calculated as ratios in relation to their respective controls (
In the field, the leaching rate of Cu2O from a commercially available paint was more than 10 μg/cm2/day after 1year [
In the field experiment conducted in this study, biofouling on test paints gradually decreased with increasing content of Cu2O in the paints, and antifouling effect was observed at concentrations of 20 wt. % Cu2O and higher. On the other hand, results of laboratory bioassays showed that the inhibiting effect of Cu2O on byssus threads production was significant from 5 wt. % Cu2O (p < 0.001) and higher. Byssus threads production ratio for Cu2O at concentration of 10 wt. % and higher (p < 0.0001) decreased below 50%. From these results, byssus threads production of mussels clearly decreased at concentrations of 10 wt. % Cu2O and higher.
To assess the antifouling property of the test paints, results of laboratory bioassay and field experiments were compared. The relationship between R and average ranks of fouling on test plates coated with varying concentrations of Cu2O are shown in
The byssus threads production ratios calculated from laboratory bioassay (▽) and the ranks of fouling observed at St. 1 (□) and St. 2 (○) field tests plotted in relation to the concentration of Cu2O in the paints. The error bars on the open triangles indicates the SD in the ratio of byssus threads production.
A method for evaluating the efficacy of biocide-releasing AF paints was investigated in order to establish a reproducible and effective laboratory bioassay with a flow-through system using
This study was conducted as part of the research project on “The study on development of ships’ structure regulations (2012)” of Japan Technology Research Association (JSTRA), which was funded by Nippon Foundation. The authors sincerely thank researchers Hiroshi Masuda, Hirohisa Mieno, Kazuki Kouzai and Eiichi Yoshikawa of Chugoku Marine Paints Co., Ltd., and Mamoru Shimada of Nippon Paint Marine Coatings Co., Ltd., for their preparation of the coated test plates, aging by the rotating cylinder device, and useful suggestion. The authors would also like to give their appreciation to the members of the working group in the research project of JSTRA for their fruitful and stimulating suggestions.