The effects of antifouling booster biocides on the GFP-rich supernatants were investigated in this study. Anemonia viridis contains different GFP molecules on their tentacles. The colour difference can be seen in the tentacles under day light and also UV-light easily (Figure 2, the photo was taken under daylight). In our research, there are 3 different regions in the tentacles of Anemonia viridis that were collected from Tuzla region. These regions are green zone (approximately 2 cm from the base) green/pink zone and pink zone (approximately 3 cm from the base). The comparison of different zones’ emission values of Anemonia viridis was given in Figure 3. The samples taken from green zone of the tentacles show the highest fluorescence intensity whereas the samples taken from pedal disk of Anemonia viridis has the lowest fluorescence intensity.
Figure 3 Comparison of different zone’s emission values of fresh Anemonia viridis (λex = 425nm; slt: 5 nm)
The changes of emission spectra of Anemonia viridis collected from different days (1st, 2nd, and 17th days) are shown in Figure 4. When the spectral properties of Anemonia viridis are concerned, these changes fit well the photoprotection of GFPs. Because it is possible by photosynthesis action which realizes between the waveband in the maximum of its spectrum and the waveband where photosynthetic pigments absorb .
Figure 4 Intra-species comparison of different fresh Anemonia viridis emission values (λex = 425nm; slt 5 nm for sample 1(1st day) and sample 2(2nd day); slt 2.5nm for Anemonia viridis sample 3-6 (collected on the 17th day)).
On the other hand, although the emission bands of some GFP are not the waveband of photosynthesis action, the energy transfers from green to green-pink region’s pigments can be occurred. Therefore, results suggest that fluorescent coupling is carried out the photoprotection function of green fluorescent pigments .
The range of irgarol concentration studied was 0.001-0.01 g/L. The effects of irgarol concentrations on the fluorescence intensity were studied and the results were given in Figure 5.
Figure 5 Effects of irgarol concentration on the relative GFP fluorescent intensity (%, Imax).
A remarkable relationship was observed between increased irgarol concentration and decreased fluorescence intensity in our samples. The experimental results show that fluorescence intensity decreased by more than 50% after 0.01 g/L irgarol concentration. GFP is very sensitive to irgarol when relative intensity begins to decrease even at low concentration between 0.001 and 0.01 g/L. The effects of irgarol on the inhibition in cell number and decrease in photosynthetic activity of marine organisms are discussed by Amara et al., 2018 . Based on the published reports on the irgarol [13- 18], it could be said that irgarol can mainly be associated with the disruption of photosynthetic electron transport chain and also it may interact with other important supramolecules in living organisms. Fernandez-Alba et al (2002) reported the range of EC50 for individual and mixture of booster biocides as 0.001 to 28.9 mg/L . They studied Vibrio fischeri and Daphnia magna as the model organisms. The toxicity of well used booster biocides, Irgarol 1051 and Sea-Nine 211, on marine alga Fucus serratus were studied by Braithwaite and Fletcher (2005) . No observable effect concentrations as 8 µg/L for both Irgarol 1051 and Sea-Nine 211 were given by the authors. Arrhenius et al (2006) studied the algal reproduction and periphyton photosynthesis in the conditions of well-known booster biocides such as TBT, diuron and irgarol . They reported the NOEC values for periphyton photosynthesis to be 32 nM, 1.8 nM and 178 nM for TBT, Irgarol and Sea Nine 211, respectively. On the other hand, NOEC values related to algal reproduction for TBT, Irgarol and Sea Nine 211 were given to be 133 nM, 2 nM and 96 nM, respectively. These low values reveal that photosynthetic organisms are remarkably affected by the booster biocides. In our research, we found the minimum concentration as 10 mg/L to decrease the fluorescent intensity as 55%. This value is well in line with the published reports discussed above. It is very important to note that the supernatant including GFPs were studied in this report compared to the published reports in which live organisms used in toxicity assays. The toxicity of booster biocides was also tested on some vertebrates. Okamura et al (2002) studied suspension-cultured fish (Oncorhynchus tshawytscha) cell line to show the toxicity of booster biocides released from antifouling paints. The authors reported low toxicity of sea nine and irgarol compared to the compounds such as copper pyrithione and zinc pyrithione in their study. As can be known from the biology of sea anemones, the anemones are consisted of symbiotic life form of zooxanthellae and anemones. Zooxanthellae settles inside the tentacles of anemones. Therefore, exposure of these photosynthetic algae to the booster biocides released from self-polishing antifouling paints may harm the population of zooxanthellae. Decreased intensity of zooxanthellae in seawater may be one of the explanations for the bleaching of anemones. However, the zooxanthellae density in seawater where the bleaching of anemones observed must be studied to explain this phenomenon.
Figure 6 Effects of seanine concentration on the relative GFP fluorescent intensity (%, Imax).
To study the effect of sea nine 211(N), the concentration ranges between 0.05-5.00 (%, v/v) was interacted with the GFP extract obtained from Anemonia viridis. According to Figure 6, no significant decrease was observed for sea nine 211(N), the structure of sea nine 211(N), contains a n-octyl, a carbonyl and two chloride groups. Although we did not observe remarkable negative effects on the fluorescence intensity of GFP, this does not mean that this chemical is safe for marine ecosystems. Many scientific papers report negative effects such as endocrine disruption and reproductive impairment , early developmental stages of the sea urchin Paracentrotus lividus [20–21], and inhibition of the detoxification system in Oryzias melastigma . A new regulation, a possible ban, on this chemical must be discussed under the lights of the scientific papers related to the effects of this chemical on marine ecosystem.
Zinc omadine, one of the well-known antifouling agents, was also studied to understand its effect on the GFP from Anemonia viridis. According to Figure 7, zinc omadine decreased the fluorescence intensity significantly.
Figure 7 Effects of zinc omadine concentration on the relative GFP fluorescent intensity (%, Imax).
Metabolic effect of the chemical was explained many years ago. Chandler and Segel (1978) reported its membrane disturbing effect . After banning of TBT based paints in the antifouling paint market, zinc omadin was used widely as one of the alternative compounds of TBT because of its easy degradation. However, many papers mentioned the resistant degradation of zinc omadin such as mentioned by Marcheselli et al (2010) . Active zinc atom in the structure might have been interacted with the 3-D structure of GFP. Reeder et al (2011) showed that zinc pyrithione shows antifungal effect by increasing cellular copper level. This causes collapsing of iron-sulphur clusters in the proteins . The toxic effects of zinc omadine on the three marine microalgae Tisochrysis lutea, Skeletonema marinoi and Tetraselmis suecica were studied by Dupraz et al (2018) . When they tested the toxicities of irgarol, zinc pyrithione and copper pyrithione individually, they found the toxicity of irgarol remarkable compared to zinc pyrithione and copper pyrithione. The authors also reported the increased efficiency in binary mixtures with copper pyrithione. Our results partially support the values of Dupraz et al (2018) . Single chemical such as zinc pyrithione may reveal different effects on the different microalgal species. Marcheselli et al (2011) studied the effects of zinc pyrithione on the marine mussel Mytilus galloprovincialis . The authors reported the bioaccumulation of zinc pyrithione in the tissues of Mytilus galloprovincialis. They also reported HSP over-expression and DNA damage in the important tissues of this marine mussel even if the samples were exposed to the non-lethal concentrations. Although we did not measure the level of zinc pyrithione in the tissues of sea anemones, it is clear that a possible zinc pyrithione bioaccumulation may be expected in the living organisms in the harbours.
The bioactive mixtures are also used in the antifouling paint manufacturing. The composition of different bioactive agents show better antifouling performance compared to the individual compounds. One of the recommended commercial products, acticide produced by Thor, was also tested in this study. The active agents in acticide are isothiazolinones, iodopropynyl butyl carbamate and a range of quaternary ammonium compounds reported by the manufacturer (https://www.thor.com/biocides.html).
Figure 8 Effects of acticide concentration on the relative GFP fluorescent intensity (%, Imax).
The range of acticide concentration studied was 0.25-3% (v/v). In order to show the relationship between acticide concentration and fluorescence intensity, Figure 8 was plotted. From Figure 8, it could be said that increased concentration of acticide decreased the fluorescence intensity from Anenomia viridis extracts. According to the literature, it has fungistatic and bacteriostatic properties. This product is generally used to prevent the microbial colonisation in the outer surfaces of the historical buildings for restoration purposes . There has been no published item in the scientific literature related to its antifouling performance of this product. Since we did not study each of the ingredients separately, each chemical in the content might have showed different effects on the GFP fluorescence intensity. Further studies can be carried out for understanding the responsible chemical in the acticide on the decreased fluorescence intensity of GFP from Anemonia viridis.