Ecotoxocicological assessment of antifouling biocides and non-biocidal antifouling paints 3. Sea-Nine3.1 Physico-chemical properties This chapter contains an ecotoxicological assessment of 4,5-dichloro-2-n-octyl-4-isothiazolin-3-on (DCOI), which is the active substance in Sea-Nine 211. 3.1 Physico-chemical propertiesTable 3.1 summarizes the physico-chemical properties of DCOI. Table 3.1
3.2 Biodegradation of DCOI in the aquatic environment3.2.1 Primary degradation in seawater Several studies have been made of the degradation of DCOI in the aquatic environment. It is stated that abiotic processes progress with half-lives of 9-12.5 days for hydrolysis and 13.4 days for photolysis. Biological processes are, however, of greater importance to the transformation of DCOI. Studies described by Shade et al. (1993) have shown that DCOI (10 µg/L) is transformed with a half-life of 11 hours in seawater with 7 × 104 bacteria/mL (total number of bacteria determined by counting in a microscope). Parallel tests with seawater samples with a lower number of bacteria (<1,000 bacteria/mL) resulted in longer half-lives for DCOI (Shade et al. 1993). These tests are not considered relevant as the biological activity of the seawater was unrealistically low. In a recent study, the transformation rate of DCOI (10 µg/L) was determined in seawater from the pleasure craft harbour of Jyllinge. The study demonstrated that 7.1% of the DCOI added remained after 72 hours at a temperature of 12°C (Jacobson and Kramer 1999). On the basis of the measured concentrations of DCOI (Jacobson and Kramer 1999), the biological half-life may be estimated at 14 hours at 12° C (Appendix 1, Section 2.4.1). 3.2.2 Mineralization and metabolites in aerobic sediment Transformation of DCOI Mineralization and metabolites A positive identification of three metabolites was achieved in a later study in which a microbial enrichment culture proved suitable for achieving higher concentrations of metabolites (Mazza 1993). The culture was enriched after dosing aquatic sediment with DCOI (5 mg/kg). A comparison between HPLC chromatograms of metabolites formed in the enrichment culture and in sediment showed that the products were almost identical. By use of the enrichment culture and more analytical methods (i.a. HPLC and GC/MS), two essential metabolites were identified as N-(n-octyl) malonamic acid and N-(n-octyl) acetamide. Furthermore, a third quantitatively less important product N-(n-octyl) b hydroxypropionamide, which is probably formed at anaerobic degradation, was identified. Table 3.2
The sediment from the aerobic biodegradation tests (Lawrence et al. 1991a) was further characterized as regards bound metabolites. Sediment samples sampled at the start of the tests and after 30 days were characterized by extraction with methylene chloride/methanol followed by extractions with HCl and NaOH (Kesterson and Atkins 1992a). Relatively water-soluble metabolites that are extracted with HCl, constituted <0.1% of the radioactivity added. Metabolites in the NaOH extract were further divided into fulvic acid and humic acid fractions containing 1.2% and 5.1%, respectively, of the 14C added after 30 days. The metabolites that were not extracted by these procedures were probably bound to humin or clay and constituted 45% of the 14C added after 30 days (Kesterson and Atkins 1992a). The results showed that the stable metabolites from DCOI were mainly bound to humic acid, humin and clay minerals in the sediment. Studies with Danish sediments Figure 3.1 Figure 3.2 In the study with the clayey sediment, water and sediment samples were sampled at the start of the incubation and after 28 and 42 days. Chemical analyses of DCOI and metabolites in these samples were made by Rohm and Haas (Spring House, Pennsylvania). The water samples turned out to have a low content of 14C (2.5-6% of the 14C added), which did not allow a more detailed characterization of metabolites. The analyses of the sediment samples from the same test showed that DCOI was transformed into compounds more polar than the parent compound and that a considerable part of the radioactivity added resisted extraction from the sediment (Table 3.3). Table 3.3
3.2.3 Mineralization and metabolites in anoxic sediment Transformation of DCOI Mineralization and metabolites Table 3.4
Water-soluble metabolites from the transformation of DCOI constituted between 3.6% and 9.3% of the radioactivity added throughout the entire test period. Metabolites that were bound to the sediment and could not be extracted with methylene chloride/methanol constituted a constantly high part of between 40% and 67% of the 14C added (Table 3.4). Further extraction with HCl and NaOH showed that relatively water-soluble metabolites constituted <0.1% while fulvic and humic acids constituted 0.6% and 3.6%, respectively, of the 14C added after 365 days. Metabolites that were still bound to the sediment, probably to humin or clay, constituted 30% of the 14C added (Kesterson and Atkins 1992b). The formation of metabolites binding to humic acids, humin and clay minerals in the sediment is in agreement with the results in the aerobic biodegradation tests (Kesterson and Atkins 1992a). Studies with Danish sediments
Figure 3.3 Water and sediment samples from the tests were sampled at the start of the test and after 28 and 56 days. Chemical analyses of DCOI and metabolites in the water samples were made by Rohm and Haas (Spring House, Pennsylvania). The analyses of water samples sampled at the termination of the test after 56 days showed that 4.0 ± 2.4% of the 14C added was present in the form of compounds with the same HPLC retention time as DCOI. In the same water samples, polar compounds constituted 13.7 ± 3.0% of the 14C added. The sediment samples were not analyzed as they contained 3-4 times less radioactivity than the sediment samples from the aerobic tests (Table 3.3). 3.2.4 Transformation and fate of DCOI in a harbour An investigation of the spread and removal of DCOI was carried out in the vicinity of a freshly painted ship and of another ship that had been painted a couple of months earlier. Both ships were lying in Korsør Harbour where the investigations were made on 26 and 27 October 1998. Those days, the temperature of the water was approx. 10°C and varied very little according the depth of the water (Steen et al. 1999). The wind was southwesterly (between approx. 240 and 255° on 26 October and approx. 200° on 27 October). The wind velocity was approx. 8-10 m/sec with wind blasts of up to 15 m/sec on 26 October and a little more on 27 October (Danish Meteorological Institute 1999). The entrance of Korsør Harbour points in a north-easterly direction why the water must be expected to have been pressed out of the harbour. The concentration of DCOI in the water phase was measured along two transects: one perpendicular to the direction of the ships and the other in north-easterly direction, i.e. in the wind direction. Most of the samples were taken on 26 October. The samples were taken over a relatively short period of time (approx. 5 hours) and the measured concentrations can thus only be considered valid for the day in question. The highest concentrations measured of DCOI were <300 ng/L close to the ships side (£1 m) and decreased to <50 ng/L at a distance of approx. 30 metres from the ship. The concentration of DCOI in a distance of 2 metres from the ships (along the transect and perpendicular to the ships) varied very little according to the water depth why the vertical mixing was considered to be total. Steen et al. (1999) have made model calculations in which Korsør Harbour was modelled as a one-dimensional box, in which the flow in and out of the harbour was neglected and in which the dispersion coefficient was varied between approx. 0.004-0.03 m2/s. This interval is stated to be the end points of the expected variation interval of the dispersion coefficient of the harbour. Apart from the spread, a first order disappearance kinetics is assumed for DCOI. The simulations were made with three different rate constants for this first order process: 0 day-1, 1 day-1, and 1 hour-1. As a result of the winds on 26 and 27 October, the dispersion must presumably have been high in the basin. By way of comparison it may be mentioned that the horizontal dispersion coefficient in Danish coastal waters typically varies between 0.04 and 5 m2/s (Harremoës and Malmgren-Hansen 1989). As regards the two transects, the best correlation between the measured and the calculated DCOI concentrations was achieved by use of a rate constant of disappearance of between 1 hour-1 and 1 day-1. With the rate constant, 1 hour-1, a good correlation was achieved between measured and calculated values close to the ships for one transect while the concentrations of the other transect was underestimated at distances of more than approx. 8 m. For both transects, the calculated concentrations are lower than the measured concentrations at larger distances from the ships (approx. 30 m). When assuming a rate constant of disappearance of 1 day-1, the calculated concentrations are higher than the measured concentrations close to the ships for both transects but lower than the measured concentrations farther away (approx. 60 m). There are thus indications that the rate constant of the disappearance of DCOI close to the ships is higher than the corresponding constant farther away from the ships. On this basis, the rate constant of disappearance of the whole basin is considered to be between 1 hour-1 and 1 day-1, which corresponds to a half-life of between approx. 0.69 and 16.6 hours. This half-life includes biological and abiotic transformation as well as processes like sorption to suspended matter, sedimentation, potential vertical mixing and potential imperfection in the calculation of the dilution in the harbour. 3.3 Bioaccumulation and aquatic toxicity3.3.1 Bioaccumulation Studies on the bioaccumulation of DCOI in fish are available but not in other types of organisms (e.g. mussels). The ability of DCOI to bioaccumulate in fish has been examined in laboratory tests over 28 days by use of [14C]DCOI. Two studies including chemical analyses of water and tissue samples have been made (Forbis et al. 1985; Derbyshire et al. 1991). In all tests, [14C]DCOI was continuously added to a flow-through system. Chemical analyses showed that, at the final part of the tests, the concentration of DCOI was considerably lower than the nominal concentration (e.g. 4.5% and 0.55% of the 14C added after 21 and 28 days, respectively (Leak 1986)) while DCOI was hardly measurable in the second test (Derbyshire et al. 1991). Presumably, the principal part of the remaining 14C activity in the water represented one or more polar metabolites. The BCF values found (measured as radioactivity) were more or less identical in the two studies. The BCF values were 130-200 for muscle tissue, 700-1,100 for internal organs and 600 for the whole fish (Forbis et al. 1985; Derbyshire et al. 1991). The chemical analyses demonstrated that only 1% of the radioactivity in the fish was intact DCOI (Leak 1986). In connection with the study by Derbyshire et al. (1991), HPLC as well as TLC was used for identifying 14C labelled substances accumulated in the tissue of the fish. These studies indicate that it was most likely a question of substances without an isothiazolone ring structure and that the substances were built into the protein of the fish. The results indicate that DCOI was transformed in the water, after which it was mainly polar and probably linear compounds that were taken up in the fish. This assumption is confirmed by the biodegradation of DCOI (cf. Section 3.2.2; Lawrence et al. 1991a). It is thus considered likely that the measured BCF values should rather be related to metabolites of DCOI but as only a few of these metabolites are identified, the importance of the recorded bioaccumulation of labelled 14C cannot be assessed. 3.3.2 Toxicity towards aquatic organisms Aquatic organisms Fresh water:
Seawater:
Furthermore, tests with one mussel and protozoans are quoted by Shade et al. (1993) and Debourg et al. (1993), respectively. In some of the tests, problems with maintaining a constant exposure concentration have been reported and not all results have been calculated on the basis of measured concentrations (see below). The result of these irregularities is an overestimation of the effect concentrations - resulting in an underestimation of the toxicity of the substance. The results, which are compiled in Appendix 4, show that there was no big difference in the sensitivity of freshwater and marine organisms. Table 3.5 summarizes the effects on the different groups of organisms. Table 3.5
The results from the algal tests performed (Forbis 1990) are calculated on the basis of the nominal concentration. The report on one of the tests shows that the concentration of DCOI decreased during the whole test period. Only 48% of the nominal concentration was left after 48 hours and, at the end of the test after 72 hours, it was only possible to measure the substance in the test vessels containing the highest concentration (Forbis 1990). The EC50 values stated are thus too high. A 21-day reproduction test with daphnids (Ward and Boeri 1990) was conducted in such a way that is difficult to draw certain conclusions. This is due to the use of various concentrations of a solvent in relation to the addition of DCOI and to large variation in the data. The NOEC value stated represents the lowest concentration tested but the way in which the test has been conducted does not exclude that effects of DCOI may have occurred at this concentration as the effect may be dimmed as an unintentional result of the solvent. As the result of this test is the lowest NOEC value found in the tests, this value forms the basis of the calculation of PNEC for DCOI. N-(n-octyl) malonamic Even with the above reservations, it must, however, be concluded that N-(n-octyl) malomanic acid is several orders of magnitude less toxic than DCOI. In connection with the investigations of N-(n-octyl) malomanic acid, QSAR calculations have been made of the toxicity of this metabolite and some substances with similar structure, which are important metabolites from the microbial transformation of DCOI. The results are given in Table 3.6. Table 3.6
The results in Table 3.6 indicate that the probable metabolites from the transformation of DCOI are neither particularly toxic nor bioaccumulative in aquatic organisms. Sediment-living Algal communities Effects of degradation of The relation between degradation and sorption of DCOI and the acute toxicity towards the marine crustacean Acartia tonsa has been examined in the present study. The tests were performed in systems with the sandy sediment and its seawater from the Sound (Appendix 2), which was also used in the biodegradation tests. DCOI was added in a concentration of 100 µg/kg to the sediment-seawater systems. Water phase and sediment were separated 20 min. after dosing and use of the water phase in tests with A. tonsa caused a mortality corresponding to 35% of the test organisms. Stationary incubation in the dark at 20-25°C resulted in the fact that there were no mortal effects on A. tonsa after day 1 (Figure 3.4). Similar results were achieved when the sediment-water systems were incubated in the light at an intensity corresponding to 340 µmol/m2 × s. Measurements made by VKI in the Sound show that, in the period from May to October 1998, the average light intensity was 420 µmol/m2 × s in a depth of approx. 1 metre. The light intensity used was thus approx. 80% of the mean value calculated on the basis of the measurements in 1998. The test results with A. tonsa show that DCOI sorbs to sediment or is transformed into metabolites with a considerably lower toxicity than the parent compound. The methods used are described in detail in Appendix 3. A parallel test was made with zinc pyrithione (cf. Section 4.4). Figure 3.4 3.4 Risk assessment of DCOICalculation of exposure
The model and the two scenarios are described in detail in Appendix 1. For the parent compound and the most essential metabolites, the following exposure concentrations were calculated for each of the two scenarios:
The three exposure concentrations were defined as the steady-state concentration of the sub-environment in question. I.e., the concentration which the calculated concentrations eventually approach when a continuous leaching of the parent compound to the water environment is simulated. The calculations of PEC have been made by use of realistic worst-case scenarios, which means that the parameters used in the model are based on realistically conservative assumptions, which results in the fact that, in practice, the calculated PEC values are seldom exceeded. The model used is not validated towards measured concentrations in harbour environments or navigation routes. More of the assumptions that form part of the simulation are of vital importance to the result of the calculations:
The biological half-life of DCOI of 14 hours, which was assumed in the simulation, is established on the basis of an experimentally determined half-life in seawater at 12°C (Jakobson and Kramer 1999). The half-life of DCOI in seawater and not in seawater and sediment was chosen as the result of the simulation is exposure concentrations at a continuous leaching of DCOI to seawater after steady state was achieved. When the pleasure craft are taken out of the water at the end of the sailing season, DCOI will probably be rapidly eliminated as DCOI is either transformed in the water phase or sorbs to the sediment, in which it is transformed with a very short half-life (cf. Sections 3.2.2 and 3.2.3). The exposure concentrations calculated for DCOI and its metabolites are approx. 50 times higher in the pleasure craft harbour than in the busy navigation route outside the harbour (Table 3.7). Table 3.7
Calculation of Predicted No-Effect Concentration (PNEC) The available studies on the aquatic toxicity of DCOI are considered representative and the data include long-term studies with fish and crustaceans. The algal test may be interpreted as a short-term as well as a long-term test (EC 1996). For DCOI, three NOEC values from long-term tests (fish, crustaceans and algae) are available, including the groups of organisms most sensitive in short-term tests (fish). On this basis, PNEC is calculated by dividing the lowest NOEC value, which is 0.00063 mg/L for crustaceans (Ward and Boeri 1990), by an assessment factor of 10 (EC 1996). This results in a PNEC of 0.00006 mg/L = 0.06 mg/L for DCOI. As already mentioned in Section 3.3.2, no unambiguous NOEC value can be derived from this long-term test with crustaceans (Ward and Boeri 1990). If this study is ignored, the results from one single long-term study with fish are available, in which NOEC was 0.006 mg/L. In this case, an assessment factor of 100 is applied, which results in a PNEC value calculated at 0.00006 mg/l = 0.06 µg/L, which is identical with the above calculated value. Calculation of PNEC for N-(n-octyl) malonamic acid is based on the lowest effect concentration. As data primarily originates from short-term tests, an assessment factor of 1,000 is used for the lowest effect concentration. For N-(n-octyl) malonamic acid, the lowest reported LC50 = 250 mg/L for rainbow trout while the value for daphnia (EC50 = 260 mg/L) as already mentioned above is a moot point. For the calculation of a PNEC for N-(n-octyl) malonamic acid, an LC50 value of 90 mg/L (towards daphnids) is used as this value is considered the actual effect concentration in the tests performed (cf. Section 3.3.2). This results in a PNEC value of 0.09 mg/L = 90 µg/L. The calculated PNEC for N-(n-octyl) malonamic acid is assumed to be representative of the other metabolites from the transformation of DCOI. The two calculations of PNEC are shown in Table 3.8. Table 3.8
Risk quotient Table 3.9
* N-(n-octyl) malonamic acid The stated risk quotients are calculated on the basis of realistic worst-case scenarios (Appendix 1), which are i.a. based on the assumption that 70% of the pleasure craft are painted with a bottom paint containing DCOI. On the basis of the assumptions made in the simulation and of the calculated PEC values, it is considered likely that a risk of chronic ecotoxic effects within the pleasure craft harbour may exist as, presumably, DCOI will constantly be applied by leaching from bottom paints. The risk quotient for DCOI out of the harbour is less than 1 and here, the risk of ecotoxic effects is considered to be low. Within as well as out of the pleasure craft harbour, a very small risk of ecotoxic effects of metabolites from the transformation of DCOI is considered to exist.
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