Survey for the occurrence of the new antifouling compound Irgarol 1051 in the aquatic environment

PII: S0043-1354(98)00501-6

Wat. Res. Vol. 33, No. 12, pp. 2833±2843, 1999 # 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/99/$ - see front matter

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SURVEY FOR THE OCCURRENCE OF THE NEW ANTIFOULING COMPOUND IRGAROL 1051 IN THE AQUATIC ENVIRONMENT D. LIU1*, G. J. PACEPAVICIUS1, R. J. MAGUIRE1, Y. L. LAU1, M M H. OKAMURA2* and I. AOYAMA2* National Water Research Institute, Burlington, Ont., Canada L7R 4A6 and 2Research Institute for Bioresources, Okayama University, Kurashiki 710, Japan

1

(First received August 1998; accepted in revised form November 1998) AbstractÐIrgarol 1051, (2-methylthio-4-tert-butylamino-6-cyclopropylamino-s-triazine), is a newly developed herbicidal additive for use in copper-based antifouling paints. It is intended as replacement for the highly toxic antifouling agent tributyltin, which has been regulated internationally. Currently, there is no information in the open literature on its environmental occurrence outside Europe. A twoyear survey was conducted in 1996±1997 to investigate the occurrence of Irgarol 1051 in Canadian and Japanese aquatic environments. Six large trade ports (Vancouver, Toronto, Montreal, Halifax, Mizushima and Kobe), 73 marinas and 13 ®shery harbors were surveyed. Irgarol 1051 was not detected in the Canadian aquatic environment, but was positively identi®ed in the enclosed coastal waters of the Seto Inland Sea in Japan. Among the six trade ports surveyed, only the Mizushima Port had low levels of Irgarol 1051, up to 19.5 ng/L. Approximately 27% of the marinas surveyed in the Seto Inland Sea were found to have been contaminated by Irgarol 1051, ranging in concentration between 12.5 and 264.2 ng/ L. Irgarol 1051 was found more frequently in ®shery harbors than in marinas, indicating that besides marinas and trade ports, ®shery harbors can also be a signi®cant source of contamination for the aquatic environment. Survey for Irgarol 1051 in the ®shery harbors has not been reported before, and it is suggested that ®shery harbors, in addition to ports and marinas, should be included in the survey list during the Irgarol 1051 monitoring study. Irgarol 1051 has been reported to be highly toxic to nontarget marine algae with the observable growth inhibition at a concentration as low as 50 ng/L, which is well within the ambient concentration levels found in some localities of the Seto Inland Sea. # 1999 Elsevier Science Ltd. All rights reserved Key wordsÐIrgarol 1051, fouling, antifouling compound, herbicide, survey, monitoring, port, marina, ®shery harbor

INTRODUCTION

Irgarol 1051 is a newly developed herbicidal additive for use in copper and zinc based antifouling paints (Gough et al., 1994). It is intended as a replacement for the highly toxic antifouling organotins biocides (e.g. tributyltin and triphenyltin) which were regulated internationally in the late 1980's and early 1990's, mainly due to their high toxicity and severe impact on the aquatic ecosystem (Beaumont and Newman, 1986; Langston et al., 1990). Irgarol 1051 (2-methylthio-4-tert-butylamino-6-cyclopropylamino-s-triazine) has recently emerged as a new aquatic contaminant. It has been found in the coastal waters of England, France, The Netherlands, Spain and Sweden (Readman et al., 1993; Gough et al., 1994; Dahl and Blanck, 1996; Ferrer et al., 1997a,b; Scarlett et al., 1997; Steen et al., 1997). It was also found in the fresh waters of Lake Geneva in Switzerland (Toth et al., 1996), thus indi*Author to whom all correspondence should be addressed. [Fax: +1-905-336-4989; e-mail: [email protected]].

cating its widespread occurrence in the European aquatic environment. Little is known to date about the long-term toxicity and degradability of Irgarol 1051 (Toth et al., 1996; Ferrer et al., 1997b). Irgarol 1051 is not considered to be readily biodegradable and its degradation in seawater and freshwater is slow, with half-lives of about 100 and 200 days, respectively (Ciba Geigy, 1995; Liu et al., 1997). It is also immune to bacterial degradation. The white rot fungus Phanerochaete chrysosporium can biotransform Irgarol 1051 via the mechanism of N-dealkylation to yield a stable metabolite M1 (2-methylthio4-tert-butylamino-6-amino-s-triazine). However, the heterocyclic ring of Irgarol 1051 remains intact after the biotransformation (Liu et al., 1997), implying the inherent persistence of Irgarol 1051 and its possible accumulation in the aquatic environment. Irgarol 1051 is not presently registered for use in Canada (under the Pest Control Products Act). In Japan, the use of antifouling agents is under the auspices of Japan Paint Manufacturers Association

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Fig. 1. Location of sampling stations in (A) Vancouver Harbor, (B) Toronto Harbor, (C) Montreal Harbor and (D) Halifax Harbor.

2834 D. Liu et al.

Antifouling compound in aquatic environment

Fig. 2. Location of sampling stations in (A) Mizushima Harbor and (B) Kobe Harbor.

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D. Liu et al.

and the Shipbuilding Research Association of Japan. In 1993 the latter had completed a toxicity study of 23 new antifouling chemicals including Irgarol 1051. To the authors' knowledge, no data are currently available in the open literature on the ambient levels of Irgarol 1051 in North American and Asian aquatic environments. In anticipation of its registration in Canada, and because it is already registered in some other countries including the OECD countries and the US, a joint long-term monitoring study between Canada and Japan was initiated in 1996 to determine whether Irgarol 1051 is present in the Canadian (i.e. via leaching from ships painted in other countries) or Japanese aquatic environment. Several large trading ports and many marinas and ®shery harbors were sampled in the study. This paper summarizes the results of our ®rst two-year study (1996±1997), focusing on the distribution of Irgarol 1051 in the dissolved phase. Irgarol 1051 in most marine waters is predominantly associated with the dissolved phase (Tolosa et al., 1996; Ferrer et al., 1997b). Such information is useful in the development of a management strategy for the control of Irgarol 1051 in the Canadian and Japanese aquatic environment. MATERIAL AND METHODS

Chemicals Irgarol 1051 [(2-methylthio-4-tert-butylamino-6-cyclopropylamino-s-triazine), identi®cation No. 84611.0] of high grade (95%) was a gift of the Ciba-Geigy Canada Ltd., Mississauga, Ont. L5M 5N3. Irgarol 1051 has a log Ko/w of 3.6 with a water solubility of 7 mg/L (Steen et al., 1997) and is also nonvolatile with a vapor pressure (258C) of 8.8  10ÿ5 Pa (Ciba Geigy, 1995). Pesticide grade organic solvents were obtained from Caledon Laboratories,

Georgetown, Ont. The sodium sulphate used for drying organic extracts was heated to 5008C for 24 h before use. All glassware were also rinsed with pesticide grade solvents before use. All other chemicals used in the experiments were reagent grade or better. Sampling sites In this joint Canadian±Japanese research project on aquatic contaminants (International Comparative Study on Toxicity Assessment of Priority Chemicals), subsurface water samples taken from six large trade ports [i.e. four Canadian (Vancouver, Toronto, Montreal, Halifax) and two Japanese (Mizushima and Kobe) harbors], 73 marinas and 13 ®shery harbors were analyzed for Irgarol 1051. All the six ports have industrial establishments and busy commercial shipping activities. The Mizushima and Kobe Harbors, located in the Seto Inland Sea, are among the top ®ve exporting ports for Japan, and their busy shipping activity would presumably facilitate the detection of Irgarol 1051 in the Japanese aquatic environment. The Vancouver, Halifax, Toronto and Montreal Harbors are also the most important importing and exporting ports for Canada. Detailed Irgarol 1051 sampling sites for each of the six trade ports are shown in Figs 1 and 2. In the ®gures each sampling site is given a numerical or alphabetical number, followed by a slash. The number before the slash indicates the site number and the one after the slash denotes the water depth (in meters) at that particular site. In the 1997 Canada survey for Irgarol 1051, ®ve more additional ports and marinas were added to the study list. They were Windsor (on the Detroit River), Port Stanley (Lake Erie), Hamilton (Lake Ontario), Kingston (on the St. Lawrence River) and Midland (Georgian Bay of Lake Huron). In the Japanese survey for Irgarol 1051, water samples were also collected from 63 marinas and 13 ®shery harbors in 10 prefectures (Fukuoka, Yamaguchi, Hiroshima, Okayama, Hyogo, Osaka, Wakayama, Tokushima, Kagawa and Ehime) along the coast of the Seto Inland Sea (Fig. 3). Sample collection and storage Subsurface (0.5 m depth) freshwater (i.e. river and lake water) and enclosed coastal water (i.e. harbors, bays and coves) samples were collected from aboard a 4-m aluminum boat which had not been painted with any antifoul-

Fig. 3. Map of the investigated marinas and ®shery harbors in the Seto Inland Sea (Japan).

Antifouling compound in aquatic environment ing paint. Samples were taken using precleaned 2-, 3- or 4L glass bottles held in a weighted stainless-steel frame ®tted with a stopper and a subsurface trigger to avoid surface contamination. In the Canadian survey for Irgarol 1051, at least four bottles (4  4 L) of water were collected from each sampling site and the collected samples were extracted on the day of collection. In the Japanese survey, one bottle (2- or 3-L) of water was taken from each sampling site, and the extraction was conducted on the day of collection. Sample preparation In the Canadian survey, only liquid±liquid extraction (LLE) procedure was used to extract Irgarol 1051 from the collected water samples, in view of the anticipated very low ambient concentrations of Irgarol 1051 in the water samples and the resulting necessity to process a large volume of water. In brief, 4 L of the water sample were transferred to a ¯at-bottom 4.3-L glass bottle containing 200 mL of dichloromethane (DCM) and a 7.5-cm magnetic stirring bar. The contents were thoroughly mixed for 30 min and then transferred to a 5-L glass separatory funnel. The extraction was repeated three times each with 200 mL of fresh DCM. For each sampling site, 16 L of water sample were processed and the pooled DCM extracts were dried through anhydrous sodium sulphate. A toluene keeper was added to the resulting extracts which were then concentrated to 5 mL on a rotary evaporator. Further concentration and solvent exchange into toluene were performed under a gentle stream of nitrogen. The toluene extract was analyzed for Irgarol 1051 and its degradation product M1 (Liu et al., 1997) by GC±NPD and GC±MS. Both solid-phase extraction (SPE) and LLE procedures were used to extract Irgarol 1051 from the enclosed coastal waters of Seto Inland Sea (Japan). In the ®rst ®eld study, 3 L of water samples were collected from each of the six sampling sites in the Mizushima Harbor [Fig. 2(a) and Table 1]. The water was ®rst passed through a glass ®ber ®lter (1 mm) and then extracted consecutively with DCM (2  300 mL). The pooled DCM extracts were reduced in volume at 408C using a rotary evaporator and a centrifugal evaporator, followed by the addition of 0.30 mL acetonitrile (MeCN) to the residue in the test tube. The MeCN solution was subject to a centrifugal ®ltration and 1 mL of the MeCN extract was used in the GC±MS analysis for Irgarol 1051. In the second and third ®eld studies, 2 L of water samples were collected from each sampling site [Fig. 2(b) and Fig. 3; Table 1] and extracted with 2  200 mL of DCM. An aliquot of 0.20 mL MeCN was used in the ®nal stage of Irgarol extraction. In the fourth ®eld study (Fig. 3, Table 1), 2 L of ®ltered (1 mm) water sample from each sampling site were passed through an ODS column (Sep Pak plus C18 ENV), and the column was eluted with 5 mL of methanol. The methanol extract was evaporated to dryness, followed by the addition of 0.20 mL MeCN to the residue. After passing through a 0.45 mm membrane ®lter (centrifugal ®ltration), the MeCN extract was directly used in the GC± MS analysis for Irgarol 1051 and its degradation product M1. Chemical analysis In the Canadian survey for Irgarol 1051, the toluene extracts were analyzed on a Hewlett Packard 5890 series II gas chromatograph equipped with a nitrogen±phosphorus detector (3008C) and a ¯ame ionization detector (3008C) utilizing an oven program with a 2-min hold at 808C and a temperature ramp of 108C/min to 1508C followed by a temperature ramp of 48C/min to 2808C and a ®nal temperature ramp of 88C/min to 3008C. The columns used were dual DB5 coated capillary columns

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(0.25 mm  27 m) which had been installed into the injector (2008C) in the splitless mode with a constant helium carrier ¯ow of 0.8 mL/min. Mass spectral analysis was performed using the same temperature program and column stationary phase (0.25 mm  30 m) on a Hewlett Packard 5971A mass selective detector (MSD) and MS Chem Station. The MSD was operated in electron impact (EI) mode with an ionization potential of 70 eV and a source temperature of 1908C. The scan range was 50± 500 amu and quanti®cation of Irgarol 1051 was obtained using selective ion monitoring (SIM). Three selective ions, m/z 182, 238 and 253, were used to con®rm the presence of Irgarol 1051. In the Japanese survey for Irgarol 1051, 1 mL of the MeCN extracts was analyzed using a Hewlett Packard 5890 series II plus gas chromatograph ®tted with an oncolumn injector (2508C) and mass detector (GC-Mate, Jeol) utilizing an oven program with a 2-min hold at 508C and a temperature ramp of 408C/min to 2008C, then followed by a temperature ramp of 108C/min to 3208C. The column used was a low bleed HP-5MS capillary column (0.25 mm  30 m) which had been installed into the injector (2508C) in the splitless mode with a constant helium carrier ¯ow of 1.3 mL/min. Throughout the study, Irgarol 1051 standard solutions of 0.1±5 ng/mL were analyzed daily during the analysis to check the instrumental conditions and the chromatographic behavior (e.g. retention time, peak shape and separation). Con®rmation of Irgarol 1051 in the MeCN extracts was based on comparing the three major ions (m/z 253, 238 and 182) with those obtained from the Irgarol 1051 standards. Quanti®cation of Irgarol 1051 in the MeCN extracts was based on the peak area of the molecular ion m/z 253. RESULTS AND DISCUSSION

A linear relationship (r = 0.996) was observed when the peak area measurements of the molecular ion m/z 253 were plotted against the corresponding measurement of Irgarol 1051 concentrations (data not shown), thus establishing the use of this quantitative method in the determination of Irgarol 1051 concentrations in the MeCN extracts of water samples taken from the Seto Inland Sea. The protonated molecular ion m/z 254 was also used in quantifying Irgarol 1051 in the Spanish coastal waters (Ferrer et al., 1997a,b). Other major ions used in the quanti®cation of Irgarol 1051 in environmental samples included ions m/z 196 (Steen et al., 1997), and m/z 182, 238 and 253 (Tolosa and Readman, 1996). In the present study GC±NPD and GC± SIM-MS with selective ions m/z 182, 238 and 253 were used to quantify Irgarol 1051 in the Canadian aquatic environment. Both methods had been used in the quantitative determination of Irgarol 1051 in the coastal waters of Monaco (Tolosa and Readman, 1996). Currently, SPE is the most commonly used method for the extraction of Irgarol 1051 from aqueous environmental samples (Readman et al., 1993; Tolosa et al., 1996; Toth et al., 1996; Zhou et al., 1996; Ferrer et al., 1997b; Scarlett et al., 1997; Steen et al., 1997). Other extraction methods include the conventional LLE procedure (Gough et al., 1994) and the newly developed enzyme-linked immunosorbent assay (ELISA) (Ferrer et al.,

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Table 1. Dissolved Irgarol 1051 in the waters of Seto Inland Sea Grid reference Survey No. 1

2

3

4

classi®cation

site

latitude N.

longitude E.

1/4 m 2/10 m 3/16 m 4/16 m 5/16 m 6/16 m

34831'16.560 34830'20.310 34831'8.120 34829'42.810 34829'30.00 34828'42.30

Mizushima 133844'26.510 133844'23.170 133844'28.710 133843'26.130 133844'18.330 133844'49.800

A/12 m B/12 m C/15 m D/15 m E/12 m F/12 m G/13 m H/14 m I/14 m J/14 m K/14 m

34840'14.370 34839'23.120 34839'25.930 34839'23.430 34839'57.810 34841'23.750 34841'3.750 34840'29.68 34840'34.680 34'40'40.93 34841'11.870

135812'18.630 135812'18.630 135814'18.160 135814'33.980 135813'45.110 135814'22.730 135815'20.30 135815'41.130 135816'25.420 135817'31.170 135816'55.660

331 340 340'

33853'40.850 33854'33.200 33855'39.760

274 275 276 279±1 279±2 279' 2790 280 281 282 282' 285 285' 2850

sampling date y.m.d

Irgarol conc. ng/L

Port trade trade trade trade trade trade

port port port port port port

96.9.16 96.9.16 96.10.21 96.10.21 96.10.21 96.10.21

19.5 0.0 6.4 3.6 0.0 0.0

Kobe Port trade trade trade trade trade trade trade trade trade trade trade

port port port port port port port port port port port

96.12.19 96.12.19 96.12.19 96.12.19 96.12.19 96.12.19 96.12.19 96.12.19 96.12.19 96.12.19 96.12.19

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Fukuoka Prefecture 130855'24.190 marina 130856'12.890 marina 130856'42.330 marina

97.7.13 97.7.13 97.7.13

0.0 0.0 0.0

34811'5.310 3486'26.870 33857'12.180 33858'45.620 33858'45.620 3480'19.680 33859'3.120 3480'0.00 33856'45.00 3385543.120 33858'13.430 3481'3.430 3481'3.750 3481'11.710

Yamaguchi Prefecture 132814'3.750 marina 132812'10.190 marina 13288'14.640 marina 131848'48.510 marina 131848'53.780 marina 131850'1.640 marina 131847'52.960 marina 131832'47.00 marina 131814'36.440 marina 131815'27.420 marina 131810'34.210 ®shery harbor 131834'59.350 marina 131835'6.210 marina 131835'4.100 marina

97.7.15 97.7.14 97.7.14 97.7.14 97.7.14 97.7.14 97.7.14 97.7.14 97.7.13 97.7.13 97.7.13 97.7.13 97.7.13 97.7.13

23.3 95.6 264.2 15.7 0.0 41.0 65.8 14.2 37.6 0.0 0.0 21.5 37.7 27.8

251±1 251±2 252±1 252±2 256 256' 265 264' 272' 273±1 273±2 273'

34821'37.180 34821'34.680 34820'16.250 34820'16.10 34822'32.570 34822'30.460 34821'58.980 34816'43.120 34815'18.430 3489'26.320 3489'26.320 3489'52.18

Hiroshima 132825'52.500 132825'55.310 13383'10.280 13383'11.250 133820'34.680 133820'36.530 132830'11.860 132845'24.960 132831'38.80 132826'24.840 132826'26.420 132827'3.690

Prefecture marina marina marina marina marina marina marina ®shery harbor ®shery harbor marina marina ®shery harbor

97.7.15 97.7.15 97.7.15 97.7.15 97.7.16 97.7.16 97.7.15 97.7.15 97.7.15 97.7.15 97.7.15 97.7.15

0.0 0.0 0.0 14.3 0.0 77.9 12.5 0.0 142.4 0.0 0.0 15.6

244 248' 249'

34831'33.590 34830'45.00 34830'1.560

Okayama Prefecture 133840'8.640 marina 133839'21.350 ®shery harbor 133830'19.330 ®shery harbor

97.7.16 97.7.16 97.7.16

0.0 13.3 0.0

216 235 235±1 235±2 235±3 235±4 235±5 237 237±1 242 245 245'

34827'13.120 34835'2.420 34829'17.810 34831'11.250 34832'47.180 34835'41.870 34835'31.250 34836'50.310 34836'45.62 34836'36.170 34843'30.00 34843'35.310

Okayama Prefecture 133854'4.920 marina 133857'47.460 marina 133857'11.600 marina 133857'52.30 marina 13480'52.550 ®shery harbor 13481'54.600 marina 133859'25.280 marina 13489'51.150 marina 13489'47.100 marina 13489'0.430 marina 134819'18.40 marina 134816'17.690 ®shery harbor

97.8.21 97.8.21 97.8.21 97.8.21 97.8.21 97.8.21 97.8.21 97.8.17 97.8.17 97.8.17 97.8.17 97.8.17

0.0 85.3 0.0 0.0 0.0 210.6 0.0 0.0 0.0 0.0 0.0 105.0

Antifouling compound in aquatic environment

2839

Table 1 (continued ) Grid reference Survey No.

classi®cation

sampling date y.m.d

Irgarol conc. ng/L

site

latitude N.

longitude E.

197±1 197±2 197' 201' 209 212

34842'24.600 34842'24.760 34843'16.560 34846'46.250 34843'20.540 34846'11.870

Hyogo Prefecture 135819'55.390 marina 135819'51.700 marina 135820'14.640 marina 134839'49.21' ®shery harbor 135819'34.740 marina 134846'13.470 marina

97.8.18 97.8.18 97.8.18 97.8.17 97.8.18 97.8.17

0.0 0.0 0.0 58.8 0.0 0.0

186 187 192±1 192±2 194

34826'1.820 34833'43.120 34820'6.560 34820'5.00 34832'47.500

Osaka Prefecture 135820'11.740 marina 135827'19.330 marina 135810'47.920 marina 135810'58.120 marina 135826'38.900 marina

97.8.18 97.8.18 97.8.18 97.8.18 97.8.18

0.0 0.0 0.0 0.0 0.0

195±1 195±2

3486'25.620 3488'25.620

Wakayama Prefecture 13588'24.140 ®shery harbor 135811'54.720 ®shery harbor

97.8.18 97.8.18

0.0 0.0

307 308 310 311

33859'21.250 3485'16.250 3482'58.430 3482'20.930

Tokushima Prefecture 134837'53.900 marina 134834'49.680 marina 134835'17.630 marina 134834'56.540 ®shery harbor

97.8.19 97.8.19 97.8.19 97.8.19

0.0 0.0 0.0 81.3

291 292' 293 294 296 297 299 302 304

3489'24.680 34823'15.310 34822'20.02 34820'10.00 34816'38.900 34819'31.250 34821'6.00 34821'15.930 34821'36.560

Kagawa Prefecture 133838'52.440 marina 13487'47.130 marina 13487'6.00 marina 134811'18.510 marina 133845'20.820 marina 133851'22.850 marina 13482'40.00 marina 13480'45.700 marina 133859'21.320 marina

97.8.20 97.8.19 97.8.19 97.8.19 97.8.20 97.8.19 97.8.19 97.8.19 97.8.19

0.0 0.0 0.0 0.0 0.0 73.5 0.0 0.0 0.0

312 313' 318 318' 3180 322

33859'12.00 33856'25.310 33854'0.780 33852'26.870 33856'11.710 3481'38.750

Ehime Prefecture 133820'18.00 marina 13386'2.460 marma 132844'8.840 marina 132842'6.150 marina 132845'53.780 marina 13382'55.780 ®shery harbor

97.8.20 97.8.21 97.8.20 97.8.20 97.8.20 97.8.21

0.0 0.0 0.0 0.0 0.0 0.0

1997a,b). In this study the relative performance of the SPE and LLE procedures was evaluated in an attempt to provide a guideline for the use of the two procedures in our ®eld survey for Irgarol 1051 in Canadian and Japanese aquatic environments. The study results (data not shown) show that both procedures worked equally well to extract the spiked Irgarol 1051 from synthetic sea water (Jones et al., 1976). Consequently, both the LLE and SPE procedures were freely used in our ®eld surveys for Irgarol 1051, with their application being dictated mainly by ®eld conditions, man-power and equipment availability. Surveillance for a new contaminant in the aquatic environment is not always easy, as many factors (e.g. availability of literature data, reagents and analytical procedures) can a€ect the outcome. Generally speaking, even with today's sophisticated analytical techniques such as GC±EI-MS, the low ng/L detection of a micro contaminant in complex natural water samples may still present some technical challenges, one of which is the requirement to process several liters of water sample in order to reach a detectable level of the target micro contami-

nant. As a result, sampling and processing can become tedious and time-consuming, particularly with the estuarine and coastal water samples, which typically have a relatively high content of suspended matter (Steen et al., 1997). Suspended particulates can easily plug the SPE ®lter disk/column, and probably for this reason the actual volume of natural water samples processed for the monitoring of Irgarol 1051 is much lower, ranging from as low as 20 mL (Ferrer et al., 1997a) to as high as 1 L (Gough et al., 1994; Zhou et al., 1996; Ballesteros et al., 1997; Scarlett et al., 1997). Under such conditions detection limits for Irgarol 1051 in natural water samples are generally in the order of 1±5 ng/ L. To enhance Irgarol detection without complicating the extraction procedure in our ®eld survey, a sampling size of 16- and 2±3-L water samples was thus processed at each site in the across Canada survey and the Japanese survey, respectively. Irgarol 1051 and its metabolite M1 were not detected at any sampling sites across Canada during the two-year (1996±1997) survey (data not shown). Several reasons may explain why Irgarol 1051 was not found in the survey. Firstly, Irgarol 1051 has

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not been registered in Canada yet and thus it is unlikely that this chemical has been utilized in the antifouling formulations in Canada. Secondly, Irgarol 1051 is a new chemical and it takes times to build up a detectable level in the Canadian aquatic environment. For example, Irgarol 1051 and copper based products have replaced the organotins con-

taining antifouling paints in Switzerland in 1990, but the detection of Irgarol 1051 in the Swiss aquatic environment was only accomplished in 1996 (Toth et al., 1996). Only Irgarol 1051 (but not its metabolite M1) was positively identi®ed in some water samples from the Seto Inland Sea in Japan. For illustration,

Fig. 4. GC±EI-MS ion chromatograms of (a) MeCN extract of the spiked Irgarol 1051 standard from synthetic sea water and (b) a water sample taken from the sampling station 3/16 m in the Mizushima Port.

Antifouling compound in aquatic environment

the mass spectrum of the MeCN extract of a water sample taken from station 3/16 m in the Mizushima Port [Fig. 2(a)] and that from a spiked Irgarol 1051 standard in synthetic sea water are shown in Fig. 4(b and a, respectively). With the aid of its retention time (12:50/12:51 min) and the analysis of its mass spectra obtained (in terms of the major ions m/z 182, 238, 253), the occurrence of Irgarol 1051 in the enclosed coastal waters of Seto Inland Sea could be established beyond any reasonable doubt. The combination of a chemical's retention time with its mass spectral data a€ords a rapid and reliable means for its identi®cation in complex environmental samples. Such an approach has been also utilized in the identi®cation of Irgarol 1051 in water, sediment, and biota samples from Lake Geneva (Toth et al., 1996). Mass spectra of Irgarol 1051 are typically characterized by major ions m/z 182, 238 and 253 and these ions alone have been successfully used in the identi®cation of Irgarol 1051 in various environmental samples (Dahl and Blanck, 1996; Tolosa and Readman, 1996; Steen et al., 1997). Table 1 summarizes our two-year (1996±1997) survey for Irgarol 1051 in the enclosed coastal waters (harbors, bays and coves) of the Seto Inland Sea in Japan. The sites surveyed comprised two large trade ports (Mizushima and Kobe), 63 marinas and 13 ®shery harbors. These sites were mainly inshore and ranged from Wakayama Prefecture on the east side to Fukuoka Prefecture on the west side of the Seto Inland Sea, covering 10 out of the 11 Prefectures along its coast. Among the total 93 sampling sites surveyed (Table 1), 26 sites (28%) had detectable levels (3.6±264.2 ng/L) of Irgarol 1051. Contamination by Irgarol 1051 of the waters of the two large trade ports Mizushima and Kobe appeared to be very low, with only three out of the 17 sampling sites (17.5%) in the two ports having detectable levels, with concentration up to 19.5 ng/ L. This ®nding is in general agreement with literature data that large trade ports usually have a

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lower level of Irgarol 1051 (Readman et al., 1993; Gough et al., 1994). At present, large vessels (e.g. cargo ships) are not required to use antifouling paint containing Irgarol 1051 and they berth only at trade ports. As a result, levels of Irgarol 1051 found in the waters of trade ports are low (Tolosa et al., 1996). On the other hand, the ambient levels (Tables 1 and 2) of Irgarol 1051 as well as the frequency of its occurrence in the marina waters of Seto Inland Sea were found to be much higher than those found at the two large trade ports Mizushima and Kobe. Among the 63 marinas surveyed, 17 had Irgarol 1051 contamination (27%), ranging between 12.5 and 264.2 ng/L. The ®nding is again in good agreement with literature data (Table 2) on the occurrence of Irgarol 1051 in the waters of marinas and ports (Readman et al., 1993; Gough et al., 1994; Tolosa et al., 1996; Steen et al., 1997). Pleasure boats mainly use Irgarol 1051 and copper based antifouling paints and they berth only at marinas. As a result the levels of Irgarol 1051 in marina waters are much higher than those found at the trade ports (Tolosa et al., 1996). To our knowledge this study is the ®rst report on Irgarol 1051 in the waters of ®shery harbors. The results of Table 1 indicate that ®shery harbors can be a signi®cant source of contamination. Among the 13 ®shery harbors surveyed in the Seto Inland Sea, six harbors (46.2%) had a detectable level of Irgarol 1051, ranging between 13.3 and 142.4 ng/L. This also means that contamination of Irgarol 1051 in the ®shery harbor waters occurs about 1.7 times more frequent than that at the marinas. The higher frequency of Irgarol 1051 detection in the ®shery harbor waters is signi®cant, particularly from the management perspective of the control of antifouling chemicals in the aquatic environment. Currently, only marinas and Ports have been considered to act as reservoirs for the contamination (Tolosa et al., 1996; Ferrer et al., 1997b). As a result, most surveys on Irgarol 1051 were mainly concentrated on monitoring the port and marina

Table 2. Irgarol 1051 in waters of ports, marinas and ®shery harbors Regime Locations Vancouver, Canada Toronto, Canada Montreal, Canada Halifax, Canada Mizushima, Japan Kobe, Japan Seto Inland Sea, Japan Cote d'Azur, France Riviera, France, Monaco Kent, Sussex, Hampshire, UK Plymouth, UK Humber, UK Fiskebackski, Sweden Port d'Ouchy, Switzerland n.d. means not detected.

coastal riverine riverine coastal coastal coastal coastal coastal coastal coastal/estuarine estuarine estuarine estuarine lake

Irgarol 1051 (ng/L) ports

marinas

n.d. n.d. n.d. n.d. n.d. ÿ 19.5 n.d.

n.d.

<5±280 13.8±264 9±14

References

®shery harbors

n.d. n.d. n.d.-264 110±1700 22±640 52±500 28±127 169±682 30±400 2.5±145

n.d. ÿ 142

this study this study this study this study this study this study this study Readman et al., 1993 Tolosa et al., 1996 Gough et al., 1994 Scarlett et al., 1997 Zhou et al., 1996 Tolosa et al., 1996 Toth et al., 1996

2842

D. Liu et al.

waters (Readman et al., 1993; Gough et al., 1994; Tolosa et al., 1996). Fishing boats require heavy applications of antifouling paint to discourage the development of fouling organisms, due to the faster wear o€ of the antifouling chemicals through their heavy use (U.S. Naval Institute, 1952). Thus, it is suggested that, besides ports and marinas, ®shery harbors should be also included in the list for Irgarol 1051 survey. Although Irgarol 1051 was not detected in the Canadian aquatic environment during our 1996± 1997 across Canada survey, our long term surveillance for this important antifouling agent will be continued. Irgarol 1051 has been found in certain localities of the Seto Inland Sea in Japan, but its ambient levels are relatively low, when compared with those found in the European aquatic environments. Irgarol 1051 contamination from the Mediterranean Sea has recently been reported at concentration levels of 10±1,700 ng/L in the marinas and ports and 1.5±17 ng/L in open coastal areas (Readman et al., 1993; Tolosa and Readman, 1996; Tolosa et al., 1996; Ferrer et al., 1997a,b). In the coast of Sweden and U.K., Irgarol 1051 was also found in the marina waters (16±682 ng/L) and in the coastal waters (2±11 ng/L)(Gough et al., 1994; Dahl and Blanck, 1996; Zhou et al., 1996; Scarlett et al., 1997). Contamination by Irgarol 1051 of the European freshwater environment was noted at levels of 1±145 ng/L (Toth et al., 1996; Steen et al., 1997). Therefore, the contamination by Irgarol 1051 in the European aquatic environment is indeed widespread. Fate and transformation pathway of Irgarol 1051 in the ambient aquatic environment are not yet fully understood. A laboratory study (Liu et al., 1997) showed that biological transformation of Irgarol was mainly via N-dealkylation at the cyclopropylamino group, which resulted in the formation of the metabolite M1. Chemical degradation of Irgarol 1051 by mercuric chloride appeared to follow the reaction of a catalyzed hydrolysis (Liu et al., 1998), and the mechanism apparently involved the formation of bidentate chelation, which weakened the cyclopropylamino bond and resulted in formation of a hydrolysis product M1. Therefore, M1 could be a major and perhaps ultimate degradation product during the biological and chemical degradation of Irgarol 1051. M1 was not detected in water samples taken during the 1996±1997 Irgarol survey and was probably due to the very low ambient concentration levels of Irgarol in these environmental samples. For this reason, a new extraction procedure employing a large XAD2 column (2.0 i.d.  28.0 cm) capable of processing 80-L water sample had been utilized in our 1998 Irgarol monitoring study, in hope of lowering the detection limit for Irgarol and M1 in environmental samples. From the perspective of toxic substances management, the reported high toxicity of Irgarol 1051 to

nontarget marine algae has great environmental concern. Both atrazine and Irgarol 1051 are triazine herbicides, but Irgarol 1051 is about 70 times more toxic to microalgal communities than atrazine (Dahl and Blanck, 1996). Growth inhibition by Irgarol 1051 of the green macroalga Enteromorpha intestinalis spores could occur at concentrations as low as 50 ng/L (Scarlett et al., 1997), and long-term e€ects on the periphyton communities in coastal water were observable at ambient levels between 63 and 250 ng/L (Dahl and Blanck, 1996), which is within the concentration range detected in some contaminated areas of the Seto Inland Sea. The detection of Irgarol 1051 in the Seto Inland Sea is signi®cant, because it is the ®rst report on the occurrence of this new antifouling compound outside Europe. Similarly, the across Canada survey for Irgarol 1051 represents the ®rst attempt to determine the ambient concentration of Irgarol 1051 in North America. Finally, it should be noted here that our 1996±1997 Irgarol study is mainly a preliminary survey, which serves as an important guide for our long-term study on new antifouling compounds. Our future survey for the occurrence of Irgarol 1051 in the aquatic environment will be expanded to include more sampling site and sampling frequency so that the true level of Irgarol 1051 in the Canadian and Japanese aquatic environments can be established statistically.

SUMMARY AND CONCLUSIONS

1. The new antifouling compound Irgarol 1051 has been surveyed for the ®rst time in Canadian and Japanese aquatic environments. 2. During the two years of surveys (1996±1997) across Canada and southern Japan, six large trade ports (Vancouver, Toronto, Montreal, Halifax, Mizushima, Kobe), 73 marinas and 13 ®shery harbors were investigated for the ambient concentration levels of Irgarol 1051. 3. Irgarol 1051 was not detected in the Canadian aquatic environment, but was positively identi®ed in the enclosed coastal waters (harbors, marinas, and ®shery harbors) of the Seto Inland Sea in Japan. Among the six large trade ports surveyed, only one port (Mizushima) had low levels of Irgarol 1051 with concentrations up to 19.5 ng/L, while the other ®ve ports had none. 4. Of the 63 marinas surveyed in the Seto Inland Sea, 17 had Irgarol 1051 contamination (i.e. 27%) at concentrations ranging between 12.5 and 264.2 ng/L. However, the occurrence of Irgarol 1051 in the ®shery harbor waters was almost twice as frequent as that in the marina waters. 5. Among the total 93 sampling sites surveyed in the Seto Inland Sea, 11 sites had Irgarol 1051 levels between 58.4 and 264.2 ng/L, which were

Antifouling compound in aquatic environment

within the reported concentration ranges a€ecting the periphyton communities in coastal water. 6. Survey on Irgarol 1051 in ®shery harbors has not been reported before in open literature and this is the ®rst study to show that ®shery harbors can be a signi®cant source of Irgarol 1051 contamination for the aquatic environment. It is recommended that ®shery harbors, in addition to ports and marinas, should be included in the survey list during the Irgarol 1051 monitoring study. AcknowledgementsÐThis work was support in part by (1) the PESTMYOP and Great Lakes 2000 funds of the Department of Environment (Canada), and by (2) a research grant provided by the Ministry of Education, Science, Sports and Culture (Japan) for the International Comparative Study on Toxicity Assessment. The authors wish to thank Ciba-Geigy Canada Ltd. (Mississauga) for the free sample of Irgarol 1051 and the three reviewers for their comments and suggestions. REFERENCES

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