Coral reefs are mostly found in shallow coastlines of tropical and subtropical areas (Suzuki and Kawahata, 2003, Inoue et al., 2004). They are among of the world’s most valuable natural resources that provide food, medicines, ecological services and source of income for the coastal residences. However, in recent years, the sustainability of the coral reef ecosystems has been thrown into doubt by a number of threats including marine pollution, global environmental changes, outbreaks of crown-of-thorns starfish (Hughes et al., 2003) and disease (Santavy and Peters, 1997). It has been estimated that about 27% of the world’s coral reefs have already been lost and 31% have been projected to be degraded by 2030 (Wilkinson, 2000). More integrated efforts and new conservation approaches are necessary to minimize the threats facing coral reefs in future (Downs et al., 2005).
Ryukyu Archipelago composed of Okinawa Islands has a unique marine ecological importance hosting about 90% of Japan’s coral reefs. However, the health of coral reefs around the Ryukyu Archipelago steadily deteriorates due hazardous chemical pollution from domestic, agricultural, industrial and shipping activities (Kitada et al., 2008, Sheikh et al., 2007, Tanabe et al., 2008), terrestrial run-off (Omija, 2004, Sakai, 2004, West and van Woesik, 2001), developmental projects (Nakano, 2004) and natural disturbance (Sano, 2001).
Diuron, [N′-(3,4-dichlorophenyl)-N,N-dimethyl-urea] is a photosystem II (PSII) herbicide derived from urea. It is considered a priority hazardous substance by the European Commission (Malato et al., 2002). Countries including the UK, Sweden, Denmark and France have restricted the use of diuron in antifouling paints (Konstantinou and Albanis, 2004, Giacomazzi and Cochet, 2004). In Japan, diuron has been extensively used in antifouling paints in shipping and agricultural activities (Okamura et al., 2003). In 2004 alone, around 11 tons of diuron was used for agricultural purposes in the Okinawa Prefecture, which was the highest amount of this toxic substance used in Japan outside of the Tokyo metropolitan region. In addition, the urban areas of Okinawa mainland, apply a significant amount of diuron as a weed control (Kitada, 2007).
As diuron blocks electron transport at photosystem II, thus inhibiting the photosynthetic process (Konstantinou and Albanis, 2004). Eco-toxicological studies have shown that diuron induces significant impacts on corals (Jones, 2005), such as the reduction of 14C incorporation in M. mirabilis (Owen et al., 2003), the reduction of ΔF/Fm’ in S. pistillatta, S. hystrix and A. Formosa (Jones et al., 2003), the loss of symbiotic algae in M. digitata and S. hystrix (Jones, 2004), and the detachment of soft tissue of juvenile of A. tenuice (Watanabe et al., 2006). Diuron has also been shown to have stronger effects than tributyltin (TBT) in the carbon metabolisms of coral G. fascicularis (Sheikh, 2008). In addition, the herbicide has been associated with serious impacts on other marine ecosystems such as mangroves dieback (Bell and Duke, 2005).
However, despite the extensive usage of diuron in Ryukyu Archipelago, and the associated toxicological implications in coral reefs and other marine ecosystems, very little is known about the baseline levels and behavior of diuron in coral reef waters around the Ryukyu Archipelago. So far, only one study has reported the diuron contents in river sediments around mainland Okinawa (Kitada et al., 2008). Yet, the risk posed by pollution of coral reef waters with the soluble fraction of toxic chemicals needs to be given priority. As such, to evaluate the impact of diuron in coral reefs, it is first necessary to elucidate its status and behavior. Therefore, this paper provides the results of a systematic monitoring study of diuron in coral reef ecosystems and adjacent environments around the Ryukyu Archipelago.
The study was conducted around Shiraho coral reefs located at Ryukyu Archipelago, southern western Japan. The Ryukyu Archipelago is located between 24 and 30oN constituting the southern part of the Nansei Islands. The Archipelago consists of more than 100 Islands lying in a chain between Kyushu (mainland Japan) and Taiwan, separating the East China Sea from the Pacific Ocean (Fig. 1).
The Shiraho reef was divided into nine transects, three sampling points were established in each transect, while ten sampling points were selected along the Todoroki River (Fig. 1). Subsurface water samples were taken using 1 L polycarbonate amber bottles. The bottles were washed with soap water, then decontaminated with 10% HNO3 solution and kept overnight. They were rinsed with Milli-Q water followed by acetone and dried before sampling. In addition, the bottles were always rinsed thoroughly with waters before sample acquisition. Total of 22 and 191 water samples were analyzed for Todoroki River and Shiraho coral reefs, respectively.
An extensive survey of diuron in the waters around the Shiraho coral reefs was done during different seasons in 2007, 2008 and 2009. In Todoroki River, waters were sampled in 2007 and 2008 (Table 2).
Diuron was extracted following the method of analysis of diuron in drinking water by the Ministry of Health, Labor and Welfare, Japan (Okinawa Prefectural Enterprise Bureau, 2003) with minor modifications. Prior to the extraction of diuron, the solid phase extraction cartridge columns (PLS-3, GL Sciences, Japan) were conditioned with 10 mL of acetonitrile, followed by methanol and Milli-Q water, respectively. About 10 mL of 0.2 M EDTA was added to 1 L of water sample and the pH of water was kept at 3.5. Diuron D-6 (C9H4Cl2D6N2O) was spiked as a surrogate standard in order to monitor the recovery of diuron. Satisfactory recoveries of more than 85% were archived for water samples that were spiked at 1 μg/L. The relative standard deviations (RSDs) of the method were below 10%. Data for the environmental samples were corrected for recovery values.
Water samples were then automatically eluted in the solid phase extraction column using a solid phase extraction controller (Shimadzu, Japan) with a flow rate of 20 mL/min. PLS-3 cartridges were then dried with nitrogen gas for 5 min. Diuron was eluted from the column by 5 mL of acetonitrile. Finally, acetonitrile was evaporated to 0.2 mL by pure nitrogen gas.
The diuron analysis was achieved using a LC–MS (Agilent 1100LC/MSD SL System) with the following operating conditions: column; Agilent ZORBAX eclipse XDB-C18 4.6 × 30 mm, 1.8 μm, column temperature; 40 °C, mobile phase A: HCO2H/water (0.1%), B: acetonitrile, gradient 95% A-(liner gradient 5 min) – 80% B (7 min), flow rate; 0.5 ml/min, injection volume; 10 μl, MS conditions: ionization mode, negative ion-ESI SIM/Scan mix mode, desolvation gas; nitrogen, 12 l/min, desolvation temperature; 350 °C, capillary voltage; 2.5 kV, SIM monitor ion, m/z 231 and 237. The detection limit of diuron in water samples was 0.02 μg/L.
The concentrations of diuron measured at Shiraho coral reefs and adjacent Todoroki River are presented in Table 1. Diuron levels of in water ranged between not detected −753 ng/L with its highest concentration found at ST1 of Todoroki River during the month of November 2008. The average concentrations of diuron were 60.2 ± 157 ng/L (n = 22) and 2.3 ± 7 ng/L (n = 191) around the Todoroki River and Shiraho coral reefs, respectively (Fig. 2).
Overall, the average levels of diuron around the Todoroki River (60.2 ng/L) were significantly higher (ANOVA, p < 0.05) compared Shiraho coral reefs (2.3 ng/L) and other coastal areas around Okinawa Island such as commercial ports; 2.1 ± 3.1 ng/L (n = 46), and Bays; 1.9 ± 1.8 ng/L (n = 42) (Fig. 2) (detailed diuron data for the Ports and Bay are not presented in this paper). A clear spatial variation among the sampling stations can be seen. In addition, it is evident that the magnitude of diuron contamination reflects the activities around the sampled sites. Todoroki River extensively receives waste waters from the adjacent sugarcane fields. Interestingly, the distribution trend contradicts the general concept that ports are always more contaminated with diuron than coral reefs. This suggests that the coral reef ecosystems around Ryukyu Archipelago face a significant risk of toxic chemicals, including diuron from land based sources.