3.1. PAH Concentrations in Organisms
In wharf roach, the highest concentration of the total of 15 PAHs (ΣPAH) was detected in the sample from Akita (96.0 ng/g-dry weight (dw); Figure 2
, Table 2
), followed by samples from Niigata, Saga, Yamagata, Fukuoka, Aomori, Ishikawa, Shimane, Hyogo, Kyoto, Nagasaki, and Yamaguchi. The detected ΣPAH concentrations (median: 48.5 ng/g-dw, detected range: 26.9 to 96.0 ng/g-dw) were similar to previously reported concentrations, except for samples that were previously collected in highly polluted areas (median: 47.0 ng/g-dw, detected range: 28.6 to 72.2 ng/g-dw) [46
]. The PAH composition was largely consistent among the 12 sampling sites (Figure 2
). The predominant PAH species was Pyr (median: 24.9 ng/g-dw), followed by Phe, Flut, and Acy (median: 7.71, 6.44, and 5.40 ng/g-dw, respectively). In a previous study, the dominant PAH species were Phe, followed by Ace and Pyr [46
]. Thus, the current results differ from previous observations. The composition differences reflect differences in pollution among the sampling sites and differences in contamination sources between sampling seasons (current study: summer, previous study: fall–winter; [47
]). High molecular weight PAHs (HMW-PAHs) (i.e., five- and six-ring PAHs) were detected at lower concentrations than low molecular weight PAHs (LMW-PAHs) (i.e., three- and four-ring PAHs), which were the most common. Three-ring PAHs were contributed at 30% and four-ring PAHs at 68% of ΣPAH concentration. It is generally believed that HMW-PAHs do not highly accumulate in organisms because of a lower intake efficiency [48
A consistent trend of high Pyr concentrations (median: 11.6 ng/g-dw) was observed in mussel tissue. The highest ΣPAH concentration was detected in a sample from Ishikawa (132 ng/g-dw; Figure 3
), followed by samples from Aomori, Hyogo, Akita, Kyoto, Fukuoka, Nagasaki, Shimane, Niigata, Yamagata, Yamaguchi, and Saga. The detected range of ΣPAH among the 12 sampling sites was 16.6 to 132 ng/g-dw. This finding is similar to that of previous studies; 16 PAHs, 87.3–361 ng/g-dw [19
]; 18 PAHs, 15.2 to 527 ng/g-dw (both values were converted from initial reported values of 2.6 to 90 ng/g-ww by using an 82.9% moisture content) [40
]. Of the 15 PAHs, Pyr was the predominant species (median: 11.6 ng/g-dw), followed by Phe and Flut (7.10 and 5.83 ng/g-dw, respectively). Additionally, the concentrations of HMW-PAHs were greater in mussels than in wharf roaches. The relative standard deviation of ΣPAH among the 12 sampling sites was larger for mussels than for wharf roaches (0.63 and 0.39, respectively). These differences reflect differences in the PAH pollution in the supralittoral and intertidal zones and suggest that wharf roaches and mussels differ in their exposure pathways and bioaccumulation capability.
3.2. PAH Concentrations in Environmental Media
The ΣPAH composition was consistent across seawater samples (Figure 4
). The detected range of ΣPAH among the 12 sampling sites was 7.79 to 19.1 ng/L (mean 13.4 ng/L). This finding is similar to that of previous studies; 13 PAHs, 6.83 to 13.81 ng/L (mean 9.4 ng/L) in the Japan Sea [49
]; 13 PAHs, 10.9 to 29.7 ng/L (mean 19.6 ng/L) in the Sea of Japan and East Sea [50
]. Phe was the predominant species (median: 5.67 ng/L). In general, there was a high contribution of three-ring PAHs to ΣPAH (87%). Comparatively, four-, five-, and six-ring PAHs had very low contributions to ΣPAH (11%, 1.5%, and 0.4%, respectively). The concentrations of LMW-PAHs were higher than those of HMW-PAHs owing to their increased water solubility [47
The detected range of ΣPAH in soil and sand among the 12 sampling sites was 2.06 to 506 ng/g-dw (median 22.0 ng/g-dw). This finding is lower than that of previous studies; 18 PAHs in sea sediment, 6.40 to 7765 ng/g-dw in Osaka Bay, Japan [12
]; 16 PAHs in wharf soil, 842 ± 203 ng/g-dw in Guangdong, China [52
]. Phe was the predominant species (median: 5.67 ng/g-dw). At seven sampling sites, that had relatively small particles and a larger soil fraction, the soil and sand samples contained a larger amount of HMW-PAHs than the seawater samples (Aomori, Ishikawa, Kyoto, Hyogo, Fukuoka, Saga, and Nagasaki; Figure 5
). The other five sites, which had relatively large sand particles, had PAH compositions similar to that of the seawater samples (Akita, Yamagata, Niigata, and Shimane). The PAH with the highest median concentration detected among the 12 sites was Pyr (4.91 ng/g-dw), followed by Flut and Chr (2.35 and 1.57 ng/g-dw, respectively). There were four-ring PAHs predominant in the ΣPAH at 61%, which was much higher than the percentage of three-ring PAHs (20%). Additionally, five- and six-ring PAHs comprised a much higher percentage of ΣPAH in soil and sand (12.1% and 6.9%, respectively) than in seawater (1.5% and 0.4%, respectively). PAH hydrophobicity is positively correlated with ring number and molecular weight. Higher molecular weight PAHs tend to partition into the particle phase. Therefore, HMW-PAHs were more likely to be found in the soil and sand samples than in the seawater samples. Additionally, the sampling sites of Ishikawa and Kyoto were located in a wharf area, while the other sites were located in beach or rocky areas. Soil samples were collected from a small ditch near a wharf in Ishikawa and Kyoto, and they contained higher concentrations of PAHs (506 and 138 ng/g-dw, respectively) compared with the other 10 sites (median: 15.7 ng/g-dw). This difference was not observed for the other analyzed samples.
Drifting seaweed is a major food source for wharf roaches. The detected range of ΣPAH in drifting seaweed among the 12 sampling sites was 11.8 to 109 ng/g-dw (mean 47.6 ng/g-dw). This finding is higher than that of previous studies; 16 PAHs in living seaweed, 1.0 to 56.4 ng/g-dw (mean 7.0 ng/g-dw) in Venice, Italy [53
]. The PAH composition in drifting seaweed was roughly separated into two groups, with two exceptions (Figure 6
) (notably, drifting seaweed samples could not be found at the Shimane site). One group was largely consistent with the PAH trends in seawater (Akita, Yamagata, Niigata, Kyoto, and Shimane). The other group was more similar to the composition found in soil and sand samples (Yamaguchi, Fukuoka, Saga, and Nagasaki). There were two exceptions: Aomori and Ishikawa. As with other environmental media, Pyr was the predominant species (median: 14.4 ng/g-dw). However, Aomori had a higher concentration and percentage of BP (6.95 ng/g-dw, 20%), and Ishikawa had a higher concentration and percentage of Flut (38%). Beyond Pyr, Flut and Phe also had substantial concentrations (6.85 and 4.11 ng/g-dw, respectively). Seaweed can accumulate PAHs from seawater during its lifecycle [53
]. Therefore, it is likely that these PAHs were transferred from the seawater and nearby soil and sand into the seaweed. Additionally, aerial deposition on drifting seaweed is a possible exposure source [54
The PCA confirmed some of the similarities found among the various sample types (Figure 7
). In the first principal component (PC) (accounting for 73.4% of the variance), wharf roach and seawater were closely related, whereas soil and sand and mussels formed a related group. The loading plot indicated that the separations in the first PC were largely driven by differences between the three-ring PAHs (i.e., Acy and Ace) and the larger molecular weight PAHs (Figure S2
). In general, concentrations of the larger five- and six-ring PAHs were higher in soil and sand, seaweed, and mussels than in wharf roach and seawater (Table S3
). By contrast, seawater samples contained greater concentrations of the smaller three-ring PAHs. These differences may account for the separation between these groups in PC1. Compared with the other samples, the relative similarities between soil and sand and mussels were expected given the hydrophobic tendency of PAHs, whereby they partition onto particles or into the high lipid content of mussels. This partitioning also explains the results of the correlation analysis, where four- and five-ring PAHs were particularly well correlated between seawater and wharf roaches. The close association between wharf roaches and seawater supports the idea that the wharf roach can be a useful proxy for assessing surface-layer PAH contamination. However, there appear to be some differences, as seen in PC2, though this accounts for only 14.6% of the variance. Although the loading plot indicated that these separations are caused by differences between the four-ring PAHs and the five and six-ring PAHs, it is not clear why these differences exist.
3.5. Correlation between PAH Concentrations in Wharf Roaches and Environmental Components
Wharf roaches were presumed to be exposed to PAHs through the soil and sand, seawater, and dietary seaweed exposure pathways. Wharf roaches are terrestrial isopods that occupy the supralittoral zone, but they take in seawater through their legs [58
]. They feed on drifting seaweed and biofilms [43
]. The correlations between PAH ring numbers and organisms and substrates were analyzed. The three-ring PAHs were significantly correlated between wharf roach and drifting seaweed and soil and sand (Spearman’s rank correlation, p
= 0.01, <0.01; rho = 0.34, 0.51, respectively); the four-ring PAHs were significantly correlated between wharf roach and drifting seaweed, seawater, and soil and sand (p
= 0.01, <0.01, 0.01; rho = 0.62, 0.59, 0.36, respectively); the five-ring PAHs were significantly negatively correlated between wharf roach and seawater (p
= 0.02; rho = −0.33). Detected concentrations of five-ring PAHs were relatively low in all analyzed samples. Therefore, it is likely that there was no significant variation in five-ring PAH concentrations to result in a significant correlation between wharf roach and seawater. Because several samples had concentrations of six-ring PAHs that were less than the LOD, correlations for these PAHs could not be analyzed (detection frequency: 80%).
For mussels, the major exposure pathway of LMW-PAHs is generally considered to be water, and that of HMW-PAHs is considered to be particles [59
]. This is explained by the hydrophobic properties of PAHs, which make them more likely to partition onto nonpolar particulate surfaces. Mussels continuously filter seawater containing organic matter, which constitutes the majority of its food. For this reason, mussels are liable to accumulate PAHs that are present in the dissolved and particulate phases. The ΣPAH concentration in mussels from Ishikawa (132 ng/g-dw) was the highest among sampling sites, likely because Ishikawa also had the highest soil and sand ΣPAH concentration (506 ng/g-dw).
However, wharf roaches are omnivorous scavengers and are not directly exposed to marine sediment. Food is generally considered the major pathway by which organisms take in PAHs [60
]. Surprisingly, the statistical analysis results indicated that LMW-PAHs (i.e., three- and four-ring PAHs) were significantly correlated between wharf roach and food source (i.e., drifting seaweed; rho = 0.34, 0.62). There was also a significant correlation between LMW-PAH concentrations in soil and sand and in wharf roach (rho = 0.51, 0.36). The LMW-PAH composition results indicated that the LMW compounds in wharf roaches had a pattern similar to that in soil and sand (especially in samples from Akita, Niigata, and Shimane; Figure 2
and Figure 5
) and to that in drifting seaweed (especially in samples from Akita, Yamagata, Niigata, Kyoto, Hyogo, and Saga; Figure 2
and Figure 6
). Some terrestrial isopods accumulate PAHs from contaminated soil [61
]. In the present study, particles of soil and sand were frequently detected in the intestines of wharf roaches (data not shown). It is possible that wharf roaches directly ingest soil and sand particles to obtain the organic matter adhered to them. This may have been a possible exposure route in the current study. Additionally, van Brummelen et al. [61
] reported a negative correlation between biota-to-soil accumulation factors and the PAH Kow
. Therefore, soil and sand may have contributed less than other routes to the HMW-PAH exposure of wharf roaches. However, the actual contribution ratio of each exposure pathway and the metabolism of PAHs in isopods remain unclear. It is possible that efficient LMW-PAH metabolism lowered their concentrations in the samples, thereby masking a detectable correlation between wharf roach and seawater.
Compared with LMW-PAHs, HMW-PAHs are metabolized more slowly in the body [63
]; however, HMW-PAHs (more than five rings) are highly hydrophobic and are found less frequently in aquatic samples. Therefore, it is difficult to evaluate the wharf roach exposure pathway to HMW-PAHs. Wharf roaches take in seawater from the ocean surface [58
] where there is often floating oil and related hydrophobic substances. Furthermore, the water surface is relatively hydrophobic compared with the underlying water. Therefore, in addition to drifting seaweed and soil and sand, wharf roaches can possibly accumulate a broad range of PAHs via seawater. However, HMW-PAH concentrations in wharf roaches were negatively correlated with those in seawater. This phenomenon may not have indicated a negative association between these two groups, rather, it could have resulted from the relatively low concentrations of five-ring PAHs in the wharf roach and seawater samples. In general, HMW-PAHs are not efficiently taken up and accumulated by organisms because of their high hydrophobicity [63
]. Although not significant, in the samples taken from Saga, the relatively high BP concentration in wharf roach may have been related to the high BP concentrations in the soil/sand and drifting seaweed samples. Therefore, it is possible that wharf roaches are exposed to HMW-PAHs through several environmental components; however, no single component has a predominant contribution because of the low intake efficiency.
Bioconcentration, biomagnification, and biota-sediment accumulation have different exposure pathways and contribution ratios that affect the total accumulation. In addition to drifting seaweed, wharf roaches feed on biofilms and other organic matter distributed throughout the supralittoral zone. Additionally, PAH metabolism varies considerably among species. Therefore, it is necessary to expose wharf roaches to several PAHs under laboratory conditions to assess the actual accumulation system and calculate the bioconcentration and biomagnification factors.