Marine ecosystems face constant exposure to environmental pollution, emphasizing the need for effective conservation measures. Persistent organic pollutants (POPs) are resistant to environmental decomposition, accumulate in living organisms, and exhibit high biological toxicity and potential for environmental contamination over long distances (Ashraf, 2017). For decades, polychlorinated biphenyls (PCBs) have attracted considerable attention because of their ubiquity and toxicity to biota and humans (Ohkouchi et al., 2016). These compounds originate from biphenyl chlorination, encompassing a theoretical range of 10–209 homologs contingent on the number and configuration of chlorine atoms (Yuan et al., 2015). In the late 1920s, PCBs were initially manufactured in various countries for use as insulating oils in electrical transformers and capacitors and as additives in paints and plastics owing to their considerable electrical insulation, chemical resistance, and non-flammability properties. However, in the majority of developed nations, including Japan, their production and utilization were banned in the 1970s, following the recognition of their environmental persistence and toxicity (Erickson and Kaley, 2011). Global initiatives for stricter regulations were initiated amidst reports of PCB contamination spreading to untouched regions, such as in the Arctic Circle. The Stockholm Convention on POPs, adopted in 2001, mandates the cessation of PCB use in any equipment by 2025 and enforces stringent management practices until 2028. Furthermore, polybrominated diphenyl ethers (PBDEs) are important environmental contaminants, which are brominated aromatic hydrocarbons with structural variations similar to PCBs and are frequently used as flame retardants in plastics (Talsness, 2008). Under the Stockholm Convention, the production, use, and import of these substances have been prohibited. Despite international endeavors, however, these substances persistently contaminate diverse biota worldwide (Domingo and Bocio, 2007; Lee and Kim, 2015).

PCBs, like most other POPs, are hydrophobic and lipophilic, and thus readily adsorb onto organic particles, such as algae, suspended sediment, and micro- or nano-plastics present in aquatic environments (Ghosh et al., 2003; Yamashita et al., 2019). They can enter the body of animals primarily via ingestion through the digestive tract and also through absorption across the body surfaces (e.g., gills and epidermis). They subsequently accumulate in adipose tissue and lipid-rich organs, such as the liver, and also accumulate throughout the food chain (Matthews and Dedrick, 1984). Exposed organisms may experience a spectrum of toxic effects of PCBs, including hepatomegaly, skin toxicity, immunosuppressive effects, reproductive and developmental toxicity, carcinogenesis, other genotoxic responses, modulation of diverse endocrine-derived pathways, neurotoxicity, and acute lethality (Safe, 1994). Among various aquatic organisms, filter-feeding bivalves are considered reliable indicators of environmental pollution because they accumulate various POPs from surrounding waters (Chiesa et al., 2018), and the uptake of PCBs by filter-feeding bivalves such as mussels and oysters has been well documented (Chu et al., 2003).

POPs are vertically transported into the deep sea via a variety of biogeochemical processes (Takahashi et al., 2014). Understanding the behavior and effects of POPs on living organisms helps determine the extent of anthropogenic impacts on deep-sea ecosystems. Organisms with a prolonged lifespan, slow growth, and slow maturation are particularly vulnerable to environmental disturbances (Nakajima et al., 2019), and some species may face extinction because they would require long periods of time to recover from such disturbances (Llodra and Billett, 2006). In recent years, reports of POPs contaminating deep-sea organisms other than fish have been presented (Ikuta et al., 2021). Chemosynthetic ecosystems in deep-sea hydrothermal and seep sites are often inhabited by non-feeding animals that rely on symbiotic bacteria colonizing their bodies for nutrition. Bivalves belonging to the genus Phreagena are deep-sea endobenthic clams, representative of non-feeding chemosynthetic symbionts, harbor obligate symbiotic bacteria in their gill cells which they inherit through maternal transmission (Ikuta et al., 2016) ( Figure 1A ). The degree of accumulation of organic contaminants in aquatic organisms depends on the biotic or abiotic rate processes involved in the uptake, distribution, and elimination of contaminants (Bjork, 1995). In aquatic environments, PCBs are either slightly dissolved in water or adsorbed onto organic particles. In some filter-feeding bivalves, the dietary uptake of contaminants associated with these particles has been suggested to be the predominant route of contamination (Bjork, 1995). However, in non-feeding chemosynthetic bivalves, gill filtration serves purely respiratory purposes, and contamination from the gastrointestinal tract does not occur. A recent report regarding PCB contamination in Phreagena clams raised concerns about potential effects on highly endemic and vulnerable deep-sea organisms, even if they are non-feeding (Ikuta et al., 2021). However, contamination pathways and the biological and physiological effects on chemosynthetic bivalves remain unclear.

Assessments of PCB contamination pathways and their effects on deep-sea organisms such as Phreagena clams are challenging because of the difficulty in maintaining these organisms in a laboratory; however, they can be inferred by assessing the contamination levels of the organism’s habitats and distribution within their bodies. In this study, we separately measured the PCB concentrations in the gills, feet, and gonads of male and female Phreagena clams collected off Hatsushima Island in Sagami Bay, where PCB contamination was reported previously (Ikuta et al., 2021). Sediments from clam colonies were collected, and the concentrations of PCBs and PBDEs were also analyzed. Because PBDEs were not detected in the most recently collected clams in our previous study (Ikuta et al., 2021), the habitat sediment was examined for the presence of PBDEs in the current study. Additionally, sediments were collected from a location at least 10 m away from the colony and sediments from the Myojin Knoll, which is located 300 miles from the populated area where PCB contamination of the resident Bathymodiolus mussels had previously been reported (Ikuta et al., 2021). Based on the results, we explored the potential effects and pathways of PCB contamination in chemosynthetic bivalves.

The gills, feet, and gonads (testes and ovaries) of male and female Phreagena clams (three each) were excised and individually analyzed for PCB content. The ovaries exhibited the highest lipid content per dry sample among all organs, averaging 23%, followed by the gills (males, 14%; females, 15%), testes (5.7%), and feet (males, 5.0%; females, 4.6%) ( Figure 1B , Table 1 ). PCBs were detected in all samples, with ovaries exhibiting the highest total PCB (ΣPCB) content among all organs, i.e., 8.8–9.6 ng/g dw (mean = 9.1 ng/g) and 30–43 ng/g lw (mean = 36 ng/g). The gills, feet, and testes exhibited significantly lower PCB content per dw compared to the ovaries, at a range of 1.5–2.8 ng/g dw (mean = 2.3 ng/g; for males, 1.9 ng/g for females), 0.79–1.5 ng/g dw (mean = 1.2 ng/g for males, 1.0 ng/g for females), and 1.6–2.7 ng/g dw (mean = 2.0 ng/g), respectively. In contrast, the gills exhibited significantly lower PCB content per lw than the ovaries, with a range of 5.3–26 ng/g lw (mean = 1.4 ng/g for both males and females) ( Figure 1C , Table 1 ). Based on the PCB homolog percentages in the ΣPCBs of each organ, tetra- (mean = 33%, dw), penta- (mean = 32%, dw), and hexa-CBs (mean = 19%, dw) dominated the biological samples, followed by tri- (mean = 11%, dw) and hepta-CBs (mean = 5%, dw) ( Figure 1D ). The amounts of other homologs were notably small, with no detection of mono-, nona-, and deca-CBs. Additionally, the percentages of the 14 coplanar PCBs in the ΣPCBs ranged from 8.1%–14% (dw) for all organ samples ( Supplementary Table 1 ).

PCBs were detected in all sediment samples, with total concentrations ranging from 0.74 to 13 ng/g dw ( Table 2 ). No statistically significant differences in the ΣPCBs were detected among samples collected from the clam colony in Sagami Bay, those collected at a distance from the colony, and those from the Myojin Knoll ( Table 2 ). The percentages of the 14 coplanar PCBs in the ΣPCBs ranged from 6.4% to 12% (dw) for all sediment samples ( Supplementary Table 2 ).

PCB homolog percentage comparisons were analyzed between sediment and biological samples using previous study data from intact Phreagena clam bodies (Ikuta et al., 2021). The results demonstrated that habitat sediments were dominated by tetra- (mean = 26%, dw), penta- (mean = 21%, dw), and tri-CBs (mean = 20%, dw), whereas the clams were dominated by tetra- (mean = 35%, dw), penta- (mean = 31%, dw), and hexa-CBs (mean = 16%, dw), indicating a trend towards higher-chlorinated PCB homologs in the clams. This trend was slightly stronger in the mussel Bathymodiolus japonicus, living sympatrically with the clams, and the PCB compositions were dominated by penta- (mean = 31%, dw), hexa- (mean = 29%, dw), and tetra-CBs (mean = 24%, dw) ( Figure 2A ) (Ikuta et al., 2021). The PCB compositions in the sediments of the clam colony, those in sediment from outside the colony, and those within the intact bodies of clams and mussels were separated by 85% similarity, with no statistically significant difference ( Figure 2B ). Additionally, a comparison of PCB compositions was conducted between the Myojin Knoll sediments and intact bodies of another mussel (B. septemdierum) from a previous study (Ikuta et al., 2021). The sediments were dominated by tetra- (mean = 39%, dw), tri- (mean = 34%, dw), and penta-CBs (mean = 10%, dw), and the mussels were dominated by penta- (mean = 32%, dw), hexa- (mean = 32%, dw), and tetra-CBs (mean = 21%, dw), indicating a tendency towards higher-chlorinated PCB homologs in the mussels. The two were separated by 80% similarity; however, no statistically significant differences were observed ( Supplementary Figure 2 ). The BSAF value of P. okutanii for the total ΣPCBs was 0.66 ( Supplementary Table 3 ).

We also analyzed the PBDE content in the sediment samples. PBDEs were detected in every sample, and the total concentrations were between 0.24–13 ng/g dw ( Table 2 ). No statistically significant differences in the ΣPBDEs were detected among samples collected from the clam colony in Sagami Bay, those collected at a distance from the colony, and the Myojin Knoll samples; however, the Myojin Knoll samples exhibited the three lowest values ( Table 2 ). Based on the PBDE homolog percentages in the ΣPBDEs, de-BDE accounted for the majority of PBDE content (mean = 83%, dw), followed by nona- (mean = 12%, dw) and octa-BDEs (mean = 2.5%, dw, Supplementary Figure 3 ).

In this study, we determined the PCB concentrations in the gills, feet, and gonads of the deep-sea chemosynthetic clam P. okutanii. A positive correlation was identified between the lipid content and PCB concentrations across all biological samples, and the ovaries exhibited the highest PCB concentrations and elevated lipid content. The buoyancy of P. okutanii eggs and numerous lipid droplets in the cytoplasm of the vegetal hemisphere (Ikuta et al., 2016) indicate that these droplets may be responsible for the accumulation of PCBs in eggs. Filter-feeding bivalves such as mussels and oysters absorb PCBs with their diet, and biomagnification of PCBs through trophic transfer has also been reported. PCBs affect various biological processes, including immune, endocrine, and reproductive functions (Ngoubeyou et al., 2022), across various organisms. Moreover, experimental studies have assessed their accumulation and effects on bivalves. For example, in Crassostrea virginica oyster, PCBs mixed with algae in the diet accumulated predominantly in the gonads, thus affecting reproductive functions (Chu et al., 2003). Similar experiments have demonstrated that PCBs accumulate in oysters and are transferred to eggs (Chu et al., 2000). In the hard clam Mercenaria, exposure to Aroclor 1254, a prevalent commercial PCB mixture primarily composed of penta- (71.44%) and hexa-CBs (21.97%), during development resulted in concentration-dependent larval morphological abnormalities (Ryan et al., 2001). Freshwater clam, Corbicula fluminea exposed to water containing Aroclor 1260, a commercial PCB mixture primarily composed of penta- (43.35%) and hexa-CBs (38.54%), exhibit pronounced gonadal atrophy, effacement of gonad architecture, and inflammation and necrosis in the digestive gland and foot processes. This confirms the considerable threat of PCB exposure to bivalve reproduction (Lehmann et al., 2007). Our previous study demonstrated that non-feeding deep-sea chemosynthetic bivalves were contaminated with PCBs (Ikuta et al., 2021). PCB contamination levels in the intact Phreagena clam bodies collected off Hatsushima Island in Sagami Bay were considerably lower compared to the significantly higher PCB concentrations detected in shallow-water mussels collected near the densely populated regions of Japan; however, this was comparable to the levels detected in shallow-water bivalves across various international sites (Ikuta et al., 2021). Additionally, this was comparable to or slightly lower than those of other deep-sea organisms at lower trophic levels collected from Sagami Bay, including pelagic fishes, crustaceans, and zooplankton (Toyoshima et al., 2009; Ikuta et al., 2021). In the present study, the PCB concentrations detected in the ovaries of Phreagena clams were considerably lower than those in the ovaries of oysters in experiments using administration of PCB-contaminated algae (3,490 ng/g, dw), as described above (Chu et al., 2003). The level of contamination that may cause harmful effects on Phreagena clams remains unclear. Examining the maturity status and PCB concentrations of clams at different growth stages will provide insight into the effects of PCBs on clam maturity. However, PCB contamination in Phreagena clams at this site has been present for over 30 years (Ikuta et al., 2021), which complicates the determination of any histological and functional abnormalities compared to uncontaminated samples. The current study revealed significantly higher levels of PCB contamination in the ovaries, indicating a potential effect on reproductive functions. Among the coplanar PCBs examined in this study, PCB118, which had the highest content in both biological and sediment samples, is known to cause reproductive abnormalities in male and female mammals (Kuriyama and Chahoud, 2004; Ge et al., 2022), although its effects on bivalves are unknown. Thus, our results raise concerns regarding a severe direct effect on the survival of this vulnerable and highly endemic species.

Our chemical analyses showed PCBs in all sediment samples (mean = 3.9 ng/g dw). This value aligns with PCB concentrations in surface sediments collected from various bays around Japan, including shallow waters (Shimizu et al., 2008) and the deep sea (Ohkouchi et al., 2016). Comparisons of each homolog composition, including intact body data from previous studies of Phreagena clams and Bathymodiolus mussels (Ikuta et al., 2021), demonstrated a distinct clustering of sediment data from the bivalve data. The bivalve bodies contained PCBs with higher-chlorinated compounds than those in the sediments, similar to those observed in shallow-water clams (Choi et al., 2010). In general, less-chlorinated PCBs are metabolized more readily than highly chlorinated congeners, which exhibit a lower tendency for in vivo metabolism and a higher tendency to remain in the animal body (Agency for Toxic Substances and Disease Registry (ATSDR) 2014). Therefore, PCBs that accumulate in organisms exhibit a higher-chlorinated composition than those in the environment. However, how extensively persistent PCBs are metabolically degraded in bivalves at low deep-sea temperatures of approximately 4°C remains to be investigated. As an alternative explanation, the examined bivalves may take up highly chlorinated PCBs. Our recent findings indicate that both deep-sea bivalves absorb plastic particles through their body surface into their gill cells by phagocytosis (Ikuta et al., 2022). Highly chlorinated and hydrophobic PCBs are more readily adsorbed onto organic particles, such as microplastics, in the environment (Colabuono et al., 2010). In line with our results, if these particles are taken up from the body surface, they may contribute to the accumulation of highly chlorinated PCBs in the bivalve bodies. The current study demonstrated that the BSAF value of P. okutanii for PCBs, which indicates the biological potential for PCB accumulation from the environment, was comparable to that of some shallow-water bivalves (Tracey and Hansen, 1996). Therefore, the continuous accumulation of microplastics and other organic particles containing PCBs and other toxic substances on the deep-sea floor can markedly affect these deep-sea clams. Substantial amounts of microplastic have already been detected in the same clam colony site, suggesting that microplastics can be carriers of POPs in this habitat, although other organic particles are also responsible for this phenomenon (Tsuchiya et al., 2023). In a previous study, no PBDEs were detected in the bodies of clams collected in 2019 (Ikuta et al., 2021); however, in the present study, PBDEs were detected in sediments from clam habitats at levels comparable to those of PCBs. The interpretation of these results is difficult because the BSAF of PBDEs in shallow-water mussels was reportedly higher than that of PCBs, indicating higher bioaccumulation of PBDEs in mussels than of PCBs (Debruyn et al., 2009). Examining PBDE concentrations in other chemosynthetic organisms, such as tubeworms living sympatrically with the clams, may provide further insight.

In conclusion, our results demonstrate that non-feeding Phreagena clams, a representative taxon in the deep-sea chemosynthetic ecosystem, take up environmental PCBs through their body surface and accumulate them predominantly in their ovaries. These results indicate a potential direct effect on their reproductive function. Recent findings have indicated a reduction in PCB contamination within Phreagena clams in recent years in Sagami Bay; however, the current study identified PCBs in the habitat sediments. Contaminated marine sediments act as a secondary source of contaminants for the benthic communities they inhabit and other organisms through food web bioaccumulation. Additionally, the increasing presence of plastics and other organic particles with adsorbed contaminants is anticipated to persistently increase and accumulate in the deep sea (Wayman and Niemann, 2021). It is necessary to take approaches such as examining PCBs associated with particles at the habitat site and PCBs dissolved in seawater to determine whether these PCB-adsorbed organic particles are the primary source of contamination. In the future, the results of the present study can serve as a foundation for monitoring the status of marine pollution in vulnerable deep-sea ecosystems.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The manuscript presents research on animals that do not require ethical approval for their study.

TI: Formal analysis, Investigation, Methodology, Visualization, Writing – original draft. HN: Funding acquisition, Resources, Writing – review & editing. RN: Formal analysis, Writing – review & editing.