Microplastics and plastic-associated chemicals in the important green turtle nesting beaches in the Northwest Pacific

ABSTRACT

It is fundamental to understand the status of microplastic contamination in various sea turtle rookeries to assess the current and future effects of microplastics on sea turtles. The Ogasawara Islands, Tokyo, Japan, are the largest rookeries for green turtles (Chelonia mydas) in the Northwest Pacific and are remotely located 1,000 km from the Japanese mainland. To assess whether microplastic contamination occurred in the important nesting beaches of green turtles in the Northwest Pacific, we investigated the abundance and polymer types of microplastics collected at the surface and the nest depth (60 cm deep) in the sand of the 14 nesting beaches of the Ogasawara Islands. Results showed that microplastics were found at both the surface and the nest depth, and no significant difference in microplastic abundance was observed between them. The presence of microplastics at nest depth may be due to oceanographic and turtle nesting processes. Polystyrene, polypropylene, and polyethylene were identified, and expanded polystyrene was predominant. Furthermore, hexabromocyclododecanes, which are frequently compounded additives for expanded polystyrene, were semi-quantitatively analyzed using a pyrolizer coupled with a gas chromatograph-mass spectrometer in half of the expanded polystyrene microplastics collected in the present study. Result showed that hexabromocyclododecanes were detected in more than 30% of the analyzed microplastics, and the microplastics from both the surface and the nest depth contained hexabromocyclododecanes. The toxicological effect of such plastic-associated chemicals on turtles is a concern if they are transferable during egg incubation. Our data provide a baseline for assessing microplastic contamination in sea turtles on the Ogasawara Islands.

INTRODUCTION

Microplastics (MPs) are defined as plastics <5 mm in diameter, such as production pellets/powders, engineered plastic microbeads, and plastics weathered and consequently fragmented from larger plastic items (Andrady, 2011). Owing to the increase in the production and use of plastics, MPs are ubiquitous in marine environments, including ocean surfaces, water columns, seafloors, shorelines, and biota (GESAMP, 2016). The direct effects of MPs on animals, such as intestinal injury and asphyxiation through ingestion, have been observed (Baulch and Perry, 2014; Alimba and Faggio, 2019). In addition, as plastics are known to contain additives and residues from the production process, such as organic compounds and metals, and adsorb those from seawater (Teuten et al., 2009; Heskett et al., 2012; Brennecke et al., 2016; Liu et al., 2020), there is concern about the transfer of plastic-associated contaminants into marine biota (Alimba and Faggio, 2019; Muñoz et al., 2021; Sala et al., 2021; Yamashita et al., 2021; Tanaka et al., 2023).

Sea turtles are marine reptiles that nest on beaches. The nesting process is as follows: they land on the beach, excavate the nest hole far from the high-tide line, lay eggs, fill the nest chamber using surface sand, camouflage the nest by throwing sand backward over the nest site, and return to the sea (Miller, 1997). Recently, there have been several reports of the presence of MPs on the surface of beach nesting areas in Cyprus (Duncan et al., 2018), the United States (Beckwith and Fuentes, 2018), and China (Zhang et al., 2021, 2022). Of these studies, two reported that MPs were present at a depth of 60 cm (Duncan et al., 2018; Zhang et al., 2022). Because 60 cm deep is the typical depth at which sea turtle eggs are found, the eggs face the risk of exposure to plastic-associated contaminants, which leached not only from surface MPs but also from nearby MPs, which could potentially transfer these contaminants into the eggs (Muñoz and Vermeiren, 2020; Sousa-Guedes et al., 2023). Moreover, the presence of MPs can alter sand permeability and temperature (Carson et al., 2011), which indirectly affects hatchling characteristics because phenotypes, such as sex, morphology, energy, and locomotor activities, are known to be affected by incubation temperature (Booth, 2017; Kobayashi et al., 2017, 2018, 2020). When assessing the current and future effects of MPs on sea turtles, it is fundamental to understand the status of MP contamination in various sea turtle rookeries.

The Ogasawara Islands, Tokyo, Japan, are the largest rookeries for green turtles (Chelonia mydas) in the Northwest Pacific, with more than 1,000 nests observed annually (Kondo et al., 2017), and are remotely located 1,000 km from the Japanese mainland (Fig. 1a). The Ogasawara Islands are located in a high-density zone of plastic debris (Eriksen et al., 2014), and many plastics are washed off on the beach. Various stakeholders, such as the government, nonprofit organizations, and volunteers, have regularly implemented cleaning activities to address plastic pollution (Tokyo Metropolitan Government, 2013). To assess whether MP contamination occurred in the important nesting beaches of green turtles in the Northwest Pacific, we investigated the abundance and polymer types of MPs collected at the surface and the nest depth in the sand of the 14 nesting beaches of the Ogasawara Islands. Furthermore, plastic-associated chemicals were semi-quantitatively analyzed using a pyrolizer coupled with a gas chromatograph-mass spectrometer (GC-MS) in some of the MPs collected in the present study. Hexabromocyclododecanes (HBCDDs) were selected as the target compounds because expanded polystyrene (EPS) was the predominant material identified in the present study (see Results), and HBCDDs are frequently compounded additives for EPS.

Fig. 1 Maps showing the study area. (a): Japanese main land and study area. A star indicates the location of the Ogasawara Islands. (b): Chichi-jima archipelago. The numbers indicate where sand sampling was performed

MATERIALS AND METHODS

Sand sampling was performed at the Chichi-jima archipelago (27° 03′–27° 20′ N, 142° 16′–142° 25′ E, Fig. 1ab) in the Ogasawara Islands, Tokyo, Japan, from August to September in 2021 and 2022, which was the nesting and hatching season of the green turtle. The Chichi-jima archipelago has approximately 30 nesting beaches (Suganuma et al., 1994). Considering the nest abundance and beach location, 14 beaches were selected to collect the sand samples (2021: n=2, 2022: n=12, Fig. 1b, Table 1). Nest abundance was confirmed using regular surveys to count the number of nests, as described by Kondo et al. (2017) (Table 1). The beach length was measured using Google Maps (https://www.google.co.jp/maps, Table 1).

Table 1 Information about beaches where sand sampling was performed

Beach	Nest abundance		Beach length (m)a	Mean distance between the sand sampling point (nesting area) and high-tide line (m)b
	2021	2022
1	43	31	96	8.7
2	34	36	70	17.1
3	73	92	288	3.2
4	91	122	292	5.2
5	96	111	156	9.9
6	156	149	157	8.3
7	69	84	423	4.3
8	26	38	157	13.0
9	87	188	97	13.2
10	41	90	128	NA
11	85	116	244	14.3
12	47	78	175	7.5
13	1	6	39	21.0
14	158	221	318	NA

^a)   Because sea turtles do not nest at the edges of the beach, the beach length was defined as the actual distance where nests were confirmed.

^b)   Distance between the sand sampling point and high-tide line was measured only in 2022.

NA=not assessed.

In the three areas where turtles frequently nest on each beach, one 100 mL batch of surface sand was collected using a 100 mL glass beaker. Additionally, sand at a depth of 60 cm (nest depth) was collected from the same sites. Three sand samples from each surface were combined in a stainless-steel container, and the samples from each nest depth were combined in another stainless-steel container. The combined sand was used as a composite sample for each beach. The 2022 survey measured the distance between the sand sampling point and the high-tide line using a walking measure (Count measure E10-S, Shinwa Rules Co., Ltd, Niigata, Japan). The sand was stored at room temperature for 1–2 weeks before being transferred to the laboratory at the Tokyo University of Agriculture and Technology. The sand was stored at 5°C until further processing.

The sand samples were freeze-dried. The dried sample was weighed (approximately 300 g) and sieved through 1- and 5-mm sieves to collect a fraction of 1–5 mm sand. The fraction was placed in a 500 mL glass beaker. Artificial seawater with 3% salinity was prepared and filtered through a 315 μm mesh nylon net (54GG-315, Tanaka Sanjiro Co., Ltd., Ogoori, Japan). MPs in this fraction were separated by adding approximately 200 mL of the artificial seawater, stirred for 1 min, and kept standing for 10 min. Subsequently, floating particles were collected and freeze-dried for further analysis.

The reproducibility and recovery of MPs extracted from sand were evaluated as follows: approximately 300 g of dry beach sand was prepared in a 500 mL glass beaker, and 20 polypropylene (PP) particles (φ2×2 mm) were spiked into the sand. The subsequent MP extraction procedure followed the method described above. Three replicate analyses were performed, resulting in a recovery rate of 100%.

Suspected MPs were identified and sorted into polymer types using Fourier transform infrared (FT-IR) spectroscopy (Nicolet iS10, Thermo Scientific, Waltham, MA, USA), and the IR absorbance at 450–4,000 cm⁻¹ was compared with standard spectra in the software library database at a similarity threshold of >70%. The color and shape (industrial, foam, fragment, sheet-like, and thread-like; Duncan et al., 2018) of each MP were recorded.

Of the MPs identified, we examined 19 EPS MPs for the presence of HBCDDs. HBCDDs in each EPS MP were analyzed on a pyrolizer (EGA/PY-3030D, Frontier Laboratories, Koriyama, Japan) coupled with a GC-MS (8860 and 5977B series respectively, Agilent Technology, Santa Clara, CA, U.S.). For thermal desorption by the pyrolizer, the furnace temperature started at 100°C, increased at 50°C/min to 200°C, and held for 3 min. The interface temperature was set at 250°C. After thermal desorption, it was separated in an HP-5MS column (30 m, 0.25 mm i.d., 0.25 μm film thickness; Agilent Technologies). The carrier gas was helium at 1 mL/min flow. The mass spectrometer was used with an electron energy of 70 eV, the source at 230°C, and the quadrupole at 150°C. The injection port was kept at 250°C. The sample was injected in split mode and the ratio was 5:1. The oven temperature started at 50°C held for 2 min, increased at 60°C/min to 240°C and then at 30°C/min to 310°C, and held for 3 min. The m/z 159 was monitored for HBCDDs semi-quantification, and m/z 161, 239, and 319 were extracted as confirmation ions. An EPS with HBCDD concentrations of 4600 mg/kg (Eguchi et al., 2021) was used as the analytical standard. The reproducibility of the HBCDD area per mg of standard EPS was within 9% in triplicate. The semi-quantification of HBCDDs was scored based on whether the amount was within a 10-fold range of the analytical standard, less than one-tenth of it, or if no peak was detected.

The MP abundance datasets were analyzed for normality using the Shapiro–Wilk test, showing a nonparametric distribution. The differences in MP abundance between the surface and nest depth samples were analyzed using the paired Wilcoxon signed-rank test. The relationships between MP abundance and the mean distance between the sampling point and the high-tide line were analyzed using the Spearman rank correlation test. Statistical analyses were performed using GraphPad Prism5 (GraphPad Software Inc., San Diego, CA, USA). p<0.05 was considered statistically significant.

RESULTS

The distance between the sand sampling point (nesting area) and high-tide line was 10.0 m (median and range=10.0 and 2.3–22.7 m, respectively; n=36). Overall, 38 MPs were found at the surface and nest depth in the nesting areas of the Ogasawara Islands (surface: median and range=3.3 and not detected–30.0 particle/kg dry sand, respectively; n=14 and nest depth: median and range=3.3 and not detected–20.0 particle/kg dry sand, respectively; n=14). No significant difference in MP abundance was observed between the surface and nest depth (p=0.45, n=14/surface or nest depth; Fig. 2). All MPs were white, and their polymer types were PS, PP, and polyethylene (PE). PS was the predominant polymer identified in the present study (PS, 82.6%; PP, 8.7%; and PE, 8.7%; Fig. 3). The shapes of each polymer were as follows: foam for PS, fragment and industrial for PP, and sheet-like and industrial for PE. All foamed PS found in the present study were EPS.

Fig. 2 Scatter dot plot of microplastic (MP) abundance at the surface and nest depth in the nesting area of the Ogasawara Islands. No significant difference in MP abundance between at the surface and the nest depth was observed (p=0.45, n=14/surface or nest depth)

Fig. 3 Abundance and polymer type of microplastics found in each beach in the nesting area of the Ogasawara Islands. (a) Surface (b) Nest depth. PS, polystyrene; PP, polypropylene; PE, polyethylene; ND, not detected

The MP abundance varied at each beach, both at the surface and nest depth (Fig. 3). Of the 14 beaches, MPs were found on the surface of eight beaches and at nest depth on 10 beaches. At beach no. 14, MPs were more abundant at a depth of 60 cm than at the surface. No significant correlation was found between MP abundance and the mean distance between the sand sampling point and the high-tide line (surface: p=0.86, Spearman r=−0.06, n=12; nest depth: p=0.84, Spearman r=−0.07, n=12).

HBCDDs were detected in more than 30% of the EPS MPs analyzed by a pyrolizer coupled with a GC-MS (Table 2). MPs from the surface and nest depth contained HBCDDs. Among the MPs with detected HBCDDs, most had HBCDDs levels comparable to those of the analytical standard.

Table 2 Semi-quantitative scoring of Hexabromocyclododecanes (HBCDDs) levels in the expanded polystyrene microplastics (MPs) found in sand of the beaches of the Ogasawara Islands

HBCDDs level	MPs (n)
++	6
+	1
−	12

Score: (++) Within a 10-fold range of the analytical standard (4,600 mg/kg), (+) less than one-tenth of the analytical standard, (−) Not detected

DISCUSSION

The present study quantified the abundance of MPs in green turtle nesting areas on the beaches of the Ogasawara Islands. The results showed that MPs were found on the surface of the nesting area at the beach and at a depth of 60 cm where turtle eggs were present. This result is consistent with those of other turtle nesting beaches (Duncan et al., 2018; Zhang et al., 2022). It should be noted that MP abundance did not statistically differ between the surface and the nest depth on the Ogasawara Islands. Duncan et al. (2018) reported that 1–5 mm MPs (similar MP size as that in the present study) were more abundant on the surface than at the nest depth in Cyprus, which is inconsistent with the results of the present study. The factors contributing to this regional difference is unclear.

MPs have been detected at deep depths on beaches in several studies that did not focus on sea turtles (Turra et al., 2014; Moreira et al., 2016; Martinelli and Monteiro, 2019). The presence of MPs at greater depths in these studies could be due to the erosion and deposition of sand due to oceanographic processes. Similarly, nesting areas on the Ogasawara Islands are occasionally affected by typhoons and high tides, raising the possibility that the presence of MPs at nest depth in the present study was due to the same process. In addition, sea turtles excavate nest holes and fill them with surface sand during nesting. Therefore, this process could also contribute to the MPs being detected at the nest depth in the present study. The high MP abundance at the nest depth on beach No. 14 (Fig. 3b) may be because of the high abundance of MPs at the surface (Fig. 3a) and high abundance of nests (Table 1).

Regarding the polymer types of MPs in the present study, most MPs in the nesting areas of the Ogasawara Islands were EPS. Two mechanisms can explain this polymer-type characteristic. First, it was thought that the predominant type of artificial debris on the Ogasawara Island beaches was EPS, reflecting the high abundance of EPS MPs. However, foamed PS accounts for only 6% of all artificial debris on Ogasawara Island beaches (Tokyo Metropolitan Government, 2013). Therefore, this mechanism is unlikely. Second, it is possible that EPS MPs preferentially accumulates in the nesting areas of green turtles. Previous studies have shown that the percentages of EPS macro-, meso-, and microplastics tend to increase upward far from the high-tide line (Heo et al., 2013; Imhof et al., 2017; Piehl et al., 2019). This phenomenon could occur because EPS is lightweight and can be easily transported by wind (Heo et al., 2013; Turner, 2020). Therefore, a second mechanism could occur, and EPS MPs in the nesting area of Ogasawara Island could be transported downward by wind and/or fragmented from EPS macro- and/or mesoplastics at the nesting areas. The high variation in the abundance of EPS MPs at the surface between beaches in the Ogasawara Islands may be due to beach direction and/or geomorphological features related to wind exposure. Anthropogenic factors, such as the frequency of cleaning activities, and environmental factors, such as local sea currents in the vicinity of the beach (Rodrigues et al., 2020) and the presence of vegetation to trap plastic debris (Imhof et al., 2017; Gallitelli et al., 2023) may also contribute to the variation of MPs in the Ogasawara Islands.

Our chemical analysis showed that HBCDDs were detected in some of the EPS MPs collected both at the surface and at a depth of 60 cm, where turtle eggs were present. Therefore, eggs face the risk of being exposed to HBCDDs that leached not only from surface MPs but also from nearby MPs. Other additives are also known to be present in EPS. For example, decabromodiphenyl ethane is an additive for EPS, serving as a flame retardant (Rani et al., 2014). Additionally, Tanaka et al. (2023) showed that dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP), which serve as phthalate plasticizers, can be retained in beached EPS. In addition, polycyclic aromatic hydrocarbons (PAHs) can be generated during the manufacturing process of EPS (Yeo et al., 2022). Furthermore, persistent organic pollutants, such as polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethanes (DDTs), and PAHs, as well as metals, are known to be adsorbed onto EPS from the surrounding environment (Van et al., 2012; Xie et al., 2022; Yeo et al., 2022). There is a concern that these contaminants may be transferred to the embryo through the eggshell and albumen during incubation (Muñoz and Vermeiren, 2020; Sousa-Guedes et al., 2023). DBP, DEHP, HBCDDs, PCBs, DDTs, PAHs, and metals have been detected in unhatched eggs and hatchlings (Keller, 2013; Ross et al., 2017; Muñoz and Vermeiren, 2020; Savoca et al., 2021). These contaminants are considered to be derived from the mother through the vitellogenic process; however, transfer via MPs may also occur. A recent study showed that the concentrations of some metals in green turtle eggshells were significantly correlated with those in the sediment surrounding the eggs (Jian et al., 2021), implicating a transfer pathway. Some of these contaminants are toxic to animals (O’Connor et al., 2002; El-Shahawi et al., 2010; Mansouri et al., 2017; Honda and Suzuki, 2020; Feiteiro et al., 2021), and their toxicological effect on turtles is a concern.

CONCLUSION

In the present study, we found MP contamination occurred in the largest green turtle rookeries in the Northwest Pacific. Predominantly EPS MPs were present not only at the surface but also at the nest depth, with HBCDDs detected in some EPS MPs. To the best of our knowledge, this is the first report on plastic-associated chemicals in MPs in a sea turtle nesting area. Our data provide a baseline for future assessments of MP contamination in sea turtles on the Ogasawara Islands. Further studies are needed to assess the effects of MPs on sea turtles, such as continuous monitoring of MPs at the surface and nest depth, measurement of plastic-associated contaminants in MPs and sand, verification of whether these contaminants can be transferred to the embryo through MP-rich sand, and investigation of the toxic effect of such contaminants on turtles.

ACKNOWLEDGMENTS

We are grateful to the Everlasting Nature of Asia (ELNA) staff and participants in the volunteer program of ELNA for helping us collect samples. We acknowledge the use of Maptool (www.seaturtle.org) to generate Fig. 1. The present study was partially supported by JSPS Grant-in-Aid study (No. 23K28248), The University of Tokyo FSI – Nippon Foundation Research Project on Marine Plastics, and the Institute of Global Innovation Research at Tokyo University of Agriculture and Technology.

AVAILABILITY OF DATA AND MATERIALS

The data that support the findings of the present study are available from the corresponding author upon reasonable request.

CONFLICTS OF INTEREST

The authors declare that they have no conflict of interest.

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