Radioactivity and radionuclides in deciduous teeth formed before the Fukushima-Daiichi Nuclear Power Plant accident

The Fukushima-Daiichi Nuclear Power Plant (FNPP) accident in March of 2011 released substantial amounts of radionuclides into the environment. We collected 4,957 deciduous teeth formed in children before the Fukushima accident to obtain precise control data for teeth formed after the accident. Radioactivity was measured using imaging plates (IP) and epidemiologically assessed using multivariate regression analysis. Additionally, we measured 90Sr, 137Cs, and natural radionuclides which might be present in teeth. Epidemiological studies of IP showed that the amount of radioactivity in teeth from Fukushima prefecture was similar to that from reference prefectures. We found that artificial radionuclides of 90Sr and 137Cs, which were believed to have originated from past nuclear disasters, and natural radionuclides including 40 K and daughter nuclides in the 238U and 232Th series contributed to the generation of radioactivity in teeth. We also found no evidence to suggest that radionuclides originating from the FNPP accident significantly contaminated pre-existing teeth. This is the first large-scale investigation of radioactivity and radionuclides in teeth. The present findings will be indispensable for future studies of teeth formed after the FNPP accident, which will fall out over the next several years and might be more contaminated with radionuclides.


Scientific Reports
| (2021) 11:10335 | https://doi.org/10.1038/s41598-021-89910-0 www.nature.com/scientificreports/ Several issues needed to be resolved before teeth formed before and after the FNPP accident could be compared. One was the yield of fission products such as 90 Sr and 137 Cs in teeth. The presence of these radionuclides in teeth has been reported in connection with past nuclear disasters [13][14][15][16][17][18][19] . Small amounts of 90 Sr and 137 Cs originated from past nuclear disasters have been found in soil samples from various locations in Japan, including Fukushima prefecture, before the accident 20 . Besides, trace amounts of 90 Sr and 137 Cs fallout are still detectable, even though atmospheric nuclear weapons testing was conducted between the 1950s and the 1970s 21 . Low levels of 90 Sr have been found not only in the natural environment but also in animal teeth that formed before the FNPP accident 22 . However, no systemic data are available on the concentration of such radionuclides in human teeth developed before the accident. Furthermore, whether radionuclide contents in teeth differ between individuals from Fukushima prefecture and those living in other areas is important to determine. Another issue is the types of the radioactivity of natural radionuclides in teeth. Teeth contain natural radionuclides such as 40 K and daughter nuclides in the 238 U and 232 Th series found in the environment 23 . However, the type and concentration of these radionuclides in teeth remain obscure.
Furthermore, even completely formed teeth might have been secondarily contaminated with radionuclides released into the environment after the accident 24 . Radionuclides could be deposited on tooth surfaces and/ or incorporated into teeth through pulp via the bloodstream. Secondary contamination could thus be a factor that increased the amount of radioactivity in teeth that formed before the accident. Therefore, whether artificial radionuclides released into the environment after the accident were incorporated into fully formed teeth needed to be determined.
We collected 4,957 deciduous teeth from children living in the Fukushima area and other areas where the effects of the FNPP accident were considered negligible between 2014 and 2016 ( Fig. 1). All teeth were considered to have been formed before the FNPP accident because the calcification of the crowns of deciduous teeth starts at the gestational age of 4 months, is completed by the age of 12 months 25 and the teeth fall out between the ages of 6 and 12 years. Thus, to collect enough deciduous teeth that formed after the accident will take several more years.
We used imaging plates (IP) to radioactivity expressed in terms of quantum levels (QLs) in deciduous teeth collected from children living in Fukushima and reference prefectures. We then epidemiologically analysed factors that contributed to the detected QL. We measured concentrations of radionuclides that emit gamma rays ( 40 K, 137 Cs, 134 Cs, and natural radionuclides in the uranium and thorium series) and beta rays ( 90 Sr) using a germanium (Ge) detector and a low-background gas-flow counter, respectively. The present findings should serve as an important foundation by providing control data for future studies on teeth developed after the FNPP accident.

Results
Radioactivity in deciduous teeth determined using IP. Figure S1  www.nature.com/scientificreports/ respectively). Most of the teeth with high QLs were filled with composite resin. Further investigation revealed that some types of composite resin emitted significant beta and gamma rays that resulted in IP with high QLs (data not shown). Since the current study's objective was to determine whether the amount of radioactivity in natural teeth increased after the FNPP accident, we eliminated all teeth with fillings and/or decayed portions on the target surface. Therefore, 3,814 deciduous teeth with healthy target labial/buccal surfaces were further investigated.

Measurement of radionuclides in teeth.
Natural radionuclides in deciduous teeth. Table 1 summarises the results of the gamma ray measurements. The 214 Pb in 238 U and 212 Pb in 228 Th series, as well as 40 K, were found within a measurement period of 1 million seconds (11.6 days). The activity concentrations were consistently higher for 40 K than the other natural radionuclides. Radioactive caesium was not detected during this study's measurement period.
Determination of 90 Sr in deciduous teeth. Table 2 shows the 90 Sr findings in deciduous teeth collected from the major cities of Fukushima, Iwaki, and Koriyama in Fukushima prefecture and the reference prefectures, Hokkaido, Niigata, Shizuoka, and Kumamoto (Fig. 1). The amount of 90 Sr radioactivity detected in teeth ranged from undetectable to 2.05 mBq/g Ca. However, 90 Sr activity concentrations in teeth did not significantly differ between Fukushima and the reference prefectures.
Determination of 137 Cs in deciduous teeth. Since no radioactive caesium was detected during the 1 million seconds (11.6 days) measurement, we increased the duration of measurement (Table 1) to 1.6-3.0 million seconds (18.5-34.7 days). By doing so, we could increase the peak counts (662 keV) and detect trace amounts of radioactive caesium. Figure 2 shows a representative gamma ray spectrum of deciduous teeth measured for 3 million seconds (34.7 days). The 137 Cs peak should occur at 662 keV. Extending the measurement period to 34.7 days reduced the signal-to-noise ratio and revealed a 137 Cs peak. Table 3 shows the amounts of 137 Cs and other natural radionuclides in teeth from the Fukushima and reference prefectures. The teeth from the Fukushima prefecture were divided into the following 4 groups according to the time they fell out: Fukushima A, before the FNPP accident; Fukushima B: 0-2 after the FNPP accident; Table 1. Radioactivity of natural radionuclides (mBq/g) in deciduous teeth. Data are shown as mean ± counting error. Parentheses show numbers of teeth in a sample. Nuclides were measured in teeth for 1 million seconds (11.6 days). When the counts did not exceed the mean + 3SD of the background, we considered the value as LTD (lower than detection limit). SD, standard deviation. www.nature.com/scientificreports/ Fukushima C, 2-4 after the FNPP accident; and Fukushima D: 4-6 years after the FNPP accident. Trace amounts of 137 Cs were detected in teeth from Fukushima and the reference prefectures. We detected 137 Cs in teeth that fell out before and after the FNPP accident in teeth in all groups except group C from Fukushima prefecture, while 134 Cs was undetectable in all samples. The natural radionuclides, 214 Pb in the 238 U series, 212 Pb in the 232 Th series, and 40 K were detected in teeth, reflecting the results obtained after measuring gamma rays for 11.6 days. The activity concentration was consistently higher for 40 K than the other natural radionuclides.
Epidemiology of the radioactivity in deciduous teeth. Table S1 shows associations between various factors and the QLs of teeth determined by multivariate multilevel regression analysis adjusted for several confounding factors.
Regional differences. Figure 3 shows that the QLs of teeth did not significantly differ between the seven districts in Fukushima and the reference prefectures. However, the QL was lower in teeth collected from the Minami-Aizu, compared with the Ken-poku district, which served as the standard. Table S2 shows the mean QLs, SDs, numbers of teeth, and average air dose rates for each district.  Table 3. Radioactivity of 137 Cs and natural radionuclides in deciduous teeth (mBq/g). Data are shown as mean ± counting error. Parentheses show numbers of teeth in a sample. When the counts did not exceed the mean + 3SD of the background, we considered the value as LTD (lower than detection limit). SD, standard deviation. Since radioactive caesium was not detected during the 1 million seconds measurement (11.6 days), we increased the duration of measurement (Table 1) to 1.6-3.0 million seconds (18.5-34.7 days) to increase peak counts (662 keV). The teeth from the Fukushima prefecture were divided into the following 4 groups according to the time they fell out: Fukushima A, before the FNPP accident; Fukushima B, 0-2 after the FNPP accident; Fukushima C, 2-4 after the FNPP accident; and Fukushima D, 4-6 years after the FNPP accident. 134 Cs was undetectable in all samples.  Figure 4 shows that the QL was the highest in a maxillary deciduous incisor, and the mean QL was significantly higher for maxillary than deciduous incisors than for any other types of maxillary or mandibular deciduous teeth (p < 0.01).
Duration that teeth remained in the oral cavity after the accident. We aimed to determine the prevalence of secondary contamination, such as radionuclide deposition on the tooth surface and/or incorporation into the pulp, caused by radionuclides in the environment after the FNPP accident. However, the amount of radioactivity did not significantly correlate with the length of time that teeth remained in the oral cavity after the accident (Fig. 5).
Effects of gender, age at the time of the accident, air dose rate, and FNPP accident-related evacuation. None of the above factors were significantly associated with the QLs of the teeth.

Discussion
We measured radioactivity in deciduous teeth collected between January 2014 and December 2016 using IP, which have become valuable radiation research tools [26][27][28] . One advantage is that IP have high sensitivity with an excellent linear response ranging over three orders of magnitude. We obtained a good linear correlation (R 2 = 0.9998), ranging from 0 to 1,000 mBq 40 K/g ( Figure S2). Corresponding to a background of 3 SD, the estimated detection limit of QL was 91.9 (equivalent to 13.1 mBq 40 K/g in the reference scale. Furthermore, > 100 teeth could be concurrently measured using one IP, thus enabling the assessment of numerous teeth without destruction. With respect to natural radionuclides, 226 Ra, a daughter nuclide in the 238 U series, has been identified in permanent teeth from Japan different concentrations among regions 18 . The activity concentration of 226 Ra is higher in teeth from inhabitants living in areas of high, compared with low natural background radiation areas in Ramsar 29 . The low-background, well-type Ge detector identified 214 Pb, a daughter radionuclide in the 238 U series, in deciduous teeth. We also found trace amounts of 212 Pb in the 232 Th series. These findings indicated that the deciduous teeth contained natural radionuclides in both the 238 U and 232 Th series. We also identified the 40 K gamma spectrum, ranging from 6.1 to 10.3 mBq/g, in deciduous teeth. The K contents in tooth enamel and dentin are 0.037%-0.30% 11,30 and 0.02%-0.04% 11 , respectively, and 0.0117% of K is radioactive (30.4 Bq 40 K/g K). Here, the values ranging from 6.1-10.3 mBq 40 K/g, (0.2-0.34 mg K/g (or 0.020-0.034%). Therefore, these K concentrations, extrapolated based on the present results, are similar to those of the previous studies.
Several studies have shown a relationship between radionuclides in teeth and the environment. For example, 90 Sr was incorporated into human teeth after liquid radioactive waste was released into the Techa River during the early 1950s, resulting in radioactive contamination of the entire Techa River region [13][14][15][16] . Additionally, 90 Sr levels are higher in the deciduous teeth of Swiss children born between 1952 and 2002 17 and in people who   1,20 . Thus, we concluded that the 90 Sr detected herein did not originate from the FNPP accident but rather from environmental contamination caused by prior atmospheric nuclear weapon tests. Like 90 Sr, 137 Cs is a common component of local and global radioactive fallout and was released following various nuclear disasters. Increased levels of 137 Cs were reported in the deciduous teeth of children from south-west Poland in 1970s 19 . However, our knowledge about the amount of 137 Cs in teeth is still limited. We detected a small amount of 137 Cs in teeth collected from Fukushima and reference prefectures after precise measurements lasting from 18.5 to 34.7 days using a low-background Ge detector. The levels of 137 Cs were one order of magnitude lower than those of 90 Sr. That 137 Cs did not originate from the FNPP accident is likely because it was detected in teeth that had fallen out before the accident and in teeth from areas where the effects of the accident were negligible. Furthermore, all examined teeth were formed before the FNPP accident, and 134 Cs was undetectable. The FNPP accident released equal amounts of 134 Cs and 137 Cs into the environment, and they might still be detectable. Some reports have suggested that the Japanese environment contained 137 Cs before the FNPP accident [33][34][35][36] . For example, the increased 137 Cs concentration in foods after atmospheric nuclear tests continues to decrease [33][34][35] . Therefore, the findings of 137 Cs and 90 Sr in teeth formed before the accident need to be considered in future studies of teeth developed after the accident.
The QLs of teeth from Fukushima and the reference prefectures did not significantly differ even though the air dose rate considerably differed among districts after the FNPP accident. However, the QL was lower in teeth from the Minami-Aizu, than other districts. The reason for this is unknown. However, geological information obtained from a map of Japan's natural radioactivity before the accident shows relatively low natural radioactivity in the area where Minami-Aizu is located 37,38 . This might explain the lower QLs of teeth from Minami-Aizu.
The QLs of upper incisors was slightly, but significantly higher than that of the other types of teeth, regardless of whether teeth were from maxillary or mandibular bones. Beta rays are the main contributors to the generation of the QL. The self-absorption of beta rays occurs to a relatively large degree due to the high density of teeth. www.nature.com/scientificreports/ Therefore, IP mainly detect beta rays that are emitted by structures proximal to the tooth surface. Hence, differences in QLs are related to differences in radiation levels at the tooth surface. The surfaces of the upper incisors are more easily exposed to the atmosphere than that of other types of teeth. Therefore, natural radionuclides, such as 222 Rn in the 238 U series, in the atmosphere could be deposited on the surface of upper incisors more easily. Radionuclides are incorporated into teeth either during tooth formation or via secondary contamination after teeth are completely formed. Secondary contamination results when radionuclides from food, water, or the atmosphere, are deposited on the tooth surface or incorporated via pulp. In fact, permanent teeth formed before the Chernobyl accident contain a large amount of radioactive strontium 24 . All teeth examined in the present study were also formed before the FNPP accident. Therefore, we presumed that the increase in the QLs was caused by secondary contamination in the present study. The QLs of teeth did not increase according to the length of time that teeth remained in the oral cavity or air dose rate. These findings suggested that teeth were not significantly affected by secondary contamination by radionuclides released into the environment after the FNPP accident. Therefore, future studies of teeth formed after the FNPP accident should consider that any increase in QLs is related to radionuclides originating from the accident.
Studies that systemically focus on radioactivity and related radionuclides in human teeth are scarce. The present findings revealed that the amount of radioactivity in human deciduous teeth exceeded the background level, even after IP were shielded with lead to minimise natural background radiation. We also detected natural radionuclides, including 214 Pb in the 238 U series, 212 Pb in the 232 Th series, and 40 K, as well as artificial radionuclides, including 90 Sr and 137 Cs. Moreover, 40 K and 90 Sr (including its daughter nuclide 90 Y) apparently played important roles in the IP response because their concentrations and beta ray energy were relatively higher than those of the other radionuclides. Therefore, when investigating radioactivity levels in teeth formed after the FNPP accident, the knowledge that teeth originally contained radioactive nuclides is important to consider.
The present study systematically measured amounts of radioactivity in thousands of teeth collected from Fukushima prefecture and reference prefectures. Our results indicated that teeth formed before the FNPP www.nature.com/scientificreports/ accident originally contained natural and artificial radionuclides unrelated to the FNPP accident. Furthermore, regional amounts and types of radioactivity in teeth collected from Fukushima and reference prefectures did not significantly differ. We found no evidence to suggest that radionuclides originating from the FNPP accident significantly contaminated pre-existing teeth. Our findings may serve as important control data for future studies on teeth formed after the FNPP accident, which will proceed over the next several years. To the best of our knowledge, this is the first large-scale epidemiological investigation of radioactivity in teeth, and the data will be important for evaluating changes in radioactive substances in teeth associated with the FNPP accident and environmental changes.

Materials and methods
Collection of teeth. We collected 4,957 deciduous teeth that had fallen out or were extracted during dental procedures from children residing in Fukushima and other prefectures between January 2014 and December 2016 (Fig. 1) Radioactivity measurements of deciduous teeth. Measurements using IP. We measured radioactivity in deciduous teeth using BAS-MS 2040 IP (FUJIFILM Corp., Tokyo, Japan). The teeth were positioned such that their labial/buccal surfaces faced the IP surface, then IP cassettes were placed in a shielding box made of lead with iron plates for 4 weeks to block natural background radiation ( Figure S2). Details of how the teeth were positioned on the IP are described in the SI. The amount of radioactivity in each tooth was determined as QLs using a Fuji FLA-7000 bio-imaging analyser (FUJIFILM Corp.) and ImageQuant TL 8.1 (GE Healthcare). We used nine IPs to measure radioactivity in thousands of samples. However, the sensitivity of the IP varied, and the QLs differed even when the same sample was measured using different IP. Therefore, we normalised the QLs obtained from different IPs by placing a reference scale of potassium chloride on each one to calibrate the radioactivity values ( Figure S2). The methods used to normalise the QLs are detailed in the SI.
Measurement of natural radionuclides and radioactive caesium. We measured gamma rays emitted from radionuclides in teeth using a low-background, well-type, CANBERRA GCW3023 Ge detector (Mirion Technologies, San Raymon, CA, USA) with a shield of 20-cm thick lead blocks 39 . Since the amount of radioactivity of individual teeth was extremely low; we measured samples of 5-8 teeth packed to fill 7-8 mm of plastic vials with a diameter of 14 mm using forceps to minimise voids. The radioactivity of 214 Pb in 238 U series, 212 Pb in 232 Th series, 40 K, 137 Cs, and 134 Cs in teeth was determined as peaks corresponding to the following: 295 and 352 keV for 214 Pb; 239 for 212 Pb; 1,461 for 40 K; 605 for 134 Cs; and 662 for 137 Cs. The measurement period was approximately 1 million seconds (11.6 days), and the estimated overall measurement errors were < 15% for the determination of 40 K.
For radioactive caesium measurements in the teeth from groups Fukushima A-D (Table 3 and Fig. 5), radionuclides were measured in teeth for 1.6-3.0 million seconds (18.5 to 34.7 days) to increase peak counts (662 keV) and detect trace amounts of radioactive caesium. Radioactive caesium in tooth samples (n = 8-23) selected from groups Fukushima A-D was determined in descending order of QLs in each group. We measured samples from 8-23 teeth packed to 35 mm high. Teeth from reference prefectures were similarly measured.
The heights of samples in the vial ranged from 7-35 mm with a density of 0.8-1.0 g/cm 3 . The height of tooth sample greatly contributes to the counting efficiency during gamma ray measurement. CaCO 3 mixed with mineral powder (CRM 42-2 standard reference material, New Brunswick Laboratory, Argonne, IL, United States) 40 was used to account for the variation in sample height. The relationship between counting efficiency and height of standard material at different energy gamma ray is shown in Figure S3. The difference in counting efficiency in regions above 200 keV, less than 35 mm in height, and a diameter of 14 mm were < 10%.
Determination of 90 Sr in deciduous teeth. Deciduous teeth do not generally contain measurable amounts of 90 Sr. Therefore, 90 Sr was determined in samples of 5-10 teeth (> 2 g per sample) as described by Koarai et al 22 . Briefly, teeth were incinerated at 450 °C, followed by chemical separation of Sr from Ca using the fuming nitric acid method. Then, Ra and Pb were chemically removed via co-precipitation with BaCrO 4 , while Y was removed via co-precipitation with Fe(OH) 3 . Thereafter, the growth of 90 Y from 90 Sr was monitored by measuring beta rays emitted from 90 Sr and its daughter 90 Y using a low-background LBC-4201B gas-flow counter (Hitachi-Aloka Medical, Ltd.). Then, activity concentration of 90 Sr was calculated. The certificated reference material (fish bone, JSAC 0471, the Japan Society for Analytical Chemistry) was used as a quality control sample for 90 Sr measurement. www.nature.com/scientificreports/ Epidemiology of radioactivity in deciduous teeth. Regional differences at the time of the FNPP accident. We investigated regional differences between the QLs of teeth from Fukushima and reference prefectures and seven administrative districts in Fukushima prefecture (Fig. 1).
Types of deciduous teeth examined. We determined whether the QL differs among incisors, canines, and molars from maxillary and mandibular bones.
Differences related to duration teeth in the oral cavity after FNPP accident. We examined teeth that were formed before the FNPP accident. Therefore, radionuclides that originated from the FNPP accident were probably not incorporated into these teeth during their formation. However, secondary contamination with radionuclides can occur even after teeth are completely formed. Therefore, we assessed whether the QLs increased depending on how long the teeth remained in the oral cavity after the accident. We referred to the report by The Japanese Society of Pedodontics 41 for the timeline of deciduous tooth eruption and deciduation.
Gender differences. We investigated the influence of gender on the QLs of teeth.
Differences in age at the time of the FNPP accident. We examined whether age at the time of the accident affected the QLs of teeth.
Air dose rate. Secondary contamination could be caused by environmental radionuclides. Therefore, we assessed correlations between the QLs of teeth and air dose rates in the areas 42 where the donor children resided. Air dose rates used in each area are described in the SI.
Evacuation due to FNPP accident. We investigated the effects of evacuation on the QLs of the teeth.

Statistical analysis.
We performed a multilevel regression analysis to determine whether the seven factors mentioned above could affect the QLs of deciduous teeth. Of the 3,814 teeth with a healthy surface on the IPs, we eliminated 7 teeth for which enough information about the seven aforementioned factors was not available. Teeth were nested in individuals in multilevel models (3,807 teeth from 2,746 children). Confounding by multiple factors was considered using a multivariate model. Values with p < 0.05 were considered significantly different. Data were statistically analysed using Stata/MP version 15 (Stata Corp., College Station, TX, USA).

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.