The effect of hydrochloric acid (HCl) on permanent molars: A scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDS) study

Abstract

It is well established that acid disposal is a potentially effective method used by criminal syndicates to hinder the identification of victims. This study documents the effects of continuous immersion in hydrochloric acid (HCl, 37%) on molars using macroscopic analysis, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS). The goal of this study is to aid in distinguishing visually unrecognizable fragments of dental remains when drastic changes in morphology have occurred as a result of acid exposure. Macroscopic, SEM, and EDS analysis were conducted on seven maxillary molars before and after HCl treatment. Molars reduced in weight relative to the length of time immersed in HCl and the dissolution time was over 40 hours longer than reported in previous studies, at just over 66 hours. SEM and EDS analysis showed acid-treated teeth exhibited morphological patterns such as cracking and layering visible at high magnification. Calcium/phosphorous ratios fell within the expected range of 1.6–2.5, indicating that HCl-treated teeth are still identifiable as osseous or dental tissue even when not visually identifiable as teeth. This is the first study to present SEM images of molar cementum before and after immersion in HCl and to present EDS results. This information can assist researchers and investigators in determining the presence of dental tissue in a forensic context associated with acid disposal.

Keywords

Acid immersion human remains destruction forensic anthropology scanning electron microscopy SEM energy dispersive X-ray spectroscopy EDS

Introduction

It is well established that using acid to dispose of bodies or to mask the possibility of identification is a method used by criminal syndicates.^1,2 In 2018, reports related to the use of acid in an attempt to destroy human remains included the murder of three film students in Mexico and disposal using sulfuric acid; the murder and attempted disposal of a woman in muriatic acid by a couple in Texas; and the attempted disposal of 2-year-old’s remains by her parents in a 5 gallon tub of acid.^3–5 Given this disposal method is still in use today, understanding how teeth react to acid immersion can provide odontologists and forensic scientists with needed information to distinguish visually unrecognizable fragments of dental remains from other materials when drastic changes in morphology have occurred.

This study investigates the effects of continuous immersion in hydrochloric acid (HCl, 37%) on seven maxillary molars using macroscopic analysis, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS). The method of continuous immersion in acid used in this study allowed for a more realistic understanding of the timing and nature of macroscopic physical changes to tooth morphology than previous studies. With SEM, new observations regarding microstructural changes to teeth before and after acid immersion were compared. Importantly, the use of EDS data in this study, the first use of EDS for HCl acid-treated dental tissues, indicated that even after continuous acid treatment had rendered a sample visually unrecognizable as human remains, it still retained the elemental signature consistent with human osseous and dental tissue. The analysis of the results provided in this study can assist in determining the presence of dental tissue in a forensic context associated with acid disposal.

Acid effects on human dentition

Previous studies have established that HCl, also known as muriatic acid, is the most destructive acid to human dentition that is easily available to the public.^2,6–9 Studies testing the effects of immersion of dental tissues in HCl are generally consistent in describing macroscopic changes.^2,6–9 The physical changes observed follow a similar pattern, where teeth first began to effervesce upon immersion, followed by transparency and gelatinous texture visible at the crown and roots, disintegration of the crown and root, then complete dissolution at approximately 24 hours.^2,6–9 The current literature has not established how teeth react to HCl when continuously submerged until dissolution, since in all cases teeth were removed from the acid bath multiple times for measurement before reimmersion.^2,6–9

Scanning electron microscope imaging and energy dispersive X-ray spectroscopy/X-ray fluorescence spectrometry

Through studies of incinerated teeth and bone, the utility of SEM has been established in the field of forensic science.^10,11 Whereas research using SEM is widely used in the field of dentistry,^12–15 a gap in research regarding SEM forensic utility is evident in examining the effects of acid on immersed human teeth. Specifically, no SEM studies have been conducted to date documenting microstructural changes of acid immersion. The microstructural changes observed using SEM could be used to help distinguish acid immersion from other taphonomic changes on very small fragments of dentition.

Forensic scientists have also begun to explore the utility of elemental analyses: EDS, also known as energy dispersive X-ray analysis (EDX), and X-ray fluorescence spectrometry (XRF).^16–19 XRF and EDS provide similar information to forensic scientists in that they are both used to detect elements present in samples, though XRF uses an X-ray to detect elements and EDS uses electron beams. Based on XRF and EDS research, forensic researchers have found human bone and teeth to have predictable calcium to phosphorous (Ca/P) ratios which are measurable with very small samples, even those which are ancient, damaged, incinerated, or chemically treated.^1,16–19 Ubelaker et al.¹⁶ used XRF to identify osseous and dental tissues using Ca/P ratios. They found that Ca/P ratios for osseous material calculated using weight percentage tend to be higher (1.82–1.95) than those reported by atomic weight (1.41–1.50). Christensen et al.¹⁷ later validated the work of Ubelaker et al.¹⁶ in their XRF study of human and nonhuman osseous and dental samples, which included unaltered, burned, weathered, ancient, and chemically altered samples (nitric acid). Vermeij et al.¹ conducted EDS analysis as part of a study of forensic case reports involving remains disposed in a mixture of different acids. They concluded the elemental presence of Ca and P was detected in small fragments after acid disposal where there was no macroscopically visibly identifiable human osseous or dental tissue. Overall, Ca/P ratios calculated using weight percentage detected for modern osseous and dental material is reported at approximately 1.6 to 2.0, or up to 2.5 if including samples which were incinerated or are archeological in nature.^16,18

Methods

Materials and sample preparation

The teeth selected for this study were intact, non-carious maxillary molars that were extracted during the course of normal orthodontic practice and made available for educational purposes. All seven dental samples used in this study were identified as maxillary molars. All samples were third maxillary molars with the exception of sample ID#3, identified as a second maxillary molar. The teeth were stored in a solution of bleach during transportation and were sterilized within a week of extraction using an autoclave (121°C, 15 lbs psi) following standard methods.²⁰ The teeth were subsequently left to dry overnight in a ventilated drying cabinet.

The samples were then measured, weighed, and photographed before and after acid treatment. Standard odontometric measurements were taken, including the buccolingual diameter, mesiodistal diameter, and crown height at mesiobuccal cusp.²¹

Each tooth was assigned a time interval for complete immersion in a commercially available formulation of 37% HCl (ACS grade). The following time intervals were used: 1 hour, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, and 48 hours. During the study, the 48-hour time interval was extended to 66 hours on the final sample molar (ID#7). The only tooth reimmersed after removal was the 48-hour sample, which was removed, rinsed, weighed, and photographed before it was returned to the acid for an additional 18 hours, for a total of 66 hours. The tooth was not dried prior to reimmersion. Complete dissolution was expected prior to 48 hours, given previous studies. Because this did not occur, sample ID#7 was returned to the acid in an attempt to better understand the timing of complete dissolution.

Each sample was completely immersed in approximately 10ml of HCl using a glass test tube. Each test tube was covered with paraffin sealing film within a fume hood in a laboratory setting with an ambient temperature ranging from 75 to 78°F. With the exception of sample ID#7, the samples were left, undisturbed, during the assigned interval. As previously described, sample ID#7 was removed at 48 hours and reimmersed for an additional 18 hours. After acid treatment, the acid was drained from the immersion test tube using a funnel and filter paper to capture the tooth and any fragments. Each tooth was then immersed in a beaker of water and stirred for approximately 30 seconds in order to cease the action of the acid. Each tooth was then placed in a ventilated drying cabinet. After drying, the teeth were then remeasured. Sample ID#7 was placed in the ventilated drying cabinet after the 66-hour removal.

Scanning electron microscope

SEM images were obtained using a JEOL-7610F SEM and EDS data were obtained with a 1040 Oxford Instruments EDX detector. Images were obtained using the secondary electron (SE) detector at accelerating voltage of 5 kV.

The EDS analysis focused on the major elements typically detected in human teeth (Ca, P). Light elements such as C, N, and O were not included in the analysis due to the difficulty of reliable measurement and detection of generated X-rays in EDS analysis. EDS spectra were collected from area averages at 100–4000× magnification. EDS analysis was performed on one sample before treatment (ID#6) and one sample after treatment (ID#7). Sample ID#7 was selected for EDS analysis after acid treatment because this sample was no longer identifiable as a human tooth.

SEM analysis was performed on samples before and after acid treatment. Images were obtained at a range of magnification levels in order to analyze morphological changes. Images of the root portion of the tooth, composed of cementum, were obtained to compare the results for all stages of disintegration and dissolution. As samples were not conductive, a high degree of charging under the electron beam was observed, limiting the time the sample was exposed to the beam. This charging was reduced by the use of a nearby conductive medium.

Results

Macroscopic analysis

The pre and post-treatment odontometric measurements, weights, and acid immersion timings for each sample are listed in Table 1. The percent reduction in weight for each tooth relative to hours immersed in acid is shown in Figure 1. Physical changes observed for each sample by acid immersion time are listed in Table 2 and selected samples are shown in Figures 2 through 5.

Table 1.

Acid immersion time, weight, and measurements for samples in the present study.

Sample^a	Acid^b(hours)	Weight before^c(g)	Weight after^d(g)	Reduction weight (%)	MD^ebefore (mm)	MD after (mm)	BL^fBefore (mm)	BL after (mm)	CH^gbefore (mm)	CH after (mm)
ID#1	1	1.56	1.15	26.14	9.12	7.00	11.44	10.12	6.34	2.70
ID#2	4	1.47	0.80	45.53	8.26	6.18	11.94	9.10	6.61	5.00
ID#3	6	2.21	1.56	29.41	10.68	8.54	11.14	10.14	8.15	2.66
ID#4	12	1.84	1.05	42.74	9.12	6.5	10.8	10.36	6.91	5.18
ID#5	18	1.92	0.59	69.23	9.94	6.34	11.04	8.74	7.07	5.66
ID#6	24	1.79	0.48	73.18	10.48	n/a^h	10.85	n/a	6.58	n/a
ID#7	48	1.99	0.31	84.34	10.36	n/a	11.69	n/a	7.72	n/a
ID#7	66	1.99	0.03	98.34	10.36	n/a	11.69	n/a	7.72	n/a

^aSample Id numbers; ID#7was treated to 48 hours, removed, rinsed, and measured, then treated again until 66 hours; ^bTreatment time in HCl; ^cBefore indicates measurement before treatment; ^dAfter indicates measurement after treatment; ^eMD is mesiodistal width; ^fBL is buccolingual width; ^gCH is crown height at the mesiobuccal cusp; ^hn/a indicates tooth could not be accurately measured due to disintegration of measurement area.

Figure 1.

Percent reduction in weight for tooth samples treated in HCl by time interval.

Table 2.

Morphological changes observed during HCl (37%) treatment.

Immersion time^a (Sample)	Changes observed to individual samples
1 hour (ID#1)	Effervescence of sample. Transparent gelatinous appearance at the roots. Disintegration and reduction in crown size and morphology. Cusps changed from a rounded to sharp appearance.
4 hours (ID#2)	Transparency at root but minimal disintegration. Enamel was almost completely disintegrated, exposing underlying dentin. Cusp shape was markedly sharp instead of rounded. Some enamel remained on the crown but white enamel residue was noted in the acid solution.
6 hours (ID#3)	Transparency and disintegration of the roots. The crown was nearly half disintegrated, and the cusps appeared sharp instead of rounded. Some enamel remained on the crown but white enamel residue was noted in the acid solution.
12 hours (ID#4)	Transparency and disintegration of the roots. The crown was over half dissolved and the pulp cavity was exposed at the occlusal surface. No enamel remained on the crown but white enamel residue was noted in the acid solution.
18 hours (ID#5)	Transparency and dissolution of one-third to half of roots. The crown was nearly one-third dissolved, and the cusps appeared sharp instead of rounded. Deep cracks were visible on the surface of the crown and the root, but the pulp cavity was not visible. No enamel remained on the crown but white enamel residue was noted in the acid solution.
24 hours (ID#6)	Transparency and dissolution of nearly half of the roots. Over a third of the crown was dissolved and the pulp cavity was exposed at the apical surface. A slight purple discoloration was observed. No enamel remained on the crown but white enamel residue was noted in the acid solution.
48 hours (ID#7)	Transparency and dissolution of over half the crown and roots. The pulp cavity of the tooth was exposed accompanied by a bright purple coloration. The sample was still identifiable as a human tooth.
66 hours (ID#7)	Near complete dissolution of the tooth except for two small fragments. Fragments were surrounded by transparent gelatinous material. Purple coloration of fragments and solution was noted. Sample was too small to be identifiable macroscopically as a human tooth.

^aObservations at 48 and 66 hours were both observed on ID#7, which was removed from acid for photography and measurement before being returned to treatment.

Figure 2.

Photograph of maxillary molar sample ID#1 (a) untreated and (b) treated in HCl at 1 hour, rinsed in water and dried in a ventilated cabinet.

Teeth varied in weight from 1.47 grams to 2.21 grams with an average weight of 1.83 grams prior to treatment. The percent reduction in weight was calculated for each sample in order to estimate the approximate amount of dissolution for each tooth relative to hours immersed in acid (Figure 1). As expected, the percentage reduction in weight of each sample increased with the length of time each sample was immersed. This effect appeared exaggerated with sample ID#2. Sample ID#2 was the lightest sample and may be considered illustrative of the relationship between weight and dissolution time. It is likely that lighter teeth have a faster dissolution time. This is more apparent when comparing the heaviest and the lightest samples. Sample ID#3, the largest tooth, had a reduction of weight after 6 hours of 29.41% while sample ID#2 had a weight reduction of 45.53% after only 4 hours.

This study did not include time to total dissolution of any sample since an intact treated tooth fragment was needed to perform the SEM/EDS analysis portion of this study. Sample ID#7 was near complete dissolution and unidentifiable as a human tooth at 66 hours, with a total reduction in weight of 98.34%, weighing 0.03 grams (Figures 3–5). The progression of macroscopic changes to the samples is consistent with the general observations in previous studies (Table 2). In this study, complete dissolution was not observed by 24 hours as expected. Instead, by 66 hours the sample had reduced in weight by over 98% (Table 1, Figures 1, 4, 5). In addition, a purple color change was observed for two samples between 24 and 66 hours (Figure 3).

Figure 3.

Photograph of maxillary molar sample ID#7 (a) untreated, (b) treated in HCl for 48 hours and rinsed.

Figure 4.

Microscopic photograph of maxillary molar sample ID#7 after 66 hours of treatment in HCl, rinsed in water.

Figure 5.

Photograph of maxillary molar sample ID#7 after 66 hours of treatment in HCl, rinsed in water and dried in a ventilated cabinet.

Scanning electron microscope

SEM images of the cementum of samples ID#1, ID#2, and ID#4 were obtained before acid treatment at a wide range of magnification, up to 150,000× (Figures 6 and 7). Small cracks were visible on untreated teeth at low levels of magnification (100×–1000×). These cracks were possibly due to sterilization procedures to prepare the samples for handling prior to imaging (Figure 6(a), Figure 8(a,b)). However, this is unknown without further research. At levels of magnification greater than 1000×, fine cracking was no longer visible (Figure 7(b–d)).

Figure 6.

SEM images of sample ID#4 cementum before treatment with increasing magnification from image (a) to (f).

Figure 7.

SEM images of sample ID#1 cementum before treatment with increasing magnification from image (a) to (d).

Figure 8.

SEM images of (a) fine cracking visible on sample ID#2 cementum before acid treatment, (b) fine cracking visible on sample ID#6 cementum before acid treatment, (c) coarse cracking visible on sample ID#1 cementum after acid treatment for 1 hour, (d) sample ID#1 after acid treatment cementum appears similar to untreated sample ID#1 seen in Figure 7(c).

SEM images of the cementum of samples ID#1 (1 hour) and ID#7 (66 hours) were taken after treatment (Figures 8, 9). Morphological changes were visible on the cementum after 1 hour of treatment in HCl in the form of large coarse cracks visible on the surface between 100× and 300× magnification (Figure 8(c)). These cracks are more prominent than those visible on untreated teeth, suggesting that the acid saturated and widened already existing small cracks. However, at higher magnification (3000×), no identifiable differences were observed for the sample treated at 1 hour compared with untreated teeth (Figure 8(d)).

Figure 9.

SEM images of Sample ID#7 cementum after acid treatment for 66 hours showing morphological appearance of lamination or layered structures.

Dramatic morphological changes were noted in sample ID#7 after 66 hours, where magnification at 2000× to 10,000× showed structures that can be characterized by an appearance of lamination or layering (Figure 9). These structures were not visible at the same magnification prior to treatment (Figures 6 and 7).

Energy dispersive X-ray spectroscopy

EDS analysis was performed on one sample before treatment (ID#6) and one sample after treatment (ID#7). Samples selected for EDS in the present study indicated the presence of Ca and P before and after treatment. However, the ratio of Ca to P reversed from greater than 1 without treatment to less than 1 after treatment. The average Ca/P ratio for five EDS measurements of varying collection size on the untreated sample was 1.2 (max = 2.6). These measurements are similar to reported values with a range of 1.6–2.6 in earlier studies.^14,16 After acid treatment, the average Ca/P ratio from eleven measurements was 0.57 (max = 1.5). There was a high degree of variation in the EDS measurements indicating a high degree of inhomogeneity in the samples based on measurement location. Due to this inhomogeneity, a larger number of measurements were taken after treatment to confirm the reversal in Ca/P ratio.

Discussion

In this study, a number of new observations were made regarding the disposal of human teeth in HCl based on macroscopic observations, SEM images, and EDS results. These observations are useful in distinguishing dental tissue altered by HCl immersion through analysis of morphological changes both macroscopically and microscopically. At the macroscopic level, HCl-treated molars are still visually identifiable as human teeth even after 48 hours of continuous immersion. Not all teeth dissolve at exactly the same rate, however, and weight differences between molars likely influenced the timing of disintegration and dissolution of each sample. In general, the results indicated that lighter teeth exhibited a greater percentage of reduction in weight in less time immersed in acid than heavier teeth, although differences in morphology such as root number, shape, and length cannot be discounted as factors influencing timing of dissolution. It can also be concluded from the results of this study that the timing expected for dissolution of molar teeth in hydrochloric acid is not fully understood. The amount of dental tissue remaining between 48 and 66 hours was surprising given the expectation of total dissolution by 24 hours given previous studies.^2–6 It is possible that the sterilization method (autoclave) employed on the extracted teeth used in this study is a contributing factor in the timing of dissolution; however, to date no study has consistently established the effects of sterilization protocols on extracted teeth. The two most commonly recommended and used sterilization methods, 10% formalin and autoclave, have been the subject of inconsistent findings regarding research outcomes.^22,23

After continuous immersion in HCl beyond 48 hours, dramatic morphological changes occured, where the dental tissues disintegrated nearly completely, leaving minute fragments (Figures 4 and 5). These fragments are so small that microscopic and elemental methods are extremely useful, if not essential, tools in determining whether or not human osseous or dental tissue is present in the context of acid disposal. The SEM images presented of untreated and HCl-treated cementum in this study are some of the first produced using a SE detector instead of a backscatter electron (BSE) detector. SE images show finer details of microstructures than BSE images due to their higher resolution.¹⁴ As a result of this study, previously unknown morphological changes were observed, such as the presence of lamination of the surfaces of the tooth between 2000× and 10,000×. These changes may tentatively be considered a morphological feature indicative of acid treatment and should be considered in future research studies.

The results of this study confirmed that EDS analysis can quickly identify an osseous or dental elemental origin for an adult maxillary molar nearly completely dissolved in HCl, a finding not reported in any other published studies regarding acid disposal. Specifically, calcium and phosphorous were readily identified as the main elemental components of all samples, both before and after acid treatment. While the Ca/P ratio of untreated samples was in the range of that expected for human osseous material, the Ca/P ratio of the acid-treated dental samples in the current study was much lower than this reported range. It is suggested that the destructive action of the HCl breaking down the hydroxyapatite of the tooth may result in a lower Ca/P ratio as the acid causes greater loss of calcium relative to phosphorus. Potentially, this finding may be useful in determining the type of acid used to destroy remains based on the Ca/P ratio of recovered fragments, although further research is needed to validate this conclusion.

Conclusion

This study is useful to the forensic community by characterizing the effects of HCl on dental remains, especially molars. Even after remains have undergone drastic morphological changes as a result of HCl immersion, microscopic residues can remain that are identifiable using SEM and EDS analysis. The use of SEM in this study has produced the first detailed SE images of the root portion (cementum) of molars, both before and after treatment with HCl. These new images and descriptions of morphological changes after chemical treatment are useful to forensic scientists in helping to establish a baseline for expected changes in microstructures of dental tissues when immersed in HCl. Likewise, the present study is the first to produce EDS results including Ca/P ratios of acid-treated dentition, which can be used as a starting point for future research using EDS to distinguish acid-treated dental fragments from other materials.

Future studies which expand current sample sizes are needed to validate the SEM/EDS findings and to better understand and explain differences in reported timing of dissolution rates of human dentition. Future studies which specifically utilize SEM images are needed to further investigate the microscopic morphology of HCl-treated dental samples using comparable images. Further research should also include differences in type of tooth, including immature or incompletely developed dentition. Through understanding the dissolution process, forensic scientists will be better able to help with complex crime scenes such as those where human remains are disposed of in acid. This study, combined with those that follow, can provide the forensic field with needed tools to identify human dentition, even when only small samples remain.

Footnotes

Acknowledgements

The authors wish to thank Linh Pham and Gunisha Sagar for their assistance in the chemistry laboratory.

Compliance with ethics statements

This manuscript has not been published and is not under consideration for publication elsewhere. This article does not contain any studies involving human participants performed by any of the authors.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Christine Jones

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