NEHA September 2025 Journal of Environmental Health

The September 2025 issue of the Journal of Environmental Health (Volume 88, Number 2), published by the National Environmental Health Association.

Environmental Health To build, sustain, and empower an effective environmental health workforce Volume 88, No. 2 September 2025 Journal of

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Environmental Health To build, sustain, and empower an effective environmental health workforce Volume 88, No. 2 September 2025 Journal of

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ABOUT THE COVER

Permanent body art has grown in popularity in recent years, with millions of individuals getting tattoos. With this decision to get a tattoo comes risk: Injecting coloring

Decoding Tattoo Inks: Multiple Analysis Techniques Reveal Discrepancies in Ingredient Composition and Elemental Content When Compared Against Label Claims............8

Special Report: Foodborne Illness Surveillance: A Model for COVID-19 and Other Future Pandemics? ............................................................................................................ 20

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compounds into the skin has been reported to cause allergies, skin inflammation, and sys- temic disorders. The goal of this issue’s cover article was to identify the pigments in a set of commercially available yellow tattoo inks. The tattoo and reference inks were examined using a range of techniques. The combined use of these techniques provides insight into ink composition without needing dicult and time-consuming sample preparation. More importantly, results indicate that the tattoo ink compositions diered from what was described on the labels. These unlabeled ingredients raise concerns about the regulation, health eects, and degradation products of tattoo inks. See page 8. Cover images © iStockphoto: Zdyma4

Development, Evaluation, and Long-Term Outcomes of Environmental Health and Land Reuse Training, Part 2—Environmental Health and Land Reuse Trainings for Environmental Health Professionals: Long-Term Follow-Up.............................................. 32

Direct From EHAC: From Classrooms to Communities: Impact Stories From Accredited Environmental Health Programs ................................................................................................. 40

The Practitioner’s Tool Kit: Field pH Monitoring 101 ................................................................. 46

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Environmental Health Calendar ...............................................................................................50

Spotlight on NEHA Resources: Emergency and Disaster Readiness ............................................ 52

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President’s Message: Let Your Light Shine—Claim Your Space! .............................................................. 6

Special Listing ........................................................................................................................... 56

NEHA Annual Financial Statement......................................................................................................58

NEHA 2026 AEC....................................................................................................................... 59

NEHA News .............................................................................................................................. 60

September 2025 • Journal of Environmental Health

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September 2025 • Journal of Environmental Health

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Open Access

 PRESIDENT’S MESSAGE

Let Your Light Shine— Claim Your Space!

Larry A. Ramdin, MPH, MA, REHS/RS, CP-FS, HHS, CHO

E nvironmental health is the second larg- est workforce in public health (Ryan et al., 2021). Despite our numbers within the public health workforce, we remain large- ly unnoticed and underappreciated. We as environmental health practitio- ners are more than the enforcers of envi- ronmental health laws and regulations (i.e., the “environmental health police”). We are educators, changemakers, and protectors of the public at every moment of their daily lives. We are involved in food safety, waste- water disposal and control, recreational waters, water quality, and air pollution, just to name a few of our areas of responsibil- ity. The animated video from the National Environmental Health Association (NEHA, 2021), Environmental Health Professionals: Your Unseen Army of Protectors , highlights our role in protecting our communities as environmental health scientists. As such, we are doers not talkers. In many societies, self-promotion is viewed negatively, but if we do not promote our- selves then who will? NEHA has embarked on renewed eorts to highlight the profession and the work we do, how we impact human lives, and how we protect the environments we live in. We all must do our part, now! Therefore, it is essential to educate our peers, principals, and communities on who we are and what we do. We need to claim our space and let our light shine. In preparing for my second column. I wondered what was special about September. September is National Disaster Preparedness Month and National Food Safety Education Month. More importantly for environmental

When I taught the communication module of the leadership workshop held as a precon- ference oering at the NEHA Annual Edu- cational Conference (AEC) & Exhibition, I encountered Wesley Nicks, director of envi- ronmental health in Placer County, California. Wesley’s oce produced a local video high- lighting all that environmental health does to protect their community, which stars his envi- ronmental health team (Placer County, 2019). In my first stint as a local public health direc- tor, I filmed two segments on food safety in the home for local cable TV. Engage with your local cable access TV station, they are always looking for content and will work with you to tell your story and educate your communities. Always be on the lookout to build relation- ships that will promote what you do. We can also look into hosting a regular program about environmental health guid- ance and tools the public can use to assist us in achieving our goals. Environmental health practitioners can bring and share their scientific knowledge to improve behav- iors and reduce environmental health risks in the community. In the post COVID-19 pandemic period, where I worked in Waterford, Massachusetts, was inundated with rodent complaints. My chief environmental ocer came to me with an idea she wanted to develop after discuss- ing it with her team. They were concerned that using natural jack-o’-lanterns (i.e., pumpkins) would provide food for rodents and draw them to homes. They wanted to recommend using artificial jack-o’-lanterns for the Halloween season, as well as provide alternative options for people who chose to

It is essential to educate our peers,

principals, and communities on who we are and what we do.

health, we celebrate World Environmental Health Day on September 26. This celebra- tion solely focuses on our profession and our- selves! How do we seize the moment? What do we do? Where do we go? We can learn from other practitioners about what they have done to promote the profession and celebrate our successes. In 2018 when I was the regional vice-president of NEHA’s Region 9, I was invited by Phyl- lis Amadio, president of the Connecticut Environmental Health Association, to pres- ent the NEHA Certificate of Merit to their board of directors. They did sterling work in getting World Environmental Health Day proclaimed as Connecticut Environmen- tal Health Day. And a year later, they con- vinced the Connecticut governor to declare the entire week as Environmental Health Week. We can take a page out of their book and contact legislators, governors, mayors, county board members, and other man- agement groups to issue proclamations for World Environmental Health Day.

Volume 88 • Number 2

https://doi.org/10.70387/001c.143996

use natural pumpkins for their jack-o’-lan- terns (e.g., displaying them 6 ft up o the ground, displaying them in a window, bring- ing them indoors at the end of the evening). They issued a press release with these ideas and it was picked up and carried by three local TV stations. Acting as an advisor instead of a regulator will have significant returns as we celebrate ourselves. NEHA is actively claiming space in print and other media to advocate for the profes- sion. You can also advocate for the profes- sion by using your scientific knowledge and writing an article for your local newspaper to educate the community about environmental health, celebrate your environmental health successes, and guide positive behaviors among your communities. The other thing you can do is host an edu- cational forum on a timely environmental health concern that needs to be addressed. Ask for time on the agenda of your legislative or supervisory boards and present on that subject, where you will have a broader com- munity impact.

Alternatively, host an open house for the community, with all your scientific test- ing equipment on display. Let visitors touch them, explain what they are used for, discuss how the data are gathered from the tests, and highlight how the data are used to prevent disease and injury. Yes, be the scientist not the regulator. Share your knowledge. Your public will respect you for it, and you can build relationships and create support with- out asking for it. Lastly, celebrate yourself. Put out a ban- ner proclaiming Environmental Health Day, recognize each other, give the deserved pat on the back to each other, hold a breakfast or luncheon (potluck or catered), and laugh and enjoy yourselves in the knowledge that you have done a good job. Be proud of who you are and let us join together in celebrating World Environmental Health Day on September 26 by letting your light shine and claiming your space! I end my column with the lyrics from We Are the World , written by Michael Jackson and Lionel Ritchie in 1985:

“There’s a choice we’re making We’re saving our own lives It’s true we’ll make a better day Just you and me”

lramdin@neha.org

References National Environmental Health Association. (2021, June 17). Environmental health professionals: Your unseen army of protec- tors [Video]. YouTube. https://youtu.be/ oLF_KS_IwkA Placer County. (2019, January 18). Meet Placer County Environmental Health [Video]. You- Tube. https://youtu.be/Pp6SYFhQbHc Ryan, B.J., Swienton, R., Harris, C., & James, J.J. (2021). Environmental health work- force—Essential for interdisciplinary solu- tions to the COVID-19 pandemic. Disaster Medicine and Public Health Preparedness , 15 (2), e1–e3. https://doi.org/10.1017/dmp. 2020.242

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September 2025 • Journal of Environmental Health

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Open Access

Decoding Tattoo Inks: Multiple Analysis Techniques Reveal Discrepancies in Ingredient Composition and Elemental Content When Compared Against Label Claims

tain various chemical compounds, includ- ing 1) vehicles such as water, glycerine, and other alcoholic derivatives; 2) additives such as surfactants, polycyclic aromatic hydrocar- bons, nanoparticles, and polymers; and 3) pigments of varying purity (Arl et al., 2019; Bäumler, 2020; Høgsberg et al., 2011; Wang et al., 2021). These chemical compounds can include substances that were designed for use in paints, non-tattoo inks, or plastics. Throughout history, the composition of tattoo pigments has evolved from natural extracts and metal salts to a mix of inor- ganic oxides, salts, inorganic pigments, and azo dyes (Barua, 2015). Historically, inorganic compounds such as mercury(II) oxide for red, cobalt(II) aluminate for blue, chromium(III) oxide for green, manganese violet for purple, titanium dioxide for white, and iron oxides for brown tones were used (Bocca et al., 2017; Poon et al., 2008; Rio et al., 2020). Often, these inorganic compounds were blended with other organic and inor- ganic components to enhance the vibrancy of the colors (Forte et al., 2009). Now, how- ever, tattoo ink manufacturers use artificial organic and organometallic pigments mixed with inorganic compounds to make tattoo inks (Negi et al., 2023), with metals still present as chromophores, shading additives, or impurities (Arl et al., 2019; Rio et al., 2020). Further, inorganic pigments based on metal salts are currently used in micropig- mentation inks in permanent cosmetics such as permanent eyebrow makeup, eyeliner, and lip color (Rio et al., 2020) due to their higher durability against light and heat, bet- ter setting capacity, and larger size—all of which make their removal more dicult. Batool A. Aljubran College of Science and Engineering, Flinders University Kirstin E. Ross, PhD College of Science and Engineering, Flinders University Ula N. Alexander, PhD College of Science and Engineering, Flinders University Claire E. Lenehan, PhD College of Science and Engineering, Flinders University

Abstract Permanent body art has grown in popularity in recent years, with millions of individuals having black/monochrome or colorful tattoos. With this decision to get a tattoo comes risk: Injecting coloring compounds into the skin has been reported to cause allergies, skin inflammation, and systemic disorders. Despite the growing number of tattooed individuals, there are currently few regulations, laws, and safety criteria for tattoo and permanent cosmetic formulations. The goal of our study was to identify the pigments in a set of commercially available yellow tattoo inks. We examined a set of previously unstudied yellow tattoo inks: lemon yellow (LY), golden yellow (GY), golden rod (GR), and bright orange (BO). We also examined reference pigments: pigment yellow 14 (PY14), pigment yellow 65 (PY65), pigment blue 15 (PB15), and pigment orange 13 (PO13). Both sets of inks were examined using a range of techniques, including Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, X-ray diraction (XRD), Raman spectroscopy, energy dispersive X-ray (EDX) spectroscopy, and inductively coupled plasma optical emission spectroscopy (ICP-OES). We report that the combined use of these techniques can provide major insights into ink composition without needing dicult and time-consuming sample preparation. Results of our study indicate that the ink compositions diered from what was described on the labels. Furthermore, we demonstrate that the tattoo inks tested included additional elements that were not listed as ingredients, such as aluminium (Al), sodium (Na), and silicon (Si). These unlabeled ingredients raise concerns about the regulation, health eects, and degradation products of tattoo inks. Keywords: tattoo ink, pigment yellow, chemical analysis, safety regulations

Introduction Body decoration by tattooing has increased in popularity in the last 10 years. It has been reported that 40% of young adults in the U.S.

and 25% of adults in Australia have at least one tattoo (Chalmers et al., 2019; Heywood et al., 2012; Lichnyi et al., 2021; Niederer et al., 2018). Tattoo ink suspensions can con-

Volume 88 • Number 2

https://doi.org/10.70387/001c.143999

Modern tattoo inks vary greatly in compo- sition and can contain hazardous ingredients not originally intended for this purpose (Negi et al., 2022) and might not have a proven track record of safety in tattooing (Lehner et al., 2011; Vasold et al., 2004). Increasingly, there are concerns about tattoo ink e ects on human health, including potential carcino- genicity (Bauer et al., 2022; Desmedt et al., 2022). Additionally, it has been reported that tattoo inks can trigger acute allergic reactions immediately or lead to hypersensitivity after long-term exposure (Senaldi et al., 2016; Renzoni et al., 2018; Wang et al., 2021). For example, Klügl et al. (2010) reported that >70% of 3,411 tattooed persons experienced issues with their skin immediately or within a few weeks after getting their tattoo. More- over, allergic responses to tattoos, especially with red inks, have been reported to persist for months or years (Serup et al., 2017). Given the growing popularity of tattoos and the possibility of dangerous ingredients in tattoo products, regulations are needed to reduce the hazards caused by inappropriate tattoo inks (Wang et al., 2021). In Australia, for example, tattoo inks are not considered therapeutic materials and are not regulated by the Therapeutic Goods Administration. Instead, the National Industrial Chemi- cals Notification and Assessment Scheme (NICNAS) regulates the chemicals found in tattoo inks but typically does not legislate the import of a chemical that is used in tat- too ink if they are listed in the Australian Inventory of Chemical Substances. Little is known about tattoo ink contamination or adulteration (Musgrave, 2014); however, there is evidence of incorrect labeling. In 2016, a NICNAS report about tattoo ink composition advised that specific tattoo inks in Australia were noncompliant with regulations, marketed with incorrect ingre- dients, or not suitable for use (National Industrial Chemicals Notification and Assessment Scheme, 2018). In addition, according to a survey conducted among tattoo artists in downtown Brisbane and Melbourne Central in Australia, tattoo art- ists were unaware of the ingredients in their inks or the possible dangers associated with them (Matsika et al., 2016). In Europe, tattoo inks are regulated by the European Union’s General Product Safety Directive. According to this directive, a pro-

ducer is required to place only safe items on the market, and a comprehensive list of con- tents must be included on the product label, which is accomplished through classification, labeling, and packaging (Minghetti et al., 2019; Negi et al., 2022). In 2011, an analysis by Hauri (2011) reported finding 34 prohibited pigments in 30 tattoo ink samples. The pigment green 36 (PG36, C.I. [color index] 74265) was declared in three green inks, but the samples were dem- onstrated to include the prohibited pigment green 7 (PG7, C.I. 74260). Furthermore, a yellow and a blue pigment were stated for one ink, but the ink was again found to contain PG7, not the stated yellow and blue pigments. The ingredient list on a violet ink was incor- rect: The ink included the pigment white (tita- nium dioxide [TiO 2 ]) and the pigment blue 15 (PB15, C.I. 74160), which when combined produce a light blue. And the violet color was shown to be created with the prohibited pig- ment violet 23 (PV23, C.I. 51319). Concerningly, mislabeling and undeclared ingredients continue to persist in commercial formulations of tattoo ink. A study by Poon et al. (2008) examining 190 tattoo inks indicated that 37% of the inks included prohibited sub- stances and 53% contained >1 of A) excessive levels of nitrosamine, B) unreported material, or C) claimed material that was not found in the ink. More than a decade later, Wang et al. (2021) reported that for 50% of the tattoo inks they tested, labeling inaccurately stated at least one pigment component. Furthermore, a study by Moseman et al. (2024) showed major discrepancies between tattoo ink composition and ingredient labels, especially for the non- pigment components (e.g., carrier, vehicle). Regarding tattoo safety and the possibility of allergic sensitization, it is likely that some pig- ments have been removed from the ink formu- lations due to EU regulations banning specific pigments in tattoo inks (Kiszla et al., 2023; Wang et al., 2021). Identifying pigments in commercial tat- too inks presents a large challenge due to the inks’ complex composition, diverse combi- nations of pigments used to achieve subtle colors, poor solubility of pigments in tradi- tional solvents, and other additives present that enhance pigment dispersion (Bauer et al., 2019). Studies have primarily relied on mass spectrometry (MS) for pigment analy- sis. Both Hauri (2011) and Wang et al. (2021)

used MALDI-TOF MS to identify pigments in tattoo inks; however, this approach has limitations. For example, Wang et al. (2021) showed that some pigments were unable to be detected using MALDI-TOF MS due to poor ionization and or low mass. Further- more, MS-based techniques often require extensive sample preparation, making the analysis time-consuming and challenging for label compliance assessments. Given the complex and varied chemical nature of tattoo ink ingredients, our study aimed to develop a novel approach for pig- ment identification by applying a combina- tion of spectroscopic techniques. Unlike previous studies that primarily analyze tattoo inks after extensive pretreatment, digestion, or extraction, our approach focused on exam- ining inks in their dried, untreated state or with minimal sample preparation. Compared with MS, spectroscopic techniques such as infrared (IR), nuclear magnetic resonance (NMR), X-ray di raction (XRD), Raman, and energy dispersive X-ray (EDX) enable rapid, minimally destructive analysis of pig- ment molecular structures, crystallinity, and elemental composition with minimal sam- ple preparation requirements (Bauer et al., 2019, 2020; Moseman et al., 2024). Although most prior research uses these techniques in isolation, our study leveraged the com- bined information from spectroscopic and elemental analysis techniques of IR, NMR, XRD, Raman, EDX, and inductively coupled plasma optical emission spectroscopy (ICP- OES) to rapidly assess the pigment composi- tion of a set of yellow inks that have not been examined previously.

Methods

Materials and Instruments INTENZE brand lemon yellow (LY), golden yellow (GY), golden rod (GR), and bright orange (BO) tattoo inks were purchased from Tattoo Direct in Victoria, Australia. Pigment yellow 14 (PY14, C.I. 21095, 97% purity); pigment yellow 65 (PY65, C.I. 11740, 98% purity); pigment white (titanium dioxide [TiO 2 ], C.I. 77891, 99.5% purity); pigment blue 15 (PB15, C.I. 74160, technical grade); pigment orange 13 (PO13, C.I. 21110, tech- nical grade); and barium sulfate (BaSO 4 , C.I. 77120, 99% purity) were purchased from AK Scientific in Union City, California. Methy-

September 2025 • Journal of Environmental Health

ADVANCEMENT OF THE SCIENCE

TABLE 1

Reported Ingredients of Tattoo Inks According to Manufacturer’s Label and Material Safety Data Sheets (MSDS)

Tattoo Ink

INTENZE: Lemon Yellow

INTENZE: Golden Yellow

MSDS

Confirmed in Study

Declaration of Ingredients *

MSDS

Confirmed in Study

Declaration of Ingredients *

2018 2022 2023

x x x x – –

x x x –

x x

TiO 2



BaSO 4 PB15 PY65 PY14 PO13 Aqua

– – – x x x x



– –

– – x x

– –



– x x

–

– – – –

–

x x x

– – –

Glycerine

Hamamelis virginiana (witch-hazel) extract

Isopropyl alcohol

–

continued 

lene chloride (99.9% purity) was purchased from RCI Labscan, Australia. Except for the tattoo inks, all chemicals used in our study were not purified further. Tattoo inks were pipetted onto microscope slides and dried in open air at ambient tem- perature for 48 hr prior to characterization. To extract the pigments from the liquid tattoo inks, we added approximately 25 mg of tattoo ink to a 50-ml conical glass tube containing 2 ml of water. The contents were mixed vig- orously and extracted 3 times with 15 ml of methylene chloride. The methylene chloride extracts were combined, dried, and analyzed as dry tattoo ink extracts. Instrumental Analysis Fourier transform infrared spectroscopy (FTIR) was used to generate an IR spectrum of the pigments and dried inks using a Perkin Elmer Spectrum 100 FTIR spectrophotom- eter equipped with an attenuated total reflec- tance (ATR) diamond crystal in the range of 400–4,000 cm -1 with a resolution of 4 cm -1 . The energy of electromagnetic radiation is expressed in wavenumbers, and the intensity is expressed as a percentage of transmittance. Solid state carbon-13 nuclear magnetic resonance ( 13 CNMR) experiments were undertaken using a Bruker Avance III 400 MHz spectrometer operating at 100 MHz

FIGURE 1

Schematic Representation of the Reference Pigments Reported to Be Present in the Lemon Yellow (LY), Golden Yellow (GY), Golden Rod (GR), and Bright Orange (BO) Tattoo Inks

Note. PB = pigment blue; PO = pigment orange; PY = pigment yellow.

for 13 C. Chemical shifts are relative to ada- mantane. Approximately 100 mg of pigment sample/dried ink extract was placed in a Bruker 4-mm rotor and spun at 5 kHz. 1 H- 13 C

cross-polarization magic angle spinning spectra were recorded using an acquisition time of 18.4 ms, a recycle delay of 2 s, and a contact time of 4 ms with a 50% ramp and

Volume 88 • Number 2

TABLE 1 continued

Reported Ingredients of Tattoo Inks According to Manufacturer’s Label and Material Safety Data Sheets (MSDS)

Tattoo Ink

INTENZE: Golden Rod

INTENZE: Bright Orange

MSDS**

Confirmed in Study

Declaration of Ingredients *

MSDS

Confirmed in Study

Declaration of Ingredients *

2018 2022 2023

TiO 2

– – – – x x x x

– – – –

x x



BaSO 4 PB15 PY65 PY14 PO13

– – – x x x x



– – x x x x x

– –



Aqua

– – –

Glycerine

Hamamelis virginiana (witch-hazel) extract

Isopropyl alcohol

–

*Declaration of ingredients according to the label on the bottles of ink that were purchased in 2019. **Golden Rod ink has the same composition according to the three MSDS from 2018, 2022, and 2023. Note. BaSO 4 = barium sulfate; PB = pigment blue; PO = pigment orange; PY = pigment yellow; TiO 2 = titanium dioxide. Source: INTENZE Advanced Tattoo Ink, 2025.

decoupling during acquisition (Spinal 64). Sideband suppression was achieved using the standard total suppression of sideband (TOSSa) sequence. XRD was recorded for pigment samples and dried tattoo inks. Data were collected using a Bruker Advanced D8 di ractometer with Co K α ( λ = 1.7889 Å, 2 θ = 10°–90°, time per step = 0.5 s). All samples were ground to a fine powder with a mortar and pestle before being loaded onto an XRD sample stage. Raman spectra were collected using a Horiba Scientific Xplora Plus Raman spec- trometer at both 786 nm and 532 nm. The analysis was performed at 200 cm -1 to 3,000 cm -1 wavenumbers, with the laser inten- sity reduced using 10% and 25% filters. For each sample, 12 scans of 20-s pulses were recorded. In all instances, dried pigment/ink powder was placed onto a glass slide, which was mounted on the Raman spectrometer’s sample holder for analysis. Scanning electron microscopy (SEM) was undertaken with an FEI F50 inspect system equipped with an Octane Pro EDX detec- tion system. Pigment samples were prepared by directly spreading pigment powder onto sticky carbon tabs. The working distance was

10 mm, and the acceleration voltage was 10 kV. The PY14, GR, and GY inks were coated with platinum with a thickness of about 2 nm to increase their electrical conductivity. ICP-OES was conducted using a Perkin Elmer Optima 8000 ICP-OES. Prior to analy- sis, approximately 100 mg of the dried ink samples were digested in 5 ml of nitric acid (HNO 3 ) using a microwave digestor. After digestion, the samples were diluted to 50 ml in Milli-Q water, giving an HNO 3 concentration of 10%, and an aliquot was diluted twice in ultrapure water (18 MΩ), giving a 5% HNO 3 concentration. Finally, the samples were fil- tered using a 0.45-µm nylon filter prior to analysis. Calibration standards (5 ppb to 1 ppm) were prepared in 5% aqueous HNO 3 .

the reported ingredients listed on the manu- facturer’s label and in the provided material safety data sheet (MSDS) of the four inks we examined. Table 1 shows the MSDS ingredient lists for the inks as of 2018, 2022, and 2023, and the declared ingredients on the label. It is evident there are some di erences in reported ingredients on the MSDS over this time. Inks used in our study were purchased in 2019, so it could be expected that the reported ingredients on the label would match those of the MSDS from 2018. Despite this expecta- tion, there are some discrepancies between each label and the corresponding 2018 MSDS. For example, the label for LY ink indicates that it contains PY65 (Figure 1), but PY65 is not listed in the 2018 MSDS. Similarly, PY14 (Figure 1) is not listed as an ingredient on the label of the LY ink, but PY14 is listed in the 2018 MSDS. The labels for GR and GY inks list that they contain PO13 (Figure 1); how- ever, PO13 is not listed in the 2018 MSDS. And lastly, PB15 (Figure 1) is listed on the label and in the MSDS for LY in 2018 and 2022, but it is no longer listed on the 2023 MSDS. Similarly, there were discrepancies on other ingredients such as BaSO 4 , which is not listed on the label of GY and BO inks.

Results and Discussion

Ink Characterization

Reported Ingredients As prior research has shown that tattoo inks ingredients are sometimes misidentified on the label (Bauer et al., 2019, 2020; Moseman et al., 2024; Wang et al., 2021), our study examined

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Pigment Characterization Reference pigments, dried inks (D-ink), and ink extracts (E-ink) were compared using FTIR, NMR, XRD, Raman, EDX, and ICP-OES to identify the likely pigments in the inks. FTIR Spectra Figure 2 depicts the FTIR spectra of pig- ments obtained from ink extracts (E-LY, E-GY, E-GR, and E-BO) and reference pig- ments (PY14, PY65, PB15, and PO13) between 1,900 cm -1 to 600 cm -1 . The full range of the spectra (4,000 cm -1 to 550 cm -1 ) and assignment of functional groups to the FTIR spectra can be found in Supplemental Figure S1 and Table S1. Moreover, PO13 had characteristic peaks at 1,653, 1,493, 1,371, 1,331, 1,235, 1,144, 1,044, 998, 907, and 682 cm -1 . These peaks were not observed in the ink extracts, which indicates that none of the ink extracts contained PO13—or if they did, PO13 presented at levels below the instrumental limit of detection. Similarly, PY65 had distinctive absorption peaks at 1,546, 1,302, 1,187, 1,135, 1,030, and 763 cm -1 that were not observed in the ink extracts. This finding indicates that PY65 was not present in any of the inks—or if it was, it was present at levels below the instru- mental limits of detection. The IR spectral of PB15 shows several peaks at 1,612, 1,464, 1,421, 1,331, 1,287, 1,166, 1,119, 1,087, 901, 877, 778, and 725 cm -1 . The absence of these peaks in LY ink indicates that the FTIR spectra could not confirm the existence of this pigment in this ink. Moreover, PY14 had peaks at 1,670, 1,515, 1,360, 1,245, 1,171, 950, 860, 782, 750, and 619 cm -1 that were correlated with the presence of C-Cl and N-C=O bonds (Bauer et al., 2020). All four ink-extracts yielded FTIR spectra that appeared very similar to the spectra from PY14, indicating that they likely contained PY14. FTIR spectra from the dried inks (D-LY, D-GY, D-GR, D-BO) were consistent with those of the extracted inks (Supplemen- tal Figure S2). NMR Analysis In case the pigments were at concentrations below the limits of detection for the FTIR study, NMR analysis was also undertaken. Figure 3 shows the 13 CNMR spectra for PY14, PY65, and PO13 along with dried inks from LY, GR, BO, and GY inks. NMR spectra of

FIGURE 2

Spectrum of Reference Pigments and Tattoo Inks Using Fourier Transform Infrared (FTIR) Spectroscopy

Note. A magnification of the infrared (IR) spectra is in the 1,900–600 cm -1 range with a resolution of 4 cm -1 to demonstrate the characteristics more accurately. IR spectra comparison of inks and pigments reveals the presence of PY14 instead of PY65 in the lemon yellow (LY) ink. The golden yellow (GY), golden rod (GR), and bright orange (BO) inks did not have PO13. E-BO = ink extract, bright orange; E-GR = ink extract, golden rod; E-GY = ink extract, golden yellow; E-LY = ink extract, lemon yellow; PB = pigment blue; PO = pigment orange; PY = pigment yellow.

FIGURE 3

Spectrum of Reference Pigments and Tattoo Inks Using 13 C Solid- State Nuclear Magnetic Resonance (NMR) Spectroscopy

Note . Spectral analysis confirms the presence of PY14 in all inks, with PO13 in the bright orange (BO) ink and PO13 absent in golden yellow (GY) and golden rod (GR) inks. NMR spectra also confirmed that the lemon yellow (LY) ink did not have PY65. C = copper; PO = pigment orange; PY = pigment yellow.

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PB15 were not collected due to paramagnetic characteristics of copper (Cu), which gener- ate local magnetic fields that might interfere with the NMR measurements. We did find distinct peaks in PY65 at approximately 38 ppm and 58 ppm, which were associated with the presence of methyl groups (CH 3 -O, CH 3 - C=O). Peaks at 145 ppm and 155 ppm were correlated to the presence of C-NO 2 and Ar-O groups, which were only found in PY65 (Blau et al., 2008). These peaks were not found in any of the inks, suggesting that none of them contained PY65. Moreover, PY14 had peaks between 130 ppm and 140 ppm and at 20 ppm and 30 ppm that can be attributed to the presence of the C-Cl and methyl group (C-CH 3 and O=C-CH 3 ). These peaks were identified in all inks, which confirmed the presence of PY14 in them. In addition, NMR characterization did not confirm the presence of PO13 in the LY, GY, and GR inks, which is because PO13 had distinguishing peaks between 150 ppm and 160 ppm that were not detected in these inks. There was also a slight chemical shift of the peak at 12 ppm, which was assigned to the CH 3 group in PO13. The presence of PO13 in the BO ink was consistent with the manu- facturer’s ingredients, with small quantities as identified in the NMR spectra. These NMR spectra for the three pigments were consis- tent with those spectra reported in the litera- ture (Feng et al., 2019). The presence of PY14 was confirmed by FTIR and NMR analyses in all three inks. The results from FTIR and NMR analyses clearly show that LY ink contains PY14 instead of PY65. This finding contradicts the manufac- turer’s label claim that LY ink contains PY65 (not PY14), but is consistent with the decla- ration in the MSDS that indicates PY14. Fur- thermore, the presence of PO13 in both the GY and GR inks could not be confirmed, as the FTIR and NMR spectra did not contain the characteristic peaks expected from PO13. The absence of PO13 in the GY ink is con- sistent with the MSDS for the ink (at time of purchase) and indicates that, once again, the ink bottle might have been mislabeled. The absence of PO13 in the GR ink is in contrast with the label and MSDS for this ink. It could be, however, that the amount of PO13 used in these inks was too small for the instrumen- tation to detect (i.e., <2 mg of PO13 in 100 mg of dried tattoo ink). This threshold was

FIGURE 4

X-Ray Diffraction (XRD) Data Analysis of Organic and Inorganic Pigments

Note. This analysis compared the spectra from the lemon yellow (LY), golden yellow (GY), golden rod (GR), and bright orange (BO) tattoo inks. Titanium dioxide (TiO 2 ) was identified in the BO and GY inks. BaSO 4 = barium sulfate; D-BO = dried ink, bright orange; D-GR = dried ink, golden rod; D-GY = dried ink, golden yellow; D-LY = dried ink, lemon yellow; PB = pigment blue; PO = pigment orange; PY = pigment yellow.

FIGURE 5

Baseline-Corrected Raman Spectra of Reference Pigments and Tattoo Inks

Note. LY = lemon yellow; GY = golden yellow; GR = golden rod; PO = pigment orange; PY = pigment yellow.

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confirmed by the control experiment to iden- tify the limits of detection of the NMR instru- ment (Supplemental Figure S3). XRD Analysis To confirm the FTIR and NMR results along with verifying the existence of inorganic ingredients, we used XRD analysis. The XRD diraction pattern of all inks revealed amor- phous and crystalline phases (Figure 4). The vehicles and organic pigments are associated with the amorphous phases of the inks. The diraction pattern of D-LY ink is similar to PY14 but not to PY65 or PB15, which further confirms the presence of PY14 (Figure 4). Furthermore, when comparing the XRD pat- tern of PO13 with that of GY and GR inks, the peaks diered, which was predicted and cor- responded with the FTIR and NMR findings. Additionally, TiO 2 has been recognized as one of the most common crystalline oxide peaks in both D-BO and D-GY inks. The intensity of TiO 2 peaks, however, was greater in D-BO ink than in D-GY ink, which might be because the quantity of TiO 2 was smaller in the D-GY ink than that in the D-BO ink, as per a November 20, 2018, clarification on the manufacturer’s website in the MSDS of these inks (INTENZE Advanced Tattoo Ink, 2025). The absence of the TiO 2 peaks in the D-LY ink, however, sug- gests that the amount of TiO 2 was smaller than the level detectable by XRD. These results from the XRD data analysis of the pigments and inks were compared with results from the refer- ence (Arl et al., 2019). The possible presence of BaSO 4 in the BO ink was evidenced by low- intensity peaks in the XRD analysis; however, this finding could not be confirmed using XRD alone, as some of these low-intensity peaks overlapped with the TiO 2 peaks. Raman Spectra Analysis Furthermore, Raman analysis was conducted to confirm the previous instrument examina- tion data and the presence of PY65, PO13, PB15, and the other inorganic ingredients (e.g., BaSO 4 and TiO 2 ), as shown in Figure 5. The full range of the spectra (2,500 cm -1 to 200 cm -1 ) can be found in Supplemental Figure S4. The Raman spectra analysis of the LY ink revealed that PY14 is a prominent component and that PY65 is not present. The challenge in resolving these separate pigments for this spe- cific shade of ink can be due to the quantity of PY14 versus PB15 (Supplemental Figure S5),

TABLE 2

Element Composition Analysis of Pigments and Inks Using EDX (Energy Dispersive X-Ray) Spectroscopy

Pigment/Ink

Element (%)

C N O Cl 66 19 14 2 62 20 14 –

Cu Ti

Ba S Si

Na Al

PY14 PY65 PB15 PO13 BaSO 4 TiO 2 D-LY E-LY D-GY E-GY D-GR E-GR D-BO E-BO

– – 2 – –

– – – –

– – – – – – 3 – – –

– – – – – – – – – – – –

76 18 6 70 21 5

– 2

– 4

– 48 – 3 70 –

– 30 19 –

– 23 –

– – – – –

76 11 7 70 16 8

3 2

– – – – – –

3 – 4 – – –

– – – – – –

65 9 18 2 65 21 10 2 67 20 10 2 69 16 11 2 60 5 20 2 63 5 18 3

– 1.0 –

–

– 10 –

– 0.7 -

0.8

–

Note . EDX spectroscopy provides supportive evidence of the presence of certain pigments in inks and establishes a match between LY ink and PY14 pigment. The elemental percentage represents the average atomic percentages from surface analysis of three spots on a sample. Al = aluminum; Ba = barium; BaSO 4 = barium sulfate; C = carbon; Cl = chlorine; CU = copper; D-BO = dried ink, bright orange; D-GR = dried ink, golden rod; D-GY = dried ink, golden yellow; D-LY = dried ink, lemon yellow; E-BO = ink extract, bright orange; E-GR = ink extract, golden rod; E-GY = ink extract, golden yellow; E-LY = ink extract, lemon yellow; N = nitrogen; Na = sodium; O = oxygen; PB = pigment blue; PO = pigment orange; PY = pigment yellow; S = sulfur; Si = silicon; Ti = titanium; TiO 2 = titanium dioxide.

primarily, which causes signals from PY14 to overwhelm signals from PB15. Many aspects of the comparatively narrow PY14 spectrum corresponded with and overlapped with the few peaks (e.g., 1,598.5 cm -1 ) derived from PB15 due to similar structural vibrations. For example, the normally noticeable 1,332 cm -1 C-C bond stretching in PB15 (Scherrer et al., 2009) is hidden by a rather weak fea- ture in PY14 at 1,310 cm -1 . Additionally, the allocated peaks from PB15 at 1,414 cm -1 and 1,136 cm -1 were shifted to 1,456 cm -1 and 1,148 cm -1 , respectively. At the same time, the BP15 peak of 584 cm -1 was masked in the D-LY ink. Therefore, Raman spectra analysis results could not confirm the presence of PB15. Furthermore, the Raman spectra analysis of the GY and GR inks was consistent with previous characterization (e.g., IR, NMR, and XRD) studies and confirmed the absence of PO13 in these inks. This finding is because PO13 had a peak at 1,588 cm -1 and this peak

was shifted to 1,600 cm -1 in the GY and GR inks. The Raman spectra analysis of TiO 2 and BaSO 4 revealed two distinct peaks at 441cm -1 and 603cm -1 (Supplemental Figure S6), which were not observed on the Raman spectra analysis of the dried inks at the same region. This result could be because the quantity of elements was inadequate to be detected by Raman analysis. As a next step, therefore, we conducted an EDX analysis. EDX Analysis The pigments and dried inks were analyzed using EDX analysis to identify the element composition (Table 2 and Supplemental Fig- ures S7–S11). As expected, the pigment refer- ence samples contained carbon (C), nitrogen (N), oxygen (O), copper (Cu), barium (Ba), sulfur (S), and chlorine (Cl). The presence of Cl in the D-LY ink spectrum is further proof that PY14 is present in this ink. It was expected that Cu (from PB15) and Ba (from

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