Our 3D printing ingredient contains acrylate (monomer of photopolymer) and 2,2-bimethoxy-2-phenylacetophenone as a photoinitiator. Except for acrylate, we also added moderate 3,4-epoxy cyclohexylmethyl to enhance the mechanical properties of the final product and overcome the drawbacks of a single photopolymer (e.g., low intensity and high anisotropy) [7].
The toxicity of our photopolymer and photoinitiator has been reported in many articles. Unpolymerized acrylate monomer is water-soluble, which means it easily leak into the aqueous medium. In consideration of the water solubility of an acrylate monomer, it may explain why our zebrafish embryos almost dead by 12 hpf and all dead by 24 hpf (Fig. 1A), in accord with the recent reports that leaching of compounds to aqueous media from plastic parts is relatively high in the first 24 h [30]. C.A. Staples et al. test the acute and chronic aquatic toxicity for acrylic acid shows that the acute LC50 (24 h) of Rainbow trout (Oncorhynchus mykiss), Sheepshead minnow (Cyprinodon variegatus), Cladoceran (Daphnia magna), and Mysid shrimp (Mysidopsis bahia) are 45 mg/l, 236 mg/l, > 110 mg/l, and 166 mg/l, respectively [31]. The investigation shows that acrylate and urethane acrylate oligomers are toxic or harmful to various species of fish, algae, and water microorganisms.
As for the photoinitiator, though 2,2-bimethoxy-2-phenylacetophenone is poorly soluble in water, it may cause damage to organs through prolonged or repeated exposure, and it is harmful to aquatic life with long-lasting effects [32]. Reactive oxygen species (ROS) are the primary metabolites of the photoinitiator, for example, peroxides and peroxy radicals [33, 34]. Otherwise, incomplete polymerization of photo-reactive resins (55–60% of polymerization under optimal conditions [35]) can cause more significant levels of toxicity and leaching rate [36]. Hence, our SLA printed parts without coating parylene C are likely to have multiple toxic effects with zebrafish, for the leaching of photopolymer and photoinitiator.
However, in this study, the parylene C coated group has almost the same level compared to control group, which imply the coating film could block the toxicity of SLA printed parts to zebrafish, despite the hatching rate of embryos exposed to the parylene C coated group mildly lower than those of control embryos at 4 dpf and parylene C coated group exhibit a higher heart rate than that of control embryos. The differences between the two groups above demonstrate that slight toxic compounds may exist leaching through the parylene C film, potentially caused by vestigital after the parylene C coating step or by molecular exchange for the compounds with lower molecular weight than 300 (e.g., acrylate 86.09, 2,2-bimethoxy-2-phenylacetophenone 256.30).
In the past decade, many studies have investigated the toxicity effects of 3D-printed parts. Methods have been exploited to modify the toxicity of 3D-printed parts, including supercritical carbon dioxide [9], sonication of the material in isopropanol [10], and exposure to ultraviolet light [11]. The recent study assessing the toxicity reduced by exposure to ultraviolet light showed a much lower survival and hatch of embryos compared to our method of coating with parylene C [11], indicating that further strategies need to be developed to totally block or reduce the toxicity of SLA printing materials.
In this study, we tested the toxicity of the SLA printed petri dishes whether or not coated with parylene C using early life zebrafish. The UT groups show a violent toxicity effect on the survival and hatch of embryos. On the contrary, the CT group has almost the same level compared to the control group, which implies the coating could significantly reduce the toxicity of STL printed parts to zebrafish. Unfortunately, it does not completely erase the toxicity of these parts. For its potential cardiotoxicity, further strategies for reducing the toxicity are deserved.
This technique can be applied in many aspects. Parylene C has been proved to be used as an inert bulk coating for metallic biomedical implants to improve their corrosion resistance [18], for it has excellent properties like low water absorption, high dielectric constant, low coefficient of friction, and moderate elongation at break [12]. Although little is known about the durability of the parylene C coated 3D-printed parts, we can still utilize this cheap and easy technique to test and improve parts that need to be implanted in vivo for the 3D printing is highly customizable. This technique can also contribute to the popularization of SLA printed products, for it can avoid toxicity from daily contact.
Using zebrafish as a model, our research provided a technique to reduce the developmental toxicity of 3D-printed parts, which currently shows an uplifting promotion to 3D printing usage. However, the whole-organism health effects of exposure to 3D-printed parts to adult zebrafish are still mysterious. The potential toxicity to whole-organism in animal models and underlying mechanisms need further investigation.