Aliivibrio fischeri as a Biosensor for Investigating the Aquatic Toxicity of Common Formulation Ingredients
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Abstract
In this thesis, A. fischeri was employed as a sensitive bioluminescent model organism to enrich the testing systems in the research group. The studies performed were divided into two parts:
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The optimization of A. fischeri cultivation and its use in ex vivo biological models.
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Assessment of aquatic toxicity (Aquatox) across various substances and formulations.
Initially, the growth and bioluminescence of A. fischeri were successfully optimized for the laboratory conditions.
The first application on ex vivo models was the establishment of a testing method to evaluate the antimicrobial activity of norfloxacin SmartFilms by cultivating the bacteria on the ex vivo gut model. A novel test was conceived, where the cultivated bacteria, treated and untreated, were applied on agar plates through cotton swabbing. A photography system was built to document the bioluminescence of the grown cultures. Through this system, the image analysis of the bioluminescence was possible, and thus, the intensity of it could successfully detect the antimicrobial activity and the efficacy of the SmartFilms. The findings conducted through this testing system achieved the aim of integrating A. fischeri on one of the ex vivo models (i.e., gut model).
The second application of A. fischeri on biological models was on the ex vivo porcine skin, where the aim was to establish an artificial microbiome layer on healthy and manipulated skin. Because the skin already contains a microbiome, Octenisept® was applied as a disinfectant. Its application did not drastically alter the biophysical properties of the skin. The bioluminescence behavior of skin vs. agar was also determined, where it was shown that agar provides better conditions than the skin for the bioluminescence. Disinfection reduced bioluminescence on agar plates but had no significant effect on skin, ensuring that it can be applied to the skin without altering the bioluminescence. This aim was advanced but not fully established, as further development is needed to replicate compromised skin conditions, as A. fischeri is not a natural inhabitant of the skin. Thus, it is still not clear how it can alter the results of experiments over time.
The most extensive part of the thesis involved the creation of a time- and concentration-dependent, high-throughput test as a modification of the Microtox® water toxicity testing system to determine the aquatic toxicity of commonly used ingredients in formulations and over-the-counter products (sunscreens, serums, and creams).
The observations of the single ingredients tested were:
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Preservatives observed as a whole group show constant bioluminescence inhibition in all phases, although the acute phase has the lowest relative bioluminescence. It is of high importance to mention that, contrary to common concerns, parabens, except Ethylparaben, showed the lowest impact on bioluminescence.
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Surfactants show initial inhibition in the acute phase. Unexpectedly, the bioluminescence rises through the chronic phase until 24 h. The obtained results suggest that the chemical composition of some surfactants (like long-chain fatty acids) might aid in the BL production in A. fischeri, as part of the bioluminescence production mechanism.
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Fragrances exhibit lower bioluminescence inhibition during the acute phase, followed by an enhancement of the rel. BL in the chronic phase, ending by inhibition at 24h.
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Antioxidants are the group showing the highest inhibition of the rel. BL, especially in the chronic phase and at 24 h. The results can be attributed to their mode of action, which might lower the oxygen availability, disrupting the bioluminescence production by impacting the responsible oxygen-dependent luciferase reaction.
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UV filters, except avobenzone, inhibited the bioluminescence. Interestingly, during the chronic phase, an increase in the rel BL was observed, followed by a potent bioluminescence inhibition at 24h. The sunscreens' analysis revealed complex, formulation-dependent toxicity behavior. Several sunscreens exhibited inhibition during the acute phase, followed by partial or complete recovery of bioluminescence at 24 hours and, in some cases, even an enhanced light output, emphasizing the importance of long-term testing. These results suggest links to formulation properties. Further statistical analyses revealed that SPF level, formulation type, and UV filter class influenced the bioluminescence production specifically:
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SPF 50 formulations inhibited the bioluminescence the most.
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Water-in-oil emulsions exhibited more potent inhibitory effects than other formulations.
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Organic filters/blockers caused lower bioluminescence in early phases. Their effect diminished over time.
Although the information obtained from the Aquatox test is discriminating and shows significant differences between the individual substances and formulations tested, a more fundamental investigation of the metabolic dynamics of A. fischeri and its response to various substances, particularly those that enhance bioluminescence, is required. Aquatox proved to be a reliable and reproducible method for assessing the aquatic effects of cosmetic and pharmaceutical ingredients and formulations. The results are of great value for both scientific research and regulatory decisions.
In conclusion, these studies conducted in this thesis achieved the integration of A. fischeri into ex vivo models and high-throughput testing systems, fulfilling the thesis aims while supporting the organism’s versatility as a biosensor.
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This item has been published with the following license: In Copyright