Larvae of Rhinellaarenarum, juveniles of Jenynsiamultidentata, and embryos of Daniorerio were used as biological models. Juvenile fish and embryos were obtained from laboratory cultures and maintained under standard aquarium conditions at approximately 25˚C. Amphibian larvae were obtained by collection in a natural environment (33˚17'44"S, 66˚20'16"W). Prior to experimentation, organisms were acclimated under laboratory conditions, and only healthy individuals showing normal behavior were selected for toxicity assays.

Embryos of D. rerio were obtained from breeding adults maintained under controlled laboratory conditions. Fertilized and viable embryos were identified under stereoscopic observation based on normal cleavage patterns, transparent chorion, and the absence of coagulation or developmental abnormalities. Unfertilized eggs and embryos showing signs of non-viability prior to exposure were discarded and were not included in the mortality analysis.

Acute toxicity assays were carried out using a commercially available electronic cigarette refill liquid. According to the product label, the formulation contained nicotine (50 mg∙mL⁻¹), propylene glycol, vegetable glycerin, and natural and artificial flavoring agents. The tested product corresponded to a sour strawberry-dragonfruit flavored formulation (batch number EC081653). No quantitative information regarding the propylene glycol/vegetable glycerin ratio was provided by the manufacturer. The product was used as received without further modification. A stock solution was prepared at a nominal concentration of 1 mL∙L⁻¹. From this stock solution, different dilutions were prepared using standardized aquarium water to obtain final exposure concentrations expressed as percentages of the stock solution.

All assays were performed under static exposure conditions at 25˚C ± 1˚C under a natural photoperiod. No aeration or feeding was provided during the exposure period. Test solutions were not renewed throughout the experiment. Organisms were maintained in standardized aquarium water prepared under laboratory conditions. The protocol was adapted from the guidelines proposed by Johnson and Finley [15] and further modified by our research group.

Juveniles of Jenynsiamultidentata and larvae of Rhinellaarenarum were exposed to 5%, 10%, 20% and 50% dilutions of the stock solution. Each experimental system contained, for fish, 1000 mL of test solution and 10 organisms, and, for amphibians, 400 mL of test solution and 10 organisms. Assays were performed in duplicate. For Jenynsiamultidentata, each treatment consisted of two independent vessels, each containing 10 organisms (n = 20 organisms per concentration). For Rhinellaarenarum, each treatment consisted of two independent vessels, each containing 10 larvae (n = 20 larvae per concentration).

For Daniorerio embryos, each concentration was tested in duplicate in two independent wells, each containing three embryos (n = 6 embryos per concentration). Mortality was recorded after 24, 48, 72, and 96 h of exposure.

For embryo assays, the methodology described in OECD Guideline 236 [16] was followed.

In parallel, acute toxicity assays were carried out using eggs of Daniorerio as early developmental-stage models. Eggs were exposed to 3%, 6%, 12.5%, 25%, and 50% dilutions of the same stock solution in wells of 5 mL. Each well contained three eggs, and all treatments were performed in duplicate. Mortality was recorded after 24, 48, and 72 h of exposure.

Control systems were maintained under the same experimental conditions without the addition of e-cigarette liquid. Organisms were considered dead when a complete absence of movement and response to external stimulation was observed.

Mortality data were expressed as percentages. Due to the exploratory nature of the study and the limited number of organisms used in some treatments, results were evaluated descriptively. Average mortality values obtained from duplicate assays were used for graphical representation and comparison among biological models.

The stock solution was prepared from a commercial electronic cigarette essence containing a declared nicotine concentration of 50 mg∙mL⁻¹. An initial dilution of 1 mL∙L⁻¹ yielded a stock solution equivalent to 50 mg∙L⁻¹ nicotine. The different exposure concentrations were subsequently prepared from this stock solution.

No mortality was observed in either Jenynsiamultidentata juveniles or Rhinellaarenarum larvae during the 96 h exposure period. All organisms survived throughout the experiment, regardless of the concentration tested (5% - 50% of the stock solution, corresponding to 2.5 - 25 mg ∙L⁻¹ nicotine). No differences were detected among treatments or between duplicate assays. These results indicate that the electronic cigarette refill liquid did not produce acute lethal effects in juvenile fish or amphibian larvae under the experimental conditions employed.

The absence of mortality at concentrations up to 25 mg∙L⁻¹ nicotine suggests a relatively low acute toxicity of the tested formulation toward juvenile fish and amphibians under the experimental conditions employed.

Unlike the results obtained for fish and amphibians, Daniorerio embryos exhibited sensitivity to the electronic cigarette essence. Mortality was observed as early as 24 h post-exposure and reached the highest values in the treatment corresponding to 50% of the stock solution (25 mg∙L⁻¹ nicotine).

In general, mortality increased with exposure concentration, although a strictly linear dose-response relationship was not observed. This variability may be associated with the limited number of embryos tested per treatment and the inherent biological variability commonly observed in exploratory embryo toxicity assays. A limitation of the present study is the relatively small number of embryos used per treatment. Therefore, the observed mortality patterns should be interpreted as preliminary evidence of embryotoxicity and should be confirmed in future studies employing larger sample sizes (See Table 1).

The highest mortality (83%) was recorded at 25 mg∙L⁻¹ nicotine, indicating that embryonic stages are considerably more sensitive to exposure than juvenile fish and amphibians. These findings are consistent with previous reports describing the vulnerability of early developmental stages to nicotine and other constituents of electronic cigarette liquids.

Table 1.Average mortality (%) of Daniorerio embryos exposed to different concentrations of electronic cigarette essence.

The observed embryotoxicity may result not only from nicotine itself but also from the combined action of other formulation components, including propylene glycol, vegetable glycerin, and flavoring agents. Therefore, the toxicity detected in this study likely reflects the overall effect of the commercial formulation rather than nicotine alone (See Figure 1Figure 1).

Figure 1.Mortality of Daniorerio embryos after 72 h of exposure to increasing concentrations of electronic cigarette essence. Concentrations are expressed as equivalent nicotine concentrations calculated from the manufacturer’s declared nicotine content (50 mg∙mL⁻¹). The highest mortality (83%) was observed at 25 mg∙L⁻¹ nicotine, indicating increased sensitivity of embryonic stages compared with juvenile fish and amphibians.

A comparison of the maximum mortality values recorded for each biological model is presented in Figure 2Figure 2. The marked contrast between fish embryos and juvenile organisms highlights the importance of developmental stage as a determinant of toxicological sensitivity.

Figure 2.Comparative mortality observed in aquatic vertebrate models exposed to electronic cigarette liquid. Values correspond to the maximum mortality recorded during the exposure period.

The results obtained in this study demonstrate marked differences in sensitivity among the biological models evaluated. While no mortality was observed in juvenile fish (J. multidentata) or amphibian larvae (R. arenarum), embryos of D. rerio exhibited evident susceptibility to the tested e-cigarette liquid.

The higher sensitivity observed in embryos is consistent with previous reports indicating that early developmental stages are generally more vulnerable to environmental contaminants because of incomplete physiological development and limited detoxification capacity. Embryonic stages are particularly sensitive to compounds capable of interfering with cellular differentiation, organogenesis, and metabolic regulation.

The absence of mortality in juvenile fish and amphibian larvae suggests that the tested formulation presents relatively low acute toxicity toward these organisms under the exposure conditions employed. However, the mortality observed in embryos indicates that early life stages may represent more sensitive bioindicators for evaluating the environmental risks associated with electronic cigarette products.

Previous studies performed by our research group using pure nicotine reported acute toxicity in fish at concentrations lower than those evaluated here [11]. These differences suggest that species, developmental stage, exposure conditions, and the composition of the tested matrix may strongly influence toxicological responses. Therefore, direct comparisons between studies should be made with caution.

Although nicotine is considered one of the principal biologically active compounds present in vaping liquids, the observed effects cannot be attributed exclusively to nicotine. Commercial formulations also contain propylene glycol, vegetable glycerin, flavoring compounds, and other additives that may contribute to the overall toxicological response. Consequently, the embryotoxic effects observed in this work likely result from the combined action of several formulation components.

These findings reinforce the importance of incorporating multiple biological models and developmental stages into ecotoxicological evaluations of emerging contaminants associated with electronic cigarettes.

It should be noted that direct comparisons among biological models should be interpreted with caution because exposure duration and concentration ranges differed among embryo, juvenile fish, and amphibian assays. Therefore, the observed differences in sensitivity provide qualitative evidence of developmental-stage susceptibility rather than a quantitative ranking of species sensitivity.

Although the concentrations evaluated in this study are higher than those typically expected in natural waters, similar exposure scenarios may occur locally following accidental spills, improper disposal of refill liquids, leakage from discarded vaping devices, or inputs from wastewater systems. Consequently, the concentrations tested represent realistic worst-case scenarios that may be relevant for assessing the environmental risks associated with increasing consumption of electronic cigarette products.