Tomislav Bolanča

Antiviral substances are considered emerging contaminants. Once released into the environment, they may affect organisms through complex and often still-unknown mechanisms. This study focuses on a class of antiviral substances with potential use in treating COVID-19 patients, aiming to identify specific structural characteristics that significantly contribute to their ecotoxicity. An empirical approach called quantitative structure–activity relationship (QSAR) was used for this purpose. The study examined 13 antiviral substances: atazanavir, daclatasvir, darunavir, emtricitabine, favipiravir, lopinavir, nirmatrelvir, oseltamivir, remdesivir, ribavirin, ritonavir, and sofosbuvir. The ecotoxicity of these antivirals was assessed using three tests: the Aliivibrio fischeri test, the Chlorella sp. test, and the Pseudomonas putida test. These three microorganisms represent different trophic levels in aquatic and soil ecosystems. Ecotoxicity was expressed as EC₂₀ and EC₅₀, and these values served as the dependent variables in the QSAR models. A large set of numerical descriptors calculated from the molecular structures of the antivirals was used as an independent variable. EC₂₀-based QSAR models offer insight into the effects of antivirals under sub-lethal exposure conditions. The results indicated that sub-lethal exposure in Aliivibrio fischeri was associated with favorable electronic properties and compact structures that promote cellular accumulation, while long-range fragments reduced toxicity. In Chlorella sp., sub-lethal exposure was driven by optimal molecular size, chain length, and specific electronic groups enabling cell penetration and biochemical inhibition. For sub-lethal exposure in P. putida, lipophilicity and reactive group geometry enhanced toxicity, while high short-range polarity mitigated it by limiting membrane permeability. Acute toxicity patterns showed similar trade-offs: strong electronic reactivity increased potency, but steric bulk, long-range polarity, or unfavorable mass distribution frequently restricted bioavailability and reduced toxic effects. Overall, the models demonstrated that antiviral toxicity results from a balance of electronic activity, structural accessibility, and physicochemical constraints, providing a mechanistic basis for predicting the environmental risk of selected antiviral substances.

Antivirals are considered emerging environmental contaminants. Unfortunately, for many of these substances, there is limited information on their occurrence, fate, and behavior in the environment, which is essential for proper risk assessment. In this study, forced degradation tests were conducted on six widely used antivirals to assess their environmental fate. These antivirals were atazanavir, emtricitabine, nirmatrelvir, oseltamivir, ribavirin, and sofosbuvir. The tests included exposure of the antivirals to (artificial) sunlight, different temperatures, water, an acidic solution (1 M HCl), an alkaline solution (1 M NaOH) and a solution containing 30% H₂O₂ as a strong oxidizing agent. Liquid chromatography with UV detection (LC-UV) was used to analyze antivirals. To monitor the conversion, an LC-UV method was developed and validated for each antiviral. According to the results of the forced degradation tests, atazanavir and emtricitabine are probably the most unstable in the aquatic environment. Oseltamivir and sofosbuvir begin to lose stability even at slightly elevated temperatures, such as 40 °C. The stability of the tested antivirals depends strongly on the medium’s pH value and the presence of an oxidizing agent. Thus, all six antivirals showed some degree of degradation under the applied alkaline and oxidative conditions, while only emtricitabine, oseltamivir, and ribavirin remained stable under the applied acidic conditions.

Trace elements are widely distributed in the environment and are considered essential when their deficiency leads to impaired biological function. This study aimed to quantify concentrations of two essential trace elements—copper (Cu) and zinc (Zn)—in the tissues of three toothed whale (Odontoceti) species: bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba), and Risso’s dolphins (Grampus griseus) found deceased along the Croatian coast of the Adriatic Sea between 1995 and 2013. A total of 190 individuals were analyzed, comprising 159 bottlenose, 25 striped, and 6 Risso’s dolphins. Concentrations of Cu and Zn were determined in liver, muscle, kidney, skin, lung, spleen, and fat tissues using inductively coupled plasma optical emission spectrometry (ICP-OES). The highest Cu concentrations were observed in the liver and kidneys of bottlenose dolphins, followed by striped and Risso’s dolphins. Zn concentrations were the highest in the skin of bottlenose and striped dolphins, whereas the liver exhibited the highest levels in Risso’s dolphins. In 14 bottlenose and 2 striped dolphins, Cu and Zn concentrations in liver tissue exceeded critical thresholds typically regulated by homeostatic mechanisms. Regression analysis indicated significant relationships between element concentrations, and both body length and body mass. In addition, trace element concentrations were positively correlated across individuals within the same tissue type, as well as among different tissues within the same individual. Overall, Cu and Zn concentrations exhibited a declining trend over the studied period across all tissue types. These findings provide important baseline data for future ecotoxicological investigations and contribute to conservation strategies for cetacean populations inhabiting the Adriatic Sea.

Trace elements are widely distributed in the environment and are considered essential when their deficiency leads to impaired biological function. This study aimed to quantify concentrations of two essential trace elements—copper (Cu) and zinc (Zn)—in the tissues of three toothed whale (Odontoceti) species: bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba), and Risso’s dolphins (Grampus griseus) found deceased along the Croatian coast of the Adriatic Sea between 1995 and 2013. A total of 190 individuals were analyzed, comprising 159 bottlenose, 25 striped, and 6 Risso’s dolphins. Concentrations of Cu and Zn were determined in liver, muscle, kidney, skin, lung, spleen, and fat tissues using inductively coupled plasma optical emission spectrometry (ICP-OES). The highest Cu concentrations were observed in the liver and kidneys of bottlenose dolphins, followed by striped and Risso’s dolphins. Zn concentrations were highest in the skin of bottlenose and striped dolphins, whereas the liver exhibited the highest levels in Risso’s dolphins. In 14 bottlenose and 2 striped dolphins, Cu and Zn concentrations in liver tissue exceeded critical thresholds typically regulated by homeostatic mechanisms. Regression analysis indicated significant relationships between element concentrations and both body length and body mass. In addition, trace element concentrations were positively correlated across individuals within the same tissue type, as well as among different tissues within the same individual. Overall, Cu and Zn concentrations exhibited a declining trend over the studied period across all tissue types. These findings provide important baseline data for future ecotoxicological investigations and contribute to conservation strategies for cetacean populations inhabiting the Adriatic Sea.

The COVID-19 pandemic has intensified the development of new antiviral agents specifically intended for the SARS-CoV-2 virus, but has also increased the use of some already known antiviral agents originally intended for other viruses. Although the pandemic has ended, the SARS-CoV-2 virus is expected to be present in the human population forever, as is the case with the influenza virus, for example. Such a scenario guarantees the continued use of SARS-CoV-2 antivirals and, accordingly, their continued release into the environment. Unfortunately, there is little or no information on the adverse potential of most of these antiviral agents. In this study, the acute toxicity of six antiviral agents used in the treatment of SARS-CoV-2 infections was determined. These are atazanavir, ribavirin, emtricitabine, nirmatrelvir, sofosbuvir and oseltamivir, sorted according to their toxicity, starting with the most toxic agent. Toxicity was determined using the marine bacterium Aliivibrio fischeri according to the ISO 11348-1:2007 standard. In addition to the toxicities of the individual antiviral solutions, the toxicities of binary antiviral mixtures were also determined. By comparing the experimentally determined toxicities of the mixtures with the values estimated by the concentration addition model and the independent action model, we analyzed the mode of joint toxic activity of these antiviral agents. Additive behavior was observed for most binary combinations. The combination of nirmatrelvir and sofosbuvir led to an antagonistic deviation from the concentration addition model, while a synergistic deviation was observed for the combinations of emtricitabine with atazanavir and with nirmatrelvir, as well as for the combinations of ribavirin with atazanavir, oseltamivir and sofosbuvir. All tested binary combinations showed a synergistic deviation from the independent action model.

The environmental impact of plastic waste is a growing global challenge, primarily due to non-biodegradable plastics from fossil resources that accumulate in ecosystems. Biodegradable polymers like polyhydroxyalkanoates (PHAs) offer a sustainable alternative. PHAs are microbial biopolymers produced by microorganisms using renewable substrates, including agro-industrial byproducts, making them eco-friendly and cost-effective. This study focused on the isolation and characterization of PHA-producing microorganisms from agro-industrial waste, including chickpeas, chickpeas with bean residues, and starch. Screening via Sudan Black staining identified PHA-accumulating strains such as Brevibacillus sp., Micrococcus spp., and Candida krusei, among others. To assess the potential for PHA biosynthesis, solid-state fermentation (SSF) was conducted using agro-industrial waste as substrates, along with a mixed culture of the isolated microorganisms. The highest observed yield was a PHA accumulation of 13.81%, achieved with chickpeas containing bean residues. Structural and thermal characterization of the PHAs was performed using Fourier-transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). FTIR-ATR spectra indicated polyhydroxybutyrate (PHB), suggesting it as the synthesized PHA type. This study highlights the potential of agro-industrial waste for sustainable PHA production and eco-friendly bioplastics.

Microplastics are an emerging class of recalcitrant organic pollutants that are of general scientific and public interest nowadays. It would be ideal to remove microplastics from the environment through biodegradation, as biodegradation is a highly ecological and economically acceptable approach. Unfortunately, the efficiency of biodegradation of conventional plastic polymers is low. The application of a suitable pretreatment could increase the efficiency of biodegradation. In this study, the applicability of UV-C/H₂O₂ and UV-C/S₂O₈²⁻ advanced oxidation processes as pretreatments for the biodegradation of polystyrene and poly(vinyl chloride) microplastics by the yeast Candida parapsilosis was investigated. For the investigated range (pH 4–10, peroxide concentration up to 20 mM and treatment duration up to 90 min), the UV-C/H₂O₂ process proved to be more effective in degrading polystyrene microplastics, while the UV-C/S₂O₈²⁻ process was more efficient at degrading poly(vinyl chloride) microplastics. Samples pretreated under optimal conditions (90 min treatment time at a pH of 5.7 and H₂O₂ concentration of 20.0 mM for polystyrene samples; 90 min treatment time at a pH of 8.6 and S₂O₈²⁻ concentration of 11.1 mM for poly(vinyl chloride) samples) were subjected to biodegradation by Candida parapsilosis. The biodegradation conditions included an agitation speed of 156 rpm and an initial pH of 5.7 for the experiments with the polystyrene samples, while an agitation speed of 136 rpm and an initial pH of 4.9 were used for the poly(vinyl chloride) experiments. The initial value of the optical density of the yeast suspension was 1.0 in both cases. The experiments showed a positive effect of the pretreatment on the number of yeast cells on the surface of the microplastics.

The global problem of microplastics in the environment is “inspiring” scientists to find environmentally friendly and economically viable methods to remove these pollutants from the environment. Advanced oxidation processes are among the most promising methods. In this work, the potential of Fenton, photo-Fenton, and Fenton-like processes for the degradation of microplastics from low-density polyethylene (LDPE), polypropylene (PP), and poly(vinyl chloride) (PVC) in water suspensions was investigated. The influence of three parameters on the efficiency of the degradation process was tested: the pH of the medium (3–7), the mass of added iron (10–50 times less than the mass of microplastics), and the mass of added H₂O₂ (5–25 times more than the mass of added iron). The effectiveness of the treatment was monitored by FTIR-ATR spectroscopy. After 60-min treatments, the PP microparticles were found to be insensitive. In the Fenton treatment of PVC and the photo-Fenton treatment of LDPE and PVC, changes in the FTIR spectra related to the degradation of the microplastics were observed. In these three cases, the treatment parameters were optimized. It was found that a low pH (3) and a high iron mass (optimal values were 1/12 and 1/10 of the mass of the microplastics for LDPE and PVC, respectively) favored all three. The degradation of LDPE by the photo-Fenton treatment was favored by high H₂O₂ concentrations (25 times higher than the mass of iron), while these concentrations were significantly lower for PVC (11 and 15 times for the Fenton and photo-Fenton treatment, respectively), suggesting that scavenging activity occurs.

Biodegradation is the most environmentally friendly and, at the same time, economically acceptable approach to removing various pollutants from the environment. However, its efficiency in removing microplastics (MPs) from the environment is generally low. The successful biodegradation of MPs requires microorganisms capable of producing enzymes that degrade MP polymers into compounds that the microorganisms can use as a source of carbon and energy. Therefore, scientists are screening and characterizing microorganisms that can degrade MPs more efficiently. These microorganisms are often isolated from sites contaminated with MPs because the microorganisms living there are adapted to these pollutants and should be able to better degrade MPs. In this study, five bacterial strains and five yeast strains were isolated from various environmental samples including activated sludge, compost, river sediment, and biowaste. Among them, screening was performed for bacteria and yeasts with the highest potential for the biodegradation of polystyrene (PS) and polyvinyl chloride (PVC) MPs, and the bacterium Delftia acidovorans and the yeast Candida parapsilosis were identified as the best candidates. Optimization of biodegradation of the selected MPs by each of these two microorganisms was performed, focusing on the influence of cell density, agitation speed and pH of the medium. It was found that within the selected experimental ranges, high values of cell density, low agitation speed, and a slightly basic medium favored the biodegradation of PS and PVC MPs by Delftia acidovorans. In the case of Candida parapsilosis, favorable conditions also included high cell density followed by a slightly higher, but not maximum, agitation speed and a weakly acidic medium. Broad spectroscopic and imaging methods indicated that Delftia acidovorans and Candida parapsilosis better adapt to PVC MPs to use it as a carbon and energy source.