Integrated Ozonation and Photocatalysis to Remove Pollutants for Reuse of Rainwater

: Rainwater is susceptible to pollutants such as sulphur dioxide, nitrogen oxides, heavy metals, and particles, posing challenges to water quality protection and soil degradation, impacting ecosystems and agriculture. The study focuses on the effectiveness of combined ozonation and photocatalysis in improving physicochemical parameters and reducing toxic substances. Integrated analyses, including ecotoxicological assessments, evaluate the impact of treatment on actual rainwater samples. The results indicate significant reductions in color, heavy metals, and organic pollutants after treatment. Microbiological analyses reveal the inactivation of E. coli , which is crucial for safe water reuse. Ecotoxicity studies show no toxicity to crustaceans, but slight toxicity to algae and bioluminescence bacteria in post-treatment samples. Genotoxicity assessments indicate that there is no detectable DNA damage. Overall, the study highlights the complex nature of rainwater pollution and the efficacy of photocatalytic ozonation in reducing contaminants, underscoring the need for more research to ensure sustainable water resource management.


Introduction
Rainwater is an important part of the water cycle and plays a key role in the recharge of water bodies.However, as urbanization progresses and industrial activities increase, rainwater becomes vulnerable to pollution, something which, in turn, creates challenges for the protection of water quality [1].Rainwater can collect pollutants from the atmosphere, such as sulphur dioxide, nitrogen oxides, heavy metals, and particles.These substances come from industrial emissions, road traffic, or fuel combustion and can cause this water to become toxic [2,3].Toxic substances from rainwater can leach into the soil, leading to the degradation of soil quality.This, in turn, affects plants, animals, and entire ecosystems, especially in agricultural areas.Heavy metals, such as mercury and lead, are particularly harmful to aquatic organisms [4,5].On the other hand, an increasing number of countries have struggled with water deficits [6].Rainwater represents a valuable resource for water supply in plant cultivation, fish farming, and replenishment of surface water and groundwater for direct and indirect potable reuse [2,7].Therefore, the allocation of resources to the treatment of rainwater constitutes an investment in a more sustainable future for our planet, contributing to circular economy opportunities [8].
Investigating toxic organic compounds in rainwater has emerged as a crucial research area, becoming more important in response to the escalating influence of human activities on the quality of stormwater.Organic compounds in rainwater come from various sources, such as industrial emissions, combustion of fuels, agriculture, or natural biological processes [9,10].Such compounds include various classes, such as hydrocarbons, pesticides, phenolic compounds, and substances of biological origin [11].To mitigate the adverse impacts of rainwater pollution, a multitude of treatment processes are available.These processes range from natural mechanisms, such as biodegradation in soil and bioremediation by plants, to advanced technological systems [12][13][14].The purpose of these processes is to eliminate or reduce the content of chemicals, particulates, heavy metals, and other contaminants that can negatively affect the quality of rainwater and the aquatic environment.Adsorption and filtration are often used in rainwater treatment, in particular to remove heavy metals and reduce color [15][16][17].The degree of elimination of impurities in adsorption processes, at the level of, e.g., heavy metals such as Cu (II), Pb (II), Ni (II), and Zn (II), is 70% to 98% [16,17], while, in relation to color, it reaches 59% to 85% [12].Other processes used are membrane techniques, which also show high efficiency.The ultrafiltration process has a total suspended solid removal efficiency of about 100%, an organic compound removal efficiency of 65%, but a low efficiency associated with heavy metal removal [18].
Conversely, these rainwater treatment processes, frequently, have limitations confined to the removal of specific contaminants, e.g., microbiological.Therefore, to ensure that treated rainwater reaches an acceptable sanitary condition and is safe to use, it is necessary to use an additional disinfection process.The disinfection process is extremely important because it allows the elimination of microorganisms such as bacteria, viruses, and other pathogens that may be present in rainwater.Various methods are used in the disinfection process, including chlorination, ozonation, UV radiation, and the use of oxidising agents [19][20][21].
Consequently, there is considerable demand for advanced technologies such as ozonation and photocatalysis, leading to a synergistic process known as photocatalytic ozonation.An additional advantage lies in the simultaneous removal of organic contaminants and heavy metals through photocatalysis.Ozone, a potent oxidant with disinfectant properties, synergizes with advanced photocatalytic materials, such as titanium dioxide, in the photocatalytic ozonation process.This synergistic effect uses light energy to initiate photocatalytic reactions, resulting in the degradation and removal of pollutants.Photocatalysis proves to be effective in reducing toxicity by breaking down harmful chemical contaminants.This occurs by activating photocatalytic processes that transform toxic substances into less harmful or even harmless components.Photocatalysis is preferred for the generation of non-toxic byproducts, the completion of mineralization, the utilization of minimal chemical components in a short time, and the removal of a broad spectrum of contaminants in aquatic environments, including contaminants of emerging concern [22][23][24][25][26][27].
This article presents detailed studies on the quality of rainwater, with particular emphasis on the evaluation of the effectiveness of the combined ozonation (O 3 ) and photocatalysis (UV/TiO 2 ) process in improving physicochemical parameters and reducing potential toxic substances.A notable aspect of this research lies in its integrated approach, encompassing physicochemical and ecotoxicological analyses assessing rainwater quality pre and post ozonation and photocatalysis treatment.Previous studies have predominantly concentrated on physicochemical parameters, neglecting the ecological impacts of toxicity on aquatic ecosystems.These studies are further distinguished by their emphasis on authentic rainwater.This involves analysing the real impact of the treatment process on genuine water samples, something which can have significant implications for the practical implementation of these methods.Moreover, they emphasize the importance of assessing the efficacy of removing toxic organics such as phenols, pesticides, and pharmaceuticals [28] and heavy metals such as mercury, lead, copper, nickel, cadmium, and arsenic [29] through the combined ozonation and photocatalysis method, which not only oxidizes contaminants but also harnesses light energy to neutralize harmful substances.

Samples of Rainwater
The research focused on two distinct types of rainwater: that originating from the highway (RW I) and that collected from the roof of a single-family house (RW II).Factors that directly affect the quality of precipitation include, among others, the intensity and duration of precipitation, the type of pollutants suspended in the air, the type and condition of the roof material, and its location.Rainwater was collected in July 2023.RW I was collected from an evaporation tank near the A4 highway in the Silesian Voivodeship in the city of Gliwice (Poland).The content of particulate matter in the air on the day of rainwater sample collection from the motorway was at the level of 15.8 µg/m 3 PM 10 and 16.7 µg/m 3 PM 2.5 [30].RW II was collected from a roof covered in tar paper, located in the Silesian Voivodeship in the city of Tychy (Poland).Near the place where water was drawn, there were a brewery, a national road, two gas stations, a housing estate of single-family houses, a grocery store, and a car showroom.The solid particle content in the air on the day in which the rainwater sample was taken from the roof was at the level of 17.6 µg/m 3 PM 10 and 17.3 µg/m 3 PM 2.5 [30].The daily concentration standards for particulate matter recommended by the World Health Organization (WHO) are 50 µg/m 3 for PM 10 and 25 µg/m 3 for PM 2.5 [30].Samples of rainwater were delivered to the laboratory within 1 h, portioned, and used immediately or frozen for later analysis.The physicochemical characteristics of the tested rainwater measured at room temperature are presented in Table 1.

Treatment of Rainwater
Ozonation with photocatalysis (UV/TiO 2 ) was carried out using a Heraeus glass batch reactor with a capacity of 0.7 L, shown in Figure 1.The reactor was equipped with a 150 W immersion medium-pressure UV lamp.The lamp was placed in a cooling jacket, which allowed a constant temperature of the process to be maintained, not exceeding 20 ± 1 • C. The photocatalysis process was carried out by irradiating the solutions with water from the highway for 5 min, 15 min, and 30 min and water from the roof for 5 min and 15 min.The exposure time of the highway water has been extended due to the high color of the water.
A catalyst, titanium dioxide (TiO 2 ), was added to the solution and stirred for 15 min to achieve homogeneity before exposure to light.The catalyst was commercial titanium dioxide P25 from Degussa (Frankfurt, Germany) at a dose of 200 mg TiO 2 /L.Titanium dioxide P25 contains a mixture of anatase and rutile (70:30, w/w).Ozone was produced from the air by an Ozone FM 500 generator with a capacity of 0.14 mg/s (WRC Multiozon, Sopot, Poland) and directed to the reactor by a ceramic diffuser.The ozone dose was 10 mg/L.
The applied photocatalytic ozonation process is further referred to as UV+O 3 .A catalyst, titanium dioxide (TiO2), was added to the solution and stirred for 15 min to achieve homogeneity before exposure to light.The catalyst was commercial titanium dioxide P25 from Degussa (Frankfurt, Germany) at a dose of 200 mg TiO2/L.Titanium dioxide P25 contains a mixture of anatase and rutile (70:30, w/w).Ozone was produced from the air by an Ozone FM 500 generator with a capacity of 0.14 mg/s (WRC Multiozon, Sopot, Poland) and directed to the reactor by a ceramic diffuser.The ozone dose was 10 mg/L.
The applied photocatalytic ozonation process is further referred to as UV+O3.

Chemical Analysis
Color and the concentrations of COD, zinc, copper, lead, nickel, nitrogen, phosphate, and ammonium were determined spectrophotometrically with Merck test kits.TOC was measured using a TOC-L series analyser (Shimadzu, Kioto, Prefektura Kioto, Japan).Conductivity and pH were monitored by a multifunctional analyser CX-461 (Elmetron, Zabrze, Poland).The errors were estimated on the standard deviation of three replicates for each analysis.The error values for all tested samples did not exceed 5%.

The Chromatographic Analysis
The extract chromatographic analysis was performed using a GC-MS(EI) 7890B gas chromatograph from Agilent Technologies (Santa Clara, CA, USA) following the detailed procedure described by Kudlek [27].

Microbiological Analysis
Enumeration of coliforms and Escherichia coli was performed using the membrane filtration method on chromogenic agar medium according to ISO 9308-1:2014-12 [31].Each bacterial enumeration was performed in triplicates.

Chemical Analysis
Color and the concentrations of COD, zinc, copper, lead, nickel, nitrogen, phosphate, and ammonium were determined spectrophotometrically with Merck test kits.TOC was measured using a TOC-L series analyser (Shimadzu, Kioto, Prefektura Kioto, Japan).Conductivity and pH were monitored by a multifunctional analyser CX-461 (Elmetron, Zabrze, Poland).The errors were estimated on the standard deviation of three replicates for each analysis.The error values for all tested samples did not exceed 5%.

The Chromatographic Analysis
The extract chromatographic analysis was performed using a GC-MS(EI) 7890B gas chromatograph from Agilent Technologies (Santa Clara, CA, USA) following the detailed procedure described by Kudlek [27].

Microbiological Analysis
Enumeration of coliforms and Escherichia coli was performed using the membrane filtration method on chromogenic agar medium according to ISO 9308-1:2014-12 [31].Each bacterial enumeration was performed in triplicates.

Ecotoxicity and Genotoxicity Tests
Growth, immobilization, enzymatic, and genotoxicity assays were performed using the green alga Desmodesmus quadricauda (CCALA 463) and plant Lemna minor, the crustacean Daphnia magna, and the bacteria Aliivibrio fischeri and Escherichia coli, respectively.Algae, duckweed, and crustaceans originated from our own cultures.Lyophilized bacteria were purchased from microbiotests' manufacturers (A.fischeri-Modern Water, E. coli-EBPI, Burlington, ON, Canada).
Growth tests with D. quadricauda were performed according to ISO 8692; growth inhibition was assessed by cell densities after 72 h of exposure to rainwater samples [32].L. minor growth tests were conducted following ISO 20079:2006 [33], with growth inhibition evaluated by both the number and total frond area after 7 days of exposure to rainwater samples.The immobilisation assay with D. magna was performed in accordance with ISO 6341 [34].The immobilised organisms were counted after 48 h of incubation with rainwater samples.Bioluminescence inhibition of A. fischeri was measured after 5 min of exposure to rainwater samples using a portable device, DeltaToxII (Modern Water, London, UK).
The ecotoxicity assessment, similarly to our previous research [21], was based on the toxicity classification system for natural waters, developed by Persoone et al. [35].In this approach, the EC 50 values obtained with each test are transformed into toxic units (TU) using the formula: TU = 1/EC 50 × 100.When the percentage effect observed in the toxicity tests was significantly higher than that of the control but below 50% (<1 TU), the TU was calculated based on the fact that the 20% effect corresponded to 0.4 TU [35].When the percentage effect was less than minus 20%, samples were evaluated for stimulation.
For each rainwater sample examined, a test score was allocated that reflected the toxic effect, according to the following scale: 0 for no significant toxic effect (a toxic effect that is not significantly higher than that in the control), 1 for a significant toxic effect (the 20% effect level, corresponding to 0.4 TU, the lowest effect to have a significant toxic impact) but <1 TU, 2 for a toxic effect (≥1 TU and <10 TU), 3 for a high toxic effect (≥10 TU and <100 TU), and 4 for a very high toxic effect (≥100 TU).
The rainwater samples were then classified into the following categories according to the highest TU value obtained (Table 2).Class weight score (CWS) and the percentage class weight score (CWS % ) were calculated for each wastewater sample: where n = number of tests performed and CWS max = maximum class weight score (the highest value of all test scores obtained for a given rainwater sample).The higher the percentage of the weight score, the more the score expressed the ecotoxicity of the rainwater sample in the concerned class.
The colorimetric assay employing the mutant strains PQ37 of E. coli was conducted following the methodology described in the implementation instructions of Environmental Bio-Detection Products Inc. [36].The quantitative assay was conducted in versions with and without metabolic activation (S9 fraction), with the application of two standard genotoxic solutions: 4NQO (4-nitro-Quinoline-1-Oxide) and 2AA (2-Amino-Anthracene).The S9 mix contained S9 extract of Aroclor-induced rat liver and was prepared according to kit instructions [27].The MB100-4A THERMO-SHAKER for microplates with rotable platform (Hangzhou Allsheng Instruments Co., Ltd., Hangzhou, China) was used for the two-hour development of the enzymatic activities, which were then measured by photometric measurement at 600 nm (genotoxic activity) and 420 nm (viability) using INNO-M microplate reader (LTEK, Seongnam, Republic of Korea).Readings were taken immediately before and after the incubation needed for color development (90 min).
Genotoxicity assessment was based on SOS induction factors-SOSIF (Table 3) was calculated according to the formula: where: A 600 S-absorbance readings at λ = 600 nm for sample wells; A 420 S-absorbance readings at λ = 420 nm for sample wells; A 600 N-absorbance readings at λ = 600 nm for negative control wells; A 420 N-absorbance readings at λ = 420 nm for negative control wells; A 600 B-averaged absorbance readings at λ = 600 nm for blank reagent wells; A 420 N-averaged absorbance readings at λ = 420 nm for reagent blank wells.Cytotoxicity assessment was based on survival rates (SR, %), which were calculated according to the formula: A survival rate of 80% was required to confirm a positive result of genotoxicity.

Results and Discussion
The physicochemical analysis of rainwater provided information on the contamination of these waters with organic compounds and heavy metals.The results of the tests, presenting the concentration of the individual heavy metals tested, are presented in Table 4, while Figure 2 presents the degree of reduction in color, conductivity, COD, and TOC in the photocatalytic ozonation.Treatment of motorway rainwater resulted in a 61% reduction in color after just 5 min of photocatalytic ozonation, with a further achievement of 75% reduction after 30 min.In the case of water from the roof, the color concentration was 7 mg Pt/L and a 57% reduction was achieved through a significant level of pigment degradation, a phenomenon welldocumented in numerous publications [38][39][40][41].
The color reduction in both cases resulted from the effective oxidation of organic pollutants present in the water.The photocatalytic ozonation process is particularly effective due to the synergy between ozone and the photocatalyst, leading to the rapid decomposition of color compounds.Differences in the level of color reduction may be due to variations in the composition of pollutants and their chemical properties in highway rainwater and roof water.Ozone (O3) is a strong oxidizing agent that can decompose complex organic compounds into simpler, less colorful compounds or into CO2 and H2O.Additionally, in the presence of UV light and a photocatalyst (e.g., TiO2), reactive oxygen species (such as hydroxyl radicals) are generated, which further oxidize organic pollutants, contributing to their decomposition [42].
Heavy metals and organic compounds come from a variety of sources, both from surface runoff and from atmospheric air.The complexity of rainwater chemistry is due to many factors, including atmospheric pollutants, the chemical composition of the air, the geography of the region through which the rain flows, and the interaction of rainwater with the surfaces through which it flows.Rain can act as a carrier for a variety of chemical pollutants, such as heavy metals, pesticides, organic compounds, and other toxic substances.These pollutants can be transported from the soil or paved surfaces to surface or groundwater, posing a risk to human health and ecosystems [43].
It is significant that most studies on this topic have been conducted on model samples, which may not fully reflect real conditions and often require longer processing times [39][40][41]44].For example, the research by Nabizadeh et al. showed that photocatalytic ozonation of a methyl blue solution resulted in 58% degradation after 20 min, compared to 39% degradation without the addition of ozone [40].However, Figueredo et al. emphasize the need for real-world trials, which may provide more reliable results [45].Their study on the treatment of real secondary wastewater showed that the degree of degradation depends on the composition of the water matrix.
Our analyses showed a high concentration of zinc ions in the water coming from the motorway, amounting to 3.95 mg/L.Zinc in the rainwater from the motorway was probably present as a result of abrasion of car tires [46][47][48].On the other hand, the water collected from the roof showed a recorded concentration that was half that of the water from the motorway.This may be related to the emission of heavy metals from the roofing material [49].A study by Chizoruo et  Treatment of motorway rainwater resulted in a 61% reduction in color after just 5 min of photocatalytic ozonation, with a further achievement of 75% reduction after 30 min.In the case of water from the roof, the color concentration was 7 mg Pt/L and a 57% reduction was achieved through a significant level of pigment degradation, a phenomenon well-documented in numerous publications [38][39][40][41].
The color reduction in both cases resulted from the effective oxidation of organic pollutants present in the water.The photocatalytic ozonation process is particularly effective due to the synergy between ozone and the photocatalyst, leading to the rapid decomposition of color compounds.Differences in the level of color reduction may be due to variations in the composition of pollutants and their chemical properties in highway rainwater and roof water.Ozone (O 3 ) is a strong oxidizing agent that can decompose complex organic compounds into simpler, less colorful compounds or into CO 2 and H 2 O. Additionally, in the presence of UV light and a photocatalyst (e.g., TiO 2 ), reactive oxygen species (such as hydroxyl radicals) are generated, which further oxidize organic pollutants, contributing to their decomposition [42].
Heavy metals and organic compounds come from a variety of sources, both from surface runoff and from atmospheric air.The complexity of rainwater chemistry is due to many factors, including atmospheric pollutants, the chemical composition of the air, the geography of the region through which the rain flows, and the interaction of rainwater with the surfaces through which it flows.Rain can act as a carrier for a variety of chemical pollutants, such as heavy metals, pesticides, organic compounds, and other toxic substances.These pollutants can be transported from the soil or paved surfaces to surface or groundwater, posing a risk to human health and ecosystems [43].
It is significant that most studies on this topic have been conducted on model samples, which may not fully reflect real conditions and often require longer processing times [39][40][41]44].For example, the research by Nabizadeh et al. showed that photocatalytic ozonation of a methyl blue solution resulted in 58% degradation after 20 min, compared to 39% degradation without the addition of ozone [40].However, Figueredo et al. emphasize the need for real-world trials, which may provide more reliable results [45].Their study on the treatment of real secondary wastewater showed that the degree of degradation depends on the composition of the water matrix.
Our analyses showed a high concentration of zinc ions in the water coming from the motorway, amounting to 3.95 mg/L.Zinc in the rainwater from the motorway was probably present as a result of abrasion of car tires [46][47][48].On the other hand, the water collected from the roof showed a recorded concentration that was half that of the water from the motorway.This may be related to the emission of heavy metals from the roofing material [49].A study by Chizoruo et al. observed a similar phenomenon, where heavy metal concentrations in rainwater were associated with the roof material [50].However, the concentrations of the other metals tested in our study, i.e., lead, nickel, and copper, in RW I and RW II were rather low.The UV/O 3 process completely removed these metals.A pivotal role was performed by the metal oxide employed as a catalyst, facilitating the adsorption of heavy metal ions [41].On the contrary, the concentration of zinc in RW I remained constant, indicating that the photocatalytic ozonation process had no significant impact on the quantity of this element.Zinc has a standard potential of −0.76 V, meaning that it is more reduced compared to other metals.This may affect its interaction with the TiO 2 surface, which may be less effective at binding Zn 2+ .The differences in metal adsorption on TiO 2 are the result of a complex interaction involving many chemical and physical agents.Zinc, due to its electrochemical and chemical properties, may not be adsorbed as effectively as other metals such as lead, copper, or nickel.Metals with a more positive standard potential (like copper) have a greater tendency to accept electrons and reduce.Zinc, having a more negative potential, may have a lower tendency to interact with other elements, a phenomenon which may explain why its adsorption is less efficient compared to that of copper, lead, or nickel.On the other hand, in rainwater, zinc ions may be present in the form of soluble complexes bound to mineral or organic ligands.Such observation indicates the complexity of the chemical composition of rainwater and suggests that chemical processes, including complexation with ligands, may affect the stability of zinc concentrations in this environment.Furthermore, it was observed that the exposure time may have been inadequate.Pan et al. [51] achieved 100% zinc removal only after 3 h of adsorption and 4 h of irradiation.More research is advisable to gain a deeper understanding of the mechanisms inherent in the process and the impact of various chemical components on the outcomes of photocatalytic ozonation [52,53].
A chromatographic analysis revealed the presence of various types of organic micropollutants in the samples tested before and after the photocatalytic ozonation process (Figure 3).concentrations in rainwater were associated with the roof material [50].However, the concentrations of the other metals tested in our study, i.e., lead, nickel, and copper, in RW I and RW II were rather low.The UV/O3 process completely removed these metals.A pivotal role was performed by the metal oxide employed as a catalyst, facilitating the adsorption of heavy metal ions [41].On the contrary, the concentration of zinc in RW I remained constant, indicating that the photocatalytic ozonation process had no significant impact on the quantity of this element.Zinc has a standard potential of −0.76 V, meaning that it is more reduced compared to other metals.This may affect its interaction with the TiO2 surface, which may be less effective at binding Zn 2 ⁺.The differences in metal adsorption on TiO2 are the result of a complex interaction involving many chemical and physical agents.Zinc, due to its electrochemical and chemical properties, may not be adsorbed as effectively as other metals such as lead, copper, or nickel.Metals with a more positive standard potential (like copper) have a greater tendency to accept electrons and reduce.Zinc, having a more negative potential, may have a lower tendency to interact with other elements, a phenomenon which may explain why its adsorption is less efficient compared to that of copper, lead, or nickel.On the other hand, in rainwater, zinc ions may be present in the form of soluble complexes bound to mineral or organic ligands.Such observation indicates the complexity of the chemical composition of rainwater and suggests that chemical processes, including complexation with ligands, may affect the stability of zinc concentrations in this environment.Furthermore, it was observed that the exposure time may have been inadequate.Pan et al. [51] achieved 100% zinc removal only after 3 h of adsorption and 4 h of irradiation.More research is advisable to gain a deeper understanding of the mechanisms inherent in the process and the impact of various chemical components on the outcomes of photocatalytic ozonation [52,53].
A chromatographic analysis revealed the presence of various types of organic micropollutants in the samples tested before and after the photocatalytic ozonation process (Figure 3).Rainwater contained alkanes, both simple alkanes (e.g., tetradecane, hexadecane, octadecane), branched alkanes (2,6,11-trimethyldodecane), and phenolic derivatives (2,4di-tert-butylphenol).In the case of precipitation, alkanes can be transferred from the atmosphere to the surface water in a process known as surface runoff.This can be the result of emissions from a variety of sources, such as the burning of fossil fuels, industrial processes, or other anthropogenic activities.However, due to their poor solubility in water, they can mostly be bound to solid particles or form a layer on the surface of the water.In the context of environmental protection, alkane contamination in water can pose a problem, especially if the amounts are large enough to affect surface water quality [54].Subsequently, phenolic derivatives were detected in both water samples, i.e., 2,4-di-tertbutylphenol, which has various applications, e.g., as an antioxidant in industrial products, preservatives, as well as a stabilizer for various chemicals.Phenolic derivatives in rainwater can come from various sources such as industry, fuel combustion, vehicle emissions, or even natural biological processes.Therefore, ensuring a healthy aquatic environment involves monitoring and controlling pollutant emissions, including alkanes [55].Similarly, the work of Schummer et al. indicate the presence of phenolic derivatives, six phenols and 14 nitrophenols in rainwater samples from eastern France.Our analyses also detected some of these contaminants, pointing to their diverse industrial use and the possibility of coming from anthropogenic emissions [56].

A Acquisition time, min
Another interesting result is the detection of benzothiazole in the highway water sample.Research by Zhang et al. also indicates the widespread occurrence of this substance in the environment, particularly in groundwater, surface water, and rainwater near industrial areas.These findings emphasize the toxicity of benzothiazole and the need for further research into its effects on health and the environment [57].Benzothiazole, detected in the highway water sample, is a common component used in the manufacturing of dyes, rubber curing accelerators, corrosion inhibitors, antibacterial agents, and antimicrobial substances in radiators [58].Benzothiazole may exhibit toxicity, underscoring the importance of studies on substance toxicity to comprehensively grasp potential health risks [58].
Table 5 shows the organic micropollutants identified in the samples corresponding to the individual peaks, and Table 6 presents the degree of removal of pollutants in the photocatalytic ozonation process.Several carboxylic acids were also detected in the water coming from the roof following 12 min and 15 min of retention, i.e., 2-methyl-2-propylpentanoic acid, 2-methyl-2-propylpentanoic acid, and 2-ethyl-2,3,3-trimethylbutanoic acid.Khwaja's research shows that the main sources of atmospheric carboxylic acids are photochemical processes and anthropogenic emissions such as car exhausts [59].
In the highway water sample, some compounds, such as benzothiazoles, 2,3,5,8tetramethyldecane, and 3,8-dimethylundecane, were completely removed after the UV+O 3 process.Other compounds, such as tetradecane, 2,4-di-tert-butylphenol, and 2,6,11-trimeth yldodecane, showed a gradual decline, while the pre-peak of hexadecane and heptadecane changed slightly.In the case of octadecane, an increase in concentration was observed in the highway rainwater sample after exposure to UV+O 3 , suggesting the possibility of forming this compound from other precursors under these conditions.The work of Beltrán et al. reports that ozonation in the presence of UV can lead to the formation of new chemical compounds through reactions between precursors present in the water [60].In contrast, in relation to the water coming from the roof, 2-ethyl-2,3,3-trimethylbutanoic acid showed a significant decrease, neodecanoic acid first increased and then decreased, and tetradecane increased after 5 min to later decrease after 15 min.Analyses showed that photocatalysis and ozonation have differentiated effects on different chemical compounds, a phenomenon which may depend on their chemical structure and reactivity in the presence of ozone and UV radiation.TiO 2 is believed to improve the treatment process due to its large specific surface area and active surface area, both of which increase the number of adsorption sites.In the process of photocatalysis, TiO 2 acts as a semiconductor which, under the influence of UV radiation, generates electron-hole pairs, leading to the formation of reactive oxygen species, such as hydroxyl radicals.These radicals are capable of effectively degrading organic pollutants, making TiO 2 an effective tool in environmental purification [61][62][63][64].Alkane molecules should also undergo an oxidation process which leads to the breakdown of larger molecules into smaller fragments, including final products that may be less harmful to the environment [65].The study revealed that, when rainwater contained numerous other compounds, the duration of the photocatalysis process was likely insufficient for the decomposition of alkanes.Considering the presence of diverse micropollutants in rainwater originating from both street runoff and roofs, it is imperative to subject rainwater to microbiological and toxicological evaluation [66].
Rainwater harvesting has been practiced over many decades for various purposes, including potable use, non-potable reuse (toilet flushing, household cleaning, clothes washing, lawn irrigation, emergency supplies for firefighting, ornamental use), irrigation of products for consumption, and cooking via installation and maintenance processes [67].The World Health Organization (WHO) drinking water guidelines state that E. coli should not be detected in a 100 mL sample of drinking water, and, if detected, immediate action should be taken to minimize human health risks [68].However, many studies have shown that rainwater contains increased levels of microorganisms originating from aerosol deposition, tree litter, and animal fecal matter, as well as following indigenous growth in biofilms and sediments [69][70][71].Several studies have also reported that roof-harvested rainwater for drinking or domestic use has been associated with disease risks [72][73][74].In our study, in both types of rainwater, the permissible level of E. coli was exceeded before treatment (Table 7).The number of coliforms and E. coli was ten times higher in the rainwater collected from the roof than in the rainwater sample collected from the highway, a phenomenon which was probably connected to the presence of bird feces on the roof.Inactivation of E. coli down to an undetectable level was observed each time in the process of photocatalytic ozonation, regardless of the time of treatment.Coliforms were inactivated down to an undetectable level in rainwater from the highway; however, the same only occurred in 62.5% (0.4 log) of the roof-harvested rainwater.This result likely stemmed from the fact that significantly higher concentrations were detected in water collected from the roof compared to the rainwater sample from the highway.Relying solely on the E. coli results, the water met the WHO standards for drinking; however, it still contained microorganisms, among which there may be pathogens.Our ecotoxicity studies indicated that rainwater samples exhibited no harmfulness toward crustaceans in the acute immobilization test, both before and after undergoing the photocatalytic oxidation treatment (Table 8).Nevertheless, rainwater collected from the highway (comprising 81.8% concentrated samples), following the longest treatment duration (30 min), induced a 42% bioluminescence inhibition in the DeltaToxII test, as measured by light loss.As per Persoone et al., rainwater from the highway should be categorized as slightly toxic to A. fischeri (Toxicity Unit, TU = 0.84, toxicity class II) [35].This same sample also resulted in a 35.3% growth inhibition of L. minor, assessed by comparing the total frond area before and after treatment, classifying the rainwater as slightly toxic (TU = 0.48).In the case of rainwater collected from the roof at the highest concentrations used in the tests, it led to a slight stimulation of algal growth (inhibition of growth equalled −24%) and bioluminescence of A. fischeri (inhibition of bioluminescence equalled −27%).However, post-treatment, this sample exhibited slight toxicity, as evidenced by the growth inhibition of algae (TU = 0.4-0.76,toxicity class II).Interestingly, the same sample stimulated duckweed growth in terms of frond numbers, with no discernible effect on the total frond area.For algae, although a slight growth inhibition was observed in most samples, sample dilution, in almost all cases, resulted in strong stimulation of algal growth, crucial information when considering using rainwater for agricultural fields irrigation.By summarizing the results from the ecotoxicity studies on rainwater samples before and after treatment, it can be concluded that the highway water sample exhibited slight toxicity, according to the classification proposed by Persoone et al. [35], irrespective of the treatment duration.Only after 15 min of photocatalytic ozonation no harm was observed toward any of the bioindicators.
Ozonation-combined techniques are used both for disinfection purposes and also to improve water organoleptic properties, reduce COD, remove pesticides, surfactants, iron, sulfur or manganese, and micropollutants [75].Ozone disinfection is also a promising method for removing residues of pharmaceuticals and personal care products and endocrine disrupting compounds [23][24][25]75,76].As mentioned earlier, a significant limitation in the use of ozonation is the formation of toxic byproducts such as brominated byproducts, aldehydes, ketones, and carboxylic acids [77,78].This may explain why, in our study, the highway rainwater sample gained ecotoxicity over the course of the treatment, a phenomenon which may be attributed to the formation of toxic byproducts during oxidation.
The composition of rainwater is influenced by the association between aerosol composition and cloud droplets, as well as depending on the geographic region and the type of land use [79].This affects the diversity of rainwater and results in its physical and chemical properties.In our study, the very important factor was also the technology of rainwater harvesting and storage, as rainwater coming from the roof was highly contaminated with fecal bacteria.All of these factors affect applicability of the proposed treatment method and possibility of safe rainwater reuse for agricultural needs.Further research is needed to validate the effectiveness of the treatment method for rainwater collected from different places and in large scale applications.
Limited research has been conducted on the influence of rainwater on the genetic material of aquatic organisms.Nevertheless, evaluating the genotoxicity of rainwater offers valuable insights into the overall safety and ecological implications of its quality, especially considering its potential use, for instance, in plant irrigation.Unlike Vlastos et al. [4], who described eco-and genotoxicity originating from heavy metals present in rainwater in Greek cities, in our studies, no genotoxicity was observed in SOS-Chromotest, meaning that all samples (before and after treatment) did not cause any detectable genotoxic damage to bacterial DNA (Table 9).

Conclusions
This study demonstrates the possible concerns associated with using rainwater as an alternative water source.Summing up, there are a number of insights which emerged from this work: 1.
The analysis of rainwater revealed its complex nature, attributed to the diversity of pollutants, encompassing alkanes, phenolic derivatives, and benzothiazole.The presence of these contaminants highlights the need for research on rainwater ecotoxicity.Investigating the emission and control of pollutants, especially alkanes, emerged as crucial for maintaining a healthy environment.

2.
The photocatalytic ozonation process demonstrated high efficiency in degrading benzothiazole, confirming its potential for the removal of organic pollutants.

3.
It is noteworthy that the photocatalytic ozonation process successfully eliminated heavy metals such as lead, nickel, and copper, employing metal oxide as a catalyst.However, the concentration of zinc did not undergo significant changes following the process.This observation may suggest the existence of specific chemical properties of zinc, underscoring the necessity for additional research to comprehend the mechanisms influencing its stability within the process.4.
The count of coliforms and E. coli bacteria was ten times higher in rainwater collected from the roof compared to the runoff from the highway.The photocatalytic ozonation process led to the inactivation of E. coli in both instances, irrespective of the duration of the process.Conversely, in the rainwater sourced from the highway, only 62.5% of coliforms were removed. 5.
Ecotoxicological studies revealed that rainwater, before and after photocatalytic ozonation, showed no toxicity to crustaceans.Highway stormwater runoff displayed a slight decrease in bioluminescence of A. fischeri and a slight reduction in total leaf area of L. minor, both before and after the process.Rainwater from the roof, at the highest concentrations, slightly stimulated algal growth and bioluminescence of A. fischeri, but post-treatment samples showed slight toxicity.
The conducted research suggests a potential variability in the influence of rainwater on aquatic ecosystems, underscoring the necessity for additional research to comprehensively grasp the mechanisms of rainwater effects on organisms under diverse environmental conditions.Ultimately, these analyses can serve as a crucial factor in decision-making concerning the sustainable and efficient use of water resources in agriculture.

Figure 2 .
Figure 2. Percentage removal of color, conductivity, COD, and TOC from rainwater in the photocatalytic ozonation process.

Figure 2 .
Figure 2. Percentage removal of color, conductivity, COD, and TOC from rainwater in the photocatalytic ozonation process.

Figure 3 .
Figure 3. Chromatograms before and after the photocatalytic ozonation process, (A)-rainwater I (RW I), (B)-rainwater II (RW II), (C)-rainwater I after 5 min of the process (RW I 5 min UV+O 3 ), (D)-rainwater I after 15 min of the process (RW I 15 min UV+O 3 ), (E)-rainwater I after 30 min of the process (RW I 30 min UV+O 3 ), (F)-rainwater II after 5 min of the process (RW II 5 min UV+O 3 ), (G)-rainwater II after 15 min of the process (RW II 15 min UV+O 3 ).

Table 1 .
Physicochemical characteristics of the tested rainwater.

Table 2 .
Toxicity assessment based on TU values.

Table 4 .
Concentration of heavy metals in rainwater before and after the photocatalytic ozonation process.

Table 5 .
Organic compounds identified in rainwater before and after the process.

Table 6 .
The percentage removal of selected compounds after the process.

Table 7 .
Number of culturable bacteria [cfu/mL] in rainwater before and after treatment (5 min, 15 min, and 30 min).Inactivation (in percentage and log) is presented in parentheses when applicable.
n.d.means not detected.

Table 8 .
Ecotoxicity assessment of rainwater before and after photocatalytic oxidation, based on the toxicity classification system for wastewater discharged into the aquatic environment.

Table 9 .
Results of SOS-Chromotest on rainwater before and after photocatalytic oxidation in the highest concentration used in the test (9.1%).