Effects of “COVID Kit” Drugs (Hydroxychloroquine, Azithromycin and Ivermectin) in the Freshwater Microalgae Desmodesmus subspicatus

Abstract

The pandemic caused by the SARS-CoV-2 virus resulted in a significant increase in the consumption of drugs aimed at preventing and treating the disease, such as hydroxychloroquine (HCQ), ivermectin (IVM) and azithromycin (AZM). As a consequence, the presence of these pharmaceuticals increased in natural aquatic environments, posing risks to non-target organisms. In this study we aimed to determine the toxic effects of these drugs (72 h), at environmentally realistic concentrations (0, 1, 5, 10, 50 and 100 µg.L^-1), isolated or mixed, on the microalgae Desmodesmus subspicatus. The effective concentration capable to inhibit microalgae growth by 50% (EC50), final biomass production, chlorophyll a and b content, the activities of catalase (CAT) and glutathione S-transferase (GST), as well as the levels of malondialdehyde (MDA) and protein carbonyl (PC) were determined. EC50 values were 510, >800 and 100 µg.L^-1, for HCQ, IVM and AZM, respectively. The highest AZM and mixture concentration inhibited microalgae growth by 35.3 and 49.8%, respectively. HCQ caused an increase in CAT activity at concentrations of 5, 10 and 100 µg.L^-1, the other treatments did not cause significant changes. The data show that HCQ alters microalgae metabolism and the EC50 results indicate that HCQ and AZM are harmful to microalgae.

Keywords:
hydroxychloroquine; ivermectin; azithromycin; EC50; biochemical biomarkers; Desmodesmus subspicatus.

HIGHLIGHTS

Microalgae were exposed to hydroxychloroquine (HCQ), ivermectin (IVM) and azithromycin (AZM).

EC50 values were 510, >800 and 100 µg.L^-1, for HCQ, IVM and AZM, respectively.

The highest AZM and mixture concentration inhibited microalgae growth.

HCQ exposure increased CAT activity.

GRAPHICAL ABSTRACT

INTRODUCTION

Contamination of natural aquatic environments by anthropogenic compounds is one of the problems arising from the human use of water resources, especially in densely populated areas [¹]. As a consequence, substances such as pesticides, metals, hydrocarbons, hygiene and personal care products, and pharmaceutical compounds (PhaCs) are frequently found in the aquatic environment [²]. Products of pharmacological origin are widely consumed by humans, and most part of them are not completely removed by conventional sewage treatment systems. As a consequence, these compounds end up entering aquatic environments, causing negative effects to resident organisms [³].

The COVID-19 pandemic recently caused by the new coronavirus, resulted in an increase in sales of a group of specific PhaCs, known in Brazil as the “COVID Kit”, composed of hydroxychloroquine (HCQ), ivermectin (IVM) and azithromycin (AZM) [⁴, ¹⁰⁶]. HCQ, belonging to the 4-aminoquinoline group and derived from chloroquine, is a drug commonly used as a treatment for malaria, rheumatoid arthritis and systemic lupus erythematosus [⁵]. IVM is an antiparasitic drug, belonging to the avermectin group, mainly used to treat diseases such as filariasis, strongyloidiasis, scabies and pediculosis [⁶]. AZM is a macrolide antibiotic used mainly to treat gram-positive bacteria such as Streptococcus pneumoniae, which causes pneumococcal pneumonia [⁷]. These three drugs were widely publicized as a form of prevention and/or treatment for COVID-19 is numerous countries, such as Brazil, even without scientific proof of effectiveness for the disease [⁸, ⁹, ¹⁰, ¹¹, ¹², ¹³, ¹⁴, ¹⁰⁴, ¹⁰⁵] causing a substantial increase their consumption by the human population [¹⁵]. According to the Brazilian Association of Distribution and Logistics of Pharmaceutical Products, the drugs present in the COVID Kit had an 857% increase in sales between April 2020 and March 2021 in Brazil [¹⁶]. Among the drugs in question, IVM presented the highest increase in sales in 2020, 557% compared to 2019. In turn, HCQ, showed an 113% increase in sales, with 963 thousand units of the drug sold in 2019 and 2 million units in 2020 [⁴].

It was estimated that due to the increased use of these medications during the pandemic period the release rates of them into the environment have increased significantly [¹⁰⁶, ¹⁰⁷]. According to Chen and coauthors [¹⁷], concentrations of AZM found in the Yangtze River, located in China, were significantly higher (up to 935 ng.L^-1), and more frequent, when compared to data from previous years, which indicated a maximus concentration of 4.3 ng.L^-1 [¹⁸]. It is important to highlight that AZM, together with other macrolide antibiotics (erythromycin and clarithromycin), was included in the European Union's watch list of emerging pollutants precisely because of its frequent detection in the aquatic environment [¹⁹].

IVM concentrations in surface water also showed a post-pandemic increase [²⁰]. Concentrations that were previously 93 ng.L^-1 [²¹] were detected in the range of 1,500 ng.L^-1 in 2020 [¹⁵]. HCQ had concentrations of 95.85 ng.L^-1 detected after the pandemic [²²]. Other drugs used to treat COVID-19, such as antivirals (Favipiravir, Lopinavir and Ribavirin) and dexamethasone, also showed an increase in environmental concentrations compared to previous years [²⁰]. In Mexico City, Durán-Álvarez and coauthors [²³] monitored the wastewater concentration of some drugs used during the period of the COVID-19 pandemic. High concentrations of dexamethasone, AZM and IVM were found in the wastewater treatment plant tributaries, with the latter two being detected in the range of 2,873.7 to 5,819.6 ng.L^-1 and 1,219.8 to 4,622.4 ng.L^-1, respectively. After treatment, removal rates were 74.4% for AZM and 88.1% for IVM.

Since the COVID-19 pandemic was a recent event, there is still no broad knowledge of the effects that the increase in these compounds in water can cause to the aquatic ecosystem [²⁴]. There is a lack of data regarding the environmental toxicity of these drugs and their mixtures, as there is the possibility of a synergistic interaction between them, further increasing the toxic effects [¹⁵, ²⁵, ¹⁰⁷]. During preparation of this study, only 11 papers were found that dealt with the potentially adverse effects of exposing aquatic organisms to chloroquine. Of these, five were carried out in fish [²⁶, ²⁷, ²⁸, ²⁹, ³⁰], three in Daphnia magna [²⁸, ³¹, ²²], one in the bivalve mollusk Mytilus edulis [³²], one in Vibrio fischeri [²⁸] and one in microalgae [²⁸]. When considering only HCQ, only eight articles were found (all published from 2020 onwards), being four on Danio rerio [³⁰, ³³, ³⁴, ³⁵], one using Physalaemus cuvieri tadpoles [²⁵], one in D. magna [²²], one with the microalgae Chlorella vulgaris and the cyanobacteria Synechococcus elongatus [³⁶], and another using mesocosmos [³⁷]. For AZM, 40 studies were found, mostly on, such as D. rerio [³⁸-⁴²] and Clarias gariepinus [⁴³, ⁴⁴] and in the microcrustacean Daphnia sp. [⁴⁵-⁴⁷]. Only four studies using microalgae as bioindicators were found [³⁶, ⁴⁵, ⁴⁸, ⁴⁹].

In order to assess the negative impacts caused by the presence of contaminants in aquatic organisms, bioindicators are used, such as microalgae [⁵⁰]. Aquatic pollutants, including PhaCs, are known to adversely affect important aspects of essential microalgae physiology, such as their growth [²⁸,³⁶, ⁷⁰,⁷¹,⁹⁶,¹⁰⁰,¹⁰³] and chlorophyl production [⁷⁰, ⁸⁵]. These effects are often a consequence of the interference of PhaCs on cellular physiology, which includes an exacerbation of the generation of reactive oxygen species (ROS) in microalgae, due to impaired aerobic metabolism and biotransformation reactions. As a consequence, increased levels of protein carbonyls (PC) and lipid peroxidation by-products, such as malondialdehyde (MDA) are formed, due to excessive oxidative reactions [⁷⁰, ⁷¹, ¹⁰⁹]. To counteract ROS effects, modulation of antioxidant enzymes, such as catalase (CAT) and glutathione S-transferase (GST) [³⁶, ⁵⁰, ⁷⁷, ⁹⁶] often occurs, as a defensive mechanism against cellular oxidative injuries. Desmodesmus subspicatus (Chlorophyta, Chlorophyceae) is a freshwater green microalga and is an example of a bioindicator largely used in toxicity tests [⁵¹, ⁵²]. Due to the marked importance of microalgae in aquatic ecosystems, as they are the main producers of oxygen and represent the basis of the food chain for several species of animals, ecotoxicological studies on these organisms are relevant [⁵³].

Considering the recent increase in the human consumption of the COVID kit drugs and the lack of information of their effects on aquatic organisms, in this study we aimed to evaluate how HCQ, IVM and AZM affects the growth, survival, chlorophyl levels and biochemical parameters related to oxidative stress (activity of the antioxidant enzymes catalase and glutathione S-transferase, lipid peroxidation and protein oxidation levels) of the microalgae D. subspicatus.

MATERIAL AND METHODS

Drugs

The drugs used in the study were hydroxychloroquine (HCQ) (CAS # 747-36-4), ivermectin (IVM) (CAS # 70288-86-7) and azithromycin (AZM) (CAS # 117772-70-0), all purchased from Sigma-Aldrich, St Louis. The drugs were diluted in distilled water, in the case of hydroxychloroquine, and in methanol (CAS # 67-56-1), in the case of the other drugs, and the solutions were stored at -20 ℃ before used.

Desmodesmus subspicatus culture

The microalgae inoculum was cultivated in Erlenmeyer flasks (250 mL), containing 100 mL of nutrient medium (CHU), prepared and autoclaved in accordance with the Brazilian standards set out in NBR 12648 - Aquatic ecotoxicology - Chronic toxicity - Test method with algae (Chlorophyceae) [⁵⁴]. The microalgae cultures were maintained in a cultivation incubator (TECNAL - TE 424) with lighting (<4500 lux provided by fluorescent lamps) and controlled temperature (20 ± 2 ℃), statically and without aeration. In order to keep it in good condition, maintaining a supply of cells in the logarithmic growth phase available for carrying out toxicity tests, replicates were carried out weekly. For this, 1 mL of inoculum in exponential growth phase was transferred to a new autoclaved Erlenmeyer flask, containing 100 mL nutrient medium (CHU), also kept in a cultivation incubator under the conditions already mentioned.

After the period of initial adaptation of the inoculum to laboratory conditions, in order to guarantee the quality of the method, a calibration curve was carried out, establishing the relationship between cell count and absorbance. For this, the microalgae were cultivated for 72 hours in test medium (DIN) at a temperature between 23 and 27 ℃, with continuous lighting above 4500 lux and continuous agitation, between 100 and 175 rpm, in accordance with NBR 12648 [⁵⁴], and then diluted to concentrations of 100, 50, 25, 12.5, 6.25, 3.12 and 1.56%. These dilutions were subjected to cell counting in a Neubauer chamber, using optical microscopy (LEICA DM500) and, in the same way, their biomass was determined through spectrophotometry with absorbance reading at 410 nm (UV/VIS Spectrophotometer - SHIMADZU 1650 PC), allowing to establish a linear regression. The wavelength used was defined based on previous tests that identified the best point for reading the culture. In these tests, three cultures had their absorbance measured at different wavelengths, with the 410 nm spectrum being defined for use in the test, which presented higher absorbance values in the three situations. Based on this calibration test, an initial density (day zero) of 10⁵ cells.mL^-1 were used to expose the microalgae to the contaminant in each of the 100 mL cultures, ensuring that the culture did not exceed a total of 10⁷ cells.mL^-1 at the end of the exposure period (72 h) to the tested PhaCs.

Finally, to guarantee the quality of microalgae cultivation for PhaCs exposure tests, sensitivity tests were carried out monthly also in accordance with NBR 12648 [⁵⁴], using a reference substance, potassium dichromate (25 mg.L^-1). To verify the sensitivity of the microalgae and calculate the effective concentration (EC50) after 72h to inhibit growth, an aliquot with a concentration of 10⁵ cells.mL^-1 was exposed to 5 concentrations of potassium dichromate solution (0.2; 0.4; 0.6; 0.8 and 1.0 mg.L^-1), in 100 mL of test medium (DIN). The test was carried out in triplicate, for 72 hours at a temperature between 23 and 27 ℃, with continuous lighting above 4500 lux and agitation, between 100 and 175 rpm, on an orbital shaking table (Lucadema, TECNAL).

At time zero, the three controls and a replicate of the highest and lowest concentration had their pH (TECNAL) and absorbance determined at 410 nm. After 72 hours of exposure, the pH of the same cultures was determined, as well as the absorbance of all concentrations and replicates. The tests were considered valid in situations where there is no variation greater than 1.5 in the pH of the controls and the microalgae growth is greater than 16 times.

Toxicity tests using environmental concentrations of the COVID kit drugs

The toxicity tests with hydroxychloroquine, ivermectin and azithromycin, were carried out under the same conditions and with the same method used for the sensitivity tests described previously. Since the drugs ivermectin and azithromycin have low solubility in water, it was necessary to use methanol or dimethyl sulfoxide (DMSO), respectively, to dilute them. Therefore, the stock solutions were prepared so that the amount pipetted into the algal culture, at the beginning of the experiment, was as small as possible, 10 μL (0.01%). As ivermectin was only soluble in methanol, when testing the effects of the mixture of the compounds, methanol was used as vehicle to dilute all compounds. To be aware of the changes caused by the solvents, we checked the effects of methanol and DMSO on algae growth. Methanol even at a volume of 0.01% caused a significant inhibition of D. subspicatus growth (average of 25.8 % from five replicates, data not shown), when compared to the growth of organisms in absence of the solvent. DMSO did not show any significant inhibition of microalgae growth. For this reason, control groups (in triplicate) also received 10 μL of the respective vehicle used for each compound: one control for hydroxychloroquine without solvent, one control for ivermectin with methanol, and one control for azithromycin with DMSO. Results from the group exposed to the mixture of compounds were compared to the control group with methanol.

Toxicity tests were done using environmentally realistic concentrations of each compound, based on data previously reported in the scientific literature. Hydroxychloroquine concentrations ranging from 13 to 420 μg.L^-1 have been reported in river water, in Spain [⁵⁵] and in wastewater treatment plants, in Northern Italy, at concentrations of 1.77 μg.L^-1 [⁵⁶]. For ivermectin, concentrations of 1.8 μg.L^-1 have already been reported in South Africa in surface water samples [⁵⁷] and concentrations of 3.2 μg.L^-1 were found in rivers in the city of Curitiba, Brazil [³⁶], while azithromycin was found at concentrations ranging from 0.01 to 3.4 μg.L^-1 in Sava river, in the Republic of Croatia [⁵⁸] and in wastewater treatment plants at concentrations ranging from 0.12 to 14.8 μg.L^-1 in the United States of America [⁵⁹]. Thus, in our study the microalgae were exposed to the compounds alone or in mixture, at concentrations of 1.0, 5.0, 10.0, 50.0 and 100.0 μg.L^-1, in triplicate, for 72 h. The mixtures were prepared using the same concentrations for each drug.

Determination of EC50 values for the COVID kit drugs

Tests to determine the effective concentration (EC50) capable to inhibit the growth of the microalgae were also performed, similar to the methods described by Marques and coauthors [³⁶]. The algae were initially exposed for a period of 72 h to the drugs in isolation at concentrations of 0, 200, 400, 600, 800 and 1000 μg.L^-1. Based on this preliminary test, changes in drug concentrations were necessary to betted determine the EC50, except for hydroxychloroquine. For ivermectin concentrations of 0, 100, 200, 400, 600 and 800 μg.L^-1 were used due to difficulty of diluting it in solvents. For azithromycin, exposure was performed using 0, 25, 50, 75, 100, 125, 150 and 175 μg.L^-1 so that the EC50 could be determined. Same solvents used to dilute the drugs in toxicity tests were used in tests to determine EC50 values, and all concentrations used were added to the aquaria in a final volume of 10 μL (0.01%). The groups without the drugs (0 μg.L^-1) received only 10 μL of the respective solvent. To check growth rate during the test, an aliquot of 1 mL of algal culture was collected after 72 hours of exposure to the respective drug and had its absorbance determined at 410 nm. The absorbance was used to calculate algae biomass in the culture medium, and this data was used to determine growth inhibition, using the formulae: Inhibition (%) 1=(Bc-Bt)/Bc ×100, where Bc and Bt represent the biomass concentrations in the control and treatment, respectively.

Chlorophyll a and b determination

Chlorophyll a and b content was quantified using the method described by Kurade and coauthors [⁶⁰] with adaptations. A 10 mL aliquot of the algal culture was centrifuged at 3,075 g (HERMLE Z 326 K) for 30 minutes. After centrifugation, the supernatant was discarded and 10 mL of 80% methanol was added to resuspend the precipitate, which was then heated in a water bath at 60 ℃ for 10 minutes, and centrifuged again at 3,075 g for 10 minutes. At the end of this process, the absorbance of the supernatant was analyzed at wavelengths 665 and 652. The values obtained were used to calculate the chlorophyll a and b contents according to Wellburn [⁶¹] and Gomaa and coauthors [⁵⁰].

Biochemical biomarkers

To determine the activity of antioxidant enzymes, at the end of 72 hours of exposure of microalgae to the drugs, a 50 mL aliquot of the culture was collected and centrifuged at 3,075 g (HERMLE Z 326 K) for 30 minutes. The precipitate formed at the bottom of the tube was stored in 200 µL of sodium phosphate buffer (50 mM, pH 7.5) at - 60 ℃.

Subsequently, the cells were disrupted by homogenization with glass beads (2 mm) [⁶²] The process was carried out using a vortex (KASVI K45-2820), where the sample remained for 30 minutes, in a cooled environment (4 ℃). The material obtained was aliquoted and stored at a temperature of - 60 ℃ until enzymatic analyzes were carried out. The supernatant of the samples was used for the analysis of the antioxidant enzymes catalase (CAT), glutathione-S-transferase (GST) as well as for the analysis of protein oxidative damage by quantifying the content of carbonyl groups and lipid peroxidation levels.

CAT activity was determined using the method proposed by Matsumura and coauthors [⁶³] 150 µL of the sample was added to 750 µL of sodium phosphate buffer (50mM, pH 7.5) and 100 µL of a hydrogen peroxide solution (200mM). The activity of the enzyme was estimated by monitoring the consumption of H2O2 by the enzyme at 240 nm (UV/VIS Spectrophotometer - SHIMADZU 1650 PC) for 60 seconds. GST activity was determined by the method of Habig and Pubst [⁶⁴], using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. 40 µL of the sample was added to 150 µL of potassium phosphate buffer (133 mM pH 6.5), followed by 20 µL of GSH (50 mM) diluted in sodium phosphate buffer (100 mM pH 7.6) and 10 µL of CDNB (20 mM) diluted in absolute ethanol. The production of CDNB-SG conjugate was monitored at 340 nm for 5 minutes in a microplate reader (FLUOstar Omega BMG LABTECH). Total protein content was determined using the Bradford method [⁶⁵] in all samples.

To access the status of oxidative damage to proteins in the algae, protein carbonyl (PC) levels were analyzed according to the method proposed by Levine and coauthors [⁶⁶], with adaptations. 200 µL of the sample was added to reaction medium containing 200 µL 2,4-dinitrophenylhydrazine (DNPH) (10 mM) diluted in hydrochloric acid (2M) and kept in a water bath for 1 hour, shaking the samples every 15 minutes. 280 µL of 28% trichloroacetic acid were added to the mixture, which was subjected to centrifugation (10,000 g for 10 min). The precipitate formed was resuspended in 250 µL of sodium dodecyl sulfate (6%) and centrifuged again (10,000 g for 10 min). The absorbance of the supernatant was read at 370 nm in a microplate reader. Results are expressed in nmol.mg protein-1.

To determine lipid peroxidation levels, another 30 mL aliquot of the culture was centrifuged at 3,075 g for 30 min. The precipitate was stored in sodium phosphate buffer (50 mM, pH 7.5) at -60 ℃ until analysis. Lipid peroxidation was estimated by measuring malondialdehyde (MDA) levels according to the method proposed by Almeida and coauthors [⁶⁷], with modifications. The sample was shaken for 30 minutes with glass beads (2 mm) in a vortex (KASVI K45-2820) in a cooled environment (4 ℃). Subsequently, 300 μL of 0.4% thiobarbituric acid (TBA, Sigma-Aldrich, Germany) diluted in HCl (0.2 M) were added to 300 μL of the homogenized sample. The mixture was heated to 90 ℃ for 40 min, and subjected to extraction with 500 μL of n-butanol (Sigma-Aldrich, USA) followed by centrifugation (5,000 g for 3 min). The supernatant was read at 540 nm on a microplate reader (FLUOstar Omega BMG LABTECH). MDA was quantified based in a tetramethoxypropane standard of curve prepared using the same procedure as for samples. Results are expressed in nmol.mg protein-1.

Statistical analyses

The data obtained in pigment quantification and biochemical analyzes were tested for normality using the Shapiro-Wilk test and for homogeneity using the Bartlett test. Differences between treatment groups and solvent control were statistically analyzed with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. In the case of non-parametric data, the Kruskal-Wallis test was used followed by the Dunn test. GraphPad Prism® software (version 8.0, GraphPad Software, San Diego, CA, USA) was used for all statistical analyses. This software was also used to determine the EC50(72h) (concentrations of the substance in which there was 50% inhibition of growth rate) for concentration-dependent relationships, using a non-linear regression curve fitting model. All tests mentioned had a significance level set at p<0.05.)

RESULTS

Effects of environmentally relevant concentrations of the COVID kit drugs on algae growth

Both IVM and HCQ did not affect D. subspicatus growth in none of the concentrations tested. In contrary, the highest concentration of AZM and MIX caused a significant inhibition (35.3 and 49.8%, respectively) of algae growth (Figure 1).

Figure 1
Number of D. subspicatus cells in 1 mL of culture medium after 72 h of exposure to the drugs IVM, AZM, HCQ and MIX, at concentrations of 0 (C - control) 1, 5, 10, 50 and 100 µg.L-1. Values are represented as the mean ± standard deviation. Different letters indicate significant difference (p<0.05) using the Tukey test.

Effects of HCQ, AZM and IVM on algae growth and determination of EC50 (72h) values.

The estimated EC50 (72h) values of HCQ and AZM were 510 and 80 μg.L^-1, respectively. Due to its low solubility at high concentrations, it was not possible to estimate the exact value of EC50 (72h) for IVM, which was higher than 800 μg.L^-1.

Chlorophyl a and b levels

Levels of chlorophyl a and b did not change in none of cultures treated with drugs in isolation in comparison to the control group (Figure 2). However, cultures exposed to the highest AZM concentration presented lower levels of both chlorophyl than the cultures exposed to 5 and 10 µg.L^-1 of same compound.

Figure 2
Chlorophyll a and chlorophyll b pigment content of D. subspicatus after 72h of exposure to the drugs IVM, AZM, HCQ and MIX, at concentrations of 0 (C - control) 1, 5, 10, 50 and 100 µg.L^-1. Values are represented as the mean ± standard deviation. Different letters indicate significant difference (p<0.05) using the Tukey (A, B, C, D, E, F, G) or Dunn (H) test.

Biochemical markers

The microalgae cultures that were exposed to IVM (Figure 3A) and to the mix (Figure 3D) did not show significant differences in CAT activity. Exposure to 50 and 100 µg.L^-1 AZM resulted in significant lower CAT activity in comparison to the groups that were exposed to 1 and 5 µg.L-1 (Figure 3B). Moreover, CAT activity was higher than control in algae exposed to 5, 10 and 100 µg.L^-1 of HCQ (Figure 3C). GST activity was not significantly altered by none of the treatments, when compared to control values. However, algae cultures that were exposed to the highest concentration of the MIX showed lower GST activity than those exposed to 5 of the MIX.

Figure 3
Activity of CAT and GST enzymes of D. subspicatus after 72h of exposure to the drugs IVM, AZM, HCQ and MIX, at concentrations of 0 (C - control) 1, 5, 10, 50 and 100 µg.L^-1. Values are represented as the mean ± standard deviation. Different letters indicate significant difference (p<0.05) using the Tukey (A, B, C, F, G, H) or Dunn (D, E) test.

MDA levels did not show significant differences between any of the treatments (Figure 4). With regard to the levels of protein carbonyls (PC), no differences were observed when comparing the control group with the cultures exposed to the drugs. However, D. subspicatus culture exposed to the highest concentration of the mix presented higher PC levels than the culture at 50 µg.L^-1.

Figure 4
Levels of MDA and carbonyl proteins of D. subspicatus after 72h of exposure to the drugs IVM, AZM, HCQ and MIX, at concentrations of 0 (C - control) 1, 5, 10, 50 and 100 µg.L-1. Values are represented as the mean ± standard deviation. Different letters indicate significant difference (p<0.05) using the Tukey (A, B, C, E, H) or Dunn (D, F, G) test.

DISCUSSION

Toxicity tests

IVM did not exert substantial toxicity to D. subspicatus, showing no growth inhibition at tested concentrations. This result agrees with Martins [⁹⁷], which also did not observe any effects of IVM on D. subspicatus growth. The EC50 test also predicted concentrations greater than 800 µg.L^-1 to cause any effect on growth. These results agree with EC50 values found by Garric and coauthors [⁴⁵] for the microalgae P. subcapitata, which was greater than 4000 µg.L^-1. For abamectin, a compound whose structure is very similar to IVM, Tišler and Eržen [⁶⁸] found an EC50 of 4400 µg.L^-1 (72 h) for the microalgae S. subspicatus, in 72 hours tests, while a value of 153,730 µg.L-1 was estimated by [³⁶] for Chlorella vulgaris. Similarly, Kołodziejska and coauthors [⁹⁸] also reported very low toxicity of doramectin, another antiparasitic drug, for the green algae Scenedesmus vacuolatus. For Daphnia sp. the EC50 concentrations of IVM (5.7 ng.L^-1; [⁴⁵]) or abamectin (5.54 µg.L-1; [⁴⁸]) were significantly lower during 48 hours of exposure, evidencing the lower IVM toxicity for microalgae, including D. subspicatus. The greater toxicity reported for microcrustaceans is probably due to the mechanism of action of IVM, as it interacts with chlorine channels allowing a greater influx of the ion and culminating in nervous hyperpolarization [⁶⁹].

AZM proved to be considerably more toxic to D. subspicatus than IVM, decreasing algae growth with an EC50 of 80 µg.L^-1. Similarly, Mao and coauthors [⁷⁰] reported an EC50 (96 h) of 41.6 µg.L^-1, for the microalgae C. pyrenoidosa, while Almeida and coauthors [⁷¹] found an EC50 (72 h) of 51 µg.L^-1 for Raphidocelis subcapitata. Guo and coauthors [⁹⁶], also showed that the macrolide antibiotic clarithromycin was able to inhibit the growth of algae Raphidocelis subcapitata and C. vulgaris, at µg.L^-1 concentrations. When comparing the toxicity of 13 different antibiotics to the freshwater green algae Pseudokirchneriella subcapitata, AZM presented the highest toxicity [¹⁰⁰]. For other organisms, such as Daphnia sp., AZM is often less toxic, having EC50 values ranging from 100 to 150 mg.L^-1. [⁷², ⁴⁶, ⁷³]. For V. fischeri, a considerably lower EC50 was reported, when compared to D. subspicatus (30.26 x 10-5 mol.L^-1; [⁷⁴], demonstrating greater resistance of the microalgae to this antibiotic. In contrast, Harada and coauthors [¹⁰³] noted a higher toxicity of AZM to algae than to bacteria. The action mechanism of AZM consists on the inhibition of bacterial protein synthesis through its binding to the 50S ribosomal subunit, acting as a bacteriostatic agent, inhibiting bacterial growth [⁷⁵, ⁷⁶]. AZM was also shown to weaken the photosynthetic activities of algae, to reduce the energy reserves and to alter the cellular structure [⁷⁰]. Other macrolide antibiotics, such as erythromycin, roxithromycin, have already had reported toxic effects on microalgae, such as genotoxicity, interfering with DNA repair genes [⁷⁷], and inhibiting the chloroplast gene translation process, which is involved in the synthesis of several proteins important for photosynthesis [⁷⁸]. These mechanisms may explain the azithromycin toxicity observed in D. subspicatus. It is important to highlight that AZM is often found in anthropized aquatic environments at concentrations higher than predicted no-effect concentration for microalgae [⁹⁹, ¹⁰¹, ¹⁰²], especially after the COVID19 pandemic.

HCQ presented an EC50 of 510 µg.L^-1 for D. subspicatus, which was much lower than values found for the microalgae C. vulgaris by Marques and coauthors [³⁶] (200,160 µg.L^-1; 72 h). These authors also tested HCQ toxicity for the cyanobacterium S. elongatus, finding an EC50 (72 h) of 6.71 µg.L^-1, evidencing the higher sensitivity of the bacteria to HCQ. For the analogous compound chloroquine, Zurita and coauthors [²⁸] also found relative high values of EC50 (48 h) for the microalgae C. vulgaris (27 mg.L^-1), for D. magna and (9 mg.L^-1), and for V. fischeri (126 mg.L^-1). Comparatively, our results demonstrate the great sensitivity of D. subspicatus to HCQ. The mechanism of action of HCQ is broad and dependent on the context associated with its use. The drug mainly interferes with lysosomes, increasing the pH inside them and inhibiting the action of their enzymes [⁷⁹]. The alkalinization effect has also been observed inside cells, in acidic organelles, limiting the uptake of iron and interfering with infection by intracellular microorganisms, such as bacteria and fungi [⁸⁰], which may be related to its toxicity to microalgae [³⁶]. Furthermore, there is evidence of a strong interaction between HCQ and DNA [⁸¹].

Regarding the mixture with the three drugs, there was considerable growth inhibition in cultures exposed to the highest concentration. It is possible that this inhibition is related only to the effect of AZM on microalgae, since the EC50 found in this study for the drug was 80 µg.L^-1. However, any potential synergistic effects of the other compounds in the mixture should not be disregarded. Agreeing with this, Marques and coauthors [³⁶] noted interactive effect of mixtures of IVM, AZM and HCQ on growth, photosynthesis and antioxidant activity of C. vulgaris. Similarly, Arreguin-Rebolledo and coauthors [¹⁰⁸] also reported important synergistic toxic effects of AZM and IVM mixture in the freshwater rotifer Lecane papuana.

Effects on biochemical parameters

With regard to biochemical analyses, only the activity of CAT in cultures exposed to HCQ showed an increase at concentrations of 5, 10 and 100 µg.L^-1, in comparison to the control group. This effect may indicate a microalgae response to increased cellular production of H₂O₂ due to intoxication [⁸². ⁸³]. The absence of changes in biomarkers of cellular injury (MDA and PC) may indicate success in neutralizing these reactive species by catalase, preventing protein carbonylation and lipid peroxidation [⁸⁴]. The lack of CAT increase at the lowest concentration (1 µg.L^-1) is consistent with results found by Marques and coauthors [³⁶], in which no change in CAT activity was observed in C. vulgaris after exposure to 2 µg.L^-1 of HCQ. However, the lack of similar CAT increase in microalgae exposed to the drug mixture is intriguing, since the mixtures contained the same HCQ concentrations. Possible antagonistic effects caused by the other chemicals in the mixture could explain this result. It should be noted that despite AZM exposure did not affect CAT activity, this enzyme was significantly lower in cultures exposed to 50 and 100 µg.L^-1, in comparison to those exposed to 1 and 5 µg.L^-1. A possible inhibitory effect of AZM on CAT would be then masking or suppressing the HCQ induction effect. Although most studies regarding the effects of antibiotics such as clarithromycin [⁷⁷, ⁹⁶] ciprofloxacin, tetracycline and amoxicillin [⁵⁰] on microalgae show an inducing effect on CAT, Marques and coauthors [³⁶] observed an inhibitory effect of AZM on CAT activity of C. vulgaris, which is consistent with our hypothesis.

Despite evidences that sulfonamide antibiotics are capable of inhibiting chlorophyll synthesis by interfering with gene expression of the chloroplasts [⁸⁵], none of the drugs altered the content of chlorophyll a and b. This finding also contrasts with the lower chlorophyll a and b content in C. pyrenoidosa after AZM exposure [⁷⁰], suggesting D. subspicatus is more resistant to this AZM effect. To the best of our knowledge, studies regarding the effects of IVM and HCQ on algae chlorophyll levels are absent in the scientific literature. Nevertheless, Sanderson and coauthors [⁸⁶] showed that IVM added to outdoor aquatic mesocosms didn’t affect chlorophyll a level.

Levels of cellular injury biomarkers (MDA and PC) and GST activity were also not affected by the drugs. Contrasting to our results, AZM exposure increased ROS generation and MDA levels in C. pyrenoidosa and R. subcapitata [⁷⁰, ⁷¹], also suggest that D. subspicatus is more resistant to toxic AZM effects in comparison to other species. Studies on the effects of IVM and HCQ on PC levels and GST activity in algae are absent in the scientific literature.

Discrepancies in responses to contaminants between different algae species are possibly due to their capacity of metabolize different chemicals. Solovchenko and coauthors [⁸⁷] reported that Desmodesmus sp. displays a relatively high tolerance to commercial formulations of pharmaceutical products (azithromycin, ibuprofen, ceftriaxone, diclofenac, amoxicillin + clavulanate potassium). Concentrations of 3, 300 and 1000 µg.L^-1, of practically all tested contaminants, had a stimulating effect on the production of chlorophyll and carotenoids in microalgae, and only azithromycin at the highest concentration, significantly reduced the pigment content. According to the author, these results were possible due to the ability of certain species of microalgae to use pharmaceutical compounds as a carbon source.

Another explanation for the resistance of D. subspicatus to contaminants is the structure of the cell wall. This microalgae species has a fourth layer of sporopollenin [⁸⁸, ⁸⁹, ⁹⁰], a biopolymer that provides resistance to the cell wall, protecting the organism from environmental stress, desiccation and UV radiation [⁹¹, ⁹²]. Thus, there is the possibility that this additional layer provides greater resistance to the toxicity of chemical compounds to the species, compared to other species. The presence of sporopollenin in the cell walls of different microalgae species is varied. The subgenus Scenedesmus has three layers of the biopolymer in its structure [⁸⁸], one layer less than D. subspicatus. The presence of sporopollenin in the cell wall of C. vulgaris is still debated [⁹³]. It is suggested that it is present in a layer, distributed in unknown quantities [⁹⁴]. Species such as C. fusca and C. sorokiniana do not have the biopolymer in their structure [⁹⁵]. Therefore, we hypothesize that the structure and thickness of the cell wall would be an important factor affecting pollutant toxic effects to algae, and further studies on this aspect are important for clarification.

CONCLUSION

Given the results presented, it was possible to establish the EC50 for the drugs IVM, AZM and HCQ for D. subspicatus, a microalgae species commonly used in standardized ecotoxicological tests. In general, IVM and HCQ were less toxic than AZM, as evidenced by the lower EC50 value estimated for AZM and the significant growth inhibition of algae culture at the highest AZM and MIX concentrations. However, effects at subcellular levels showed an increase of CAT in algae exposed to HCQ, which would represent a higher stressful cellular condition due to increased production of H₂O₂. According to European Regulation, substances that have an EC50, for microalgae, lower than 1 mg.L^-1 are considered very toxic to the aquatic environment, therefore, both AZM and HCQ fit in this classification. Taking together, our results points to potential negative effects of the COVID-19 kit to freshwater algae, especially if considering that the effects reported here occurred in short-term exposure experiments (72 h). More intense effects could be predicted if prolonged exposure periods are considered, which would imply in important ecological negative impacts.

• Funding:
  This work has the financial support of the “Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC, 1594/2021). E.A. Almeida is a recipient of productivity fellowship by CNPq (313503/2021-4)..

Acknowledgments:

None

Data Availability Statement:

Research data are only available upon request for corresponding author

REFERENCES

• ¹ Akhtar N, Syakir Ishak MI, Bhawani SA, Umar K. Various natural and anthropogenic factors responsible for water quality degradation: A Review. Water [Internet]. 2021;13(19):2660. Available from: https://www.mdpi.com/2073-4441/13/19/2660/htm
  » https://www.mdpi.com/2073-4441/13/19/2660/htm
• ² Peña-Guzmán C, Ulloa-Sánchez S, Mora K, Helena-Bustos R, Lopez-Barrera E, Alvarez J, et al. Emerging pollutants in the urban water cycle in Latin America: A review of the current literature. J Environ Manage [Internet]. 2019;237:408-23. Available from: http://dx.doi.org/10.1016/j.jenvman.2019.02.100
  » http://dx.doi.org/10.1016/j.jenvman.2019.02.100
• ³ Pereira A, Silva L, Laranjeiro C, Lino C, Pena A. Selected pharmaceuticals in different aquatic compartments: Part I-source, fate and occurrence. Molecules [Internet]. 2020;25(5):1026. Available from: http://dx.doi.org/10.3390/molecules25051026
  » http://dx.doi.org/10.3390/molecules25051026
• ⁴ Conselho Federal de Farmácia - “Survey shows how fear of Covid-19 impacted drug sales” [Internet]. site.cff.org.br. Available from: https://site.cff.org.br/
  » https://site.cff.org.br/
• ⁵ Danza Á, Graña D, Goñi M, Vargas A, Ruiz-Irastorza G. “Hydroxychloroquine in the treatment of systemic autoimmune diseases”. Rev Med Chil [Internet]. 2016;144(2):232-40. Available from: http://dx.doi.org/10.4067/s0034-98872016000200012
  » http://dx.doi.org/10.4067/s0034-98872016000200012
• ⁶ Ashour DS. Ivermectin: From theory to clinical application. Int J Antimicrob Agents [Internet]. 2019;54(2):134-42. Available from: http://dx.doi.org/10.1016/j.ijantimicag.2019.05.003
  » http://dx.doi.org/10.1016/j.ijantimicag.2019.05.003
• ⁷ Bakheit AHH, Al-Hadiya BMH, Abd-Elgalil AA. Azithromycin. Profiles of drug substances, excipients, and related methodology. 2014;39:1-40. Available from: https://doi.org/10.1016/B978-0-12-800173-8.00001-5
  » https://doi.org/10.1016/B978-0-12-800173-8.00001-5
• ⁸ Bansal P, Goyal A, Cusick A IV, Lahan S, Dhaliwal HS, Bhyan P, et al. Hydroxychloroquine: a comprehensive review and its controversial role in coronavirus disease 2019. Ann Med [Internet]. 2021;53(1):117-34. Available from: http://dx.doi.org/10.1080/07853890.2020.1839959
  » http://dx.doi.org/10.1080/07853890.2020.1839959
• ⁹ Skipper CP, Pastick KA, Engen NW, Bangdiwala AS, Abassi M, Lofgren SM, et al. Hydroxychloroquine in nonhospitalized adults with early COVID-19. Ann Int Med. 2020; ;173;623-631. Available from: https://doi.org/10.7326/M20-4207
  » https://doi.org/10.7326/M20-4207
• ¹⁰ Tang W, Cao Z, Han M, Wang Z, Chen J, Sun W, et al. Hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial. BMJ. 2020;m1849. Available from: https://doi.org/10.1136/bmj.m1849
  » https://doi.org/10.1136/bmj.m1849
• ¹¹ López-Medina E, López P, Hurtado IC, Dávalos DM, Ramirez O, Martínez E, et al. Effect of ivermectin on time to resolution of symptoms among adults with mild COVID-19. JAMA [Internet]. 2021;325(14). Available from: https://doi.org/10.1001/jama.2021.3071
  » https://doi.org/10.1001/jama.2021.3071
• ¹² Marcolino MS, Meira KC, Guimarães NS, Motta PP, Chagas VS, Kelles SMB, et al. Systematic review and meta-analysis of ivermectin for treatment of COVID-19: evidence beyond the hype. BMC Infec Dis. 2022;22(1):639. Available from: https://doi.org/10.1186/s12879-022-07589-8
  » https://doi.org/10.1186/s12879-022-07589-8
• ¹³ Yousafzai ADK, Bangash AH, Asghar SY, Abbas SMM, Khawaja HF, Zehra S, et al. Clinical efficacy of Azithromycin for COVID-19 management: A systematic meta-analysis of meta-analyses. Heart & Lung. 2023;60:127-132. Available from: https://doi.org/10.1016/j.hrtlng.2023.03.004
  » https://doi.org/10.1016/j.hrtlng.2023.03.004
• ¹⁴ Yuan Y, Jiao B, Qu L, Yang D, Liu R. The development of COVID-19 treatment. Front Immunol [Internet]. 2023 14:1125246. Available from: https://doi.org/10.3389/fimmu.2023.1125246
  » https://doi.org/10.3389/fimmu.2023.1125246
• ¹⁵ Tarazona JV, Martínez M, Martínez MA, Anadón A. Environmental impact assessment of COVID-19 therapeutic solutions. A prospective analysis. Sci Total Environ. 2021;778:146257. Available from: https://doi.org/10.1016/j.scitotenv.2021.146257
  » https://doi.org/10.1016/j.scitotenv.2021.146257
• ¹⁶ Abradilan - Associação Brasileira de Distribuição e logística de produtos farmacêuticos. “Covid-kit: sales of medicines in Brazil increased by 857%” Abradilan. URL https://www.abradilan.com.br/ (accessed 3.19.24).
  » https://www.abradilan.com.br/
• ¹⁷ Chen X, Lei L, Liu S, Han J, Li R, Men J, et al. Occurrence and risk assessment of pharmaceuticals and personal care products (PPCPs) against COVID-19 in lakes and WWTP-river-estuary system in Wuhan, China. Sci Total Environ. 2021;792:148352.Available from: https://doi.org/10.1016/j.scitotenv.2021.148352
  » https://doi.org/10.1016/j.scitotenv.2021.148352
• ¹⁸ Zhou LJ, Li J, Zhang Y, Kong L, Jin M, Yang X, et al. Trends in the occurrence and risk assessment of antibiotics in shallow lakes in the lower-middle reaches of the Yangtze River basin, China. Ecotoxicol Environmental Saf [Internet]. 2019;183:109511. Available from: https://doi.org/10.1016/j.ecoenv.2019.109511
  » https://doi.org/10.1016/j.ecoenv.2019.109511
• ¹⁹ Sharma L, Siedlewicz G, Pazdro K. The toxic effects of antibiotics on freshwater and marine photosynthetic microorganisms: state of the art. Plants [Internet]. 2021;10(3):591. Available from: https://doi.org/10.3390/plants10030591
  » https://doi.org/10.3390/plants10030591
• ²⁰ Morales-Paredes CA, Rodríguez-Díaz JM, Boluda-Botella N. Pharmaceutical compounds used in the COVID-19 pandemic: A review of their presence in water and treatment techniques for their elimination. Scie Total Environ [Internet]. 2022;814:152691. Available from: https://doi.org/10.1016/j.scitotenv.2021.152691
  » https://doi.org/10.1016/j.scitotenv.2021.152691
• ²¹ Rodriguez-Gil JL, San Sebastián Sauto J, González-Alonso S, Sánchez Sanchez P, Valcarcel Y, Catalá M. Development of cost-effective strategies for environmental monitoring of irrigated areas in Mediterranean regions: Traditional and new approaches in a changing world. Agric Ecosyst Environ. 2013;181:41-9. Available from: https://doi.org/10.1016/j.agee.2013.09.007
  » https://doi.org/10.1016/j.agee.2013.09.007
• ²² Pol Dominguez-García, Reinerio Rolando Rodríguez, Barata C, Cristian Gómez-Canela. Presence and toxicity of drugs used to treat SARS-CoV-2 in Llobregat River, Catalonia, Spain. Environ Sci Pollut Res. 2023;30(17):49487-97. Available from: https://doi.org/10.1007/s11356-023-25512-9
  » https://doi.org/10.1007/s11356-023-25512-9
• ²³ Durán-Álvarez JC, Prado B, Zanella R, Rodríguez M, Suhaila Díaz. Wastewater surveillance of pharmaceuticals during the COVID-19 pandemic in Mexico City and the Mezquital Valley: A comprehensive environmental risk assessment. Sci Total Environ. 2023;900:165886-6. Available from: https://doi.org/10.1016/j.scitotenv.2023.165886
  » https://doi.org/10.1016/j.scitotenv.2023.165886
• ²⁴ Farias DF, Souza T, Souza JACR, Vieira LR, Muniz MS, Martins RX, et al. COVID-19 Therapies in Brazil: should we be concerned with the impacts on aquatic wildlife? Environ Toxicol Chem. 2020;39(12):2348-50. Available from: https://doi.org/10.1002/etc.4888
  » https://doi.org/10.1002/etc.4888
• ²⁵ da Luz TM, Araújo AP da C, Estrela FN, Braz HLB, Jorge RJB, Charlie-Silva I, et al. Can use of hydroxychloroquine and azithromycin as a treatment of COVID-19 affect aquatic wildlife? A study conducted with neotropical tadpole. Sci Total Environ [Internet]. 2021;780(146553):146553. Available from: http://dx.doi.org/10.1016/j.scitotenv.2021.146553
  » http://dx.doi.org/10.1016/j.scitotenv.2021.146553
• ²⁶ Macphee C, Ruelle R. Lethal effects of 1888 chemicals upon four species of fish from western North America. Research technical compeltion report, project A-013-IDA [Internet]. Idaho Waters Digital Library. 1969 [cited 2024 Jul 30]. Available from: https://www.lib.uidaho.edu/digital/iwdl/items/iwdl-196913.html
  » https://www.lib.uidaho.edu/digital/iwdl/items/iwdl-196913.html
• ²⁷ Tojo JL, Santamarina MT. Oral pharmacological treatments for parasitic diseases of rainbow trout Oncorhynchus mykiss II:Gyrodactylus sp. Dis Aquat Organ [Internet]. 1998 [cited 2024;33(3):187-93. Available from: https://www.int-res.com/abstracts/dao/v33/n3/p187-193/
  » https://www.int-res.com/abstracts/dao/v33/n3/p187-193/
• ²⁸ Zurita JL, Jos Á, Peso A del, Salguero M, López-Artíguez M, Repetto G. Ecotoxicological evaluation of the antimalarial drug chloroquine. Aquat Toxicol. 2005t;75(2):97-107. Available from: https://doi.org/10.1016/j.aquatox.2005.07.009
  » https://doi.org/10.1016/j.aquatox.2005.07.009
• ²⁹ Ramesh M, Anitha S, Poopal RK, Shobana C. Evaluation of acute and sublethal effects of chloroquine (C18H26CIN3) on certain enzymological and histopathological biomarker responses of a freshwater fish Cyprinus carpio Toxicol Rep. 2018;5:18-27. Available from: https://doi.org/10.1016/j.toxrep.2017.11.006
  » https://doi.org/10.1016/j.toxrep.2017.11.006
• ³⁰ Davis SN, Wu P, Camci ED, Simon JA, Rubel EW, Raible DW. Chloroquine kills hair cells in zebrafish lateral line and murine cochlear cultures: Implications for ototoxicity. Heari Res. 2020;395:108019. Available from: https://doi.org/10.1016/j.heares.2020.108019
  » https://doi.org/10.1016/j.heares.2020.108019
• ³¹ Rendal C, Kusk KO, Trapp S. The effect of pH on the uptake and toxicity of the bivalent weak base chloroquine tested on Salix viminalis and Daphnia magna Environ Toxicol Chem [Internet]. 2011;30(2):354-9. Available from: http://dx.doi.org/10.1002/etc.391
  » http://dx.doi.org/10.1002/etc.391
• ³² Moore MN, Viarengo A, Donkin P, Hawkins AJS. Autophagic and lysosomal reactions to stress in the hepatopancreas of blue mussels. Aquat Toxicol. 2007;84(1):80-91. Available from: https://doi.org/10.1016/j.aquatox.2007.06.007
  » https://doi.org/10.1016/j.aquatox.2007.06.007
• ³³ Mendonça-Gomes JM, da Costa Araújo AP, da Luz TM, Charlie-Silva I, Braz HLB, Jorge RJB, et al. Environmental impacts of COVID-19 treatment: Toxicological evaluation of azithromycin and hydroxychloroquine in adult zebrafish. Sci Total Environ. 2021;790:148129. Available from: https://doi.org/10.1016/j.scitotenv.2021.148129
  » https://doi.org/10.1016/j.scitotenv.2021.148129
• ³⁴ Luo M, Xie D, Lin Z, Sun H, Liu Y. Toxicology evaluation of overdose hydroxychloroquine on zebrafish (Danio rerio) embryos. Sci Rep. 2022;12(1): 18259. Available from: https://doi.org/10.1038/s41598-022-23187-9
  » https://doi.org/10.1038/s41598-022-23187-9
• ³⁵ Silva IF, Enes KP, Rocha GM, Varotti FP, Barbosa LA, Thomé RG, et al. Toxicological effects of hydroxychloroquine sulfate and chloroquine diphosphate substances on the early-life stages of fish in the COVID-19 pandemic context. J Environ Sci Health A Tox Hazard Subst Environ Eng [Internet]. 2023;58(10):825-30. Available from: http://dx.doi.org/10.1080/10934529.2023.2238587
  » http://dx.doi.org/10.1080/10934529.2023.2238587
• ³⁶ Marques RZ, Kochi LY, Guzman-Tordecilla M, Malage L, Juneau P, Gomes MP. COVID-19 pharmaceuticals in aquatic matrices: The threatening effects over cyanobacteria and microalgae. Sci Total Environ [Internet]. 2023;892(164309):164309. Available from: http://dx.doi.org/10.1016/j.scitotenv.2023.164309
  » http://dx.doi.org/10.1016/j.scitotenv.2023.164309
• ³⁷ Ben Ali M, Hedfi A, Almalki M, Karachle PK, Boufahja F. Toxicity of hydroxychloroquine, a potential treatment for COVID-19, on free-living marine nematodes. Mar Pollut Bull [Internet]. 2021;167(112361):112361. Available from: http://dx.doi.org/10.1016/j.marpolbul.2021.112361
  » http://dx.doi.org/10.1016/j.marpolbul.2021.112361
• ³⁸ Thiripurasundari M, Sathya K, Uma A, Srinivasan M, Rajasekar P. A comparative study on the toxicity of ivermectin in zebra fish and catla fish models. Indo Am J Pharm Res [Internet]. 2014:4(9):3683-3688; Available from: https://www.semanticscholar.org/paper/004f608ec3aaac84b3236e9f48ff2754e58fb836
  » https://www.semanticscholar.org/paper/004f608ec3aaac84b3236e9f48ff2754e58fb836
• ³⁹ Domingues I, Oliveira R, Soares AMVM, Amorim MJB. Effects of ivermectin on Danio rerio: a multiple endpoint approach: behaviour, weight and subcellular markers. Ecotoxicol [Internet]. 2016;25(3):491-9. Available from: http://dx.doi.org/10.1007/s10646-015-1607-5
  » http://dx.doi.org/10.1007/s10646-015-1607-5
• ⁴⁰ Oliveira R, Grisolia CK, Monteiro MS, Soares AMVM, Domingues I. Multilevel assessment of ivermectin effects using different zebrafish life stages. Comp Biochem Physiol C Toxicol Pharmacol [Internet]. 2016;187:50-61. Available from: http://dx.doi.org/10.1016/j.cbpc.2016.04.004
  » http://dx.doi.org/10.1016/j.cbpc.2016.04.004
• ⁴¹ Azevedo VC, Kennedy CJ. P-glycoprotein inhibition affects ivermectin-induced behavioural alterations in fed and fasted zebrafish (Danio rerio). Fish Physiol Biochem. 2022;48(5):1267-83. Available from: https://doi.org/10.1007/s10695-022-01111-2
  » https://doi.org/10.1007/s10695-022-01111-2
• ⁴² Di Paola D, Iaria C, Marino F, Enrico Gugliandolo, Piras C, Crupi R, et al. Environmental impact of pharmaceutical pollutants: synergistic toxicity of ivermectin and cypermethrin. Toxics. 2022;10(7):388-8. Available from: https://doi.org/10.3390/toxics10070388
  » https://doi.org/10.3390/toxics10070388
• ⁴³ Ogueji E, Nwani C, Mbah C, Iheanacho S, Nweke F. Oxidative stress, biochemical, lipid peroxidation, and antioxidant responses in Clarias gariepinus exposed to acute concentrations of ivermectin. Environ Sci Pollut Res. 2020;27(14):16806-15. Available from: https://doi.org/10.1007/s11356-019-07035-4
  » https://doi.org/10.1007/s11356-019-07035-4
• ⁴⁴ Ogueji EO, Nwani CD, Mbah CE, Nweke FN. Acute hematological toxicity of ivermectin to juvenile Clarias gariepinus Toxicol Environ Chem. 2019;101(3-6):300-14. Available from: https://doi.org/10.1080/02772248.2019.1691554
  » https://doi.org/10.1080/02772248.2019.1691554
• ⁴⁵ Garric J, Vollat B, Duis K, Péry A, Junker T, Ramil M, et al. Effects of the parasiticide ivermectin on the cladoceran Daphnia magna and the green alga Pseudokirchneriella subcapitata. Chemosphere. 2007;69(6):903-10. Available form: https://doi.org/10.1016/j.chemosphere.2007.05.070
  » https://doi.org/10.1016/j.chemosphere.2007.05.070
• ⁴⁶ Li Y, Ma Y, Yang L, Duan S, Zhou F, Chen J, et al. Effects of azithromycin on feeding behavior and nutrition accumulation of Daphnia magna under the different exposure pathways. Ecotoxicol Environ Saf. 2020;197:110573-3. Available from: https://doi.org/10.1016/j.ecoenv.2020.110573
  » https://doi.org/10.1016/j.ecoenv.2020.110573
• ⁴⁷ Sands B, Noll M. Toxicity of ivermectin residues in aged farmyard manure to terrestrial and freshwater invertebrates. Insect Conserv Divers [Internet]. 2022;15(1):9-18. Available from: http://dx.doi.org/10.1111/icad.12526
  » http://dx.doi.org/10.1111/icad.12526
• ⁴⁸ Braun AS, Novelli A, Vieira BH, Espindola G. Ecotoxicological effects of Vertimec® 18EC on plankton. Ecotoxicol Environ Contam. 2012;7(2):23-9. Available from: https://doi.org/10.5132/jbse.2012.02.004
  » https://doi.org/10.5132/jbse.2012.02.004
• ⁴⁹ Karuppasamy S, Musale AS, Soni B, Bhadra B, Gujarathi N, Sundaram M, et al. Integrated grazer management mediated by chemicals for sustainable cultivation of algae in open ponds. Algal Res. 2018;35:439-48. Available from: https://doi.org/10.1016/j.algal.2018.09.017
  » https://doi.org/10.1016/j.algal.2018.09.017
• ⁵⁰ Gomaa M, Zien-Elabdeen A, Hifney AF, Adam MS. Phycotoxicity of antibiotics and non-steroidal anti-inflammatory drugs to green algae Chlorella sp. and Desmodesmus spinosus: Assessment of combined toxicity by Box-Behnken experimental design. Environ Technol Innov. 2021;23:101586. Available from: https://doi.org/10.1016/j.eti.2021.101586
  » https://doi.org/10.1016/j.eti.2021.101586
• ⁵¹ de Vasconcelos EC, Dalke CR, de Oliveira CMR. Influence of select antibiotics on Vibrio fischeri and Desmodesmus subspicatus at μg L^-1 concentrations. Environ Manage [Internet]. 2017;60(1):157-64. Available from: http://dx.doi.org/10.1007/s00267-017-0841-4
  » http://dx.doi.org/10.1007/s00267-017-0841-4
• ⁵² Doležalová Weissmannová H, Pavlovský J, Fišerová L, Kosárová H. Toxicity of diclofenac: cadmium binary Mixtures to Algae Desmodesmus subspicatus Using Normalization Method. Bulletin of Environmental contamination and toxicology. 2018;101(2):205-13. Available from: https://doi.org/10.1007/s00128-018-2384-7
  » https://doi.org/10.1007/s00128-018-2384-7
• ⁵³ Bai X, Acharya K. Algae-mediated removal of selected pharmaceutical and personal care products (PPCPs) from Lake Mead water. Sci Total Environ. 2017;581-582:734-40. Available from: https://doi.org/10.1016/j.scitotenv.2016.12.192
  » https://doi.org/10.1016/j.scitotenv.2016.12.192
• ⁵⁴ ABNT, 2018. Associação Brasileira de Normas Técnicas. NBR 12648 - “Aquatic Ecotoxicology: Chronic toxicity - Algae test method”. (Chlorophyceae).
• ⁵⁵ Gwenzi W, Selvasembian R, Offiong NAO, Mahmoud AED, Sanganyado E, Mal J. COVID-19 drugs in aquatic systems: a review. Environ Chem Let. 202215;20(2):1275-94. Available from: https://doi.org/10.1007/s10311-021-01356-y
  » https://doi.org/10.1007/s10311-021-01356-y
• ⁵⁶ Cappelli F, Longoni O, Rigato J, Rusconi M, Sala A, Fochi I, et al. Suspect screening of wastewaters to trace anti-COVID-19 drugs: Potential adverse effects on aquatic environment. Sci Total Environ. 2022;824:153756. Available from: https://doi.org/10.1016/j.scitotenv.2022.153756
  » https://doi.org/10.1016/j.scitotenv.2022.153756
• ⁵⁷ Fatoki OS, Opeolu BO, Genthe B, Olatunji OS. Multi-residue method for the determination of selected veterinary pharmaceutical residues in surface water around Livestock Agricultural farms. Heliyon. 2018;4(12):e01066. Available from: https://doi.org/10.1016/j.heliyon.2018.e01066
  » https://doi.org/10.1016/j.heliyon.2018.e01066
• ⁵⁸ Bielen A, Šimatović A, Kosić-Vukšić J, Senta I, Ahel M, Babić S, et al. Negative environmental impacts of antibiotic-contaminated effluents from pharmaceutical industries. Wat Res. 2017;126:79-87. Available from: https://doi.org/10.1016/j.watres.2017.09.019
  » https://doi.org/10.1016/j.watres.2017.09.019
• ⁵⁹ Bhandari A, Close LI, Kim W, Hunter RP, Koch DE, Surampalli RY. Occurrence of ciprofloxacin, sulfamethoxazole, and azithromycin in municipal wastewater treatment plants. practice periodical of hazardous, toxic, and radioactive waste management. 2008;12(4):275-81. Available from: https://doi.org/10.1061/(ASCE)1090-025X(2008)12:4(275)
  » https://doi.org/10.1061/(ASCE)1090-025X(2008)12:4(275
• ⁶⁰ Kurade MB, Kim JR, Govindwar SP, Jeon BH. Insights into microalgae mediated biodegradation of diazinon by Chlorella vulgaris: microalgal tolerance to xenobiotic pollutants and metabolism. Algal Res. 2016;20:126-34. Available from: https://doi.org/10.1016/j.algal.2016.10.003
  » https://doi.org/10.1016/j.algal.2016.10.003
• ⁶¹ Wellburn AR. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol. 1994;144(3):307-13. Available from: https://doi.org/10.1016/S0176-1617(11)81192-2
  » https://doi.org/10.1016/S0176-1617(11)81192-2
• ⁶² Melegari SP, Perreault F, Costa RHR, Popovic R, Matias WG. Evaluation of toxicity and oxidative stress induced by copper oxide nanoparticles in the green alga Chlamydomonas reinhardtii Aquat Toxicol. 2013;142-143:431-40. Available from: https://doi.org/10.1016/j.aquatox.2013.09.015
  » https://doi.org/10.1016/j.aquatox.2013.09.015
• ⁶³ Matsumura T, Tabayashi N, Kamagata Y, Souma C, Saruyama H. Wheat catalase expressed in transgenic rice can improve tolerance against low temperature stress. Physiol Plant. 2002;116(3):317-27. Available from: https://doi.org/10.1034/j.1399-3054.2002.1160306.x
  » https://doi.org/10.1034/j.1399-3054.2002.1160306.x
• ⁶⁴ Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem [Internet]. 1974;249(22):7130-9. Available from: https://doi.org/10.1016/S0021-9258(19)42083-8
  » https://doi.org/10.1016/S0021-9258(19)42083-8
• ⁶⁵ Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem [Internet]. 1976;72(1-2):248-54. Available from: https://doi.org/10.1016/0003-2697(76)90527-3
  » https://doi.org/10.1016/0003-2697(76)90527-3
• ⁶⁶ Levine RL, Williams JA, Stadtman ER, Shacter E. [37] Carbonyl assays for determination of oxidatively modified proteins. Elsevier eBooks. 1994 Jan 1;346-57. Available from: https://doi.org/10.1016/S0076-6879(94)33040-9 from: https://hero.epa.gov/hero/index.cfm/reference/details/reference_id/4939694
  » https://doi.org/10.1016/S0076-6879(94)33040-9» https://hero.epa.gov/hero/index.cfm/reference/details/reference_id/4939694
• ⁶⁷ de Almeida EA, Miyamoto S, Bainy ACD, de Medeiros MHG, Di Mascio P. Protective effect of phospholipid hydroperoxide glutathione peroxidase (PHGPx) against lipid peroxidation in mussels Perna perna exposed to different metals. Mar Pollut Bull. 2004;49(5-6):386-92. Available from: https://doi.org/10.1016/j.marpolbul.2004.02.020
  » https://doi.org/10.1016/j.marpolbul.2004.02.020
• ⁶⁸ Tišler T, Kožuh Eržen N. Abamectin in the aquatic environment. Ecotoxicol. 2006;15(6):495-502. Available from: https://doi.org/10.1007/s10646-006-0085-1
  » https://doi.org/10.1007/s10646-006-0085-1
• ⁶⁹ Campbell WC. Ivermectin and Abamectin. Springer eBooks. Springer Nature; 1989. Available from: https://doi.org/10.1007/978-1-4612-3626-9
  » https://doi.org/10.1007/978-1-4612-3626-9
• ⁷⁰ Mao Y, Yu Y, Ma Z, Li H, Yu W, Cao L, et al. Azithromycin induces dual effects on microalgae: Roles of photosynthetic damage and oxidative stress. Ecotoxicol Environ Saf. 2021;222:112496. Available from: https://doi.org/10.1016/j.ecoenv.2021.112496
  » https://doi.org/10.1016/j.ecoenv.2021.112496
• ⁷¹ Ana Catarina Almeida, Gomes T, Antonio J, Lillicrap A. Specific toxicity of azithromycin to the freshwater microalga Raphidocelis subcapitata Ecotoxicol Environ Saf. 2021;222:112553-3. Available from: https://doi.org/10.1016/j.ecoenv.2021.112553
  » https://doi.org/10.1016/j.ecoenv.2021.112553
• ⁷² Sanderson H, Thomsen M. Comparative analysis of pharmaceuticals versus industrial chemicals acute aquatic toxicity classification according to the United Nations classification system for chemicals. Assessment of the (Q)SAR predictability of pharmaceuticals acute aquatic toxicity and their predominant acute toxic mode-of-action. Toxicol Let. 2009;187(2):84-93. Available from: https://doi.org/10.1016/j.toxlet.2009.02.003
  » https://doi.org/10.1016/j.toxlet.2009.02.003
• ⁷³ Bawa-Allah KA, Ehimiyein AO. Ecotoxicological effects of human and veterinary antibiotics on water flea (Daphnia magna). Environ Toxicol Pharmacol. 2022;94:103932. Available from: https://doi.org/10.1016/j.etap.2022.103932
  » https://doi.org/10.1016/j.etap.2022.103932
• ⁷⁴ Dong Y, Fang Z, Xu Y, Wang Q, Zou X. The toxic effects of three active pharmaceutical ingredients (APIs) with different efficacy to Vibrio fischeri Emerg Contam. 2019;5:297-302. Available from: https://doi.org/10.1016/j.emcon.2019.08.004
  » https://doi.org/10.1016/j.emcon.2019.08.004
• ⁷⁵ Parnham MJ, Haber VE, Giamarellos-Bourboulis EJ, Perletti G, Verleden GM, Vos R. Azithromycin: Mechanisms of action and their relevance for clinical applications. Pharmacol Therap. 2014;143(2):225-45. Available from: https://doi.org/10.1016/j.pharmthera.2014.03.003
  » https://doi.org/10.1016/j.pharmthera.2014.03.003
• ⁷⁶ Sandman Z, Iqbal OA. Azithromycin [Internet]. PubMed. Treasure Island (FL): StatPearls Publishing; 2023. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557766/
  » https://www.ncbi.nlm.nih.gov/books/NBK557766/
• ⁷⁷ Guo J, Bai Y, Chen Z, Mo J, Li Q, Sun H, et al. Transcriptomic analysis suggests the inhibition of DNA damage repair in green alga Raphidocelis subcapitata exposed to roxithromycin. Ecotoxicol Environ Saf. 2020;201:110737. Available from: https://doi.org/10.1016/j.ecoenv.2020.110737
  » https://doi.org/10.1016/j.ecoenv.2020.110737
• ⁷⁸ Liu B, Nie X, Liu W, Snoeijs P, Guan C, Tsui MTK. Toxic effects of erythromycin, ciprofloxacin and sulfamethoxazole on photosynthetic apparatus in Selenastrum capricornutum Ecotoxicol Environ Saf. 2011;74(4):1027-35. Available from: https://doi.org/10.1016/j.ecoenv.2011.01.022
  » https://doi.org/10.1016/j.ecoenv.2011.01.022
• ⁷⁹ Schrezenmeier E, Dörner T. Mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology. Nat Rev Rheumatol. 2020;16(3):155-66.Available from: https://doi.org/10.1038/s41584-020-0372-x
  » https://doi.org/10.1038/s41584-020-0372-x
• ⁸⁰ Rolain JM, Colson P, Raoult D. Recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the 21st century. Int J Antimicrob Ag. 2007;30(4):297-308. Available from: https://doi.org/10.1016/j.ijantimicag.2007.05.015
  » https://doi.org/10.1016/j.ijantimicag.2007.05.015
• ⁸¹ Bazoni RF, Moura TA, Rocha MS. Hydroxychloroquine exhibits a strong complex interaction with DNA: unraveling the mechanism of action. J Phys Chem Let. 2020;11(22):9528-34. Available from: https://doi.org/10.1021/acs.jpclett.0c02590
  » https://doi.org/10.1021/acs.jpclett.0c02590
• ⁸² Cirulis JT, Scott JA, Ross GM. Management of oxidative stress by microalgae. Can J Physiol Pharmacol [Internet]. 2013;91(1):15-21. Available from: http://dx.doi.org/10.1139/cjpp-2012-0249
  » http://dx.doi.org/10.1139/cjpp-2012-0249
• ⁸³ Pikula KS, Zakharenko AM, Aruoja V, Golokhvast KS, Tsatsakis AM. Oxidative stress and its biomarkers in microalgal ecotoxicology. Curr Op Toxicol. 2019;13:8-15. Available from: https://doi.org/10.1016/j.cotox.2018.12.006
  » https://doi.org/10.1016/j.cotox.2018.12.006
• ⁸⁴ Ugya AY, Imam TS, Li A, Ma J, Hua X. Antioxidant response mechanism of freshwater microalgae species to reactive oxygen species production: a mini review. Chem Ecol. 2019;36(2):174-93. Available from: https://doi.org/10.1080/02757540.2019.1688308
  » https://doi.org/10.1080/02757540.2019.1688308
• ⁸⁵ Chen S, Wang L, Feng W, Yuan M, Li J, Xu H, et al. Sulfonamides-induced oxidative stress in freshwater microalga Chlorella vulgaris: Evaluation of growth, photosynthesis, antioxidants, ultrastructure, and nucleic acids. Sci Rep. 2020;10(1):8243. Available from: https://doi.org/10.1038/s41598-020-65219-2
  » https://doi.org/10.1038/s41598-020-65219-2
• ⁸⁶ Sanderson H, Laird B, Pope L, Brain R, Wilson C, Johnson D, et al. Assessment of the environmental fate and effects of ivermectin in aquatic mesocosms. Aquat Toxicol. 2007;85(4):229-40. Available from: https://doi.org/10.1016/j.aquatox.2007.08.011
  » https://doi.org/10.1016/j.aquatox.2007.08.011
• ⁸⁷ Alexei Solovchenko, Vasilieva SG, Zaitsev PV, Lukyanov A, Skripnikova E, Antal TK. Approaches to rapid screening of pharmaceutical xenobiotic effects on microalgae via monitoring of photosynthetic apparatus condition. J Appl Phycol. 2022;34(1):353-61. Available from: https://doi.org/10.1007/s10811-021-02660-4
  » https://doi.org/10.1007/s10811-021-02660-4
• ⁸⁸ An SS, Friedl T, Hegewald E. Phylogenetic relationships of Scenedesmus and Scenedesmus-like coccoid green algae as inferred from ITS-2 rDNA Sequence Comparisons. Plant Biol. 1999;1(4):418-28. Available form: https://doi.org/10.1111/j.1438-8677.1999.tb00724.x
  » https://doi.org/10.1111/j.1438-8677.1999.tb00724.x
• ⁸⁹ Hegewald E. New combinations in the genus Desmodesmus (Chlorophyceae, Scenedesmaceae). Arch Hydrobiol Suppl Algol Stud [Internet]. 2000;96:1-18. Available from: http://dx.doi.org/10.1127/algol_stud/96/2000/1
  » http://dx.doi.org/10.1127/algol_stud/96/2000/1
• ⁹⁰ Machado L, Carvalho G, Pereira RN. Effects of innovative processing methods on microalgae cell wall: prospects towards digestibility of protein-rich biomass. Biomass [Internet]. 2022;2(2):80-102. Available from: https://doi.org/10.3390/biomass2020006
  » https://doi.org/10.3390/biomass2020006
• ⁹¹ He X, Dai J, Wu Q. Identification of sporopollenin as the outer layer of cell wall in microalga Chlorella protothecoides Front Microbiol. 2016;7:1047. Available from: https://doi.org/10.3389/fmicb.2016.01047
  » https://doi.org/10.3389/fmicb.2016.01047
• ⁹² Grienenberger E, Quilichini TD. The Toughest Material in the Plant Kingdom: An Update on Sporopollenin. Frontiers in Plant Science. 2021 Sep 3;12. Available from: https://doi.org/10.3389/fpls.2021.703864
  » https://doi.org/10.3389/fpls.2021.703864
• ⁹³ Safi C, Zebib B, Merah O, Pontalier PY, Vaca-Garcia C. Morphology, composition, production, processing and applications of Chlorella vulgaris: a review. Renew Sustain Energy Rev [Internet]. 2014;35:265-78. Available from: https://doi.org/10.1016/j.rser.2014.04.007
  » https://doi.org/10.1016/j.rser.2014.04.007
• ⁹⁴ Weber S, Grande PM, Blank LM, Klose H. Insights into cell wall disintegration of Chlorella vulgaris PloS One [Internet]. 2022;17(1):e0262500. Available from: https://doi.org/10.1371/journal.pone.0262500
  » https://doi.org/10.1371/journal.pone.0262500
• ⁹⁵ Biedlingmaier S, Wanner G, Schmidt A. A correlation between detergent tolerance and cell wall structure in green algae. Zeitschrift für Naturforschung C. 1987;42(3):245-50. Available from: https://doi.org/10.1515/znc-1987-0313
  » https://doi.org/10.1515/znc-1987-0313
• ⁹⁶ Guo J, Peng J, Lei Y, Kanerva M, Li Q, Song J, Guo J, Sun H. Comparison of oxidative stress induced by clarithromycin in two freshwater microalgae Raphidocelis subcapitata and Chlorella vulgaris Aquat Toxicol. 2020 Feb;219:105376. doi: 10.1016/j.aquatox.2019.105376.
  » https://doi.org/10.1016/j.aquatox.2019.105376.
• ⁹⁷ Martins JM. Efeito da ivermectina no crescimento da microalga Raphidocelis subcapitata (= Pseudokirchneriella subcapitata) (Bachelor's thesis, Universidade Federal do Rio Grande do Norte). 2022.
• ⁹⁸ Kołodziejska M, Maszkowska J, Białk-Bielińska A, Steudte S, Kumirska J, Stepnowski P, Stolte S. Aquatic toxicity of four veterinary drugs commonly applied in fish farming and animal husbandry. Chemosphere. 2013. 92(9):1253-9. doi: 10.1016/j.chemosphere.2013.04.057.
  » https://doi.org/10.1016/j.chemosphere.2013.04.057.
• ⁹⁹ Ranjan N, Singh PK, Maurya NS. Pharmaceuticals in water as emerging pollutants for river health: A critical review under Indian conditions. Ecotoxicol Environ Saf. 2022 Dec 1;247:114220. doi: 10.1016/j.ecoenv.2022.114220.
  » https://doi.org/10.1016/j.ecoenv.2022.114220.
• ¹⁰⁰ Fu L, Huang T, Wang S, Wang X, Su L, Li C, Zhao Y. Toxicity of 13 different antibiotics towards freshwater green algae Pseudokirchneriella subcapitata and their modes of action. Chemosphere. 2017 Feb;168:217-222. doi: 10.1016/j.chemosphere.2016.10.043.
  » https://doi.org/10.1016/j.chemosphere.2016.10.043.
• ¹⁰¹ Zhou H, Ying T, Wang X, Liu J. Occurrence and preliminarily environmental risk assessment of selected pharmaceuticals in the urban rivers, China. Sci Rep. 2016 Oct 7;6:34928. doi: 10.1038/srep34928.
  » https://doi.org/10.1038/srep34928.
• ¹⁰² Osorio V, Larrañaga A, Aceña J, Pérez S, Barceló D. Concentration and risk of pharmaceuticals in freshwater systems are related to the population density and the livestock units in Iberian Rivers. Sci Total Environ. 2016 Jan 1;540:267-77. doi: 10.1016/j.scitotenv.2015.06.143.
  » https://doi.org/10.1016/j.scitotenv.2015.06.143.
• ¹⁰³ Harada A, Komori K, Nakada N, Kitamura K, Suzuki Y. Biological effects of PPCPs on aquatic lives and evaluation of river waters affected by different wastewater treatment levels. Water Sci Technol. 2008;58(8):1541-6. doi: 10.2166/wst.2008.742.
  » https://doi.org/10.2166/wst.2008.742.
• ¹⁰⁴ Sansone NMS, Boschiero MN, Marson FAL. Efficacy of Ivermectin, Chloroquine/Hydroxychloroquine, and Azithromycin in Managing COVID-19: A Systematic Review of Phase III Clinical Trials. Biomedicines. 2024 Sep 27;12(10):2206. doi: 10.3390/biomedicines12102206
  » https://doi.org/10.3390/biomedicines12102206
• ¹⁰⁵ Choudhary R, Sharma AK. Potential use of hydroxychloroquine, ivermectin and azithromycin drugs in fighting COVID-19: trends, scope and relevance. New Microbes New Infect. 2020 Apr 22;35:100684. doi: 10.1016/j.nmni.2020.
  » https://doi.org/10.1016/j.nmni.2020.
• ¹⁰⁶ Fernandez R, Colás-Ruiz NR, Bolívar-Anillo HJ, Anfuso G, Hampel, M. Occurrence and Effects of Antimicrobials Drugs Aquatic Ecosys Sust. 2021;13:13428. ttps://doi.org/10.3390/su132313428
• ¹⁰⁷ Efrain Merma Chacca D, Maldonado I, Vilca FZ. Environmental and ecotoxicological effects of drugs used for the treatment of COVID 19. Front Environ Sci. 2002,10:940975. https://doi.org/10.3389/fenvs.2022.940975
  » https://doi.org/10.3389/fenvs.2022.940975
• ¹⁰⁸ Arreguin-Rebolledo U, Morales-Romero LA, Arzate-Cárdenas MA, Páez-Osuna F, Betancourt-Lozano M, Rico-Martínez R. Contrasting toxicity response to a mixture of azithromycin and ivermectin between a freshwater and a euryhaline rotifer. Environ Sci Pollut Res. 2024;31(37):49905-15. DOI: 10.1007/s11356-024-34406-3
  » https://doi.org/10.1007/s11356-024-34406-3
• ¹⁰⁹ Güneş E, Aydin H, Nizamlioğlu HF. Investigation of the protective effect of acetazolamide and SLC-0111 on carbon tetrachloride-induced toxicity in fruit fly. Toxicol Rep. 2021;8:1300-4. https://doi.org/10.1016/j.toxrep.2021.06.014
  » https://doi.org/10.1016/j.toxrep.2021.06.014

• Editor-in-Chief:
  Alexandre Rasi Aoki

• Associate Editor:
  Marcos Pileggi

Publication Dates

• Publication in this collection
  01 Sept 2025
• Date of issue
  2025

History

• Received
  06 Aug 2024
• Accepted
  24 June 2025