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Article

Toxicity of the Antiretrovirals Tenofovir Disoproxil Fumarate, Lamivudine, and Dolutegravir on Cyanobacterium Microcystis novacekii

by
Gabriel Souza-Silva
1,*,
Mariângela Domingos Alcantara
1,
Cléssius Ribeiro de Souza
1,
Carolina Paula de Souza Moreira
2,
Kenia Pedrosa Nunes
3,
Cíntia Aparecida de Jesus Pereira
4,
Marcos Paulo Gomes Mol
2,* and
Micheline Rosa Silveira
2
1
Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil
2
Fundação Ezequiel Dias, Departamento de Pesquisa e Desenvolvimento, Belo Horizonte 30510-010, MG, Brazil
3
Department of Biomedical Engineering and Science, Florida Institute of Technology, Melbourne, FL 32901, USA
4
Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil
*
Authors to whom correspondence should be addressed.
Water 2025, 17(6), 815; https://doi.org/10.3390/w17060815
Submission received: 11 February 2025 / Revised: 7 March 2025 / Accepted: 8 March 2025 / Published: 12 March 2025
(This article belongs to the Special Issue Fate, Transport, Removal and Modeling of Pollutants in Water)
Versions Notes

Abstract

:
Antiretrovirals (ARVs) have become one of the most prescribed groups of drugs, and these residues are found in the environment. Among them, the most widely used in HIV treatment are tenofovir (TDF), lamivudine (3TC), and dolutegravir (DTG). This study aimed to evaluate the toxicity of ARVs TDF, 3TC, and DTG on the cyanobacterium Microcystis novacekii and estimate their environmental risk. DTG showed the highest toxicity among the drugs tested, inhibiting cyanobacteria cell growth and metabolic activity at low concentrations. TDF and 3TC alone were less toxic, with more pronounced adverse effects in long time exposures at high concentrations. However, the combination of ARVs, especially TDF and 3TC, showed a synergistic effect, significantly increasing toxicity compared to the drugs alone. Excipients found in commercial formulations of ARVs, such as sodium lauryl sulfate, also influenced toxicity. Although DTG showed the highest risk to cyanobacteria, the environmental risk assessment indicated that TDF and 3TC, although less toxic to M. novacekii, may pose moderate-to-high environmental risks at typical environmental concentrations. These results reinforce the need for strict regulation and monitoring of the release of ARVs into the environment, and the development of effective treatments for removing these residues in sewage treatment plants. This study contributes to understanding the ecotoxicological impacts of ARVs and highlights the importance of long-term assessments to adequately estimate the environmental risks of ARVs and their commercial formulations.

1. Introduction

The number of people living with the human immunodeficiency virus (PLHIV) has increased over the years, and more than 39 million PLHIV were accounted for worldwide in 2023 [1]. Thus, antiretrovirals (ARVs) have become one of the most commonly prescribed groups of drugs and these residues are now found in the environment [2,3,4,5,6,7,8,9]. tenofovir (TDF), lamivudine (3TC), and dolutegravir (DTG) are the most commonly described [1] among the ARVs used in the treatment of HIV.
TDF and 3TC are nucleotide analog reverse transcriptase inhibitors (NRTIs) with a mean daily dose of 300 mg each. DTG is an HIV integrase inhibitor (INI) with a daily dose of 50 mg, recently indicated for the treatment of HIV [10]. Due to their widespread use and inefficient removal by wastewater treatment plants, residues of these drugs are frequently detected in aquatic environments [2,3,4,5,6,7,8,9].
Thus, ARVs are reported in different places around the world, and the presence of these residues in the environment has been reported in South Africa [4,9], Romania [11], Croatia [11], Portugal [12], Germany [13], Kenya [6,8,14], and Zambia [5]—in concentrations ranging from ng/L to µg/L. Although environmental concentrations are considered to pose no direct risk to human health [15,16], concentrations of 50 µg/L are sufficient to trigger multidrug resistance in human pathogenic bacteria such as Bacillus cereus and Escherichia coli [17].
A growing body of evidence has indicated that ARV residues are likely to affect the survival, physiology, behavior, and reproduction of aquatic organisms at environmentally relevant concentrations [18,19,20,21,22]. Therefore, assessing the ecotoxicological effects of ARVs in water bodies is important for environmental and public health. Furthermore, understanding the toxic effects of ARV residues on lower trophic level organisms, such as cyanobacteria, is crucial to understanding their impacts on the ecosystem and environmental balance [23].
Another important factor is the ecotoxicological impact of the excipients used in the various pharmaceutical forms. Studies have shown that excipients, although they have no pharmacodynamic effects, can be toxic to the environment, including organisms at different trophic levels [24,25,26].
Cyanobacteria have a cosmopolitan distribution, present in Europe, Oceania, South America, the Middle East, and Asia, and can easily adapt to adverse environmental conditions [27,28]. In addition to being at the base of the food chain of various higher organisms such as crustaceans, mollusks, and fish, they are essential for photosynthesis and have the potential to degrade pollutants through bioaccumulation and biotransformation mechanisms [29,30].
Among the cyanobacteria, Microcystis novacekii stands out, which can be found in rivers, lakes, reservoirs, and tanks, whether they are polluted or not [31,32]. Due to their importance in ecosystems, their capacity for bioaccumulation and release of substances, in addition to being an important indicator of pollution in aquatic environments, these organisms have the potential for consistent assessment of pollution levels of different substances and structured use in effluent treatment plants [29,33,34].
Studies demonstrate the ability of cyanobacteria of the genus Microcystis sp. to degrade drugs and pesticides in the aquatic environment without generating active or toxic metabolites [29,33,35,36]. Although it has the ability to metabolize or store pollutants, depending on the type of substance and concentration, oxidative stress and photosynthetic damage are the main toxic mechanisms of several pollutants for cyanobacteria, triggering harmful effects on the aquatic ecosystem [37].
Therefore, this study aims to evaluate the toxicological effects and environmental risk of active pharmaceutical ingredients (APIs) and ARV drugs based on TDF, 3TC, and DTG on the aquatic microorganism M. novacekii in order to understand the effects of these residues in the environment.

2. Materials and Methods

2.1. Drugs

The following drugs were used in this study: (i) DTG (Blanver Farmoquímica e Farmacêutica S.A., São Paulo—Brazil); (ii) TDF + 3TC (Cristália Produtos Químicos e Farmacêuticos Ltd.a., São Paulo—Brazil); and (iii) 3TC (Fundação para o Remédio Popular (FURP), São Paulo—Brazil); and (iv) TDF (Ezequiel Dias Foundation (FUNED), Minas Gerais—Gameleira Biosynth, Brazil). We assumed that the actual ARV concentrations are those reported by the manufacturer in the formulation, considering that the drugs (API + excipients) were thoroughly tested and approved by a thorough pharmaceutical quality control system for commercial use.
The DTG drug, used in this study, consists of 50 mg of the active pharmaceutical ingredient (API) DTG; microcrystalline cellulose 101; mannitol 25 C; povidone K30; sodium starch glycolate; sodium stearyl fumarate; and Opadry® II Yellow (partially hydrolyzed polyvinyl alcohol, titanium dioxide, macrogol/PEG, talc, yellow iron oxide). The average weight of the DTG drug tablets was 307.3 ± 3.0 mg, with the amount of API being 50 mg, corresponding to approximately 16.2% of the tablet composition.
The 3TC drug, used in this study, consists of 150 mg of the active pharmaceutical ingredient 3TC; silicon dioxide; magnesium stearate; sodium starch glycolate; microcrystalline cellulose; and Opadry white YS-1-7003 (hypromellose, titanium dioxide, macrogol, and polysorbate 80). The average weight of the 3TC drug tablets was 226.3 ± 2.6 mg, with the amount of active ingredient being 150 mg, corresponding to approximately 66.3% of the tablet composition.
The TDF drug, used in this study, consists of 300 mg of the active pharmaceutical ingredient TDF; croscarmellose sodium; starch; lactose monohydrate; microcrystalline cellulose; sodium lauryl sulfate; magnesium stearate; hypromellose; macrogol; titanium dioxide; and indigotine aluminum lake blue dye. The average weight of the TDF drug tablets was 687.1 ± 6.4 mg, with the amount of API being 300 mg, corresponding to approximately 43.7% of the tablet composition.
The TDF + 3TC drug used in this study is composed of 300 mg of the active ingredient (TDF); 300 mg of the active ingredient (3TC); lactose monohydrate; microcrystalline cellulose; starch; croscarmellose sodium; sodium starch glycolate; magnesium stearate; hypromellose; and Opadry® II Yellow (part-hydrolyzed polyvinyl alcohol, titanium dioxide, macrogol/PEG, talc, yellow iron oxide). The average weight of the TDF + 3TC tablets was 1064.5 ± 53.0 mg, with the amount of active ingredient being 600 mg (300 mg of TDF + 300 mg of 3TC), corresponding to approximately 56.4% of the tablet composition.

2.2. Active Pharmaceutical Ingredients

The following APIs were used in this study: (i) DTG (purity ≥ 95.0%) (Biosynth, Staad, Switzerland); and (ii) TDF (purity ≥ 99.8%) (FUNED, Gameleira, Brazil) and 3TC (purity ≥ 99.5%) (FUNED, Gameleira, Brazil). Information regarding the physicochemical properties of these substances is described in Table 1.

2.3. Test Organism Culture

Cultures of M. novacekii (Komárek) Compère (isolated from water samples collected from Lagoa Dom Helvécio, in the Rio Doce State Park (42°, 35′, 595″; 19°, 46′, 419″), Minas Gerais, Southeastern Brazil) kept under controlled conditions in the Water Laboratory in germination chambers at 23.0 °C ± 2 °C with a 12/12 h photoperiod (light/dark) under light intensity (45 ± 5 μmoL/m/s) were used in the tests of this study. The medium used for the cultivation and assays of M. novacekii was ASM-1 (pH = 8.0 ± 0.2) [38].

2.4. Preparation of Test Substances

All APIs and drugs were solubilized in the ASM-1 culture medium to ensure osmotic equilibrium between the biological model and the test substance. Due to the TDF and 3TC APIs’ good solubility (Table 1), using solvents for solubilization was unnecessary. To solubilize drugs TDF and 3TC, they were initially manually pulverized using a pestle and porcelain mortar. Then, the material was transferred to a glass beaker and solubilized using a hot plate with magnetic stirring (IKA RT 15, Staufen/Germany) under controlled temperature conditions (35 ± 1 °C), stirring (500 rpm), and time (10 min).
After reaching room temperature, each solution was individually filtered through a qualitative filter, and the pH was adjusted to (pH = 8.0 ± 0.2) with 0.1 M sodium hydroxide (NaOH) solution. The APIs were solubilized in the same way without the pulverizing step. The final concentration of APIs and drug solutions was 600 mg/L (TDF) and 600 mg/L (3TC).
For the API and DTG, solubilization occurred in the ASM-1 culture medium with the addition of solvent dimethyl sulfoxide (DMSO) at a maximum concentration of 1%. DTG was manually pulverized using a porcelain pestle and mortar and homogenized. Then, the material was transferred to a 50 mL glass beaker, and 10 mL of the DMSO solvent was added. The solution was homogenized using a hot plate with magnetic stirring under controlled conditions of temperature (35 ± 1 °C), stirring (500 rpm), and time (10 min).
The solution was then filtered through qualitative filter paper, and the pH was adjusted (pH = 8.0 ± 0.2) with 0.1 M sodium hydroxide (NaOH) solution, with a final volume of 1 L. The final concentration of the API and drug solutions was 100 mg/L (DTG). The concentrations used in the assays are shown in Table 2.
These concentrations were defined according to a preliminary test to determine the concentration ranges applicable to each biological model, providing a more accurate effect concentration at 50% of the test organism values (EC50). The concentrations of medication are given based on the IFA concentration in their composition.

2.5. Solvent Control

Due to the DTG API’s low solubility in water, DMSO, an efficient aprotic solvent used as a drug solvent in (eco)toxicological investigations [39], was adopted to increase its solubility in the ASM-1 culture medium. For this purpose, toxicity tests were performed with DMSO before exposing the cyanobacteria to the drugs or API, per the OECD protocol N° 201 of 2006 [40], at concentrations ranging from 0.01 to 5.0% DMSO.
One mL aliquots of the cultures exposed to the solvent at different concentrations were collected daily (for 14 days) in triplicate to evaluate the impact of the DMSO solvent on the growth of cyanobacteria. The experiments performed with DTG showed no significant difference (p > 0.05) between the results of the negative control group and the solvent control (1% DMSO). This excludes the possibility of solvent effects on the toxicity results with the cyanobacterium M. novacekii within 14 days of exposure.
Furthermore, a solvent control was performed in triplicate with a maximum concentration of 1% DMSO for all toxicity tests using DTG. To approve the test using the solvent, the difference in cell growth between the solvent control and the negative control must be less than 5%, and there must be no significant difference, i.e., p > 0.05.

2.6. Acute and Chronic Toxicity Test

With modifications, the 2011 OECD protocol N° 201 [40] was adopted for the acute and chronic growth inhibition tests of M. novacekii. Initially, 250 mL Erlenmeyer flasks were prepared with the concentrations of interest, and cell cultures were added, obtaining an initial cell density of 106 cells per milliliter.
They were then incubated in triplicate at 22.0 °C ± 1.0 °C with a 12/12 h photoperiod (light/dark) and constant agitation for 96 h for acute exposure and 14 days for chronic exposure. The ASM-1 culture medium was used as a negative control for the experiments performed in triplicate. Based on the means of five growth curves previously established for M. novacekii, the culture concentrations in cells per milliliter (y) were calculated from the cell density (X) using the formula:
y = 107  • X − 254,076 (R2 = 0.9737)
Then, we built the mean growth rates and growth inhibition curves as a function of the concentrations of the test substances. The growth rate coefficient (µ) was calculated at 96 h and 14 days of exposure. The optical densities of the M. novacekii cultures were determined by spectrometry (Spectroquant–Merck Millipore, Darmstadt/Germany) at a wavelength of 680 nm. The sample’s cell concentration per milliliter was determined by microscopy (Nikon Eclipse E200, Tokyo/Japan) using a Neubauer Chamber.

2.7. Metabolic Activity Assay—MTT

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) is commonly used in cell viability assays, where it is reduced by cellular enzymes (mainly mitochondrial dehydrogenases) into a colored product, usually a formazan. Although MTT is not directly a “dehydrogenase assay”, it is related to the concept, as the reduction process of MTT involves mitochondrial dehydrogenases, which play an essential role in cellular energy production. The metabolic activity assay was performed using MTT (Sigma Aldrich—St. Louis, MO, USA) with the cyanobacterium M. novacekii following the method described by Barmshuri et al. [41] with modifications.
MTT solutions (5 mg/mL) were prepared in an ASM-1 culture medium using vortexing to solubilize the dye. The solution was filtered through a 0.22 μm filter, and all its preparation was conducted in a dark environment. Cyanobacteria were exposed to APIs at no-observed-effect concentrations, i.e., 10 mg/L TDF, 100 mg/L 3TC, or 0.1 mg/L DTG, for 96 h under the same conditions applied for the growth inhibition assay. After cell exposure, 1 mL of each group was transferred to 1.5 mL microtubes in triplicate, and 50 μL of MTT was added. The microtubes were incubated at 37 °C for 4 h in the dark. After incubation, the microtubes were centrifuged at 10,000 rpm for 5 min at room temperature, and the supernatants were discarded.
Then, 200 μL of dimethyl sulfoxide (DMSO) solvent (Sigma Aldrich—St. Louis, MO, USA) was added to the pellets in the microtubes after incubation for 5 min at room temperature. After this time, the microtubes were rigorously vortexed for 10 s. The microtubes were centrifuged at 10,000 rpm for 10 min at room temperature, and the supernatants were transferred to a 96-well plate reader (Thermo Scientific Multiskan FC—Waltham, MA, USA) to measure the samples’ absorbance at 570 nm. The blank in this experiment consisted of the culture medium (ASM-1) and reagents (MTT and DMSO) without the cyanobacteria.

2.8. Toxicity Classification on the Test Organism

Typically, ecotoxicological tests using algal species express the results of aquatic toxicity of a given substance in EC50 value at 72 or 96 h of exposure. However, chronic toxicity data are less available than acute data, and the range of test procedures is less standardized, hampering chronic risk classification. In this study, the “hazardous to the aquatic environment” classification (Table 3) of Annex 2.28 (b) of the Globally Harmonized System of Classification and Labeling of Chemicals [42] was used to determine the toxicity of ARVs on the cyanobacterium M. novacekii.

2.9. Environmental Risk Assessment (ERA)

The environmental risk assessment was performed using the Ecological risk assessment (ERA) tool. In this study, the ERA was performed based on the calculation of Risk Quotients (RQs) (Equation (2)): the relationship between the measured environmental concentration (MEC) and the concentration of antiretrovirals (tenofovir, lamivudine, and dolutegravir) that does not cause an effect (Predicted Non-effect Concentration—PNEC). The PHARMS-UBA database of The Umweltbundesamt [43] was used to obtain the ARVs’ environmental concentration data.
RQs = MEC/PNEC
The ecological risk of each target substance was classified into four degrees per the RQs value, as follows: RQs < 0.01, insignificant risk; 0.01 ≤ RQs < 0.1, low risk; 0.1 ≤ RQs < 1, medium risk; and RQs ≥ 1, high risk. The PNEC was defined as EC50 values (data obtained from the literature and/or in the present study) divided by an evaluation factor of 1000 for each organism [44].

2.10. Statistical Analysis

Statistical analyses were performed using R software (version 4.2.1). Initially, the Shapiro–Wilk test was performed to verify data normality. Then, the results obtained in each treatment were statistically compared with the negative control group, using analysis of variance for nonparametric data (Wilcoxon).
Values of p < 0.05 (95% CI) were considered significantly different. Dose-response models were also performed using log-logistic, log-normal, and Weibull models, which were tested using the “drc” extension package in the R statistical software to estimate the best fit function [45]. Dose-response regression statistical models represent the relationship between the independent variable (dose or concentration) and the dependent variable (response or effect).

3. Results and Discussion

From this study, it was possible to observe that most of the tests conducted showed that acute exposure (4 days) of M. novacekii to ARVs is more toxic than chronic exposure (14 days). This effect may be a consequence of a possible hormesis effect of the organisms to ARVs, either through adaptation mechanisms, resistance, or even the biodegradation of ARVs by the organism, biotransforming them into potentially less toxic byproducts. In other cases, this effect was not observed, possibly due to the cell’s inability to adapt to the ARV or even its greater stability against degradation processes, as in the case of DTG, where increased exposure time resulted in higher observed toxicity.
Among the ARVs tested, DTG showed the most significant toxicity, TDF had moderate toxicity, and 3TC evidenced the lowest toxicity on M. novacekii. Table 4 presents the compiled toxicity results of the API and commercial drugs based on DTG, TDF, and 3TC to cyanobacteria.

3.1. DTG Toxicity

DTG drug showed estimated EC50 values of 27.6 ± 5.4 and 19.5 ± 1.8 mg/L for acute and chronic exposure, indicating that this ARV can be classified as “low toxic” [42]. Considering that the exposure time directly influenced the observed response (inhibition of cell growth), leading to a significant toxicity increase (p < 0.05), cyanobacterium M. novacekii was unable to recover from intoxication by DTG at concentrations higher than 0.5 mg/L (DTG API in drug) (Table 5).
DTG API’s toxicity on the cyanobacterium M. novacekii was approximately 15 times greater than that of DTG drug. Exposure to API led to significant inhibition (p < 0.05) of cell growth at concentrations higher than 0.1 mg/L of DTG. The EC50 value was estimated at 1.7 ± 0.3 mg/L and 0.9 ± 0.4 mg/L at 96 h and 14 days of treatment, respectively, and was considered toxic to M. novacekii [42].
Although the 0.1 mg/L DTG API concentration did not cause significant cell growth inhibition compared to the control (p > 0.05), the cellular metabolic activity of cyanobacteria was significantly affected at this concentration (p < 0.05). Regarding the control (0.0 mg/L), we observed a 56% reduction in the cell’s capacity to convert the MTT dye into formazan crystals in the treatment group (0.1 mg/L). This decline indicates that the cellular metabolic activity was affected by the DTG API exposure at concentrations lower than 0.1 mg/L, although its cell growth was not compromised at this same concentration (Table 6).
This is the first study to assess the toxicological effects of the DTG ARV (drug and API) on aquatic organisms. Although there are ecological modeling studies [46], due to the lack of toxicity studies on DTG, we could not reliably estimate the risks of this ARV to the environment. Moreover, using other biomarkers, such as metabolic activity, may estimate these environmental effects.
DTG’s toxicity is associated with its molecular structure and dissociated forms (cationic, neutral, or anionic), which can influence its bioavailability in the absorption process and consequently reduce or increase its toxicity to aquatic organisms [21]. The ionization state is controlled by the pH of the solution and its pKa values. In this study, the pH of the solution used for cyanobacteria exposure to API and DTG drug was 8.0 ± 0.2. Based on the DTG pKa values (−0.5/10.1) and the culture medium pH, ARV is probably mainly in its cationic form.
Furthermore, DTG has lipophilic properties, which may increase toxicity in more complex aquatic organisms, such as mollusks, amphibians, and fish. This means that DTG can associate with fats and lipids in the tissues of aquatic organisms, allowing greater absorption and storage in the organisms, where its degradation may be slow, resulting in bioaccumulation, as with other pharmaceutical residues with lipophilic properties [47,48,49].
In this study, we observed that, although excipients may increase toxicity in some cases, others may result in reduced toxicity, as with the DTG API. Stabilizers and complexing agents such as Mannitol and Povidone can result in complexation or binding to DTG and consequently can form less toxic products, as observed in the growth inhibition test of cyanobacterium M. novacekii. These compounds can encapsulate the API, reducing its bioavailability and toxicity in aquatic organisms [24,50,51].

3.2. TDF Toxicity

For the TDF drug, both acute and chronic exposure did not show toxicity in M. novacekii [41]. Even the highest concentrations of the drug, 256 mg/L, were insufficient to inhibit the 10% cell growth (EC10). On the other hand, the organisms under treatment with TDF grew significantly more at concentrations below 16 mg/L when compared to the negative control group (p < 0.05), observed in acute and chronic exposure.
The TDF drug showed positive activity for the growth of cyanobacteria. Moreover, a significant increase (p < 0.05) in the growth of M. novacekii was observed when the dilution factor of the solutions was augmented, indicating a supposed dose-response dependent hormesis effect. This effect observed at low doses was previously reported for different aquatic organisms, such as microalgae Raphidocelis subcapitata [21,52], crustacean Daphnia magna [53,54] and different plant species [55] in the presence of nitrogen-containing organic molecules, as is the case of the TDF drug and the ARV zidovudine (ZDV) [21].
Exposure to the TDF API led to significant inhibition (p < 0.05) of M. novacekii cell growth at API concentrations above 16 mg/L. The EC50 value was estimated at 130.7 ± 5.8 mg/L and 147.0 ± 7.3 mg/L at 96 h and 14 days of treatment, respectively, and was considered practically non-toxic to M. novacekii [42]. Considering that the increased exposure time caused a significant reduction (p < 0.05) in the inhibition of cell growth, the cyanobacteria recovered from API poisoning at high TDF concentrations (up to 256 mg/L) after 14 days of exposure (Table 7).
Although the EC50 value obtained is considered practically non-toxic [42], concentrations of 10 mg/L of TDF API were sufficient to significantly alter (p < 0.05) the cellular metabolic activity of cyanobacterium M. novacekii. Regarding the control (0.0 mg/L), we observed a 12% reduction in its metabolic activity in the treatment group (10 mg/L), indicating that the cell can suffer effects even when there is no reduction in its cellular growth (Table 6).
This study’s TDF API toxicity results corroborate those of Silva et al. [36], who estimated the EC50 value at 161.0 mg/L in M. novacekii exposed to TDF API after 96 h of exposure. However, when evaluating the TDF drug, our study showed that the excipients found in its formulation probably reduced the toxicity of the TDF API, and we could not observe growth inhibition even at concentrations of 256 mg/L of TDF. Furthermore, Russo et al. [52] reported that exposure of R. subcapitata for 72 h to ARVs resulted in a slight inhibition of microalgae growth for the ARVs stavudine (STV) and zidovudine, belonging to the same NRTI group as TDF.
Another study by Gomes et al. [20] estimated EC50 values at 594.4 and 671.1 mg/L for Synechococcus elongatus and Chlorococcum infusionum after 96 h of exposure, indicating low TDF API toxicity on cyanobacteria and microalgae, respectively. In addition to these organisms, Artemia salina was also resistant to TDF API exposure, with an EC50 value for organism immobility estimated at 111.8 mg/L [36].
Another organism resistant to TDF exposure is the mollusk Biomphalaria glabrata, which resists concentrations of 300 mg/L without causing significant mortalities compared to the negative control. However, when evaluating the mollusk’s immune system after 21 days of exposure to the TDF API, we observed a reduction in the viability of hemocytes, the mollusk’s defense cells, with an estimated EC50 of 2.7 mg/L, coupled with the increased metabolic activity of these cells, with an estimated EC50 of 1.6 mg/L [56].
However, exposure of the TDF API to the bioluminescent bacterium Aliivibrio fischeri resulted in toxicity, with an estimated EC50 value of 14.8 mg/L after 15 min of exposure. Regarding the TDF drug, this toxicity was even more significant (EC50 = 8.20 mg/L) than for the isolated API [36]. This difference in toxicity between the drug and the isolated API can be explained by excipients found in the formulation. Among these, sodium lauryl sulfate is a highly toxic surfactant, with EC50 values below 10 mg/L. This high toxicity may result from the bacteriostatic properties of sodium lauryl sulfate and its ability to create pores in bacterial membranes, leading to cell death [24].
However, as observed in the present study, the toxicity values of individual compounds for aquatic organisms do not directly correspond to the toxicity of pharmaceutical products. They cannot be extrapolated to other biological models. Even though it is a gram-negative bacterium, excipient sodium lauryl sulfate did not cause the death of M. novacekii. Thus, even with formulations of potentially toxic compounds, the effect may be non-toxic, and synergistic/antagonistic interactions between different excipients may occur [24].
The influence of pharmaceutical excipients on the API toxicity is evident when compared with the same API in different marketed drugs. The toxicity of five drugs with the same API (fluoxetine) showed different toxicities on the microalgae Chlorella vulgaris, ranging from EC50 0.25 to 15.0 mg/L. Considering that the drugs have the same API and that all test conditions were controlled and kept constant, the only variable is the excipients found in the drugs [25].
As observed, even when using API from different batches, it is possible to produce reproducible results with ARVs. In this study, the EC50 value for M. novacekii was estimated at 130.7 mg/L, while the study conducted by Silva et al. [36], using the same organism and method but API from different batches, observed an EC50 value of 161.0 mg/L. However, the reproducibility of drug toxicity results (excipients + API) is low. While our study did not observe toxicity of M. novacekii exposed to TDF (EC50 > 256.0 mg/L), Silva et al. [36] estimated the EC50 value at 89.0 mg/L. This variation can be explained by the difference between producers, composition, and the different excipients used in the TDF drug.
Although a very stable molecule at acidic pH (2 to 3) [57], TDF can undergo hydrolysis when solubilized in an ASM-1 medium since the experiment was conducted under controlled conditions, such as pH 8.0, which favors molecule de-esterification. Moreover, TDF is biodegradable by the cyanobacterium M. novacekii [58], justifying API and drug low toxicity under the cyanobacterium.

3.3. 3TC Toxicity

The 3TC drug showed low toxicity to cyanobacteria with an estimated EC50 value of 60.3 ± 2.7 and 18.2 ± 3.5 mg/L for acute and chronic exposure, respectively [42]. Considering that the increase in exposure time caused a three-fold increase in toxicity (p < 0.05), the cyanobacterium could not recover from intoxication by drug 3TC at concentrations higher than 5 mg/L (Table 8).
The 3TC API did not evidence toxicity to M. novacekii after acute exposure. The EC50 value was estimated at 184.8 ± 22.1 mg/L at 96 h of exposure. However, no significant difference in cell growth inhibition was observed (p > 0.05) after chronic exposure of the cyanobacterium to API when compared to the negative control group. Therefore, M. novacekii managed to recover from intoxication by 3TC API at concentrations of up to 400 mg/L after 14 days of exposure.
Among the three ARVs evaluated in this study, 3TC was the API with the lowest toxicity to the cyanobacterium M. novacekii, with EC50 values considered practically non-toxic [42]. Even after exposure to high concentrations (100 mg/L), they were not sufficient to significantly alter (p > 0.05) the cellular metabolic activity of cyanobacterium M. novacekii (Table 6). These results corroborate those found in the cell growth assay, reinforcing the “practically non-toxic” classification since exposure to the 3TC API did not impact cell growth or metabolic activity.
This low toxicity observed corroborates the results found by Gomes et al. [20], where cyanobacterium Synechococcus elongatus and the microalgae Chlorococcum infusionum revealed EC50 values of 570.3 and 571.1 mg/L after 96 h of exposure to 3TC API, respectively. However, other studies reported high toxicity of 3TC to microalgae R. subcapitata and microcrustacean Ceriodaphnia dubia with estimated EC50 values of 3.013 and 1.345 mg/L after 96 h of exposure, for growth inhibition and immobility, respectively [21]. Furthermore, in a study by Omotola et al. [22], environmentally relevant concentrations of 3TC were sufficient to cause immobility in Daphnia magna, with an estimated EC50 value of 34.1 µg/L and 12.3 µg/L for 24 and 48 h of exposure, respectively.
In this study, the 3TC drug evidenced toxicity three times greater than the API during acute exposure. Furthermore, an even more significant difference was observed in chronic exposure, with an estimated EC50 value of 18.2 mg/L for the drug and greater than 400 mg/L for the API, in which it was impossible to estimate the EC50 value since the highest concentration tested (400 mg/L) could not inhibit 10% of cell growth. A study by Turek et al. [24] revealed that commonly used excipients increased the toxicity of the APIs Valsartan (VAL), Losartan potassium (LOS-K), and Telmisartan (TEL). In A. fischeri, the estimated EC50-15 min value was 234.8 mg/L for the VAL API, while the estimated EC50-15 min value for the drug was 19.4 mg/L, a 12-fold increase in toxicity to the organism [24].
These findings suggest that the excipients in the 3TC drug evaluated in this study appear to increase API toxicity in the environment, especially in low-trophic level organisms, such as producers. Among the excipients of these drugs, microcrystalline cellulose may have increased the API toxicity on the organism. This excipient’s particle size is reduced due to its industrial processing, which may increase absorption properties, bioaccumulate, and disrupt cellular functioning in cyanobacteria [24].

3.4. ARVs’ Association Toxicity

The drug exposure (composed of two APIs TDF + 3TC) evidenced low toxicity to the cyanobacterium, with an estimated EC50 value of 51.6 ± 2.2 mg/L for acute exposure and 68.7 ± 3.7 mg/L for chronic exposure. Thus, mixing the two APIs increased the 3TC toxicity by 14% (EC50 = 60.3 mg/L—isolated) and caused a drastic increase in TDF (EC50 > 400 mg/L—isolated).
On the other hand, we observed an increase in toxicity when evaluating the toxicity of the combination of the two TDF and 3TC drugs compared to a single drug composed of the two APIs for the cyanobacterium, with an estimated EC50 value of 5.6 ± 0.3 mg/L for acute exposure and 4.5 ± 0.2 mg/L for chronic exposure, with a reduced EC50 value of 9.2 and 15.2 times, respectively. Furthermore, the ability to recover from cyanobacterial poisoning was not observed with the combination of two TDF and 3TC drugs since toxicity increased over time (EC50-4d > EC50-14d), unlike the single drug composed of two APIs, which showed reduced toxicity (EC50-4d < EC50-14d) with increasing exposure time.
A significant difference (p < 0.05) was observed between the means of inhibiting the growth of cyanobacteria exposed to the drug composed of two APIs (TDF + 3TC) when compared to the same combination, but with the combination of two TDF and 3TC drugs. The drug, composed of two APIs (TDF + 3TC) in a single tablet, was classified as low toxic, as it returned an EC50 value between 10 and 100 mg/L. However, combining two APIs (TDF + 3TC) was classified as toxic since the EC50 value found was between 1 and 10 mg/L [42].
The principal difference between the two exposures is in the excipients used in the formulation of the drugs since the concentration and the APIs were the same in both tests. When analyzing the excipients, we observed that the mixture of the two drugs shows all the excipients that comprise the drug composed of two APIs. However, in addition to containing the same excipients, these drugs also include colloidal silicon dioxide and sodium lauryl sulfate, excipients not found in the drug composed of two APIs.
Among these excipients, sodium lauryl sulfate is more toxic than colloidal silicon dioxide to A. fischeri [24]. Although the isolated TDF drug containing sodium lauryl sulfate revealed low toxicity to M. novacekii, together with the presence of the 3TC drug, this excipient may be increasing the toxicity of the mixture of these two drugs (TDF and 3TC) since it has bacteriostatic properties [24].
However, as observed in the present study, the toxicity values of individual compounds for aquatic organisms do not correspond directly to the toxicity of pharmaceutical products. They cannot be extrapolated to other biological models. Even though it is a gram-negative bacterium, excipient sodium lauryl sulfate did not cause the death of M. novacekii. Thus, even with formulations of potentially toxic compounds, the effect may be non-toxic, and synergistic/antagonistic interactions between different excipients may occur.
Although the mixture of the two drugs (TDF and 3TC) showed toxicity, exposure to the TDF and 3TC APIs did not show toxicity on the cyanobacterium even after 14 days of exposure. This result reinforces the need for studies with drugs (API + excipients) and not only with the API alone since the estimated acute and chronic EC50 values were higher than 256 mg/L for M. novacekii for the mixture of the TDF and 3TC APIs.
The mixture of DTG and 3TC drugs evidenced little toxicity to M. novacekii. The estimated EC50 value of the mixture was 22.4%, corresponding to 10.9 ± 0.7 mg/L of DTG and 65.5 ± 3.9 mg/L of 3TC in a 4-day exposure. The isolated DTG drug showed an EC50 value of 27.6 mg/L, 2.5 times higher than that found in the mixture DTG + 3TC, suggesting a possible synergistic effect between the two APIs. However, this difference was negligible in the isolated 3TC drug, with an EC50 value of 60.3 mg/L (isolated) and 65.5 mg/L (mixture). After 14 days of exposure, none of the tested concentrations were significantly different from the control, suggesting these substances’ capacity for recovery from cyanobacterial poisoning.
The mixture of DTG and 3TC APIs is classified as toxic to the cyanobacterium since the EC50 value was estimated at 2.9 ± 1.5%, corresponding to 10.5% during acute exposure. We observed that the EC50 value was higher during chronic exposure, estimated at 19.2 ± 2.0%, corresponding to 9.6 ± 1.0 mg/L of DTG and 77.0 ± 7.8 mg/L of 3TC. As seen in the drug, we noted a synergistic effect between the DTG and 3TC APIs. However, the results of chronic exposure revealed a recovery capacity from cyanobacterial poisoning to this ARV combination. Notwithstanding this, the EC50 value of this combination is still classified as “toxic” for M. novacekii, even with recovery [42].
Cyanobacteria exposed to the combination of DTG and TDF inhibited cell growth, with an estimated EC50 value of 9.57%. This value corresponds to a concentration of 4.79 ± 0.73 mg/L of DTG and 28.71 ± 4.35 mg/L of TDF in a 4-day exposure. In chronic exposure (14 days), we observed a 2.5-fold reduction in toxicity, with the EC50 of the mixture estimated at 24.14%, corresponding to 12.07 ± 1.34 mg/L of DTG and 72.42 ± 8.01 mg/L of TDF.
Although it showed more significant toxicity in acute exposure (4 days), the cells recovered from poisoning during chronic exposure, mainly in concentrations below 10% of the mixture. Based on the results obtained in this study, we can hypothesize that the (physical and/or biological) degradation byproducts of DTG and TDF drugs may be less toxic than their unchanged products since the acute toxicity (EC50 = 9.6%) is 2.5 times greater than the chronic toxicity (EC50 = 24.1%).
The DTG and TDF APIs test results reinforce this hypothesis. From the cyanobacteria M. novacekii exposure to this mixture, the EC50 value was estimated at 2.3 ± 0.9% after 4 days of exposure, 4.2 times higher than the combination of drugs based on these APIs. This value corresponds to a concentration of 1.1 ± 0.4 mg/L of DTG and 11.7 ± 4.4 mg/L of TDF. However, during chronic exposure (14 days), the cell recovery capacity was higher (approximately 1.7 times) in the API than in the drug, with an estimated EC50 value of 41.9 ± 6.4%, corresponding to 21.0 ± 3.2 mg/L of DTG and 214.6 ± 32.8 mg/L of TDF.
This cell ability to recover from poisoning by the association changed the DTG and TDF toxicity classification, initially (4 days of exposure) classified as “toxic” and later (14 days of exposure) classified as “low toxic” since the values left the range of 1–10 mg/L and went to the range of 10–100 mg/L [42], a similar behavior observed in the exposure of the DTG and TDF drugs.
These results show that although acute cyanobacteria exposure to these APIs is considered toxic, these cells can recover from poisoning. Furthermore, the lack of excipients reduces toxicity, as evidenced in chronic exposure since no agents in the medium stabilize and distribute the APIs like the excipients in commercial DTG and/or TDF drugs.
The combination of three ARVs was evaluated, in addition to the combinations of two drugs. Initially, we observed high acute toxicity of the combination of drugs, with an estimated EC50 value of 11.7 ± 1.3%, i.e., 5.6 ± 0.6 mg/L for DTG and 14.9 ± 0.2 mg/L for TDF and 3TC. Regarding chronic exposure, increasing the exposure time led to reduced toxicity, with an estimated EC50 of 33.2 ± 0.4%, or 14.9 ± 0.2 mg/L for DTG and 89.5 ± 1.1 mg/L for TDF and 3TC [42], indicating that, for the drugs combination, while more toxic in the short period, cyanobacterium M. novacekii recovered from poisoning.
Finally, for the association of the DTG, TDF, and 3TC APIs, the results of their toxicity on the cyanobacterium M. novacekii were classified as “very toxic” per Annex 2.28 (b) of the Globally Harmonized System of Classification and Labeling of Chemicals [42] since they returned an EC50 value of less than 1 mg/L during acute exposure. In this test, the concentrations of the different APIs were equal, i.e., 100, 10, 1, 0.1, and 0.01 mg/L. From the exposure of the cells to this mixture, the EC50 value was estimated at 0.9 ± 0.1 mg/L after 4 days of exposure and 3.5 ± 0.5 mg/L after 14 days of exposure.
The study by Gomes et al. [20] evaluated the toxicity of three ARVs (TDF, 3TC, and Efavirenz) on the aquatic photosynthetic species Synechococcus elongatus (Cyanobacterium) and Chlorococcum infusionum (Chlorophyta). The toxicity tests indicated that, at environmental concentrations, TDF and 3TC did not show significant risks to the species studied. However, Efavirenz was toxic, reducing growth rate and affecting photosynthesis, respiration, and oxidative metabolism.
The combination of TDF with Efavirenz caused synergistic effects, leading to a significant reduction in the rate of photosynthesis and the activity of the enzyme NADH-cytochrome c oxidoreductase. Furthermore, oxidative stress in cells increased, evidenced by elevated oxidative stress markers, such as hydrogen peroxide and lipid peroxidation [20].
Although 3TC alone did not show significant toxicity at the environmental concentrations tested, its presence in combination with Efavirenz intensified oxidative damage in cells of both species. Combining all antiretrovirals resulted in even higher levels of hydrogen peroxide and lipid peroxidation [20].
The results of the present study corroborate those found by Gomes et al. [20], where, at environmental concentrations, both TDF and 3TC did not evidence risks to cyanobacterium M. novacekii. Furthermore, synergistic effects between the antiretrovirals DTG + TDF, TDF + 3TC, and TDF + DTG + 3TC were also reported in our study.
The results of the drug combinations in this study reinforce the importance of conducting long-term tests (chronic exposure) exposing bacteria to isolated ARVs. Chronic toxicity tests with cyanobacteria are reliable since these organisms’ action and adaptation mechanisms are observed in longer exposures [59]. Our results reinforce this need since they identified that DTG and 3TC alone have more significant toxicity in chronic exposures when compared to the results of acute toxicity. However, the effect was the opposite for the combinations of DTG and 3TC, with recovery from drug poisoning in the long term.
Chronic exposures of these organisms, especially at environmentally relevant concentrations, allow for assessing the development of resistance to the API they are exposed to and other classes. Wallace et al. [17] demonstrated that the ARVs didanosine, lamivudine, stavudine, zidovudine, raltegravir, dolutegravir, and efavirenz, even at low concentrations between 10 and 100 µg/L, can produce resistance in bacteria such as E. coli and B. cereus. Furthermore, developing resistance to one of these ARVs favors the development of resistance to other ARVs, even in different classes.
Furthermore, the authors strongly recommend conducting tests with pharmaceutical combinations, especially with similar drugs or drugs used in combination, such as the ARVs in this study, to assess ecotoxicological effects since there is no known standard yet on the relationship between the toxicity of drugs and APIs. This is because, like other contaminants, pharmaceutical products are not isolated in the environment and are typically found as combinations, justifying studies with pharmaceutical combinations of drugs and APIs [60].

3.5. Environmental Risk Assessment

Antiretrovirals can reach the aquatic environment through improper disposal of medications, effluents from sewage treatment plants, and human excreta containing drug residues. ARVs have been detected worldwide in different bodies of water, mainly in surface water, groundwater, domestic, hospital, and industrial effluents, and treated and untreated effluents. Table 9 shows the environmental risk assessment of ARVs DTG, 3TC, and TDF on the aquatic organism M. novacekii (Table 9).
DTG is found in very low amounts in the environment, possibly below the detection limit, hindering its measurement, even with an excretion rate of 95% [61]. However, its environmental concentration in different effluents in Portugal was estimated, through consumption, as between 0.004 and 0.071 µg/L [12]. In South Africa, the mean annual consumption of an adult is 18 kg of DTG, of which 53% is released unchanged in feces and 25% as by-products in urine [62].
In Belo Horizonte, Brazil, the annual estimate of ARV DTG elimination in the environment was 6561.4 kg and 2020. The growing elimination over the years derived from the drug’s implementation in the HIV treatment protocol in Brazil in 2017 [63].
Although DTG was the API with the most significant toxicity in M. novacekii among the ARVs evaluated in this study, classified as “toxic” for M. novacekii [42], considering its environmental concentration, it has low environmental risk. However, this risk may be greater if the impact that DTG on cellular metabolism is considered since low concentrations (0.1 mg/L) were sufficient to reduce cellular activity.
TDF is an antiretroviral widely used worldwide. As a result, residues of these ARVs have been identified in surface waters in South Africa at concentrations ranging from 0.110 to 0.243 µg/L [4,9] and were detected in surface waters in Romania and Croatia [11]. Its elimination is relatively higher than that of other ARVs consumed worldwide.
In Belo Horizonte, Brazil, the estimated annual elimination of the ARV TDF into the environment was approximately 1000 kg in 2020, about 8 times more than DTG in the same year. This amount of ARV eliminated into the environment results from its high elimination in unchanged form in urine (80%), corresponding to 31% of all ARV released in the city [63]. In South Africa, the mean annual consumption of an adult is 110 kg of TDF, 6 times greater than the consumption of DTG [62].
The TDF API is practically non-toxic to M. novacekii [42]. Furthermore, based on the environmental concentrations reported in the literature, the environmental risk of TDF is virtually non-existent or low. Even with the significant change in cellular metabolic activity caused by exposure to ARV, the required concentration was 10 mg/L of the TDF API, making this substance of low toxicity to M. novacekii and low risk to the environment.
Like TDF, 3TC is widely used globally and identified in different concentrations ranging from 0.016 to 228.3 µg/L in Germany [13], Kenya [6,8,14], Zambia [5], South Africa [9,22], and Belgium [64].
Its elimination is similar to TDF since they are administered in the same dosage and a single tablet in many places worldwide. In Belo Horizonte, Brazil, the estimated annual elimination of the 3TC ARV in the environment was approximately 983 kg in 2020, corresponding to 45% of all ARV released in the city [63]. In South Africa, the mean annual consumption of 3TC by an adult is equal to the consumption of TDF, 110 kg, of which 70% is eliminated in the urine unchanged and 5% as metabolites [62].
Environmental data on 3TC are more abundant in the literature. It is detected in low concentrations in Germany (0.02–0.06 µg/L) and in high concentrations in Kenya (0.7–228.3 µg/L) [22]. Because of this high variation in its environmental concentration, the environmental risk also becomes variable. In Africa (South Africa and Kenya), 3TC has a high environmental risk, while based on the EC50 values obtained in this study, it is classified as practically non-toxic to M. novacekii.
In this way, considering the possible environmental impacts, the data from this study may influence measures for regulations and the development of pharmaceutical waste removal techniques in wastewater treatment plants (WWTPs). The implementation of more effective and representative methods can significantly contribute to reducing environmental contamination and ensuring long-term water safety. Thus, investments in research and innovation are essential to address the challenges associated with chemical residues in wastewater [65,66].
Furthermore, although not explored in this study, the simultaneous presence of antiretrovirals (ARVs) and microcystins (MCs) produced by cyanobacteria (such as M. novacekii) in aquatic environments represents an environmental and public health challenge. Cyanobacteria of this genus can produce and release MCs, toxins that can negatively affect aquatic organisms and pose risks to human health, especially through the consumption of contaminated fish. The coexistence of ARVs and MCs can potentiate toxic effects, increasing the complexity of ecotoxicological impacts and complicating mitigation strategies. Furthermore, considering that ARVs are essential drugs for the treatment of HIV, their presence in water bodies raises concerns about persistent environmental contamination and chronic exposure of aquatic and human biota to these substances. Thus, understanding the interactions between ARVs and MCs becomes crucial to assess risks and develop effective strategies for monitoring and controlling this contamination [67].

4. Conclusions

The presence of ARVs in the environment and their toxicity to aquatic organisms such as M. novacekii highlights the ecotoxicological risks caused by dolutegravir, tenofovir, and lamivudine. In this study, DTG showed the greatest toxicity among the compounds tested, with significant effects on the inhibition of cell growth and metabolic activity of cyanobacteria, even at low concentrations. The results suggest that the toxic effects of DTG may be related to its molecular structure and lipophilic characteristics. On the other hand, TDF and 3TC evidenced lower toxicity in isolation, with adverse effects observed mainly in chronic exposures and at high concentrations.
However, the toxicity of ARVs increased significantly when combined, indicating a synergistic effect between the different compounds, especially in the combination of TDF and 3TC. This synergistic effect highlights the importance of evaluating ARV combinations since these drugs are rarely found alone in the aquatic environment. Furthermore, we underscore the role of excipients in commercial formulations of ARVs as they can increase and reduce the toxicity of the active substances. The identified excipients, such as sodium lauryl sulfate, have shown the potential to increase the toxicity of commercial formulations compared to the active pharmaceutical ingredients alone.
Although DTG revealed a greater risk to the cyanobacterium, the environmental risk assessment indicated that the TDF and 3TC ARVs, which had low toxicity to M. novacekii, when considering their environmental concentration, can be classified as substances with medium and high risk to the environment. Furthermore, continued use and inadequate disposal of these compounds may pose long-term risks to aquatic biodiversity and ecosystem integrity. These results reinforce the need for stricter regulation and monitoring strategies to control the release of ARVs into the environment, and the development of efficient treatments to remove ARV residues in wastewater treatment plants.
This study contributes to understanding the ecotoxicological impacts of the DTG, TDF, and 3TC ARVs and underscores the importance of running long-term toxicity assessments at environmentally relevant concentrations. Moreover, it highlights the need for further research to understand the effects of commercial formulations of ARVs on aquatic organisms, considering the potential impact of excipients and synergistic interactions between compounds.

Author Contributions

All authors contributed to the study’s conceptualization and design. Material preparation, data collection, and analysis were performed by G.S.-S. and M.D.A. The first draft of the manuscript was written by G.S.-S. and all authors (G.S.-S., M.D.A., C.R.d.S., C.P.d.S.M., K.P.N., C.A.d.J.P., M.P.G.M. and M.R.S.) commented on previous versions of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was founded by the Minas Gerais State Agency for Research and Development FAPEMIG (process n. APQ-03069-23 and BIP-00012-24) and National Council for Scientific and Technological Development CNPq (process n. 403853/2023-0).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We are grateful to the National Council for Scientific and Technological Development (CNPq) and the Minas Gerais State Research Support Foundation (FAPEMIG) for the essential funding that made this work possible. We recognize the importance of continued investment in scientific research for environmental preservation and public health, and we are grateful for their trust and encouragement in our work.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Physicochemical properties of the active pharmaceutical ingredients dolutegravir, tenofovir disoproxil fumarate, and lamivudine.
Table 1. Physicochemical properties of the active pharmaceutical ingredients dolutegravir, tenofovir disoproxil fumarate, and lamivudine.
InformationDTGTDF3TC
CAS number1051375-19-9147127-20-6134678-17-4
Molecular weight441.4 g/moL519.5 g/moL229.3 g/moL
Chemical formulaC20H18F2N3NaO5C19H30N5O10PC8H11N3O3S
pKa−0.5/10.14.1/18.64.3/14.3
Log KOW1.6−1.9−0.9
Water solubility269 mg/L13,400 mg/L70,000 mg/L
Melting point190–193 °C113–115 °C160–162 °C
Chemical structureWater 17 00815 i001Water 17 00815 i002Water 17 00815 i003
Notes: DTG = dolutegravir; TDF = tenofovir disoproxil fumarate; 3TC = lamivudine; CAS = Chemical Abstracts Service; pKa = acid constant logarithm; Log KOW = logarithm of the n-octanol-water partition coefficient.
Table 2. Test concentrations of active pharmaceutical ingredients and medication based on dolutegravir, tenofovir disoproxil fumarate, and lamivudine used in toxicity tests with the cyanobacterium M. novacekii.
Table 2. Test concentrations of active pharmaceutical ingredients and medication based on dolutegravir, tenofovir disoproxil fumarate, and lamivudine used in toxicity tests with the cyanobacterium M. novacekii.
Test substanceClassificationARV Test Concentrations (mg/L) **API Purity
DTGMedication45; 35; 25; 15; 10; 5; 0.5 and 0.1INOA
TDF + 3TC *Medication256; 128; 64; 32; 16; 8; 4; 2 and 1INOA
3TCMedication300; 200; 100; 50; 25; 10; 5 and 1INOA
TDFMedication256; 128; 64; 32; 16; 8; 4; 2 and 1INOA
DTGAPI50; 40; 30; 20; 10; 1; 0.1 and 0.01>95.0%
3TCAPI400; 300; 200; 100; 50; 25; 10; 5 and 1>99.5%
TDFAPI512; 256; 128; 64; 32; 16; 8; 4 and 2>99.8%
Notes: * = Single medication composed of two drugs; DTG = dolutegravir; TDF = tenofovir disoproxil fumarate; 3TC = lamivudine; ** = effective concentration tested; API = active pharmaceutical ingredient; INOA = information not openly available.
Table 3. Risk classification based on EC50 values determined from ecotoxicological tests with microalgae per the criteria established by Annex 2.28 B of the Globally Harmonized System of Classification and Labeling of Chemicals.
Table 3. Risk classification based on EC50 values determined from ecotoxicological tests with microalgae per the criteria established by Annex 2.28 B of the Globally Harmonized System of Classification and Labeling of Chemicals.
EC50 ValueToxicity Classification
≤1 mg/LHigh toxicity
>1–≤10 mg/LToxic
>10–≤100 mg/LLow toxicity
>100 mg/LVirtually non-toxic
Table 4. Results of the ecotoxicological assessment of the Active Pharmaceutical Ingredient (API) and commercial medication of different antiretrovirals on the cyanobacterium Microcystis novacekii through the cell growth inhibition assay.
Table 4. Results of the ecotoxicological assessment of the Active Pharmaceutical Ingredient (API) and commercial medication of different antiretrovirals on the cyanobacterium Microcystis novacekii through the cell growth inhibition assay.
ARVTimeEC50Statistical ModelClassification *
Active Pharmaceutical Ingredient (API)
DTG4 days1.7 ± 0.3 mg/LWeibullToxic
DTG14 days0.9 ± 0.4 mg/LWeibullToxic
TDF4 days130.7 ± 5.8 mg/Llog-normalVirtually non-toxic
TDF14 days147.0 ± 7.3 mg/LWeibullVirtually non-toxic
3TC4 days184.8 ± 22.1 mg/LWeibullVirtually non-toxic
3TC14 days>400 mg/Llog-logisticVirtually non-toxic
DTG + TDF4 days1.1 ± 0.4/11.7 ± 4.4 mg/Llog-normalToxic
DTG + TDF14 days21.0 ± 3.2/214.6 ± 32.8 mg/LWeibullLow toxicity
DTG + 3TC4 days1.4 ± 0.8/11.4 ± 6.2 mg/LWeibullToxic
DTG + 3TC14 days9.6 ± 1.0/77.0 ± 7.8 mg/Llog-normalToxic
TDF + 3TC4 days>256 mg/LWeibullVirtually non-toxic
TDF + 3TC14 days>256 mg/Llog-logisticVirtually non-toxic
DTG + TDF + 3TC4 days0.9 ± 0.1 mg/Llog-logisticHigh toxicity
DTG + TDF + 3TC14 days3.5 ± 0.5 mg/LWeibullToxic
Commercial medication
DTG4 days27.6 ± 5.4 mg/LWeibullLow toxicity
DTG14 days19.5 ± 1.8 mg/LWeibullLow toxicity
TDF4 days>256 mg/LWeibullVirtually non-toxic
TDF14 days>256 mg/Llog-normalVirtually non-toxic
3TC4 days60.3 ± 2.7 mg/Llog-normalLow toxicity
3TC14 days18.2 ± 3.5 mg/LWeibullLow toxicity
DTG + TDF4 days4.79 ± 0.73/28.71 ± 4.35 mg/Llog-logisticToxic
DTG + TDF14 days12.07 ± 1.34/72.42 ± 8.01 mg/LWeibullLow toxicity
DTG + 3TC4 days10.9 ± 0.7/65.5 ± 3.9 mg/Llog-logisticLow toxicity
DTG + 3TC14 days>100 mg/LWeibullVirtually non-toxic
TDF + 3TC4 days5.6 ± 0.3 mg/Llog-logisticToxic
TDF + 3TC14 days4.5 ± 0.2 mg/Llog-logisticToxic
TDF + 3TC **4 days51.6 ± 2.2 mg/Llog-logisticLow toxicity
TDF + 3TC **14 days68.7 ± 3.7 mg/Llog-normalLow toxicity
DTG + TDF + 3TC4 days5.6 ± 0.6/14.9 ± 0.2 mg/Llog-normalToxic
DTG + TDF + 3TC14 days14.9 ± 0.2/89.5 ± 1.1 mg/LWeibullLow toxicity
Notes: * = Toxicity classification criteria established by the guidelines of the Globally Harmonized System of Classification and Labeling of Chemicals [42]; ** = Commercial medication composed of two active pharmaceutical ingredients in the same tablet.
Table 5. Result expressed as an average of three experiments, in triplicate, of the absorbance of groups of cyanobacteria M. novacekii exposed to the active pharmaceutical ingredient (API) and the commercial medicine based on DTG at different concentrations.
Table 5. Result expressed as an average of three experiments, in triplicate, of the absorbance of groups of cyanobacteria M. novacekii exposed to the active pharmaceutical ingredient (API) and the commercial medicine based on DTG at different concentrations.
ClassificationTest Concentrations (mg/L)Absorbance (680 nm)
0 d4 d14 d
NC (0.0)0.1190.2612.027
Medication0.10.1180.2602.043
0.50.1200.2591.771
5.00.1170.2331.168
10.00.1190.2190.806
15.00.1200.1980.466
25.00.1190.1700.207
35.00.1180.0580.076
45.00.1200.0520.057
NC (0.0)0.1220.2722.066
API0.010.1180.2622.068
0.10.1200.2692.061
1.00.1170.1820.502
10.00.1160.1420.155
20.00.1180.0720.077
30.00.1120.0770.057
40.00.1220.0580.076
50.00.1260.0520.057
Table 6. Result of the metabolic activity assay using the MTT dye for the exposure of the cyanobacterium M. novacekii to the active pharmaceutical ingredients (API) dolutegravir (DTG), tenofovir disoproxil fumarate (TDF), and lamivudine (3TC) at concentrations of 0.1, 10, and 100 mg/L, respectively.
Table 6. Result of the metabolic activity assay using the MTT dye for the exposure of the cyanobacterium M. novacekii to the active pharmaceutical ingredients (API) dolutegravir (DTG), tenofovir disoproxil fumarate (TDF), and lamivudine (3TC) at concentrations of 0.1, 10, and 100 mg/L, respectively.
ConcentrationGroupAbsorbance (570 nm)Metabolic Inhibition
123Mean
0.0 mg/LControl0.6350.6080.6620.6350.0%
0.1 mg/LDTG0.2840.3020.2550.28055.85%
10 mg/LTDF0.5780.5550.5420.55812.07%
100 mg/L3TC0.6470.6330.6240.6350.05%
Table 7. Result expressed as an average of three experiments, in triplicate, of the absorbance of groups of cyanobacteria M. novacekii exposed to the active pharmaceutical ingredient (API) and the commercial medicine based on TDF at different concentrations.
Table 7. Result expressed as an average of three experiments, in triplicate, of the absorbance of groups of cyanobacteria M. novacekii exposed to the active pharmaceutical ingredient (API) and the commercial medicine based on TDF at different concentrations.
ClassificationTest Concentrations (mg/L)Absorbance (680 nm)
0 d4 d14 d
NC (0.0)0.1940.3042.108
Medication1.00.1950.3162.131
2.00.1950.3102.094
4.00.1940.3102.132
8.00.1960.3132.125
16.00.1950.3092.142
32.00.1950.3122.127
64.00.1940.3112.135
128.00.1940.3182.127
256.00.1960.3082.107
NC (0.0)0.1790.2862.000
API2.00.1800.2881.993
4.00.1800.2881.997
8.00.1800.2881.996
16.00.1810.2821.970
32.00.1790.2711.961
64.00.1810.2641.822
128.00.1790.2251.179
256.00.1800.1280.089
512.00.1790.0550.056
Table 8. Result expressed as an average of three experiments, in triplicate, of the absorbance of groups of cyanobacteria M. novacekii exposed to the active pharmaceutical ingredient (API) and the commercial medicine based on 3TC at different concentrations.
Table 8. Result expressed as an average of three experiments, in triplicate, of the absorbance of groups of cyanobacteria M. novacekii exposed to the active pharmaceutical ingredient (API) and the commercial medicine based on 3TC at different concentrations.
ClassificationTest Concentrations (mg/L)Absorbance (680 nm)
0 d4 d14 d
NC (0.0)0.1390.2161.888
Medication1.00.1400.2191.922
5.00.1400.2171.885
10.00.1380.2080.493
25.00.1390.1970.371
50.00.1400.1730.298
100.00.1400.1590.125
200.00.1380.1410.115
300.00.1400.1010.088
NC (0.0)0.1350.3312.108
API1.00.1330.3362.121
5.00.1340.3192.111
10.00.1320.3182.081
25.00.1310.2962.097
50.00.1340.2932.112
100.00.1310.2332.107
200.00.1360.2142.103
300.00.1320.1272.123
400.00.1320.1132.107
Table 9. Assessment of the acute environmental risk in surface waters of the antiretrovirals dolutegravir (DTG), tenofovir (TDF), and lamivudine (3TC) on the aquatic organism Microcystis novacekii based on the EC50 values of the isolated active ingredients.
Table 9. Assessment of the acute environmental risk in surface waters of the antiretrovirals dolutegravir (DTG), tenofovir (TDF), and lamivudine (3TC) on the aquatic organism Microcystis novacekii based on the EC50 values of the isolated active ingredients.
ARVLocalConcentration (µg/L)PNEC (µg/L)RQs
DTG *PortugalMin. = 0.004
Max. = 0.071		1.720.002 [2]
0.004 [2]
TDFSouth AfricaMin. = 0.110
Max. = 0.250		130.72<0.001 [1]
0.002 [2]
3TCKenyaMin. = 0.700
Max. = 228.30		184.820.004 [2]
1.235 [4]
3TCSouth AfricaMin. = 0.040
Max. = 33.99		184.82<0.001 [1]
0.183 [3]
3TCU.S.AMin. = 0.016
Max. = 0.150		184.82<0.001 [1]
<0.001 [1]
3TCGermanyMin. = 0.020
Max. = 0.060		184.82<0.001 [1]
<0.001 [1]
Notes: RQ = Risk Quotient; PNEC = Predicted No-Effect Concentrations; * environmental estimation data; Min. = Minimum concentration of ARV detected; Max. = Maximum concentration of ARV detected; [1] = Negligible risk; [2] = Low risk; [3] = Medium risk; [4] = High risk.
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Souza-Silva, G.; Alcantara, M.D.; Souza, C.R.d.; Moreira, C.P.d.S.; Nunes, K.P.; Pereira, C.A.d.J.; Mol, M.P.G.; Silveira, M.R. Toxicity of the Antiretrovirals Tenofovir Disoproxil Fumarate, Lamivudine, and Dolutegravir on Cyanobacterium Microcystis novacekii. Water 2025, 17, 815. https://doi.org/10.3390/w17060815

AMA Style

Souza-Silva G, Alcantara MD, Souza CRd, Moreira CPdS, Nunes KP, Pereira CAdJ, Mol MPG, Silveira MR. Toxicity of the Antiretrovirals Tenofovir Disoproxil Fumarate, Lamivudine, and Dolutegravir on Cyanobacterium Microcystis novacekii. Water. 2025; 17(6):815. https://doi.org/10.3390/w17060815

Chicago/Turabian Style

Souza-Silva, Gabriel, Mariângela Domingos Alcantara, Cléssius Ribeiro de Souza, Carolina Paula de Souza Moreira, Kenia Pedrosa Nunes, Cíntia Aparecida de Jesus Pereira, Marcos Paulo Gomes Mol, and Micheline Rosa Silveira. 2025. "Toxicity of the Antiretrovirals Tenofovir Disoproxil Fumarate, Lamivudine, and Dolutegravir on Cyanobacterium Microcystis novacekii" Water 17, no. 6: 815. https://doi.org/10.3390/w17060815

APA Style

Souza-Silva, G., Alcantara, M. D., Souza, C. R. d., Moreira, C. P. d. S., Nunes, K. P., Pereira, C. A. d. J., Mol, M. P. G., & Silveira, M. R. (2025). Toxicity of the Antiretrovirals Tenofovir Disoproxil Fumarate, Lamivudine, and Dolutegravir on Cyanobacterium Microcystis novacekii. Water, 17(6), 815. https://doi.org/10.3390/w17060815

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