Degradation Of Hormone 17β-Estradiol by Photolysis (UV) and Advanced Oxidative Process (Uv/H2O2), and Subsequent Toxicity Analysis

DOI : 10.17577/IJERTV11IS110032
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Degradation Of Hormone 17β-Estradiol by Photolysis (UV) and Advanced Oxidative Process (Uv/H2O2), and Subsequent Toxicity Analysis

Bruna Costa1, Lucélia Hoehne2

1 Undergraduation in Chemical Engineering – UNIVATES

2 Biotechnology Graduate Couse – UNIVATES

Abstract: – Population growth has led to the development of several industry sectors, which helps in the significant increase in the volume of waste generated at risk of contamination of the environment. Within this context, the theme of micropollutants was born, which researchers from all over the world began to conduct studies on their incidence and possible effects. The literature shows that micropollutants are found in different environmental matrices (water, soil, air) in concentrations of microgram or nanogram per liter, and its main characteristic is persistence in the environment in which it is located. This persistence generates a low rate of removal of these contaminants in conventional water and effluent treatment plants, which makes the theme receive even more attention. Although these substances are found at low concentrations, they are able to deregulate the biological balance of the ecosystem through chronic and acute effects in several organisms. Substances such as 17-Estradiol (E2), a female natural hormone with great potential to act as an endocrine disruptor in different organisms. Several studies on the toxicity of this compound show that it has harmful effects, including changes in gonads, hermaphroditism and feminization of fish, turtles and mussels, as well as prostate cancer in mammals. Therefore, the objective of this work was to evaluate the degradation of the hormone 17-Estradiol using photolysis (UV) and advanced oxidative process (UV/H2O2) in batch reactor, and toxicity analysis against Artemia salina microcrustacean. The tests were performed from solutions containing 100 mg/L of E2 in all tests, and in the tests containing H2O2, the oxidant was used at concentrations of 70, 100 and 300 mg/L. The tests containing only UV radiation showed about 4.45% of contaminant removal, while the highest concentration tested with oxidant showed removal of 72% of E2. Toxicity tests showed that the diluted test solutions did not present toxicity to the bioindicator. The results obtained suggest that the UV/H2O2 assay is capable of promoting a greater removal of E2 as the oxidant concentration increases and is capable of increasing the quality of the treated effluent.

Keywords: Micropollutants. Estrogen hormones. Effluent treatment. Ecotoxicity.


Population growth tied to the constant search for improvement in quality of life has led to the development of several industrial sectors, and thereby increasing the volume of waste generated with high risk of contamination of the environment. The theme of these contaminants, classified as emerging contaminants or micropollutants, has gained prominence since the last decade, which has caused many researchers around the world to approach this theme in different aspects. These compounds are found in several environmental matrices (soil, water, air) in concentrations of nanogram (ng/L) or microgram per liter (g/L) and can be both of anthropic origin (domestic, industrial, hospital) and natural effluents. Although these substances are found in low concentrations, they are capable of disrupting or deregulating the biological balance of the ecosystem (MONTAGNER; VIDAL; ACAYABA, 2017; KEYS, 2018; SERVIEN et al., 2022).

Within this group of contaminants are endocrine disruptors (EDs), which is found in many types of substances such as natural and synthetic hormones, drugs, pesticides, phthalates, phytoestrogens, bisphenols, among others. Its main feature is the ability to assume identical function to a natural hormone and thereby interfere in the synthesis, secretion, transport, action or elimination of natural hormones that are responsible for maintaining the body. Included in this group, the natural estrogen 17- Estradiol (E2) and the synthetic 17-Ethinylestradiol (EE2) are the EDs that are most concerned both by the influence and by the amount introduced into the environment (BLEDZKA et al. 2010; BIRTH, 2015; KEYS, 2018).

The possible harmful effect on human and ecological health of endocrine disruptors has been mentioned since the 1960s, but received significant attention from the international community only in the 1990s, when there were reports on the occurrence of decreased sperm quality and feminization of fish in contact with wastewater effluents from treatment plants (ANKLEY; TYLER, 2013). A study using “paulistinha” fish (Danio rerio) showed that chronic exposure of young males at low concentrations (1.5 ng/L) of EE2 reversed the sex of males in phenotypic females (PAIVA; DE SOUZA; VAN HAANDEL, 2011). The literature also reports that the presence of EE2 can cause prostate cancer in humans, while E2 may be responsible for the change in the functions of digestive glands in mussels (CANESI et al., 2008; TESKE; ARNOLD, 2008).

Since EDs are persistent compounds, research shows that their removal in effluent treatment plants may not be complete, as conventional stations are not designed for removal of these organic compounds. It follows that, there is a need to develop efficient methods of removal and or degradation, such as nanofiltration, adsorption in activated carbon, photolysis or advanced oxidative processes (AOPs). The latter have been effective in the degradation of different micropollutants, having a variety of combinations, such as ultraviolet (UV) and H2O2, Fe+2 and H2O2, Fe+2 and UV/H2O2, heterogeneous systems, among others, with the UV/H2O2 combination being the most prominent for degradation of organic compounds (BLEDZKA, 2010; DAYS, 2014;

PISHARODY et al., 2022).

However, there are few reports in the literature on studies for hormone degradation, along with toxicity analysis in the final treatment. Thus, it is important to develop effective AOPs that ensure a safe and effective treatment (DIAS, 2014; MENON et al., 2021). Based on this, the objective of this work was to evaluate the degradation of the hormone 17-Estradiol using photolysis (UV) and advanced oxidative process (UV/H2O2) in batch reactor, and toxicity analysis against the microcrustacean Artemia salina.

Preparation of solutions


The stock solution with concentration of 1000 mg/L of E2 (Sigma-Aldrich CAS 50-28-2) was prepared in a solvent system containing ultrapure water (Milli-Q, Millipore, USA) and ethanol (Scientific Êxodo) in the ratio of 1:1 v/v and 1 mL of a sodium hydroxide solution (Scientific Êxodo) 10 mol/L.

For the degradation tests, the stock solution was diluted in ultrapure water at a concentration of 100 mg/L. In the UV/H2O2

assays, the H2O2 concentration was adjusted with a 33% hydrogen peroxide solution (Analytical Standard, Scientific Exodus).

For residual peroxide assays, 0.06 mol/L ammonia metavanadade solutions were prepared and a stock solution containing 5000 mg/L of H2O2 was prepared, according to the Nogueira, Oliveira and Peterlini method (2005).

For the ecotoxicity tests, positive, negative and test solution solutions were prepared, according to NBR ABNT 16530/2016. The positive control was performed with copper sulfate pentahydrate solution (Synth) at a concentration of 1 g/L, the negative control was performed with a solution containing 1.5 g/L of sea salt and the test solutions were prepared from the solution that presented the highest degradation during the tests. The concentrations of the test solutions were 100%, 50%, 25% and 12.5%, bth diluted with the negative control solution.

Reactor assembly

The reactor used for the photolysis and AOP tests was adapted from Cordeiro et al., (2021). It is a batch bench reactor with a maximum sample capacity of 250 mL, as illustrated in Figure 1. Inside the reactor was inserted a quartz tube with 14 cm long and 4 cm in diameter, and inside the quartz tube a mercury vapor lamp (Osram, 125W, max=254 nm) of 125 W as a source of UVC radiation. The system was kept under agitation, and the temperature was maintained at 22 ºC.

Figure 1 – Reactor structure for degradation testing

Photolysis test

Source: Cordeiro et al. (2021).

The conditions for the photolysis test were adapted by Liu and Liu (2004). The E2 test solution was prepared from the stock solution at a concentration of 100 mg/L. 100 mL of the test solution was inserted into the reactor, and it was irradiated for 60 minutes. To evaluate the degradation, aliquots of 3 mL were removed at different time intervals, and each aliquot was immediately inserted in a Molecular Absorption Spectrophotometer in the UV/Visible region and its absorbance was read at 297 nm.

UV/H2O2 Test

The tests using advanced oxidative process were performed under the conditions established in Table 1, both in triplicate.

Table 1 – Conditions established for UV/H2O2 assays

Essay Concentration E2 (mg/L) Concentration H2O2 (mg/L) pH Irradiation time (min) Sample volume (mL)
1 100 70 12 60 100
2 100 100 12 60 100
3 100 300 12 60 100

Source: Authors (2022).

Residual peroxide test

The tests to evaluate the residual peroxide content of the samples after irradiation tests were performed based on the colorimetric method of Nogueira, Oliveira and Peterlini (2005). The method consists of the reaction between the vanadate ion with hydrogen peroxide in acid medium, which leads to the formation of peroxivanadium cation, responsible for the reddish staining of the solution.

Ecotoxicity test

Ecotoxicity tests were performed based on adaptations based on ABNT NBR 16530/2016. Initially, the cysts of A. salina submitted to hatching for 24 hours in an aquarium containing sea salt solution with a concentration of 30 g/L. The aquarium was kept under constant aeration, with luminosity and temperature of 24±5 ºC.

After the period necessary for cyst hatching, the definitive assay was performed on plates of 24 culture wells with a capacity of 3 mL of solution. For this, 10 microcrustacean nauplii were transferred to each test well containing 5 negative control replicas, 5 positive control replicas and 5 replicas of each concentration of test solution used.

The plates were left in incubation for a period of 24 hours, and then the counting of dead or unmobile organisms was performed.

Photolysis test


The photodegradation of E2 occurs probably through the absorption of photons of ultraviolet radiation, which causes it to become reactive (LIU et al., 2017). Figure 2 shows that the assay was able to remove about 4.45% of the initial E2 concentration after 60 minutes of irradiation. The literature shows that this hormone may present low degradation by photolysis, depending on the conditions and time submitted to the test. Liu et al. (2017), evaluated that the half-life of E2 is 5.32 h in the presence of light intensity of 500 mW/cm².

Figure 2 – E2 concentration profile during photolysis. Conditions: C0 E2 = 100 mg/L; pH = 12; Temperature = 22 ºC; UVC irradiation.



C/C0 E2






Source: Authors (2022).


0 10 20 30 40 50 60

Time (min)

In 2018, Chaves evaluated the degradation of solutions containing 100 g/L of E2 by photolysis, and obtained about 40% of contaminant removal after 60 minutes, reaching 85% degradation only after 120 minutes of testing. Liu and Liu (2004) performed photolysis tests on solutions containing 100 mg/L of E2 with conditions similar to this work, and achieved a 40% removal rate after 60 minutes of testing. With this, it can be observed that, although photolysis tests present a certain degradation, they are still not able to promote a high removal of E2 within 60 minutes of testing, further motivating the search for other methods.

UV/H2O2 Test

The use of combined UV/H2O2 systems are widely used in advanced oxidative processes in order to increase the efficiency of contaminant removal in effluents. Hydroxyl radicals formed from the photolysis of H2O2 have high oxidation capacity, besides having low selectivity, which makes it versatile for the removal of several classes of organic contaminants (LIN et al., 2022).

Figure 3 shows that there was degradation of the hormone E2 over time in all concentrations of H2O2 tested according to Table 1. It can be observed that the higher the concentration of hydrogen peroxide present in the test solution, the greater the degradation of the hormone, reaching approximately 72% of E2 removal in the condition containing 300 mg/L of H2O2.

Figure 3 – E2 concentration profile during UV/H2O2 test. Conditions: C0 E2 = 100 mg/L; pH = 12; Temperature = 22 ºC; UVC irradiation.

1 2 3












0 10 20 30 40 50 60 70

Time (min)


C/C0 E2


Source: Authors (2022).

Similar results were obtained by Ma et al. (2022) when evaluating the removal efficiency of hormone E2 using UV and H2O2 at different concentrations. During 60 minutes of testing, they obtained about 80% degradation of a solution containing 50 g/L of E2 and 15 mg/L of H2O2. The same work observed that when the dose of H2O2 was increased, removal efficiencies also increased, since there was a greater amount of hydroxyl radicals available to react with the hormone. In 2021, Bohrer evaluated E2 degradation in a system containing 8 mg/L of contaminant and 1.8 g/L of H2O2, and obtained about 95% e2 removal during 60 minutes of testing.



Although the above-mentioned studies observed an increase in removal efficiency when there was an increase in the concentration of H2O2, it is important to observe the proportion between contaminant and oxidant concentrations. This is because when in excess, The H2O2 acts as a sequester of hydroxyl radicals and when reacting, forms the hydroperoxyl radical (HO ), which is less reactive and thus impairs the efficiency of the process (CHAVES, 2018).

Hansen and Andersen (2012) evaluated the removal of E2 using UV/H2O2 for concentrations of 10, 30, 60 and 100 mg/L of H2O2 and concluded that the increase in oxidant concentration from 10 to 60 mg/L was accompanied by increased contaminant removal, but by adding 100 mg/L of H2O2 the compounds have been reduced in their removal rate compared to the results of 60 mg/L.

Table 2 shows the results obtained through residual peroxide analyses. It is observed that both tests consumed about 93% of the initial peroxide concentration. This indicates that possibly the removal efficiency has been stabilized due to the low availability of free hydroxyl radicals for reaction. Chaves (2018) evaluated the degradation of a solution containing 100 g/L of E2 and 3 mg/L of H2O2 and reached 100% removal after 20 minutes of testing, however, only 23% of the available hydrogen peroxide was consumed during the process.

Table 2 – Residual H2O2 content of the tested samples

Test Contaminant Concentration (mg/L) H2O2Initial(mg/L) H2O2 Residual (mg/L) Consumed (%)
1 100 70 4.505 ± 0.631 93
2 100 100 6.323 ± 0.175 93
3 100 300 20.263 ± 0.350 93

Source: Authors (2022).

Therefore, it is noted that there is a tendency to increase the efficiency of removal of E2 in the conditions of the present study, if there is an increase in the concentration of H2O2 in the system.

Ecotoxicity test

The main objective of advanced oxidative processes is the degradation of persistent substances with toxic potential to organisms that are exposed, however, the degradation of these compounds does not indicate that their mineralization was complete or that their sub-products have lower toxicity. In addition, the identification of intermediate compounds formed during reactions is challenging, making toxicity tests accessible to provide information about the risks that degraded substances can cause (WANG; WANG, 2019).

Table 3 represents the results obtained from the exposure of the microcrustacean A. salina control solutions and test solutions. It is observed that the treatment used to E2 presents toxicity to the bioindicator at its highest concentration of exposure, and as the concentration decreases and the dilution of the test solution increases, the toxic effect decreases until it does not present toxic effect, as observed in the lower concentration of test solution.

Table 3 – Average number and standard deviation of organis ms killed or without mobility.
Group Concentration (%) Average ± Standard deviation
Negative control 100 0.00 ± 0.00
Positive control 100 10.00 ± 0.00
100 10.00 ± 0.00
Test solution 50 7.00 ± 0.00
25 1.00 ± 1.00
12,5 0.00 ± 0.00

Source: Authors (2022).

In 2021, Bohrer evaluated the toxicity of E2 solutions degraded by different advanced oxidative processes, and observed that the tests at the concentration of 100% showed high toxicity (10.00 ± 0.00), but in the first dilution containing 50% the solutions no longer presented toxicity, and remained so in the other concentrations.

Although the literature does not present too many reports of toxicity tests with solutions containing E2 before or after degradation tests, the importance of conducting toxicity tests even with intact molecules is emphasized. A study conducted by Lima (2019) showed that solutions containing acetylsalicylic acid (200 mg/L) and paracetamol (95 mg/L) showed toxicity against the bioindicator A. salina in all tested conditions. The use of this microcrustacean as a bioindicator is useful in the preliminary toxicity assessment of a given sample, however, the assay presents only acute response information, that is, in a short period of time. Thus, it is necessary to use complementary tests, as well as the variation of the species of the tested organisms in order to determine, in addition to immediate effects, the chronic effects of these substances (SILVEIRA et al., 2020).


It was observed that photolysis and the advanced oxidative process UV/H2O2 are able to degrade solutions containing E2 at different removal rates. Photolysis showed reduced efficiency in the degradation of the evaluated compound, presenting about 4.45% of total removal.

The UV/H2O2 process showed low degradation at the lowest Concentrations of H2O2, however, in the highest concentration used reached the rate of 73% of E2 removal. Residual peroxide assays showed that in all tests there was a consumption of 93% of the available H2O2, indicating that the removal rate may have stabilized due to the low availability of hydroxyl radicals for reaction.

The ecotoxicity assays showed that the greater the dilution of the degraded solution, the lower the mortality effect against the Artemia salina bioindicator. Thus, it can be concluded that the UV/H2O2 process did not form toxic by-products for the microcrustacean under the tested conditions.


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